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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY <PCT) 



{51) International Patent Classification 6 : 
C12N 5/00, 5/02 



Al 



(11) International Publication Number: WO 97/40137 

(43) International Publication Date: 30 October 1997 (30.10.97) 



(21) International Application Number: PCT/US97/06433 

(22) International Filing Date: 17 April 1997 (17.04.97) 



(30) Priority Data: 
60/016,245 
60/029,838 



19 April 1996 (19.04.96) US 
28 October 1 996 (28. 1 0.96) US 



(60) Parent Applications or Grants 

(63) Related by Continuation 
US 

Filed on 
US 

Filed on 



60/016,245 (CIP) 
19 April 19% (19.04.96) 
60/029.838 (CIP) 
28 October 1996 (28.10.96) 



(71) Applicant (for all designated States except US): OSIRIS 

THERAPEUTICS, INC. (US/US]; 2001 Aliceanna Street, 
Baltimore, MD 21231-2001 (US). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): KADIYALA, Sudha 
(IN/US]; 1531 Lancaster Street, Baltimore, MD 21231 
(US). BRUDER, Scott, P. [US/US]; 3698 Ashley Way, 
Owings Mills, MD 21 117 (US). MUSCHLER, George, F. 



[US/US]; 2270 Chatfield Road, Cleveland Heights, OH 
44106 (US). 

(74) Agents: HERRON, Charles, J. et al.; Carella. Byrne, Bain, 
Gilfillan, Cecchi, Stewart & Olstein, 6 Becker Farm Road. 
Roseland, NJ 07068 (US). 



(81) Designated States: AU, CA, JP, US, European patent (AT. BE, 
CH, DE, DK, ES, FI, FR, GB, GR, IE, IT. LU, MC NL 
PT, SE). 



Published 

With international search report. 



03 

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m 

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(54) Title: REGENERATION AND AUGMENTATION OF BONE USING MESENCHYMAL STEM CELLS 
(57) Abstract 

/uK*c/?l SCl °^f d m com P° sitions and methods for augmenting bone formation by administering isolated human mesenchymal stem cells 
(hMSCs) with a ceramic material or matrix or by administering hMSCs; fresh, whole marrow; or combinations thereof in a resorbable 
biopolymer which supports their differentiation into the osteogenic lineage. Contemplated is the delivery of (i) isolated, culture-expanded 
human mesenchymal stem cells; (ii) freshly aspirated bone marrow; or (iii) their combination in a carrier material or matrix to provide 
for improved bone fusion area and fusion mass, when compared to the matrix alone. The material or matrix can be a granular ceramic 
or mrecKhmensionally formed ceramic implant. The material or matrix can also be a resorbable biopolymer. The resorbable biopolymer 
is an absorbable gelatin, colagen or cellulose matrix, can be in the form of a powder or sponge, and is preferably a bovine skin-derived 
gelatin. The implants can be shaped as a cube, cylinder, block or an anatomical site. The compositions and methods can further include 

^Tfo r wi a . bl0 ! C Il^ faCt0r Such aS a svmhetic glucocorticoid, like dexamethasone, or a bone morphogenic protein, like BMP-2 BMP-3 
BMP-4, BMP-6 and BMP-7. ' " ' 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify Stales party to the PCT on the front pages of pamphlets publishing international applications under the PCT. 



At 


Albania 


ES 


Spam 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


Fl 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


PR 


France 


LV 


Ijjxcmbourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


CE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


Gil 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


IIU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


vz 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzsian 


NO 


Norway 


ZW 


Zimbabwe 


CI 


Cote d'lvoirc 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






CU 


Cuba 


KZ 


Kaz&ksian 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


KU 


Russian Federation 






DE 


Germany 


LI 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


IJC 


Sri Lanka 


SE 


Sweden 






EE 


Esionia 


LR 


Liberia 


SG 


Singapore 







WO 97/40137 



PCT/US97/06433 



REGENERATION AND AUGMENTATION OF BONE 
USING MESENCHYMAL STEM CELLS 

This application is a continuation-in-part of U.S. provisional application serial 
no. 60/016,245, filed April 19, 1996 and U.S. provisional application serial no. 
60/029,838 filed October 28, 1996. 

Autologous, culture-expanded, bone marrow-derived MSCs have now been 
shown to regenerate clinically significant bone defects. Using techniques for 
isolating and cultivating human MSCs, it should be possible to implement therapeutic 
strategies based on the administration of a patient's own cells which have been 
harvested by a simple iliac crest aspiration. This method may provide an alternative 
to autogenous bone grafting, and will be particularly useful in clinical settings such 
as ageing and osteoporosis, where the number and/or function of endogenous MSCs 
have been reduced. 

The repair of large segmental defects in diaphyseal bone is a significant 
problem faced by orthopaedic surgeons. Although such bone loss may occur as the 
result of acute injury, these massive defects commonly present secondary to 
congenital malformations, benign and malignant tumors, osseous infection, and 
fracture non-union. The use of fresh autologous bone graft material has been viewed 



WO 97/40137 



PCT/US97/06433 



as the historical standard of treatment but is associated with substantial morbidity 
including infection, malformation, pain, and loss of function (72,149). The 
complications resulting from graft harvest, combined with its limited supply, have 
inspired the development of alternative strategies for the repair of clinically 
significant bone defects. The primary approach to this problem has focused on the 
development of effective bone implant materials. 

Three general classes of bone implants have emerged from these 
investigational efforts, and these classes may be categorized as osteoinductive, 
osteoinductive, or directly osteogenic. Allograft bone is probably the best known 
type of osteoconductive implant. Although widely used for many years, the risk of 
disease transmission, host rejection, and lack of osteoinduction compromise its 
desirability (76). Synthetic osteoconductive implants include titanium fibermetalsand 
ceramics composed of hydroxyapatite and/or tricalcium phosphate. The favorably 
porous nature of these implants facilitate bony ingrowth, but their lack of 
osteoinductive potential limits their utility. A variety of osteoinductive compounds 
have also been studied, including demineralized bone matrix (34,45), which is known 
to contain bone morphogenic proteins (BMP). Since Urist's original discovery of 
BMP (138), others have characterized, cloned, expressed, and implanted purified or 
recombinant BMPs in orthotopic sites for the repair of large bone defects 
(44,126,146,147). The success of this approach has hinged on the presence of 
mesenchymal cells capable of responding to the inductive signal provided by the 
BMP (74,129). It is these mesenchymal progenitors which undergo osteogenic 
differentiation and are ultimately responsible for synthesizing new bone at the 
surgical site. 

One alternative to the osteoinductive approach is the implantation of living 
cells which are directly osteogenic. Since bone marrow has been shown to contain 
a population of cells which possess osteogenic potential, some have devised 
experimental therapies based on the implantation of fresh autologous or syngeneic 
marrow at sites in need of skeletal repair (26,27,49,105,113,144,145). Though 
sound in principle, the practicality of obtaining enough bone marrow with the 
requisite number of osteoprogenitor cells is limiting. 



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Summary of the Invention 

The present invention provides compositions and methods for directing MSCs 
cultivated in vitro to differentiate into specific cell lineage pathways prior to, and/or 
at the time of, their implantation for the therapeutic treatment of elective procedures 
or pathologic conditions in humans and other species. The use of both autologous 
and allogenic MSCs is contemplated in this invention. 

The investigations reported here confirm the in vitro and in vivo osteogenic 
potential of MSCs; demonstrate the in vivo osteogenic potential of MSCs when 
implanted at an ectopic subcutaneous site; and illustrate that purified, culture- 
expanded MSCs can regenerate a segmental bone defect which would otherwise 
result in a clinical non-union. These experiments compared the healing potential of 
MSCs delivered in an osteoinductive, osteoinductive or other appropriate resorbable 
medium. We also show de novo formation of bone at the site of a desired fusion, 
e.g. spinal or joint fusions. 

The invention provides a method for augmenting bone formation in an 
individual in need thereof by administering isolated human mesenchymal stem cells 
with a matrix which supports the differentiation of such stem cells into the osteogenic 
lineage to an extent sufficient to generate bone formation therefrom. The matrix 
is preferably selected from a ceramic and a resorbable biopolymer. The ceramic can 
be in paniculate form or can be in the form of a structurally stable, three 
dimensional implant. The structurally stable, three dimensional implant can be, for 
example, a cube, cylinder, block or an appropriate anatomical form. The resorbable 
biopolymer is a gelatin, collagen or cellulose matrix, can be in the form of a powder 
or sponge, and is preferably a bovine skin-derived gelatin. 

Particularly, the invention provides a method for effecting the repair or 
regeneration of bone defects in an animal or individual in need thereof. Such defects 
include, for example, segmental bone defects, non-unions, malunions or delayed 
unions, cysts, tumors, necroses or developmental abnormalities. Other conditions 
requiring bone augmentation, such as joint reconstruction, cosmetic reconstruction 
or bone fusion, such as spinal fusion or joint fusion, are treated in an individual by 



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WO 97/40137 PCT/US97/06433 

administering, for example into the site of bone in need of augmentation, fresh whole 
marrow and/or isolated human mesenchymal stem cells or combinations thereof in 
the gelatin, cellulose or collagen based medium to an extent sufficient to augment 
bone formation therefrom. The composition can also contain one or more other 
components which degrade, resorb or remodel at rates approximating the formation 
of new tissue. 

The invention also contemplates the use of other extracellular matrix 
components, along with the cells, so as to achieve osteoconduction or osteoinduction. 
In addition, by varying the ratios of the components in said biodegradable matrices, 
surgical handling properties of the cell-biomatrix implants can be adjusted in a range 
from a dimensionally stable matrix, such as a sponge or film, to a powder. 

The above method can further comprise administering to the individual at 
least one bioactive factor which induces or accelerates the differentiation of 
mesenchymal stem cells into the osteogenic lineage. The MSCs can be contacted 
with the bioactive factor ex vivo and are preferably contacted with the bioactive 
factor when the MSCs are in contact with the matrix. The bioactive factor can be, 
for example, a synthetic glucocorticoid, such as dexamethasone, or a bone 
morphogenic protein, such as BMP-2, BMP-3, BMP-4, BMP-6 or BMP-7. The 
bone morphogenic protein can be in a liquid or semi-solid carrier suitable for 
intramuscular, intravenous, intramedullary or intra-articular injection. 

The invention further provides acomposition for augmenting bone formation, 
which composition comprises a matrix selected from the group consisting of 
absorbable gelatin, cellulose and collagen in combination with at least one of fresh 
bone marrow and/or isolated mesenchymal stem cells. The composition can be used 
in the form of a sponge, strip, powder, gel or web. The invention also provides a 
method for augmenting bone formation in an individual in need thereof by 
administering to said individual a bone formation augmenting amount of the 
composition. 



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PCT/US97/06433 



More particularly, the invention provides a method for effecting the repair 
of segmental bone defects, non-unions, malunions or delayed unions in an individual 
in need thereof by administering into the bone defect of said person isolated human 
mesenchymal stem cells in a porous ceramic carrier, thereby inducing the 
differentiation of such stem cells into the osteogenic lineage to an extent sufficient 
to generate bone formation therefrom. Preferably, the porous ceramic carrier 
comprises hydroxyapatite and, more preferably, the porous ceramic carrier further 
comprises /3-tricalcium phosphate. The porous ceramic carrier may also contain one 
or more other biodegradable carrier components which degrade, resorb or remodel 
at rates approximating the formation of new tissue extracellular matrix or normal 
bone turnover. 

The invention also provides for the use of other extracellular matrix 
components, or other constituents, so as to achieve osteoinductive or osteoinductive 
properties similar to natural extracellular matrix. The composition is an absorbable 
gelatin, cellulose and/or collagen-based matrix in combination with bone marrow 
and/or isolated mesenchymal stem cells. The composition can be used in the form 
of a sponge, strip, powder, gel, web or other physical format. The composition is, 
for example, inserted in the defect and results in osteogenic healing of the defect. 

In addition, by varying the ratios of the components in said biodegradable 
matrices, surgical handling properties of the cell-biomatrix implants xxn be adjusted 
in a range from a porous ceramic block or a moldable, putty-like consistency to a 
pliable gel or slurry. 

More particularly, the invention comprises a rigid cell-matrix implant for 
large segmental defects, spinal fusions or non-unions, gel or slurry cell-matrix 
implants, or infusions for stabilized fractures and other segmental bone defects. 
Custom cell-matrix implants containing autologous or allogeneic MSCs can be 
administered using open or arthroscopic surgical techniques or percutaneous 
insertion, e.g. direct injection, cannulation or catheterization. 



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PCT/US97/06433 



In a preferred embodiment, a composition of human mesenchymal stem cells 
(hMSCs) is obtained from either homogeneous, culture-expanded preparations 
derived from whole-marrow (or other pre-natal or post-natal source of autologous 
or allogeneic hMSCs), or from enriched or heterogenous cultures containing an 
effective dose of hMSCs. The key to effective clinical outcomes using MSC therapy 
is to provide that number of mesenchymal stem cells to the patient which repairs the 
bone or other tissue defect. This is referred to as the "Regenerative MSC 
Threshold", or that concentration of MSCs necessary to achieve direct repair of the 
tissue defect. The Regenerative MSC Threshold will vary by: 1) type of tissue 
(i.e., bone, cartilage, ligament, tendon, muscle, marrow stroma, dermis and other 
connective tissue); 2) size or extent of tissue defect; 3) formulation with 
pharmaceutical carrier; and 4) age of the patient. In a complete medium or 
chemically defined serum-free medium, isolated, culturally-expanded hMSCs are 
capable of augmenting bone formation. In an osteoinductive or other optimized 
medium, such as a resorbable biopolymer, fresh whole bone marrow containing 
about 10 4 MSCs per ml of marrow is also capable of augmenting bone formation. 
Combinations of these techniques are also contemplated. 

In another aspect the invention contemplates the delivery of (i) isolated, 
culture-expanded, human mesenchymal stem cells; (ii) freshly aspirated bone 
marrow; or (iii) their combination in a carrier material or matrix to provide for 
improved bone fusion area and fusion mass, when compared to the matrix alone. 
Particularly preferred is the delivery of a composition comprising purified 
mesenchymal stem cells and fresh bone marrow aspirates delivered in a carrier 
material or matrix to provide for improved bone fusion area and fusion mass. 

One composition of the invention is envisioned as a combination of materials 
implanted in order to effect bone repair, osseous fusion, or bone augmentation. The 
components of this implanted material include, in part, porous granular ceramic, 
ranging in size from 0.5 mm to 4 mm in diameter, with a preferred size ranging 
from 1.0 to 2.5 mm in diameter. The composition of the ceramic may range from 
100% hydroxyapatite to 100% tricalcium phosphate, and in the preferred form, 
consists of a 60/40 mixture of hydroxyapatite and tricalcium phosphate. The ceramic 



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WO 97/40137 PCT/US97/06433 

material may be uncoated, or coated with a variety of materials including autologous 
serum, purified fibronectin, purified laminin, or other molecules that support cell 
adhesion. The granular ceramic material can be combined with MSCs ranging in 
concentration from 10 thousand to 30 million cells per cc of ceramic, with a 
preferred range between 3 and 15 million cells per cc. It is also envisioned that the 
cells may be in the form of fresh marrow obtained intraoperatively, without ex vivo 
culture-expansion. 



Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, 
rib or other medullary spaces. Other sources of human mesenchymal stem cells 
include embryonic yolk sac, placenta, umbilical cord, periosteum, fetal and 
adolescent skin, and blood. The cells are incubated at 37 °C with the ceramic for 0 
to 5 hours, preferably 3 hours. Prior to implant, the cell-loaded granules can be 
combined with either fresh peripheral blood, human fibrin, fresh bone marrow, 
obtained by routine aspiration, or other biological adjuvant. These final 
combinations are allowed to form a soft blood clot which helps to keep the material 
together at the graft site. Implant or delivery methods include open or arthroscopic 
surgery and direct implant by injection, e.g. syringe or cannula. Finally, these 
implants may be used in the presence or absence of fixation devices, which 
themselves may be internally or externally placed and secured. 

The composition can also contain additional components, such as 
osteoinductive factors. Such osteoinductive factors include, for example, 
dexamethasone, ascorbic acid-2-phosphate, ^-glycerophosphate and TGF superfamily 
proteins, such as the bone morphogenic proteins (BMPs). The composition can also 
contain antibiotic, antimycotic, antiinflammatory, immunosuppressive and other 
types of therapeutic, preservative and excipient agents. 



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PCTYUS97/06433 



Brief Description of the Drawings 

Figures 1A-1D. Phase contrast photomicrographs of rat MSC cultures at 
various stages of development. 

Figure 1 A. A MSC colony at day seven of primary culture is composed 
of uniformly spindle-shaped cells. 

Figure IB. Passage one rat MSCs are distributed evenly across the 
surface of the dish 4 days after replating. 

Figure 1C. Rat MSCs grown in Control Medium for twenty-eight days 
become confluent and multi-layered, but do not form mineralized nodules. APase 
staining (dark gray) reveals a fraction of cells which are positive. 

Figure ID. Rat MSC cultures grown in the presence of Osteogenic 
Supplements for twenty-eight days form mineralized nodules which stain black by 
the von Kossa method. Cell cultures were stained by APase and von Kossa 
histochemical techniques as described below (Unstained (a,b), Alkaline 
phosphatase histochemistry and von Kossa (c,d), all x45). 

Figure 2. Light micrograph of a representative histological section from a 
MSC-loaded HA/TCP implant placed ectopically in subcutaneous tissue. MSCs 
were loaded and the sample was implanted as described below, harvested at eight 
weeks, decalcified, and processed in paraffin for microscopy. Only remnants of 
the HA/TCP ceramic <c) remain, while the pores of the implant are filled with 
bone (b), blood vessels (v), and hematopoietic elements including adipocytes 
(Toluidine blue-O, x70). 

Figure 3A-3H. High resolution radiographs showing the healing of the 
segmental defect at four and eight weeks with various implants. The radiographs 
were obtained on a Faxitron imaging system immediate following sacrifice. The 
polyethylene fixation plate is on the top of the bone in each radiograph. The four 
week radiograph is on the left, and the eight week radiograph is on the right for 



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PCT/US97/06433 



each group. The radiodensity of the HA/TCP material reveals the porous nature 
and the central canal of each implant. 



Figures 3A and 3B. Defects left empty; 

Figures 3C and 3D. Defects fitted with HA/TCP carrier alone; 

Figures 3E and 3F. Defects fitted with a MSC-loaded HA/TCP carrier; 

Figures 3G and 3H. Defects fitted with a marrow-loaded HA/TCP 
carrier. Defects left empty following segmental gap resection undergo reactive 
bone formation at the cut ends of the bone, leading to a classical non-union in this 
well established model. At four weeks, the MSC-loaded samples have begun to 
fill the pores of the implant material. No union is evident in any implant type at 
four weeks. By eight weeks, modest union of the host-implant interface has 
occurred in the carrier (d) and carrier plus marrow groups (h), but complete 
integration and bone bridging is evident in the carrier plus MSC group (0. Total 
filling of the pores with bone in the MSC-loaded sample is also evident in panel 
F. (xl.5) 



Figure 4A-4F. Light micrographs showing representative healing of the 
segmental defect at four and eight weeks with various implant types. Intact limbs 
were harvested, fixed, dehydrated, cleared, embedded in polymethylmethacrylate, 
cut, and ground to 100 micron thickness prior to staining. Some animals received 
India ink injections to allow visualization of the vascular tree, present here in 
panels B, C, D, and E as black staining. The HA/TCP material artifactually 
appears black in these photomicrographs as a result of undecalcified processing. 
The cut edges of the host conices are noted by arrowheads in a and b, and 
similar sections are presented in all other panels. 

Figures 4A and 4B. Defects fitted with HA/TCP carrier alone at four and 
eight weeks, respectively; 



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WO 97/40137 PCT7US97/06433 

Figures 4C and 4D. Defects fitted with a MSC-loaded HA/TCP carrier at 
four and eight weeks, respectively; 



Figures 4E and 4F. Defects fitted with a marrow-loaded HA/TCP at four 
and eight weeks, respectively. New bone present within the pores, or at the host- 
implant interface appears blue or violet in these specimens. Importantly, only 
samples containing a MSC~loaded implant effectively heal the defect, as noted by 
the substantial amount of bone present within the implant and at the interface with 
the host in panels c and d. See text for further details (Toluidine blue-O, x8). 

Figure 5A-5B. High power light micrographs showing bone regeneration 
at eight weeks in segmental gaps fitted with a MSC-loaded HA/TCP implant. 
Panel a shows the cut edge (arrowheads) of the host cortex with new bone in 
direct apposition. New bone at this host-implant interface is contiguous with 
bone formed in the pores of the HA/TCP carrier. Panel b shows both lamellar 
and woven bone (blue) filling the pores of the HA/TCP carrier. The carrier 
appears black in these images as an artifactual result of undecalcified specimen 
preparation. Blood vessels (v) which orient the secretory activity of osteoblasts 
are evident within the pores (Toluidine blue-O, x75). 

Figure 6. Osteogenic differentiation of human MSCs in vitro. Phase 
contrast photomicrographs (a, b) of human MSC cultures under growth and 
osteogenic conditions. 

Figure 6A. First-passage MSCs display characteristic spindle-shaped 
morphology and are distributed evenly across the surface of the dish after 
replating. 

Figure 6B. MSC cultures grown in the presence of OS for 16 days form 
mineralized nodular aggregates which stain gray for APase and black for 
mineralized matrix (Unstained (a) xl8, APase and von Kossa histochemistry (*>), 
x45). 



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PCT/US97/06433 



Figure 6C. APase activity and calcium deposition in MSC cultures grown 
in Control or OS Medium on days 4, 8, 12 and 16, Samples were harvested at 
the indicated days, and APase activity, cell number, and calcium deposition were 
determined as described in Materials and Methods. The results represent the 
mean ± SD of triplicate cultures from first passage cells, */ > <0.05, t/ > < 0.005 
(compared to Control). 

Figure 7. Light micrograph of a representative histological section from a 
human MSC-loaded HA/TCP implant placed ectopically in subcutaneous tissue of 
an athymic rat. MSCs were loaded into the ceramic, implanted as described in 
Materials and Methods, harvested at 12 weeks, decalcified and processed in 
paraffin for microscopy. Only remnants of the HA/TCP ceramic (c) remain, 
while the pores of the implant are filled with bone (b), blood vessels (arrow) or 
fibrous tissue (0. Cuboidal osteoblasts are seen lining the surface of the 
developing bone. (Toluidine blue-O, x75). 

Figure 8. Segmental gap defect model and radiography, (a) A 
polyethylene fixation plate is positioned on the lateral aspect of this representative 
rat femur. Four bicortical screws and 2 cerclage wires are used to secure the 
plate in place. An 8 mm segment of bone is removed along with its adherent 
periosteum, and a ceramic implant, with or without cells, is placed into the defect 
site. The overlying muscles are returned to their proper anatomic position, and 
the skin is closed with resorbable sutures. High resolution radiographs obtained 
immediately following sacrifice show the extent of healing of the segmental defect 
at 12 weeks with the 2 implant types {b, c). While total integration of the 
implant at the host-ceramic interface is evident in the carrier plus MSC group (b) y 
only modest union is observed in the cell-free implants (c). The pores of the 
MSC-loaded implant are filled with bone throughout the gap, but the cell-free 
carrier contains little bone and several cracks. 

Figure 9. Histologic representation of bone regeneration in segmental 
femoral defects. Immunohistochemical staining with antibody 6E2 (a) 
demonstrates that 4 weeks following implantation of a MSC-loaded sample, the 



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WO 97/40137 PCT/US97/06433 

cells within the pores of the carrier are reactive on their surface, and therefore of 
human origin, while cells outside the ceramic are not immunoreactive. In phase 
contrast microscopy (b) f the ceramic is black, and cells in the pores and 
surrounding the outside of the implant are evident. The ceramic material itself 
adsorbs fluorescent secondary antibody and appears green (a, b, x75). Light 
micrographs showing representative healing of a segmental defect implanted with 
HA/TCP carrier alone (rf), or carrier plus MSCs {c,e t J) y 12 weeks after 
implantation. Limbs were harvested, fixed, dehydrated, cleared, embedded in 
polymethylmethacrylate, cut, and ground to a thickness of 100 for staining. 
The ceramic appears black in these photomicrographs as an artifact of 
undecalcified specimen preparation, and bone present within the pores or at the 
host-implant interface appears blue-violet. The MSC-loaded specimen shown 
here was subjected to destructive mechanical torsion testing, and was 
subsequently processed for histology in two separate pieces. Repositioning 
photomicrographs of the two pieces approximates the appearance of the femur 
prior to testing (c). The actual fracture plane is denoted by the double arrows 
above and below the implant. The cut edges of the host cortices are noted by 
arrowheads in c. d, and e. Only samples containing a MSC-loaded implant 
effectively heal the defect. Higher power micrographs demonstrate the substantial 
amount of bone present at the host-implant interface (e) and within the body of 
the implant (/). (Toluidine blue-O, (c, d) x7, (e) x31, (/) x45). 

Figures 10A-10B. 3-D coordinate system, defined by the right-hand rule, 
will be centered at the midpoint of the line between the neural foramen markers 
at the level of interest, which defines the X axis. The Z direction is defined by a 
line through the midpoints of the lines between neural foramens at the levels 
above and below the level of interest (i.e. between orange points). The positive 
Z direction is cervical to lumbar. The positive Y direction is dorsal. The base of 
the volume of interest is defined by the X-Z plane. The volume of the fusion 
mass is defined by the outline of all bone density voxels in the positive Y axis of 
the X-Y plane between z = -7.5 mm to z = +7.5 mm. Complete fusions will 
measure 20-30 mm wide in the X-dimension and 10-15 mm in the height in the 
Y-dimension. 



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Figure 1 1 . The grid system used to assign union score is shown in 
schematic cross-section at the level of the facet joints. One half point is assigned 
for union in each of the box areas. 

Detailed Description of the Invention 

Bone grafting procedures are widely used to treat acute fractures, fracture 
non-unions, bone defects, and to achieve therapeutic arthrodesis. Autogenous 
cancellous bone is the current "gold standard" for clinical bone grafting. 
Contemporary dogma attributes this effectiveness to three primary intrinsic 
properties: osteoconduction, osteogenic cells, and osteo induct ion (76,96), which 
can be defined as follows: 

Osteoconduction - The scaffold function provided by the transplanted 
extracellular bone matrix which facilitates cell attachment and migration, and 
therefore the distribution of a bone healing response throughout the grafted 
volume. This property is likely dependent on adhesion molecules within bone 
matrix such as: collagens, fibronectin, vitronectin, osteonectin, osteopontin, 
osteocalcin, proteoglycans and others. Growth factors in the matrix may also 
play a role. 

Osteogenic cells - Those cells in the autograft derived from bone or bone 
marrow which survive transplantation and go on to proliferate and/or undergo 
osteoblastic differentiation. 

Osteoinduction - The bioactive property of autogenous bone derived from 
the presence of growth factors or other elements in the graft which stimulate the 
proliferation and/or differentiation of osteoblastic progenitors. Many growth 
factors have been identified in bone matrix including: bone morphogenetic 
proteins (BMPs), transforming growth factor-beta (TGF-/3), basic fibroblast 
growth factor (bFGF), and insulin-like growth factor (IGF). Transplanted 
non-osteogenic cells in bone marrow may also elaborate factors which contribute 



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to a bone healing response (140,48) endothelial cells have been specifically 
implicated (141). 

The marrow or isolated mesenchymal stem cells can be autologous, 
allogeneic or from xenogeneic sources, and can be embryonic or from post-natal 
sources. Bone marrow cells may be obtained from iliac crest, femora, tibiae, 
spine, rib or other medullary spaces. Other sources of human mesenchymal stem 
cells include embryonic yolk sac, placenta, umbilical cord, periosteum, fetal and 
adolescent skin, and blood, in order to obtain mesenchymal stem cells, it is 
necessary to isolate rare pluripotent mesenchymal stem cells from other cells in 
the bone marrow or other MSC source. 

The present invention provides a composition for the repair of bone 
defects by the rapid regeneration of healthy bone. The composition is an 
absorbable gelatin, cellulose and/or collagen-based matrix in combination with 
bone marrow and/or isolated mesenchymal stem cells. The composition can be 
used in the form of a sponge, strip, powder, gel, web or other physical format. 
The composition is, for example, inserted in the defect and results in osteogenic 
healing of the defect. 

The composition can also contain additional components, such as 
osteoinductive factors. Such osteoinductive factors include, for example, 
dexamethasone, ascorbic acid-2-phosphate, ^-glycerophosphate and TGF 
superfamily proteins, such as the bone morphogenic proteins (BMPs). The 
composition can also contain antibiotic, antimycotic, antiinflammatory, 
immunosuppressive and other types of therapeutic, preservative and excipient 
agents. 

The invention also provides a method for treating a bone defect in an 
animal, particularly a mammal and even more particularly a human, in need 
thereof which comprises administering to the bone defect of said animal a bone 
defect-regenerative amount of the composition of the invention. 



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The investigations reported here confirm the in vivo healing potential of 
fresh whole marrow or MSCs delivered in the matrix alone, or in the matrix in 
combination. 

The invention also contemplates the use of other extracellular matrix 
components, along with the cells , so as to achieve osteoinductive or 
osteoinductive properties. In addition, by varying the ratios of the components in 
said biodegradable matrices, surgical handling properties of the cell-biomatrix 
implants can be adjusted in a range from a dimensionally stable matrix, such as a 
sponge or film, to a moldable, putty-like consistency to a pliable gel or slurry to 
a powder. 

The marrow or isolated mesenchymal stem cells can be autologous, 
allogeneic or from xenogeneic sources, and can be embryonic or from post-natal 
sources. Bone marrow cells may be obtained from iliac crest, femora, tibiae, 
spine, rib or other medullary spaces. Other sources of human mesenchymal stem 
cells include embryonic yolk sac, placenta, umbilical cord, periosteum, fetal and 
adolescent skin, and blood. In order to obtain mesenchymal stem cells, it is 
necessary to isolate rare pluripotent mesenchymal stem cells from other cells in 
the bone marrow or other MSC source. 

In a particularly preferred embodiment, the composition of the invention 
comprises an absorbable implant, containing whole marrow and/or isolated MSCs 
for repair of segmental defects, spinal fusions or non-unions and other bone 
defects. Custom cell-matrix implants containing autologous, allogeneic or 
xenogeneic bone marrow and/or MSCs can be administered using open surgical 
techniques, arthroscopic techniques or percutaneous injection. 

Human mesenchymal stem cells (hMSCs) can be provided as either 
homogeneous, culture-expanded preparations derived from whole-marrow (or 
other pre-natal or post-natal source of autologous or allogeneic hMSCs), from 
hMSC-enriched or heterogenous cultures or fresh, whole marrow (when combined 
with an osteoinductive or other optimized medium) containing an effective dose of 



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at least about 10\ preferably about 10 4 , MSCs per milliliter of the composition. 
The key to effective clinical outcomes, in this embodiment using MSC therapy, is 
to provide that number of enriched or culture-expanded mesenchymal stem cells 
to the patient, or about the same number in an optimized medium, which repairs 
the bone or other tissue defect beyond that in a volume of whole marrow 
equivalent to that of the defect. This is referred to as the "Regenerative MSC 
Threshold", or that concentration of MSCs necessary to achieve direct repair of 
the tissue defect. The Regenerative MSC Threshold will vary by: 1) type of 
tissue (i.e., bone, cartilage, ligament, tendon, muscle, marrow stroma, dermis 
and other connective tissue); 2) size or extent of tissue defect; 3) formulation with 
pharmaceutical carrier; and 4) age of the patient. 

In a preferred embodiment, the method further comprises administering at 
least one bioactive factor which further induces or accelerates the differentiation 
of such mesenchymal stem cells into the osteogenic lineage. Preferably, the cells 
are contacted with the bioactive factor ex vivo, while in the matrix, or injected 
into the defect site at or following the implantation of the composition of the 
invention. It is particularly preferred that the bioactive factor is a member of the 
TGF-/3 superfamily comprising various tissue growth factors, particularly bone 
morphogenic proteins, such as at least one selected from the group consisting of 
BMP-2, BMP-3, BMP-4, BMP-6 and BMP-7. 

In the embodiment which uses a gelatin-based matrix, an appropriate 
absorbable gelatin sponge, powder or film is cross-linked gelatin, for example, 
Gelfoam* (Upjohn, Inc., Kalamazoo, Ml) which is formed from denatured 
collagen. The absorbable gelatin-based matrix can be combined with the bone 
reparative cells and, optionally, other active ingredients by soaking the absorbable 
gelatin sponge in a cell suspension of the bone marrow and/or MSC x^Hs, where 
the suspension liquid can have other active ingredients dissolved therein. 
Alternately, a predetermined amount of a cell suspension can be transferred on 
top of the gelatin sponge, and the cell suspension can be absorbed. 



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In the embodiment which uses a cellulose-based matrix, an appropriate 
absorbable cellulose is regenerated oxidized cellulose sheet material, for example, 
Surgicel® (Johnson & Johnson, New Brunswick, NJ.) which is available in the 
form of various sized strips or Oxycel® (Becton Dickinson, Franklin Lakes, NJ) 
which is available in the form of various sized pads, pledgets and strips. The 
absorbable cellulose-based matrix can be combined with the bone reparative cells 
and, optionally, other active ingredients by soaking the absorbable cellulose-based 
matrix in a cell suspension of the bone marrow and/or MSC cells, where the 
suspension liquid can have other active ingredients dissolved therein. Alternately, 
a predetermined amount of a cell suspension can be transferred on top of the 
cellulose-based matrix, and the cell suspension can be absorbed. 

In the embodiment which uses a collagen-based matrix, an appropriate 
resorbable collagen is purified bovine corium collagen, for example, Avitene® 
(MedChem, Woburn, MA) which is available in various sizes of nonwoven web 
and fibrous foam, Heiistat* (Marion Merrell Dow, Kansas City, MO) which is 
available in various size sponges or Hemotene* (Astra, Westborough, MA) which 
is available in powder form. The resorbable collagen-based matrix can be 
combined with the bone reparative cells and, optionally, other active ingredients 
by soaking the resorbable collagen-based matrix in a cell suspension of the bone 
marrow and/or MSC cells, where the suspension liquid can have other active 
ingredients dissolved therein. Alternately, a predetermined amount of a cell 
suspension can be transferred on top of the collagen-based matrix, and the cell 
suspension can be absorbed. 

The above gelatin-based, cellulose-based and collagen-based matrices may, 
optionally, possess hemostatic properties. 

Preferred active ingredients are those biological agents which enhance 
wound healing or regeneration of bone, particularly recombinant proteins. Such 
active ingredients are present in an amount sufficient to enhance healing of a 
wound, i.e., a wound healing-effective amount. The actual amount of the active 
ingredient will be determined by the attending clinician and will depend on 



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various factors such as the severity of the wound, the condition of the patient, the 
age of the patient and any collateral injuries or medical ailments possessed by the 
patient. Generally, the amount of active ingredient will be in the range of about 
1 pg/cm 2 to 5 mg/cm 2 . 



Exam ple 1 
Rat Gap Defect Repair 

Materials & Methods 
Materials 

Dexamethasone (Dex), sodium ^-glycerophosphate (0GP), antibiotic 
penicillin/streptomycin, and alkaline phosphatase histochemistry kit #85 were 
purchased from Sigma Chemical Co. (St. Louis t MO), DMEM-LG (DMEM) 
tissue culture medium from GIBCO Laboratories (Grand Island, NY), and L- 
ascorbic acid-2-phosphate (AsAP) from Wako Chemical (Osaka, Japan). Fetal 
bovine serum (FBS) was purchased from GIBCO following an extensive testing 
and selection protocol (80). Porous hydroxyapatite/0-tricalcium phosphate 
(HA/TCP) ceramic, mean pore sire 200-450 /xm, was generously provided by 
Zimmer, Inc. (Warsaw, IN). All other routine reagents used were of analytical 
grade. 

MSC Isolation and Cultivation 

MSC isolation and culture expansion was performed according to 
previously published methods (32). Briefly, male Fisher F344 rats (200-275 g) 
were sacrificed by pentobarbital overdose. The tibias and the femurs were 
recovered by dissection under sterile conditions, the metaphyseal .ends of the 
bones were cut, and the marrow plugs were flushed out*y passing saline through 
a needle inserted into one end of the bone. Pooled marrow clots were dispersed 
by gentle pipetting, followed by sequential passage through a series of smaller 
needles yielding a single-cell suspension. The cells were then centrifuged for ten 
minutes at 900 x£, and resuspended in DMEM containing 10% FBS (Control 
Medium). Fifty million nucleated cells were plated onto petri-dishes (sixty cm 2 ) 
in seven milliliters of Control Medium, and grown at 37°C in the presence of 5% 



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C0 2 . Non-adherent cells were removed at the time of the first medium change, 
four days post plating, and cells were routinely fed twice weekly thereafter. 
These primary cultures approached confluence typically at thirteen days, were 
then released by a five minute exposure to 0.25% trypsin containing one 
millimolar EDTA, and subcultivated at a density of 10* cells/cm 2 . Cells for 
implantation were derived from these first passage cultures ten days after 
replating, at which time they were approximately 85% confluent. 

In Vitro Osteogenic Assays 

At the end of first passage, MSCs were replated into six-well plates at a 
density of 10 4 cells/cm 2 in Control Medium. The following day (Day 0), fresh 
Control Medium was provided, and the cells were grown in the absence or 
presence of Osteogenic Supplements (OS) (100 nanomolar Dex, 0.05 millimolar 
AsAP and ten millimolar j8-GP) (64). Media changes were performed twice 
weekly, and at days seven, fourteen, twenty-one, and twenty-eight, cultures were 
assayed for cell number, alkaline phosphatase (APase) histochemistry, and 
mineralized matrix production utilizing techniques previously described (64). 

Implant Preparation 

HA/TCP blocks were shaped into cylinders approximately four millimeter 
in diameter and eight millimeter in length. A central canal roughly one 
millimeter in diameter was bored through the length of the entire cylinder using 
an eighteen gauge hypodermic needle. Cylinders were cleaned by sonication and 
rinsing in distilled water, and then sterilized by 220°C dry heat for five hours. 
The cylinders were subsequently coated with human plasma fibronectin (Cal- 
Biochem, Irvine, CA) by soaking in a 100 microgram per milliliter solution for 
sixteen hours at 4°C. The implants were then air dried at room temperature 
overnight in a sterile biosafety cabinet, and stored at 4°C. HA/TCP cubes, 
measuring three millimeter per side, were similarly prepared and coated with 
fibronectin as described above for use in the ectopic osteogenesis assay. 

HA/TCP implants, both in cube and cylinder form, were loaded with 
MSCs using a modification of a technique previously described (32,83). Briefly, 

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implants were placed in a suspension of MSCs (7.5 x 10 6 cells/ml) in serum free 
DMEM. The loading vessel was capped, and the implants were subjected to a 
vacuum in three bursts of five seconds each to remove air present within the 
pores of the HA/TCP, and to facilitate fluid flow into the pores. The loading 
vessels were capped loosely, placed in a tissue culture incubator for two hours, 
and gently agitated every thirty minutes until the time of surgery. Cell-free 
control cylinders were treated identically, with the notable exception that the 
serum free DMEM contained no cells. The third implant group was designed to 
generously approximate the clinically relevant control of a fresh bone marrow 
aspirate. Just prior to implantation, fresh marrow cell suspensions were obtained 
as previously described, centrifuged for ten minutes at 900 xg, and resuspended 
in a volume of serum free DMEM which would coat each cylinder with the 
number of bone marrow cells derived from one entire femur, approximately fifty 
million (144,145). The HA/TCP implants were loaded with this fresh marrow by 
rolling them in the congealed marrow suspension. 

Surgical Model and Experimental Design 

The rat femoral gap model described here is a modification of one used 
extensively to study long bone repair (34,37,61,83,105,126,129,144,145,147). 
Briefly, both femurs of male F344 rats (300-350 g) were exposed by an 
anterolateral approach. Soft tissue and muscle was elevated while keeping the 
periosteum intact along the surface of the bone. A polyethylene fixation plate 
(four by four by twenty-three millimeters) (Hospital for Special Surgery, New 
York, NY) was secured to the anterolateral aspect of each femur by four threaded 
Kirschner-wiVes and two cerclage wires (Zimmer, Warsaw, IN). An eight 
millimeter transverse segment of the central diaphysis, along with its adherent 
periosteum, was removed by a rotary osteotomy burr under saline irrigation. 
These stabilized segmental defects were either left empty, or replaced with a cell- 
free HA/TCP cylinder, a MSC-loaded cylinder, or a cylinder loaded with a fresh 
marrow cell suspension. Implants were secured by placing two 4-0 Vicryl 
(Ethicon, Somerville, NJ) sutures around the ceramic and the fixation plate. The 
muscles were apposed, and the fascia and skin were closed in a routine layered 
fashion. Rats implanted with MSC-loaded cylinders also received subcutaneous 



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implants of the MSC-Ioaded HA/TCP cubes to correlate the ectopic osteogenesis 
assay with orthotopic bone regeneration and the in vitro osteogenic potential of 
syngeneic MSCs. Rats implanted with marrow-loaded cylinders similarly 
received subcutaneous implants of marrow-loaded cubes. The animals were 
allowed full activity in their cages postoperatively. No animals experienced 
failure of fixation or other post-operative complications. At least six limbs were 
used for each of the implant groups, randomly selected between left or right. 
Upon sacrifice at four and eight weeks, the vascular tree of some animals was 
perfused with India ink, and the entire femur and surrounding soft tissue was 
carefully dissected. Specimens were immediately evaluated radiographically, and 
subsequently processed for undecalcified histology. 

Radiographic Analysis 

The specimens were radiographed using a high resolution Faxitron 
Imaging system (Buffalo Grove, IL) with an exposure of thirty-five kVP for thirty 
seconds. The radiographs were independently evaluated by two of the authors 
who were blinded with respect to the duration and type of implant. Bone 
formation was scored on a semiquantitative scale with ranges as follows: distal 
host-implant union (0-2); proximal host-implant union (0-2); and implant core 
density (0-4). The union scores and the core density scores were added to give a 
maximum possible score of eight for each implant. Results from both examiners 
were averaged to give final scores. 

Histology and Histomorphometry 

Following fixation in 10% buffered formalin, the femurs were dehydrated, 
cleared, and embedded in polymethylmethacrylate. Longitudinal sections were 
cut on a water-cooled Isomet saw (Buehler, WI), and a central section of each leg 
was ground to 100 micrometer thickness, polished, and stained with Toluidine 
blue-O. Leica Quantimet 500MC (Cambridge, UK) image analysis software was 
used to determine the area of HA/TCP implant, bone, and soft tissue in the 
diaphyseal defect region of each section. The data were analyzed by one-way 
analysis of variance (ANOVA) (Sigmastat, Jandel Scientific). Further analyses 
were performed according to post hoc Student-Newman-Keuls tests. 



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Subcuianeously implanted ceramic cubes were similarly fixed in formalin, then 
decalcified, dehydrated, embedded in paraffin, serially sectioned, and stained with 
Toluidine blue-O. 

Results 

MSC Cultivation and Osteogenic Differentiation in vitro 

Rat MSC cultures were established from syngeneic animals and, by seven 
days, formed characteristic colonies on the surface of the culture dish (Fig. 1A). 
Several hundred MSC colonies arose from the fifty million nucleated cells seeded 
on each sixty cm 2 dish. On the basis of this observation, rat MSCs, like human 
MSCs (13,54), appear to be present at a frequency of approximately one in 10 5 
nucleated marrow cells. Primary MSC cultures subcultivated on day fourteen 
attached uniformly to the surface of new dishes, and were allowed to divide for 
roughly ten days, or until the dishes became -85% confluent. Passaged cells 
also demonstrate a characteristic morphology (Fig. IB), and uniformly divide 
upon the dish resulting in an even distribution of MSCs throughout the plate. 
Cells derived from this first passage were used for preparing implants as 
described above, and an aliquot was used to confirm the in vitro osteogenic 
potential of rat MSCs. 

Seven days after replating for the osteogenic assay, both Control and OS- 
treated cultures were composed of spindle-shaped cells, 40-50% of which were 
stained for APase. During the next twenty-one days, Control cells remained 
fibroblastic, increased their cell surface APase, but never underwent the 
morphologic changes associated with the development of mineralized bone 
nodules (Fig. 1C). By contrast, OS-treated cultures began to form aggregates of 
polygonal and cuboidal cells intensely stained for APase, and by day twenty-one, 
the cultures had formed characteristic bone-like nodules which contained von 
Kossa stained mineral deposits. Further mineralization of these nodules through 
day twenty-eight (Fig. ID) was accompanied by a decrease in APase staining, 
especially within the internodular regions. 



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MSC-Mediated Osteogenesis in Ectopic HA/TCP Implants 

All MSC-loaded HA/TCP cubes implanted in the host rats had ample 
evidence of osteogenesis by four weeks. At the eight week time point, a 
substantial amount of bone, and occasionally cartilage, was present within the 
pores of the cubes. A representative section from a MSC-loaded cube harvested 
eight weeks following implantation is shown in Figure 2. The unstained granular 
areas reflect the former regions of ceramic material which have been removed 
during the decalcification step of specimen preparation. As seen in the 
photomicrograph, bone formation occurs within the pores of the cubes, and is 
associated with vascular elements which penetrate the implant. Such angiogenesis 
is obligatory to new bone formation since the secretory activity of osteoblasts is 
an oriented phenomenon guided by vasculature. Both woven and lamellar bone 
can be seen depending on the duration of implantation, and the precise region 
examined. Most of the pores are filled with bone and small islands of 
hematopoietic elements, with the remainder being filled with a loose connective 
tissue. In contrast to these MSC-loaded samples, cubes loaded with fresh marrow 
contained negligible osseous tissue at four weeks, and only slightly more even at 
eight weeks. As previously demonstrated (32,54), cubes implanted without MSCs 
or marrow contained no bone, but were filled with fibrous tissue and blood 
vessels. 

Radiographic Evaluation 

High resolution Faxitron radiographs provided sufficient clarity and detail 
to discern subtle changes occurring within the implant and the surrounding host 
bone. Figure 3 shows representative radiographs of the femurs from each of the 
groups recovered at four and eight weeks post- implantation. As demonstrated in 
these radiographs, the fixation remained intact in all the samples and there were 
no fractures in any of the femurs. In animals whose femoral defects were left 
empty, reactive bone formation at the transversely cut edges of the host femur 
was observed at four weeks (Fig. 3A). By eight weeks, slightly more bone was 
present within the gap, however, most of this bone appeared to form along the 
edge of the fixation plate which was in contact with the periosteum (Fig. 3B). 
Every specimen which was left empty resulted in the formation of a radiographic 



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non-union. Some limbs, irrespective of the group, also contained an eccentric 
spicule of bone which was usually on the outside of the defect opposite the 
fixation plate. 

When the HA/TCP cylinder was implanted, negligible reactive bone 
formation occurred at the cut edges of the femur. Due to the mineral content of 
the implant material (HA/TCP), one can appreciate the structural details of the 
implant itself upon radiographic evaluation. The details of the central canal and 
pores are clearly visible in the four week radiographs (Fig. 3C), and serve to 
provide an important baseline for comparison to the other radiographic images. 
Blurring of the pore margins can be appreciated by eight weeks (Fig. 3D) in these 
cell-free implants. Importantly, the lack of union between the implant and the 
host is manifested as a clear zone of radiolucency between the implant itself and 
the cut edges of the femur in all animals at four weeks. In contrast to the four 
week carrier alone, animals which received MSC-loaded HA/TCP cylinders 
demonstrated substantial new bone formation within the pores of the implant by 
four weeks (Fig. 3E). Increasing radiodensity, and obliteration of the apparent 
pore structure, was used as an indication of new bone formation within the core 
of the implant. Although integration of the implant, or union, was not observed 
by four weeks, the subsequent formation of a radiodense bone bridge between the 
implant and the host completely masked the interface. By eight weeks, the MSC- 
loaded implant was contiguous and completely integrated with the normal host 
bone (Fig. 3F). HA/TCP implants which were loaded with fresh marrow did not 
appear to produce radiodense bone within the pores at either time point, although 
modest integration with the cut ends of the host bone was evident by eight weeks 
(Figs. 3G and 3H). 

The average of the radiographic scores at each time point for each implant 
group is provided in Table 1. 



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Table 1 



Average of Radiographic Scores for Each Implant Group 





Four Weeks 


Eight Weeks 


oiiiy 




C+M 


\_ onjy 




C+M 


Proximal Union 


1.5 


0.8 


0.2 


1.2 


1.3 


1.0 


Distal Union 


0.5 


1.4 


0.7 


1.2 


2.0 


1.6 


Core Density 


0.7 


2.0 


0.3 


0.6 


3.7 


0.7 


Total Score 


2.7 


4.2 


1.2 


3.0 


7.0* 


3.3 



Table 1, Average of radiographic scores for each implant group at each 
time point. C = carrier, M = marrow. Radiographs were evaluated and scored 
by two independent observers blinded to the identity of each implant. Union was 
scored both proximal I y and distally on a scale of 0-2. Core density was scored 
on a scale of 0-4. n=3 for each group at each time point. The maximum 
possible total score is 8. One-way analysis of variance at the two different time 
points, with cell loading (none, MSCs, and marrow) as the independent variable 
showed significant difference between groups at 8 weeks (F = 10.9, p = 0.01) 
but were not significantly different at four weeks. * = significantly greater (p < 
0.05) than other groups at the corresponding time point (according to post hoc 
Student- Newman- Keuls tests). 

In the case of the defects filled with the HA/TCP carrier alone, the low 
scores indicate the absence of any radiodense material within the pores, and 
minimal union of the implant with the host bone. Loading the HA/TCP implant 
with fresh marrow did not result in an improvement in the healing of the defect, 
and the low scores reflect the similarity of this group to that of the carrier alone. 
However, loading the HA/TCP carrier with MSCs produces a vigorous 
osteogenic response. Even at four weeks, pore filling was observed and is 
reflected in the considerably higher scores of these implants. Interestingly, even 
in this case the host-implant union was modest compared to controls. By the 
eight week time point, the pores of the implant were filled with new bone and the 
host-implant union was well established. 



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Histologic and Histomorphometric Evaluation 

Histologic evaluation of the samples confirmed the observations made by 
radiography. In the empty defects, reactive bone formation appeared to emanate 
from the cut ends of the host cortices and endosteum. Even at eight weeks there 
was no bridging across the defect, and a fibrous non-union had formed at the 
center of the segmental gap. Photomicrographs of representative sections of the 
implant groups recovered at four and eight weeks are shown in Figure 4. In 
defects fitted with the HA/TCP carrier alone, the pores of the implant were filled 
with fibrous tissue (Fig. 4A) and were well vascularized as determined by India 
Ink injection. No bone could be seen within the pores of the implant and there 
was limited integration with the host. Even at eight weeks, most of the pores 
were devoid of any bone despite significant vascularization evident in this 
photomicrograph (Fig. 4B). A small amount of new bone was present at the 
host- implant interfaces, and at one end of this representative implant, host-derived 
endosteal bone appears to be advancing into the medullary canal of the implant. 
Bone formation in samples loaded with fresh marrow was very similar to that of 
the HA/TCP carrier alone (Figs. 4E and F). However, a modest amount of new 
bone could be seen within the pores of the implant at eight weeks, correlating 
with the results of the ectopic implants. Union of these implants was similar to 
that observed with cell-free implants; reactive bone formation slightly penetrated 
the pores at the ends of the implant. 

In contrast to the sparse osteogenesis resulting from the addition of fresh 
marrow to the HA/TCP, most of the pores of the implants loaded with MSCs 
contained considerable new bone by four weeks (Fig. 4C). Again, there was still 
a clear demarcation between the cut edges of the host bone and the ends of the 
implant. At eight weeks nearly every pore was filled with new bone, except in 
some discrete areas where loading of the MSCs may have been suboptimal. 
Interestingly, substantial new bone formation occurred at the interface between 
the host and the implant, leading to a continuous span of bone across the defect 
(Fig. 4D). Furthermore, a periosteal callus was also present in samples loaded 
with MSCs (Fig. 4D), but not in other implant types. The bone formed within 
the pores and at the ends of these implants represents de novo bone formation, is 



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highly cellular, and is presented in higher magnification photomicrographs in 
Figure 5. New woven and lamellar bone can be seen in intimate contact with the 
cut edge of the host cortex at eight weeks (Fig. 5A). Importantly, this region of 
union is directly contiguous with bone formed throughout the pores of implant. 
In regions deeper within the HA/TCP, filling of the pores with new bone is 
evident, as is the association of vasculature which orients the secretory activity of 
the differentiating osteoblasts (Fig. 5B). 

The results qualitatively described above are mirrored in the 
histomorphometric data presented in Table 2. 



Table 2 



Carrier alone 


Carrier + MSCs 


Carrier + Marrow 


2.3 ± 1.5 


19.3 ± 3.7* 


2.9 ± 1.7 


10.4 ± 2.4 


43.3 ± 7.7* 


17.2 ± 6.0 



Table 2. Bone fill in HA/TCP implants as a percentage of 
available space. Histomorphometric measurements were obtained 
on the bone formed within the confines of the segmental resection, 
excluding the implant material itself and the medullary canal. The 
values are reported as means of three samples along with standard 
deviations from the mean. One-way analysis of variance at the two 
different time points, with cell loading (none, MSCs, and marrow) 
as the independent variable showed significant difference between 
MSC-loaded samples at both 4 weeks (F = 43.3, p < 0.001) and 
8 weeks (F = 26.2, p < 0.002). * = significantly greater (p < 
0.01) than other groups at the corresponding time points (according 
to post hoc Studem-Newman-Keuls tests). No difference was 
observed between marrow and carrier alone at either time point (p 
> 0.1). 



The cell-free HA/TCP implants had a bone fraction of only 2.3% and 
10.4% at four and eight weeks, respectively. Importantly, this fraction of bone at 
eight weeks correlates with previously published results (126). These fractions 
primarily represent the bone ingrowth from the cut ends of the host cortices. The 
marrow-loaded HA/TCP cylinders did exhibit modest osteogenesis within the 

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body of the implant and consequently had a slightly higher value of 17.2% at 
eight weeks. Importantly, by four weeks, the MSC-loaded samples exceeded the 
eight week value for the other two groups. The 19.3% bone fill at this four week 
time point is most likely attributable to MSC-mediated osteogenesis. The average 
bone fraction within the implant increased over time, reaching 43% by eight 
weeks. One-way ANOVA performed on the data along with the Student- 
Newman-Keuls tests showed that at both four and eight weeks, the MSC 
treatment was significantly better than the carrier alone or the marrow-loaded 
carrier (p < 0.01). No significant difference between carrier alone and marrow- 
loaded implants was detected. The volume fraction of the HA/TCP carrier 
remained constant, and served as an internal control for the histomorphometry 
system. Even though the empty defects had 34% bone fill by eight weeks, there 
was no bridging across the defect, and thus would be classified as a clinical non- 
union. 

Discussion 

In the present study, we have demonstrated that purified, culture-expanded 
syngeneic progenitor cells are capable of healing a clinically significant bone 
defect in a well established animal model. These progenitor cells are referred to 
as mesenchymal stem cells since they give rise not only to bone (11,32,54,64), 
but to cartilage (32,66,80,142), muscle (121,143), tendon (23), and a stromal 
tissue which supports hematopoietic differentiation (87). While the osteogenic 
potential of both animal and human MSCs has been proven via subcutaneous 
implants in ectopic assays, rigorous and quantitative studies establishing the 
ability of culture-expanded MSCs to regenerate large segmental bone defects have 
not been reported to our knowledge. The combination of MSCs with a porous 
HA/TCP implant material are shown in the present study to be an effective 
strategy for healing large segmental bone defects. The current investigation 
further substantiates that compared to fresh marrow, MSCs produce significantly 
more bone when placed in either an ectopic or an orthotopic site. With these 
results as a foundation, we may begin to refine our approach to autologous cell 
therapies for the regeneration of skeletal defects. 



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To further characterize the cells used in this study, we cultured them in 
the presence and absence of a medium which induces osteogenic differentiation in 
vitro. As has been reported in numerous other laboratories (77,88,89,1 18,135), 
these rat marrow-derived cells develop along the osteogenic lineage in response to 
dexamethasone, eventually forming mineralized nodules of bone-like tissue on the 
surface of the dish. Such differentiation is evident in our photomicrographs (Fig. 
1), and serves to document that the cells used in these implants indeed possess the 
ability to form bone, one of the inherent properties of MSCs. Furthermore, the 
bone and cartilage formed in cubes implanted subcutaneously not only confirms 
the osteochondral potential of the MSCs, but acts as an internal control to verify 
that every host rat was capable of providing an environment which could support 
osteogenesis within these combined celhmatrix implants. Additional experiments 
documenting the multilineage potential of these cells were not included as part of 
the current study because previous publications have focused on describing such 
potential in greater detail (32,79,80,121,143). The isolation and selection 
procedures for rat MSCs are similar to those used for human MSCs (32,54,80), 
and result in the formation of characteristic primary colonies illustrated in Figure 
1A. These cells are mitotically expanded to yield a morphologically 
homogeneous population which divides uniformly across the dish. Both human 
and rat MSCs have been shown to possess multilineage potential, and the details 
of in vitro osteogenic differentiation of human MSCs has recently been reported 
(33,64). Conditions for the isolation and culture expansion of human MSCs 
without lineage progression have been optimized (13,54,80), and the development 
of a serum free medium for human MSC growth has been completed (58). 

The radiographic findings in this study establish a precedent for obtaining 
non-invasive evidence of bone regeneration in animals, or humans, which receive 
MSCs in an orthotopic location. Given the porous nature of the HA/TCP 
implants, new bone which forms within the interstices of the material is readily 
apparent radiographically by four weeks, in spite of the inherent radiodensity of 
the HA/TCP material. The progressive increase in radiodensity evident by eight 
weeks correlates well with the histological observations of processed limbs. 
Interestingly, despite the presence of new bone within the core of implants by 



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four weeks, integration at the host-implant interface was not observed until eight 
weeks. The mean radiographic scores for the three implant groups document a 
significant (p < 0.05) difference between MSCs and either marrow-loaded and 
ceramic implants at eight weeks, while no significant difference was observed 
between marrow-loaded and ceramic implants at either time point. 

The histologic studies demonstrate appositional bone growth on the surface 
of the HA/TCP throughout the core of the implant, consistent with previous 
observations of osteogenesis in ectopic implants loaded with MSCs (32,47,54). 
The bone which is formed at four and eight weeks in MSC-loaded samples is 
woven in many areas, but lamellar bone can also be appreciated (Figs. 5A and 
5B). It is critical to note that in the process of regenerating this osseous defect, 
bone formation occurs by a direct conversion of mesenchymal cells into 
osteoblasts rather than by an endochondral sequence. As regeneration of the bone 
at the defect site continues, the pores of the ceramic are filled with significantly 
more bone, which is laid down upon the walls of the implant or existing bone, 
and oriented by the invading vasculature. These blood vessels, visualized by 
India inking of animals immediately prior to sacrifice, also provide a portal for 
the entry and establishment of new marrow islands which contain hematopoietic 
elements, as well as host-derived MSCs. The process of bone remodeling ensues, 
and eventually the donor bone is replaced by host bone (47). At the edge of the 
defect, integration of the implant is achieved with direct continuity between the 
cut edge of the host cortex and the new bone formed upon the surface of the 
implant (Fig. 5A). Since only minimal host-implant union occurs in rats provided 
with either marrow-loaded or cell-free ceramics, the advanced integration 
observed in MSC-loaded ceramics likely reflects the combined contributions of 
implanted MSCs and host-derived cells. The lack of early union in all samples 
was surprising in light of the fact that defects which were left empty underwent a 
substantial amount of reactive bone formation at the cut edges of the cortices. It 
is possible that the presence of an implant in the defect site inhibits migration 
and/or prolapse of the surrounding loose mesenchyme which contributes to the 
reactive bone formation in the empty defects. Furthermore, micromotion of the 
implanted cylinders would likely hinder stable union at the interface. 



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The ability of MSCs to regenerate a large segmental defect in this 
experimental model compares favorably with other investigations testing implants 
such as demineralized bone matrix, bone marrow, purified or recombinant BMPs, 
allograft, ceramics, and fibermetals (34,37,74,83,105,126,129,144,145,147). 
While the use of recombinant BMP has received considerable attention, the 
precise mechanism of action has only recently been appreciated. These powerful 
inductive molecules act on undifferentiated mesenchymal cells to initiate the 
endochondral cascade, ultimately resulting in the formation of bone. Studies of 
undifferentiated rat marrow stromal cells confirm that BMP-2 acts to directly 
stimulate osteoblast development, and that this stimulation is enhanced by the 
addition of dexamethasone (77). Others have shown that bone formation occurs 
in an orthotopic site when fresh marrow alone is added, but the rate and extent of 
healing is a function of the amount of marrow and the number of osteoprogenitor 
cells residing therein (26,49,129,144). An important set of experiments by 
Takagi and Urist (129) demonstrate that the addition of BMP is not effective at 
healing segmental defects when access to the medullary canal and the marrow 
stroma is prevented, thus indicating an absolute requirement for the cellular 
constituents of marrow in BMP-mediated bone repair. These results were 
bolstered by studies indicating that the implantation of fresh marrow along with 
BMP in a rat segmental gap model is more effective than either component 
implanted alone (74). One may conclude from all of the above that marrow- 
derived mesenchymal progenitors, or MSCs, are the target for endogenous 
osteoinductive molecules, such as BMPs, which are released during normal bone 
healing. It therefore follows that one must have an adequate supply of MSCs in 
order to respond to the normal (or exogenously supplied) signals of bone repair, 
or healing will be effete. 

The histomorphometric data generated in this study provides a basis for 
comparison to other investigations. When fresh marrow from one femur 
equivalent is loaded on an HA/TCP implant, no significant difference in bone 
formation is observed when compared to implants which receive no cells. This is 
true for both time points in our study, and likely reflects an inadequate number of 
MSCs in the volume of marrow applied. Had we loaded the implants with 



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considerably more marrow, we and others would predict that greater healing of 
the bone defect would have occurred (74,144). Nevertheless, an appropriate 
clinically relevant control is generously approximated by applying the total cell 
population obtained from one long bone since removing all the marrow from 
multiple long bones for the repair of a focal defect is contradictory to sound 
clinical judgment. Perhaps most importantly, MSCs produced a bone fill of 
19.3% and 43.2%, respectively, at four and eight weeks. When purified BMP 
was applied to an identical carrier in the same experimental model, the bone fill 
was 21 % at four weeks, and only 22% by eight weeks (126). These BMP-coated 
HA/TCP implants did not achieve a bone fill of 43 percent until 16 weeks 
following implantation. While similar amounts of bone resulted from both 
implant types at four weeks, MSCs produce twice as much bone as BMP by the 
eight week time point. In this formulation, it took BMP sixteen weeks to form 
the same amount of bone which MSCs produce in only eight weeks. On this 
basis, it appears that MSCs offer a considerable advantage to the use of BMP 
alone, although some combination of BMP and MSCs could provide an even 
faster, more vigorous bone repair as discussed above. 

Since the number of progenitor cells present at the site of repair is a 
critical factor, it is obligatory to estimate how the MSC-loaded implants compare 
with marrow-loaded implants in this regard. The number of nucleated marrow 
cells which were placed on an implant was approximately fifty million; the same 
number harvested from one long bone. Another fifty million cells were used to 
initiate the MSC culture which eventually provided cells for one implant. From 
these fifty million cells, roughly 500 MSC colonies develop, and these cells are 
mitotically expanded to three million by the end of first passage. This represents 
a 6,000-fold increase in MSC number due to approximately twelve population 
doublings. Using the current technique to load these type of implants, it appears 
that only about 150,000 cells become adherent following incubation with the MSC 
suspension (32). Nevertheless, the local administration of 150,000 purified MSCs 
would increase the number of progenitor cells 300 times over the number 
normally present in fifty million unfractionated marrow cells. On the basis of 
these calculations, the advantage which this technique offers over other bone 



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regeneration strategies is direct delivery of the cellular machinery required for 
bone formation. This approach would have an extraordinary advantage in settings 
where the number of endogenous progenitor cells is reduced, such as that which 
occurs in ageing, osteoporosis, or a variety of other pathologic conditions 
(33,72,82,118,128,135). Other investigators have pursued this logic by 
attempting to deliver more progenitor cells simply by concentrating the marrow, 
by crude fractionation and removal of red blood cells, or by cultivating the 
stromal cells in vitro (26,83,103,105,144). Now that techniques and conditions 
have been established which support the expansion of purified human MSCs in 
culture as much as one billion fold without a loss in osteogenic potential (13), 
analogous clinical protocols for regenerating human bone defects are not far 
away. It will be possible to further expedite the healing process by directing 
these culture-expanded MSCs ex vivo to enter the osteogenic lineage prior to 
implantation, thus decreasing the in situ interval between implantation and their 
biosynthetic activity as osteoblasts. Additional efforts are underway to develop 
cell delivery vehicles which will provide more flexibility to the surgeon, including 
materials which can be shaped to fit any type of defect. By combining a 
pharmacologic stimulus, such as BMP, with an even better delivery vehicle, we 
will be able to offer patients therapeutic options which have never before been 
available. 



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Large Segmental Canine Femoral Defects Are Healed 
With Autologous Mesenchymal Stem Cell Therapy 

This study demonstrates that culture-expanded, autologous mesenchymal stem 
cells can regenerate clinically significant bone defects in a large animal model. 

Recently, the ability of syngeneic bone marrow-derived mesenchymal stem 
cells (MSCs) to repair large segmental defects in rodents was established (68). 
These MSCs may be isolated from marrow or periosteum, expanded in number ex 
vivo, and delivered back to the host in an appropriate carrier vehicle. Studies in rats 
demonstrated that the amount of bone formed 8 weeks following implantation of 
MSCs was twice that resulting from BMP delivered in the same carrier (68,126). 
In order to demonstrate clinical feasibility of this technology, our objective was to 
regenerate segmental bone defects in a large animal amenable to stringent 
biomechanical testing. To achieve this goal, we developed a canine femoral gap 
model to compare radiographic, histologic, and biomechanical data following 
implantation of an MSC-loaded carrier, carrier alone, and cancellous autograft bone. 

Materials and Methods 

MSC Cultivation and Manipulation 

A 15cc bone marrow aspirate was obtained from the iliac crest of each 
animal, according to an lACUC-approved protocol, and shipped on ice by overnight 
courier to the cell culture facilities. Isolation of canine MSCs was achieved by 
centrifuging whole marrow aspirates over a Percoll cushion, using procedures 
analogous to those developed for human MSC isolation (54). Tissue culture flasks 
(185 cm 2 ) were seeded with 10 7 nucleated cells isolated from the cushion, and 
cultured with DM EM containing 10% fetal calf serum from a selected lot {80). 
Cells were passaged at 8 x 10 3 cells/cm 2 , and transported back to the veterinary 
hospital where they were maintained until the time of implantation. Cell-loaded 
implants were prepared by incubating fibronectin-coated porous hydroxyapatite- 
tricalcium phosphate (HA/TCP) cylinders (Zimmer, Inc.) in a 7.5 x 10 6 cells/ml 
suspension of MSCs for 3 hr at 37°C, The interval between marrow harvest and 
implantation was 16 days. An aliquot of cells from each preparation was also 



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cultured under osteoinductive conditions to quantify aspects of osteoblastic 
differentiation. 



Canine Femoral Gap Model 

A unilateral segmental femoral defect model was developed for this study 
following IACUC approval. Under general anesthesia, thirty-six skeletally mature 
female purpose-bred hounds (20 kg) underwent resection of a 21 mm long 
osteoperiosteal segment from their mid-diaphysis. A 4.5 mm Synthes* 8-hole 
lengthening plate was contoured to the lateral aspect of the bone, and secured with 
bicortical screws. The defect was filled with one of three materials; 1) a cell-free 
HA/TCP cylinder, 2) an MSC-loaded HA/TCP cylinder, or 3) cancellous bone 
harvested from the iliac crest. HA/TCP implants were secured by placing two 
sutures around the implant and the plate. Animals received peri-operative 
antibiotics, and analgesics were administered for three days postoperatively. 

Radiographic and Histologic Analyses 

Standard radiographic images were obtained at pre-op, immediately post-op, 
and at 4 week intervals until termination of the study. All samples contained a 
radiodensity step wedge to provide a basis for comparing changes over time, and 
between dogs. Upon sacrifice, specimens were subjected to high resolution Faxitron 
radiography, and subsequently processed for biomechanical evaluation. Following 
torsion testing, undecalcified longitudinal sections will be processed for quantitative 
histomorphometry. 

Biomechanical Testing 

Sixteen weeks after implantation, animals were sacrificed for torsion testing 
of femurs. The fixation plate, screws, and adherent soft tissue were removed, and 
the metaphyses of the bones were embedded. The specimens will be externally 
rotated in a custom torsion test apparatus, failure load and stiffness recorded, and the 
data analyzed by one way ANOVA according to post hoc Student-Newman-Keuls 
tests. 



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Results 

All animals tolerated the surgical procedure well, with no incidence of infection, 
implant rejection, or failure of fixation. Two modes of repair were apparent in the MSC- 
loaded samples; first, considerable callus formation occurred at both host-implant interfaces; 
and second, a substantial collar of bone surrounding the implant itself developed. Cell-free 
implants did not possess either of these features. Autograft samples underwent a traditional 
consolidation sequence, with the majority of bone laid down in the medial aspect of the gap 
defect. MSC-loaded samples not only became fully integrated at the host implant interface, 
but the periosteal collar extended proximally and distally beyond the cut edges of the gap. 
Furthermore, the diameter of new bone at the mid-diaphysis was greater in MSC-loaded 
implants than either autograft samples or intact limbs. Biomechanical analysis of harvested 
samples is currently in progress, in vitro analyses of the osteogenic potential MSCs from 
each animal demonstrate the development of alkaline phosphatase-positive cells which deposit 
significant mineralized extracellular matrix. 

Preliminary histomorphometrical data from MSC-loaded (n=2) and cell-free (n=l) 
HA/TCP carrier shows that bone fill as percentage of available space is 39% and 7% 
respectively. In the case of the MSC-loaded samples, in addition to the considerable amount 
of bone in the confines of the ceramic block, there was also a fairly large mineralized 
periosteal callus. Also, the marrow space was reestablished within the defect. Whereas in 
the cell-free HA/TCP cylinders, most of the bone present was in the endosteal space with 
some penetration into the implant. 

Torsional testing of the samples (n=6 per group) showed that the MSC-loaded 
samples were almost twice as strong as the cell-free samples, but were only a third as strong 
as autograft controls. 

Discussion 

The present study demonstrates that MSCs from a large animal may be culture- 
expanded, and implanted for the successful repair of large diaphyseal bone defects. 
Radiographic and histologic evidence indicates that not only do the MSCs form bone within 
and around the implant directly, but their presence elicits a response in the host periosteum 
to form additional bone. The mechanism of this is currently not known, but is consistent 



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with our observation that MSCs undergoing osteogenic differentiation secrete a paracrine 
factor(s) which is osteoinductive (63). The conspicuous lack of callus formation and 
periosteal reaction in the cell-free implants was an unexpected finding. Radiographic 
evidence suggests that MSC-mediated bone regeneration is faster than autograft throughout 
the study period. In addition to establishing a new standardized model for large animal bone 
repair, this study illustrates the feasibility of translating autologous stem cell therapy from 
the laboratory into the clinic. 

Example 3 

In vivo Bone Formation Using Human Mesenchymal Stem Cells 
Although rat MSCs have been shown to synthesize structurally competent bone in 
an orthotopic site (68), human MSCs have only been shown to form bone in vitro (12,64) 
and in an ectopic implantation site in immunodeficient mice (55). Since fracture healing 
and bone repair depend on the ability to amass enough cells at the defect site to form a 
repair blastema, one therapeutic strategy is to directly administer the precursor cells to 
the site in need of repair. This approach is particularly attractive for patients who have 
fractures which are difficult to heal, or patients who have a decline in their MSC 
repository as a result of age (72,1 18), osteoporosis (128), or other metabolic 
derangement. With this in mind, the goal of the current study was to show that purified, 
culture-expanded human MSCs are capable of regenerating bone at the site of a clinically 
significant defect. 

Materials and Methods 

Human MSC Cultivation and Manipulation 

Isolation and culture-expansion of human MSCs from a bone marrow aspirate 
obtained from a normal volunteer after informed consent was conducted as previously 
described (54,52). Following initial plating in Dulbecco's Modified Eagle's Medium 
(Sigma) containing 10% fetal bovine serum (BioCell) from a selected lot (80), non- 
adherent cells were removed on day 3 at the time of the first medium change, and fresh 
medium was replaced twice weekly thereafter. Adherent MSCs represent approximately 
1 in 10 5 nucleated cells originally plated. When culture dishes became near-confluent, 
cells were detached and serially subcultured. 



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In Vitro Osteogenic Assays 

Human MSCs were replated into six-well dishes at a density of 3x10 s cells/cm 2 . 
The following day {Day 0), fresh medium was provided, and the cells were grown in the 
absence or presence of Osteogenic Supplements (OS) (12,64). Media changes were 
performed twice weekly, and at days 4, 8, 12 and 16, cultures were assayed for cell 
number, alkaline phosphatase (APase) biochemistry and histochemistry, and mineralized 
matrix production utilizing techniques previously described (64). 

Implant Preparation 

Porous hydroxyapatite//3-tricalcium phosphate (HA/TCP) ceramic blocks, mean 
pore size 200-450 jim (Zimmer, Inc., Warsaw, IN), were shaped into cylinders 
approximately 4 mm in diameter and 8 mm in length with a 1 mm central canal, or cut 
into cubes 3 mm per side. MSC-loaded implants were prepared by incubating human 
fibronectin-coated HA/TCP cubes and cylinders in a 7.5 x 10 6 cell/ml suspension of first 
passage MSCs for 2 hr at 37 °C as previously described (68). Cell-free control cylinders 
were prepared identically. 

Athymic Rat Femoral Gap Model 

The femoral gap surgical model employed here has been used extensively in 
euthymic rats to study long bone repair (68,129,34,147). Briefly, both femurs of Harlan 
Nude (Hsd:Rh-/7iw) rats (325 g) were exposed by an anterolateral approach. A 
polyethylene fixation plate was attached to each femur by four Kirschner wires, and an 8 
mm transverse segment of the central diaphysis, along with its adherent periosteum, was 
removed by using a rotary osteotomy burr under saline irrigation. Each animal then 
received a cell-free HA/TCP cylinder in one femoral defect, an identical cylinder loaded 
with human MSCs in the contralateral defect, and a subcutaneous implant of a MSC- 
loaded HA/TCP cube along the dorsum. 

Radiography 

Immediately after sacrifice at each time point, all specimens were radiographed in 
a lateral position using a high resolution Faxitron Imaging system with an exposure of 35 
kVP for 30 sec. 



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Quantitative Histomorphoroetry and Immunochemistry 

Upon sacrifice at 4, 8, and 12 weeks, a minimum of 3 specimens of each type 
were processed for undecalcified histology following radiography. Longitudinal sections 
were cut, stained with Toluidine blue-O, and quantitative assessment of bone formation 
was performed using Leica Quantimet 500MC image analysis software as previously 
described (68). The data were analyzed by Student's t-test. Subcutaneously implanted 
samples were fixed in formalin, decalcified, embedded in paraffin, serially sectioned, and 
similarly stained. Limbs from one animal at each time point were also prepared for 
immunostaining by monoclonal antibody 6E2, which distinguishes human cells from rat 
cells (54). Undecalcifled cryosections were incubated with 6E2 supernatant, or an 
irrelevant primary monoclonal antibody control (SB-1) (10), followed by FITC-conjugated 
goat anti-mouse IgG secondary antibody (GIBCO) diluted 1:500 in phosphate-buffered 
saline. 

Biomechanical Testing 

Twelve weeks after implantation, 7 experimental animals and 6 un opera ted control 
animals were sacrificed for torsion testing of femurs as previously described (81). Tlie 
fixation plate and adherent soft tissue were removed, and the metaphyses of the bones 
were embedded. The specimens were externally rotated in a custom torsion test 
apparatus, failure load and stiffness recorded, and the data analyzed by one way ANOVA 
with post hoc Student-Newman-Keuls tests. 

Results 

MSC Cultivation and Osteogenic Differentiation In Vitro 

Human MSC cultures were established and, by 7 days, formed characteristic 
colonies on the surface of the culture dish. Primary colonies which were subcultivated on 
day 14 attached uniformly to the surface of new dishes, and were allowed to divide for 
another 7 days until they became ~ 85 % confluent. Passaged cells demonstrated their 
characteristic spindle-shaped morphology (Fig. 6A), and uniformly divided resulting in an 
even distribution of MSCs throughout the plate. Cells derived from this first passage 
were used for preparing implants as previously described, and an aliquot was used to 
confirm their osteogenic potential in vitro. 



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As described in previous studies (12,64,13), MSCs cultured with OS underwent a 
dramatic change in cellular morphology from that of spindle-shaped to cuboidal, which 
was accompanied by an increase in APase activity and production of an extracellular 
matrix rich in bone hydroxyapatite (Fig. 6B). A significant increase in APase activity 
was observed after 4 days of OS treatment with maximal activity occurring on day 8, 
followed by a decline through day 16 (Fig. 6C). This late decrease in APase activity of 
OS cultures correlates with increasing mineral deposition and terminal differentiation of 
cells into osteocytes. While no calcium deposition was detected either by Von Kossa 
staining or the sensitive colorimetric quantitative calcium assay in Control cultures, 
Figure 6C illustrates that MSCs grown with OS deposited a significant amount of calcium 
by days 12 (60 ± 5.1 M g/dish) and 16 (98 ± 5.0 M g/dish). 

MSC-Mediated Osteogenesis in Ectopic HA/TCP Implants 

Human MSC-loaded HA/TCP cubes implanted in the subcutaneous space of 
athymic rats displayed evidence of osteogenesis by 4 weeks, but considerably more bone 
was present within the pores at 8 and 12 weeks. A representative section from a MSC- 
loaded cube harvested 12 weeks following implantation is shown in Figure 7. Bone 
formation occurs within the pores of the cubes, and is associated with vascular elements 
which penetrate the implant. Such angiogenesis is obligatory to new bone formation since 
the secretory activity of osteoblasts is an oriented phenomenon guided by vasculature 
(25). As previously demonstrated (32,54), cubes implanted without MSCs never 
contained bone but were filled with fibrous tissue and blood vessels only. 

Osteotomy Model and Radiography 

Figure 8A illustrates the segmental defect model used in this study. The 
polyethylene fixation plate on top of the femur provides stability following creation of the 
8 mm diaphyseal defect. No animals experienced failure of fixation or other post- 
operative complications throughout the course of study. Previous studies have established 
that femoral defects that are not implanted with a bioactive material give rise to a fibrous 
non-union devoid of bone (68,34,147). High resolution Faxitron radiographs provided 
sufficient clarity and detail to discern subtle changes occurring within the implant and the 
surrounding host bone. Representative radiographs of the femurs from the 2 groups 
recovered 12 weeks post-implantation demonstrate substantially more bone in animals 



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which received MSC-loaded HA/TCP cylinders (Fig. 8B) versus cell-free cylinders (Fig. 
8C). Increasing radiodensity, and obliteration of the apparent pore structure, was used as 
an indication of new bone formation within the core of the implant. Although integration 
of the implant, or union, was not generally observed by 4 weeks, the subsequent 
formation of a radiodense bone bridge between the implant and the host at 8 weeks 
completely masked the interface. By 8 weeks, the MSC-loaded implant contained 
considerable bone within the pores and was integrated with the host bone at the ends of 
the implant. At 12 weeks, union was complete and additional bone was evident in the 
pores. Callus formation along the fixation plate was observed in some samples, as was 
an occasional eccentric spicule of bone usually present along the medial aspect of the 
femur. Some specimens, both with and without cells, contained cracks within the core of 
the implant. 

Immunocytochemical Evaluation 

Immunocytochemical staining with antibody 6E2 demonstrates that, at 4 weeks, 
virtually all the cells within the pores of the implant were reactive on their surface and 
were, therefore, of human origin (Fig. 9A). Along the immediate periphery of the 
implant, the host rat cells were intermingled with the human donor cells, but as the 
distance away from the surface of the implant increased, the representation of donor cells 
precipitously declined. The presence of these peripheral cells which are not 
immunostained also serves as a negative control for this established antibody. The 
ceramic material itself, which appears black in the phase contrast micrograph <Fig. 9B), 
displays a high level of background fluorescence. The exquisite sensitivity of the 6E2 
amigen.antibody interaction necessitated that we use unfixed frozen sections which, 
unfortunately, limited our ability to process these calcified tissue specimens for 
immunostaining. While we were able to obtain satisfactory cryosections of 4 week 
samples (shown here), we were unable to prepare sections from later samples which 
contained substantially more bone. 

Histologic Evaluation 

Analysis of the Toluidine blue-O-stained samples confirmed the observations made 
by radiography. Photomicrographs of representative sections of the implant groups 
recovered at 12 weeks are shown in Figure 9. Most of the pores of the implants loaded 



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with MSCs contained substantial new bone by 8 weeks, and this process of bone 
regeneration continued through the 12 week assessment period (Fig. 9C). At 8 weeks 
nearly all pores contained new bone, except in some discrete areas where loading of the 
MSCs may have been compromised. Evaluation of limbs following biomechanics testing 
indicates that fractures were of a transverse or spiral nature, and were generally 
propagated through a central region of the implant containing cartilage or a modest 
amount of bone, as seen in Figure 9C. During the regenerative process, substantial new 
bone formation occurred at the interface between the host and the implant, leading to a 
continuous span of bone across the defect. New woven and lamellar bone can be seen in 
intimate contact with the cut edge of the host cortex at 12 weeks (Fig. 9E), and this 
region of union is directly contiguous with bone formed throughout the pores of implant. 
In regions deeper within the HA/TCP (Fig. 9F), filling of the pores with new bone and 
vasculature is evident. 

In defects fitted with the HA/TCP carrier alone, the pores of the implant were 
predominantly filled with fibrous tissue even at 12 weeks (Fig. 9D). Many samples had 
evidence of modest integration at the host-implant interfaces, and at one end of this 
representative implant (Fig. 9D), host-derived endosteal bone appears to be advancing 
into the medullary canal of the carrier as a result of osteoconduction. None of the cell- 
free ceramic carriers contained bone throughout the pores of the implant. 

Histomorphometric Evaluation 

The results qualitatively described above are mirrored in the histomorphometric 
data presented in Table 3. 



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Table 3 

Bone Fill in HA/TCP Implants as a Percentage 
of Available Space 





Four Weeks 


Eight Weeks 


Twelve Weeks 


Carrier Alone 


1.89 ± 1.00 


11.47 ± 7.08 


29.51 ± 8.93 


Carrier plus MSCs 


1.95 ± 1.92 


26.46 ± 3.60* 


46.61 ± 14.83* 



Table 3. Longitudinal sections through the segmental defect of a thymic 
rats implanted with ceramic carriers, with and without human MSCs, were 
evaluated histomorphometrically for bone content . The results represent 
the mean ± SD of 3 experimental limbs of each group at 4 and 8 weeks, 
and 8 limbs of each group at 12 weeks. *P<0.05 compared to the carrier 
alone at each time point. 

Bone present in the cell-free HA/TCP implants primarily represents the bony 
ingrowth from the cut ends of the host cortices. At 4 weeks and beyond, the MSC-loaded 
samples contained significantly more bone than the cell-free group, and the average bone 
fraction within the implant increased over time, reaching 26.5% and 46.6% by the 8 and 
12 week time points, respectively. This increased bone fraction at 8 weeks is 2.3-fold 
higher than that measured in cell-free implants at the same time, and by 12 weeks, is over 
23-fold higher than that observed in either condition at 4 weeks. The volume fraction of 
the HA/TCP carrier remained constant, and served as an internal control for 
histomorphometry. 

Mechanical Testing 

Twelve experimental and 1 1 intact femora from age and weight-matched control 
animals were tested in torsion 12 weeks after implantation. Two experimental limbs were 
not tested because they were extremely fragile. Gross inspection of the healed defects 
revealed a distal varus rotation deformation in most specimens. Table 4 summarizes the 
mechanical testing results in terms of torsional strength, stiffness, and total energy 
absorbed. 



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Table 4 

Mechanical Properties of Rat Femora 12 Weeks after Implantation 





intact Control 


Carrier Alone 


Carrier + Macs 


Strength(N mm) 


409 ± 71 


74 ± 63 


159 ± 37 


Stiffness(N mm/deg) 


39 ± 5.5 


6.6 ± 4.2 


16.2 ± 4.0 


Energy(N mm x deg) 


2.6 ± 0.7 


0.6 ± 0.4 


1.3 ± 0.8 



Table 4. Mechanical testing data on rat femur samples from 
unoperated age matched controls (Intact Control), or animals whose 
segmental defects were implanted with the HA/TCP carrier alone (Carrier 
alone) or the MSC-loaded HA/TCP (Carrier + MSCs). These results 
represent the mean ± SD of 6 limbs from each experimental implant 
group, and 11 limbs from control animals. Twelve weeks after 
implantation, each specimen was harvested, the ends of the bone were 
embedded, and the samples were tested in external rotation at 6 
degrees/second along the longitudinal axis until failure. One-way ANOVA 
on each of the parameters showed a significant difference between the 
groups at P< 0.0001. Furthermore, each of the groups were significantly 
different from the other for strength and stiffness (/ > <0.05), as determined 
by post hoc Student-Newman-Keuls tests. 

These results demonstrate a 115%, 145% and 112% increase in strength, stiffness 
and torsional energy absorbed, respectively, in MSC- loaded samples compared to cell-free 
carrier samples. All three groups were found to be statistically different from each other 
in failure torque and stiffness. 



Discussion 

The results presented here demonstrate that purified, culture-expanded human 
MSCs are capable of healing a clinically significant bone defect in a well-established 
model for bone repair. While the osteogenic potential of human MSCs has been proven 
by neo-osteogenesis in subcutaneous implants (54), as well as in studies of isolated MSCs 
in vitro (12,64), this is the first demonstration that human MSCs can form bone at an 
orthotopic site in need of repair. The combination of MSCs with a porous HA/TCP 
carrier possesses regenerative potential which is histomorphometrically and 
biomechanically superior to the carrier alone. This investigation paves the way for the 



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clinical application of autologous MSC-therapy for the treatment of orthopedic defects in 
man. 

The progressive increase in radiodensity of the healing bone at 8 weeks parallels 
the histological observations of processed limbs. Immunocytochemistry proves that the 
cells associated with the ceramic at 4 weeks are of human origin, and that the cells 
surrounding the implant are from the host. At 8 weeks and beyond, bone is laid down by 
the donor MSCs and eventually resorbed and replaced by bone derived from host cells 
through the normal remodeling sequence (24,47). It is important to note that in the 
process of regenerating this osseous defect, bone formation occurs by a direct conversion 
of mesenchymal cells into osteoblasts rather than by an endochondral cascade. This 
observation is consistent with previous studies of osteogenesis in implants loaded with 
animal or human MSCs (32,70,54,68). As the regenerative process continues, the pores 
of the ceramic are filled with an increasing amount of bone, which is laid down upon the 
walls of the implant or existing bone, and oriented by the invading vasculature that 
provides a portal for the entry and establishment of new marrow islands containing 
hematopoietic elements and host-derived MSCs. 

The rate of bone regeneration is lower than that observed in euthymic rats 
implanted with syngeneic MSCs (68), suggesting that immunocompromised rats are not 
the ideal hosts to assess the bone-forming potential of human MSCs. This may be due in 
pan to the xenogeneic nature of the implant and the increased natural killer cell activity, 
which may be a compensatory mechanism for the animal to cope with its deficient T-cell- 
mediated immunity (123). Nevertheless, a significantly higher amount of bone was 
formed in the defect which received MSCs compared to those limbs receiving the carrier 
only. The extent of host-implant union was greater in the MSC-loaded implants, which 
likely reflects the combined contributions of implanted MSCs and host-derived cells. 

The ability of human MSCs to regenerate bone in this experimental model 
compares favorably with other investigations testing implants such as demineralized bone 
matrix, bone marrow, purified or recombinant bone morphogenic proteins (BMP), 
allograft, ceramics, fibermetals and gene-activated matrices (129,34,147). In addition to 
forming a substantial amount of histologically normal bone, the biomechanical data 



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demonstrate that torsional strength and stiffness at 12 weeks were -40% that of intact 
control limbs, which is more than twice that observed with the cell-free carrier, and also 
twice that achieved in a similar study of bone repair using fresh autograft in a primate 
long bone defect model (29). 

Recently, growth factors such as recombinant human BMP have been implanted in 
experimental bone defect models in an effort to stimulate bone repair (147,78,29). 
Although recombinant BMPs are capable of inducing the endochondral cascade in ectopic 
implants (146), their ability to reproducibly direct bone formation at orthotopic sites has 
been hampered by the problems associated with the design and selection of an appropriate 
carrier. In contrast to the mechanical data showing significant bone regeneration in a 
MSC-loaded ceramic, BMP delivered in the same HA/TCP carrier did not increase 
implant strength over the carrier alone (126). The brittle nature of this ceramic, 
combined with its slow resorption and complex porous structure, may explain why even 
in the presence of significant bone formation mechanical strength remains less than intact 
limbs. In addition, stress shielding of the new bone, as a result of the load-bearing 
fixation plate, also restricts the strength of the healing defect. We believe, as has been 
previously suggested (16), that the use of an osteosupportive HA/TCP cylinder may not 
be the ideal matrix for replacement of diaphyseal defects . Efforts at designing the 
optimal biomatrix carrier for the delivery of MSCs is an active area of investigation. 

Implantation of culture-expanded autologous MSCs offers the advantage of directly 
delivering the cellular machinery responsible for synthesizing new bone, and 
circumventing the otherwise slow steps leading to bone repair. Even in patients with a 
reduced ability to regenerate connective tissue, presumably due to a low titer of 
endogenous MSCs (72,128,144,11), these rare MSCs may be isolated and culture- 
expanded over one billion-fold without a loss in their osteogenic potential (13), thus 
restoring or enhancing a patient's ability to heal tissue defects. The studies presented 
here suggest that MSC-based cell therapies will be useful for the reconstruction of a 
variety of tissue defects in man. 



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Example 4 

Effect of Coating on the Osteogenic Response of 
MSC-Loaded HA/TCP Cubes 
This experiment was performed in an attempt to establish that uncoated HA/TCP 
cubes are equivalent to fibronectin- or autologous serum-coated HA/TCP cubes in 
supporting MSC-mediated osteogenesis. 

Materials & Methods 

Standard HA/TCP cubes coated with either fibronectin, 1 % autologous serum, 
10% autologous serum or those left uncoated, were loaded with MSCs and implanted 
subcutaneously into athymic mice. The cubes were retrieved six weeks post-implantation 
and inspected for the level of osteogenesis by decalcified histological methods. The 
experiments were done with multiple human and canine donors, and were performed in 
duplicate mice. 

Results & Conclusion 

MSC-Ioaded cubes from all treatment groups showed a significant amount of bone 
formation at six weeks. The coating of HA/TCP cubes with either fibronectin or serum 
had no effect on the level of MSC-mediated osteogenesis within the cube. As expected, 
the cell-free control HA/TCP cubes did not have osteogenesis. Based on the above 
results, we conclude that uncoated HA/TCP is a viable carrier for the delivery of MSCs 
to effect bone repair/augmentation. 



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Example 5 

The Comparison Of Bone Grafting Materials 
Using a Canine Spinal Fusion Model 
The data reported here provides important information regarding the value of fresh 
bone marrow and of culture expanded purified mesenchymal stem cells as a means of 
improving existing graft materials for spinal fusion applications. This technology may 
result in a significant improvement in the efficacy of bone grafting materials and 
procedures. Furthermore, use of bone grafting procedures which do not require harvest 
of autogenous bone graft will significantly reduce the morbidity of bone grafting 
procedures. 

Materials & Methods 

We developed the canine posterior segmental spinal fusion model specifically for 
evaluation and comparison of developmental bone grafting materials which had proven 
efficacy in small animals. Canine bone is a better model for human bone than rodents 
(35), and avoids many of the limitations associated with defect models discussed herein. 
Use of a larger graft volume and a common site for clinical graft procedures (the spine) 
are significant advantages. Testing three materials in each animal allows control for 
interanimal variation and reduces the number of animals required. The model allows 
each fusion site to be mechanically tested without artifacts from internal fixation. Union 
score of the cross sectional area of the fusion mass at the site of failure has also proven to 
be a sensitive means for comparison between materials. Quantitative CT image analysis 
of each fusion site prior to destructive testing was developed in response to the first 
review. This now allows quantitative analysis of fusion mass volume, mean electron 
density (mineralization), and cross sectional area. 

Using this model, we have shown that a collagen/ceramic (CC) composite 
composed of Type I bovine fibrillar skin collagen and 0.5-1.0 mm diameter granules of a 
biphasic calcium phosphate ceramic (60% hydroxyapatite, 40% tri-calcium phosphate), is 
ineffective as a graft material. Used alone it is no better than an ungrafted defect. 
Addition of an extract of bone matrix proteins or adding autogenous bone improved 
efficacy (p<0.01), but the result remained inferior to pure autogenous bone 
(p<0.01)(55). We also showed that adding this collagen/ceramic composite to autogenous 



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bone as a "bone graft expander" significantly reduced autograft performance 
(p< 0.01)(58). Interestingly, successful fusions resulting from the CC composite had 
similar mechanical properties to fusions resulting from autograft, indicating that residual 
unresorbed ceramic granules had no evident adverse effect on the material properties of 
the fusion mass. 

We have evaluated bone marrow processing as a means of enhancing graft 
performance in a spinal fusion study, using the Collagen Corporation collagen/ceramic 
composite as a delivery system for a purified matrix protein, Osteoinductive Factor 
(OIF). This matrix was mixed 50:50 with either autogenous cancellous bone (AB), 
freshly aspirated bone marrow (ABM), or fresh bone marrow nucleated cells which had 
been concentrated ten fold by cemrifugation and buffy coat isolation (BMC). 
Unfortunately, as the study was completed, it was discovered that OIF was not an active 
cytokine. Earlier preparations of OIF had been inductive due to contamination with 
BMPs. As a result, the overall fusion rate was low. However, there was a strong trend 
for improved results from marrow concentration when ABM and BMC are compared 
(p=0.06, Logistic Regression Model for Clustered Data). Unfortunately, our ability to 
answer this important question was limited by the poor baseline performance of the 
matrix. This highlights the importance of selecting an effective and reliable matrix for 
evaluation of bone marrow grafts in this project. The incidence of union scores from 0 to 
4 for each material in this experiment are presented in Table 5. 



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Table 5 

Spinal Fusion Union Score for Selected Imp in g Mg teriak 





Union Score 


Total Score 


Materials 


N 


4 


3 


2 


1 


0 




AB+OIF/CC 


11 


2 


1 


1 


2 


5 


15 


BMC+OIF/CC 


11 


0 


2 


2 


1 


6 


11 


ABM+OIF/CC 


11 


0 


2 


0 


0 


9 


6 



These studies show that the posterior segmental canine spinal fusion model is a 
sensitive and reliable tool for comparison of graft materials. Ungrafted sites heal poorly 
with a low union score. Autogenous cancellous bone graft is very effective, but does not 
heal every graft site. This spectrum of performance for autogenous bone makes it 
possible to evaluate materials which may perform as well as or better than autogenous 
cancellous bone. Both union score and mechanical testing can and have been used 
effectively to compare materials. The use of high resolution CT image analysis adds an 
important nondestructive assessment tool which is flexible, quantitative and reproducible. 
This model sets a new standard for the evaluation of bone grafting materials. 

Posterior Segmental Canine Spinal Fusion Model 

The specific aims of this project address the comparison of three graft materials: 
Granular Ceramic matrix loaded with autogenous mesenchymal stem cells; Granular 
Ceramic matrix loaded with fresh bone marrow; and Granular Ceramic matrix loaded 
with fresh bone marrrow and autogenous mesenchymal stem cells. 

Twelve male beagle dogs (age 10-14 months, 12-14 kg) are used for the 
experiment. Localized fusions are performed at three spinal fusion sites, Ll-2, L3-4, and 
L5-6. Each site is internally fixed using dual plates immobilizing adjacent spinous 
processes, separated by one mobile segment. Each animal is grafted with one of the three 
materials under evaluation. To limit the potential for surgical bias and to insure 
distribution of materials at each of the three graft sites, twelve cards, two sets of the six 



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possible combinations of the three materials in three sites, are prepared at the beginning 
of each experiment and placed in an envelope. Site assignments are then made 
intraoperatively by blind drawing after site preparation is complete. Internal fixation is 
applied to each segment using plates placed on either side of the spinous processes. No 
external immobilization is used. AH animals are euthanized twelve weeks 
post-operatively lateral faxitron radiographs of each excised spine are obtained to assess 
the integrity of fixation. After removal of plates, high resolution CT images are acquired 
of all segments from L1-L6 in each spine. Individual fusion segments are mechanically 
tested to failure and physically evaluated for union. Comparison between materials is 
made based on union score, quantitative image analysis of CT data, and the mechanical 
properties observed for each fusion. 

Three weeks prior to spinal fusion each animal undergoes aspiration of bone 
marrow from the left iliac crest using sterile techniques under short acting IV sedation. 
These samples will then be transported to isolate and proliferate mesenchymal stem cells. 

On the day of surgery, each animal in the study undergoes spinal fusion at three 
separate levels. Mesenchymal stem cells are provided, and fresh bone marrow will be 
harvested again, this time from the right iliac crest. Each material composite is then 
prepared intraoperatively. Following preparation of the surgical bed and application of 
internal fixation, a 2 cc volume of each material is grafted at one level in each animal. 
In this way each animal has one fusion site for each of the materials. The materials are 
distributed according to a randomization protocol to prevent surgical bias and insure 
uniform distribution of materials by site. 

Initial Bone Marrow Aspiration and MSC Preparation 

Following sterile skin preparation, a small (3 mm) stab incision is made using a 
#11 blade. A Lee-Lok bone marrow aspiration needle (Lee-Lok Inc., Minneapolis, MN), 
is advanced into the bone cavity. The obturator is removed. A 2 cc volume of bone 
marrow is then aspirated promptly into a 10 cc syringe containing 1 cc of heparinized 
saline-(1000 units/ml). The syringe is detached and inverted several times to 
insure-mixing. Subsequent aspirates are taken using identical technique through the same 
skin incision and the same cortical window, but redirecting the needle tip to 



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intramedullary sites separated by at least 1 cm. Pressure is held on the site for 3-5 
minutes to insure hemostasis. No dressing is necessary. No post-operative 
immobilization or restriction is required. 

MSC Isolation and Cultivation 

Iliac crest marrow aspirates (10 ml) were obtained from 15-25 kg random source canines 
under Propofol anesthesia (Diprivan 1%, Stuart Pharm., DE). The marrow was drawn 
into a 10 ml syringe containing 7500 units of Heparin (Elkins-Simm, Cherry Hill, NJ) to 
prevent clotting. For some canines, the marrow was placed on ice and shipped overnight 
to the cell culture facility. The marrow was mixed with two volumes of Complete media, 
consisting of 10% FBS and antibiotics (100 U/ml penicillin G, 100 jxg/ml streptomycin 
sulfate, and 0.25 /xg/ml amphotericin B) in low-glucose DMEM. The nucleated cell 
fraction of the marrow was enriched for MSCs based on density separation on a 1.063 
g/cc Percoll cushion. Two hundred million nucleated cells in 5 ml of media were 
carefully layered over 20 ml of Percoll (Sigma, St. Louis, MO). Separation was achieved 
by centrifugation at 400 x g for 20 minutes. Cells collecting at the media-Percoll 
interface were then washed and plated in 100 mm petri-dishes at 1.6 x 10* cells/cm 2 in 7 
ml of Complete media, or in T-185 culture. flasks at 5.4 x 10 4 cells/cm 2 in 32 ml of 
Complete media. The cells were incubated at 37 °C in a humidified 5% C0 2 
environment. On day 4 of culture, the non-adherent cells were removed along with the 
culture media. The cultures were fed twice weekly and passaged between days 10-13 by 
releasing the cells with 0.05% Trypsin 0.53 mM EDTA exposure for 5 minutes. The 
cells were replated at 8 x ^cells/cm 2 for all subsequent passages. For some 
experiments, additional passaging was performed when cells approached confluence, 
typically 5-7 days after plating. 

Spinal Fusion Surgical Procedure 

Under general endotracheal anesthesia a mid-line posterior longitudinal incision is 
made from T10 to L7. Cutting cautery is used to outline the tips of spinous processes LI 
through L6 and subperiosteal elevation of paraspinal muscles is performed. The 
imerspinous ligament and interlaminar tissue are excised at the Ll-2, L3-4, and L5-6 
interspaces preserving the ligamentum flavum. A dental burr is used to excise facet 
cartilage to the level of subchondral bone and to perform a superficial decortication of the 



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adjoining laminar surfaces under continuous saline irrigation. Entry into the neural canal 
is avoided. When site preparation is complete, a saline soaked gauze is placed in each 
fusion site, the wound is covered; and an unscrubbed assistant blindly selects a card 
containing the material/site assignment for that animal. 

Porous hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic material, supplied 
from Zimmer Inc. as a 60/40 combination, is lightly crushed and sieved to select for a 
particle size ranging from 1.0 to 2.S mm in diameter. Following sterilization of the 
granules, 15 million MSCs are incubated with the 1 cc of ceramic for 3 hours at 37 
degrees Celsius with agitation every 30 minutes. As noted above, implant groups consist 
of 1) ceramic granules combined with autologous MSCs, 2) ceramic granules combined 
with autologous MSCs and fresh bone marrow obtained by aspiration, and 3) ceramic 
granules combined with fresh bone marrow alone. 

Following preparation of all graft materials, materials are carefully placed in their 
appropriate sites. Approximately 1 cc of each graft is used to fill the area of the excised 
facets and the interlaminar space. The remainder of each graft is layered over the dorsal 
surface of the adjoining lamina. 

Fixation at each site is then applied. A 1 mm burr is used to place a hole in the 
central region of the distal spinous process at each site. 316L stainless steel plates (0.125" 
x 0.4" x 1.4") are placed on either side of the adjoining spinous processes and fixed to 
the caudal spinous process using a stainless steel bolt and nut (size 2-56, 0.5" long). 
Four symmetrical holes in each plate allow selection among three potential fixation 
lengths to accommodate variation in the interspinous distance at individual sites (0.75", 
0.90\ and 1.05"). The fixation to the cranial spinous process at each level is 
accomplished by drilling through the appropriate hole in the plates to create a hole in the 
spinous process and by passing a second bolt and nut. Both bolts are tightened firmly 
without crushing the spinous processes. A second locking nut is then applied at each site 
to prevent loosening. The spinal wound and the autograft donor site is then closed using 
O-Dexon Plus sutures in deep fascia, interrupted subcutaneous sutures of 2-0 Dexon Plus, 
followed by staples in the skin. 



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Following sterile skin preparation, a small (3 mm) stab incision is made using a 
#11 blade. A Lee-Lok bone marrow aspiration needle (Lee-Lok Inc., Minneapolis, MN), 
is advanced into the bone cavity. The obturator is removed. Bone marrow is then 
aspirated promptly into a 10 cc syringe, and mixed with the granular ceramic matrix and 
allowed to set for 10-15 minutes to allow clot formation. Excess fluid is then pressed out 
with a gauze sponge and a 2 cc volume of the matrix impregnated with fresh bone 
marrow is implanted. 



Animal Care 

Study animals are cared for in accordance with the Principles of Laboratory Care 
and The Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 
1985. 

Each animal receives prophylactic antibiotics of Penicillin G 500,000 units IM pre- 
operatively and Ampicillin 250 mg po per day for five days post-operatively. 
Acepromazine and Tylenol are used for perioperative pain. No external immobilization is 
used. Animals are housed in cages for three days post-operatively and then moved to 
runs where they are exercised daily. 



Medications 

Premedication: 

Sedation for 1st Aspirate 

Induction 

Anesthesia 

Post-op 

Distress/pain 

Euthanasia 



- Atropine (0.02 mg/lb) |M 

- Penicillin G 1.2 million U IM 

- Pentobarbital 20-25mg /kg IV 

- Na Thiamylal (Surital) 6-8 mg/lb IV 

- Closed hallothane and oxygen 

- Penicillin G 250 mg IM qd x 5d 

- Acepromazine 2.2 mg/10 kg IV prn 

- Pentobarbital 50 mg IVP 



Specimen Harvest 

After twelve weeks animals are euthanized by pentobarbital overdose and the 
lumbar spine is harvested intact. Lateral Faxitron radiographs are obtained to document 
the integrity of fixation. After removal of the plates individual segments are meticulously 



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cleaned of soft tissue, taking care not to damage the fusion mass and to prevent 
dehydration of the specimen. Segments are then potted in Orthodontic Resin (Meer 
Dental Supply, Cleveland, OH) and frozen in double sealed plastic bags at -20°C 

Quantitative CT Image Analysis of Fusion Sites 

Quantitative assessment of the fusion mass is performed using helical X-ray 
Computed Tomography (CT) and automated 3-dimensionaI image processing techniques. 
Quantitative measures at each fusion site includes: 1) fusion mass volume, 2) area 
measured at the mid-disk cross-sectional plane, and 3) mineralization density of the fusion 
mass. 

Scanning is done on a Somatome Plus 40 CT scanner (Siemens Medical Systems). 
The specimen is placed with the cranial-caudal axis perpendicular to the direction of table 
travel. In this orientation the intervertebral spaces are oriented perpendicular to the scan 
plane. This reduces the chance of missing features of nonunions occurring in the 
transaxial plane and allows the midline of the intervertebral space to be identified more 
accurately. Scans will be done at 120 kVp, 210 mA, 1 sec helical mode, 2 mm 
collimation, and table speed 2 mm/sec, for 30 sees. This will produce images with 2 mm 
section thickness and 1 mm 2 pixel area. Images are then reconstructed using the bone 
algorithm and an image-to-image overlap of 1 mm. This provides the highest possible 
spatial resolution available in three dimensions (i.e. a voxel size of 1 mm 3 ). A Siemens 
bone mineral density phantom is placed beneath each specimen to provide a reference for 
the quantitation of bone mineralization density. 

Bone and soft tissue CT values are easily different iable (CT bone - 150-1000, CT 
soft tissue - 0 — 20). Therefore, three-dimensional segmentation of the reconstructed 
volume data sets is performed using a basic automated threshold algorithm in each two 
dimensional slice followed by a connectivity algorithm between slices. Once the 
three-dimensional data set has been segmented, the volume of the fusion mass is 
measured in a specified region of interest, defined by anatomic fiducial markers. The 
user selects two points which define the dorsal most points of the left and right neural 
foramen at the level of interest and one level above and below the site of interest. The 
user also identifies the midpoint of the intervertebral disk. The interactive tools for 



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manipulating and visualizing the 3-D data sets are developed using the GL library and Iris 
Inventor Toolkit available on a Silicon graphics workstation. 

The volume of the fusion mass is calculated by summing the segmented voxels 
(bone) within the specified region of interest and multiplying this sum by the appropriate 
voxel volume (1 mm 3 ). The area of the fusion mass will be measured at the mid-fusion 
cross-sectional plane by summing the number of segmented pixels (bone) in the 
mid-fusion slice and multiplying the sum by the appropriate pixel area (1 mm 2 ). A 
mean-mineralization density is calculated for the entire fusion mass and referenced to the 
density of the phantom. A 2-D topographic map of bone mineral density at the mid-disk 
cross-sectional plane is provided. The original volume of bone and bony cross section 
from the region of interest for each segment can be estimated by measuring the same 
region in the adjacent normal segment(s). This is used to normalize data from each 
fusion segment. 

Software for this quantitative analysis requires four modules: 3-D segmentation, 
animation to view cross- sectional planes and mark fiducial points, calculation of 
rectangular region of interest and quantitative measures of bone fusion, and an interactive 
display module to view reconstructed data in 3-D. 

Mechanical Testing 

Prior to testing, each specimen is thawed for 24 hours at room temperature. 
Testing is performed on an MTS TESTAR Bionix System using a custom four point 
bending device. After three sinusoidal conditioning cycles, load displacement data are 
collected nondestructively in flexion-extension and left-right bending. Failure testing is 
then performed in right bending using the ramp function at 8 mm/sec. Bending has been 
selected as the mode of failure because bending stiffness was found to be most closely 
correlated with union status in prior studies (94). Load-displacement curves are used to 
derive stiffness, maximum load, displacement to failure, and total energy to failure. 

Union Score 

Immediately after mechanical testing, the surfaces of the fractured specimen are 
examined using a metal probe. By comparing both sides of the fracture surface, the 



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degree of union is scored from zero to four based on a regional grid system (Figure 11). 
A score of four is defined as complete fusion of both facet joints and the entire lamina. 
One half point is added for union in either half of each facet joint or any of four 
quadrants of the adjoining laminar surfaces. Therefore, scores of 0, 1, 2, 3 and 4 
represent union of roughly 0, 25, 50, 75 and 100 percent of the cross-sectional area of 
the grafted volume, respectively. Scoring is done by consensus of two observers who 
examine the specimens, together and are blinded with respect to the material grafted at 
each site. 

Statistical Analysis of Spinal Fusion Union Score and CT Data 

Union score and CT generated data are analyzed to determine whether outcome 
differs statistically due to site and/or material effects. The data set is considered 
"incomplete" since only one material can be tested at each site within an animal. 
Therefore, both site and material are repeated factors but with missing observations. To 
accommodate the missing data profile of the experimental design as well as the possible 
correlation of observations within the same animal, a regression model for repeated 
measures data is used. This modeling strategy uses the Generalized Estimating Equation 
(GEE) approach described by Zeger et al. (150) and Ou et al. (108). Since union scores 
for a given dog are potentially not independent of each other, a working covariance 
structure of independence and a robust variance estimate are used in fitting the model. 
The covariance structure cannot be ignored without impacting inferences on the regression 
coefficients for site and material effects. T-tests using the robust variance estimates are 
used for specific comparisons between materials and sites as described by Paik (112). 
Union score remains the primary outcome parameter, however, distributional properties 
of the CT data will eventually dictate the statistical modeling strategy. 

Statistical Analysis of Spinal Fusion Mechanical Testing Data 

Mechanical parameters are strongly influenced by union score. Therefore, 
mechanical testing is used in this model as a secondary outcome parameter, primarily as a 
means to compare the material properties of bone formed in complete fusions (Large 
cross sectional area-Union Score 3.5 to 4.0) induced by different materials. Selection of 
complete unions allows comparison of fusions having similar moments of inertia. The 
complexity of bony geometry in partial fusions and wide local variation in the quality of 



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ununited soft tissue in the graft site prevents useful analysis of partial unions. Choice of 
this strategy reflects our practical belief that unless union score is high, the material 
properties of the bone formed by a given graft material will not be clinically important. 
The Student t-test has been used for first order comparisons. Paired analysis is most 
appropriate, but is not possible unless the union rates of the two materials to be compared 
are high. 

Interim Results of Spinal Fusion Study in Large Canines 

When 15 million MSCs are incubated per 1 cc of granular ceramic, using the 
techniques for creating MSC- loaded ceramic granules described in this application, we 
have found that greater than 95% of MSCs provided are adherent to the ceramic material 
after a 3 hour incubation period at 37 degrees Celsius. The MSC-ceramic material fits 
nicely into the surgical defect site created, without evidence of any migration of the 
implanted material up to 12 weeks post-operatively. Samples, either with or without 
MSCs, that were combined with fresh marrow at the time of implantation formed a soft 
clot which loosely covered the ceramic. No technical problems were encountered in the 
preparation or implantation of any samples. Analysis of the cells derived from each 
animal demonstrated osteogenic potential in vitro and significant bone formation in the 
standard in vivo ectopic implantation assay. 

All animals were terminated 12 weeks postoperatively. Plain film radiographs 
demonstrate the presence of osseous union in nearly all animals receiving MSC-based 
implants. Three-dimensional reconstruction from 2mm thick CT scans obtained at 1 mm 
intervals through the surgical site illustrate a larger fusion mass in those regions 
containing implants with MSCs compared to implants containing fresh marrow alone. 
Fusion scores of samples containing MSCs were also higher than those containing only 
fresh marrow. Furthermore, these fusion scores were also higher than that achieved in a 
previous study using samples prepared in a similar fashion containing recombinant human 
BMP-2. 



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Exam ple 6 
Bone Defect Repair Using Bone Marrow 
in an Absorbable Collagen-Containing Sponge 

The objectives of this study were to demonstrate efficacy of bone marrow and/or 
mesenchymal stem cells (MSCs) in healing clinically significant bone defects in an 
established animal model. 

Materials & Methods 

In the study, Fisher 344 rats (Charles River Laboratories, Wilmington, MA) of 
approximately 325 grams in weight were used. A bilateral femoral gap 8 mm in length 
was created in each femur. This length is approximately towards the diameter of the 
mid-diaphysis of the femur. An internal fixation plate was applied with four Kirschner 
wires. The groups for comparison were separately treated with one of the following: 

(1) Gelfoam® sterile sponge (Upjohn - Kalamazoo, MI); 

(2) Gel foam® sterile powder; 

(3) Peripheral blood clot; 

(4) Peripheral blood clot plus marrow derived from four bones; 

(5) Gelfoam® sponge containing marrow derived from four bones. 

(6) Gelfoam® sponge plus varying amounts of marrow from one bone down to 
one-half of one bone in the presence and absence of fresh peripheral blood to provide 
clot. 

In this animal system, fresh marrow from four bones yields approximately 150 
million cells while fresh marrow from one-half of one bone yields approximately 20 
million nucleated cells. Each group consisted of a minimum of three animals, all of 
which were sacrificed six weeks post-operatively to obtain the desired end-points. Some 
animals received high-resolution Faxitron radiographs at an intermediate point three 
weeks after implantation. At the six-week time point when all animals were sacrificed, 
the limbs were removed, radiographed, and prepared for undecalcified histological 
evaluation. 



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Handling properties of the Gelfoam® sponge, in combination with fresh marrow in 
the presence or absence of fresh peripheral blood clot t was desirable and nearly 
equivalent. 

Results 

Evaluation of radiographs following sacrifice of the animals at 6 weeks revealed 
no bone in the defect region of those animals implanted with either Gelfoam® sponge 
alone or those animals implanted with fresh marrow and a peripheral clot. Minimal 
endosteal spiking of new bone at the cut edges of the defect was observed, as is the case 
with the historical control of no implant alone. By contrast, those animals receiving 
Gelfoam® sponge plus marrow from four bones or one bone, in the absence or presence 
of peripheral clot, demonstrated a robust osteogenic healing response in the region of the 
implant. Those animals implanted with Gelfoam® sponge and marrow from one-half of 
one bone in the presence of peripheral clot demonstrated only modest amounts of bone 
formation. Finally, those animals implanted with Gelfoam® sponge and marrow from 
one-half of one bone in the absence of fresh peripheral clot demonstrated no bone in the 
defect region. Histologic analysis of all of the specimens confirms the observations made 
based on high-resolution radiographs. The formation of neocortices in samples of 
Gelfoam® sponge loaded with marrow cells was impressive. Histologic evaluation also 
indicates that no residual Gelfoam® material was retained at the site of the implant six 
weeks following surgery. Samples of Gelfoam® sponge loaded with marrow from one 
bone demonstrated islands of developing hemaetopoietic elements in the medullary canal. 
Host-implant interfaces appear to be intact. 

In summary, significant osteogenic response of syngeneic marrow in«tch of the 
recipient rats which were implanted with Gelfoam® sponge indicates the suitability of this 
cell and matrix combination implantation for the repair of significant bone defects. 



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118. Quarto, R.; Thomas, D.; and Liang, T.: Bone progenitor cell deficits and the 
age-associated decline in bone repair capacity. Calcif. Tissue Int. 56:123-129, 
1995. 

119. Ragni P, Lindholm TS, and Lindholm TC: Vertebral fusion dynamics in the 
thoracic and lumbar spine induced by allogenic de mineralized bone matrix 
combined with autogenous bone marrow. An experimental study in rabbits. 
Italian Journal of Orthopaedics & Traumatology 13:241-51, 1987. 

120. Rejda BV, Peelen JG, and de GK.: Tri-calcium phosphate as a bone substitute. 
Journal of Bioengineering 1:93-7, 1977. 

121. Saito, T.; Dennis, J.E.; Lennon, D.P.; Young, R.G.; and Caplan, A.I.: 
Myogenic expression of mesenchymal stem cells within myotubes of mdx mice in 
vitro and in vivo. Tissue. Engin. l(4):327-343, 1995. 

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122. Salama R, and Weissman SL.: The clinical use of combined xenografts of bone 
and autologous red marrow. A preliminary report. Journal of Bone & Joint 
Surgery British 60:1 1 1-5, 1978. 

123. Schuurman, H.-J., Hougen, H.P., and van Loveren, H. (1992) ILAR Journal. 
34(1-2), 3-12. 

124. Simmons DJ, Ellsasser JC, Cummins H, and Lesker P.: The bone inductive 
potential of a composite bone allograft-marrow autograft in rabbits. Clinical 
Orthopaedics & Related Research 97:237-47, 1973. 

125. Stabler CL, Eismont FJ, Brown MD, Green BA, and Malinin Ti.: Failure of 
posterior cervical fusions using cadaveric bone graft in children. Journal of Bone 
& Joint Surgery American 67:371-5, 1985. 

126. Stevenson, S.; Cunningham, N.; Toth, J.; Davy, D.; and Reddi, A. H.: The 
effect of osteogenin (a bone morphogenic protein) on the formation of bone in 
orthotopic segmental defects in rats. J. Bone Joint Surg. 76(11): 1676- 1687. 1994. 

127. Stevenson S, Hohn RB, and Templeton JW.: Effects of tissue antigen matching on 
the healing of fresh cancellous bone allografts in dogs. American Journal of 
Veterinary Research 44:201-6, 1983. 

128. Tabuchi, C; Simmon, D.J.; Fausto, A.; Russell, J.; Binderman, I.; and Avioli, 
L.: Bone deficit in ovariectomized rats. J. Clin. Invest. 78:637-642, 1986. 

129. Takagi, K.; and Urist, M. R.: The role of bone marrow in bone morphogenic 
protein-induced repair of femoral massive diaphyseal defects. Clin. Orthop. ReL 
Res. 171:224-231, 1982. 

130. Thomas ED, and Storb R.: Technique for human marrow grafting. Blood 36:507- 
15, 1970. 



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131. Thomas 1, Kirkaidy WW, Singh S, and Paine KW.: Experimental spinal fusion in 
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132. Tiedeman, J.J.; Connolly, J.F.; Strates, B.S.; and Lippiello, L.: Treatment of 
nonunion by percutaneous injection of bone marrow and demineralized bone 
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133. Tiedeman, J.J.; Huurman, W.W.; Connolly, J.F.; and Strates, B.S.: Healing of a 
large nonossifying fibroma after grafting with bone matrix and marrow. Clin. 
Orthop. ReL Res. 265:302-305, 1991. 

134. Tomford WW, Starkweather RJ, and Goldman MH.: A study of the clinical 
incidence of infection in the use of banked allograft bone. Journal of Bone & 
Joint Surgery American 63:244-8, 1981. 

135. Tsuji, T.; Hughhes, F.J.; McCulloch, C.A.; and Melchher, A.H.: Effect of 
donor age on osteogenic cells of rat bone marrow in vitro. Mech. Ageing Dev. 
51:121-132, 1990. 

136. Tuli SM, and Singh AD.: The osteoninductive property of decalcified bone 
matrix. An experimental study,. Journal of Bone & Joint Surgery British 
60:116-23, 1978. 

137. Turksen K, and Aubin JE.: Positive and negative immunoselection for enrichment 
of two classes of osteoprogenitor cells. Journal of Cell Biology 114:37384, 1991. 

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139. Urist MR, and Dawson E.: Intertransverse process fusion with the aid of 
chemosterilized autolyzed antigen-extracted allogeneic (AAA) bone. Clinical 
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140. Urist MR, DeLange RJ, and Finerman GA.: Bone cell differentiation and growth 
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thickness defects of articular cartilage J. Bone Joint Surg. 76A:579-592, 1994. 

143. Wakitani, S.; Saito, T.; and Caplan, A.I.: Myogenic cells derived from rat bone 
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Qualitative and quantitative analysis of orthotopic bone regeneration by marrow. 
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149. Younger, E.M.; and Chapman, M.W.: Morbidity at bone graft donor sites. J. 
Orthop. Trauma. 3:192-195,1989. 

150. Zeger SL, Liang KY, and Albert PS.: Models for longitudinal data: a generalized 
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Orthopaedic Research 10:562-72, 1992. 



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What Is Claimed Is: 

1. A method for augmenting bone formation in an individual in need thereof 
by administering to said individual isolated human mesenchymal stem cells with a 
medium which supports the differentiation of such stem cells into the osteogenic lineage 
to an extent sufficient to generate bone formation therefrom. 

2. The method of claim 1 wherein the medium is a porous ceramic or 
resorbable biopolymer. 

3. The method of claim 2 wherein the ceramic is selected from the group 
consisting of hydroxyapatite, 0-tricalcium phosphate and combinations thereof. 

4. The method of claim 2 wherein the ceramic is in particulate form. 

5. The method of claim 2 where the ceramic is a structurally stable, three 
dimensional implant. 

6. The method of claim 5 where the structurally stable, three dimensional 
implant is a cube, cylinder, block or in the shape of an anatomical form. 

7. The method of claim 2 wherein the resorbable biopolymer is selected from 
the group consisting of a gelatin, collagen and cellulose. 

8. The method of claim 7 wherein the medium is a powder, sponge, strip, 
film, gel or web or a structurally stable, three dimensional implant in the form of a cube, 
cylinder or block or in the shape of an anatomical form. 

9. The method of claim 7 wherein the gelatin is a bovine skin-derived gelatin. 

10. The method of claim 1 which further comprises administering to said 
individual at least one bioactive factor which induces or accelerates the differentiation of 
such mesenchymal stem cells into the osteogenic lineage. 



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11. The method of claim 2 which further comprises administering to said 
individual at least one bioactive factor which induces or accelerates the differentiation of 
such mesenchymal stem cells into the osteogenic lineage, 

12. The method of claim 7 which further comprises administering to said 
individual at least one bioactive factor which induces or accelerates the differentiation of 
such mesenchymal stem cells into the osteogenic lineage. 

13. The method of claim 10 wherein the cells are contacted with the bioactive 
factor ex vivo. 

14. The method of claim 11 wherein the cells are contacted with the bioactive 
factor ex vivo. 

15. The method of claim 12 wherein the cells are contacted with the bioactive 
factor ex vivo. 

16. The method of claim 13 wherein the cells are contacted with the bioactive 
factor when in contact with the matrix which supports the differentiation of such stem 
cells into the osteogenic lineage to an extent sufficient to generate bone formation 
therefrom. 

17. The method of claim 14 wherein the cells are contacted with the bioactive 
factor when in contact with the matrix which supports the differentiation of such stem 
cells into the osteogenic lineage to an extent sufficient to generate bone formation 
therefrom. 

18. The method of claim 15 wherein the cells are contacted with the bioactive 
factor when in contact with the matrix which supports the differentiation of such stem 
cells into the osteogenic lineage to an extent sufficient to generate bone formation 
therefrom. 



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19. The method of claim 10 wherein the bioactive factor is a synthetic 
glucocorticoid. 

20. The method of claim 1 1 wherein the bioactive factor is a synthetic 
glucocorticoid. 

21. The method of claim 12 wherein the bioactive factor is a synthetic 
glucocorticoid. 

22. The method of claim 19 wherein the synthetic glucocorticoid is 
dexamethasone. 

23. The method of claim 20 wherein the synthetic glucocorticoid is 
dexamethasone. 

24. The method of claim 21 wherein the synthetic glucocorticoid is 
dexamethasone. 

25. The method of claim 10 wherein the bioactive factor is a bone morphogenic 
protein. 

26. The method of claim 1 1 wherein the bioactive factor is a bone morphogenic 
protein. 

27. The method of claim 12 wherein the bioactive factor is a bone morphogenic 
protein. 

28. The method of claim 25 wherein the bone morphogenic protein is in a 
liquid or semi-solid carrier suitable for intramuscular, intravenous, intramedullary or 
intra-anicular injection. 



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29. The method of claim 26 wherein the bone morphogenic protein is in a 
liquid or semi-solid carrier suitable for intramuscular, intravenous, intramedullary or 
intra-articular injection. 

30. The method of claim 27 wherein the bone moiphogenic protein is in a 
liquid or semi-solid carrier suitable for intramuscular, intravenous, intramedullary or 
intra-articular injection. 

31. The method of claim 25 wherein the bone morphogenic protein is selected 
from the group consisting of BMP-2, BMP-3, BMP-4, BMP-6 and BMP-7. 

32. The method of claim 26 wherein the bone morphogenic protein is selected 
from the group consisting of BMP-2, BMP-3, BMP-4, BMP-6 and BMP-7. 

33. The method of claim 27 wherein the bone morphogenic protein is selected 
from the group consisting of BMP-2, BMP-3, BMP-4, BMP-6 and BMP-7. 

34. A composition for augmenting bone formation, which composition 
comprises a porous ceramic in combination with at least one of fresh bone marrow and 
isolated mesenchymal stem cells. 

35. The composition of claim 34 wherein the porous ceramic is in particulate 

form. 

36. The composition of claim 34 wherein the porous ceramic is a structurally 
stable, three dimensional implant. 

37. A composition for augmenting bone formation, which composition 
comprises a resorbable biopolymer selected from the group consisting of gelatin, cellulose 
and collagen in combination with at least one of fresh bone marrow and isolated 
mesenchymal stem cells. 



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38. The composition of claim 37 wherein the resorbable biopolymer is in 
particulate form. 

39. The composition of claim 37 wherein the resorbable biopolymer is a 
sponge, strip, film, gel or web or a structurally stable, three dimensional implant. 

40. A method for augmenting bone formation in an individual in need thereof 
which comprises administering to said individual thereof a bone formation augmenting 
amount of the composition of claim 34. 

41. A method for augmenting bone formation in an individual in need thereof 
which comprises administering to said individual thereof a bone formation augmenting 
amount of the composition of claim 35. 

42. A method for augmenting bone formation in an individual in need thereof 
which comprises administering to said individual thereof a bone formation augmenting 
amount of the composition of claim 36. 

43. A method for augmenting bone formation in an individual in need thereof 
which comprises administering to said individual thereof a bone formation augmenting 
amount of the composition of claim 37. 

44. A method for augmenting bone formation in an individual in need thereof 
which comprises administering to said individual thereof a bone formation augmenting 
amount of the composition of claim 38. 

45. A method for augmenting bone formation in an individual in need thereof 
which comprises administering to said individual thereof a bone formation augmenting 
amount of the composition of claim 39. 



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Fl G. 4C 




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SUBSTITUTE SHEET (RULE 26) 



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F! G. 5 B 




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SUBSTITUTE SHEET (flUL€ 26) 



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FIG. 1 1 

POSTERIOR 




SUBSTITUTE SHEET (RULE 26) 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US97/06433 



A. CLASSIFICATION OF SUBJECT MATTER 
IPC(6) :C12N 5/00, 5/02 

US CL :435/ 325, 366, 384, 395; 514/44; 424/ 93.21 
According to International Patent Classification (IPC) or to both national classification and IPC 



FIELDS SEARCHED 



Minimum documentation searched (classification system followed by classification symbols) 
U.S. : 435/ 325, 366, 384, 395; 514/44; 424/ 93.21 



Documentation searched other than minimum documentation to the extent that such documents arc included in the fields searched 



Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) 
MEDLINE, EMBASE, BIOSIS, CAPLUS, WPIDS, APS 

search terms: bone formation, mesenchymal stem cells, ceramic, bone morphogenetic protein, bmp, osteoblasts, 



matrix 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 1 



Citation of document, with indication, where appropriate, of the relevant passages 



Relevant to claim No. 



NADE et al. Osteogenesis after bone and bone marrow 
transplantation. The ability of ceramic materials to sustain 
osteogenesis from transplanted bone marrow cells: 
preliminary studies. Clinical Orthopaedics and Related 
Research. December 1983, No. 181, pages 255-263, see 
entire document. 

GOSHIMA et al. The origin of bone formed in composite 
grafts of porous calcium phosphate ceramic loaded with 
marrow cells. Clinical Orthopaedics and Related Research. 
August 1991, No. 269, pages 274-283, see entire 
document. 



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fx] Further document! are listed in the continuation of Box C. Q See patent family i 



Spec Mi 



docunMtt dermis*, <» town! eute of the an whicfa m iie< conakJered 
to be of ptfticukr relevance 

earlier docai 



MiUabed oe or after the mternaiional ftliaj dale 

(which mmy ttuow doubt oo priority cbum(a) or which m 
cited lo catabliah Che pubbcaboa date of mother ccuuoa or other 
-i , <M ^ecfeeo) -Y- 



l"r?!?"" I * fm * f 10 *■ ond 4mckm *^ eaJimitioo or other 

d nrwt p. b Uah uJ prior to the ■f rrw oo*) fUinf dale but bier than 
the priority caumed 



bier document pobUahed after the mteroational filing date or priority 
date and oof in conflict with the appucatioo b* cited to undemaod the 
principle or theory uodcriykg the mvcstioa 

document of particular relevance; the claimed invention cannot be 
considered novel or cannot be considered to involve an mventivcetep 
when the document m taken aione 

document of particular relevance; the claimed mveotxm cannot be 
considered to involve an inventive aiep when the document m 
combined with one or more other auch document*, such c 
being obvious to a pcraoo afcilled in the art 

the aame patent family 



Date of the actual completion of the international search 
14 JULY 1997 



Date of mailing of the international search report 

04 AU&1997 




Name and mailing address of the ISA/US 
Commissioner of Patent* and Trademarks 
BoiPCT 

Washington, D.C. 20231 
Facaimile No. (703) 305-3230 



Authorized officer] 

JILL D. SCI 
Telephone No. (703) 308-0196 



Form PCT/ISA/210 (second aheet)(July 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US97/06433 



C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 



Citation of document, with indication, where appropriate, of the relevant passages 



Relevant to claim No. 



SAKATA et aJ. Effect of bone marrow mononuclear phagocytes 
on the bone matrix-induced bone formation in rats. Clinical 
Orthopaedics and Related Research. July 1987, No. 220, pages 
253-258, see entire document. 

GOSHIMA et al. The osteogenic potential of culture-expanded rat 
marrow mesenchymal cells assayed in vivo in calcium phosphate 
ceramic blocks. Clinical Orthopaedics and Related Research. 
January 1991, No. 262, pages 298-311, see entire document. 

GOSHIMA et al. Osteogenic potential of culture-expanded rat 
marrow cells as assayed in vivo with porous calcium phosphate. 
Biomaterials. March 1991, Vol. 12, No. 2, pages 253-258, see 
entire document. 

HA YNESWORTH et al. Characterization of cells with osteogenic 
potential from human marrow. Bone. 1992, Vol, 13, pages 81- 
88, see entire document. 

LIND et al. Bone morphogenetic protein-2 but not bone 
morphogenetic protein-4 and -6 stimulates chemotactic migration 
of human osteoblasts, human marrow osteoblasts, and U2-OS 
cells. Bone. January 1996, Vol. 18, No. 1, pages 53-57, see 
entire document. 



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Form PCT/ISA/210 (continuation of second sheet XJuly 1992)* 



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