WORLD INTELLECTUAL PROPERTY ORGANIZATION
INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (POT)
(51) International Patent Classification ^ :
C12N 5/00, A61L 27/00
(11) international Publication Number: WO 97/45532
(43) International Publication Date: 4 C^ember 1997 (04.12.97)
(21) International Application Number: PCT/US97/O9067
(22) International Filing Date: 28 May 1997 (28.05.97)
(30) Priority Data:
28 May 1996 (28.05.96)
(71) Applicant: BROWN UNIVERSITY RESEARCH FOUNDA-
TION [US/US]; 42 Charksncld Street, P.O. Box 1949, Prov-
idence, Rl 02912 (US).
(72) Inventors: VALENTINI. Robert, F.; 28 Selkirk Road.
Cranston, Rl 02905 (US). KIM. Hyun, D.; Apartment 4A.
140 Prospect Street, Providence, Rl 02906 (US).
(74) Agent: KINDREGAN, Helen; Wolf, Greenfield & Sacks. PC,
600 Atlantic Avenue, Boston, MA 02210 (US).
(81) Designated Stales: CA, JP. European patent (AT, BE, CH, DE,
DK, ES. FI, FR, <5B, GR, IE, IT, LU. MC. NL, PT, SE).
With international search report.
Before the expiration of the time limit for amending the
claims and to be republished in the event of the receipu of
(54) Title: HYALURONAN BASED BIODEGRADABLE SCAFFOLDS FOR TISSUE REPAIR
A hyaluronic acid derivitized scaffold and method of forming arc disclosed. The scaffolds are useful for various medical purposes
such as tissue repair, tissue reconstruction and wound healing. In order to enhance these processes the scaffolds may be engineered to
tncoiporate biologically active molecules such as BMP.
FOK THE PURPOSES OF INFORMATION ONLY
codes used to identify Sutes party .o Ae PCT on .he froa. pages of pamphlet publishing international applications under the PCT.
Bosnia and Heraegovina
Central African Reinrt>lic
Republic of Korea
Republic of Korea
Republic of Moldova
The foimer Yugoslav
Republic of Macedonia
Trinidad and Tobago
United States of America
wo 97/45532 PCT/US97/09067
HVAMIRONAN BASED BIODEGRADABLE SCAFFOLDS FOR TISSUE REPAIR
Background Of The Invention
5 Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of alternating
glucuronic acid and N-acetyl D-glucosamine monomers. It is present in connective tissues and
plays a vital role in many biological processes such as tissue hydration, proteoglycan
organization in the extra-cellular matrix, and cell differentiation. Because of its important
biological roles, hyaluronic acid has been widely exploited in medical practice for use in treating
10 many different conditions. Several hyaluronic acid containing products are currently marketed
for pharmaceutical or veterinary use, including a product for intra-ocular injection during eye
surgery, synovitis agents for veterinary use, and coated gauzes for wound dressings.
Through the esterification of carboxyl groups of hyaluronic acid with various
therapeutically inactive and active alcohols, it has been possible to synthesize biopolymers with
15 medically desirable properties that are significantly different from those of hyaluronic acid itself.
The biocompatibility of these altered molecules appears to be quite good. For example, a
chemically modified form of hyaluronic acid "HYAFF-1 1" prevents fast enzymatic degradation
in vivo and degrades slowly in concert with new tissue formation. The HYAFF -1 1 material is
commercially available as skin repair products for wound and bum patients,
20 The following depicts the structure of hyaluronic acid.
H i r^ OH
The carboxyl group of hyaluronic acid can be replaced with various moieties, including
30 molecules such as ethyl, propyl, pentyl, benzyl or larger molecules such as hydrocortisone or
methyl prednisolone. Reaction conditions can be controlled so as to influence the degree of
substitution on the hyaluronic acid molecule, thereby further influencing the properties of the
Summary n f the Invention
The invention utilizes derivatives of hyaluronic acid as raw material to fabricate porous,
5 degradable scaffolds for a variety of medical purposes, including, but not limited to, tissue repair
and reconstruction and wound healing. These scaffolds are biocompatible and have degradation
products that are substantially non-toxic. These products are an improvement oyer prior art
materials such as poly lactide, poly glycolide, their copolymers, and the like which, although
biocompatible, render acidic degradation products that are not necessarily optimal for tissue
The porous scaffolds of tiie invention can be fabricated to any size or shape and can be
produced to virtually any desired predetermined pore size, depending upon the application. The
scaffolds of the invention can be adapted to promote host cells of different varieties to migrate,
adhere, proliferate, differentiate, and synthesize new tissue inside the pores. The invention can
1 5 accelerate the infiltration and integration of host tissue, while degrading slowly in concert with
new tissue formation. In addition, the porous scaffold can be used as a substrate for covalent or
non-covalent attachment of bioactive molecules such as cytokines, peptides, proteins, etc. that
have specific effects on ingrowing cells or surrounding tissue. Depending upon the bioactive
molecules selected, these effects on ingrowing cells can be directed to enhancing cell migration,
20 adhesion, commitment, proliferation and/or differentiation.
According to one aspect of the invention, a method for forming a substrate for cell growth
is provided. A water-insoluble hyaluronic acid derivative is dissolved in a first solvent. A
mixture of the first solvent, the water-insoluble hyaluronic acid derivative and a pore forming
agent is formed, wherein the pore forming agent is insoluble in the first solvent. The mixture
25 then is contacted with a second solvent, wherein the water-insoluble hyaluronic acid derivative is
insoluble in the second solvent, but the pore forming agent is soluble in the second solvent,
whereby the first solvent and the pore forming agent are extracted from the mixture to produce a
porous scaffold of the water-insoluble hyaluronic acid derivative.
The pore forming agent preferably is sized so as to leave pores sufficient to permit cell
30 ingrowth into the scaffold when the pore forming agent is extracted fi-om the mixture. The
resulting scaffold is a three-dimensional structure of interconnected pores which permit cell
ingrowth and, eventually, tissue replacement of the scaffold. In one particularly preferred
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embodiment, the pore forming agent is particles having a diameter between 10-1000 micrometers
with optimal tissue ingrowth at 1 06 and 600 micrometers.
It further is preferred to vacuum dry the scaffold from a wet state at a temperature of
between 4°C and 30°C, most preferably at ambient or room temperature. This results in a
5 scaffold that is non.brittle, handleable, and capable of being auloclaved with steam or gas
without undesirably affecting the pore interconnectivity of the scaffold. Most preferably the
water-insoluble hyaluronic acid derivative is hyaluronic acid esterified with a benzyl moiety.
In further embodiments, the method involves agents which are attached to the scaffold.
The attachment may be covalent or non-covalent attachment. The attachment may be directly to
10 the hyaluronic acid derivative in advance of the scaffold formation, or may be applied covalently
or non-covalently after the formation of the scaffold, such as by a coating. The agent also may
be blended with the dissolved hyaluronic acid derivative in the first solvent, to cause the agent to
be intermixed with and part of the formed scaffold structure. The kinds of agents contemplated
for attachment to the scaffold include drug agents for being released to surrounding tissues,
15 antipathogens for inhibiting pathogenic invasion of the scaffold, cell stimulating agents for
causing, for example, cell migration, adhesion, commitment, proliferation and/or differentiation
in, on or within the scaffold, and bioerodable coatings or blending agents for influencing the
bioerodability of the scaffold and/or for containing any of the foregoing agents such as
antipathogen agents, drug agents or cell stimulating agents.
20 According to another aspect of the invention, a substrate for cell growth is provided. The
substrate is a scaffold of vvater-insoluble derivatized hyaluronic acid defining interconnected
pores of sufficient size to permit mammalian cell ingrowth into the pores, wherein the
derivatized hyaluronic acid is a covalent conjugate of hyaluronic acid and a water-insoluble
moiety that renders the conjugate insoluble in water. The preferred features of the scaffold are as
25 described above in connection v^th the methods. All the various products resulting from the
foregoing methods are intended to be embraced by this aspect of the invention. For example,
scaffolds coated non-covalently or covalently with bioactive agents are contemplated by the
invention. Likewise, scaffolds coated with or blended with bioerodable polymers are
contemplated by the invention. The preferred covalent conjugate of hyaluronic acid is esterified
30 with benzyl moieties, most preferably wherein 100% of the carboxyl moieties of the hyaluronic
acid are esterified with benzyl moieties. Scaffolds composed of hyaluronic acid esterified with
other moieties (e.g. drugs, peptides) may also be employed.
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According to another aspect of the invention, a two-phase scaffold is provided. The iv/o:
phase scaffold is prepared by adding a hydrogel, a biodegradable polymer such as poly lactic acid
(PLA) or polyglycolic acid (PGA), or a ceramic such as hydroxyapatite or iricalcium phosphate
to a hyaluronic acid solution. Preferably the hyaluronic acid solution is HYAFF and the two-
phase scaffold is prepared with a hydrogel The pores of the two-phase scaffold are filled with
the hydrogel. The two-phase scaffold has all of the preferred features as described above in
connection with the single-phase scaffold of the invention.
According to another aspect of the invention, methods for growing cells are provided.
These cells are contacted with the scaffold of the invention as outlined above, and permitted to
grow upon and/or into the pores of the scaffold.
According to another aspect of the invention, methods of stimulating cell differentiation
are provided by contacting cells with the scaffold of the invention.
According to another aspect of the invention, methods for tissue culture are provided by
contacting cells with the scaffold of the invention.
According to still other aspects of the invention, methods for treating a variety of
conditions are provided, including methods for reconstituting or repairing bone, methods for
accelerating wound healing, methods for repairing cartilage as well as methods for reconstituting
tissues in the ectodermal, mesodermal and endodermal layers that require replacement or
Scaffolds with cells seeded upon the scaffolds also represent an aspect of the invention.
One particularly important aspect of the invention is the stimulation of bone cells isuch as
osteoblasts and pre-osteoblasts, and precursors thereof, to grow within a scaffold which can be
precoated, dipped or filled vnih bone morphogenetic proteins in order to induce bone growth and
differentiation from bone precursor cells.
These and other aspects of the invention will be described in greater detail below.
firief Descript ion Of The Drawings
Figure 1 is a graph depicting the release of BMP from Hyaluronic Acid scaffolds,
prepared as a scaffold alone (HA S), as a scaffold precoated with BMP and dried (HA P), as a
scaffold in which BMP and collagen are gelled together (HA SCB), and a scaffold in which the
scaffold is precoated with BMP and then collagen (HA PC); and
Figure 2 is a graph depicting the release of BMP from Poly-lactic Acid scaffolds,
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prepared as a scaffold alone (PLA S), as a scaffold precoated with BMP and dried (PLA P), as a _
scaffold in which BMP and collagen are gelled together (PLA SCB), and a scaffold in which the
scaffold is precoated with BMP and then collagen (PLA PC)
5 Detailed Description Of T he Invention
The invention involves three-dimensional biodegradable scaffolds of hyaluronic acid
derivatives for tissue reconstruction and repair. The porous scaffold has interconnected pores
that permit cells to grow into the scaffold, preferably completely penetrating the scaffold with
cells, and thereby, eventually replacing the scaffold with tissue. The scaffold can be fabricated to
10 be virtually any shape, size or thickness, and can be produced to various porosities and pore
sizes, depending upon the application. The scaffold is degradable, so that eventually it can be
completely replaced by tissue. The scaffold degrades slowly in concert with new tissue
formation. Such a scaffold offers the advantage of promoting host cells to migrate, adhere,
proliferate and synthesize new tissue inside the pores, accelerating, for example, wound healing.
15 Void volumes for the scaffold according to the invention can range from 40-90%. Pore
sizes for the scaffold of the invention can range from 10-1000 micrometers.
The invention requires the use of hyaluronic acid derivatives that are water-insoluble, but
are soluble in a first solvent. The water-insoluble hyaluronic acid is dissolved in that first
solvent, together with a pore forming agent that is insoluble in the first solvent. That mixture
20 then is contacted with a second solvent in which the hyaluronic acid derivative is insoluble but in
which the pore forming agent is soluble. In this manner, the first solvent is replaced/extracted by
the second solvent in which the hyaluronic acid is insoluble, bringing the hyaluronic acid
derivative out of solution and forming a scaffold. Likewise, the pore forming agent is soluble in
the second solvent and is extracted/dissolved, thereby leaving a porous scaffold of the water-
25 insoluble hyaluronic acid derivative.
The water-insoluble hyaluronic acid derivatives are known to those skilled in the art and
described in numerous publications. For example, because hyaluronic acid is a polycarboxylic
acid, its water-insoluble esters may be prepared using standard methods for the esterification of
carboxylic acids, such as the treatment of free hyaluronic acid with the desired water-insoluble
3D moieties in the presence of appropriate catalysts. Alternatively, the esters may be prepared by
treating a quaternary ammonium salt of hyaluronic acid with an esterifying agent in a suitable
aprotic solvent. Details of this latter method have been described in European Patent Application
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No. EP216453, April 1, 1987, the disclosure of which is incorporated herein by reference.
Esterification of hyaluronic acid with suitable water-insoluble moieties may be achieved also by
the use of linking groups interposed between the hyaluronic acid and the water-insoluble moiety.
Likewise, hyaluronic acid may be derivatized via amide bonds, as will be clear to those
5 skilled in the art. Such hyaluronic acid derivatives are described in the following PCT
publications, the disclosure of which is incoiporated herein by reference. W095/24429 discloses
highly reactive esters of carboxy polysaccharides, including hyaluronic acid. PCT Patent
applications W095/24497 and W095/041 32 disclose methods for preparing high molecular
weight hyaluronic acid derivatives.
10 Hyaluronic acid is a linear polysaccharide. Many of its biological effects are a
consequence of its ability to bind water, in that up to 500 ml of water may associate with 1 gram
of hyaluronic acid. Esterification of hyaluronic acid with uncharged organic moieties reduces the
aqueous solubility. Complete esterification with organic alcohols such as benzyl renders the
hyaluronic acid derivatives virtually insoluble in water, these compounds then being soluble only
15 in certain aprotic solvents.
When films of hyaluronic acid are made, the films essentially are gels which hydrate and
expand in the presence of water (hydrogels). By esterifying the hyaluronic acid and making it
insoluble in water, the scaffolds of the present invention then are possible. The scaffolds are not
hydrated in the presence of water and maintain their overall structure, permitting cell ingrowth.
20 Thus, the hyaluronic acid derivatives useful according to the invention are those sufficiently
derivatized such that the hyaluronic acid derivative will not form a hydrogel. Those of ordinary
skill in the art can easily test whether sufficient derivitization with an uncharged moiety has
occurred so as prevent the formation of a hydrogel. The preferred hyaluronic derivative is 100%
esterified hyaluronic acid-benzyl covalent conjugates, sold under the trade name HYAFF by
25 Fidia Advanced Biopolymers, Abano Terme, Italy.
Solvents for the water-insoluble derivatized hyaluronic acid molecules include
dimethylsulfoxide (DMSO), N-methyl-pyrrolidone (NMP), 1, 1. 1, 3, 3, 3-hexafluoro-2-propanol
(HFIP) and dimethylacetamide (DMAC). Other appropriate solvents will be known to those of
ordinary skill in the art. NMP is the prefened solvent.
30 Non-solvents for the derivatized hyaluronic acid useful in the invention include water,
ethanol, isopropanol, glycerol, ethyl acetate, tetrahydrofuran, and acetone. Other non-solvents
will readily be known to those of ordinary skill in the art: To be clear, the non-solv«nt ("second
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solvent") is used to replace the solvent and cause the extraction of the first solvent such as NMP_
or DMSO, thereby causing the formation of the scaffold and to dissolve the pore forming agent,
thereby producing pores in the scaffold.
The pore forming agents usefiji in the invention are particles of a desired size that arc
insoluble in the first solvent but that are soluble in the second solvent The particles preferably
are sized and are present in sufficient concentration so as to create pores of a sufficient size to
permit a plurality of mammalian cells to grow into and throughout the interconnected pores. In
one particularly preferred embodiment involving bone grow^th, the particles are between 1 00 and
600 micrometers in diameter. The pore forming agents may be any of a variety of materials,
depending on the particular selection of the solvent and non-solvent. Examples include: salt
crystals such as NaCl, KCL, MgClj, CaCl. and BaS04; soluble proteins such as albumin,
globulins, and the like; soluble dextrans such as dextran and dextransulfates, and the like; soluble
hydrogels such as agarose, alginate, chitosan, cellulose, carboxymethylcellulose, and the like;
and microspheres of polylactic acid, polyglycolic acid, and the like. Those of ordinary skill in
the art will readily be able to select usefiil pore forming agents. Tables 1 , 2, and 3 in the
Examples provide examples of the use of different sizes and concentrations of NaCl as well as
various lyophilization techniques to produce a variety of pore sizes and shapes.
As mentioned above, the scaffolds may be coated with a variety of materials, including
bioactive agents, and bioerodable agents. Bioactive agents include antipathogenic agents such as
antibiotics, antivirals, and antifungals, antiinflammatory agents, immunomodulators, cytokines,
etc. Virtually any bioactive compound useful in the scaffold or in the environment of the
scaffold may be coated onto the scaffold. In one particularly important embodiment, bioactive
molecules that have specific effects on ingrowing cells are coated onto the scaffold. Such
molecules can be those that effect cell migration, cell adhesion, cell conunitment, cell -
proliferation, cell differentiation, etc. Such molecules include interlukins, interferons, bone
morphogenetic factors, growth factors including platelet-derived growth factor, epidermal
growth factor, transforming growth factor and fibroblast growth factor and colony stimulating
factors. In one important aspect of the invention, the scaffold is coated with bone morphogenetic
proteins (BMPs) or growth and differentiation factors (GDFs) in order to induce the formation of
differentiated bone cells from bone precursor cells.
The coating also can be a biodegradable polymer which is added to influence the
degradation rate of the scaffold. Biodegradable polymers useful according to the invention
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include polylactic acid, polyglycolic acid, polylactic-polyglycolic copolymers, polycaprolactone,
polyphosphazenes and polyorthesters. Other biodegradable polymers are well known to those of
ordinary skill in the art and are described in great detail in the art relating to tissue implants and
sustained release polymeric devices.
Instead of coating the scaffold with the foregoing polymer materials a two-phase scaffold
may be prepared, in which the scaffold pores may be filled with the foregoing materials or a
hydrogel or ceramic. The two-phase scaffold may be prepared as described below in Example 5.
The two-phase scaffold has all of the preferred features as described above in connection with the
single-phase scaffold of the invention.
As mentioned above, the materials may be non-covalently coated on the scaffolds or
covalently attached to the scaffolds. If covalently attached to the scaffolds, such covalem
attachment may be carried out prior to the formation of the scaffold or may be carried out after
formation of the scaffold. Drugs may be incorporated in a gel which solidifies within the
scaffold (e.g. collagen type I).
Many of the objects and advantages described above in connection with coatings may be
achieved by blending such materials in solution with the water-insoluble hyaluronic acid
derivative prior to formation of the scaffold. For example, biodegradable polymers may be
included in such solutions, with the resulting scaffold being a blend of the derivatized hyaluronic
acid and the biodegradable polymer. Likewise, bioactive molecules may be blended with the
water-insoluble hyaluronic acid derivative prior to formation of the scaffolding. When a
biodegradable polymer is blended with the hyaluronic acid derivatives of the invention, then it is
prefeired that the biodegradable polymer comprise less than 50% of the total material of the
scaffold, and more preferably 10% or less of the total material of the scaffold. In any event, the
nature of the biodegradable polymer and amount must be adjusted, depending upon the
hyaluronic acid derivative selected and the desired characteristics of the end product so that the
final scaffold has the characteristics desired for the particular application. The biodegradable
polymer of this embodimem, however, should be chosen so that it is not dissolved by the water
or other non-solvent. If the biodegradeable polymer is soluble, it may be chemically modified to
make it insoluble. Techniques for chemical modification are well known to those of skill in the
It is preferred that the scaffold be dried from the wet state by lyophilization without
freezing. In other words, a vacuum pressure is applied to dry the scaffold. It is prefened that the
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vacuum pressure be applied at about ambient or room temperature, because doing so at either an_
elevated temperature or by freeze-drying adversely affects the interconnectivity of the pores and
the overall structure of the scaffold. According to the methods of the invention, scaffolds are
produced with not only desirable porosity for cell ingrowth, but also with a structural integrity so
that they may be sterilized using steam or gas sterilization without adversely affecting the
scaffold structure and characteristics.
The scaffolds of the invention have a variety of clinical uses. One important ex^ple is
in the repair of bone defects caused by trauma, bone tumor resection, in the case of joint fusion
and spinal fusion for non-healing fractures and osteoporotic lesions. It is noted that the scaffold
may be seeded with bone cells (osteoblasts and osteocytes) and bone cell precursors
(mesenchymal stem cells from bone marrow, periosteum, endosteum, etc.) before implantation.
The scaffolds also may be used in treating tooth and jaw defects in cases of trauma, bone loss,
tooth loss, gum disease and the like. The scaffold again can be seeded with cells of the foregoing
type for such purposes. The scaffolds also are useful in treating cartilage defects such as those
which result from rheumatoid arthritis, osteoarthritis and trauma. Cells useful for seeding in
such circumstances are chondroblasts and chondrocytes and cartilage cell precursors such as the
cell precursors described above in connection with bone. The scaffolds also may be used to
repair defects and damage in skin, muscle and other soft tissues such as results from trauma,
bums, ulcers (diabetic ulcers, pressure sores, venus, stasis ulcers, etc.). In this case, scaffolds can
be seeded with, for example, dermal fibroblasts, keratinocytes, and skeletal muscle cells.
Likewise, damage to visceral organs including liver damage, heart attack damage, and damage
resulting from intestinal cancer or intestinal ulcer may be treated with the scaffolds of the
invention. In these instances, the scaffolds can be seeded with cells such as hepatocytes, cardiac
muscle cells, intestinal cells, etc.
The invention also pertains to in vitro culture of cells with the purpose of creating tissue
constructs for repairing tissues and organs in vivo. The scaffolds may be used to promote tissue
culture of committed cells and/or differentiation of precursor cells. Thus, the scaffolds of the
invention can be used in virtually all instances when it is desirable to provide a substrate for the
growth of cells onto or into a tissue replaceable matrix. Scaffolds can also be used with
autografts, allografts, and xenografts associated with bone grafts, cartilage grafts, and joint
resurfacing implants and are particularly important applications of the present invention.
F^ample 1 : Pry pamtion of Polvmer ScaffoWs-
/. Scaffold preparation:
n\ 1 Scaffold Fahrication Techniqw:
HYAFF-1 1, a 100 % esterified derivative of hyaluronic acid (commercially available
from Fidia Advanced Biopolymers, Abano Terme, Italy) was dissolved in N-melhyl pyrrolidone
for 12 hours at room temperature to make a 1 0 % (w/v) solution. Presieved NaCl crystals (Fisher
Scientific, or Sigma) were mixed for 1 0 minutes at 20°C with the polymer solution at salt to
polymer dry weight ratio of 9:1 or 1 5:1 (w/w) to create a slurry or a paste-like mixture. The size
and quantity of NaCI used determines the porosity, pore distribution, and interconnectivity of the
final scaffold product. The quantity used in each experiment, therefore, was adjusted in order to
produce a scaffold having the desired porosity, pore distribution, and interconnectivity. The
average porosity, pore distribution, and interconnectivity produced at particular concentrations of
NaCI is depicted in Table 1 . The final scaffold pore size is dependent on the size of the NaCl
crystals used and, therefore, the size of the NaCl crystals to be used was determined based on the
desired pore size. The desired size of NaCl used ranged anywhere between 1 06 and 600 urn.
The effect of different lyophilization techniques on pore formation is presented in Table 2.
Average pore size produced at particular concentrations of NaCl is depicted in Table 3.
Status of Pores
Salt size (nm)
Pore size i\xm)
The paste was then formulated into scaffolds utilizing either of the two following
(1). The paste was packed into rubber molds which were submerged in a 1 liter bath of
distilled water for 48 hours at 20**C and stirred gently. The water was changed frequently
(preferably everj' 8 hours). The water serves both as a nonsolvent which is capable of absorbing
organic solvent and as a pore former which dissolves the NaCl, leaving pores in the scaffold.
15 The resulting wet scaffold was lyophilized at room temperature for 24 hours, without drying, in
order to obtain maximum pore interconnectivity with characteristic ultrathin septa between pores.
It was observed that freezing or air drying greatly diminishes the ability of the scaffold to exhibit
optimal pore characteristics. The dry scaffolds were then trimmed and cut to desired shape and
20 (2). The paste was packed into rubber molds which were submerged in 1 liter of 1 00 %
ethanol at 20T, a nonsolvent, for 24 hours with frequent change of ethanol. Ethanol only
absorbs the organic solvent while leaving the salt crystals intact. The scaffold was then cut or
pressed into the desired shape or size (alternatively the scaffold was cut and shaped after the
incubation with water). Pore formation was achieved next by submerging the scaffold in 1 liter
25 distilled water for 24 hours. The water was changed every 8 hours. It was observed that the
scaffold begins to float during the final stages of salt leaching. The resulting wet scaffold was
lyophilized to dryness.
The final scaffold was then sterilized either by standard^thylene oxide gas sterilization or
steam autoclave at 250T for 30 minutes with a 15 minute dry cycle, after which it was used for
30 in vitro cell seeding or in vivo implantation.
h) HYAFF-1 Id75 Scaffold Fabrication Techniow:
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Polymer scaffolds are prepared from HYAFF-1 lp75, a 75% esterified form of hyaluronic
acid using the following technique.
A known amount of HYAFF-1 lp75 is dissolved (12 hours, room temp.) in an organic
solvent (preferably dimethyl sulfoxide, DMSO) to make a 10% (w/v) solution. Presieved NaCl
crystals of desired size or size ranges (narrow or wide ranges, anywhere between 1 06-600 urn)
are mixed (5 min., room temp.) With the polymer solution at salt to polymer dry wejght ratio of
9: 1 or 1 5 : 1 (w/w) to create a slurry or a paste-like mixture. The paste is packed into rubber
molds and submerged in 100% ethanol for 48 hours with frequent change of EtOl I. Pore
formation is achieved next by submerging the scaffolds in distilled water (1 L) with stirring for
30 minutes with frequent change of water. Water treatment is stopped when the foams have
floated and expanded slightly. HYAFF-1 lp75 loses integrity and structure if left in water for
extended periods of time, but 30 minutes has been found to be enough time for complete salt
leaching while maintaining scaffold structure and integrity. The slight expansion causes NaCl to
be leached out much faster than HYAFF-1 1 scaffolds. The resulting wet scaffold is lyophilized
2. Treated Scaffold preparation:
In some experiments, the scaffold was dipped, injected, chemically immobilized, or
sprayed with drugs, peptides, proteins, cytokines, growth factors, oligonucleotides, antisense
oligonucleotides, DNA, or polymers prior to in vitro or in vivo application. This process
formulated a hybrid structure that, depending on the molecule or polymer used, targeted cell
migration, adhesion, commitment, proliferation, or differentiation. The following non-limiting
examples of treated scaffolds were prepared.
a) Bone morphogenic protein scaffolds: Scaffolds were produced as described above
except that lO^g of recombinant human bone morphogenic protein (rhBMP) was adsorbed on to
the scaffold by dip coating the scaffold with a 0.1 - 1000 Mg/ml BMP solution and air drying for
30 minutes in a laminar flow hood. Alternatively, 0. 1 - 1 000 \iglm\ BMP in collagen type I
suspension at 4"C was added to the scaffold and then gelled at 37''C for 1 hour.
b) Dipped protein scaffolds: The preformed scaffold was dipped in a solution containing
a drug. A small volume of fluid (e.g. solvent, such as water, DMSO. etc.) containing a known
amount of drug was coated or sprayed onto the scaffold. The solvem was evaporated by air-
drying, application of vacuum, lyophilization,€tc.
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c) Chemical linking of drug to scaflfold: A drug was chemically linked to HYAFF
scaffold. HYAFF surface is activated with coupling agents which bind to OH, NH2» SH or
COOH groups on the HYAFF. The coupling agent which was bifunctional was reacted with OH,
NH2, SH or COOH groups on a drug to achieve binding. In one example, CDI (a
heterobifunctional coupling agent) in acetone or EDC (a heterobifunctional coupling agent
similar to CDI except that it is soluble in water) in water was used to attach to OH groups on the
HYAFF. The other end of the EDC or CDI reacted with NH. or COOH groups on the drug or
peptide to achieve covalent linkage.
d) A drug that was soluble in DMSO, NMP, etc. (but insoluble in the non-solvent water,
ethanol, etc.) was mixed with the HYAFF/DMSO or NMP, etc. solution so as to achieve
incorporation of drug or peptide in the bulk of the HYAFF material. A carrier molecule or
excipient (albumin, dextran, carboxymethyl cellulose, etc.) was also incorporated.
e) A drug was added to a preformed HYAFF scaffold by the use of a gel-forming
material. For example growth factors, BMPs, etc. were added to liquid collagen gels maintained
at temperature. The growth factor/liquid gel was coated, dipped or sprayed onto the HYAFF
scaffold. The scaffold/gel construct was then warmed to room temp. (20'*C) or higher (body
temp. 37°C) for between 20 minutes and two hours in order to effect gelling and entrapment of
the drug or peptide.
J) In some experiments, HYAFF scaffold was also sprayed, dipped or coated with a
second polymer including HYAFF, PLLA, PGA, etc., that contained a drug or peptide. This
enabled release of the drug and tailoring of scaffold degradation rate.
g) In some experiments, the HYAFF scaffold was also made with degradable
microspheres comprised of PLLA, PGA, PLGA, etc., which slowly degraded to form pores. As
the pores opened, tissue ingrowth occurred. This approach allowed staged tissue invasion in
cases where early ingrowth was to be discouraged (e.g. infection, -etc.).
FiTftm ple 1: In vitro ceil growth- pr nUfcration and differentiation on polvmer scaffolds.
Polymer scaffolds were fabricated as described in Example 1, using sieved NaCl -crystals
(212 - 600 urn) and both 9:1 and 15:1 salt to polymer dry weight ratios. The scaffolds were
trimmed to produce cylinders having a 5 mm diameter by 3 mm thickness and autoclaved for
sterility. The scaffolds were then prewet in 70 % ethanol, and rinsed in sterile PBS. Primary rat
calvarial osteoblasts isolated from 7 day pups by sequential enzymatic digestion in 1 .37
wo 97/45532 PCT/US97/09067
mg/ml/collagenase/0.25% trypsin were seeded onto scaffolds at a density of 100,000 cells / 20 uj
media / scaffold. The cells were allowed to penetrate the pores for 30 minutes, prior to the
addition of Ham's F12 + 1 0 % FBS (Gibco or Sigma) alone or supplemented with 50 ug/ml
ascorbic acid (Sigma) and 10 mM p-glycerophosphate (Sigma). The cells were maintained in the
scaffold apparatus under tissue culture conditions for up to 11 weeks. Cell viability and
attachment were assessed by fluorescent microscopy using a Live / Dead Eukolight viability kit
(Molecular Probes, Inc., Eugene, OR). Under scanning electron microscopy (SEM) and light
microscopic evaluation, the polymer scaffolds showed interconnecting pores 200 to 600 microns
in diameter. The 15:1 polymer scaffolds showed greater pore interconnectivity and a thinner
septum between pores than the 9:1 scaffolds. Pilot studies showed that autoclaving did not alter
scaffold geometry or induce degradation. Osteoblasts seeded onto polymer scaffolds remained
viable at 1 , 6, and 11 weeks as evidenced by greater than 95 % fluorescence staining for live
versus dead cells. Increased fluorescence intensity at later lime points also suggested significant
cell proliferation, although this was not quantified. Scaffolds seeded with osteoblasts cultured in
ascorbic acid / phosphate supplemented media showed nodules of calcification at 4 weeks and
beyond, suggesting that HYAFF scaffolds support differentiation.
Fnamplc 3: J« vivo cell m iprafinti and growth on imnlanted scafffolds.
In vivo results demonstrated that HYAFF scaffolds can support bone formation. In one
study, scaffolds (4 mm diameter, 1 mm thickness) were implanted into 4 mm full thickness rat
cranial defects and harvested after 3 weeks. Empty rat cranial defect sites served as negative
controls. Upon macroscopic examination after sacrifice, the control empty defect site remained
largely devoid of tissue. Histological analysis revealed a thin fibrous connective tissue sheath
adjacent to the dural surface and continuous with the original cranial bone. For HYAFF scaffold
sites, the implant felt semi-rigid upon examination and was intimately connected with the
surrounding cranial bone. Histologically, the entire implant was filled with invading tissue. The
polymer scaffold showed minimal signs of degradation. The scaffold pores were completely
invaded and contained numerous tightly-packed fibroblastic cells with occasional blood vessels.
The newly formed tissue in the pores stained positive for collagen and mineral, as evidenced by
fast green and von Kossa staining. In some larger pores islands of bone trabecule were
observed. Trabeculae consisted of a row of osteoblast-like cells over an osteoid seam adjacent to
darkly stained mineral matrix. These results provide concrete evidence that HYAFF scaffolds
wo 97/45532 PCT/US97/09067
alone are osteoconductivc (ability to support bone ingrowth), in bony defects.
Example 4: Peptide treated scaffolds capable of
inducing and sustaining cellular differentiation.
Addition of exogenous factors to the scaffold, such as drugs, peptides, or proteins, can
additionally enhance target tissue formation, especially for large, non-healing, critical-sized
defects. Examples of such drugs, peptides, or proteins are provided in Example 1 above.
Bone morphogenetic proteins (BMPs) are members of the transforming growth factor
10 beta (TGFp) superfamily proteins involved in the induction of cartilage and bone. These
osteogenic and chondrogenic proteins are capable of committing undifferentiated mesenchymal
stem cells into bone and cartilage forming cells. Scaffolds adsorbed with 10 ug bone
morphogenetic protein (rhBMP-2) were implanted into rat 8 mm diameter critical-sized cranial
defects to assess bone formation at a bony site. In addition, similar scaffolds were implanted into
15 subcutaneous tissue to assess ectopic bone formation. After 3 wrecks, scaffolds at both sites
displayed significant bone formation in pores that were filled with mineralizing tissue, as
evidenced histologically with von Kossa staining. BMP-2 scaffolds exhibited more tissue
ingrowth and mineralization than untreated scaffolds.
Example S: Preparatio n of a two-phase scaffold,
A two-phase scaffold was prepared by adding a hydrogel to the solution instead of adding
a soluble crystal, protein, or microsphere to the HYAFF solution. For example, HYAFF was
dissolved in DMSO and preformed microspheres were added. Agarose microspheres were
formed through a hot-melt techniques and sieved to a size of 100-700 microns. The HYAFF
25 solution and microsphere were mixed and molded into a prescribed shape. The resulting mold
was then immersed in a bath of water or ethanol which did not dissolve the microspheres but did
extract the solvent. Similarly, the solvent was extracted by drying under ambient conditions,
with slightly elevated temperature (<70C) and/or with a gentle vacuum. After or during solvent
evaporation/air drying the construct was lyophilized to affect f\ill solvent removal and scaffold
30 formation. The resulting scaffold contained entrapped microspheres which constituted 40-90%
of the scaffold volume. 70-90% of the void volume contained microspheres with the remainder
being empty due to microsphere loss. Following lyophilization, the microspheres were
dehydrated and shrank to 5-20% of their original size. The microspheres were rehydrated by
immersion in water or placement in tissue. The rehydration resulted in microspheres which were
90-95% of their original starting size (e.g. after initial fabrication). Microspheres were fabricated
from a range of hydrogel materials including agarose, alginate, chitosan, collagens type I, IV,
etc Matrigel,laminin.etc. The hydrogel microspheres were also covalemly linked with drugs
and peptides using the coupling strategy described above. Likewise, drugs or peptides were
™xed with the hydrogel during or after microsphere formation. The advantage of a two-phase
scaffold is that a range of microspheres containing a range of drugs or peptides (including several
difTerem drugs and peptides together) can be incorporated in the scaffold for various purposes
(e.g. antibiotics to treat infection, growth factors to stimulate tissue growth, BMPs to stimulate
bone or cartilage induction).
jf^^r-rlr^^: rnmparis>ri ^ft^ntinn of BMP in Wvahir^niy (HA)
, ... f.„Pn,rira.ior, : Poly-L-lactic acid (PLLA, MW 100kD;Polysciences,lnc.) and
5 derivatized hyaluronic acid (HA. Fida Advanced Biopolymers, Italy) scaffolds were prepared
using solvent casting/particulate leaching and phase inversion/particulate leaching techniques,
respectively. A 20% solution of PLLA in methylene chloride and a 10% solution of HA in N-
methyl pyrrolidone were each mixed with sodium chloride crystals (106-600 um) at a salt to
polymer ratio (w/w) of 15:1 . The PLLA mixture was molded, air dried, washed in distilled
,0 water and dried, all for 24 hours. The HA mixture was washed in distilled water and
lyophilized, each for 48 hours. Scaffolds were trimmed to a thickness of 1 .5 mm and a diameter
of 5 mm, and sterilized by steam autoclaving.
. p^^^..c,,ff-,frfr..e,.„.r/>....r.n-o«: Recombinant human bone morphogenetic protein-2
25 (rhBMP2, a gift from Genetics Institute, 4.4,g) was coated on HA and PLLA scaffolds in 3
ways- 1) BMP precoating and drying (HA P or PLA P), 2) BMP and collagen gelled together m
situ (HA SCB or PLA SCB), and 3) BMP precoating followed by the addition of collagen (PC).
For all three preparations, scaffolds were prewet in ethanol and rinsed in sterile saline and loaded
with 4.4 ug rhBMP2. For collagen containing scaffolds, 25 ul collagen 1 (Vilrogen, Collagen
30 Corp.) was used. Scaffolds (HA S or PLA S) alone served as a control.
order to assess the amount and bioactivity of rhBMP2 released from scaffolds, an alkahne
wo 97/45532 PCT/US97/09067
phosphatase assay was used. C3H10T1/2 murine embryonic fibroblasts (ATCC) were cultured ,
in Basal Eagle media (Sigma) with 10% FBS. Scaffolds were placed on membrane inserts (3.0
um pore size, Fisher) and incubated in 24 well plates containing 12,500 cells/cm'. Scaffolds
were incubated with cells for either 24 or 48 hours and transferred to freshly plated cells after
5 each time point for up to 48 days. Cells that had been incubated with scaffolds containing BMP
were cultured for a total of 4 days. At each time point, cells were lysed in 0.1% Triton X-100
buffer and assayed for alkaline phosphatase activity using p-nitrophenol phosphate and read
spectrophotometrically at 410 nm. Specific activity was normalized by total protein and
expressed as ug/hr/mg protein. Scaffold alone (S), cells alone (control), and collagen gel alone
10 (collagen) served as negative controls, while one time dose of soluble 1 ug/ml BMP served as
4. Results: The release of rhBMP-2 by the various scaffolds was determined by the ability of the
scaffold to stimulate stem cell induction. Hyaluronic acid scaffolds released minimal levels of
rhBMP-2 as assessed by their inability to stimulate stem cell induction (Fig. 1), Even after 14
days in vitro, little induction was seen. In contrast, PLLA scaffolds and collagen gels released
significant levels of BMP for up to 2 weeks (Fig. 2). This level of induction was comparable to
that seen with 1 ug soluble BMP. These results demonstrate that scaffolds can be engineered to
locally sequester BMP and suggest that hyaluronic acid scaffolds are superior to poly-L-lactic
acid or collagen in their ability to retain BMP.
Scaffolds that sequester BMP at the repair site may show superior bone healing or fusion.
Increased BMP concentrations within the scaffold should promote more vigorous cell invasion
and bone induction. BMP is available within the scaffold to act locally due to decreased
diffusion of BMP out of the scaffold and into the surrounding tissues or bloodstream. Loss of
BMP due to diffusion may not only decrease the scaffold's potency, but also lead to potential
bone formation at other unwanted sites. Enhanced BMP retention in HA may be attributed in
part to ionic interactions where the negatively charged side groups in HA interact with the
positively charged N-terminal region of rhBMP2. These results demonstrate that scaffold
chemistry is important in sequestering BMP and that hyaluronic acid scaffolds are superior to
poly-L-laetic acid or collagen in their ability to retain BMP.
Each of the foregoing patents, patent applications and references is herein incorporated by
wo 97/45532 PCT/US97/09067
reference in its entirety. Having described the presently prefened embodiments, in accordance _
with the present invention, it is beHeved that other modifications, variations and changes will be
suggested to those skilled in the art in view of the teachings set forth herein. It is, therefore, to be
understood that all such variations, modifications, and changes are believed to fall within the
5 scope of the present invention as defined by the appended claims.
1 . A method for forming a substrate for cell growth comprising:
5 dissolving a water-insoluble hyaluronic acid derivative in a first solvent,
forming a mixture of the first solvent, the water-insoluble hyaluronic acid
derivative and a pore forming agent that is insoluble in the first solvent, and
contacting the mixture with a second solvent, wherein the water-insoluble
hyaluronic acid derivative is insoluble in the second solvent but the pore forming agent is soluble
10 in the second solvent, whereby the first solvent and the pore forming agent are extracted from the
mixture to form a porous scaffold of the water-insoluble hyaluronic acid derivative.
2. The method of claim 1 , wherein the pore forming agent is sized so as to leave
voids sufficient to permit cell ingrowth into the scaffold when the pore forming agent is extracted
15 from the mixture.
3. The method of claim I further comprising dr>'ing the scaffold under vacuum at a
temperature between 4°C and 3 7T.
20 4. The method of claim 3 wherein the scaffold is dried at ambient temperatures.
5. The method of claim 1 wherein the water-insoluble hyaluronic acid derivative is a
covalent conjugate of hyaluronic acid^sterified with a benzyl moiety.
25 6. The method of claim 1 fiarther comprising coating the scaffold with a bioerodable
7. A substrate for cell growth comprising:
a scaffold of water-insoluble derivatized hyaluronic acid defining interconnected
30 pores of sufficient size to permit mammalian cell ingrowth into the pores, wherein the
derivatized hyaluronic acid is a covalent conjugate of hyaluronic acid and a water-insoluble
moiety that renders the conjugate insoluble in water.
wo 97/45532 PCT/US97/09067
8. The substrate of claim 7 wherein the covalent conjugate comprises an ester bond,
via a carboxy group of the hyaluronic acid,
9. The substrate of claim 7 wherein the covalent conjugate comprises an amide bond
5 via a carboxy group of the hyaluronic acid.
10. The substrate of claim 7 wherein the covalent conjugate is hyaluronic acid
esterified with a benzyl moiety.
10 11. The substrate of claim 1 0 wherein the scaffold is coated with a bioerodible
12. The substrate of claim 7 further comprising mammalian cells within at least some
pores of the scaffold.
1 3 . The substrate of claim 7, wherein the scaffold is a two-phase scaffold
14. The methods of claim 1 further adding a compound selected from the group
20 consisting of drugs, growth factors, peptides, proteins, cytokines, oligonucleotides, antisensc
oligonucleotides, DNA and polymers.
15. The method of claim 1 4, wherein the compound is added by coating the porous
scaffold with the compounds.
1 6. The method of claim 14, wherein the compound is added by covalent attachment
to the porous scaffold.
T"' — I — ' I • I — — ■ — I ■ 1 ■ I • — I ■ I
12 16 20 24 28 32 36 40 44 48
I ' I ' I ■ I ■ I ■ I ■ 1 ■ I ■ I ■ I ' I
8 12 16 20 24 28 32 36 40 44 46
INTERNATIONAL SEARCH REPORT
tnte. .ional Application No
CLASSIFICATION OF SUBJECT MATTER
PC 6 C12N5/00 A61L27/0O
Aocording to International Patent ClaMtfication (IPC) or to both national ctawifioatton and IPC
Minimum documentation Marched (claaaitication »y»!em foltowod by dasaifioation aymbote)
PC 6 C12N A61L
DocumenUUon •earchwi othar than minimum dooumanlation to the eirtent that.uch documentt are included in the fields tearohed
Eleotronic data base oonauKed during the international search (name of data bate and, where practical. »eareh ter me ueed)
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category • Citation of document, with indication, where appropriate, of the relevant paeiaget
Releveflt to claim No.
WO 96 10426 A (YAMANOUCHI PHARMACEUTICAL
CO. LTD.) 11 April 1996
-& EP 0 784 985 A (YAMANOUCHI
PHARMACEUTICAL CO. LTD.) 23 July 1997
see page 4, column 5, line 13 - column 6,
line 30; claims
see page 7, column 12, line 46 - line 51
WO 94 17840 A (FIDIA ADVANCED BIOPOLYMERS
S.R.L.) 18 August 1994
see claims; examples 1-4
WO 93 11803 A (M.U.R,S.T.) 24 June 1993
see claims; examples 12,14
Further documenta are beted in the continuation of box C.
Patent family members are litted in annax.
* Speoial oategoriee of cited dooumenta :
■A' document defining the general atale of the art which m not
oonaidered to be of paftioular ivlevanoe
'E* earlier docurrwnt but publiahed on or aftar the htemational
V document which may throw doubti on priorily ctalmfe) or
whioh i» cted to eetablieh the publication date of another
citalion or other ipeoial reaaon <ai specified}
'O* document refenvtg to an oral dwolosufie, use, exhibitton or
'P* document pubfshed prior to the international filing date but
later than the priority date claimed
"T" later dooumcnl pUbliBhed after the international liKng date
or priority date and not in confliel with the appfeoatbn but
cited to undervtand the principle or theory underlying the
•X' document of particular relevance; the claimed invention
cannot be considered novel or cannot be eonaidared to
involve an inventive step when the document is taken alone
*Y* document of particular relevanoe; the claimed invention
oannot be considered to involve an inventive step when the
document is oombined wih one or more other such docu-
ments, suoh oombination being cbmus to a peraon sKMIed
in the art.
*&' dooumamt member of the same patent family
Date of the aolual completion of theintemational eearoh
6 October 1997
Date of mailing of the international search report
2 9. 10. 97
Name and mailing address of ttte ISA
European Patent Office, P.B. SB18 Patentlaan 2
NL - 2280 HV RijawiiK
Tel. (♦3170) 340*2040, Tx. 31 651 epo nl.
Fonn PCT/lSA/310 (second thost) (July 19S2)
INTERNATIONAL SEARCH REPORT
tnformaUon on patenl family membets
Inte «onat Application No
cited in search report
wo 961G426 A
WO 9417840 A
AU 3577895 A
CA 2200052 A
EP 0784985 A
IT 1263144 B
AU 6001494 A
EP 0682534 A
JP 8506497 T
WO 9311803 A
Foim PCTrtSA^IO (paten* »«T>rty aniwK) <July 1992)
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