WO2008067655A1

WO2008067655A1 - Biocompatible hydrogel-based scaffolds

Info

Publication number: WO2008067655A1
Application number: PCT/CA2007/002179
Authority: WO
Inventors: Mehrdad Rafat; Fengfu Li; Malcolm Austin Latorre
Original assignee: Q6 Biomaterials Inc.
Priority date: 2006-12-05
Filing date: 2007-12-05
Publication date: 2008-06-12

Abstract

Biocompatible scaffolds are disclosed which comprise an oxidized polysaccharide selected from the group consisting of chondroitin sulfate, alginate, hyaluronic acid, water-soluble starch and its derivatives, and water-soluble gum and its derivatives, and an amine-containing polymer selected from collagen, chitosan, their derivatives, and a mixture thereof, crosslinked to said oxidized polysaccharide.

Description

BIOCOMPATIBLE HYDROGEL-BASED SCAFFOLDS

FIELD OF THE INVENTION

This invention relates to biocompatible hydrogels useful as scaffolds.

BACKGROUND OF THE INVENTION

During the last two decades, significant advances have been made in the development of biocompatible and biodegradable materials for biomedical and tissue engineering applications. One such application is the development of scaffolds. Scaffolds, also known as matrices, are artificial three-dimensional structures capable of containing cells, drugs or bioactive agents, and/or fluids, such as cell growth medium. Scaffolds can serve multiple purposes including supporting three-dimensional tissue formation, allowing cell attachment and migration, delivery and retention of cells and biochemical factors, enabling diffusion of vital cell nutrients and expressed products, and exerting certain mechanical and biological influences to modify the behaviour of the cell phase.

Scaffolds may be formed either in vitro or in situ. In vitro scaffolds can be prefabricated into a defined shape and architecture. Thereafter, they can, for example, be seeded with cells, and either implanted directly into the tissue of interest, or first cultured in vitro to generate new growth and then implanted into the tissue. For some applications scaffolds that gel in situ (which includes in vivo) are more desirable. With in situ gelling scaffolds, desired components, such as cells and/or other biological materials like cell adhesive motifs and cell growth nutrients, can be combined with a liquid solution of the polymer, and injected into the desired regions, such as into irregularly shaped tissue defects. Once injected, the liquid gels (solidifies), encapsulating the cells and/or other materials within the scaffold.

Some of the most promising systems for in situ gelling are poly (N- isopropylacrylamide-derived (pNIPAAM-derived) systems and PEO/PPO block copolymer systems. pNIPAAM polymer exhibits a soluble hydrophilic state below its lower critical solution temperature (LCST), usually about 32⁰C. pNIPAAM has been grafted onto some natural polymers such as chitosan and gelatin. The resulting gelatin-g-pNIPAAM and chitosan-g-pNIPAAM, also have a phase transition above about 32⁰C. They have shown some promising results for cell encapsulation. However, because pNIPAAM is non- biodegradable and its toxicity is unknown, pNIPAAM-derived thermoresponsive biomaterials, pose problems for commercialization.

PEO/PPO block copolymer, i.e. poly (ethylene oxide-co-propylene oxide-co- polyethylene oxide) (PEO-PPO-PEO) (Commercial name: Poloxamer™ (ICI)) is water soluble and forms gels around body temperature, i.e., 37⁰C. PEO-PPO-PEO has been widely investigated for drug delivery and has been used in some marketed products at low molecular weight. However, PEO-PPO-PEO has not been widely used in medicine because of its toxicity (found in animal trials) and its non-biodegradability.

Naturally occurring polymeric biomaterials, such as proteins and polysaccharides, have the advantage that they generally have good biocompatibility and low immunogenicity. US patent 6,953,784 discloses an injectable biodegradable carrier for drug delivery, comprising a first and second polysaccharide crosslinked together, wherein the first polysaccharide is oxidized to provide the functional groups necessary for the crosslinking reaction. Exemplified are carriers in which the first polysaccharide is hyaluronic acid and the second polysaccharide is hyaluronic acid or chondroitin sulphate.

US patent 6,514,522 discloses an oxidized polysaccharide, arabinogalactan, cross- linked with the polyamine, chitosan, in the form of microspheres. This microsphere scaffold is formed in vitro and is intended to be used in wound healing, tissue adhesion prevention, and as a surgical homeostatic material. US patent 5,866,165 discloses crosslinking collagen with oxidized polysaccharides such as hyaluronic acid to form a scaffold potentially useful for the repair of bone and cartilage. However, this scaffold is not particularly cannot be used as an in situ gelling scaffold.

SUMMARY OF THE INVENTION

It is desirable to develop a polymer in the form of a hydrogel that is useful as a scaffold.

In a first aspect, the present invention provides a biocompatible and biodegradable hydrogel scaffold comprising: an oxidized polysaccharide, said polysaccharide being selected from the group consisting of chondroitin sulfate, alginate, hyaluronic acid, water- soluble starch and its derivatives, and water-soluble gum and its derivatives, and an amine- containing polymer selected from collagen, chitosan, their derivatives, and a mixture thereof, crosslinked to said oxidized polysaccharide. The hydrogel scaffolds of the invention may be used to make in situ gels and in vitro matrices. Uses of the scaffolds include the sustained release of stem cells and bioactive molecules, such as medications and growth factors. They may also be used as matrices for the regeneration of tissue, such as corneal tissue. In one aspect the scaffolds of the present invention may be optically clear, permitting them to be used, for Example in the cornea.

In one aspect, the scaffolds of the present invention may be injectable, thus avoiding the need to perform surgery. In another aspect, hydrogel scaffolds of the invention may be preformed in vitro. Advantageously, in certain aspects, scaffolds of the invention can be made under a wide range of pH conditions, allowing for a diversity of scaffolds to be fabricated. By varying the pH, one can affect the porosity, the interaction with cells, and the transparency of the scaffold.

Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way of Example only, with reference to the attached Figures, wherein:

Fig. 1 is a graph showing the cell count for in vitro growth of corneal epithelial cells beside scaffolds in accordance with aspects of the present invention; and

Fig. 2 is a graph showing the swelling behaviour of scaffolds in accordance with certain aspects of the invention. Fig. 3 is a graph showing the in vitro growth of DRG nerves (neurites) on selected scaffolds in accordance with aspects of the present invention.

DETAILED DESCRIPTION

Generally, the present invention provides a biocompatible and biodegradable hydrogel scaffold comprising (i) an oxidized polysaccharide, said polysaccharide being selected from the group consisting of chondroitin sulfate, alginate, hyaluronic acid, water- soluble starch and its derivatives, and water-soluble gum and its derivatives, and (ii) an amine-containing polymer selected from collagen, chitosan, their derivatives, and a mixture thereof, crosslinked to said oxidized polysaccharide.

Depending on the application, the scaffold may gel in vitro or in situ. A hydrogel is a three-dimensional network of hydrophilic polymer chains that are crosslinked through either chemical or physical bonds. Because of the hydrophilic nature of polymer chains, hydrogels absorb water to swell in the presence of abundant water. By definition, water constitutes at least 10% of the total weight (or volume) of a hydrogel. When the content of water exceeds 95% of the total weight (or volume), the hydrogel is called a superabsorbent.

The Oxidized Polysaccharide

The oxidized polysaccharide of the invention may be any of oxidized chondroitin sulfate, alginate, hyaluronic acid, water-soluble starch and its derivatives, or water-soluble gum and its derivatives. The term water-soluble starch includes water-soluble starches that are known to one skilled in the art. Derivatives of water-soluble starches include water-soluble starches that have been oxidized and/or PEGylated.

The term water-soluble gum includes water-soluble gums that are known to one skilled in the art. Examples include xanthan gum, guar gum, dextran gum, acacia gum, carrageenan gum, locust bean gum, and scleroglucan gum. Derivatives of water-soluble gums include water-soluble gums that have been oxidized and/or PEGylated.

The method of preparing the oxidized polysaccharide comprises opening sugar rings on the polysaccharide and oxidizing hydroxyl groups to aldehydes using a selective oxidizing agent. Any selective oxidizing agent, which oxidizes a hydroxyl group to an aldehyde, can be used, such as specific sugar oxidases, sodium periodate, and potassium periodate. In one embodiment sodium periodate or potassium periodate is used. Preferably, the oxidizing agent is one in which a residue thereof would not be harmful to a patient or it can be easily extracted out.

The number of aldehyde groups (AD) produced in this manner can be stoichiometrically controlled, and characterized in terms of oxidation degree (OD), given in mols AD per mol of polysaccharide. A method for determining the degree of oxidation using hydroxylamine hydrochloride titration is provided in Zhao et al., Pharmaceutical Research 8:400-402 (1991 ). For instance, the oxidized polysaccharide may have an OD ranging from 52 to 261 mol aldehyde per mol polysaccharide.

OD may also be stated as % (mol AD/total mol OH) representing:

number of OH groups converted to aldehyde total number of oxidizable OH groups (prior to oxidization).

Thus, for choindroitin sulphate (with a molecular weight of 6OkDa, approximately two oxidizable OH's per repeating unit, and a repeating unit with a molecular weight of 503 Da), an OD of 52 to 261 mol aldehyde per mol polysaccharide is equivalent to approximately 20 to 100% mol AD/total mol.

Oxidizable OH groups include, for instance, adjacent OH groups. Oxidation of adjacent hydroxy! groups is known in the art (see for example, Methods in Carbohydrate Chemistry. 1962 1 : 432-435. Academic Press, New York, Ed. Whistler Wolfrom; and Johann MG Reyes et al. Invest. Ophthalmol. Vis. Sci. 2005 46: 1247-1250).

The oxiized polysaccharide of the invention may have an OD of from 1 to 100%, preferably it has an OD of 20-100%, more preferably 80-100%. Higher ODs (i.e. the more aldehydes) can result in higher the cross-linking density. In turn, when used for sustained release, this will slow down the release rate. Higher ODs also result in slowing down absorption of the hydrogel by the body.

In one aspect the oxidized polysaccharide is an oxidized chondroitin sulphate, also referred to herein as chondroitin sulfate aldehyde (CS-A). Typically, the oxidized chondroitin sulphate is prepared by dissolving chondroitin sulphate and sodium periodate together in deionized water in the dark and allowing them to react for a period of time sufficient to achieve the desired oxidation degree (OD).

Amine-Containing Polymer

The amine-containing polymer is collagen, chitosan, their derivatives, and a mixture thereof. Typically, the average molecular weight of the polymer is about 200 to 600 kDa, but lighter and heavier polymers may be used.

The collagen may be obtained from conventional sources. It may also be a recombinant form, which is particularly useful when very high purity is desirable. The term collagen derivatives includes gelatin. Chitosan is a linear polysaccharide composed of randomly distributed beta(1 ,4) linked D-glucosamine and N-acetyl-D-glucosamine. The term chitosan includes both acid-soluble and water-soluble chitosan. In one embodiment, the chitosan is acid-soluble chitosan. Chitosan derivatives includes N,O-carboxymethyl chitosan, N-carbosymethyl chitosan, and O-carboxymethyl chitosan, and polyethylene glycol (PEG)-derivatized chitosan.

The amine-containing polymer may be a combination of both chitosan and collagen. The mass ratio of collagen to chitosan may be varied depending on the desired physical and biological characteristics of the scaffold. In one embodiment the mass ratio of collagen to chitosan ranges from 0 to 20, more preferably 1 to 8. The ratio may also be 0 to 8 or 0.5 to 8. In one aspect, the ratio is 4. Typically, if a more transparent and/or homogeneous scaffold is desired, a higher collagen to chitosan mass ratio will be used. Use of a combination of chitosan and collagen results in a hydrogel that has a variety of different functional sites (e.g. -COOH, -OH, -NH₂, -CH..etc.), meaning that one has more flexibility in terms of incorporating a variety of drugs, biological agents... etc.

Cross-linking

The aldehyde groups on the oxidized polysaccharides are reacted with the amino groups on the amine-containing polymer, forming covalent imine crosslinks, via a Schiff-base reaction. Generally, the reaction proceeds by cross-linking in aqueous solution at a pH of about 3 to 10. The reaction may also proceed at pH 3-7.4, pH 9-10, or at a pH of 5 or 7.4. The ratios of oxidized polysaccharide to amine-containing polymer can be varied, depending on what physical and biological characteristics are desired. For instance the weight percentage of the oxidized polysaccharide to the amine-containing polymer may be from 5 to 100%, 15 to 95 %, 20 to 30 %, or 24%. In terms of moles of aldehyde in the oxidized polysaccharide to moles of amine in the amine-containing polymer, this may be from about 0.01 to 12, 0.03 to 11 , 0.04 to 3.6, and about 0.75. Scaffolds with a low solid content or high swelling and high water content are preferred for injectable gels; they are also typically more porous. On the other hand, scaffolds with a high solid content, low swelling and low water content are preferred as skin constructs or grafts. High swelling is typically achieved by using less cross-linker (i.e. less of the oxidized polysaccharide, or using a polysaccharide with a lower OD) or using less concentrated solutions of all the reactant. Scaffolds with a lower weight percentage of oxidized polysacchardie to amine-containing polymer are more transparent and homogeneous. The transparency of the gels may be controlled. The transparency of the scaffold will depend on the chemical composition of the scaffold as well as the pH at which it is made. Typically, in tha basic pH range, the higher the pH, the more transparent the scaffold. All the scaffolds made at pH 9-10 shown in the following examples were clear. The properties of the scaffolds also will depend on the proximal conditions, whether in vitro or in situ.

To make scaffolds in vitro, the oxidized polysaccharide and the amine-containing polymer are mixed together and transferred into a scaffold mold after mixing, and the cross- linking reaction is allowed to proceed. For instance, the scaffolds may cure over a period about 12 to 24 hours at room temperature and for 12 to 24 hours at 37°C. Finally, the scaffolds are washed to extract out any reaction by-products. Sodium cyanoborohydride (Na(CN)BH₃) was not used, as it would be toxic if any residue remained in the hydrogel.

To make the scaffolds in situ, the oxidized polysaccharide and the amine-containing polymer are combined together with any desired materials (e.g. cells, growth factors,... etc.), maintained at a temperature well below their phase transition until injection, and then injected into the desired location.

In another aspect, the scaffold excludes constructs comprising hyaluronic acid and only collagen as the polyamine-containing polymer.

Examples

Synthesis of Oxidized Polysaccharide

Example 1.1 : Synthesis of chondroitin sulfate aldehyde with an OD of 52 mol AD/mol

CS (CS-A-52): 4 g of chondroitin sulfate C (Sigma-Aldrich) and 0.5 g of sodium periodate (NaIO₄, Sigma-Aldrich) were dissolved together in 66 ml of deionized water and were protected from light. The reaction was allowed to continue for 16 hours in the dark, with vigorous stirring. The insoluble by-products were removed with a 0.45 μrπ filter, and the product was loaded into a dialysis membrane (7 Spectra/Por Membrane, MWCO=2000, OD=29mm) against _ddH₂O in the shell side by which the product was purified of water-soluble by-products and un-reacted small molecules. The product, CS-aldehyde, was obtained by lyophilization at -65°C for 8 hrs. The determination of the degree of oxidation was performed by hydroxylamine hydrochloride titration (H. Zhao et al., Pharmaceutical Research, 8: 400- 402 (1991 )). The OD was determined as 52 mol AD/mol CS. Example 1.2: Synthesis of chondroitin sulfate aldehyde with an OD of 94 mol AD/mol CS (CS-A-94): Two grams of chondroitin sulfate C (CS, Sigma-Aldrich) and 2 g of sodium periodate (NaIO₄, Sigma-Aldrich) were dissolved together in 33 ml of deionized water and were protected from light. The reaction was processed as for Example 1.1. The OD was determined as 94 mol AD/mol CS.

Example 1.3: Synthesis of Chondroitin Sulfate Aldehyde with an OD of 261 mol AD/mol CS (CS-A-261): Two grams of chondroitin sulfate C (CS, Sigma-Aldrich) and 4 g of sodium periodate (NaIO₄, Sigma-Aldrich) were dissolved together in 33 ml of deionized water and were protected from light. The reaction was processed as for Example 1.1. The OD was determined as 261 mol AD/mol CS.

Preparation of Amine-Containing Polymers Example 1.4: Preparation of Solutions of Chitosan, Collagen, and mixtures thereof :

Solutions of amine-containing polymers were prepared. Chitosan solutions were prepared containing 2-10% (w/v) chitosan by dissolving chitosan flakes (M_w 40OkDa obtained from Fluka) in 0.2 N hydrochloric acid (HCI) and stirring at 4⁰C. Collagen solutions were prepared containing 1-20% (w/v) collagen type I; freeze dried porcine collagen powder that was obtained from Nippon Meat Packers, lnc (Japan) was dissolved in cold water (sterile _dd H₂O) and stirred at 4°C. Mixtures of chitosan and collagen were prepared by mixing the above mentioned solutions of chitosan and collagen together in a syringe system at predetermined ratios to make a homogeneous blend containing 2-10% (w/v) chitosan and 1-20% (w/v) collagen type I.

Preparation of Scaffolds

Various scaffolds in accordance with aspects of the invention are shown in Table 1. The w/v %'s given for the acid-soluble chitosan and the collagen columns refer to pre-mixing %'s. The collagen to chitosan mass ratio refers to the ratios upon mixing and throughout the crosslinking reaction.

The amine-containing polymers were cross-linked using chondroitin sulfate aldehyde (CS-A). Generally, the 200 series scaffolds (collagen concentration ranges from 2-20% w/v) were prepared by mixing the amine-containing polymer with the oxidized polysaccharide (the crosslinker) at a pH of about 3 using 0.6 M 2-(N-Morpholino) ethanesulfunic acid (MES) buffer or a pH of about 5 to 7.4 using sodium hydroxide or various concentrations of phosphate buffered saline (PBS). The 300 series scaffolds (collagen concentration is 1% w/v) were prepared as described.

Example 2.1 : Preparation of BCI-206 scaffold: 1 ml of 5% chitosan solution was transferred to a 2.5 ml Luer tip glass syringe. The chitosan solution was then mixed with 0.3 ml MES buffer using a plastic Tefzel Tee-piece. The mixture was then mixed with 0.0181 g CS-A-261 cross-linker agent [CS-A to chitosan mass ratio of 0.31] that was dissolved in 0.2 ml MES buffer, at about 0°C-4°C. The components were thoroughly mixed by repeated pumping action between the first and second syringes through the Tee. This process was performed under iced water to prevent premature crosslinking and air bubbles formation and entrapment.

The homogeneous mixture was immediately dispensed into 500 microns glass flat moulds (or plastic in vitro moulds) and cured first at room temperature for 24 hours, and then at 37⁰C for 24 hours, in 100% humidity environments at both temperatures. Each final scaffold sample was carefully separated from its mould after immersion in phosphate buffered saline (PBS) for 1 hour. Finally, the cross-linked scaffolds were immersed in PBS solution (0.5% in PBS, containing 1 % chloroform) at room temperature to extract out reaction by-products.

Example 2.2: Preparation of BCI-207 scaffold: This scaffold was prepared as for Example 2.1 using 1 ml of 5% chitosan solution and 0.0091 g CS-A-261 cross-linker agent [CS-A to chitosan mass ratio of 0.18].

Example 2.3: Preparation of BCI-210 scaffold: This scaffold was prepared as for Example 2.1 using 1 ml of 5% chitosan solution and 0.0091 g CS-A-52 cross-linker agent [CS-A to chitosan mass ratio of 0.18].

Example 2.4: Preparation of BCI-211 scaffold: 0.5 ml of 5% chitosan solution was added to 0.25 ml of a 4% collagen solution [collagen:chitosan mass ratio of 0.5] in a 2.5 ml Luer tip glass syringe. The composition was then mixed with 0.35 ml MES buffer (or PBS buffer) using a plastic Tefzel™ Tee-piece. The mixture was then mixed with 0.0141 g CS-A-261 cross-linker agent [CS-A to chitosan/collagen mass ratio of 0.47] that was dissolved in 0.35 ml MES buffer (or PBS buffer), at about 0°C-4°C. The remainder of the reaction was processed as for Example 2.1.

Example 2.5: Preparation of BCI-212 scaffold: This scaffold was prepared as for Example 2.4 using 0.25 ml of 5% chitosan solution and 0.5 ml of a 4% collagen solution [collagen:chitosan mass ratio of 2.0] and 0.0072 g CS-A-261 cross-linker agent [CS-A to chitosan/collagen mass ratio of 0.18].

Example 2.6: Preparation of BCI-213 scaffold: This scaffold was prepared as for

Example 2.4 using 0.5 ml of 5% chitosan solution and 0.25 ml of a 4% collagen solution [collagen:chitosan mass ratio of 0.5] and 0.0282 g CS-A-261 cross-linker agent [CS-A to chitosan/collagen mass ratio of 0.94], which was synthesized according to Example 1.3.

Example 2.7: Preparation of BCI-214 scaffold: This scaffold was prepared as for

Example 2.4 using 0.25 ml of 5% chitosan solution and 0.5 ml of a 4% collagen solution [collagen:chitosan mass ratio of 2.0] and 0.0145 g CS-A-261 cross-linker agent [CS-A to chitosan/collagen mass ratio of 0.51].

Example 2.8: Preparaton of BCI-217 scaffold: 0.7 ml of 4% collagen solution was transferred to a 2.5 ml Luer tip glass syringe. The collagen solution was then mixed with 0.3 ml MES buffer using a plastic Tefzel Tee-piece. The mixture was then mixed with 0.0193 g CSA-261 cross-linker agent [CSA to collagen mass ratio of 0.69] that was dissolved in 0.3 ml MES buffer, at about 0°C-4°C. The remainder of the reaction was processed as for Example 2.1.

Example 2.9: Preparation of BCI-218 scaffold: 0.17 ml of 5% chitosan solution was added to 0.75 ml of a 4% collagen solution [collagen:chitosan mass ratio of 4.2] in a 2.5 ml Luer tip glass syringe. The composition was then mixed with 0.3 ml MES buffer (or PBS buffer) using a plastic Tefzel Tee-piece. The mixture was then mixed with 0.0089 g CS-A-261 cross-linker agent [CS-A to chitosan/collagen mass ratio of 0.24] that was dissolved in 0.3 ml MES buffer (or PBS buffer), at about 0°C-4°C. The remainder of the reaction was processed as for Example 2.1. Example 2.10: Preparation of BC I -221 scaffold: This scaffold was prepared as for Example 2.9 using 0.145 ml of 3% chitosan solution and 0.75 ml of a 2% collagen solution [collagen:chitosan mass ratio of 4.2] and 0.0092 g CSA-261 cross-linker agent [CSA to chitosan/collagen mass ratio of 0.49].

Example 2.11 : Preparation of BCI-222 scaffold: This scaffold was prepared as for Example 2.9 using 0.3 ml of 5% chitosan solution and 0.50 ml of a 20% collagen solution [collagen:chitosan mass ratio of 8.0] and 0.0184 g CSA-261 cross-linker agent [CSA to chitosan/collagen mass ratio of 0.16].

Example 3.1 : Preparation of BCI-300 scaffold: 1 ml of 1 % type I collagen was added to a 4 ml vial. This vial was cooled on ice for 30min. 200μl of CSA-52 (12 mg CSA/ml water) was added causing a translucent gel to be formed. The final pH of the mixture was 4. The gel was incubated for a further 15h at room temperature and washed with water.

Example 3.2: Preparation of BCI-301 scaffold: 1 ml of 1 % type I collagen was added to a 4 ml vial. This vial was cooled on ice for 30min. 50μl of CSA-52 (12mg CSA/ml water) was added causing a translucent gel to be formed. The final pH of the mixture was 4. The gel was incubated for a further 15h at room temperature and washed with water.

Example 3.3: Preparation of BCI-302 scaffold: 1 ml of 1 % type I collagen was added to a 4 ml vial, and this vial was cooled on ice for 30min. Then 10μl NaOH (2N) was added and mixed. 200μl of CSA-52 (12mg CSA/ml water) was added, forming a transparent gel. The final pH of the gelling mixture was 10. The gel was incubated for a further 15h at room temperature and then washed with water overnight.

Example 3.4: Preparation of BCI-303 scaffold: 1 ml of 1 % type I collagen was added to a 4 ml vial, and the vial was cooled on ice for 30min. 10μl NaOH (2N) was added and mixed. Then, 30μl of CSA-94 (22.67mg CSA/ml water) was added forming a transparent gel. The final pH of the gelling mixture was 10. The gel was incubated for a further 15h at room temperature and then washed with water overnight. Example 3.5: Preparation of BC I -304 scaffold: 1 ml of 1 % type I collagen was added to a 4 ml vial, and the vial was cooled on ice for 30min. 10μl NaOH (2N) was added and mixed. Then, 150 μl of CSA-94 (22.67mg CSA/ml water) was added forming a transparent gel. The final pH of the gelling mixture was 10. The gel was incubated for a further 15h at room temperature and then washed with water overnight.

Table 1: Summary of compositions for chitosan/collagen/cs.ald scaffolds

co

NPM: Collagen suppliers name: Nippon Meat Packers, Inc, Japan ^* CS-ald: Chondroitin sulfate aldehyde OD: Oxidation degree (mole CHOs per mole CS) : CHO to NH₂ molar ratio: The NH₂ refers to the total amount of calculated NH₂ in chitosan and the NH₂ of lysine residuals in collagen

Example 4: Growth of human corneal epithelial cells (HCEC) on the invented materials for cytotoxicity evaluation (In Vitro Biocompatibility Assessment): The scaffolds produced according to Examples 2 and 3 were assessed to determine the short-term (7 days) biocompatibility of the scaffolds with respect to the HCEC line. Scaffolds were washed 2 x 3 hours in PBS and treated with antibiotics for 24 hours. Scaffolds were cut into pieces if necessary and placed on the bottom of a 24-well tissue culture plate (n=3). Human corneal epithelial cells (passage #1235) were seeded at a density of 5x10³ cells per scaffold in KSFM culture medium (Invitrogen Cat# 17005-042) supplemented with 1x penicillin/streptomycin (Sigma Cat# P4333). Cultures were photographed on Days 2, 4 and 7 post-seeding. Quantified results were obtained by analyzing all photographs. An area of scaffold was selected for counting, and viable-looking cells within that area were counted using Northern Eclipse 6.0 software. The number of cells counted was normalized to the area of scaffold selected, and multiplied by a constant. The counts were expressed as cells per unit surface area. In order to see the cells and morphology of the cells' nuclei 4'-6-Diamidino- 2-phenylindole (DAPI) staining was used for less transparent scaffolds. All materials supported excellent or good cell growth on and beside the scaffolds compared to culture dish control surfaces. They were all confluent by Day 7.

The number of epithelial cells beside the scaffolds were quantified for days two, four, and seven post-seeding and shown in Figure 1. As we can see in this Figure, growth rate of the cells was found to be similar and comparable to the control. A confluent epithelium was observed for all scaffolds and control on day 7.

Example 5: Water Uptake (Swelling) Test: The scaffolds were tested to determine their swelling properties with respect to a PBS solution. The swelling behaviour of the scaffolds was measured by swelling the dry scaffolds in phosphate buffered saline (1 % PBS) media. Scaffolds were dried by placing them in cold chamber purged by filtered dry air at 4 ⁰C. Pre- weighed dry hydrogel films were then immersed in buffer solutions at pH 7.4 at room temperature. The immersion time was 24 hrs. The films were withdrawn from the solution and their wet weights were determined after first blotting with a filter paper to remove the surface water and immediately weighing the films. The swelling ratio was calculated using the equation:

Sf? (w%) = ((W_S - W_d) / W_d) * 100 (1) where SR represents the water absorption or uptake (w%) of the films, W_d and W_s are the weights of the samples in the dry and swollen states, respectively.

The crosslinking index (Cl) was defined and calculated from the following equation:

C/ = (1/SR) x 700 (2)

where Cl represents the cross-linking Index or extend of crosslinking.

The solid content was also determined using the following equation:

SC (w%) = (W _d / W_s) x 700 (3)

All swelling behaviours of scaffolds are plotted on the average of three trials in Figure 2. The BCI-12 scaffold had the highest swelling ratio and the lowest crosslinking index (Cl), while BCI-13 had the lowest swelling ratio and the highest crosslinking index. These results are in good agreement with the concentration of the crosslinking agent (CHCVNH₂ ratio) as summarized in Table 1. The lower the crosslinking agent concentration, the lower was the Cl and the higher was the swelling ratio. All scaffolds exhibited high degree of swelling behaviour specific to hydrogels.

Example 6: Evaluation of surface neurite extension of dorsal root ganglia (DRG) on scaffolds: Selected scaffolds were tested for in vitro biocompatibility to nerve cells using dorsal root ganglia (DRG) from chick embryos (E 8.0). DRGs were isolated from 8 day old chick embryos and adhered to the surface of washed hydrogels with collagen matrix. DRGs were then covered with DRG medium. Neuhtes were grown for a total of 7 days, with DRG medium being changed for every 2 days. Samples were fixed on day 7 post-adhering using 4% paraformaldehyde followed by staining at 4°C overnight for the presence of neurofilament using mouse anti-NF200 antibody (Sigma Cat# N5389) diluted 1 :40 in TCT. Neurofilaments were visualized the following day using donkey anti-mouse-Cy2 (Amersham Pharmacia Cat# PA42002, 1 :200 in TCT). Whole-mounts were imaged using an Axiovert microscope. As shown in Fig. 3, DRG nerve overgrowth was observed for BCI-221 , and BCI-218 scaffolds. Despite the capability of BCI-221 and culture dish control, BCI-218 better supported the nerve cells and DRG neuritis. All references referred to herein are hereby incorporated by reference.

The above-described embodiments of the present invention are intended to be examples only. Alterations, modifications and variations may be effected to the particular embodiments by those of skill in the art without departing from the scope of the invention, which is defined solely by the claims appended hereto.

Claims

CLAIMS:

1. A biocompatible and biodegradable hydrogel scaffold comprising:

an oxidized polysaccharide, said polysaccharide being selected from the group consisting of chondroitin sulfate, alginate, hyaluronic acid, water-soluble starch and its derivatives, and water-soluble gum and its derivatives, and

an amine-containing polymer selected from collagen, chitosan, their derivatives, and a mixture thereof, crosslinked to said oxidized polysaccharide.

2. The scaffold of claim 1 , wherein the oxidized polysaccharide is oxidized chondroitin sulfate.

3. The scaffold of claim 2, wherein the amine-containing polymer is collagen, chitosan, or a mixture thereof.

4. The scaffold of claim 1 , wherein the oxidized polysaccharide is oxidized chondrointin sulfate and the amine-containing polymer is a mixture of collagen and chitosan.

5. The scaffold of any one of claims 1 to 4, wherein the oxidized polysaccharide has an oxidation degree of 1 to 100%, 20 to 100%, or 80 to 100%.

6. The scaffold of any one of claims 1 to 5, wherein the weight percentage of the oxidized polysaccharide to the amine-containing polymer is from 5 to 100%, 15 to 95 %, 20 to 30 %, or 24%.