READY TO USE BIODEGRADABLE AND BIOCOMPATIBLE CELL-BASED NERVE CONDUIT FOR NERVE INJURY AND A METHOD OF PREPARATION THEREOF

Abstract

An artificial tissue construct for nerve repair and regeneration includes a biocompatible and biodegradable nerve guidance matrix comprising a plurality of biopolymers that include chitosan, gelatin, collagen and hyaluronic acid. A cross-linker includes glutaraldehyde. The nerve guidance matrix is formed as a three-dimensional scaffold polyelectrolyte complex (PEC). A subconfluent and grown monolayer of at least one of human mesenchymal stem cells, mesenchymal stem cells, differentiated Schwann cells and neuronal cells is on the biocompatible and biodegradable nerve guidance matrix for direct implantation or delivery. A method of making the artificial tissue construct is disclosed.

Claims

1-20. (canceled)

21. An artificial tissue construct for nerve repair and regeneration, comprising: a biocompatible and biodegradable nerve guidance matrix comprising a plurality of biopolymers that include chitosan, gelatin, collagen and hyaluronic acid, and a cross-linker comprising glutaraldehyde, wherein said nerve guidance matrix is formed as a three-dimensional scaffold polyelectrolyte complex (PEC); and a subconfluent and grown monolayer of at least one of human mesenchymal stem cells, mesenchymal stem cells, differentiated Schwann cells and neuronal cells on the biocompatible and biodegradable nerve guidance matrix for direct implantation or delivery.

22. The artificial tissue construct according to claim 21, wherein said biocompatible and biodegradable nerve guidance matrix further comprises at least one of polycaprolactone (PCL), polypyrrole, polyurethane (PU), polyallylamine, polyethyleneglycol 200 (PEG 200), gum acacia, guar gum and partially denatured collagen.

23. The artificial tissue construct according to claim 21, wherein said nerve guidance matrix comprises 1%-10% w/v of gelatin, 0.5%-2.5% w/v of chitosan, 0.1%-2% w/v of hyaluronic acid, 0.1%-10% w/v of collagen, and 5%-50% w/v of glutaraldehyde solution.

24. The artificial tissue construct according to claim 23, wherein said nerve guidance matrix further comprises gelatin having a 50-300 bloom strength and DAC (dialdehyde cellulose) chitosan ranging from 75%-95%.

25. The artificial tissue construct according to claim 21, wherein said subconfluent and grown monolayer of at least one of human mesenchymal stem cells, mesenchymal stem cells, differentiated Schwann cells and neuronal cells on the biocompatible and biodegradable nerve guidance matrix are seeded at cell density of 0.5×10.sup.5 to 0.8×10.sup.5 cell/cm.sup.2.

26. The artificial tissue construct according to claim 21, wherein said three-dimensional scaffold PEC is freeze-dried and said subconfluent and grown monolayer of at least one of human mesenchymal stem cells, mesenchymal stem cells, differentiated Schwann cells and neuronal cells on the biocompatible and biodegradable nerve guidance matrix are 80% to 100% confluent.

27. The artificial tissue construct according to claim 21, comprising a semi-solid transport medium on which the biocompatible and biodegradable nerve guidance matrix is supported, wherein the semi-solid transport medium comprises an agar medium comprising HEPES at a concentration of 2-3 gm/l and sodium bicarbonate at a concentration of 2-3.5 gm/l.

28. An artificial tissue construct for nerve repair and regeneration, comprising: a biocompatible and biodegradable lyophilized nerve guidance matrix comprising a plurality of biopolymers that include chitosan, gelatin, collagen and hyaluronic acid, and a cross-linker comprising glutaraldehyde, said nerve guidance matrix formed as a three-dimensional scaffold polyelectrolyte complex (PEC); a subconfluent and grown monolayer of at least one of human mesenchymal stem cells, mesenchymal stem cells, differentiated Schwann cells and neuronal cells on the biocompatible and biodegradable nerve guidance matrix for direct implantation or delivery; and a semi-solid transport medium on which the biocompatible and biodegradable nerve guidance matrix is supported.

29. The artificial tissue construct according to claim 28, wherein the semi-solid transport medium includes nutrients.

30. The artificial tissue construct according to claim 28, wherein the semi-solid transport medium comprises a 1% to 3% agar medium.

31. The artificial tissue construct according to claim 28, wherein the semi-solid transport medium comprises an agar medium comprising HEPES at a concentration of 2-3 gm/l and sodium bicarbonate at a concentration of 2-3.5 gm/l.

32. The artificial tissue construct according to claim 28, wherein said nerve guidance matrix comprises 1%-10% w/v of gelatin, 0.5%-2.5% w/v of chitosan, 0.1%-2% w/v of hyaluronic acid, 0.1%-10% w/v of collagen, and 5%-50% w/v of glutaraldehyde solution.

33. The artificial tissue construct according to claim 32, wherein said nerve guidance matrix further comprises gelatin having a 50-300 bloom strength and DAC (dialdehyde cellulose) chitosan ranging from 75%-95%.

34. The artificial tissue construct according to claim 28, wherein said subconfluent and grown monolayer of at least one of human mesenchymal stem cells, mesenchymal stem cells, differentiated Schwann cells and neuronal cells on the biocompatible and biodegradable nerve guidance matrix are seeded at a cell density of 0.5×10.sup.5 to 0.8×10.sup.5 cell/cm.sup.2.

35. A method of making an artificial tissue construct, comprising: forming a biocompatible and biodegradable nerve guidance matrix by adding 0.5%-2.5% w/v of chitosan to 1%-10% w/v of gelatin solution, followed by adding 0.1%-2% w/v of hyaluronic acid and 0.1%-10% w/v of collagen and homogenizing to form a mixture, and cross-linking the mixture with 5%-50% w/v of glutaraldehyde solution to form a three-dimensional scaffold polyelectrolyte complex (PEC); and growing a subconfluent monolayer of at least one of human mesenchymal stem cells, mesenchymal stem cells, differentiated Schwann cells and neuronal cells on the biocompatible and biodegradable nerve guidance matrix.

36. The method according to claim 35, comprising seeding the at least one of human mesenchymal stem cells, mesenchymal stem cells, differentiated Schwann cells and neuronal cells on the biocompatible and biodegradable nerve guidance matrix at a cell density of 0.5×10.sup.5 to 0.8×10.sup.5 cell/cm.sup.2.

37. The method according to claim 35, comprising lyophilizing the three-dimensional PEC after cross-linking.

38. The method according to claim 35, supporting the biocompatible and biodegradable nerve guidance matrix on a semi-solid transport medium.

39. The method according to claim 38, wherein the semi-solid transport medium comprises an agar medium comprising HEPES at a concentration of 2-3 gm/l and sodium bicarbonate at a concentration of 2-3.5 gm/l.

40. The method according to claim 35, wherein the gelatin has a 50-300 bloom strength and adding DAC (dialdehyde cellulose) chitosan ranging from 75%-95%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 shows the Design and fabrication of biopolymer-based nerve guidance Nerve conduit/Matrix Form.

[0046] FIG. 2 shows Degradation behavior of Nerve conduit/Matrix in Trypsin and collagenase.

[0047] FIG. 3 shows Swelling ratio of Nerve conduit/Matrix in 1×PBS.

[0048] FIG. 4 shows the FTIR spectra of glutaraldehyde cross-linked nerve conduit/matrix.

[0049] FIG. 5 shows Surface morphology of Nerve conduit/Matrix by SEM analysis (A) Cross-sectional view (100×) and (B) Surface view (200×).

[0050] FIG. 6 shows MTT assay showing the biocompatibility of Nerve conduit/Matrix with L929 cells. (PC-plain control; SC-static control; DC-dynamic control).

[0051] FIG. 7 shows Fluorescence images showing viability of MSCs on Nerve conduit/Matrix on day 7 in culture.

[0052] FIG. 8 shows Biocompatibility of Nerve conduit/Matrix for MSCs. (A) MSCs pre-stained with PKH26 growing uniformly across the scaffold; (B) Calcein-AM stained MSCs on nerve conduit/matrix scaffold; (C) Nuclear DAPI stained; (D) Merge PKH and DAPI; (E) Merge PKH, DAPI and Calcein-AM (10× magnification).

[0053] FIG. 9 shows Scanning electron micrographs of MSCs seeded on the surface of Nerve conduit/Matrix.

[0054] FIG. 10 shows Fluorescence images showing Calcein-AM staining on different cross-sectional sections of Nerve conduit/Matrix.

[0055] FIG. 11 shows Calcein-AM staining on revived Nerve conduit/Matrix (stored at −80° C. after lyophilization) cryopreserved in cryomedia A, cryomedia B, cryomedia C and cryomedia D on day 4.

[0056] FIG. 12 shows Bright field images showing morphological changes in MSCs during their differentiation into Schwann cells at different time intervals.

[0057] FIG. 13 shows Immunostaining for the Schwann cell markers CD56 (red), p75NGFR (green), CD 104 (red), S100 (green) after 14 days of differentiation. Cell nuclei were stained with DAPI (blue). Scale bars, 100 sm.

[0058] FIG. 14 shows In vivo degradation of nerve conduit and matrix form implanted subcutaneously on the dorsal site of rats at 4, 8, 12, 16, 20 and 27 weeks after implantation.

[0059] FIG. 15 shows Dissection (A, B) and surgical reconstruction (C) of experimental sciatic nerve injury.

[0060] FIG. 16 shows Histological examination of regenerated sciatic nerves after 8 weeks of implantation. (A) H&E staining shown the overview of nerve morphology in each group; (B) Myelination of regenerated nerves revealed by toluidine blue staining (10× magnification).

STATEMENT OF THE INVENTION

[0061] Accordingly, the present invention provides novel and unique technique of culturing human mesenchymal stem cells, mesenchymal stem cells differentiated schwann cells and nerve cells into a proliferating, sub-confluent layer on a lyophilized biocompatible conduit/matrix prepared from plurality of composite polymers by using glutaraldehyde as a cross-linker without any integrated harmful chemicals for direct implantation or delivery of the said human mesenchymal stem cells, wherein in the said invention, the said cells are transferred while in a proliferative state and the final product obtained is transported in semi-solid medium. The said semi-solid medium is agar medium 1% to 3% and cell culture medium with essential growth factors including HEPES 2-3 gm/l and sodium bicarbonate 2-3.5 gm/l. suitable for grafting and provides a better, efficient, easy to use, cost effective ready to use biodegradable and biocompatible artificial nerve conduit/matrix for nerve repair and regeneration with sensory and motor function in a synergistic manner wherein the grafts can be prepared within 12 days.

DETAILED DESCRIPTION OF THE INVENTION

[0062] It should be noted that the particular description and embodiments set forth in the specification below are merely exemplary of the wide variety and arrangement of instructions which can be employed with the present invention. The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. All the features disclosed in this specification may be replaced by similar other or alternative features performing similar or same or equivalent purposes. Thus, unless expressly stated otherwise, they all are within the scope of present invention. Various modifications or substitutions are also possible without departing from the scope or spirit of the present invention. Therefore it is to be understood that this specification has been described by way of the most preferred embodiments and for the purposes of illustration and not limitation.

[0063] The present invention provides a novel and unique technique of nerve conduit/matrix preparation and culturing of human mesenchymal stem cells, schwann cells and neuronal cells and their proliferation on a biocompatible conduit/matrix suitable for nerve implantation/grafting.

[0064] The present invention provides a ready to use biodegradable and biocompatible tissue construct with autologous/allogeneic human stem cells based product.

[0065] The present invention also provides a reconstructive procedure to meet the specific requirements necessary to achieve satisfactory healing of nerve injury and restore functional integrity in the least time and with the least complications and morbidity. The nerve conduit/matrix as provided by the present invention has tissue like properties and is capable of being used for nerve regeneration and repair.

[0066] The present invention is directed to bioengineered tissue constructs of cultured cells and endogenously produced extracellular matrix components without the requirement of exogenous matrix components or network support or scaffold members. The invention can thus advantageously be made entirely from human cells, and human matrix components produced by those cells, for example, when the bioengineered tissue construct is designed for use in humans.

[0067] The present invention is also directed to methods for producing tissue constructs by stimulation of cells in culture, such as Mesenchymal stem cells, and Mesenchymal stem cells differentiated into schwann cells and nerve cells to produce extracellular matrix components without the addition of either exogenous matrix components, network support, or scaffold members to helpful in nerve repair and regeneration.

[0068] In the present invention, further, this tissue construct can be made by seedings of Mesenchymal stem cells and Mesenchymal stem cells differentiated schwann cells and nerve cells to produce a cultured tissue construct that mimics the cell composition and tissue structures for signal transduction, nerve repair and regeneration as native tissues. The tissue constructs of the invention are useful for clinical purposes such as nerve grafting to a patient with tissue or organ defect, such as peripheral nerve injury or any other type of nerve injury, or for in vitro tissue testing or animal grafting such as for safety testing or validation of pharmaceutical, cosmetic, and chemical products.

[0069] The present invention uses proliferative/preconfluent Mesenchymal stem cells (MSCs), schwann cells, nerve cells, mesenchymal stem cells differentiated schwann cells and mesenchymal stem cells differentiated neuronal cells whereby cells are transferred from culture to the ready to use living nerve conduit/matrix.

[0070] In an embodiment, the cells are grown directly on the polymeric conduit/matrix (scaffold) for direct implantation or delivery. The cells with scaffold can therefore be transferred as such to the patient thus avoiding the potential damage occurring in the conventional enzymatic separation from the culture vessel.

[0071] In an embodiment, the cells are transferred while in a proliferative state. In some embodiments, the use of preconfluent cells aids in the adherence of such cells to the application site as they express an integrin profile different from fully differentiated, terminal cells.

[0072] In an embodiment, the interactive component of the invention is provided by the use of actively proliferating Mesenchymal stem cells, schwann cells, nerve cells (unipolar or bipolar or multipolar). During nerve repair and regeneration of damaged nerve, a number of cytokines, growth factors etc. are released at the application site, that will helpful in signal transduction and nerve regeneration.

[0073] In an embodiment, the cells at the application site express molecules that have both an autocrine as well as a paracrine effect.

[0074] In an embodiment, the uses of an artificial nerve conduit graft substitute are useful for both repair and regeneration of damaged nerve in a synergistic manner. Repair indicates the process that a tissue undergoes to completely regenerate/reform. Allogeneic Mesenchymal stem cells, Schwann cells and nerve cells used in this nerve conduit/matrix will helpful in repair and regeneration of the damaged nerve.

[0075] In another embodiment, the process involves the optimization of scaffolds onto which cells are seeded to form a uniform tissue with scaffolds that provide physical and chemical cues to guide the process. Scaffolds may be selected from a group comprising of natural biopolymers such as chitosan, gelatin, collagen and hyaluronic acid.

[0076] In another embodiment, the Scaffolds take forms ranging from sponge like sheets to gels to highly complex structures with intricate pores and channels made with new materials processing technologies. The spatial and compositional properties of the scaffold, the porosity of the scaffold and interconnectivity of the pores are all required to enable cell penetration into the structure as well as the transport of nutrients and waste products. Differential porosity will helpful in the cells attachment and signal transduction.

[0077] In an embodiment, the sequential timed patterned physico-chemical treatment of the four or more polymers is carried on to get lyophilized 3D scaffold of polyelectrolyte complex (PEC) and also at the same time using a specifically designed aspect ratio of a system for agitation/homogenization. The sequential timed patterned physico-chemical treatment of polymers can be as dissolution of gelatin at temperature 35-75° C., preferably at 60° C. using 5% of gelatin, wherein the process comprises:

[0078] Stirring of the gelatin solution at 2000-3200 rpm at temp 15-30° C. for 15-25 min.

a) Adding of 1% chitosan solution (in 0.5-2.5% glacial acetic acid solution) dropwise in gelatin solution at temp 15-30° C. and stir the solution with homogenizer for 20-30 min.
b) Adding of hyaluronic solution (in milli Q water) preferably 0.1-1% dropwise in mixture and stir for 10 minutes.
c) Adding of collagen solution type 1 or type 4 (in glacial acetic acid solution) preferably 0.1-1% dropwise in mixture and stir for 10 minutes.
d) Adding of glutaldehyde solution preferably 25-50% dropwise at final concentration of 0.1-0.5%.

[0079] In an embodiment, once the above method of physico-chemical treatment of polymers is complete then the process of freeze drying of the composite solution is carried on. The composite was freeze at −80° C. for 12 hrs and then lyophilize for 72 hrs at 0° C. and 500 motor vacuum.

[0080] In an embodiment, after freeze drying conduit/matrix was neutralized with ammonia fumes (25% ammonia solution fumes) for 12 hrs in closed chamber inside the fume hood.

[0081] In an embodiment, further mesenchymal stem cells, schwann cells and nerve cells are seeded onto biocompatible scaffold at cell density of 0.5×10.sup.5 to 0.8×10.sup.5 cell/cm.sup.2. The cells are monolayer and 80% to 100% confluent at the final stage of product formulation. The cells used for seeding is passage 2 to passage 5. The mesenchymal stem cell, schwann cells and nerve cells used for seeding is human mesenchymal stem cells, schwann cells is differentiated from human mesenchymal stem cells and nerve cells is differentiated from human mesenchymal stem cells and only pure population. The mesenchymal stem cells, schwann cells and nerve cells have secrete several growth factors and cytokines (extracellular matrix) helpful in nerve repair and regeneration.

[0082] In an embodiment, the final product obtained will transport in semi-solid medium. The semi-solid medium is agar medium 1% to 3% and cell culture medium with essential growth factors. The agar medium contains HEPES 2-3 gm/l and sodium bicarbonate 2-3.5 gm/l. The semi-solid medium contains agar medium and cell culture medium in the ratio of 5:5, 6:4, 7:3 and 8:2 or any one of them respectively. The semi-solid medium maintains the cell viability of matrix between 60% to 90% at the temperature 4° C. to 37° C. for 28 days.

EXAMPLES

[0083] The following examples are for the purposes of illustration only and therefore should not be construed to limit the scope of the invention:

Example 1: Design and Fabrication of Biopolymer-Based Nerve Guidance Conduit/Matrix

[0084] Preparation of the Nerve Conduit/Matrix:

[0085] In an embodiment, take 50 ml of 5% gelatin solution and homogenize it for 15 min at 2000 rpm. Add 25 ml of 1% chitosan solution dropwise into the gelatin solution. Continue stir this mixture for 30 min to form a homogenous blend. Dropwise add 500 μl of 0.1% HA solution with stirring for 10 min. To this blend, add 1 ml of 0.1% collagen solution. After 10 min of continuous stirring, add 200 μl of 50% glutaraldehyde solution for crosslinking. Once the mixture is homogenized cast sample in trays/conduits and freeze it down at −80° C. for 12 hr followed by lyophilization (cycle 72 hrs, drying at 0° C., vacuum 500 mtorr) to form porous scaffolds.

[0086] Mesenchymal Stem Cells, Schwann Cells and Nerve Cells Inoculation and their Culture:

[0087] In an embodiment, 0.5×10.sup.5 cells were seeded in the pre acclimatized scaffold (scaffold soaked in cell culture medium) and culture the cells at the day 12-15. After/between the day 12-15 scaffolds were completely filled with mesenchymal stem cells, schwann cells and nerve cells and rich of growth factors and nutrients as shown in FIG. 1.

Example 2: In Vitro Degradation Behavior of Nerve Conduit/Matrix

[0088] In an embodiment, the in vitro degradation of Nerve conduit/Matrix was studied by incubating them in an enzymatic solution and then monitoring their weight-losses at different time points.

[0089] In an embodiment, Scaffold samples were incubated in 1×PBS containing trypsin (0.25 mg/ml) & collagenase (0.1 mg/ml) at 37° C. in a shaker incubator at 60 rpm for various periods of up to 21 days.

[0090] In an embodiment, at predetermined time intervals, the scaffolds were removed from the incubation medium, washed with deionized water, then subsequently oven dry for final weight measurement. Weight-loss was then determined as a difference between dry mass of sample before and after the incubation, normalized to dry mass of sample before the incubation.

[0091] In an embodiment, Scaffolds incubated in trypsin had the highest weight reduction after two weeks of incubation compared to incubation in collagenase. In addition, in vitro degradation of scaffolds in PBS, saline and media at 37° C. was also performed to check long term mechanical stability and degradation of scaffold material.

[0092] In an embodiment, Scaffolds were not degraded in PBS, saline and media up to four months indicating controlled degradation behavior of nerve conduit/matrix as depicted in FIG. 2.

Example 3: Swelling Test of Fabricated Nerve Conduit/Matrix

[0093] In an embodiment, to determine the percentage of water absorption, swelling studies were performed by immersion of scaffolds in 1×PBS. The dry weight of the scaffold was determined before immersion (Wd). Scaffolds were placed in PBS buffer solution and after a predetermined time points, the scaffolds were taken out and surface adsorbed water was removed by filter paper and their wet weight were recorded (Ww) as disclosed in FIG. 3. The ratio of swelling was determined using equation:

[00001]$\mathrm{Swelling}\mathrm{ratio}=\frac{\left(\mathrm{Ww}-\mathrm{Wd}\right)}{\mathrm{Wd}}$

Example 4: Fourier Transform Infrared (FTIR) Analysis

[0094] In an embodiment, the chemical structure of the fabricated Nerve conduit/Matrix was analyzed by Fourier transform infrared spectroscopy (FTIR). The infrared spectra of the scaffold was measured over a wavelength range of 4000-400 cm.sup.−1 as disclosed in FIG. 4.

Example 5: Morphological Characterization of Nerve Conduit/Matrix Using SEM

[0095] In an embodiment, scanning electron microscopy (SEM) was performed to study the surface and cross-sectional morphology of nerve conduit/matrix. The SEM image (FIG. 5) showed that the nerve conduit/matrix possessed a well-defined and well integrated porous structure that is essential for cell attachment, proliferation and survival. The images at higher magnification indicate the presence of open and interconnected pores of different sizes on the surface of scaffolds. Thus, nerve conduit/matrix possess sufficient porous morphology beneficial for vascularization and nutrient exchange between the lumen and the outer environment.

Example 6: Evaluation of Nerve Conduit/Matrix Cytotoxicity by MTT Assay

[0096] In an embodiment, in vitro cytotoxicity assays were performed to test the biocompatibility of nerve conduit/matrix. This assay is based on the measurement of viability of cells via metabolic activity. Yellow water-soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid) is metabolically reduced in viable cells to a blue-violet insoluble formazan. The number of viable cells correlates to the colour intensity determined by photometric measurements after dissolving the formazan in DMSO. The cytotoxicity of the scaffolds was evaluated according to ISO guidelines 10993-5-2009, using 24 hr extraction period at dynamic condition.

[0097] To calculate the reduction of viability compared to the blank, following equation was used:

[00002]$\%\mathrm{Viability}=\frac{\mathrm{Absorbance}\mathrm{Extract}\mathrm{Concentration}\left(570\mathrm{nm}\right)}{\mathrm{Absorbance}\mathrm{Blank}\left(570\mathrm{nm}\right)}$

[0098] Clearly, MTT data (FIG. 6) revealed that scaffold nerve conduit/matrix did not induce any cytotoxic response at any of the tested extract concentrations. Overall, nerve conduit/matrix displayed significant biocompatibility and cell viability to L929 cells.

Example 7: Evaluation of Cell Attachment and Proliferation of MSCs on Nerve Conduit/Matrix

[0099] In an embodiment, calcein-AM staining was performed to check the nerve conduit/matrix support for cell adhesion and proliferation of MSCs. Sterile scaffold was equilibrated it in culture medium overnight before seeding. MSCs were seeded at a density of 5×10.sup.4 cells per scaffold followed by incubation at 37° C. in a CO.sub.2 incubator. After 5 days of culture, media was removed from scaffold and washed with serum free media to completely remove the serum esterase activity. 2 μM Calcein-AM staining solution was prepared by adding 1 μl of 2 mM Calcein-AM to 1 ml of serum free media. Sufficient volume of Calcein-AM staining solution was added to cover the scaffold surface. Cells were incubated for 30 min at 37° C. Labeled cells were imaged using fluorescence microscopy to check cell adhesion & proliferation as disclosed in FIG. 7.

Example 8: PKH26 Staining of MSCs on Nerve Conduit/Matrix

[0100] In an embodiment, PKH26 staining was performed to track the labelled MSCs on nerve conduit/matrix after 48 hrs of culture. MSCs pre-stained with PKH26 grown uniformly across the scaffold and exhibits adequate fluorescent signal after 48 hrs in culture. Calcein staining further confirmed the biocompatibility of scaffold nerve conduit/matrix for MSCs as disclosed in FIG. 8.

Example 9: Morphological Observation of Cell Adhesion Through SEM

[0101] In an embodiment, MSCs were cultured at the concentration of 0.5×10.sup.5 cells on nerve conduit/matrix scaffold. After 5 days of cell culture, the morphology of cells seeded on the composite scaffolds was observed using SEM. As shown in FIG. 9, the MSCs were well attached and spread out, showing more cell outgrowths. In addition, cell invasion indicates the porous nature of conduit, which provides enough space and 3D environment for cell growth and migration.

Example 10: Sectioning of Nerve Conduit/Matrix Scaffold to Check MSCs Proliferation and Migration

[0102] In an embodiment, Nerve conduit/matrix were into different sections to check MSCs cells adherence and viability on the lumen and outside surface of conduit wall using Calcein-AM stain. Conduit images were captured (FIG. 10) in both vertical and horizontal sections in order to ensure the cells migration.

Example 11: Cryopreservation and Freeze-Drying of Cell Seeded Nerve Conduit/Matrix

[0103] In an embodiment, to check cell survival efficiency after freeze-drying (FIG. 11), trehalose was used at a single concentration of 0.5 M in combination with three different concentration of Bovine Serum Albumin (BSA) i.e. 12.5%, 10%, 9.5%. MSCs seeded scaffolds were cryopreserved using freezing media consisted of alpha MEM with 10% FBS along with A) alpha MEM+20% FBS+0.5 M Trehalose dehydrate+12.5% BSA; B) alpha MEM+20% FBS+0.5 M Trehalose dehydrate+10% BSA; C) alpha MEM+20% FBS+0.5 M Trehalose dehydrate+9.5% BSA; D) alpha MEM+20% FBS+0.5M Trehalose dehydrate alone. Petri dishes containing scaffolds were placed in a freezing solution containing isopropyl alcohol that provided a 1° C./min cooling rate when stored at −80° C. Freeze-drying was done by transferring frozen samples from −80° C. to the temperature-controlled shelves of a lyophilizer. Lyophilization cycle: Lyophilization cycle: Drying 0°, Vacuum 500 mtorr, 48 hrs. cycle. Cryopreserved scaffolds under lyophilized condition in cryomedia media A, B, C, D were revived successfully as shown by calcein staining performed after 4.sup.th day post revival.

Example 12: In Vitro Differentiation of MSCs into Schwann-Like Cells

[0104] In an embodiment, for differentiation, several reagents and trophic factors were applied to induce MSCs into cells with a phenotype similar to that of Schwann cells (FIG. 12) by sequential treatment: first with β-mercaptoethanol (BME), followed by all-trans-retinoic acid (ATRA) treatment, and then culturing the cells in the presence of forskolin (FSK), bFGF, PDGF, and HRG. Phase-contrast microscopy revealed that the differentiated MSCs were morphologically different from the original undifferentiated MSCs. Cells cultured in the differentiation media changed from a fibroblast-like morphology to an elongated spindle shape (FIG. 12), similar to that of Schwann cells.

[0105] In an embodiment, in order to confirm the successful SC differentiation, immunocytochemistry of S-100, p75, CD104 and CD56, all known as markers of Schwann cells was performed. After induction, most of the differentiated SCs were positive for S100, CD56, CD104 and p75 (FIG. 13), in contrast to undifferentiated MSCs.

Example 13: In Vivo Degradation Study of Nerve Conduit/Matrix

[0106] In an embodiment, for in vivo degradation study, the nerve conduit/matrix in both conduit and sheet form were implanted subcutaneously on the back of SD rats (FIG. 14). At predefined time points (4, 8, 12, 16, 20 and 27 weeks) after implantation, the test scaffolds were photographed and harvested together with surrounding tissue for histological evaluation. The nerve conduit/matrix was not degraded throughout the implantation period, and remained stable upto 27 weeks post-implantation.

Example 14: In Vivo Implantation to Check the Efficacy of Nerve Conduit/Matrix Pre-Seeded with Differentiated Schwann Cells

[0107] In an embodiment, the in vivo study was conducted to assess the efficacy of nerve conduit/matrix pre-seeded with mesenchymal stem cells (MSCs) and MSCs differentiated Schwann cells to repair peripheral nerve defect in rat sciatic nerve transection model. The rat sciatic nerve transection model has been commonly used for the evaluation of tissue engineered NGCs in promoting peripheral nerve regeneration in vivo. Before in vivo implantation, in vitro biocompatibility of nerve conduit/matrix with MSCs was investigated. Our initial pilot study to optimize the in vivo experiments was conducted on 15 Sprague Dawley rats. The right sciatic nerve of each animal was transected and a 10 mm segment of the nerve removed thus creating a gap that was bridged with (1) nerve conduit/matrix seeded with either differentiated SCs (nerve conduit/matrix+DSC) or (2) both MSCs and differentiated SCs (nerve conduit/matrix+B2+DSC). The left sciatic nerve remained intact and used later as a control. The efficacy of nerve conduit/matrix was investigated based on the results of walking track analysis, electrophysiology, and histological assessment (FIG. 15).

Example 15: Histological Analysis of Implanted Nerve Conduit/Matrix

[0108] In an embodiment, for the histological evaluation of nerve regeneration, harvested nerve tissue was sectioned and stained with hematoxylin-eosin and toluidine blue. Morphological observations were carried out at 8.sup.th weeks post-operatively, detected that regenerated myelinated fibers were smaller and showed a thinner myelin sheath in comparison to normal nerves. The nerve conduit/matrix+DSC group showed distribution of nerve fibers with myelin and fibrous connective tissue (FIG. 16). This group showed irregular shaped perineurium and epineurium, which was largely occupied by fibrous connective tissue with center showing distorted axons with wavy plasma and myelin sheath. In addition, numerous blood vessels were observed around the regenerated nerves. Regeneration of fibrous myelinated nerve is considered important factor in terms of nerve regeneration of damaged nerve. The number of regenerated nerve fibers distal to the NGCs seeded with differentiated SC was fewer in comparison to midpoint of conduit. The nerve conduit/matrix+B2+DSC group displayed growing nerves with less myelinated fibers and thinner myelin sheaths in comparison to nerve conduit/matrix+DSC group. In this group, however, far fewer nerve fibres but a large quantity of connective tissues were visible between the nerve stumps. Besides, infiltration of inflammatory cells was found in the proximal and middle portions of the graft, surrounded by fibrous connective tissue fibers and nerve cell bodies.

[0109] In an embodiment, the prepared nerve conduit/matrix is potential substitute in nerve regeneration and repair and it helps in faster restoration of motor and sensory function in nerve injury (peripheral nerve injury, spinal cord injury and any other type of nerve injury). Various experiments were conducted to check the said efficacy of bioengineered nerve conduit/matrix. Cell viability, fluorescence microscopy and scanning electron microscopy results confirm the cell growth and distribution of the cells uniformly in matrix. In vivo studies confirmed the potential nerve regeneration and repair property of conduit.

[0110] In an embodiment, the obtained results of the potential and properties of the product of this invention were found considerably efficacious. It clearly indicates the technical advancement as compared to prior art.

[0111] Thus, the present invention provides novel and unique technique of culturing human mesenchymal stem cells, mesenchymal stem cells differentiated schwann cells and nerve cells into a proliferating, sub-confluent layer on a lyophilized biocompatible conduit/matrix prepared from plurality of composite polymers by using glutaraldehyde as a cross-linker without any integrated harmful chemicals for direct implantation or delivery of the said human mesenchymal stem cells, wherein in the said invention, the said cells are transferred while in a proliferative state and the final product obtained is transported in semi-solid medium. The said semi-solid medium is agar medium 1% to 3% and cell culture medium with essential growth factors including HEPES 2-3 gm/l and sodium bicarbonate 2-3.5 gm/l. suitable for grafting and provides a better, efficient, easy to use, cost effective ready to use biodegradable and biocompatible artificial nerve conduit/matrix for nerve repair and regeneration with sensory and motor function in a synergistic manner wherein the grafts can be prepared within 12 days.

[0112] So accordingly, the present invention provides an improved biodegradable, biocompatible, high porosity three-dimensional artificial nerve conduit/matrix based scaffold polyelectrolyte complex (PEC) with autologous/allogeneic human stem cells for nerve repair and regeneration, and a method of preparing thereof, said scaffold comprising of plurality of composite polymers and using glutaraldehyde as cross-linker, wherein said scaffold is non-adherent, has differential porosity, is able to grow cells directly on the polymeric conduit/matrix (scaffold) for direct implantation or delivery.

[0113] In an embodiment, said grafts are prepared within 12 days.

[0114] In another embodiment, said scaffolds take forms ranging from sponge like sheets to gels to highly complex structures with intricate pores and channels made with new materials processing technologies such that the spatial and compositional properties of the scaffold, the porosity of the scaffold and interconnectivity of the pores enables cell penetration into the structure as well as the transport of nutrients and waste products with differential porosity helping in the cells attachment and signal transduction.

[0115] In another embodiment, said method is comprising a unique technique of culturing human mesenchymal stem cells, mesenchymal stem cells, differentiated schwann cells and nerve cells into a proliferating, sub-confluent layer on a lyophilized biocompatible conduit/matrix, without any integrated harmful chemicals for direct implantation or delivery of the said human mesenchymal stem cells, wherein the cells are transferred while in a proliferative state and the final product obtained is transported in semi-solid medium.

[0116] In another embodiment, said nerve conduit/matrix provides repair and regeneration in a synergistic manner.

[0117] In another embodiment, said plurality of polymers are preferably selected from but not limited to gelatin, chitosan, collagen, hyaluronic acid, polyvinyl alcohol (PVA), poly caprolactone (PCL), poly pyrrole, poly urethane (PU), poly allyl amine, poly ethylene glycol 200 (PEG 200), gum acacia, guar gum and partially denatured collagen.

[0118] In another embodiment, said polymers are in the range of gelatin 1%-10% w/v, chitosan 0.5%-2.5% w/v, hyaluronic acid 0.1%-2% w/v, collagen 0.1/−10% w/v and glutaraldehyde solution 5%-50% v/v with gelatin of 50-300 bloom strength, DAC (dialdehyde cellulose) chitosan ranging from 75%-95%.

[0119] In another embodiment, said semi-solid medium is agar medium in the range of 1% to 3% and cell culture medium with essential growth factors including HEPES 2-3 gm/l and sodium bicarbonate 2-3.5 gm/l suitable for grafting which results in a better, efficient, easy to use, cost effective, ready to use biodegradable and biocompatible artificial nerve conduit/matrix for nerve repair and regeneration with sensory and motor function, in a synergistic manner.

[0120] In another embodiment, said method of preparing the scaffold comprises physico-chemical treatment; and lyophilization of freeze-dried scaffold.

[0121] In another embodiment, said method comprises sequential timed patterned physico-chemical treatment of the four or more polymers to get 3D scaffold of polyelectrolyte complex (PEC) and also at the same time using a specifically designed aspect ratio of a system for agitation/homogenization.

[0122] In another embodiment, said method comprises stabilizing the scaffold by cross linking with glutaraldehyde solution and freeze drying.

[0123] In another embodiment, said obtained freeze-dried 3D scaffold is stabilized and neutralized by ammonia fumes (5%-25%) for 12-24 hrs in closed chamber to make the stable and functional scaffold for cell seeding.

[0124] In another embodiment, said obtained scaffold is freeze-dried to make the stable scaffold for seeding of autologous or allogeneic mesenchymal stem cells (MSCs derived from bone marrow or umbilical cord) Schwann cells and neuronal cells (differentiated form mesenchymal stem cells).

[0125] In another embodiment, said mesenchymal stem cell, Schwann cells and neuronal cells are seeded onto biocompatible scaffold at cell density of 0.5×10.sup.5 to 0.8×10.sup.5 cell/cm.sup.2.

[0126] In another embodiment, said cells are monolayer and 80% to 100% confluent at the final stage of product formulation.

[0127] In another embodiment, said cells seeded on scaffold are cultured-in with serum and without serum medium.

[0128] In another embodiment, said mesenchymal stem cells are autologous or allogeneic or both.

[0129] In another embodiment, said mesenchymal stem cells, schwann cells and neuronal cells secrete several growth factors and cytokines (extracellular matrix) helpful in nerve regeneration and repair.

[0130] In another embodiment, the final product is transported in semi-solid medium and/or liquid medium or in frozen condition, such that the semi-solid medium/liquid medium provides nutrients and support to matrix and maintains the cell viability of matrix between 70% to 95% at the temperature 4° C. to 37° C. for 15 days.

[0131] In another embodiment, said conduit/scaffold is in sheet form and/or hollow cylindrical conduit form.

Advantages of the Invention

[0132] The scaffold of the present invention helps in the nerve regeneration and repair. [0133] The present invention comprises of improved healing. [0134] Can be manufactured in any size and shape as per the requirement. [0135] Easy to handle. [0136] Environment friendly as it is degradable easily. [0137] It ensures rapid healing in peripheral nerve injury. [0138] It ensures faster recovery and repair of motor and sensory function. [0139] The grafts can be made within 12 days. [0140] It is economical and offers an alternative treatment to the standard nerve injury treatment methods. [0141] There is a dramatically reduced risk of transmission of infectious disease due to rigorous process controls. [0142] It helps in restoration of motor and sensory function of damaged tissue.