EXTRACELLULAR MATRICES WHICH CAN BE USED AS SCAFFOLD FOR LIVING CELLS

Abstract

The present invention discloses an extracellular matrix comprising a modified polysaccharide consisting of repeating disaccharide units whereby in at least 11% of the disaccharide units one primary alcohol group is oxidized into a carboxylic acid.

Claims

1-14. (canceled)

15. A material comprising: a modified agarose having an at least partial β-sheet structure and comprising repeat disaccharide units, wherein at least 11% of the repeat disaccharide units are carboxylic acids, and wherein the modified agarose has a shear modulus G′ in the range of from 10 Pa to 10.sup.7 Pa.

16. The material of claim 15, wherein 20-90% of the repeat disaccharide units are carboxylic acids.

17. The material of claim 15, wherein 50-95% of the repeat disaccharide units are carboxylic acids.

18. The material of claim 15, wherein the modified agarose further comprises an unmodified polysaccharide.

19. The material of claim 18, wherein the unmodified polysaccharide is selected from the group consisting of agarose, a member of the carrageenan family, hyaluronic acid, heparin sulfate, dermatan sulfate, chondroitin sulfate, alginate, chitosan and pullulan.

20. The material of claim 18, wherein the unmodified polysaccharide comprises agarose.

21. The material of claim 18, wherein the modified agarose is obtained from agarose exhibiting an alpha-helix structure.

22. The material of claim 18, further comprising a peptide sequence covalently coupled to the carboxylic acids.

23. The material of claim 18, further comprising a nucleic acid sequence covalently coupled to the carboxylic acids.

24. The material of claim 15, wherein the material has a shear modulus G′ in the range of from 1 Pa to 100 kPa.

25. The material of claim 15, wherein the material has a shear modulus G′ in the range of from 1 Pa to 50 kPa.

26. The material of claim 15, wherein the material has a shear modulus G′ in the range of from 10 Pa to 10 kPa.

27. The material of claim 15, wherein the material is an implant.

28. The material of claim 15, wherein the material is a regenerative implant.

29. The material of claim 30, wherein the regenerative implant is an artificial skin.

30. The material of claim 15, wherein the material is injectable into a subject.

31. The material of claim 15, wherein the material further comprises living cells.

32. The material of claim 15, wherein the material is implanted as skin in a subject.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0039] FIG. 1 shows a mechanism for polysaccharide modification.

[0040] FIGS. 2A-2B show examples of polysaccharides.

[0041] FIG. 3 shows a scheme illustrating the strategies for preparation of modified polysaccharides. In the box on the left hand side modifications made by the supplier are shown. In the box on the right hand side two blendings made by the final user are represented.

[0042] FIGS. 4A-4D: FIG. 4A shows a reaction mechanism of the D-Galactose primary alcohol oxidation into a carboxylic acid; FIG. 4B shows the FTIR spectrum of the blue native Agarose and red modified Agarose; FIG. 4C shows the MAS-NMR of the red modified Agarose and blue native Agarose; FIG. 4D shows the percentage of modification determined by FTIR plot against the amount of base added to the reaction as a mean of 12 reactions, error bars represent the standard deviation.

[0043] FIGS. 5A-5H: FIG. 5A shows the temperature sweep of native Agarose, red and 60% modified Agarose, blue dotes represent G′ and triangles G″; FIG. 5B shows the frequency sweep of native Agarose, red and 60% modified Agarose, blue dotes represent G′ and triangles G″; FIG. 5C shows the temperature of gelation of Agarose of different percentage of modification; FIG. 5D shows the shear modulus of Agarose of different percentage of modification. In all charts, the error bars represent the standard deviation, FIGS. 5E-5H show pictures of 2% w/v gels respectively 1 native Agarose, 2 28% modified Agarose, 3 60% modified Agarose, 4 93% modified Agarose.

[0044] FIGS. 6A-6F: FIG. 6A shows the circular dichroism of red: native Agarose and blue: 93% modified Agarose,; FIG. 6B shows the circular dichroism of dashed: 93% Agarose at 5° C. and plain 93% modified Agarose at 90° C.; FIG. 6C shows the circular dichroism of different modified gel of Agarose at 203 nm, FIG. 6D shows the circular dichroism of dashed red : native Agarose, dashed blue: 93% modified Agarose, plain red native K-carrageenan, plain blue 93% modified k-carrageenan; FIG. 6E shows the zeta potential of different modified Agarose; FIG. 6F shows blue: size and red: polydispersity measured by light scattering of diluted solution of different modified Agarose. In all charts, error bars represent the standard deviation.

[0045] FIGS. 7A-7H: FIG. 7A shows RMSD from the first frame for red native Agarose and blue totally modified Agarose; FIG. 7B shows the cumulated H bonds between the two strand of polysaccharide; FIG. 7C shows the Ramachandran plot of native Agarose AG link in chain 1 and GA link in chain 2; FIG. 7D shows the Ramachandran plot of totally modified Agarose AG link chain 1 and GA link in chain 2; FIG. 7E shows frame 1 of the MD simulation for native Agarose; FIG. 7F shows the frame token from the middle of the simulation; FIG. 7G shows frame 1 of the MD simulation for totally modified Agarose; FIG. 7H shows the frame token from the middle of the simulation.

[0046] FIGS. 8A-8O: FIGS. 8A-8E show native polysaccharide, FIGS. 8F-8H show 28% of modification, FIGS. 8I-8K show 60% of modification, FIGS. 8L-8P show 93% of modification; Row 1 is the ESEM of 2% w/v freeze dried Agarose gel; Row 2 is the AFM picture of the height of Agarose gels, Row 3 is the 3D reconstruction of AFM pictures; Row 4 is the ESEM of 2% w/v freeze dried k-carrageenan gels at low magnification; Row 5 is the ESEM of 2% w/v freeze dried k-carrageenan at high magnification, FIG. 8Q shows the surface roughness of the AFM sample of Agarose gels against the percentage of modification.

PREFERRED EMBODIMENTS OF THE INVENTION

[0047] First the Agarose has been prepared by following protocols already published. The addition of NaOH during the reaction maintain the optimal pH of the reaction and compensate the acidification of the solution by the creation of carboxylic acids with the oxidizer TEMPO ((2,2,6,6-Tetramethyl-piperidin-1-yl)oxyl) reactivated with NaOCl and catalyst by the KBr, FIG. 4A. The NaOH is used to maintain the optimal pH of 10.8 for the reaction, but also gives a quantitative characterization of the amount of carboxylic acid formed as it compensates the formation of the acidic moiety. Therefore a relation between the volume of base added and the quantity of carboxylic acid formed can be considered, equation 1. The probable side reaction that consist of the oxidation of the carboxylic acid down to the aldehyde is neutralized at the end of the oxidation by adding NaBH.sub.4 which reduce the freshly reacted aldehyde back into a primary alcohol.

[00001]$\begin{array}{cc}\frac{m_{\mathrm{Agarose}}}{M_{\mathrm{Agarose}}}\times {\mathrm{DPI}}_{\mathrm{Agarose}}=n_{\mathrm{NaOH}}& \left(1\right)\end{array}$

[0048] In order to have precise and reliable result, the characterization of the synthesis has been conducted by using different techniques. As a first qualitative analysis the .sup.13C MAS-NMR shows the appearance of a peak at 180 ppm, characteristic of the carboxylic acid, and the vanishing of the peak at 55 ppm characteristic of the primary alcohol. The quantitative method, FTIR, shows the increase of the ratio between the peaks at 1650 cm.Math.1: vibration band of the double bond carbon oxygen and 1360 cm.sup.−1: the vibration band of the carbon oxygen from the primary alcohol, FIG. 4 B. The relative amount of carboxylic acid and primary alcohol can be calculated by integrating the peak of interest and using the equation (2).

[00002]$\begin{array}{cc}\%\mathrm{Oxidation}=\frac{\int v_{C=O}}{\int v_{C=O}+\int v_{C-O}}\times 100& \left(2\right)\end{array}$

[0049] The formation of carboxylic acid along the polysaccharide backbone is monitored by the amount of NaOH during the reaction and afterwards by the quantitative analysis using FTIR spectrometry. In order to compare both of the technique, the amount of NaOH added to the reaction against the calculated modification using FTIR has been plotted given a linear relation that show the accuracy of the both methods but also the reproducibility of this synthesis, FIG. 4 D.

[0050] It is assumed that the incorporation of ligands at the primary alcohol of the C6 position on the D-galactose of the Agarose repetition unit leads to a lower shear modulus and a lower temperature of gelation. In order to characterize in which manner the introduction of carboxylic groups on the polysaccharide backbone induces the change of mechanical and gelation properties a systematic rheological study of the gels has been conducted.

[0051] The gelation point, also named temperature of gelation, is determined by the equality of the shear storages to the loss moduli at a constant shear frequency and deformation with decreasing the temperature as shown in FIG. 5A. Compared to the technique of the inverted bottle the temperature sweep is an objective measure that gives a more precise value. The increase of modification lowered the gelation temperature in a linear way. The difference of gelation temperature from native agarose of 40° C. to 93% modified down to 5° C. gives interesting features that can be used to tune the gelation temperature of the gel, see FIG. 5 C. Moreover the agarose forms a hydrogel that has a hysteretic behavior, i.e. the formation of physical crosslinked points happened at a lower temperature as the breakdown of the gel, which gives a particular thermal stability that makes it an interesting material for biological application.

[0052] The gel stiffness can be characterized by doing a frequency sweep, shear of the gel at a constant deformation and temperature with a frequency increase. In rheology a gel state can be defined as state where the shear and loss moduli are both independent of the shear frequency, FIG. 5 C. The modification of the polysaccharide backbone induces a lower shear and loss moduli which result of a weaker gel. This weakening of the polymer network seems to be related to the incorporation of carboxylic group, as the loss of primary alcohol provokes a linear loss of stiffness (FIG. 5 D).

[0053] The versatile stiffness of the gel can be compared to the human tissue stiffness. The different mechanical properties of the cell surrounding have been reporting to vary from 10.sup.5 Pa for bones down to 10.sup.1 Pa for nerves. The magnitude order of the gel stiffness covered by the different proportion of modification goes from 10.sup.4 Pa for a 2% w/v gel of native agarose down to 10.sup.2 Pa for a 93% modified gel of the same concentration.

[0054] It is known that the agarose backbone folds in an alpha-helix and that the gel is formed by the aggregation of these helices. The loss of rigidity of the different gels for a given concentration could be attributed to a loss of aggregation of the alpha helix. Moreover, the loss of the gel turbidity attributed to the amorphous structures formed by the aggregates reinforce the hypothesis of a loss of crosslinked point (see FIG. 5 E 1 to E 4).

[0055] It has been suggested in the literature that the physical crosslinked point formed in the agarose hydrogel can be assimilated as spherical nanoparticles and then be characterized with SLS (static light scattering). It appears that the increase of the modification along the polymer backbone dramatically decreases the size of this crosslinked point but also their polydispersity resulting in smaller aggregates of a smaller size distribution, see FIG. 6 F. These results support the hypothesis of less aggregation of alpha-helix, but the loss of polydispersity is also a quantification of the organization of the aggregate. The decrease of the polydispersity reveals that the aggregates are organized in a more regular shape.

[0056] On the other hand, the measurement of the zeta-potential gives information of the mobile charges present on the polymer surface. It appears that the increase of the modification is linearly proportional to the increase of the zeta potential, see FIG. 6 E. The increase of the zeta potential transcript the incorporation of repulsion charges between the polymer chains that might disrupt the aggregation of the alpha-helix domains.

[0057] In the past, the alpha helix of the agarose has been characterized by using circular dichroism (CD). The native agarose CD is composed of a single peak at 185 nm that is characteristic of an alpha-helix conformation of the secondary structure. In order to understand the gelation mechanism of the modified Agarose, CD has been measured for different modified agarose preparations. It appears that the peak characteristic of an alpha-helix is shifted to higher wavelength and a new peak appears at 203 nm, see FIG. 6 A. The hypothesis of a lower number of alpha-helix aggregate will have lead to a reduced ellipticity in the alpha-helix area of the spectrum, but the shift of the spectrum to another area suggest a change of the folding of the modified agarose. The analogy of the conformation of polysaccharides with the folding of protein gives an indication of the new secondary structure of the modified agarose. Indeed the area of 203 nm correspond to the organization of proteins in beta-sheet. The consistent study of different modified gel highlight that the appearance of the new peak is linearly proportional to the amount of carboxylic acid along the polysaccharide backbone, see FIG. 6 C. The modification of the primary alcohol into a carboxylic acid clearly induces a change of the secondary structure into the agarose that is still able to form a gel at low temperature. The CD spectrum of the modified agarose has been done at two different temperatures, at 5° C. where the modified agarose is below its sol-gel transition temperature and at 90° C. above its gel-sol transition temperature. It appears that at high temperature the new peak at 203 nm vanished. The agarose made a gel that is temperature dependent, the temperature dependency of the new peak suggest that the gel dependency is now driven by this new organization and not anymore by the aggregation of the alpha-helix.

[0058] Agarose is not the only polysaccharide which exhibits an alpha-helix structure. Indeed the family of the Carrageenan is also known to organize in alpha-helices and form also physical gels that are temperature dependent. The same spectroscopy study has been followed to characterize the k-Carrageenan that has been oxidized at the same position, according the same protocol than the agarose. The peak of the unmodified k-carrageenan and unmodified agarose are of opposite sign due to the different helix rotation, but both of them are in the same wavelength. The CD curves obtained for the modified k-carrageenan exhibit the same new peak at the same wavelength of 203 nm, see FIG. 6 D. The possibility to obtain the same CD behavior for two polysaccharides of the same family implies that the modification of this primary alcohol in theses polymers can be a general method to induce a beta-sheet like structure in polysaccharides.

[0059] In order to validate these hypotheses a molecular dynamic simulation of two polysaccharides backbones over 15 ns has been ran. Past studies on polysaccharide have demonstrated the relevance of the molecular dynamic simulation. It is of high importance to validate the model of the new organization of the modified agarose. The root mean square deviation of the geometry from its initial position show that after 2 ns a stable conformation is obtained and will remain stable until the end of the simulation, see FIG. 7A. In order to characterize the distance between the two polysaccharides strands the formation of h-bond between these two strands has been calculated. The formation of an h-bond is driven by the distance of an electronegative atom to a hydrogen atom. Each h-bond on each frame has been summed and this sum has been plotted against the time. Therefore a stable h-bond sum transcript the non presence of any h-bond, see FIG. 4 B. This calculation shows that the native agarose is able to form h-bond all along the simulation, but the totally modified agarose strands do not form any h-bonds after 5 ns, highlighting that the two polymer chains are too far from each other in order to interact together in this manner. The non interaction of the two polymer strands confirms the data obtained from the zeta potential that show repulsion forces between the two strands.

[0060] The main question that has to be answered by the simulation is the physical and geometrical possibility to form a beta-sheet structure. In past studies the conformation of the sugar cycles of the polymer chain has been analyzed using a Ramachandran plot. The dihedral angle of the glycosylic linkage can be plotted and compared with the cartography obtained for protein folding. The native agarose has its dihedral angle formed by the anhydro-galactopyranose/galactose bound of the chain 1 and galactose/anhydrogalactopyranose bound of the chain 2 in the alpha helix domain, see FIG. 7 C. The same bounds on the modified agarose have their dihedral angles in the beta sheet area of the plot, see FIG. 7 D. The change of area of the dihedral angles that switch from the alpha-helix position, to the beta-sheet location show that this suggested organization from the other analysis is possible. The animations created by the software give an idea about the polymers chains position along the time of the simulation. The conformation of the polymer of the first frame has been obtained from the x-ray data of the agarose. The two polymers have been modeled with the same initial conformation, see FIG. 7 E1 and F1. It appears that the chains of the native agarose are staying together along the simulation time, see FIG. 7 E2, but the chains of the modified agarose are repulsed from each other and position their carboxylic acid opposite from each other, see FIG. 7 F2.

[0061] The molecular dynamic simulation attests the possibility of this beta sheet conformation. As for the CD data, the plot of the dihedral angles is based on the theory developed for protein. The use of the specific area of the Ramachadran plot in order to describe the folding of protein has only by used so far, for protein. As the organization of the chains of polymer changes from an aggregation of alpha-helix to a beta-sheet, the impact on the macrostructure of the material should be non negligible.

[0062] The investigation of the macrostructure of the gel has been done by using an ESEM, see FIG. 7 A1 to D1. The highest difficulty in the study of gels is the difficulty to reveal the hydrated structure of the polymer network. Therefore freeze dried sample have been used for the electron microscope. The change of structure between the native and the modified agarose shows a radical change of their structure. The unorganized fibers of the native agarose leave the place to a highly ordered fibril structure as the percentage of modification of the polymer backbone is increased. The unorganized fibers are coupled with small ordered area in the 28% modified sample. The 60% modified gel is more organized and reveals still small domains of unorganized fibers, but the 93% modified gels is totally organized in sheets.

[0063] This new organization of polysaccharide is surprising. These pictures validate the hypothesis of a new folding structure but moreover confirm the beta sheet organization of the polymer chains. The circular dichroism has shown that the modification of the primary alcohol of the D-galactose of the agarose repetition unit lead to the same change of folding of the secondary structure. The structure of the k-carrageenan hydrogels has been imaged by following the same protocol as for the agarose. The ESEM picture reveals that the modification of the k-carrageenan primary alcohol leads to the same highly organized structures forming thin sheets of the same kind as for 93% modified agarose gels. The same change of organization is shown for two different polysaccharide of the same family, which encourages the possibility of a general method for modifying polysaccharide organization and predict their secondary structure based on the protein models already existing, see FIG. 8 A4, A5, D4 and D5.

[0064] Cells which are cultivated on two-dimensional substrate show a dependence on the roughness of the substrate. Therefore the surface of the gel has been characterized in a semi-dry state by using an AFM. It can reveal the roughness of the surface. The height pictures highlight a loss of the relief, as the modification is increased, see FIG. 8 from A2 to D2. The tri-dimensional reconstruction allows visualizing clearly the loss of the roughness, see FIG. 8 from A2 to D2. The mathematic calculation of the roughness of the gel surface appears to be linearly related to the amount of carboxylic acid on the polymer backbone, see FIG. 8 E. The loss of the roughness can be explained by the smaller crosslinked points, aggregates calculated with the light scattering experiment, that are formed in the modified gels. The surface is smoother and the organization in sheets shown by the ESEM correlate the explanation of a smoother gel that has fibers more organized in a unilateral direction.

Example 1

a) Modification of Agarose

[0065] Agarose type I has been obtained from Calbiochem. TEMPO (((2,2,6,6-Tetramethylpiperidin-1-yl)oxyl), NaOCl, NaBH4, NaBr, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)), MES buffer (2-(N-morpholino)ethanesulfonic acid) have been obtained from Sigma Aldrich and used as received. Solution of 0.5 M NaOH have been freshly made every three-month as well as solution of 5 M HCl. Peptide GGGGRGDSP (SEQ ID NO. 3) has been obtained from Peptide International. Ethanol technical grade was used without any further purification. Deionized water was used for non sterile synthesis.

[0066] Agarose was modified under sterile conditions: all the chemicals were dissolved in autoclaved water and filtered with a 0.2 μm filter. All the glassware was autoclaved and the reaction was conducted under a laminar flow. Agarose (1 g) was autoclaved in MilliQ water. Autoclaved agarose was poured into a 3 necked round bottom flask. A mechanical stirrer was adapted to one of the neck. A pH-meter was adapted on the round bottom flask. The reactor was then cooled down to 0-5° C. and vigorously stirred. TEMPO (0.160 mmol, 20.6 mg) was added, NaBr (0.9 mmol, 0.1 g) and NaOCl (2.5 ml, 15% solution) was as well poured inside the reactor. The solution was adjusted to pH=10.8 with HCl and NaOH. The pH was maintained at 10.8 by adding NaOH. At the end of the reaction NaBH.sub.4 (0.1 g) was added and pH=8 was reached. The solution was stirred for 1 hour and NaCl (0.2 mol, 12 g) and ethanol (500 ml) was added. The agarose was precipitated and extracted in a funnel. The two layers were then filtered on a frit glass. The agarose was then dialyzed in Spectra Pore 4, MWCO=12-14000 for 2 days and the water was changed two times. Prior dialysis in the membrane were left overnight in a 70% ethanol solution, 2 hours before use they were rinsed in autoclaved water. Finally the product was put on a Christ LO 2-8 LO plus at 0.1 mbar for the main drying and at 0.001 mbar during the desorption phase. Samples were put in round bottle flask and frozen in liquid nitrogen bath on a Rotary evaporator modified for this purpose. Thin layer of frozen solution were obtain on the flask wall reducing the lyophilization time.

b) Analytical Methods were Performed as Follows

aa) Infrared Spectroscopy

[0067] FTIR spectra have been recorded on a Brucker vector 22. Sample were prepared with 2 mg of substance in 200 mg of KBr, then grinded and pressed under a 10 tons press for 10min.

bb) Nuclear Magnetic Resonance

[0068] Magic Angle Spinning NMR spectra were recorded in the solid state using a Brucker Avance ORX 500 spectrometer, freeze dried samples were put in a ceramic holder and spin at 7500U/s.

cc) Rheology

[0069] Rheology experiments were performed with an Anton Paar Physica MCR 301 equipped with a Peltier temperature cell. Sample were prepare as 2% w/v in deionized water, heated at 90° C. and stirred for 10 min until a clear solution was obtain. The liquid was then poured on the rheometer plate, pre-heated at 80° C., using a pipette. The solution was able to stabilize for 10 min before starting to record. A plate tool from Anton Paar: PPR25 was used for all the experiment. Sol-gel transition and frequency sweep were made using the same sample in a single cycle: 10 min equilibrium at 80° C., cooling down to 5° C. in 30 min and record of G′ and G″ every 1.5° C. at 1 rad/s with a deformation of 10%, stabilized at 5° C. for 30 min, heated up at 37° C. and stabilized for 30 min then the frequency sweep was recorded at 37° C. by increasing the rotation frequency from 0.01 rad/s up to 10 rad/s over 30 min with a deformation of 10%. Sol-gel transition temperature was calculated as the temperature where tane=0.

Circular Dichroism

[0070] Circular dichroism spectra were obtained using a Jasco spectropolarimeter J-810 equipped with a Peltier temperature cell Jasco PFD-425S. Solution of 0.15% w/v of agarose was made in Milli-Q water at 90° C. for 15 min then solution have been cooled down at 5° C. in the CD chamber for 30 min prior measurement. Each spectrum has been recorded three times and summed together. Each spectrum for a given modification is a mean of three different syntheses.

ee) Dynamic Light Scattering

[0071] DLS has been done on a Beckman Coulter Delsa Nano C particle analyzer with a polystyrene cuvette of 1 cm. Agarose has been dissolved in Milli-Q water at 90° C. for 15 min to obtain a 0.15% w/v solutions that have been cooled down at room temperature for a day. The light scattering has been done at 15° C. and samples have been equilibrated for 30 min prior to measurement. For each measurement three measures have been done and an average has been calculated. Each point is a mean of three different syntheses.

ff) Zeta Potential

[0072] Zeta potential has been measured on a Beckman Coulter Delsa Nano C particle analyzer. The same solutions have been used as for the light scattering experiment. Measurements have been made in a flow cell that has been aligned with the laser prior every measurement. Each measurement has been made three times and an average has been calculated, each spectrum for a given modification is a mean of three different syntheses.

gg) ESEM

[0073] SEM pictures were obtain with a ref agarose gels of 2% w/v were prepared and 2 ml of this solution was frozen dried for 24 hours under 0.1 mbar vacuum in a 5 ml glass vial. The sample has then been vertically cut and the inside of the sample has been imaged at different magnification. Images shown here are representative of different areas of a given sample at different magnification, which have been reproduced with three different gels prepared from different batches.

hh) AFM:

[0074] AFM pictures were obtained with a Veeco Dimension 2100. Samples were prepared on a 3 mm microscopic glass holder that has been passivated. The glass slide was washed with 0.1 M NaOH and dry in the oven. The dry slides are then passivated with few drops of dichloromethylsilane. Two slides are sandwiched together to have a uniform passivation. After 10 min the slides are washed with water and the excess of dichloromethylsilane is washed with soap and the slides are dried. Slides side was prepared in a hydrophobic way. Agarose samples are prepared as 2% w/v gels and 25 μl of the solution is poured on an unmodified glass slide, a dichloromethylsilane passivated slide is then adjusted on top of the solution. Slides of 0.5 mm are put as spacer between the hydrophobic and the normal glass slide, the whole montage is then allowed to gel for 30 min at 4° C. The upper slide (hydrophobic) is after that removed and a thin layer of agarose gel is obtained, this gel is then allowed to stabilized at room temperature for 30 min before measurement in order to avoid any shrinkage or dilatation of the gel during the measurement.

ii) Molecular Dynamic

[0075] MD simulations have been done using the Desmond package of the Maestro version 8.5 from Schrödinger. Initial conformation has been obtained from the x-ray structure of the agarose that has been downloaded from the PDB library. Modified agarose has been drawn from the PDB file directly inside the Maestro software. Implicit water model has been build using the Desmond tool, resulting in a 10 A square box build by following the TIP3 solution model. The simulations have been run in the model NPV at 300° K at atmospheric pressure for 15 ns. Analysis of the results was done using the VMD software and the tools available in the standard package.

Example 2

Peptide Modification of the Agarose

[0076] Functionalization of agarose with the G.sub.4RGDSP peptide has been done by EDC peptide coupling. Agarose (30 mg, 0.25 μmol) was dissolved in autoclaved water; all the chemical and buffer were sterilized on a 0.2 pm filter, MES buffer was added and the solution reached a pH=4. The peptide was added (500 μg) and then EDC was added (200 mg) and the solution was stirred for two hours minutes at 40° C. to avoid any gel formation, The solution was then dialyzed in Spectra Pore 4 MWCO=12-14 kDa, water was changed three times and the sample was freeze dried. Prior dialysis; the membranes were left overnight in a 70% ethanol solution, 2 hours before use they were rinsed in autoclaved water.

Elementary Analysis Samples have been analyzed on an Elementar Vario EL

Example 3: Use of Modified Extracellular Matrix for Biological Tests

Cell Culture

[0077] Modified gels were prepared at a 2% w/v concentration in DMEM media and heated at 60° C. for 30 minutes in order to avoid any degradation of the RGD peptide. The temperature was adjusted to 37° C. and the culture media was completed with usual nutrients and cytokines. Human chondrocytes were obtain from ATCC and used between passage 3 and 5. Solution of agarose was mixed with the cells and then seeded in a 48 wells plate. The plate was then store at 4° C. for up to 30 min in order to allow the sol-gel transition to occur. The plates were then cultivated in a incubator at 37° C., 4% CO.sub.2 for two weeks.

Cell Shape Factor

[0078] All pictures have been taken on an Axio Observer A1, from Carl Zeiss, equipped with a DIC filter. Pictures were taken after 1, 5 and 14 days at different magnification in different area of the sample. The cell perimeter and cell area were then measured and the CSF was calculated. The results are a mean of three different batch of peptide modified Agarose which has been reproduce two times, which represent a total of 6 wells. Per well more than 100 cells were measured in order to have a meaning full CSF.

Real Time PCR

[0079] At 7 days, 14 days and 21 days of cell culture in the gel, the media was removed and the gels were frozen at −80° C. overnight. The solids obtained were then crushed and digested with a CT AB buffer. Then extract using the Qiagen kits.