COMPLEMENT ACTIVE FRAGMENT-LOADED THREE-DIMENSIONAL BIOMATERIAL FOR DENTAL AND/OR OTHER TISSUE REGENERATION

Abstract

A three-dimensional biomaterial including particles encapsulating at least one complement active fragment dispersed therein. Also, a process of manufacturing of the three-dimensional biomaterial with at least one biocompatible and/or resorbable polymer and the particles. The three-dimensional biomaterial is useful for tissue regeneration, especially with regards to dental pulp, dentin, periodontal and gingival tissues, as well as bones tissues.

Claims

1.-15. (canceled)

16. A biomaterial comprising: a three-dimensional matrix made of at least one biocompatible and/or resorbable polymer, and particles encapsulating a complement component, a complement active fragment, or a combination thereof; said particles being dispersed in the three-dimensional matrix.

17. The biomaterial according to claim 16, wherein the biocompatible and/or resorbable polymer is selected from polysaccharides, proteins and any combinations or copolymers thereof.

18. The biomaterial according to claim 16, wherein the biocompatible and/or resorbable polymer is selected from collagen, chitosan, alginate and any combinations or copolymers thereof.

19. The biomaterial according to claim 16, wherein the encapsulating particles are polymeric microspheres.

20. The biomaterial according to claim 19, wherein the polymer of the microspheres is selected from biocompatible and/or biodegradable polyesters.

21. The biomaterial according to claim 19, wherein the polymer of the microspheres is selected from polycaprolactone (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polyhydroxybutyrate (PHB), poly(3-hydroxy valerate), poly(ethylene succinate) (PESu), poly(butylene succinate) (PBSu) and any combinations or copolymers thereof.

22. The biomaterial according to claim 19, wherein the polymeric microspheres are made of poly(lactic-co-glycolic acid) (PLGA).

23. The biomaterial according to claim 16, wherein the complement component and complement active fragment are selected from complement component C1, complement component C2, complement component C3, complement component C4, complement component C5, complement active fragments thereof and any combinations thereof.

24. The biomaterial according to claim 16, wherein the complement component and complement active fragment are selected from complement component C5 or C3.

25. The biomaterial according to claim 16, wherein the complement component and complement active fragment are selected from complement active fragment C5a or C3a.

26. The biomaterial according to claim 16, comprising PLGA microspheres encapsulating complement active fragment C5a, said microspheres being dispersed in a collagen three-dimensional matrix.

27. The biomaterial according to claim 16, further comprising a second phase made of a three-dimensional matrix made of at least one biocompatible and/or resorbable polymer which is free of particles encapsulating a complement component, a complement active fragment, or a combination thereof.

28. The biomaterial according to claim 16, wherein the three-dimensional matrix is porous.

29. The biomaterial according to claim 16, wherein the three-dimensional matrix is a hydrogel.

30. A process for manufacturing the biomaterial according to claim 16, said process comprising contacting particles encapsulating a complement component, a complement active fragment, or a combination thereof, with at least one biocompatible and/or resorbable polymer to form a three-dimensional matrix made of the at least one biocompatible and/or resorbable polymer in which the particles are dispersed.

31. The process according to claim 30, wherein the particles encapsulating a complement component, a complement active fragment, or a combination thereof, are contacted with the biocompatible and/or resorbable polymer by impregnation in a hydrogel prepared by dissolution of said polymer in an aqueous solution followed by homogenization.

32. The process according to claim 31, further comprising a step of water removal to provide the biomaterial under the form of a sponge.

33. A method of tissue repair and/or tissue regeneration for a patient in need thereof, comprising contacting the tissue to be repaired and/or regenerated with the biomaterial according to claim 16.

34. The method according to claim 33, wherein the tissue is selected from dental pulp, dentin, periodontal and gingival tissues.

35. The method according to claim 34, wherein the tissue is selected from bones, nervous tissues and skin tissues.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0144] FIG. 1 is a perspective view of a cylindrical biphasic biomaterial of the invention.

[0145] FIG. 2 is a series of photographs showing monophasic (a) and biphasic [0146] (b) C5a-microspheres-loaded collagen matrices according to the invention and a magnification (c) showing microspheres dispersed in the matrix.

[0147] FIG. 3 is a series of schemes representing the dentin-pulp regeneration process in a direct pulp capping or pulpotomy procedure. On the left side (a) of schemes (1) to (4) is represented a classic direct pulp capping or pulpotomy procedure, while on the right side (b) is represented a direct pulp capping or pulpotomy procedure using the biomaterial of the invention under biphasic form. Scheme (1) represents a damaged tooth with a carious lesion, wherein a part of dentin and pulp are removed. Scheme (2) shows the placement of a material filling the cavity in dentin and pulp: (a) a classic direct pulp-capping material and (b) the biphasic biomaterial of the invention, with the loaded phase on the upper part. Scheme (3) shows the migration of dental pulp stem cells. Scheme (4) shows the final state of the tooth after the direct pulp capping or pulpotomy procedure.

[0148] FIG. 4 is a series of schemes representing the bone regeneration process. FIG. 4(1) shows a bone tissue with a bone defect. FIG. 4(2) shows the placement of a monophasic biomaterial of the invention filling the cavity. FIG. 4(3) shows the migration of bone progenitor cells which colonize the biomaterial, enabling to regenerate the bone tissues as represented on FIG. 4(4).

[0149] FIG. 5 is a series of 3 photographs (a), (b) and (c), showing PLGA microspheres encapsulating C5a complement active fragment. The indicated scale is 10.00 μm.

[0150] FIG. 6 is a graph representing the cumulated percentage of C5a release overtime from microspheres encapsulating C5a placed in DMEM.

[0151] FIGS. 7a and 7b are graphs showing the dental pulp fibroblasts viability overtime for different materials. The control is the DMEM medium. Collagen sponges were tested, either unloaded collagen sponges or C5a-encapsulating-microspheres loaded collagen sponges, either monophasic or biphasic. FIG. 7a reports the results of the direct evaluation, while FIG. 7b reports the results of the indirect evaluation.

[0152] FIG. 8 is a graph representing the migration overtime of dental pulp fibroblasts in presence of the biomaterial of the invention.

[0153] FIG. 9 is a graph representing the C5a release overtime from a collagen sponge loaded with C5a encapsulated in microspheres.

[0154] FIG. 10 is a graph representing the C5a release overtime from a collagen sponge loaded with free C5a.

EXAMPLES

[0155] The present invention is further illustrated by the following examples.

Abbreviations

[0156] BSA: bovine serum albumin,
DCM: dichloromethane,
DMSO: dimethylsulfoxide,
h: hour,
min: minute,
PLGA: poly(lactic-co-glycolic acid);
PVA: polyvinyl alcohol,
rpm: round per minute.

Material

[0157] C5a (R&D system, France); Kit ELISA C5a (R&D system, France). DMEM (Dulbecco's Modified Eagle's Medium) and all cell culture materials and reagents: Fischer Scientific (PAA laboratories)—France. Magnetic beads: Dynal Biotech, Oslo—Norway. Primary antibodies: R&D Systems, Lille—France. Collagen (Fibrillar collagen provided by Datascope, Getinge Group, USA), Chitosan (Sigma), Alginate (Septodont, France), PLGA (EXPANSORB® DLG 50-2A provided by PCAS, France), PVA (Merck, Germany).

I. Particles Encapsulating Complement Active Fragments

I.1. Manufacturing of Polymer Microspheres Encapsulating C5a

[0158] Microspheres of PLGA were prepared by a water/oil/water (w/o/w) double-emulsion and solvent extraction/evaporation method adapted from the method previously described by Kalaji et al. (Kalaji N. et al., J. Biomed. Nanotech., 2010, 6(2), pp. 1-11). In brief, PLGA was dissolved in DCM to form the oil phase. This oil phase was then emulsified using a high-speed mixing apparatus (Ultrathurax®, T25 basic, IKA® Werke/Germany) with an internal aqueous phase of purified water containing C5a always with BSA to form a w/o emulsion. All preparations were performed at room temperature. The resulting emulsion was added to 50 mL of external aqueous solution containing 0.1% (w/v) PVA and emulsified with Ultrathurax® in order to produce the double w/o/w emulsion. The double emulsion was then poured into a large volume of water (100 mL) under magnetic stirring for 2 hours to allow removal of the organic solvent. Finally, the resulting microspheres were centrifuged, washed twice with 50 mL of deionized water, lyophilized and stored at −20° C.

I.2. Characterization of polymer microspheres encapsulating C5a

[0159] Microspheres' morphology was analyzed using a Keyence VHX-5000 digital microscope. This device allows to automatically reconstruct a sharp and deep image, thanks to its intuitive software making measurements of microspheres size in a very short time.

[0160] The particles obtained by the w/o/w double-emulsion and solvent extraction/evaporation method described above in example I.1. are under the form of microspheres. The outer surface of the microspheres has a rough aspect, probably reflecting on their porosity, as illustrated in FIG. 5. The microspheres have mean size of 25.51 (+9.83) μm in diameter.

I.3. Encapsulation efficiency

[0161] The encapsulation efficiency test aims at determining the quantity complement component and/or complement active fragment (for example C5a) present in the particles.

[0162] This is particularly important to adjust the quantity of complement component and/or complement active fragment in the biomaterial.

[0163] It is important that the encapsulation efficiency is high in order to limit the required amount of particles in the biomaterial in order to avoid the presence of a too important amount of PLGA which might be toxic.

[0164] Two methods were used to measure the encapsulation efficiency: [0165] Method 1 (direct determination) consists in measuring the amount of complement active fragment entrapped in the microspheres after extraction. The extraction protocol consists in dissolving about 5 mg of microspheres in 1 mL of DMSO, then adding 9 mL of DMEM (Dulbecco's modified eagle medium) and analyzing by ELISA the quantity of complement active fragment trapped in the microspheres (described below in section 111.2). [0166] Method 2 (indirect determination) comprises determining the lost quantity of complement active fragment in the aqueous supernatant at the end of the microspheres preparation (after solvent evaporation, including rinsing water) using an ELISA quantification. The lost quantity is compared to the initial amount of complement active fragment involved in the encapsulation process to determine the encapsulated amount.

[0167] The encapsulation efficiency of the microspheres encapsulating C5a obtained in example I.1 was determined. The two methods, direct and indirect, are concordant. The encapsulation efficiency varies from 70% to 79%, with an average of 74.79 (+3.72) %.

1.4. Liberation Assay

[0168] The liberation assay aims at determining the amount of complement component and/or complement active fragment (for example C5a) that can be released from the encapsulating microspheres.

[0169] Polymeric microspheres encapsulating C5a were placed in a fixed volume of DMEM at 37° C., 5% CO.sub.2. At 1, 7, 14, 21 and 31 days, the medium is removed and stored at −20° C. and fresh medium is added. At each time point a dosage of liberated C5a is performed using ELISA assay (described in section 111.2).

[0170] The liberation of C5a from the microspheres encapsulating C5a obtained in example I.1 was analyzed. As shown in FIG. 6, an important burst release of C5a was observed the first day of the liberation assay. During this burst release, 53% of the C5a present in the microspheres is released. This initial burst release was followed by a slow release of C5a during 21 days where 91% of the encapsulated C5a has been released.

II. Biomaterial of the Invention: Manufacturing and Characterization

II.1. Manufacturing of Unloaded Matrices

[0171] Porous solid matrices were prepared using a freeze-drying method. First a hydrogel was prepared by dissolution of the chosen biocompatible and/or resorbable polymer (for example collagen, chitosan or alginate—see table 1 below) in an aqueous solution and homogenized. The gel was then distributed in a mold to fit the size requirement (3 mm in diameter and 5 mm in height). The gel was then frozen for 12 h at −40° C. and freeze dried using the following parameters: sublimation at −40° C., 12 mbar, during 24 h, to provide a matrix under the form of a sponge.

TABLE-US-00001 TABLE 1 Composition of the gel used for matrix preparation. Amount in the Homogenization Polymer gel (w/w) Dissolution Speed Duration Collagen 1.5% Purified water 14000 rpm 10 min (fibers) Chitosan   1% Acetic acid 0.25%  300 rpm 1 h (powder) (w/w) aqueous solution Alginate   1% Purified water  300 rpm 1 h (powder)

[0172] In the case of a mixed matrix (for example chitosan 1%/collagen 1% or alginate 1%/collagen 1%), each gel is prepared individually and then mixed for 30 min at 300 rpm before distribution.

II2. Manufacturing of Matrices Loaded with C5a-Microspheres

[0173] Microspheres-loaded porous solid matrices were prepared as described in example II.1, with a further step of loading of the gel with polymer microspheres encapsulating C5a of example I.1 (4 mg of microspheres per mL of gel) before distribution in the molds. After 30 min magnetic stirring at 500 rpm, the gel is distributed in the mold, frozen and lyophilized using the parameters previously described, to provide a loaded matrix under the form of a sponge.

[0174] In the case of biphasic porous solid matrix preparation (i.e. a material comprising a first phase of microspheres-loaded matrix and a second phase of unloaded matrix), the microspheres-loaded gel is prepared in the same way, with a concentration of microspheres of 30 mg of microspheres per mL of gel and addition of 0.5 mg of patent blue V dye was added. The microspheres-loaded gel is first distributed in the mold and then the unloaded gel is distributed to complete the filling of the mold. Freeze-drying then enables obtaining a biphasic sponge. In one example, 10 μl of loaded gel were distributed in the mold, followed by 60 μl of unloaded gel.

[0175] For comparative purposes, matrices loaded with free C5a were also prepared. Free C5a-loaded collagen sponges were prepared as described in example II.1, with a further step of loading of the gel with free C5a (4 μg of C5a per mL of gel) before distribution in the molds. After 30 min magnetic stirring at 500 rpm, to allow an even distribution of C5a in the collagen gel, the gel is distributed in the mold, frozen and lyophilized using the parameters previously described, to provide a free C5a-loaded collagen sponge.

II.3. Loading Rate of C5a in the Three-Dimensional Matrix

[0176] The determination of the loading rate of C5a in the biomaterial enable to know the amount of C5a present in the biomaterial.

[0177] For matrices loaded with free C5a, an indirect approach was used to determine the C5a loading rate: the C5a lost during the freeze drying and in the foils used for transportation was measured using ELISA assay. This amount of C5a lost during the manufacturing process was deduced from the amount of C5a engaged in the experiment (4 μg/mL of gel), enabling to calculate the loading rate.

[0178] Results. For the free C5a loaded scaffold obtained in example 11.2, it was measured that 1 μg of C5a was lost for 1 mL of collagen gel. The loading rate in C5a of the material is thus of 75% in weight of the total weight of C5a used in the biomaterial manufacturing process.

[0179] For the microspheres loaded matrices, an indirect approach was also used to determine the C5a loading rate: in this case, the microspheres are trapped in the collagenic matrix and cannot be lost during transportation or freeze drying. Using the encapsulation efficiency calculated for the microspheres, it is possible to calculate the amount of C5a loaded in the matrix using the concentration of microspheres loaded in the gel.

[0180] Results. For the microspheres loaded matrices, the encapsulation efficiency of the microspheres is of 75% (average), as determined in example 1.3. The microspheres are dispersed in the gel, and the C5a is not lost during this step. Therefore, the loading rate in C5a of the material is thus of 100% in weight of the total weight of C5a used in the biomaterial manufacturing processes and of 75% is the encapsulation step is also considered.

II.4. Biomaterial Morphology Characterization by Digital Microscopy

[0181] Porous solid matrices' morphology was analyzed using the Keyence VHX-5000 digital microscope.

[0182] The monophasic and biphasic microspheres-loaded matrices obtained in example 11.2 were analyzed and the morphology of the loaded matrices can be seen in FIG. 2. Especially, FIG. 2(a) shows a monophasic microspheres-loaded collagen sponge obtained as described in example 11.2; and a magnification (scale 100 μm) of the sponge structure. FIG. 2(b) shows a biphasic microspheres-loaded collagen sponge obtained as described in example 11.2. The dotted line shows the demarcation between the loaded phase at the bottom and the unloaded phase on the top. The triangles in the magnification (scale 100 μm) on the right point some of the encapsulated microspheres.

[0183] FIG. 2(c) is a further magnification (scale 20.0 μm) of encapsulated microspheres dispersed in a collagen sponge matrix.

II.5. Water Uptake Assay—Absorption Factor

[0184] The water uptake test aims at simulating the blood absorption and assessing the water uptake by the porous network. The test is inspired from Hui-Min Wang works on collagen/hyaluronic acid/gelatin sponges (Hui-Min Wang et al., PLOS ONE, 2013, 8(6), e56330).

[0185] More specifically, the dry material is weighted before being plunged into a beaker of fluid. Once the fluid spontaneously penetrates the matrix, bubbles of water are removed by pressing the material. This step is critical since air bubbles constitute an unused volume and can influence slightly the water intake. The material is then removed from the fluid with pin tweezers. At this step, the material must not be damaged or squeezed. Water is allowed to drain from the material, without squeezing. Once no more drops form, the wet material is weighted.

[0186] Absorption factor calculated using the formula below:

[00001]$\mathrm{Absorption}\mathrm{factor}=\frac{\mathrm{wet}\mathrm{material}\mathrm{weight}-\mathrm{dry}\mathrm{material}\mathrm{weight}}{\mathrm{dry}\mathrm{material}\mathrm{weight}}$

Results

[0187] The absorption factor allows the comparison of the water uptake of the different biomaterials. The porosity of the biomaterial influences the absorption factor calculated using the formula above. The biomaterial comprising matrices of collagen 1.5%, collagen 1%—chitosan 1% and collagen 1%—alginate 1% have similar absorption factor.

[0188] The absorption factor of unloaded sponges obtained as described in example II.1 was determined as explained above (table 2):

TABLE-US-00002 TABLE 2 Absorption factor of unloaded sponges. 3D matrix Absorption factor Collagen 1.5% 33 Collagen 1%-Chitosan 1% 35 Collagen 1%-Alginate 1% 33

[0189] These results show that all tested sponges have a similar absorption of water. This is important since the sponge needs to absorb the blood in order to allow the migration of the cells necessary for the regeneration.

II6. Degradation Assay

[0190] The degradation test is inspired from Davidenko et, al. works (Davidenko et al., Acta Biomaterialia, 2015, 25, pp. 131-142) and aims at determining the degradation of the material in water at 37° C.

[0191] For each tested material, 4 sponges of known masses are placed on a beaker in 20 mL of water at 37° C. in order to monitor their dissolution as a function of the incubation time by quantifying their mass loss. At each time t.sub.x of monitoring, the sample is taken from the dissolution medium, freeze-dried and weighted. The mass loss is calculated as follows:

[00002]$\%\mathrm{mass}\mathrm{loss}=\frac{\mathrm{dry}\mathrm{sponge}\mathrm{mass}\left(\mathrm{at}t0\right)-\mathrm{dry}\mathrm{sponge}\mathrm{mass}\left(\mathrm{at}\mathrm{tx}\right)}{\mathrm{dry}\mathrm{sponge}\mathrm{mass}\left(\mathrm{at}t0\right)}*100$

[0192] The total duration of the test is defined depending on the sample state in the beaker, according to whether it is still present or already totally degraded.

Results

[0193] Unloaded sponges obtained as described in example II.1 were tested. The collagen sponge has a slow degradation rate, and following the degradation assay, it was shown that after 14 days, only 33% of the scaffold is lost. The mixed sponges (collagen—chitosan or collagen—alginate) adopt the degradation rate of the collagen.

Part III. Biological Evaluation

III.1. Cells Preparation and Characterization

[0194] Collection of Molar Teeth. Human immature third molars freshly extracted for orthodontic reasons and carious teeth were obtained in compliance with French legislation (including informed consent and institutional review board approval of the protocol used).

[0195] Primary Pulp Cell Culture. Human pulp cells were prepared from the immature third molars at the two-thirds root formation stage by the explant outgrowth method. Briefly, pulp is extracted from the teeth. The pulp is then mechanically cut to form small pulp explants. Each explant is placed in a small volume of DMEM in a petri dish. After 24 h, the petri dish is filled with medium. Cells outgrowing of the explants are collected constituting a pulp cell population.

[0196] Magnetic Cell Sorting. Pulp progenitor cells were directly sorted from primary pulp cell cultures at passages 1 to 5 with mouse anti-human STRO-1 IgM with immune magnetic beads according to the manufacturer's protocol (Dynal, Oslo, Norway). Briefly, magnetic beads are coated with a mouse anti human STRO-1 IgM. Cells are then added on the prepared beads. Once the STRO-1 antigen is fixed to its specific antibody, the cell populations are separated using the beads. The non-fixed population, or negative fraction, is a pulp fibroblasts population. The fixed population, or positive fraction, is a pulp stem cell population. On this latter population, beads need to be washed once the cells adhere to the culture flask.

[0197] Pulp Cell Characterization. This test is used to confirm what cells are present after magnetic cell sorting. STRO-1 sorted and non-sorted pulp cells were grown in 8-well glass culture chambers up to 70% confluence. In these 2 cultures, cells were rinsed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde during 15 min at 4° C. for 1 h. While non-sorted pulp cells were incubated for 1 h with primary antibody against fibroblast surface protein (FSP; 2 μg/mL), STRO-1 sorted cells were incubated with primary antibodies against STRO-1 (5 μg/mL) and CD44 (2.5 μg/mL), CD90 (2.5 μg/mL), CD105 (2.5 μg/mL), CD146 (2.5 μg/mL), or CD166 (2.5 μg/mL). Controls were incubated with the respective primary antibody isotypes. After washing, cells were incubated for 45 min with their respective secondary antibody, Alexa Fluor 488 or Alexa Fluor 594 (2 μg/mL), and with DAPI (1 μg/mL) for fluorescence microscopy detection.

III.2. C5a Concentration Determined by ELISA Assay

[0198] C5a concentration is determined by an Enzyme-Linked ImmunoSorbent Assay (ELISA) according to the manufacturer's instructions (R&D Systems, Lille, France). Briefly, a 96 wells plate was coated with a primary antibody against C5a protein over-night at 4° C. After 1 h in a blocking solution, the samples are placed in the wells and incubated for 2 h at room temperature. A biotinylated secondary antibody against C5a is then added for 2 h at room temperature. Streptavidin coupled with HRP enzyme will then be added to the wells. The revelation occurs by the addition of TMB that is transformed by the HRP enzyme into a blue colored substrate. Plate are read at 650 nm wavelength.

III.3. Toxicity Assay

[0199] Toxicity of the biomaterial was assayed in vitro using dental pulp fibroblasts (STRO-1 negative fraction of dental pulp cells after magnetic sorting).

[0200] Cells are seeded in 96 wells plate and culture to subconfluency. Media containing biomaterials' eluates, were prepared by incubating samples in DMEM for 24 h. Fibroblasts were incubated with conditioned media for 24 and 72 h (indirect method).

[0201] The biomaterial has also been placed directly in contact with the cells (direct method). At each time point a MTT test was performed. MTT is placed on the cells, at a concentration of 1 mg/mL and incubated for 2 h, allowing the formation of formazan crystal. After 2 h incubation, the crystals are dissolved using DMSO. The plate is read at 550 nm wavelength.

[0202] Results. The cell viability with the different materials of the invention has been evaluated. The control was the DMEM medium alone and gave a cell viability was 100%. The toxicity of the encapsulating microspheres was evaluated with the MTT test.

[0203] The results showed that the encapsulating microspheres (whether loaded with C5a or unloaded) were not toxic to the cells.

[0204] The toxicity of the unloaded collagenic matrix and of monophasic or biphasic C5a—encapsulating-microspheres-loaded collagen sponges was also evaluated directly and indirectly with the MTT test, as presented in the graphs on FIGS. 7a and 7b, respectively. The results showed that there is no toxicity of the loaded or unloaded collagen matrices to the dental pulp cells.

[0205] Chitosan, collagen—chitosan and collagen—alginate matrices, loaded and unloaded, have also been tested via indirect toxicity assay. The results showed no toxicity of these matrices.

III.4. Migration Assay

[0206] To assess the activity of the C5a released by the biomaterial, a migration assay was performed. Indeed, the C5a protein is known for its role in mobilizing immune and stem cells and this function is the most important one for the regeneration of the dentin-pulp complex.

[0207] Migration was assayed in 24-well plates equipped with transwell inserts with an 8-μm pore-size membrane. STRO-1 sorted cells (10.sup.4 cells/well) were resuspended in serum-free MEM and seeded in the upper chamber. The lower chambers were loaded with conditioned media. After 24 h, non-migrating STRO-1 sorted cells on the upper side of the insert membrane were wiped off using a cotton bud, and STRO-1 sorted cells on the lower surface of the same membrane were fixed with ethanol and stained with hematoxylin. The number of migrating STRO-1 sorted cells to the lower surface of the membrane was counted in 5 random fields under a light microscope (×200).

[0208] Results. In the control condition of this experiment (DMEM medium), only few dental pulp stem cells migrated. The presence of C5a released from the biomaterial of the invention significantly increased the migration of dental pulp stem cells until day 7 as reported in FIG. 8.

III.5. C5a Liberation Assay

[0209] An encapsulated C5a-loaded matrix is placed in a fixed volume of DMEM at 37° C., 5% CO.sub.2. At 1, 7, 14, 21 and 31 days, the medium is removed and stored at −20° C. and fresh medium is added. At each time point a dosage of liberated C5a is performed using ELISA assay (described in section 111.2).

[0210] Results. As showed on FIG. 9, the incorporation of C5a-encapsulating-microspheres in collagen matrix allows a slow and controlled release of C5a in the conditions of the test. On the first day, an important burst release of C5a is observed. During this burst release, 42% of the C5a present in the biomaterial is release. After this initial burst release, a slow and controlled release of C5a was obtained until 31 days. Results are expressed in percentage of C5a present in the scaffold.

[0211] Comparative example—free C5a-loaded matrix. A collagen sponge loaded with free C5a (example 11.2) was tested in the same conditions of liberation assay. It was observed that 90% of the C5a is liberated at 1 day in a high burst release (FIG. 10). After this initial burst release, no more C5a is released from the matrix. In the conditions of the assay, the liberated C5a remained stable and active for 7 days. Nevertheless, it is known that C5a has a short half-life in vivo, namely of less than 1 hour.