	Example
Buffering/Thermo-gelling	contents
agents	(% w/v)	Remarks

Glycero-phosphate salts	4-10
Glucose-phosphate salts	6-12
Fructose-phosphate salts	1-6
Histidine	2-10	Histidine and glucosamine used as co-
Glucosamine	2-10	buffering agent with glycero-phosphate.
BIS-TRIS	1-8	Used alone or mixed with glycero-
		phosphate.
MES (sulfonate) salt	1-4	Used alone or mixed with glycero-
		phosphate.
HEPES (sulfonate) salt	1-4	Used alone or mixed with glycero-
		phosphate.
TES (sulfonate) salt	1-4	Used alone or mixed whh glycero-
		phosphate.

Glycerophosphate (GP) salts act herein as buffering/thermo-gelling agents for the chitosan solution. Other buffering/thermo-gelling phosphate sources may be used, typically organic monobasic phosphate salts, such as glucose-phosphate or fructose-phosphate salts. Other buffering agents may be also associated with glycerophosphate salts so as to enhance the buffering/thermo-gelling action such as amino acids or organic sulfonate salts. Table 1 summarizes the potent composition of liquid components. Histidine was typically admixed with GP in the chitosan solution (Ex: 1.5% w/v chitosan, 4.0% w/v GP+4.0% w/v histidine). BIS-TRIS may be used alone as a buffering/ thermo-gelling agent (Ex: 1.5% w/v chitosan+2.0% w/v BIS-TRIS). HEPES, TES or MES sulfonate agent may be used alone as a buffering/thermo-gelling agent (Ex: 1.5% w/v chitosan+2.0% w/v HEPES, TES or MES).[0195]

a) Addition of a Second Water-Soluble Polymer in the Liquid Component[0196]

A second water-soluble polymer may be dissolved in the chitosan-GP aqueous solution. Table 2 gives the composition of liquid component consisting in chitosan-GP plus a water-soluble polymer. Glycerophosphate may be added prior to the dissolution of the second polymer, or after the dissolution of the second polymer. Thermosensitive polymers such as the methyl cellulose, hydroxypropyl methylcellulose or Pluronic® were found to be the more sensitive to the concentration in glycerophosphate salts. Those salts were found to affect the gelling or precipitating temperature of the polymer, thus leading to a precipitation of the chitosan/GP/polymer(2) system (FIG. 6).[0197]

All polymers (2) were dissolved in a prepared chitosan-GP solution, except for collagen, and more generally other polycationics, that are dissolved in combination with the chitosan.[0198]

TABLE 2


Liquid component compositions having an admixed second water-
soluble polymer.

	Polymer	Chitosan	GP
	content	content	content
Polymer (2)	(w/v)	(w/v)	(w/v)	Remarks

Hydroxyethyl	1.0%	2.0%	8.0%	Form gels.
Cellulose
Hydroxypropyl	0.55%	1.0%	4.5%	Form gels.
Methyl Cellulose
Polyethylene glycol	1.0%	2.0%	8.0%	Form gels.
Methyl Cellulose	1.0-2.0%	1.0-2.0%	4.0-6.0%	Form gels. Precipitation
				may occur with higher GP
				contents.
Pluronic ®	1.0%	2.0%	2.0%	Form gels. Precipitation
				may occur with higher GP
				contents.
Collagen (type I)	1.0%	1.0%	8.0%	Form gels.

b) Addition of Water-Soluble Ingredients of Interest[0199]

Some organic molecules that are soluble or miscible with water may be added to the chitosan-based liquid component to give modified or improved physico-chemical characteristics, mechanical or handling performances, or biological properties. This includes without limitation polyols, sugars, amino-acids, saccharides, and polysaccharides.[0200]

Polyols & Sugars Of particular interest may be the polyols such as polyols having diol hydrocarbon moieties which may be useful for the processing or the performances of the liquid component (see Table 3). Among those polyols, sugar-polyols, saccharide-polyols and glycols are preferred: glycerol, mannitol, sorbitol and ethylene glycol compounds such as the triethylene glycol, tetraethylene glycol were found to be good representative examples, being attractive and bringing modifications or improvements to the liquid component or resulting thermo-formed gel. Sugars such as fructose, glucose, etc may be used similarly.[0201]

TABLE 3


Liquid component compositions having added water-soluble non-
polymeric ingredient (Polyols)

	Ingredient	Chitosan	GP
	content	content	content
Polymer (2)	(w/v)	(w/v)	(w/v)	Remarks

Glycerol	0.1-1.0	2.0%	8.0%	Form gels. Changed rheological
				parameters of gels. Stabilize
				chitosan sol viscosity.
Sorbitol	0.1-1.0	1.0%	4.0%	Form gels. Changed rheological
(Mannitol)				parameters of nonsterile gels.
				Stabilize chitosan sol viscosity.
Ethylene	0.1-1.0	2.0%	8.0%	Form gels. Changed rheological
glycol				parameters of nonsterile gels.
				Stabilize chitosan sol viscosity.
Tri- (Tetra-)	0.1-1.0	2.0%	8.0%	Form gels. Changed rheological
Ethylene				parameters of gels. Stabilize
glycol				chitosan sol viscosity.

Other water-soluble (bio)chemical ingredients may be of interest to be added to the chitosan-GP liquid component. However, such ingredients must not disturb the chitosan-GP composition (ingredient) and its thermo-gelling property. Glycoaminoglycans may be added to the chitosan-GP solution to a certain extent. It must be taken care of not inducing precipitation of chitosan. In Table 4, heparin was used as the GAGs to be added. Chitosan solutions were 4.0% w/v chitosan (deacetylation 95%) in 0.19M HCl. GP solutions were 54,6% w/v in water. Heparin in water solutions were at 1 mg/mL (A), 0.1 mg/mL (B), 10 μg/mL (C) and 1 μg/mL (D).[0202]

TABLE 4


Liquid component compositions having added water-soluble non-
polymeric ingredient (Heparin)

#	Composition	pH	Remarks

1.	500 μL chitosan + 150 μL GP	7.04	Gels.
	250 μL water + 100 μL Heparin (B)
2.	500 μL chitosan + 250 μL water	7.01	Gels.
	150 μL GP + 100 μL Heparin (C)
3.	500 μL chitosan + 250 μL water	6.87	Gels.
	150 μL GP + 100 μL Heparin (B)
4.	500 μL chitosan + 250 μL water	6.97	Reduced precipitation.
	150 μL GP + 100 μL Heparin (A)		Gels.

c) Addition of a Second Water-Soluble Phosphate Source to the Liquid Component[0203]

An acidic chitosan aqueous solution (2.0-4.0 w/v) was made with a chitosan deacetylated at 83-85%, filtered and dialyzed, and was prepared from a 0.1M HCl solution.[0204]

A chitosan-GP aqueous solution was prepared from a pre-cooled (4° C.) chitosan in HCl solution and a 54-55% (w/v) disodium glycerophosphate (GP) in distilled water solution. The pH of the resulting liquid chitosan-GP solution was measured at 21° C. A phosphate solution (1) was prepared with 0.144 g/l KH[0205]₂PO₄.7H₂O potassium dihydrogen phosphate hydrated) and 0.795 g/l Na₂HPO₄disodium hydrogen phosphate) and had a pH of 7.4 at 20° C. Amounts of the chitosan-GP solution and phosphate solution (1) were admixed homogeneously, then the pH of the resulting solutions was measured. The solutions were finally disposed at 37° C. for gelation, all signs of precipitation being noted. In Table 5, all chitosan-GP+phosphate solution (1) (80:20 to 50:50, vol) showed no signs of precipitation, and gelled within 30 minutes at 37° C.

TABLE 5


Composition of liquid components supplemented with a second
source of water-soluble phosphate.

				Gelling
		Chitosan:GP	[Phosphate]	time
Composition	pH	(% w/v)	mol/l	(Initial)	Precipitation

10 ml chitosan-GP	7.0	2.0:8.2	0	30	No
				minutes
8 ml chitosan-GP +	6.9	1.6:6.6	0.0014	30	No
2 ml phosphate (1)				minutes
7 ml chitosan-GP +	6.9	1.4:5.7	0.0021	30	No
3 ml phosphate (1)				minutes
5 ml chitosan-GP +	6.9	1.0:4.1	0.0035	30	No
5 ml phosphate (1)				minutes
8 ml chitosan-GP +	6.9	1.6:6.6	0.04	Slow	No
2 ml phosphate (2)				gelation
1:1
7 ml chitosan-GP +	6.9	1.4:5.7	0.06	Slow	No
3 ml phosphate (2)				gelation
1:1
5 ml chitosan-GP +	6.95	1.0:4.1	0.001	30-40	No
5 ml phosphate (2)				minutes	precipitation
1:100

All presented concentrations (%, mol/l) are final.[0206]

A concentrated phosphate solution (2) was prepared from 283.92 g/l of Na[0207]₂HPO₄(0.2 mol/l disodium hydrogen phosphate) and 239.96 g/l of NaH₂PO₄(0.2 mol/l sodium dihydrogen phosphate) and had a pH of 7.4 at 37° C. This phosphate solution was used with dilutions at 1:1, 1:10, 1:100 and 1:1000. Equal volumes (50:50) of the diluted to concentrated phosphate solution (2) and chitosan-GP solution were mixed homogeneously. The pH of the resulting solutions was measured, and the solutions disposed at 37° C. for gelation, all signs of precipitation being noted. All chitosan-GP/phosphate (2) gelled with various rates at 37° C.

A more concentrated phosphate solution (3) was prepared: 0.5 mol/l NaH[0208]₂PO₄(600 g/l) and 0.5 mol/l Na₂HPO₄(709.8 g/l). Volumes of the concentrated phosphate solution (3) was added to chitosan-GP solutions, and mixed homogeneously. In Table 6, the pH of the resulting solutions was measured, and the solutions disposed at 37° C. for gelation, all signs of precipitation being noted. Chitosan-GP is fully compatible with 5 mM PBS solution at pH 7.2-7.4. Compatibility depends upon the phosphate content (Tables 1a-1b): the addition of highly concentrated phosphate solutions (especially dibasic phosphates) renders the chitosan-GP system more turbid and prone to precipitation or heterogenous gelation.

TABLE 6


Composition of liquid components supplemented with a second
source of water-soluble phosphate.

10 ml chitosan-GP	7.0	4.0:8.2	0	30	No
				minutes
9 ml chitosan-GP +	6.7	3.6:7.4	0.05		More turbid
1 ml phosphate (3)
8 ml chitosan-GP +	6.7	3.2:6.6	0.1		Highly turbid;
2 ml phosphate (3)					Gels
					hetero-
					geneously

d) Addition of a Water-Soluble Carbonate Source to the Liquid Component[0209]

The chitosan-GP solutions were prepared as in Example 1c.[0210]

A carbonate solution was prepared from a 0.2 mol/l solution of monosodium carbonate, having a pH of 8.16 at 21° C. Equal volumes (50:50) of the diluted (1/10) to concentrated carbonate solution and chitosan-GP solution were mixed homogeneously. A carbonate (0.1 mol/l)+phosphate (0.1 mol/l) solution was also used. The pH was measured, and the solutions were disposed at 37° C. for gelation, all signs of precipitation being noted (see Table 7). Chitosan-GP systems are fully compatible with carbonate buffer such as a 5 mM phosphate/carbonate buffer at a pH of 8.8. But, this compatibility will decline for too high carbonate contents.[0211]

TABLE 7


Composition of liquid components supplemented with a source of
water-soluble carbonate.

		[Carbonate]	Gelling time
Composition	pH	mol/l	(initial)	Precipitation/Remarks

10 ml chitosan-GP	7.0	0	30 minutes	No
8 ml chitosan-GP +	7.0	0.04	30 minutes	No
2ml carbonate
6 ml chitosan-GP +	7.1	0.08	30 minutes	No. Some sparse
4 ml carbonate				complexes may occur by
				the surface.
5 ml chitosan-GP +	7.1	0.10	30 minutes	No.Fibrous complexes
5 ml carbonate				occur sparsely at the
				upper level.
5 ml chitosan-GP +	6.95	0.01	30 minutes	No
5 ml carbonate 1:10

In Example 1d, liquid chitosan-GP formulations supplemented with water-soluble phosphates and/or carbonates present a reduced shell-life and stability, even at low temperatures (4° C.). This is dose-dependent, the more concentrated is the content of water-soluble phosphate and/or carbonate in the chitosan-GP formulation, the less stable is the resulting solution.[0212]

e) Typical Preparation of Sterile Liquid Component[0213]

Sterilization Method of Liquid Components[0214]

Sterilization of liquid component can be performed during the preparation and processing of the chitosan-GP solutions. The chitosan-GP systems can not be sterilized by energizing methods, due to unexpected and undesirable thermal gelling. Chitosan solutions (no GP) and GP solutions (no chitosan) are to be sterilized separately. GP aqueous solutions have no viscosity and are sterilized by filtration in all cases, without any noticeable adverse effects. Chitosan materials (solid) or chitosan solutions (acidic aqueous medium) must be sterilized while avoiding the occurrence of significant degradative effects on both chitosan polymer and chitosan-GP systems.[0215]

The preferred methods of sterilization of chitosan comprises autoclaving chitosan powder or solution, and gamma-irrdiation of nonliquid chitosan material (including frozen solution)(see Tablee 8).[0216]

TABLE 8


Effects of sterilizing on Chitosan-GP systems (no additive)

	Effects on chitosan	Effect of thermo-gelling Chitosan
Chitosan sterilization	biopolymer	GP systems.

Autoclaving of chitosan	Controlled	Gels (slightly decreased gelling rate).
solutions in acidic	modification;
media.
Autoclaving of chitosan	Controlied	Modify gel properties.
suspension in water.	modification;
Irradiation of chitosan	Controlled	Gels (slightly decreased gelling rate).
materials (4° C.).	modification;
Irradiation of chitosan	Stronger	Gels (affect the gelling rate).
materials (20° C.).	modification;
Irradiation of chitosan	No to nonsignificant	Gels (no changes).
solutions (−80° C.).	modification;

EXAMPLE IIIncorporation of Bone Healing and/or Inductive Agents into the Liquid Component

a) Incorporation and Compatibility of Whole Blood and Bone Marrow in the Liquid of Mineral-Polymer Composites.[0217]

Blood component or whole blood from patient to be treated is often used to mix with bone filling materials (Ex: calcium phosphate or carbonate granules mixed with autologous blood).[0218]

A fresh blood sample was collected from auricular arteries and from subchondral defects of rabbits. Blood was admixed with the liquid component of the mineral-polymer composites (chitosan-GP). Bone marrow was collected in rabbit from tibia and femur, and used fresh. Upon complete and rapid homogenization, the gelling of this blood containing liquid component was monitored at 37° C. by rheological analyses (FIG. 17). The gel has an elastic modulus (G′) about 800-1000 Pa after 9 hrs at 37° C.[0219]

This demonstrated that the thermo-gelling of the liquid component is not affected by blood or marrow and enables to incorporate blood and its components, and bone marrow, within the thermo-gelling hybrid formulation.[0220]

b) Incorporation and Release of Proteins (albumin) in the Liquid and/or Solid Component of Mineral-Polymer Composites[0221]

Bovine serum albumin (BSA) was the protein used to model the protein incorporation and release from hybrid formulations, either by the liquid and/or solid component.[0222]

Incorporation of BSA in the liquid component was performed by incorporating directly the BSA to the liquid component formulation. Using a 2-syringe procedure, a chitosan solution and a glycerophosphate solution are admixed homogeneously, then the chitosan-GP solution is similarly admixed with a BSA in PBS (×1) solution. The final chitosan content in these gels were 1.44, 1.8 and 2.16% w/v. The final BSA content in these gels was 250 μg/ml. Gels are molded in petri dishes (constant surface area) (37° C., 1 h), then covered with 10 ml of PBS (×1). At the desired time, a 1000 μl solution speciment is collected for titrating the BSA content, and replaced by 1000 μl of fresh PBS solution. The BSA content is determined spectrophotometrically by using a Coomassie Plus Protein assay (Pierce). The BSA released from the chitosan-GP gels is determined at 1, 3, 6, 24, 48h, 4, 6 days, etc. The BSA release from chitosan-GP gels is rapid, and by 24 hrs, at least 50% of the BSA is totally released from the gels. Incorporation of BSA in the solid component was performed by contacting Biphasic Calcium Phosphate granules (2 sizes: <150 μm, 150-250 μm) with Bovine Serum Albumin (BSA) solutions. The BCPIBSA weight ratio was 50 mg of BCP for 500, 1000 or 2500 μg of BSA. Initial BSA solutions were prepared in Phosphate Buffered Solution (PBS×1) at BSA concentrations about 1, 2 or 5 mg/ml. A 500 μl of BSA solution was poured onto 50 mg of the BCP granules. Adsorptions were performed in centrifugation filters (Nanosep MF Microconcentrator) with a filter size of 0.45 μm. Adsorption was done at 37° C. for 24 hrs. Following adsorption, BCP granules were collected by ultracentrifugation for 10 minutes at 13000 rpm. The remaining BSA solution is collected. BCP granules were disposed in cell culture inserts (0.4 μm filter) of a 24-well plate, and covered with 1.5 ml of PBS (×1). At the desired time, a 500 μl solution speciment is collected for titrating the BSA content, and replaced by 500 μl of fresh PBS solution. The BSA content is determined spectrophotometrically by using a Coomassie Plus Protein assay (Pierce). The BSA released from the BCP granules is determined at 1, 3, 6, 24, 48 h, 4, 6 days, etc. The BSA release from BCP granules is progressive.[0223]

The BSA releases are equivalent for both sizes, and correspond to a total release about 40 micrograms after 700 hours (20 micrograms after 200 hours).[0224]

c) Incorporation and Release of Bone Proteins (BP) in the Liquid Component of Mineral-Polymer Composite.[0225]

The osteogenic agent was a bone protein (BP), in fact a pool of bone morphogeneic proteins that were solubilized in an acidic aqueous environment. The chitosan-GP aqueous solution was prepared as described in Example 1. A BP solution was made by dissolving the proteins in a 0.01M HCl (6.0 mg/ml). Three volumes of the BP solution were sampled, and carefully admixed to chitosan-GP solutions to give clear homogeneous chitosan-GP+BP compositions having distinct contents in proteins. The volume of all prepared compositions was completed with sterile water to 12.5 ml as required. All operations were performed at 4° C. under sterile conditions. The final BP content of the three compositions was 33, 100 and 330 μg/ml. Gel formation of all BP containing compositions was reached within few minutes at 37° C. The incorporation of BPs did not interfer with the gel formation. Gelled chitosan-GP/BP samples were tested in vitro by activity/release assays, and evaluated in vivo by a rodent subcutaneous assay. A rodent subcutaneous assay was used: Long-Evans rats (20) were anesthetized with sodium pentobarbital (400 μg). Gel-containing samples (200 μl) were injected sub-cutaneously in the absence of an incision. The gel formed a sphere structure. Each group contained 5 animals. The gel compositions were compared using variable BP doses (0, 10, 30 μg). The in vivo subcutaneous assay demonstrates that the BP is active in the chitosan-GP formulation, thus leading to induction of bone and cartilage neo-tissues.[0226]

Bone healing factors that are useful for fracture healing were selected as representative bone healing agents to be incorporated into the liquid component.[0227]

c) Incorporation and Release of Fibroblast Growth Factor (FGF) in the Liquid Component of Mineral-Polymer Composite[0228]

Fibroblast growth factor (basic, bFGF) was used here as a Growth Factor member of the TGF-□ GF family to be incorporated in the liquid component and hybrid materials bFGF and bFGF with Heparin were added to the chitosan-GP solutions, bFGF and bFGF/Heparin alone being used as controls. Heparin in the chitosan-GP solution was used at 10 μg/mL while bFGF was at 1 μg/mL. Chitosan-GP (1 mL) solutions were disposed in dishes, with PBS and 50 μg/mL bovine serum albumin, at 37°[0229]C. A 1 mL of the medium was collected at 0, 6, 24, 48, and 72 hours. The bFGF is quantified by ELISA (Quantikine FGF basic immunoassay). The bFGF in Chitosan-GP gels (no heparin) is mainly released by 24 hrs. When incubated with heparin, bFGF (+heparin) remained within chitosan-GP gels, and the release is clearly slowed down.

Additionally to the liquid component, bFGF and other GFs can be immobilized by the way of the solid component, or incorporated directly in the hybrid compositions.[0230]

d) Incorporation of Human Demineralized Bone Powder (HDBP) to the Mineral-Polymer Composite.[0231]

Demineralized bone powder (DBP) is a demineralized bone matrix (DBM) material transformed into solid powder, and is composed of the non-mineral non-living organic bone components (coliagen, bone proteins . . . ). Demineralized bone matrix is osteoinductive. A cortical demineralized bone powder (CDBP) (Community Tissue Services, OH & Impladent, N.Y., USA) from human allografts was selected for typical examples. It is an allogenic cortical demineralized bone matrix that has been crushed in particles about 0.3-0.5 mm in size. CDBP was sterilized by irradiation and freeze-dried. As for allografts, CDBP was tested negative for HBsAG, anti-HBc, anti-HCV, STS, HIV ½, HTLV-1 and HIV-1 antigen. A chitosan-GP formulation basis was obtained first by preparing a 2.3% w/v chitosan (84% deacetylation) in 0.10M HCl solution, and then by admixing the desired amount of a 55% w/v disodium glycerophosphate in distilled water solution. Final concentrations in the clear liquid chitosan-GP formulation were around 1.8-2.0% w/v chitosan and 8.0% w/v glycerophosphate.[0232]

CDBP was incorporated in a solid state to the chitosan-GP solution, thus resulting in a CDBP in chitosan-GP suspension. Loading of chitosan-GP with CDBP (Impladent Ltd., NY, USA) was determined by initial solid to liquid weight or volume ratio. The chitosan-GP/CDBP compositions were gelled at 37° C., entrapping homogeneously the CDBP.[0233]

EXAMPLE IIISelf-Forming Hybrid Composition and Material

Different calcium phosphate materials were used to form self-forming (gelling) hybrid compositions and materials (FIG. 1). All selected calcium phosphates were recognized (clinically) as being osteoconductive. Table 9 gives the corresponding compositions and S/L ratios.[0234]

TABLE 9


Corresponding Compositions And S/L Ratios

Liquid	Chitosan-	Chitosan-	chitosan-	chitosan-	Chitosan-	chitosan-
Component	GP	GP	GP	GP	GP	GP

Solid	Durapatite	β-TCP	HA	Apafill-G	Coralina	BCP
Component	(<125 pm)		Resorb	(100-400	(400-800
	(212-250		(300-400	μm)	μm)
	μm)		μm)
Composition	HA	β-TCP	Synthetic	HA	Coralline	HAP/β-TCP
			HA		HA
S/L ratio	0:10-7:10	0:10-7:10	0:10-3:10	0:10-3:10	0:10-3:10	0:10-5:10
(load)

a) Composition with Durapatite hydroxyapatite[0235]

A chitosan-GP formulation basis was obtained first by preparing a 2.3% w/v chitosan (84% deacetylation) in 0.1 M HCl solution, and then by admixing the desired amount of a 55% w/v disodium glycerophosphate in distilled water solution. Final concentrations in the clear liquid chitosan-GP formulation were 2.0% w/v chitosan and 8.0% w/v glycerophosphate. Loading in solid Durapatite particles was determined by solid to liquid weight ratio, e.g. 1:10, 3:10 & 5:10 S/L loading ratio. Typical experiments were conducted with a 3:10 Ca-P loading (Table 9).[0236]

Durapatite particles were selected by sieving in 0-125 μm diameter, 212-250 μm diameter and >250 μm diameter size groups. Homogeneous mixing of Ca—P ceramics in chitosan-GP was achieved by manual kneading and magnetic stirring during 2-3 hours. The resulting slurries generally show greater extrudability or flowing properties which is observed by the viscosimetric analysis. It shows that higher shear rates result in lowering the viscosity (<10,000 mPa.s), the higher viscosity being observed at low shear rates (up to 60,000 mPa.s).[0237]

All hybrid compositions rheologically demonstrate a self-gelling behaviour that is typical of the liquid component property. In nearly all cases, the Durapatite loading induces more elevated viscoelastic parameters such as typically the elastic and viscous moduli (G′ and G″). Durapatite (212-250 μm) incorporation seems to be the most efficient in improving G′ and G″. To some extent, the G′ and G″ increasing rates seems to be lineraly correlated with the S/L ratio. Mechanical performances expressed by compression modulus (transient and equilibrium) are not markedly enhanced by the Durapatite incorporation although {fraction (3/10)} to {fraction (5/10)} loading seemed to provide hybrid gels with higher compressive strengths (FIGS. 2, 4,[0238]5,7,8, and12).

b) Hybrid Materials with Tricalcium Phosphate (TCP)[0239]

The selected chitosan-GP solution was the same as previously described in Example 3a above. Loading in tricalcium phosphate (TCP) particles as determined by solid to liquid weight ratio varied, (1:10, 3:10 & 5:10 S/L). Typical experiments were conducted with a 3:10 Ca—P loading (Table 9). All chitosan-GP/TCP compositions were monitored by viscosimetric and rheologic methods, and formed solid hybrid gels at 37° C. (FIGS. 3 and 9).[0240]

c) Hybrid Materials with Synthetic Nonceramic Hydroxyapatite (HA Resorb Osteogen)[0241]

The selected chitosan-GP solution was the same as previously described in Example 3a above. Loading in synthetic nonceramic hydroxyapatite HA resorb Osteogen (Impladent Ltd., NY, USA) particles was 1:10 & 3:10 S/L (Table 9). Osteogen hydroxapatite is a dental hydroxyapatite material, about 300-400 μm in size. All chitosan-GP/Osteogen compositions were monitored by viscosimetric and rheologic methods, and formed solid hybrid gels at 37° C. (FIGS. 10 and 13).[0242]

d) Hybrid Materials with Hydroxyapatite Ceramic Dense Granules (Apafill-G)[0243]

The selected chitosan-GP solution was the same as previously described in Example 3a above. Loading in dense Apafill-G hydroxyapatite (BIOMAT, Habana, Cuba) particles was 3:10 S/L (Table 9). Apafill-G hydroxapatite is an orthopaedic and dental hydroxyapatite material, about 100-400 μm in size. All chitosan-GP/Apafill compositions were monitored by viscosimetric and rheologic methods, and formed solid hybrid gels at 37° C.[0244]

e) Hybrid Materials with Coralline Hydroxyapatite Granules (Coralina)[0245]

The selected chitosan-GP solution was the same as previously described in Example 3a above. Loading in Coralina hydroxyapatite (Laboratorio de Biomaterales, Cuba) particles was 3:10 S/L (Table 9). Corallina hydroxapatite is produced from marine coral materials, and is used as an orthopaedic and dental nonresorbable hydroxyapatite material, about 400-2400 μm in size. All chitosan-GP/Corallina compositions were monitored by viscosimetric and rheologic methods, and formed solid hybrid gels at 37° C.[0246]

f) Hybrid Materials with Biphasic Calcium Phosphate (BCP)[0247]

The selected chitosan-GP solution was the same as previously described in Example 3a above. Particles of HAP/β-TCP (35-40:65-60) biphasic calcium phosphate (Clarkson Chromatographic Products Inc., PA, USA; and Teknimed, Vic-en-Bigorre, FRANCE) were loaded at a solid to liquid weight ratio from 1:10 to 5:10 (Table 9). BCP is about 40:60 (wt.) powder mixture of a hydroxyapatite and a β-tricalcium phosphate. All chitosan-GP/BCP compositions were monitored by viscosimetric and rheologic methods, and formed solid hybrid gels at 37° C. All materials from examples 2a to 2f gelled into solid hybrid gels or showed endothermic signs of gelation for loadings ranging typically from {fraction (0/10)} to {fraction (10/10)}, and preferably from {fraction (0/10)} to {fraction (7/10)} (0 to 1 g/mL). Ideal Ca-P loading (injectability, gel uniformity . . . ) seemed generally to be around {fraction (3/10)} to {fraction (5/10)}, however changed slightly with the Ca—P chemical nature and granulometry (size, density) (FIGS. 11, 14,[0248]15, and16).

g) Osteoinductive Bone Hybrid Compositions and Bio-Materials[0249]

A self-forming bone hybrid composition and bio-material was composed of chitosan-GP, hydroxyapatite and CDBP. The chitosan-GP was prepared to 1.5% (w/v) of chitosan and 8.2% (w/v) of GP. A 1.0 ml of human CDBP powder was admixed with a 10 ml of chitosan-GP solution, then loaded with 3.0 grams of Durapatite particles (200-250 μm).[0250]

The flowable resulting suspension was carefully mixed and homogenized (manually and mechanically). Gel formation was reached and controlled at 37° C.[0251]

EXAMPLE IV

In vivo Study of Bone Compositions, Bio-Materials and Related Components[0252]

a) Subcutaneous Injections of Pure Liquid Components[0253]

Liquid phases consisting in aqueous thermo-gelling solutions of chitosan (2.0% w/v) and glycerophosphate (8.2% w/v) were prepared sterile, by autoclaving chitosan solutions and filtering glycerophosphate solutions. Adult albino Sprague Dawley rats were anesthezied for 40 minutes by intra-peritoneal injections of 2.7 ml/kg of a Hypnorm® and Midazolal (Versed®) mixture, made by mixing 1 ml of Hypnorm® and 1 ml of 5 mg/ml Midazolal with 2 ml of sterile water. Sub-cutaneous injections about 0.2 ml/injections were performed on each side of the spine mid-line. The animals were returned to normal activities. Sacrifices by carbon dioxide overdose were done at 3, 7, 14, 21, 30, 60, 90 and 120 days. Implants were collected, macroscopically examined and analyzed by histology (H&E staining on thin slides). Gross observations showed that chitosan-GP systems formed in situ cohesive matrices having spherical to ovoid shapes. Implants did not travel subcutaneously, at any times. Histological analyses showed a reduced acute inflammation, and a moderate chronic inflammation that was associated to the implant erosion. Chitosan-GP systems were found to give cohesive, shaped, and resident implants with a moderate associated inflammatory reactions. Erosion in situ of the chitosan-GP implants was progressive and peripheric. The implants and materials resided in situ for 120 days.[0254]

b) Intramuscular Injections of Pure Liquid Components[0255]

Intra-muscular injections in rats were performed with the materials and protocols that were described in Example 4a, except that injections were intramuscularly in the muscular tissues of lower limbs. Gross observations showed that chitosan-GP systems formed in situ cohesive matrices having spherical to ovoid shapes. Implants did not travel subcutaneously, at any times. Histological analyses showed a reduced acute inflammation, and a moderate chronic inflammation that was associated to the implant erosion. Chitosan-GP systems were found to give cohesive, shaped, and resident implants with a moderate associated inflammatory reactions. Erosion in situ of the chitosan-GP implants was progressive and peripheric. The implants and materials resided in situ for 80 days.[0256]

c) Osseous Injections of Pure Liquid Components[0257]

Osseous injections in a rat trancondylar model were performed with the materials and protocols described in Example 4a, except that thermo-gelling materials were disposed in transcondylar cylindrical defects.[0258]

Gross observations showed that chitosan-GP systems formed in situ cohesive matrices. Histological analyses showed a reduced acute inflammation, and a moderate chronic inflammation that was associated to the implant erosion.[0259]

d) Subcutaneous Injections of Hybrid Materials with HA Resorb[0260]

Subcutaneous injections in rats were performed with the materials and protocols that were described in Example 4a. The hybrid compositions were aqueous, and containing 2.0% w/v of chitosan, 8.2% w/v of glycerophosphate, and 30% w/v (about 3 grs crystal per 10 grs solution) of HA Resorb synthetic hydroxyapatite crystals. Chitosan-GP was prepared sterile (chitosan autoclaved; GP filtered 0.22 μm). HA Resorb crystals were sterile at admixing in the solution. Each injection was about 200 μl in volume. Hybrid implants formed in situ were cohesive, infiltrated and did not travel in situ. The implants remained in place for a period of at least 80 days.[0261]

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.[0262]