when combined with blood or blood components the polymer could be in an aqueous solution or in an aqueous suspension , or in a particulate state , the essential characteristics of the polymer preparation being that 1 ) it is mixable with blood or selected components of blood , 2 ) that the resulting mixture is injectable or can be placed at or in a body site that requires tissue repair , regeneration , reconstruction or bulking and 3 ) that the mixture has a beneficial effect on the repair , regeneration , reconstruction or bulking of tissue at the site of placement . a preferred embodiment is shown in example 5 where a solution of the natural polysaccharide , chitosan , was used at a concentration 1 . 5 % w / v and in 0 . 135 moles / l disodium glycerol phosphate buffer at ph = 6 . 8 . this solution was mixed with peripheral rabbit blood at a ratio of 1 part polymer solution to 3 parts blood . the polymer / blood mixture was then injected into a surgically prepared articular cartilage defect in the rabbit where it solidified within 5 minutes ( fig2 ). histological observations of the healing process revealed a stimulated repair that resulted in hyaline cartilage after 6 – 8 weeks ( fig2 ). control defects that did not receive the polymer / blood mixture were incompletely healed or healed with non - functional fibrous or fibrocartilagenous tissue ( fig2 ). this example demonstrates that the use of a polymer / blood mixture can result in more effective healing and greater functionality of repaired tissue than simply inducing bleeding at the wound site . trivial modifications of this invention are evident to those skilled in the art . other polymers and other formulations of polymers or polymer blends may be substituted for the chitosan solution providing they retain the three characteristics cited in the previous paragraph . and clearly , this approach may be trivially applied to the repair of tissues other than cartilage such as meniscus , ligament , tendon , bone , skin , cornea , periodontal tissues , abscesses , resected tumors , and ulcers . applications in tissue bulking and reconstruction are also evident . we present examples and evidence to teach possible mechanisms of action of this invention including 1 ) inhibition of the typical platelet - mediated contraction of a blood clot by mixing blood with the polymer prior to solidification ( fig1 ) 2 ) the resulting maintained full - volume scaffold and therefore better defect filling for tissue repair ( fig1 ) 3 ) adherence of the solidified polymer / blood mixture to the surrounding tissues ( fig2 a ) 4 ) a slower release of chemotactic and mitogenic protein factors from the polymer / blood mixture than from a simple blood clot ( fig2 ) 5 ) maintenance of leukocyte and platelet viability in the polymer blood / mixture ( fig2 ) and 6 ) provision of a polysaccharide environment in the repair site that is more conducive to cartilage formation than is a purely proteinaceous matrix ( fig2 – 6 , 8 – 10 , 24 ). these phenomena are demonstrated to occur in our examples . their demonstration does not , however , reject the possibility that other important events occur such as those involving the kinetics of cellular degradation of the polymer , and binding / concentration of endogenous factors by the chitosan . a second preferred embodiment of this invention is shown in examples 1 and 2 where a thermogelling chitosan solution was used to deliver primary chondrocytes to subcutaneous regions in mice or to culture chambers in vitro . in this case the absence of blood components necessitates a gelling capability on the part of the chitosan solution alone , and this property is endowed via a particular preparation of the chitosan solution using glycerol phosphate and other similar buffers . in our examples we demonstrate that the polymer solution may be mixed with cells and the polymer / cell solution injected in vivo or in vitro whereupon it gels , maintaining functionality and viability of the cells ( fig1 - 11 ). the cells may be resuspended in a physiological buffer , or other cell carrier suspension such as cellulose in an isotonic buffer , prior to mixing with the chitosan solution . we show data demonstrating the formation of cartilage tissue in vitro ( fig2 – 6 ) and in vivo ( fig8 – 11 ) when primary chondrocytes are injected with this polymer solution . trivial modifications and extensions of this embodiment of the invention are also evident to those skilled in the art where , for example , other cell types may be used and concentrations of the chitosan and the buffer may be changed to achieve the same result . the present invention will be more readily understood by referring to the following examples , which are given to illustrate the invention rather than to limit its scope . mixing of thermogelling chitosan solution with primary chondrocytes for in vitro growth of cartilage chitosan ( 0 . 22 g , 85 % deacetylated ) as an hcl salt powder was sterilized by exposure to ultraviolet radiation in a biological laminar flow hood and then dissolved in 7 . 5 ml h 2 o resulting in a ph near 5 . 0 . d (+)- glucosamine ( 0 . 215 g , mw 215 . 6 ) was dissolved in 10 ml of 0 . 1m naoh and filter sterilized using a 22 μm pore size disk filter . glycerol phosphate ( 0 . 8 g , mw 297 including 4 . 5 mole water per mole glycerol phosphate ) was dissolved in 2 . 0 ml of h 2 o and filter sterilized using a 22 μm pore size disk filter . 2 . 25 ml of the glucosamine solution was added drop - by - drop under sterile conditions to the chitosan solution with agitation at a temperature of 4 ° c . then 1 ml of the glycerol phosphate solution was added under the same conditions . this final solution is still a liquid and remains so for an extended period ( i . e . days ) if the temperature is kept low , i . e . near 4 ° c . the ph of this solution is physiological at 6 . 8 and the osmolarity is also physiological , around 376 mosm / kg - h 2 o . it is of critical importance to retain these two parameters within the limits required to maintain cell viability . these limits vary with cell type but are generally 6 . 6 & lt ; ph & lt ; 7 . 8 and 250 mosm / kg - h 2 o & lt ; osmolarity & lt ; 450 mosm / kg - h 2 o . a solution is prepared by dissolving 150 mg hydroxyethyl cellulose ( fluka ) and 6 ml dmem ( dulbecco &# 39 ; s modified eagles medium ), and filter sterilized using a 22 μm pore size disk filter . a cell pellet is resuspended with 2 ml of hydroxyethyl cellulose - dmem solution , and admixed into the chitosan - glycerol phosphate solution . as a negative control , the chitosan solution mixed with 2 ml of hydroxyethyl cellulose - dmem solution with no cells was generated . when this solution is heated to 37 ° c . it transforms into a solid hydrogel similarly to the thermogelling solution disclosed in a previous invention ( chenite et al . patent wo 99 / 07416 ). most importantly , this previous invention did not demonstrate that cell viability was maintained throughout the thermogelllng process in this chitosan solution , and thus did not enable the use of this chitosan solution for cell delivery , tissue repair and tissue regeneration . the above solution in the liquid state at 4 ° c . was mixed with enzymatically isolated primary chondrocytes ( buschmann et al ., 1992 ) and then poured into a plastic culture dish ( fig1 a to 1f ). in fig1 a , a cell pellet is resuspended and admixed ( fig1 b ) into the liquid chitosan gel solution at 4 ° c . in fig1 c and 1d , the liquid solution is poured into a tissue culture petri and allowed to solidify at 37 ° c . for 30 minutes , after which the solid gel with cells is washed with dmem , and discs cored using a biopsy punch . in fig1 e , 1000 μm pore mesh grids are placed in 48 - well plates . in fig1 f , the chitosan gel discs with cells are placed in culture in individual wells . a gel harboring cells formed after a 20 minute incubation at 37 ° c . using a biopsy punch , 6 mm diameter 1 mm thick discs were cored from the gel and placed in culture for up to 3 weeks . discs were cultured individually in 48 - well tissue culture plates with sterile nylon 1000 μm meshes beneath to allow media access to all surfaces . over 90 % of the encapsulated cells were viable immediately after encapsulation , and throughout the culture period ( fig2 a to 2c ). samples were incubated in calcein am and ethidium homodimer - 1 to reveal live ( green ) and dead ( red ) cells . freshly isolated chondrocytes ( fig2 a ) were encapsulated in the gel , solidified and tested immediately for viability ( fig2 b ), or after 20 days of culture in the gel ( fig2 c ). fig2 c shows cells with typical chondrocyte morphology from the middle of the gel . several distinct cell types exhibited the same high degree of viability after encapsulation and cell culture , including rat - 1 , cos , 293t , and de - differentiated bovine articular chondrocytes , confirming that the gelation process maintained cell viability , and could thus be used to deliver cells in vivo by injection . toluidine blue staining of the gel with cells after 22 days of culture revealed a metachromatic ring of staining surrounding encapsulated primary chondrocytes , indicating the build - up of proteoglycan , or gag , which was beginning to fuse between closely adjacent cells ( fig3 d ). these regions also stained with antibodies raised against aggrecan , type ii collagen and link protein . the chitosan gel matrix was also found to bind toluidine blue ( fig3 c ). this property enabled to observe the lattice structure of the gel , after employing an aldehyde fixation . interestingly , the pericellular ring of gag observed around the chondrocytes contained little chitosan matrix , the latter appearing to have been degraded by chondrocyte - produced factors ( fig3 d ). primary calf chondrocytes were encapsulated in chitosan gel at 2 × 10 7 primary chondrocytes per ml and cultured as 6 mm discs for up to 20 days . primary calf chondrocytes were encapsulated and cultured in 2 % agarose and analyzed in parallel . day 0 and day 20 cultures were processed by paraffin sectioning and toluidine blue staining for agarose gel cultures ( fig3 a and 3b ) and chitosan gel cultures ( fig3 c and 3d ). at day = 0 , nuclei stain dark blue ( fig3 a and 3c ) whereas accumulated pericellular gag stains metachromatic blue - violet ( fig3 b and 3d , large arrows ). these pericellular regions were immunopositive for aggrecan , collagen ( ii ) and cartilage link protein . at a magnification of 40 × in fig3 e , quantitative biochemical analysis of gag present at days 0 , 14 , and 20 of culture using the dmmb assay revealed a similar accumulation of gag in chitosan gel compared with agarose gel . rna analysis of type ii collagen and aggrecan mrna expressed by the encapsulated chondrocytes revealed high levels at 14 and 22 days of culture ( fig4 , lanes 4 and 5 ) that were comparable to those levels observed in articular chondrocytes in cartilage ( fig4 , lane 6 ). a mixture of antisense 32 p - labeled rna probes complementary to bovine type ii collagen , aggrecan , and gapdh was hybridized with trna ( lane 1 ), or total rna , from bovine kidney ( lane 2 ), from primary chondrocytes ( 10 7 / ml ) cultured in chitosan gel for 0 days ( lane 3 ) 14 days ( lane 4 ) or 20 days ( lane 5 ), or adult bovine articular cartilage ( lane 6 ). samples were treated with rnase a and t1 , then submitted to electrophoresis and autoradiography . protected bands showing the presence of individual transcripts are as indicated . the maintenance of the chondrocyte phenotype in the chitosan / glycerol - phosphate gel is shown by the continued expression of aggrecan and type ii collagen . western analysis of proteins produced by encapsulated cells showed an accumulation of cartilage matrix link protein between 2 and 3 weeks in culture ( fig5 ). total proteins were extracted , separated by sds - page , and immunoblotted with antisera recognizing vimentin , pcna , the c - propeptide of type ii collagen , or cartilage link protein . samples analyzed include chitosan gel with no cells ( lane 1 ), bovine kidney ( lane 2 ), duplicate samples of primary chondrocytes ( 10 7 / ml ) cultured in chitosan gel at day = 0 ( lanes 3 and 4 ), day = 14 ( lanes 5 and 6 ), or day = 20 ( lanes 7 and 8 ), 2 - week calf articular cartilage ( lane 9 ), or adult bovine cartilage ( lane 10 ). results show the accumulation of cartilage - specific proteins cp2 and link at 14 and 20 days , as well as the persistence of pcna expression through culture day 20 , as a marker for cell proliferation . discs containing primary bovine articular chondrocytes were mechanically evaluated at days 4 and 13 of culture using uniaxial unconfined compression stress relaxation tests . by comparing to control gels with no cells , a significant , cell - dependent degree of stiffening was observed even at day 4 and became much more dramatic at day 13 ( fig6 ). discs (˜ 5 mm diameter ) from days 4 , 13 and 19 of culture were mechanically tested in unconfined compression by applying 5 ramps of 10 % the disk thickness (˜ 1 . 5 mm ) during 10 seconds and holding that displacement during subsequent stress relaxation ( the 2nd ramp from 10 – 20 % is shown in the graph ). the gel discs without cells displayed a weak behavior while cell - laden gels became evidently stiffer with time in culture and more characteristically viscoelastic , like articular cartilage . by analyzing these data with a composite poroelastic model ( soulhat et al ., 1999 ) a doubling of the non - fibrillar matrix modulus ( 2 . 5 → 5 kpa ) was found , a 5 × increase in the fibrillar matrix modulus ( 100 → 500 kpa ) was also found together with a near 100 × reduction in hydraulic permeability ( 5 → 0 . 08 × 10 – 12 n - s / m4 ) due to the presence of primary chondrocytes in these gels during only 13 days of culture in vitro . taken together , these results demonstrate that the chitosan gel is cytocompatible and cytodegradable , conducive to maintenance of the chondrocyte phenotype , and permits the elaboration of a neo - cartilage matrix with a significant increase in mechanical stiffness in vitro . mixing of thermogelling chitosan solution with primary chondrocytes and subcutaneous injection for in vivo growth of cartilage to demonstrate that this in situ gelling system can be employed in animals , athymic mice ( cd1 nu / nu ) were subjected to dorsal , subcutaneous injections of 100 to 300 μl of chitosan gel described in example 1 , containing 10 million calf articular chondrocytes per ml ( fig7 ). a cell pellet of primary calf chondrocytes was admixed with liquid chitosan gel at 4 ° c . to achieve a concentration of 1 to 2 × 10 7 cells / ml , and injected in liquid form as 100 μl subcutaneous dorsal implants in anesthetized nude mice . in situ gelling was apparent by palpation 5 to 10 minutes post - injection . control mice were similarly injected with chitosan gel alone . a palpable gel was formed within 10 minutes of injection . implants were recovered at 21 , 48 , and 63 days post - injection . toluidine blue staining revealed the gross production of gag - rich extracellular matrix by the implants containing cells ( fig8 a ). no gag accumulation was seen in implants of chitosan gel alone ( fig1 b ). primary calf chondrocytes at 2 × 10 7 cells / ml liquid chitosan gel were injected in liquid form as 100 μl subcutaneous dorsal implants in anesthetized nude mice . control mice received 100 μl subcutaneous dorsal implants of liquid chitosan gel alone . 48 days after injection , implants were harvested and processed for paraffin histology and toluidine blue staining . metachromatic violet staining reveals the accumulation of gag in the implant with chondrocytes ( fig8 a ). no gag accumulation is detected in the implant with chitosan gel only ( fig8 b ). cartilage - specific mrna expression , collagen type ii and aggrecan , was detected in the in vivo implants with primary chondrocytes at day 48 post - injection ( fig9 ). no type ii collagen or aggrecan expression was detected in implants of chitosan gel alone ( fig9 ). a mixture of antisense 32 p - labelled rna probes complementary to bovine type ii collagen , aggrecan , and gapdh were hybridized with trna ( lane 1 ), or total rna , from bovine kidney ( lane 2 ), from day = 48 in vivo nude mouse implants with chitosan gel only ( lane 3 ) or day = 48 in vivo implants of chitosan gel with primary chondrocytes at 2 × 10 7 cells / ml ( lane 4 ), or adult bovine articular cartilage ( lane 5 ). samples were treated with rnase a and t1 , then submitted to electrophoresis and autoradiography . protected bands showing the presence of individual transcripts are as indicated . the maintenance in vivo of the chondrocyte phenotype in the chitosan / glycerol - phosphate gel is shown by the expression of aggrecan and type ii collagen . cartilage - specific proteins were detected in in vivo implants with primary chondrocytes from days 48 and 63 post - injection ( fig1 ). no cartilage - specific proteins were detected in implants with chitosan gel only ( fig1 ). total proteins were extracted , separated by sds - page , and immunoblotted with antisera recognizing vimentin , pcna , the c - propeptide of type ii collagen , or cartilage link protein . samples analysed include chitosan gel with no cells ( lane 1 ), bovine kidney ( lane 2 ), two distinct in vivo nude mouse implants of chitosan gel only at day 63 ( lanes 3 and 4 ), of in vivo implants of chitosan gel with 2 × 10 7 calf chondrocytes per ml gel at days 48 ( lane 5 ) or day 63 ( lane 6 ), 2 - week calf cartilage ( lane 7 ), or adult bovine cartilage ( lane 8 ). results show the accumulation of cartilage - specific extracellular matrix proteins cp2 and link , in only those chitosan gel implants carrying chondrocytes . the acronym pcna means “ proliferating cell nuclear antigen ”. cp refers to type 2 collagen c pro - peptide and link refers to cartilage link protein . the in vivo implants with no cells had a pasty consistency , whereas the implants with cells could be cored into 3 to 5 mm discs and subjected to mechanical testing to reveal a high mechanical stiffness not found in an in vitro disc without cells ( fig1 ). these data indicate that chondrocytes can be delivered in situ , via injection , with the chitosan thermogelling solution as a carrier . the injected chondrocytes remain viable , and synthesize and assemble significant levels of a proteoglycan - rich extracellular matrix that stiffens over time to form a functional cartilaginous tissue . in fig1 , primary calf chondrocytes at 2 × 10 7 cells / ml liquid chitosan gel were injected in liquid form as 100 μl subcutaneous dorsal implants in anesthetized nude mice . control mice received 100 μl subcutaneous dorsal implants of liquid chitosan gel alone . 48 days after injection , implants were harvested . implants of chitosan gel only had a paste - like consistency , and could not be mechanically tested . implants with primary chondrocytes had the appearance of cartilage , and a 3 mm biopsy was cored from the center of the implant , and tested in unconfined compression using 2 . 5 % thickness compression with a relaxation criteria of 0 . 05 g / min . the equilibrium modulus at 20 % and 50 % compression offset is shown for the 48 day implant containing cells compared to a control disk left in vitro during a 42 day period . the in vivo grown chondrocyte laden gel has developed substantial mechanical stiffness during 48 days due to the synthesis and assembly of a functional cartilage matrix ( fig8 a ). one of the most significant advantages of this chitosan thermogelling formulation for cartilage repair is its ability to conform and adhere to irregular cartilage defects and other irregularly shaped cavities in the body that require tissue repair , regeneration , reconstruction or bulking . many current tissue repair procedures suffer drastically in this respect . chitosan - glycerol phosphate liquid gel without cells was delivered ex vivo to porcine femoral condylar intra - chondral ( not involving bone ) defects . disc - shaped defects in the articular cartilage were created using a biopsy punch ( fig1 a ) and the chitosan solution described in example 1 was injected into these defects and allowed to solidify in an incubator at 37 ° c . the articulating cartilage surface was opposed and simulated joint motions were performed after which the gel was observed to remain in the cartilage defect ( fig1 b ). the gel not only remained in the defect but also adhered to the surrounding bone and cartilage surfaces and did not contract . in fig1 a and 12b , liquid chitosan gel was deposited in 6 mm diameter full - thickness cartilage defects ( fig1 a ) and allowed to solidify at 37 ° c . for 30 minutes in a humidified incubator . the joint was then closed , and joint motion simulated for several minutes . the chitosan gel adhered to and was retained in all of the defects after simulated joint motion ( fig1 b ). in vivo filling of intrachondral defects was also performed on the patellar groove of rabbits . a rectangular ( 4 mm × 5 mm ) defect was created by shaving off cartilage down to the harder calcified cartilage layer with a microsurgical knife . several microfracture holes were introduced using a 16 - gauge needle , the thermogelling chitosan solution described in example 1 was injected into this defect and allowed to solidify for 5 minutes ( fig1 a ) and the rabbit knee joint sutured up . the rabbit was allowed to ambulate freely and the following day it was euthanised and the treated knee joint prepared for histological analysis ( fig1 b ). a live new zealand white rabbit was anesthetized , and a 3 × 4 mm chondral - only defect created in the trochlea of the femoral patellar groove . several microfracture holes were introduced with a 16 gauge needle . liquid thermogelling chitosan was loaded into the defect and allowed to gel for 5 minutes in situ ( fig1 a ). the joint was closed , and the rabbit allowed to recover with unrestricted motion for 24 hours before sacrifice and joint dissection ( fig1 b ). histological analysis ( fig1 ) revealed the retention of this thermogelling chitosan gel in the very thin cartilage layer of the rabbit ( only about 0 . 8 mm thick ). the gel adhered firmly to surrounding bone and cartilage tissue , demonstrating good retention , thereby enabling its use as an injectable thermogelling polymer delivery vehicle for the repair of cartilage and other tissues . the joint and defect shown in fig1 b ( filled with thermogelling chitosan , and residing 24 hours in vivo ) was fixed , embedded in lr white plastic resin , sectioned , and stained with toluidine blue . a cross - section of the defect reveals retention of the chitosan gel in situ , as well as adherence to cartilage and bone surfaces in the defect . several distinct mixing methods were employed to admix blood with an aqueous polymer solution ( fig1 a ). blood and polymer are admixed in a recipient , resulting in a homogenous liquid blend of blood and polymer . in general , 3 volumes blood was mixed with 1 volume of 1 . 5 % polysaccharide in an isotonic and iso - osmolaric solution . in the case of chitosan gel , 1 . 5 % chitosan was dissolved in 70 mm hcl and 135 mm β - glycerol phosphate . in the first blood / polymer mixing method , one , 1 cc syringe was loaded with 750 μl whole peripheral blood , and a second 1 cc syringe was loaded with 250 μl liquid polymer solution . the syringes were interconnected , and mixed by pumping the two phases back - and - forth 40 times , until apparently homogenous . in the second mixing method , 625 μl of liquid polymer solution was deposited in a 2 . 0 ml cryovial ( corning ) with several 3 mm – 6 mm steel balls . the cryovial was filled with 1 . 875 ml whole blood , the cap screwed on , and the vial shaken vigorously for 10 seconds . in the third mixing method , 2 ml of liquid polymer solution was deposited in a sterile 12 ml glass borosilicate vial ( interglass 5 cc serological vial ). the vial closed with a rubber stopper and metal crimper , and a 25 ml air vacuum was drawn in the vial with a 10 ml syringe and 20 - gauge needle . using proper phlebotomy techniques , peripheral blood from either rabbit artery , or human or equine vein was drawn into a sterile 10 ml syringe . a 20 - gauge needle was attached to the syringe , and inserted through the rubber stopper of the vial . 6 ml of peripheral blood was admitted to the vial . the vial was vortex mixed for 10 seconds at full speed . following any of these mixing techniques , the resulting mixture was deposited into a 4 ml borosilicate glass vial at room temperature , a plastic vial at 37 ° c ., or an agarose well ( fig1 b and 15c ), or an articular cartilage defect ex vivo . as a control , the same treatment was performed with peripheral whole blood only . as another control , a vacutainer vial of edta - treated blood was drawn to measure cbc and platelet number . all blood samples tested displayed normal cbc and platelet counts for the respective species . regardless of the species , the prepared blood / polymer , solidified and adhered strongly to the walls of the glass vial within 2 . 5 to 18 minutes after mixing ( fig1 ). mixed whole peripheral blood solidified in general more slowly compared to blood / chitosan gel ( fig1 ). separate samples of blood , with or without liquid chitosan gel , were mixed and solidification time was measured by the number of minutes elapsed between mixing , and achieving a solid adherent mass in the original mixing vial , or secondary recipient . testing of additional blood / polymer solutions , including blood / hyaluronic acid , blood / hydroxyethyl cellulose , and blood / alginate , revealed that these mixtures also solidify in a time period that is comparable to blood alone ( fig1 a ). here it was concluded that admixture of chitosan liquid gel into whole peripheral blood accelerates clot formation , and that blood / chitosan gel solidification time is acceptable for clinical application . contraction was tested on mixed fresh peripheral rabbit blood , or rabbit blood mixed with pbs or various 1 . 5 % polysaccharide solutions including chitosan in glycerol phosphate buffer . fresh blood without mixing was also analyzed . a heparin blood / chitosan in glycerol phosphate buffer mixture was also analyzed . 500 μl of each sample was deposited into a 4 ml glass tube at 37 ° c . at distinct time points , all excluded plasma was removed from each tube and weighed , to determine the amount of clot contraction . all samples except blood / chitosan glycerol phosphate mixtures contracted to 30 – 50 % of their original volume . blood / chitosan mixtures contracted minimally maintaining approximately 90 % of their initial volume . to test for the degree of contraction of solidified blood / polymer mixes relative to coagulated whole blood , a clot contraction test was performed on an array of blood / polymer samples , using several controls ( fig1 a , 17 b and 17 c ). one group of controls consisted of non - agitated whole peripheral blood , or agitated whole peripheral blood , or whole peripheral blood agitated 3 : 1 ( volume : volume ) with phosphate - buffered saline . these samples were compared with experimental samples containing 3 volumes whole peripheral blood agitated with 1 volume of distinct 1 . 5 % polysaccharide solutions dissolved in pbs ( alginate , hydroxyethyl cellulose , or hyaluronic acid ). another sample consisted of 3 volumes whole peripheral blood mixed with 1 volume chitosan - glycerol phosphate solution . at intervals up to 18 hours after solidification , the excluded serum for each condition was measured in triplicate , as an indication of degree of contraction . samples with peripheral blood , ± pbs , contracted to 30 % of the original mass ( fig1 a ). peripheral blood admixed with the polysaccharides alginate , hydroxyethyl cellulose , or hyaluronic acid contracted to 40 %– 50 % of the original mass ( fig1 a ). the blood / chitosan gel samples showed negligible contraction , with contraction to 90 % of the original mass ( fig1 a ). the heparinised blood / chitosan gel samples also resisted contraction , to 85 % of the original mass ( fig1 a ). from these data it was concluded that blood / chitosan gel resists contraction , and provides a more space - filling fibrin scaffolding inside the cartilage defect . in fig1 b and 17c , samples shown include blood ( 1 ), or mixed blood ( 2 ), blood / pbs ( 3 ), blood / chitosan in glycerol - phosphate ( 4 ), heparin blood / chitosan ( s ), blood / alginate ( 6 ), blood / hydroxyethyl cellulose ( 7 ), and blood / hyaluronic acid ( 8 ). to test whether anti - coagulated blood could be used to generate blood / polysaccharide in situ solidifying implants , 3 volumes of blood treated with 1 . 5 mm edta , 0 . 38 % citrate , acid - 0 . 38 % citrate dextrose , or sodium heparin ( becton dickinson ) was mixed with 1 volume chitosan - glycerol phosphate solution . chitosan - glycerol - phosphate solution was able to reverse heparin - ( fig1 ), edta -, and citrate - mediated anti - coagulation . 1 . 5 % chitosan in glycerol - phosphate solution , or three distinct 1 . 5 % polysaccharide solutions , were admixed at a ratio of 1 volume polysaccharide solution , to 3 parts whole peripheral blood . 500 μl of each sample was deposited in a glass borosilicate tube and allowed to solidify for 60 minutes at 37 ° c . different polysaccharides include hyaluronic acid - pbs ( 1 ), hydroxyethyl cellulose - pbs ( 2 ), alginate - pbs ( 3 ), and chitosan - glycerol phosphate ( 4 ). as a control , heparin blood only was analyzed ( 5 ). after 60 minutes , the tubes were laid horizontally and photodocumented . only the mixture of chitosan - glycerol phosphate and heparinised blood became solid . other heparin blood / polysaccharide mixtures using hydroxyethyl cellulose , alginate , or hyaluronic acid , failed to solidify ( fig1 ). from these data it was concluded that blood / chitosan in situ solidifying implants can be generated using anti - coagulated blood . histological sections of solid blood / polymer samples showed that mixtures were homogenous , that red blood cells did not hemolyse after mixing or solidification , and that platelets became activated and were functional ( as evidenced by the generation of a dense fibrin network ) ( fig1 a to 19c ). a solidified mixture of blood / chitosan was fixed , embedded in lr white plastic , sectioned , and stained with toluidine blue . ( in fig1 a , at 20 × magnification , global homogeneous mixing is apparent . in fig1 b , at 100 × magnification , intermixed pools of red blood cells and chitosan hydropolymer is apparent . at 2000 × magnification ( by environmental electron scanning microscopy ) the presence of fibrin fiber network throughout the blood / chitosan composite is evident . some leukocytes remained viable a number of hours following mixing and solidification ( fig2 ). peripheral whole blood was mixed with chitosan gel and allowed to solidify . in fig2 a , 60 minutes post - solidification , the plug was placed in viability stain with calcein am / ethidium homodimer - 1 to reveal live white blood cells ( green cells , large arrows ), live platelets ( green cells , small arrows ), and dead white blood cells ( red nuclei ). in fig2 b , a distinct sample was fixed at 180 minutes post - solidification , embedded in lr - white , and submitted to transmission electron microscopy . active phagocytosis by peripheral monocytes ( arrow head ), reflecting cell viability , is evident in tem micrographs at 3 hours post - mixing and solidification . an analysis of the total serum proteins lost from either blood or blood / chitosan following solidification was performed . equal volumes of blood , or blood / chitosan gel were solidified in agarose wells . the discs were transferred to individual wells of a 48 - well plate containing 1 ml pbs and incubated at 37 ° c . for 3 hours . the discs were successively changed into fresh pbs solution at 37 ° c . at 4 , 5 , 7 , and 19 hours . pbs washes were lightly centrifuged to remove any cells prior to analysis . several discs were extracted for total protein after 3 or 19 hours in pbs . total proteins present in the discs , or pbs washes , were analysed by sds - page and total protein stain with sypro orange . serum proteins were released more slowly more sustained from the blood / chitosan samples compared with blood samples ( fig2 ). these data suggest that blood and platelet - derived proteins involved in wound healing are released in a more sustained and prolonged manner from blood / chitosan - filled defects , compared with blood clot - filled defects . solid discs of blood / chitosan gel , or blood only , were generated from 150 μl initial liquid volume . resulting discs were washed in 1 ml pbs for 3 hours , then transferred successively at 4 , 5 , 7 , and 19 hours for a total of four additional 1 ml pbs washes . after 3 or 19 hours of washing , representative discs were extracted with gucl to solubilise total retained proteins . soluble proteins were precipitated from equal volumes of gucl extracts or pbs washes , separated on sds - page gels , and stained for total proteins using sypro orange . comparatively , more proteins were retained in the blood / polymer discs than the blood discs throughout the 19 hour wash period . comparatively , a slower and more prolonged release of serum proteins into the pbs washes was seen for blood / chitosan than blood over the 19 hour wash period . preparation , mixing and injection of blood / polymer mixture to improve healing of articular cartilage defects chondral defects with perforations to the subchondral bone were treated with a peripheral blood / chitosan - glycerol phosphate mixture that was delivered as a liquid , and allowed to solidify in situ ( fig2 a to 22c ). in fig2 a , a full - thickness cartilage defect , 3 × 4 mm square , was created in the femoral patellar groove of an adult ( more than 7 months ) new zealand white rabbit . four , 1 mm diameter microdrill holes were pierced to the bone , until bleeding was observed . in fig2 b , liquid whole blood was mixed at a ratio of 3 volumes blood to 1 volume chitosan in glycerol phosphate solution , and deposited to fill the defect . in fig2 c , after 5 minutes in situ , the blood / chitosan implant appeared to solidify . the capsule and skin were sutured , and the animal allowed to recover with unrestricted motion . a similar treatment in human patients is schematized in fig2 d , where prepared cartilage defects receive an arthroscopic injection of liquid blood / polymer that solidifies in situ . alternatively , an arthroscopic injection of liquid polymer is mixed with bone - derived blood at the defect site ( fig2 e ). in fig2 d , the patient blood is mixed with the polymer ex vivo , and delivered to a prepared defect by arthroscopic injection , or ( fig2 e ) the polymer is delivered arthroscopically or during open knee surgery and mixed at the defect site with patient blood issuing from the defect . as a proof - of - concept study , the effects of blood / chitosan gel treatment were tested in rabbits . adult , skeletally mature new zealand white rabbits ( 7 months and older ) were anesthetized , with xylazine - ketamine followed by isofluorene / oxygen gas anesthesia . the trochlea of the femoral patellar groove was exposed by a parapatellar incision and patellar displacement . a full - thickness cartilage defect , up to 4 × 5 mm , in the trochlea of the femoral patellar groove , was produced with a microsurgical knife . four , 4 mm deep , 1 mm diameter bone - penetrating holes were generated by either microdrill with constant irrigation with 4 ° c . pbs , or by puncture with a custom - made awl and hammer . the defect was flushed with pbs , and depending on the degree of bleeding , up to 200 μl of sterile epinephrine ( 2 μg / ml ) in phosphate buffered saline was injected into the bleeding holes . the cartilage defect was covered with a sterile gauze soaked with pbs . rabbit peripheral blood was removed from the central artery of the ear with a vacutainer ™ needle and untreated , siliconized glass 4 cc vacutainer ™ vials from becton dickison . in one treatment , 750 μl blood was drawn into a sterile 1 cc syringe . a second syringe holding 250 μl of chitosan - glycerol phosphate solution ( 1 . 5 % chitosan / 70 mm hcl / 135 mm β - glycerol phosphate ) was interconnected with the blood - containing syringe with a sterile plastic connector . the syringes were pumped back - and - forth 40 times . the mix was drawn into one syringe , to which a 20 - gauge needle was attached . after purging half of the mix , one drop ( about 25 μl ) was deposited into the defect . in a separate treatment , 2 ml blood was added to a polypropylene cryovial tube containing 667 μl 1 . 5 % chitosan / 70 mm hcl / 135 mm β - glycerol phosphate and 6 sterile 3 . 2 mm diameter stainless steel beads . the tube was capped , and shaken for 10 seconds , rigorously ( around 40 to 50 actions ). the resulting liquid blood / chitosan mix was removed from the vial with a sterile 1 cc syringe , and a 20 g needle was attached to the syringe . after purging 200 μl from the syringe , one drop ( about 25 μl ) was deposited to fill the cartilage defect . the blood / chitosan mixture was allowed to solidify for 5 minutes , after which the capsule and skin were sutured , and the wound disinfected . rabbits were sacrificed at 1 week ( n = 1 , male ) or at 51 or 56 days ( n = 2 , 1 male , 1 female ). joints were fixed , decalcified , embedded in lr / white plastic , sectioned , and stained with toluidine blue . blood / chitosan - treated defects at 1 week of healing revealed large numbers of chemotactic cells migrating towards the blood / chitosan - filled zone ( fig2 a ). untreated defects had a relatively weak chemotactic response ( fig2 b ) towards the blood clot at the top of the defect . a chondral defect with microdrill holes was created in both femoral patellar grooves of an adult new zealand white rabbit , one of which was filled with blood / chitosan gel , and another left untreated . one week after healing , the joints were fixed , processed in lr - white , and toluidine blue stained . at 2 to 3 mm below the surface of the cartilage , a large number of cells migrating towards the defect filled with blood / chitosan were evident ( fig2 a ), whereas fewer migrating cells were seen at the same region of the untreated defect ( fig2 b ). after 5 to 8 weeks healing , the blood / chitosan - treated defect was filled with hyaline repair tissue in 2 rabbits ( 1 male , 1 female )( fig2 a ). this blood / chitosan - based repair tissue had the appearance of hyaline , gag - rich cartilage repair tissue . the repair tissue from untreated , or blood - only treated microfracture defects , had the appearance of fibrocartilage ( fig2 b ). there was no histological evidence of blood / chitosan or blood clot persisting within the defect site at or beyond 3 weeks post - delivery . a chondral defect with microdrill holes was created in both femoral patellar grooves of an adult new zealand white rabbit , one of which was filled with blood / chitosan gel , and another left untreated . at 51 or 56 days after healing , the joints were fixed , processed in lr - white , and toluidine blue stained . in fig2 a , repair tissue from the blood / chitosan - treated defect had the appearance of metachromatically staining hyaline cartilage , which adhered to the defect surfaces , and filled the defect . in fig2 b , repair tissue from the untreated defect had the appearance of fibro - cartilage , with practically no metachromatic staining for gag , and only partial defect filling . while the invention has been described with particular reference to the illustrated embodiments , it will be understood that numerous modifications thereto will appear to those skilled in the art . accordingly , the above description and accompanying drawings should be taken as illustrative of the invention and not in a limiting sense . for example , we have demonstrated that mixing chitosan in solution with blood allows the formation of polymer / blood clot that does not contract significantly , demonstrates a slowed release of chemotactic and mitogenic blood proteins , maintenance of blood cell viability , and a dramatically improved repair of articular cartilage defects . it is obvious to those skilled in the art that the chitosan solution could be prepared differently to achieve the same result . examples include : 1 ) altered chitosan concentration and mixing ratio with blood 2 ) altered choice of aqueous solution by changing buffer type and species concentration 3 ) an aqueous suspension of chitosan aggregates 4 ) a particulate chitosan powder combined with a proper mixing technique to distribute these particle throughout the blood and partly dissolve them . other polymers may be used such as 1 ) another polysaccharide like hyaluronan if its anti - 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the present invention relates to a new method for repairing human or animal tissues such as cartilage , meniscus , ligament , tendon , bone , skin , cornea , periodontal tissues , abscesses , resected tumors , and ulcers . the method comprises the step of introducing into the tissue a temperature - dependent polymer gel composition such that the composition adhere to the tissue and promote support for cell proliferation for repairing the tissue . other than a polymer , the composition preferably comprises a blood component such as whole blood , processed blood , venous blood , arterial blood , blood from bone , blood from bone - marrow , bone marrow , umbilical cord blood , placenta blood , erythrocytes , leukocytes , monocytes , platelets , fibrinogen , thrombin and platelet rich plasma . the present invention also relates to a new composition to be used with the method of the present invention .