Autologous Cartilage Microparticle In A Composite Biomimetic Hydrogel To Repair Focal Articular Cartilage Defect

IntroductionThe articular cartilage(AC)is distributed at each end of long bones,its physiological function is weight bearing,low friction interface,and wear resistance.But AC has a very limited capacity of self-repair due to its avascular and aneural nature.Chondral injury in knee is very common.Cartilage damage is difficult to repair and leads to osteoarthritis,causing pain or joint disability of patients and eventually leading to severe mental and physical discomfort.There are many factors,such as rheumatoid arthritis,osteoarthritis,acromegaly,hemophilia and joint fracture and so on,causing AC damage.Cartilage tissue engineering uses scaffold with cell and cell factor to repair cartilage defect,the mechanism of which is the extracellular matrix secreted by cells gradually replaces the scaffold,and integrates with surrounding normal cartilage,achieving the purpose of cartilage repair.Autologous chondrocyte transplantation(ACI),a technique of cartilage tissue engineering,has undergone three development stages.The first generation of ACI from Sweden medical scientist Peterson et al,who first reported in rabbit AC defect model with absorbable suture autogenous periosteal suture in the periphery of the cartilage defect,and then injected amplification of autologous cartilage cell suspension in the defects and postoperative 16 weeks confirmed tissue repair is a transparent cartilaginous tissue.The second regeneration of ACI technique was introduced by Gooding,who used I/III type collagen films instead of periosteumin in 2006.After postoperative 2 years follow-up the repair effect of periosteum group had no significant difference compared with treated group,the advantage is to avoid the increase additional trauma,but there are still some cell leakage and membrane shedding.To overcome the shortcoming of ACI technology,researchers expanded chondrocytes after seeded into I / III collagen scaffold,then co-cultured with biological glue fixed on the defect,which is the third generation of ACI technology(namely matrix induced autologous chondrocyte transplantation).However,these methods are not desirable for repairing cartilage defect.The reasons are:(1)The limited proliferation capacity of chondrocytes when expanded in vitro.The ACI needs to expand cell in vitro,which may result in a loss of chondrocyte phenotype and dedifferentiation of chondrocyte(2)There are many differences between tissue engineering and nature cartilage matrix.ACI technology applies for chondrocyte suspension and lacks of extracellular matrix of fixing and supporting,which leads to regeneration tissue with poor biomechanical and wear-resisting lasting.MACI technology uses type I/III collage as scaffold materials,while the nature cartilage matrix is type II collage.So structure and matrix component of repair tissue have difference to the natural cartilage.In this study,we aimed to make type II collage-chondroitin sulfate-hyaluronic acid as biomimetic hydrogel and make it biosafe.We combined the cartilage microparticles with biomimetic hydrogel to use it repair the knee articular cartilage defect in Gui Zhou mini pig.Methods1.Chondroitin sulfate was oxidized by sodium periodate.Using infrared spectroscopic analysis the oxidized chondroitin sulfate to detect whether it contain aldehyde or not.Using different ratio of type II collage-oxidized chondroitin sulfate-hyaluronic acid to make biometic hydrogel and screen out the best optimize ratio of hydrogel.2.We selected the New Zealand white rabbits as experimental animals.Cartilage microparticles were prepared by microparticle acquisition device and made it mixed with biomimetic hydrogel.The viability was evaluated after the compound cultured with 3d,7d,14 d,21d,25 d,30d.3.Fifteen Gui Zhou mini pigs,which have thirty keen joints were randomly divided into two groups,were selected as experimental animals to take grinding microparticles of cartiage and create full –thickness chondral defect,of which diameter is 0.8 mm in the femoral tochlear of knee joint.Mixed the microparticles with the biomimetic hydrogel with the ratio 3:1,which embedded in the cartilage defect.At 1,3,6 months after surgery,the animals were sacrificed,For the macro-and microscopic evaluation,knee joints were opened and photographed.They were evaluated using the modified ICRS macroscopic grading.Histomorphological evaluation of the repair tissue quality was carried out according to the modified O'Driscoll score..Results1.Accroding to the FITR,the oxidized CS have newabsorption peak in wavelength in the 1750 cm,which is the C = O absorption peak of aldehyde.Mixed the micropartical with the biomimetic hydrogel with the ratio 3:1,4:1,5:1 and finally the optimization ratio is 3:1.2.Cultured the cartilage microparticles and biomimetic hydrogel compound in 3 d,7 d,14 d,21 d,25 d,30 d and taken specimens to do HE staining,observed the chondrocyte were alive in the microparticles.3.After 1 month postoperatively,there was only little healing seen in control group,the calcified layer was uncontinuous and the subchondral bone was exposed and collapsed.While there was a nearly complete filling of the defects in treatment group,the regenerated tissue displayed normal color.After 3 and 6 months of postoperatively,the complete filling was seen in both groups.However,macroscopic observations the gross and histologically showed that,in control group,the repaired tissue had rough surfaces,discernible edges,irregular calcified layer,and disordered subchondral bone;better repaired tissues were observed in treatment group,which had smooth surfaces,decent integration with the surrounding cartilage,continuous calcified layer,and homogeneous subchondral bone.Modified International Cartilage Repair Society(ICRS)scores of treatment and control groups were 14±1.22 and 0.6±0.89 at 1 month,17.8±1.31 and 12.4±2.4 at 3 months(n=5,p < 0.05),and 19.4±0.89 and 18.2±0.44 at 6 months(n=5,p < 0.05),respectively.Modified O'Driscoll scores of treatment and control groups were 13±1.22 and 2.2±0.45 at 1month(n=5,p < 0.05),15.8±1.30 and 5.2±0.84 at 3 months(n=5,p < 0.05),and17.2±0.84 and 7.2±0.44 at 6 months(n=5,p < 0.05),respectively.Conclusion1.Chondroitin sulfate was converted in to its aldehyde derivative by periodate oxidation and used as a cross-linking agent for collagen giving rise to a new class of hydrogels.Which name is shiff-base reaction.2.OCS has capacity of crosslinking with COLII-HA to formed a new class of biomimetic hydrogel.Which mixed with cartilage microparticles and cultured 1 month.We observed the chondrocyte were alive in the microparticles to support theoretical basis for our animal research.3.Cartilage microparticle composite biomimetic hydrogel implanted cartilage defect to repair articular cartilage of the knee in Guizhou mini pig,in each repair tissue had smooth surface,and desirable integration with the surrounding cartilage.After 6 month postoperative,Sirius red staining suggested that the matrix of repair tissue was mainly type II collagen.The repair results of each time point in the treatment groups were better than that of the control groups(P<0.05).