OHA-COL ? Biomimetic Gel Combined With Autologous Concentrated Bone Marrow Cell For Cartilage Defect Repair Of Minipig

BackgroundArticular cartilage is a thin dense connective tissue which covered gliding surfaces of the synovial joint,it provides a low-friction surface,with strong compressive strength and is known to be wear-resistant.Articular cartilage is an avascular,aneural and alymphatic structure,thus,it has a limited self-repair potential.While,the incidence of cartilage defect is very high in clinical settings,Widuchowski reported that the overall incidence of chondral injury in knee arthroscopy is approximately 60% in a retrospective study of 25,124 knee arthroscopies.It is one of the most important reason of pain,dysfunction even disablity of effected extremity.Long term outcomes of current clinical treatments,such as joint cavity debridement,bone marrow stimulation,autologous cartilage tissue implant and autologous chondrocyte implantation are less than satisfactory.Cartilage tissue engineering is a promising approach for cartilage defect repair,however,there are still a lot of problems exist in this technology,such as: the program is complicated,poor mechanical properties of repaired tissue,bad integration with host tissue and fibrocartilaginous degeneration.Thus,it is of great significance to exoplore a more convenient repair strategy with better repair effect.Thus,to simplify the operation sequence while increase the repair effe ct of cartilage defect,we proposed a new one-step strategy to repair the cartilage defect by oxidized hyaluronic acid(OHA)-type ? collagen(COL ?)biomimetic gel combined with autologous concentrated bone marrow cell.Hyaluronic acid and type ? collagen are both normal composition of articular cartilage,thus,them were choiced to construct the biomimetic gel.Then,the gel was mixture with autologous concentrated bone marrow cell,and it was injected into cartilage defect by one-step method.Under the unique microenvironment of articular cavity and surrounding hyaluronic acid-type ? collagen matrix,the implanted cells would differentiate into chondrocytes,and effectively repair the chondral defects.After that,we used animal experiments to test the effectiveness and safety of this repair strategy.Concentrated bone marrow cell was extracted at the beginning of the surgery,and mixed with pre-prepared type ? collagen gel.Then,8 milimeter diameter full-thickness cartilage defect in the femoral trochlea of Guizhou minipig was created.Next,oxidized hyaluronic acid was added,followed by cartilage defect injection,the gel was cross-linked by Schiff's base reaction.At 1,3,and 6 months postoperatively,the regenerated tissue was evaluated by magnetic resonance imaging(MRI),macro-and microscopic observation,and histological analysis,respectively.Our data confirmed the feasibility and effectiveness of this one-step repair strategy,and implied its potential in clinical application.Research contentSection ?: The fabrication of oxidized hyaluronic acid-type ? collagen biomimetic gel and it's in vitro testIn this section,type ? collagen hydrogel was extracted from fresh knee cartilage of minipig by pepsin digestion method,and its component identification was carried out by SDS-PAGE.Oxidized hyaluronic acid was fabricated by periodic acid oxidize,component was identificated by fourier infrared spectrum analyzing and toxicity testing was carried out by CCK-8 cytotoxic test.After that,type ? collagen hydrogel was mixed with concentrated bone marrow cell,then oxidized hyaluronic acid was added,type ? collagen cross-linked with oxidized hyaluronic acid by Schiff's base reaction.Finally,the oxidized hyaluronic acid-type ? collagen-bone marrow mesenchyme concentrated cells(BMSCs)complex was constructed.The complex was cultured in vitro to preliminary confirme the feasibility of this strategy.Materials and Methods:1.Fabrication of type ? collagen hydrogel of minipig knee cartilage(1)Acquisition of dry cartilage powderi.Acquisition of fresh knee hyaline cartilage of minipig;ii.Frozen drying of hyaline cartilage;iii.Hyaline cartilage was pulverized into powder by liquid nitrogen cryogenic pulverization.(2)Extraction of type ? collagen hydrogeli.Cartilage powder digested by guanidine hydrochloride;ii.Pepsin digestion,type ? collagen salting out and dialysis.2.Type ? collagen hydrogel identification(1)Molecular weight and purity identification of collagen ? hydrogel by SDS-PAGE;(2)Sterility test of collagen ? hydrogel.3.Fabrication,identification and toxicity test of oxidized hyaluronic acid(1)Periodic acid oxidize hyaluronic acid;(2)Frozen drying of oxidized hyaluronic acid;(3)Fourier infrared spectrum analyzing of oxidized hyaluronic acid;(4)Toxicity test of oxidized hyaluronic acid.4.Oxidized hyaluronic acid-type ? collagen in vitro sol-gel transition and culture(1)Extraction and identification of bone marrow mesenchyme concentrated cellsi.Bone marrow aspiration at crista iliaca of Guizhou mini-pig;ii.Acquisition of concentrated bone marrow mesenchyme cell by Percoll density gradient centrifugation;iii.Isolation,cultivation and expansion of bone marrow mesenchyme stem cells from concentrated bone marrow cell;iv.Identification of differentiation potential of bone marrow mesenchyme stem cells by osteogenesis,adipogenesis and chondrogenesis induction.(2)Oxidized hyaluronic acid-type ? collagen in vitro gelling and testi.Oxidized hyaluronic acid-type ? collagen in vitro gelling;ii.Frozen drying of oxidized hyaluronic acid-type ? collagen gel;iii.Scanning electron microscopy observation of frozen dried gel;iv.Swelling and dissolution test of oxidized hyaluronic acid-type ? collagen gel.(3)In vitro cultured with bone marrow mesenchyme stem cellsi.Fabrication of oxidized hyaluronic acid-type ? collagen-BMSC complex;ii.Harvested after 14 and 28 days' in vitro culture;iii.HE staining and scanning electron microscopy observation.Rsults:SDS-PAGE showed the main component of hydrogel was type ? collagen,no cytotoxicity was found by periodic acid oxidized hyaluronic acid.Oxidized hyaluronic acid-type ? collagen had good gelling ability.Concentrated bone marrow cell can be effectively separate by Percoll density gradient centrifugation.Osteogenesis,adipogenesis and chondrogenesis induction culture showed good differentiation potential of concentrated bone marrow cell.In vitro culture of oxidized hyaluronic acid-type ? collagen-bone marrow mesenchymal stem cell complex showed: seed cells encapsulated in complex displayed round shape and chondrocyte-like morphology,the complex did not show any sign of degradation or change in cell density,indicatin good biocompatibility in oxidized hyaluronic acid-type ? collagen hydrogel.Section ?: Biomimetic gel combined with concentrated bone marrow cell for cartilage repairIn this section,we aimed to test the effectiveness and safety of oxidized hyaluronic acid-type ? collagen-concentrated bone marrow cell complex for cartilage repair via large animal experiments.Eight-milimeter diameter cartilage defect in the femoral trochlea of Guizhou minipig was created.During the repair process,concentrated bone marrow cell was extracted at the beginning of the surgery,and mixed with pre-prepared type ? collagen sol,followed by addition of oxidized hyaluronic acid.Then the mixture was injected into the cartilage defect by one-step method.At 1,3,and 6 months postoperatively,the regenerated tissue was evaluated by magnetic resonance imaging(MRI),macro-and microscopic observation,and histological analysis,respectively.Our data confirmed the feasibility and effectiveness of this one-step repair strategy,and implied its potential in clinical application.Materials and Methods:1.Experimental animals grouping(1)Forty Guizhou mini-pigs(sex: 20 males and 20 females,age: 10-12 months,weight: 40-50kg)were divide into A,B two groups randomly.Twenty knees for each group,40 knees in total;(2)Group A was blank control group(no treatment group),while,group B was experimental group(treated with oxidized hyaluronic acid-type ? collagen-concentrated bone marrow cell complex);(3)Five knees from each group were sampled and analysed at 3 different time point(1,3 and 6 months post-operatively).2.Fabrication of oxidized hyaluronic acid-type ? collagen-concentrated bone marrow cell complex(1)Bone marrow aspiration from minipig at crista iliaca of Guizhou mini-pig;(2)Acquisition of concentrated bone marrow cell by Percoll density gradient centrifugation;(3)Concentrated bone marrow cell mixed with type ? collagen gel;(4)Addition of oxidized hyaluronic acid.3.Animal operation(1)Creation of 8 milimeter diameter cartilage defect in the femoral trochlea of Guizhou minipig;(2)Oxidized hyaluronic acid-type ? collagen-concentrated bone marrow cell complex injection.4.Effect test of cartilage repair(1)MRI scan: MRI scaning(Siemens Tim Trio,Erlangen,Germany)was performed using T1 and T2-weighted,fat suppressed gradient echo sequences.The MRI results were evaluated using a modified MOCART scoring system;(2)Macroscopic observation: to find out if there was joint effusion,synovium edema,patellofemoral joint cartilage abrasion and repair condition of cartilage injure;(3)Specimens harvest and fix: all specimens were fixed in 10% neutral formalin and decalcified by 4% ethylenediaminetetraacetic acid(EDTA)decalcifying solution;(4)Tissue slice and staining: fast green-safranin O staining,masson trichrome staining,toluidine blue staining,immunohistochemical(IHC)for Col-? and Sirius red staining were performed;(5)Semi-quantitative histomorphological evaluation of the quality of the repair tissue was carried out according to a modified O'Driscoll score and modified ICRS macroscopic grading system(Visual assessment scale);(6)Cell imaging: fluorescence in situ hybridization(FISH)of Y-chromosome was performed to detect the if implanted cells stayed in the defect.Results:MRI scoring,macroscopic observation and histomorphological evaluation all showed that experimental group has better cartilage injure repair effect than blank control group.Experimental group has better results in repair tissue cell distribution,surface evenness,integration with adjacent normal cartilage tissue,type ? collagen content,GAG content and better morphology in calcified cartilage zone and subchondral bone zone.Cell imaging showed embedded cells were still in the defect and have self renewing capacity.ConclusionIn conclusion,here we have shown that oxidized hyaluronic acid-type ? collagen combined with concentrated bone marrow cell can be a promising option in the treatment of large full thickness chondral defects,the repair tissue was hyaline cartilage.Embedded concentrated bone marrow cell has self-renewal and chondrogenic differentiation capacity that participated in cartilage defect repair.In future studies,we will investigate the mechanism of this strategy at cellular level,and transfer this protocol into clinical trial when we get the approval of the clinical use of Col-? gel.