Repair Of Rabbit Knee Joint Osteochondral Defects With Multilayer PLGA Scaffolds And Platelet Rich Plasma

Articular cartilage and subchondral bone and its border histology structure are different,at the junction of bionic design conforms to the cartilage and bone tissue regeneration need stents may be achieved the integration of bone cartilage reconstruction is an important research content.Research gradually confirmed the platelet rich plasma(platelet rich plasma,PRP)in tissue regeneration of bone,cartilage,ligaments,and autologous origin,the advantages of high safety and easy to obtain,platelet rich plasma is favor by related researchers.Study confirms that PRP promote progenitor cells within the subchondral bone of emigration and differentiation into cartilage;Ectomesenchymal stem cells can also promote the proliferation and differentiation into cartilage cells;PRP ectomesenchymal cells can form joint bone tissue.PRP liquid characteristic determines its weakness is a lack of mechanical strength,if three layers can be different from synthetic aperture PLGA scaffolds and multi-directional differentiation potential of bone marrow mesenchymal stem cells between jointly build osteochondral composite,and put it in a joint osteochondral defect model,could be more beneficial to regenerative repair of the organization.By optimizing the design of three layers PLGA scaffolds may be conducive to seek a higher degree of bionic bone cartilage scaffold for bone tissue engineering cartilage defect repair methods.Objective1.According to the rabbit knee joint cartilage and border area without hole glue layer thickness design PLGA scaffolds.Upper PLGA scaffolds for cartilage layer thickness(0.3 mm),in the middle of the simulated osteochondral PLGA membrane boundary layer thickness(0.3 mm),bone of the lower thickness(3.4 mm).PLGA scaffolds without hole glue layer height 4 mm.Rabbit experiment preparation is difficult to repair 4 mm in diameter of cartilage and subchondral bone defect.Preparation of osteochondral defect in the depth of 4 mm.PRP was prepared and was load on PLGA scaffolds,building PLGA/PRP complex and injecting PRP to repair osteochondral defect in knee joint.Related tests in order to investigate nonporous glue layer PLGA and PRP effect in the New Zealand rabbit model of osteochondral defect repair;Preliminary clarify PRP role in the repair of rabbit knee joint osteochondral defect model.2.Through the acquisition of New Zealand rabbit bone marrow between bone marrow mesenchymal stem cells after amplification in vitro as the seed cells.Using improved centrifugal tube,by several times with the centrifugal method of vaccination in nonporous adhesive layer PLGA scaffolds building PLGA/BMSCs complex,preliminary exploration by several times with centrifugal inoculation of adhesive layer of PLGA scaffolds vaccination in feasibility;A preliminary exploration of BMSCs induced into cartilage environment in the body and the osteogenesis effect;Clarify nonporous glue layer PLGA scaffolds/BMSCs complex and PRP injection whether enhance the regeneration of the subchondral bone and cartilage repair.3.By adjusting the aperture of PLGA scaffolds glue layer,porous PLGA scaffolds and nonporous glue layer PLGA scaffolds,preliminary discussion more porous PLGA scaffolds and nonporous glue layer PLGA of intra-articular injection of PLGA scaffolds combined PRP effect differences in bone cartilage defect repair.Method1.Normal temperature molded particle leaching preparation technique was used to prepare nonporous glue layer PLGA scaffolds.PGA and PLG materials(75:25)soluble in methylene chloride with NaCl crystal hole agents after mixing.Put stents mould,mould pressing forming,mold release.Nonporous glue layer PLGA scaffolds for height is 4 mm;Bracket consists of three layers;Respectively the upper layer of cartilage thickness(0.3 mm),in the middle of the simulated osteochondral PLGA membrane boundary layer thickness(0.3 mm),bone of the lower thickness(3.4 mm).PLGA scaffolds for 4 mm in diameter.The preparation of adhesive layer of PLGA scaffolds of cartilage and bone porosity is 92%,cartilage segment aperture 50-100 microns,bone segments of pore size is 300-450 microns.75%of the alcohol sterilization stents.Preoperative one hour before the second centrifugation preparation of PRP,manual counting the platelet concentration.Infiltrating method preparation of the drug-loaded PLGA/PRP complex,activate the PRP ClCa2.Within New Zealand rabbit bilateral femoral condyle in the preparation of osteochondral defects(4 mm in diameter,depth of 4 mm)model.Experimental group(PLGA load PRP + articular cavity injection PRP),control group 1(blank PLGA implant group)and control group 2(defect of articular cavity injection of PRP.4 weeks,12 weeks and 24 weeks after surgery in morphology,histology,Q-polymerase chain reaction(PCR)on Collagen type ?,Aggrecan,Collagen type I,Collagen type X expression,Micro-CT scan evaluation.2.Bone marrow bone marrow puncture method of experimental animals,gradient centrifugation BMSCs,adherent culture,extend,using three generations of BMSCs as seed cells.Preparation of 3-4 x106/ml BMSCs suspension,and by using fractional centrifugal method will glue layer cells inoculated with no hole PLGA scaffolds layers of cartilage and bone morphogenic section,electron microscope scanning cell adhesion,small animal imaging testing the distribution of cells within the PLGA scaffolds.Experimental animals of autologous PRP infiltrating hole without glue layer PLGA/BMSCs complex,joint puncture injection of PRP.After 12 weeks of gross morphology,toluidine blue and red O-solid green,type ? Collagen staining histology detection,such as the Q-polymerase chain reaction(PCR)on Collagen type ? expression,Micro-CT scan evaluation.3.The nonporous glue layer PLGA scaffolds preparation with the above method.Preparation process of porous glue layer PLGA scaffolds specific as follows:if the above method,PLGA scaffolds for cylindrical height is 4 mm;Cartilage and subchondral section with the height of the support structure,pore size and porosity.In the middle of the porous structure of aperture is 100-150 microns,the preparation into one of the three layers of PLGA scaffolds.Electron microscope scanning glue layer structure.With the PRP preparation of the drug-loaded PLGA/PRP complex + PRP articular cavity injection,the implanted into New Zealand rabbit bone cartilage defects,followed up for 4 weeks,12 weeks and 24 weeks postoperatively,gross appearance assessment;His red-solid green,toluidine blue O,? collagen dyeing histological staining assessment;Micro-CT scan evaluation.Result1.According to the method of experimental nonporous glue layer PLGA scaffolds.Platelet concentrations in PRP was 1648.19±76.84 x103/mu l,platelet concentration for preparation before the whole blood platelet concentration of 5.1 times.Gross score:4 w:PLGA/PRP + PRP injection group is 9.67±0.58,PLGA group is 8.00±1.00,PRP injection group is 7.00±1.00.12 w:PLGA/PRP + PRP injection group was 13.67±0.58,PLGA group is 9.33± 0.58,PRP injection group was 8.67± 0.58.24 weeks:PLGA/PRP + PRP was 14.67 ± 0.58;PLGA/PRP + PRP group were higher than that of PLGA and PRP injection group all time points(4 weeks,12 weeks,24 weeks)(p<0.05).Histologic evaluation:4 weeks:Histological score of PLGA/PRP + PRP group was 18.33±0.58,histological grading of PLGA group was 14.67±0.58,histological grading of PRP injection group was 16.67±0.58.12 weeks:Score of PLGA/PRP + PRP group was 22.00±1.00,Score of PLGA group was 16.33±0.58,Score of PRP injection group was17.67±0.58.24 w:Score of PLGA/PRP + PRP injection group was 23.08±1.00;Score of PLGA group was 18.00±1.00;Score of PRP injection group was 19.33±0.58.Each time point(4 weeks,12 weeks,24 weeks)score in PLGA/PRP+PRP group were higher than that of PLGA and PRP injection group,type ? collagen expression in PLGA/PRP + PRP group was most obvious.Q-PCR detection:Expression of type ? collagen and aggrecan PLGA/PRP + PRP group was higher than the PRP injection group at each time point(12 w and 24 w).Micro-CT scan:The number of mineralized tissue in subchondral bone area increased in 24 weeks.12 w:BV/TV in PLGA/PRP + PRP injection group was(20.50+1.29)%,BV/TV in PRP injection group and PLGA group is(15.75+1.71)%and(17.0 + 2.94)%.BV/TV of PLGA/PRP + PRP group was obviously higher than the other two groups(P = 0.000).24 w:BV/TV of PLGA/PRP + PRP injection group was(42.25 + 2.63)%,BV/TV of PRP injection group and PLGA group were(34.75 + 5.73)%and(28.75 + 5.96)%.2.Cell culture and vaccination:three generations of BMSCs for long shuttle line pit shape distribution.Cartilage layer of adhesive layer of PLGA scaffolds and osteogenesis period of centrifugal inoculation cell percentage respectively(83.5±2.20)%and(80.5±2.50)%.Cartilage layer and the distribution of section by inoculation cell count to 3.34 x105 x106/ml and 3.22 ml.Electron microscope visible cartilage layer and the distribution of section material inside.There are a large number of cells and adhesion(figure).PKH26 fluorescent tags of BMSCs have focused on PLGA scaffolds.General assessment:A large number of new tissue was observed at 12 weeks after surgery.The surface was relatively smooth and the defect boundary was fuzzy.The score of PLGA/BMSCs complex embedded joint defect after intra-articular injections of PRP was 14±0.58 at 12 weeks after surgery.Histologic evaluation:Fibrocartilage was visible in the central region of the defects.pit defect more peripheral cartilage,cartilage area gradually reduced from edge of defects to the center.The migration trend of cartilage cells was obvious.Cartilage matrix gradually reduced,the center area is type II collagen expression.Scaffolds are still residual in the middle area.Histological score of PLGA/BMSCs complex embedded PRP injections group was 21.80±0.58.The relative expression level of type II collagen were no obvious difference between PLGA/PRP +PRP group and PRP injection group.Micro-CT scan:BV/TV was(30.0+3.37)%in PLGA/BMSCs +PRP injection group at 12 weeks and it was more than the group of PLGA/PRP complex +PRP injection group(20.50+1.29)%(P = 0.002).In accordance with the above method for porous and nonporous glue layer of two different specifications of the PLGA scaffolds.Cartilage aperture 50-150 microns,bone segments of pore size is 300-450 microns;Glue layer is respectively 100-150 microns the PLGA membrane structure of porous structure and has no holes.General evaluation:The defect on the surface of the two groups was smoothed at 4 weeks.There was part of surface subsidence.The boundary is more obvious.The defect surface of nonporous glue layer group was smooth at 12 weeks.The defect surface of defect repair in porous glue layer group was not smooth.The eage was more obvious.There was collapse in centere and margin for new transparent cartilage in eage of nonporous glue layer group at 24 weeks.New tissue at the center of the subsidence is less in the group of porous glue layer group.The histologic score:The histologic score was 10.67±0.58 in the nonporous glue layer group.The histologic score was 10.33±0.58 in the nonporous glue layer group at 4 weeks.The histologic score was 13.67±0.58 in the porous glue layer group at 12 weeks.The histologic score was 13.33±0.58 in the porous glue layer group at 12 weeks.At 24 weeks,the histologic score was 11.67±0.58 in the nonporous glue layer group and 12.33±0.58 in the porous glue layer group.There was no difference between the two groups.The porous glue layer group score tend to be higher than nonporous glue layer group.Histologic evaluation:The cartilage and subchondral bone tissue regeneration repair gradually gets better in the PLGA/PRP+PRP injection over time.Porous glue layer material residual gradually reduce and subchondral bone area decreased significantly at 24 weeks.The residual early(4 weeks)to maintain hole defect area level in performance is a bit poor in the porous glue laye group.Histologic grading:At 4 weeks,the score was 14.00±1.00 in the nonporous glue layer group and was 13.33±0.58 in the porous glue layer group.At 12 weeks,the score was 15.67±0.58 in the nonporous glue layer group and was 15.67±0.58 in the porous glue layer group.At 24 weeks,the score was 22.67±0.58 in the nonporous glue layer group and was 21.67±0.58 in the porous glue layer group.No obvious difference was found between two groups(P = 0.101).Type II collagen expression level relatively follow-up test results found that the type II collagen by defect edge toward the center area increased gradually.The distribution of type II collagen was uniform.The trend of two groups was basically identical.There are still a little residual of PLGA membrane in the nonporous glue layer group at 24 weeks.Micro-CT scan:The mineralized tissue is less in the center area between the two groups.Two groups of the subchondral bone area of bone regeneration are relatively few.Conclusion1.Injection of PRP can promote repair articular cartilage and subchondral bone defect.The nonporous glue layer PLGA scaffolds loaded autologous PRP and the injection of RPP can repair the defect of articular cartilage and subchondral bone,and the regenerative effect can be further strengthened.2.By several times with centrifugal inoculation method is a suitable method for the nonporous glue layer PLGA scaffolds and the inoculation efficiency was highe.The nonporous glue layer PLGA scaffolds with autologous BMSCs and PRP can further enhance the subchondral bone defect area of freshman mineralization ability of the organization.3.The nonporous glue layer PLGA and the porous glue layer PLGA scaffolds loaded with PRP promote the regeneration of the articular cartilage.The degradation of PLGA nonporous glue layer was slower.The mechanical support strength of the porous glue layer PLGA scaffold was poor at the early stages and was easy to collapse.The glue layer of PLGA scaffold need to be further optimized.Porous PLGA scaffolds osteogenesis layer should be optimized to enhance bone regeneration.