| BackgroundOsteoarthritis(OA)is a degenerative joint disease characterized by the destruction of the articular cartilage,causing chronic pain and the loss of joint function.OA is the most common disabling factor in the elderly,and there is no cure currently.The clinical treatments nowadays include pain control and the use of supplements.Since OA is a progressive disease,joint replacement surgery is needed when drug treatment cannot relieve joint pain,but the cost of surgery is high and the service life of artificial joints is limited.Therefore,it is urgent to find a new treatment to delay the progression of OA.Extracellular matrix(ECM)is the product of resident cells,containing bioactive substances related to cell proliferation,migration,and adhesion.Decellularized extracellular matrices(d ECM)can be obtained by removing cellular components from tissues in physical,chemical,and other methods.The d ECM has lower immunogenicity,which is beneficial to implant it into allogeneic body.Studies have shown that because d ECM contains most of the structure and proteins from the original tissue,it may be beneficial to damaged tissue and promote vascular endothelial cells growth.At present,cartilage decellularized matrix is mainly used for repairing cartilage defects or scaffolding materials for tissue engineering,but there is little such a research on OA.In this study,we injected the d ECM of cartilage into the knee joint of rabbit OA model to evaluate the efficacy of the knee repair through gross and histology analysis.Objective1.To explore a modified preparation method of cartilage decellularized matrix and evaluate its properties.2.To explore the repaired effect of cartilage acellular matrix on rabbit knee osteoarthritis.Methods1.Rabbit knee cartilage was taken and decellularized by a combination of physical,chemical,and enzymatic methods.Hematoxylin-eosin(HE)staining,4’,6-diamidino-2-phenylindole(DAPI)staining,toluidine blue staining,and immunohistochemical staining were used to evaluate decellularized cartilage.The contents of glycosaminoglycan(GAG)and deoxyribonucleic acid(DNA)before and after decellularization of cartilage were quantitatively detected,and the changes of internal structure before and after decellularization were observed by scanning electron microscope.2.The decellularized cartilage was ground into granules using a tissue grinder,and the size of the cartilage particles was assessed by Alcin blue staining.3.The knee OA model was made by cutting the anterior cruciate ligament of 36 New Zealand rabbits,and six rabbits were randomly selected to verify the OA model 6 weeks later.After successful modeling,the remaining 30 rabbits were randomly divided into five groups(group A,B,C,D,and E).Rabbits in group A,group B,group C,group D,and group E were injected with 200μl of normal saline,200μl of 10%(w/v)cartilage decellularized suspension,200μl of 20%(w/v)cartilage decellularized suspension,200μl of 40%(w/v)cartilage decellularized suspension,and 200μl of 50%(w/v)cartilage decellularized suspension into the knee joints,respectively.Macroscopic and microscopic assessments on the degree of osteoarthritis were performed 12 weeks later.Results1.The decellularized cartilage was similar to the untreated cartilage in appearance and size.HE staining and DAPI staining indicated that the cell and DNA were almost completely removed by the decellularization protocol.The toluidine blue staining indicated that the contents of GAG in acellular group and unacellular group were similar.The immunohistochemical staining indicated that the staining intensity of cartilage collagen II after decellularization was similar to that of undecellularized cartilage which showed that there was no significant change in type II collagen after cartilage decellularization.Quantitative results of GAG and DNA showed that the GAG content per mg of cartilage before and after decellulation were 5.68±0.53μg/mg and5.07±0.16μg/mg,respectively,and there was no statistical difference between them(P>0.05).The DNA contents were 0.90±0.08μg/mg and 0.17±0.02μg/mg,respectively,and the differences between the two groups were statistically significant(P<0.05).Scanning electron microscopy results also confirmed that the cellular components inside the cartilage were lost and the surface structure became loose after decellularization.Its pore density had increased(before decellularization:994.93 n/mm~2,after decellularization was 1615.19 n/mm~2),and the pore diameter had also increased(before decellularization:7.25μm,after decellularization:7.57μm).Imag J software was used to measure the diameter of the particles,and the diameter of the cartilage decellularized particles was 27.97±3.96μm.2.After 6 weeks of modeling,we found that the cartilage continuity of the knee joint in the anterior cruciate ligament group was significantly damaged,and osteophytes were formed on the femoral side,which initially proved that the osteoarthritis model was successfully manufactured.After 12 weeks we found that the osteophytes and cartilage erosion on the cartilage surface of the femoral side of the knee joints in groups B,C,D and E were significantly less than in group A.Histologically,compared with groups B,C,D,and E,it was obvious that the cartilage surface of group A was damaged,the chondrocytes were arranged disorderly,and voids were formed at the boundary between bone and cartilage.Conclusion1.The cartilage decellularization scheme we adopted can almost completely remove chondrocytes and DNA,while more GAG and type II collagen content can be retained,which can be used as an ideal tissue engineering material.2.The cartilage decellularized matrix has a certain therapeutic effect on osteoarthritis,which delays the progress of osteoarthritis and provides a new idea for the treatment of osteoarthritis. |
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