Effect Of Bone Marrow Mesenchymal Stem Cells-derived Extracellular Matrix Scaffold On Chondrogenic Differentiation Of Marrow Clot After Microfracture Of Bone Marrow Stimulation In Vitro

ObjectiveTo evaluate the feasibility and validity of chondrogenic differentiation of marrowclot after microfracture of bone marrow stimulation combined with bone marrowmesenchymal stem cells (BMSCs)-derived extracellular matrix (ECM) scaffold invitro.MethodsBMSCs were obtained and isolated from20New Zealand white rabbits (5-6months old). The3rd passage cells were cultured and induced to osteoblasts,chondrocytes, and adipocytes in vitro, respectively. ECM scaffold was manufacturedusing the3rd passage cells via a freeze-dying method. Microstructure was observedby scanning electron microscope (SEM). A full-thickness cartilage defect (6mm indiameter) was established and5microholes (1mmin diameter and3mm in depth)were created with a syringe needle in the trochlear groove of the femur of rabbits toget the marrow clots. Another20rabbits which were not punctured were randomlydivided into groups A (n=10) and B (n=10): culture of the marrow clot alone(group A) and culture of the marrow clot with transforming growth factor β3(TGF-β3)(group B). Twenty rabbits which were punctured were randomly dividedinto groups C (n=10) and D (n=10): culture of the ECM scaffold and marrow clotcomposite (group C) and culture of the ECM scaffold and marrow clot composite with TGF-β3(group D). The cultured tissues were observed and evaluated by grossmorphology, histology, immunohistochemistry, and biochemical composition at1,2,4and8weeks after culture.ResultsCells were successfully induced into osteoblasts, chondrocytes and adipocytes invitro. Highly porous microstructure of the ECM scaffold was observed by SEM. Thecultured tissue gradually reduced in size with time and disappeared at8weeks ingroup A. Soft and loose structure developed in group C during culturing. Chondroidtissue with smooth surface developed in groups B and D with culture time. Thevolume of groups C and D were significantly larger than that of group B at4and8weeks (P <0.05); group D was significantly larger than group C in size (P <0.05).Few cells were seen, and no glycosaminoglycan (GAG) and collagen type IIaccumulated in groups A and C; many cartilage lacunas containing cells wereobserved and more GAG and collagen type II were synthesized in groups B and D.The contents of GAG and collagen increased gradually with time in groups B and D,especially in group D, and significant difference was found between groups B and Dat4and8weeks (P <0.05).ConclusionThe BMSCs-derived ECM scaffold combined with the marrow clot aftermicrofracture of bone marrow stimulation is effective in TGF-β3-inducedchondrogenic differentiation in vitro.