USE OF EX VIVO-GENERATED CARTILAGE DERIVED FROM HUMAN BONE MARROW MESENCHYMAL STEM CELLS EXPANDED IN MEDIUM SUPPLEMENTED WITH FGF2 IN TREATING SURGICALLY-INDUCED CHONDRAL DEFECTS IN ATHYMIC RAT KNEE: A STEP TOWARDS TREATING HUMAN OSTEOARTHRITIS

Aim and objectives: The degeneration of articular cartilage in joints leads to osteoarthritis that in turn causes life longpain in joints and, impair the mobility of patients in many cases. The available remedies to treat osteoarthritis are thedrugs to reduce the pain, and or joint replacement. The success of second option is not always easy and comes with a heavy price tag and withpost-surgical complication, in some cases. The resurfacing of knee joint or hip joint cartilage defects with bone marrow-derived mesenchymalstem cells (MSCs) can be a promising opportunity for joint cartilage repair to treat osteoarthritis. The long-term advantage of this research is thattransplantation of cartilage tissue generated ex vivo from MSCs isolated from bone marrow aspirated from the same patient to treat the patient'sknee or hip osteoarthritis, i.e., autologous transplantation that assures low rejection rate. Since MSCs can be expanded through multiplepassages, the cell source can be enormous and the cells can be frozen in liquid nitrogen for long time to coordinate the timing of patient'streatment. To further enhance the MSCs chondrogenic differentiation efciency and transplantability of derived chondrogenic tissue, MSCsmay be pre-treated with certain growth factors, FGF2, or FGF2+WNT3Abefore chondrogenesis begins.Methods: MSCs were isolated from human bone marrow and were characterized as described earlier. Bone marrow was aspirated from patientsundergoing total knee arthroplasty or total hip arthroplasty. Cartilages were generated ex vivo from chondrogenic differentiation of MSCs.Before chondrogenesis began, MSCs were expanded in MSCs medium only (abbr. MSCs_MO group) or MSCs medium containing FGF2 (abbr.MSCs_FGF2 group) or MSCs_FGF2+WNT3A (abbr. MSCs_FGF2+WNT3A group) for one passage. Expanded MSCs were than placed forchondrogenesis for 4 weeks in complete chondrogenesis medium (CCM) on COL II-coated transwell inserts in 24-well plates at high density.CCM was changed every 48hr. Cartilages were characterized by special stainings and immunohistochemistry on parafn-embedded sections,and transmission electron microscopy (TEM) as described earlier (Juneja et al., 2016). Cartilage from each group was transplanted in twofemoral trochlear defects created surgically in the right knee of athymic nude rat (n=7 rat in each group). Knee defects transplanted with bringlue served as control. At 8-weeks post surgery, rats were sacriced, knee decalcied, parafn-embedded sections of knee were assessed forrepair of knee defects using Safranin O staining and COLII immunostaining.Results: Ex vivo-generated cartilages differed in their properties. Hardness of cartilage was in this order in cartilage groups:MSCs_FGF2+WNT3A > MSCs+FGF2 > MSCs_MO. Thickness of cartilage was in this order: MSCs_FGF2+WNT3A > MSCs+FGF2 >MSCs_MO (P<0.001).Proteoglycans in cartilage were in this order: MSCs_FGF2+WNT3A > MSCs+FGF2 > MSCs_MO as determined by toluidine blue staining(P<0.001) as well as by Safranin O staining. All the groups of cartilages showed the presence of positive chondrogenic markers (COLII, COLVI,aggrecan and lubricin) and absence of hypertrophic chondrocyte marker (COL X) as shown by immunohistochemistry. There was nomineralization and apoptosis in cartilages as shown by von Kossa and apoptosis tunnel assay, respectively. TEM showed that chondrocytes incartilages were from 'best to worst' in this order: MSCs_FGF2 > MSCs_FGF2+WNT3A> MSCs_MO. Chondrocyte quality was assessed by thepresence of number of lipid droplets and ovalness of the chondrocyte's nucleus. More the number of lipid droplets in chondrocyte, worst thequality of chondrocyte. More the indented and lobulated nucleus in chondrocyte, worst the chondrocyte. More oval chondrocyte was consideredas best chondrocyte. The transplantation success of cartilages was in order from 'best to worst': MSCs_FGF2 > MSCs_FGF2+WNT3A >MSCs_MO groups. Cartilage transplantation success was based on COLII staining and Safranin O staining of defects region.Conclusion: Ex vivo-generated cartilage, differentiated from MSCs derived from human bone marrow, was able to repair induced-chondraldefects in athymic nude rat knee. Using this method, human osteoarthritis can be treated using patient's own bone marrow derived MSCs.