Subhash C. Juneja


Aim and objectives: The degeneration of articular cartilage in joints leads to osteoarthritis that in turn causes life long
pain in joints and, impair the mobility of patients in many cases. The available remedies to treat osteoarthritis are the
drugs 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 with
post-surgical complication, in some cases. The resurfacing of knee joint or hip joint cartilage defects with bone marrow-derived mesenchymal
stem cells (MSCs) can be a promising opportunity for joint cartilage repair to treat osteoarthritis. The long-term advantage of this research is that
transplantation of cartilage tissue generated ex vivo from MSCs isolated from bone marrow aspirated from the same patient to treat the patient's
knee or hip osteoarthritis, i.e., autologous transplantation that assures low rejection rate. Since MSCs can be expanded through multiple
passages, the cell source can be enormous and the cells can be frozen in liquid nitrogen for long time to coordinate the timing of patient's
treatment. To further enhance the MSCs chondrogenic differentiation efciency and transplantability of derived chondrogenic tissue, MSCs
may be pre-treated with certain growth factors, FGF2, or FGF2+WNT3A before chondrogenesis begins.
Methods: MSCs were isolated from human bone marrow and were characterized as described earlier. Bone marrow was aspirated from patients
undergoing 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 for
chondrogenesis 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 parafn-embedded sections,
and transmission electron microscopy (TEM) as described earlier (Juneja et al., 2016). Cartilage from each group was transplanted in two
femoral trochlear defects created surgically in the right knee of athymic nude rat (n=7 rat in each group). Knee defects transplanted with brin
glue served as control. At 8-weeks post surgery, rats were sacriced, knee decalcied, parafn-embedded sections of knee were assessed for
repair of knee defects using Safranin O staining and COL II 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). paragraphs 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 (COL II, COL VI, aggrecan and lubricin) and absence of hypertrophic chondrocyte marker (COL X) as shown by
immunohistochemistry. There was no mineralization and apoptosis in cartilages as shown by von Kossa and apoptosis tunnel assay, respectively.
TEM showed that chondrocytes in cartilages were from 'best to worst' in this order: MSCs_FGF2 > MSCs_FGF2+WNT3A > MSCs_MO.
Chondrocyte quality was assessed by the presence of number of lipid droplets and ovalness of the chondrocyte's nucleus. More the number of
lipid droplets in chondrocyte, worst the quality of chondrocyte. More the indented and lobulated nucleus in chondrocyte, worst the chondrocyte.
More oval chondrocyte was considered as 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 COL II 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-chondral
defects in athymic nude rat knee. Using this method, human osteoarthritis can be treated using patient's own bone marrow derived MSCs.

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