Studies On Multipotency Of Human Subacromial Bursa Mesenchymal Stem Cells And The Role Of PEDF During Osteogenic Differentiation

Mesenchymal stem cells (MSCs) have generated much interest inregenerative medicine due to their multipotential they possess. However,with no specific marker available to identify the cells, the biology ofMSC is still not understood very clearly.Mesenchymal stem cells were found and isolated in a lot of tissues,including bone marrow, tendon, adipose and synovium tissues. Thecharacteristic makes the cells attractive in regenerative medicine. Theapplication of mesenchymal stem cells in regenerative medicine willrequire isolation and identification of the cells from a cell mixture withother cell types. Numerous surface markers have been shown to beexpressed on MSCs, including CD44, CD73, CD105and so on. Thestandard markers for distinguishing these cells from other cell types havenot been established. However, we can use several cell markers togetherto identify mesenchymal stem cells. So, even no specific makers, it can’taffect the applification of mesenchymal stem cells in regenerativemedicine either. Great strides have recently been made in organregeneration utilizing stem cells. As reported, MSCs can be grown on ascaffold or combined with some factors and then inserted into the animalto induce the repair and regeneration of the tissue or organ needed.Rotator cuff injuries are a common clinical problem either as a resultof overuse or aging. Biological approaches to tendon repair that involveuse of scaffolding materials or cell-based approaches are currently beinginvestigated. The cell-based approaches are focused on applyingmultipotent mesenchymal stem cells (MSCs) mostly harvested from bonemarrow. In the present study, we focused on characterizing cells harvested from tissues associated with rotator cuff tendons based on anassumption that these cells would be more appropriate for tendon repair.We isolated MSCs from bursa tissue associated with rotator cuff tendonsand characterized them for multilineage differentiation in vitro and invivo. Human bursa was obtained from patients undergoing rotator cuffsurgery and cells within were isolated using collagenase and dispasedigestion. The cells isolated from the tissues were characterized forosteoblastic, adipogenic, chondrogenic, and tenogenic differentiation invitro and in vivo. The results showed that the cells isolated from bursatissue exhibited MSCs characteristics as evidenced by the expression ofputative cell surface markers attributed to MSCs. The cells exhibited highproliferative capacity and differentiated toward cells of mesenchymallineages with high efficiency. Bursa-derived cells expressed markers oftenocytes when treated with bone morphogenetic protein-12(BMP-12)and assumed aligned morphology in culture. Bursa cells pretreated withBMP-12and seeded in ceramic scaffolds formed extensive bone, as wellas tendon-like tissue in vivo. Bone formation was demonstrated byhistological analysis and immunofluorescence for DMP-1in tissuesectionsmade fromthe scaffolds seeded with the cells. Tendon-like tissueformed in vivo consisted of parallel collagen fibres typical of tendontissues. Bursa-derived cells also formed a fibrocartilagenous tissue in theceramic scaffolds. Taken together, the results demonstrate a new sourceof MSCs with a high potential for application in tendon repair.Bone diseases are a type of common diseases in clinical treatment.Bone diseases can be caused by malnutrition, cancer, infection or genetics.The most common bone disease is osteoporosis. The underlyingmechanism in all cases of osteoporosis is an imbalance between boneresorption and bone formation. An interplay of these mechanismsunderlies the development of fragile bone tissue. Osteogenesis Imperfecta (OI) is a genetic disorder characterized by bones that break easily, oftenfrom little or no apparent cause.Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor found in a wide variety of tissues. Recent findingsindicated that lack of PEDF leads to osteogenesis imperfecta (OI) type VIwhose hallmark is a defect in mineralization. We investigated the effectsof PEDF on human mesenchymal stem cells (hMSCs) and signalingpathways through which PEDF displays its activities in hMSCs. hMSCsincubated in a medium supplemented with PEDF induced expression ofosteoblastic related genes. In addition, PEDF induced alkalinephosphatase activity (ALP) in MSCs at14days of incubation inmaintenance medium; hMSCs incubated in osteogenic medium inpresence of PEDF expressed19%more ALP activity (35.655±1.827U/mg protein, P=0.0.041than cells incubated in the same mediumwithout PEDF supplementation (29.956±2.100U/μg protein). hMSCsincubated in osteogenic medium in presence of PEDF deposited50%more mineral (2.108±0.306OD/ml/well/1x104cells/cm2,, P=0.017) thanMSCs incubated in absence of the protein (1.398±0.098OD/ml/well/1x104cells/cm2) as determined by Alizarin Redquantitation. Reduction in PEDF expression in MSCs by siRNA led todecreased ALP activity (33.552±2.009U/ng protein) of knockdowngroup VS39.269±3.533U/ng protein of scrambled siRNA group,P=0.039) and significant reduction in mineral deposition (0.654±0.050OD/ml/per well/1x104cells/cm2of knockdown group VS1.152±0.132OD/ml/well/1x104cells/cm2of wild type group, P=0.010). DecreasedALP activity and mineral deposition were restored by supplementationwith exogenous PEDF protein. PEDF activated ERK and AKT signalingpathways in MSCs to induce expression of osteoblastic related genes. These data suggest that PEDF is involved in MSCs osteoblasticdifferentiation，mainly on mineral deposition.In summary, the results demonstrate human bursa mesenchymalstem cells(HBDMSC) is a new MSC source with a high potential forapplication in tendon repair. PEDF can regulate osteogenic differentiationmainly on mineral deposition through ERK and AKT signalling pathwayin MSC.