Mircro-Injury Repair Of Proliferation And Differentiation Of Rat Tendon Stem Cells Induced By Platelet-Rich Plasma

Tendinopathy is one of common diseases of orthopedics surgery, sports medicine andprofessional injury. According to statistics, there are at least30,000,000tendinopathy casesper year. A great deal of research shows that tendon micro injury induced by overloadingand its immature repair constitutes the direct reason for tendinopathy, but the exactpathogenesis is still not clear. Clinical treatment for tendinopathy mainly includes physicaltreatment, repair and reconstruction. Repair surgeries include local lesions debridement,etc.; Reconstruction surgeries are mainly aimed at unsuccessful tendinopathy repair, andreconstruction measures at present have authotransplantation, homologous transplantationand tissue engineering of tendon, etc. The common treatment problem lies in lack for cellsof strong repairing strength and micro environment promoting tendon repair.Recently, with the development of research on cells and stem cells treatment, celltherapy and stem cell therapy haven been administered to promote tendon repair. These cellsinclude fibroblast in tendon, ESCs, BMSCs, and ADSCs and so on. In2007, Bi et al.identified a unique cell population, termed tendon stem/progenitor cells (TSCs), withuniversal stem cell characteristics. In physical condition, TSCs can be differentiated andproliferated into tenocytes; in pathological condition, TSCs can be differentiated intoosteocyte, chondrocyte, adipocyte and tenocyte; TSCs plays an essential role in repairingtendon injury and maintaining tendon micro environment; the discovery of TSCs definitelyprovides the possibility of applying stem cell therapy to treat tendinopathy.Tendons are bands of connective tissues that function to transmit muscular forces tobones, thus enabling joint movements. As a result, tendons are subjected to largemechanical loads and are frequently injured, especially in athletic settings. Because they areliving tissues, tendons respond to mechanical loads by changing their metabolism, and withtime this mechanobiological response leads to changes in their structural and mechanical properties. Therefore, it is of great interest to understand how the newly discovered TSCsrespond to mechanical loading so that how these loading-induced physiological changesoccur can be better understood.The autologous platelet-rich plasma fraction of blood contains a myriad of growthfactors with the potential for tissue healing, and many, including growth factors likeplatelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforminggrowth factor-beta1(TGF-β1), and insulin-like growth factor (IGF-I), are increased intendons during healing. Autologous platelet-rich plasma (PRP) has been used to promotethe healing of tendons or ligaments. Active releasate of PRP (PRCR) can stimulate tendonstem cell proliferation and collagen production in vitro, in addition to increasing thedeposition of a collagen-rich extracellular matrix. Collectively, these studies havedemonstrated that PRCR treatment in vivo can increase tenocyte number and the productionof collagen type I and III, the main components of tendon. Treatment with PRCR followinglarge mechanical loading stress may also regulate the differentiation of TSCs towardnon-tenocyte lineages known to impede healing, but this has not been directly tested.Objectives1. To find that TSCs have the universal characteristics of stem cells, including clone,proliferation and multi-directional differentiation, by the way of separating and identifyingrat TSCs;2. To make clear the influence of mechanical stretch on TSCs proliferation anddifferentiation;3. To investigate the influence of platelet-rich plasma on TSCs proliferation anddifferentiation;4. To analyze effect of conbining platelet-rich plasma and TSCs on tendon acutechronic injury;5. To provide a new approach for tendinopathy repair through combining platelet-richplasma and TSCs.Methods1. Isolation and characterization of mltipotent rat tendon stem cells: Tendon stem cells(TSCs) were isolated from the Achilles tendons of Sprague Dawley rats8-week-old.Growth curves of passage0were tested. Then we investigated their clonogenicity, and also the change in cell morphology at different passages. Expression of stem cell markers ormarkers of different cell lineages was examined by fluorescence-activated cell sorting(FACS) analysis and immunocytochemical staining. The osteogenic, adipogenic, andchondrogenic differentiation potential of cells were investigated using histology,immunohistochemical staining, and gene expression analysis.2. Mechanobiological response of tendon stem cells: Firstly, we designed a new cyclicstretching system of TSCs in vitro according to the orientation and stressing type of TSCsin vivo and evaluate its effects. Using this in vitro system, we systemically investigatedTSCs morphology change, proliferation, cycle and apoptosis, following intense mechanicalstretch. Morever, the expression of cell marker genes was investigated by quantitativereal-time RT-PCR analysis.3. Effects of PRP for proliferation and differentiation of TSCs: We measuredproliferation, differentiation, and gene expression in cultured adult rat tendon stem cells(TSCs) following intense mechanical stretching and incubation in control medium ormedium supplemented with PRP (2%or10%). Western blotting was used to measure theexpression of the tenocyte markers collagen type I and collagen type III, the adipocytemarker peroxisome proliferator-activated receptor gamma (PPARγ), the chondrocytemarker Sox9, and the osteocyte marker Runx2. In addition, the cell fractions expressingPPARγ, Sox9, or Runx8were determined by flow cytometry. Release of TGF-β1and VEGFwas measured by ELISA.4. Injured rat Achilles tendons were treated with botulism toxin to create amechanically unloaded condition (unloaded) or left untreated (loaded), and then treatedwith phosphate-buffered saline (PBS), plasma-rich platelets (PRP, tendon stem cells (TSCs),or a combination (TSCs+PRP). mRNA and protein expression of collagen I, collagen III,tenascin C, and Smad8were determined by real time PCR and immunostaining,respectively.5. Chronic tendinopathy was created in the left Achilles tendon of26rats by overuserunning. Twenty-four rats were randomly allocated into three groups of6and receivedcontrol treatment; PRP; TSCs; and PRP+TSCs. For cell tracking in vivo, the remaining twoanimals were injected with TSCs infected with lentivirus-GFP. Rats were sacrificed at4and8weeks after the therapeutic injection, and tendon tissue was analyzed by histology, immunostaining, and biomechanical testing to evaluate tissue repair.Results1. We have successfully isolated a unique cell population with stem cell characteristicsfrom adult rat Achilles tendon tissue. These tendon-derived cells had universal MSCcharacteristics, including clonogenicity, high proliferative potential at low seeding density,MSC marker expression, and multidiffer-entiation potential including osteogenesis,adipogenesis, and chondrogenesis.2. In response to4%or8%mechanical stretching, TSCs became highly elongated andaligned along the microgrooves in the axis of stretching. Mechanical stretching increasedTSCs proliferation in a stretching magnitude-dependent manner. Moreover, low mechanicalstretching at4%promoted differentiation of TSCs into tenocytes, whereas large stretchingat8%induced differentiation of some TSCs into adipogenic, chondrogenic, and osteogeniclineages, as indicated by upregulated expression of marker genes for adipocytes,chondrocytes, and osteocytes.3. PRP markedly increased TSC number and the expression of collagen type I,collagen type III, and TGF-β1, indicating that PRP treatment following mechanical stresspromoted tenocyte differentiation. In contrast, the cell fractions expressing PPARγ, Sox9, orRunx2were significantly reduced by PRP treatment. Moreover, PRP reduced lipidaccumulation in TSC-derived adipocytes, mucoid formation in TCS-derived chondrocytes,and mineral deposition in TSC-derived osteocytes.4. TSCs+PRP treatment has synergistic effects on the healing of acute tendon injuryunder loading conditions. Loading promotes the proliferation and differentiation of TSCsinto tendon cells, and improve tendon repair, and type I, III collagen and Smad8expressionare increased.5. TSCs+PRP treatment improved tendon remodeling, histological outcomes, collagencontent, and tensile strength of tendinopathic Achilles tendons. Injected TSCs+PRP wereintegrated into tendon matrix and could be tracked up to8weeks in vivo.Immunohistochemistry showed that it improved type I and III collagen expression inrepaired tendon.Conclusion1. The successful isolation of tendon-derived stem cells under the optimized growth and differentiation conditions was useful for future stem-cell-based tissue regenerativestudies as well as studies on their roles in tendon physiology, healing, and disorders usingthe rat model.2. Low mechanical stretching may be beneficial to tendons by enabling differentiationof TSCs into tenocytes to maintain tendon homeostasis. However, large mechanical loadingmay be detrimental, as it directs differentiation of TSCs into non-tenocytes in tendons, thusresulting in lipid accumulation, mucoid formation, and tissue calcification, which aretypical features of tendinopathy at later stages.3. Platelet-rich clot releasate may promote tendon healing in vivo by inducing tenocytedifferentiation while suppressing the adipocyte, chondrocyte, and osteocyte lineagesbelieved to impede healing.4. TSCs+PRP may be a useful treatment of Achilles tendinopathy.