The Biological Effects Of Platelet-rich Plasma(PRP)on Tendon Stem Cells

The tendinopathy is very common in orthopedic department, sports medicine, rehabilitation, and occupational medicine. Tendinopathy is serious impacted on people’s daily lives, especially affected the athletes on their competitive level by forcing top athletes to retire early. The main clinical manifestations of tendinopathy are pain, tenderness and even rupture in tendon or tendon-bone conjunction, and limited range of motion in the local joint. The most common sites of tendinopathy are the patellar tendon, Achilles tendon, the supraspinatus tendon rotator cuff, the deep flexor tendon and extensor total tendon BDC in humeral epicondyle. All of Plantar fasciitis, Achilles tendinitis, patellar tendinitis, tennis elbow, golf elbow, supraspinatus tendinitis are tendinopathy. Tendinopathy is so common, however, its pathogenic mechanics remain unclear. People always think that acute and chronic injuries of the ligaments and tendons are closely related to tendinopathy.Newly discovered tendon stem/progenitor cells (TSPCs), also known as tendon stem cells (TSCs) or tendon-derived stem cells (TDSCs), with the adult stem cells’ potential ability of self proliferation and adipogenic, chondrogenic, osteogenic differentiation, are the precursor cells of tendon cells because they can directly become tenocytes after culture. Study show that TSCs involve the healing process of acute and chronic tendon injuries and TSCs are the key cells to the repair of tendons and ligaments. Different doses of stimulation of repeatitive cyclic mechanical stress can affect the mechanical biological behaviors of tendon stem cells. Long term repeatitive cyclic mechanical stress may eventaully lead to tendinopathy. Researches show that4%of the mechanical stretching can increase the TSCs proliferation, promot TSCs become tenocytes, while8%of mechanical stretching, however, may induce TSCs differentiate into fat, cartilage and bone cells. Thus, small mechanical stretch (orderly and moderate physical exercise) can promote the the TSCs proliferation and promot TSCs becoming tendon cells, resulting in maintaining the healthy tendons or ligaments. However, large amount of mechanical loading (such as overuse) is harmful to induce TSCs differentiate into non-tendon cells, leading to the accumulation of fat, mucus formation and tendon calcification, which are the typical pathological changes of tendinopathy.The natural mechanism of tendinopathy is still unclear. As a result, the clinical treatment ot tendinopathy is often empirical. The treatments include:rest, reducing the burden of training, massage, cryotherapy, eccentric training, oral medicine of non-steroidal anti-inflammatory drugs (NSAIDs), local injection of platelet-rich plasma (PRP), cell therapy, extracorporeal shock wave therapy (ESWT), and injection of glucocorticoid. Surgical treatment may be given to the patients who are unsatificiant with the conservative treatment or clinical symptoms persist heavier. Among These treatments, local injection of PRP, because of its autologous source, simplely prepair, PRP gel, the safety of clinical use, convenient usage, and economic advantages, are welcomed by both doctors and patients. Therefore, we should pay more attention to the clinical application of PRP injection to treat "tendinopathy".We obtain PRP by centrifugation of whole blood and platelet-rich plasma contains concentrated platelets over baseline levels. Generally, only at least3-5times platelets contration in plasma can be named as PRP. When PRP is activated, the platelets in PRP release a large amount of growth factors, including platelet-derived growth factor (PDGF), transforming growth factor-(3(TGF-β), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF),endothelial cell growth factor (EGF), platelet-derived endothelial cell growth factor (PDEGF), insulin-like growth factor (IGF). These growth factors play very important roles in the proliferation and differentiation of cells, which can stimulate nurovascularogenosis of the injured tissue, increase the blood supply and nutrition to repair or regenerate the injured tissue.In vitro studies have found that The PRP promotes the TSCs proliferation and accerate tendon cells formation. Animal studies have also confirmed the results, and some clinical trials have verified that PRP can promote the healing of acute and chronic injuries of the tendons and ligaments, and effectively treat tendinopathy. However, the method of PRP local injection to treat acute/chronic tendon/ligament injuries and tendinopathy is not always satisfactory, a number of clinical trials have found that PRP treatment is useless in repair of acute/chronic tendons/ligaments injury and tendinopathy. Therefore, the clinical application of PRP in treatment of tendinopathy needs further basic and clinical research.Actually, we know that PRP has no strict definition and PRP is individually prepared. As a result, PRP varies from person to person, and in some circumstances, PRP cannot be prepared in some patients. After carefully analyzing, we know that there are4kinds of different PRP in clinical using. Two of them are with leukocytes while the other two are without leukocytes. They are P-PRP, P-PRF, L-PRP, and L-PRF, respectively. Some researchers indicated that leukocyte-rich PRP caused a much greater acute inflammatory response than leukocyte-poor PRP. Therefore, we hypothesizes that L-PRP retards the process of tendon and ligament healing based on the leukocytes in PRP have negative effects on TSCs.This study is divided into three parts, including (1) tendon stem cells isolation, culture and identification;(2) The preparation of P-PRP and L-PRP and the effects of P-PRP and L-PRP on TSCs proliferation, differentiation and cell mechanics;(3) Construction of P-PRP gel-TSCs complex and tracking TSCs in vitro and in vivo. We confirm the above hypothesis in order to better apply PRP to treat acute or chronic tendon injuries and tendinopathy, theoretically and clinically.Part one Rabbit tendon stem cells isolation, culture and identificationIsolation and identification of rabbit tendon stem cells (TSCs) and assess their stem cell properties in order for preparation of the next experiment. The study includes:(1) TSCs of isolation and culture;(2) the ability of colony-forming and proliferation of TSCs;(3) flow cytometry tests and immunofluorescence identification of TSCs;(4) determination of adipogenic, chondrogenic osteogenic differentiation potentials of TSCs.The protocol for obtaining TSCs from rabbits was approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh (IACUC Protocol number:1105545A). The cells were isolated from3months old healthy male New Zealand rabbits and cultured at37℃in5%CO2. Then cell culture, colony formation, stem cell identification and potentials abilities of adipogenic, chondrogenic, osteogenic diferentiation of P3cells were observed.Clones formation of the tendon-derived cells (TDCs) was seen in7days culture after isolation. TDCs were strongly expressed the stem cell surface markers of CD90and CD44by flow cytometry. The stem cell markers (NS, Nanog, Oct-4, SSEA4) were detected by immunofluorescence microscopy. The results showed that80%of the TDCs cells were positive stainning immunofluorescence method. The proliferation of TDCs became slow, and lipid droplets were gradually found in TDCs after adipogenic induction. After adipogenic induction for4weeks, the lipid droplets increased and oil red-O staining was seen in red in the TDCs. After chondrogenic inducing differentiation for4weeks, the TDCs are stained in red by Safranin-O staining. After osteogenic differentiation for4weeks, the calcium deposition formation and the mineralized nodules of the calcium deposits of TDCs were seen in red by Alizarin red S staining.The TDCs in this study were tendon stem cells (TSCs), because they had colony forming and the rapid proliferation abilities; positive expression of cell stem cell surface antigens of CD90, and CD44, while negative expression of CD34; positive expression of stem cell-specific markers of NS, Nanog, SSEA4and OCT-4; and multiple differentiation potentials of adipogenic, chondrogenic and osteogenic differentiation in the condition of adipogenic, chondrogenic, osteogenic induction. These TSCs can be used for the next step research. Part two Preparation of P-PRP and L-PRP and the effects of P-PRP and L-PRP on TSCsIn order to clarify whether the efficacy of PRP were impacted by leukocytes in PRP, we prepared P-PRP and L-PRP, and analysed the role of P-PRP and L-PRP on TSCs.We withdrew fresh whole blood in heart of3-month-old healthy male New Zealand rabbit right after euthanasia (IACUC Protocol number:1105545A) and keep this fresh blood in tubes containing3.8%sodium citrate at0℃. Carefully inserted6mL blood in the tube contained6mL Polymorphprep (Accurate Chemical&Scientific Crop NY) and centrifuged (400g,30min) to prepare P-PRP and L-PRP. The leukocytes in P-PRP and L-PRP were counted with an automated cell counter (Nexcelom Bioscience LLC, Lawrence, Massachusetts). The P-PRP and L-PRP were activated by22mM CaCL2and stored at4℃until use. P-PRP medium, L-PRP medium and normal medium were prepaired. The media contained the same ingredients (20%fetal bovine serum,100U/ml penicillin,100U/ml streptomycin, low sugar DMEM) apart from the deferent contration of P-PRP or L-PRP. The cytokines in the three kind of media were analysed by ELISA. TSCs were seeded in6-well plate (10000per well) and cultured in the complete media containing different concentration of P-PRP, L-PRP (0%,2%,5%,10%) at37℃in5%CO2.4days later, count the TSCs with an automated cell counter (Nexcelom Bioscience LLC, Lawrence, Massachusetts) and calculate the PDT of different groups and observe the stem cell characteristic surface markers of each groups by flow cytometry. Multi-differentiation potentials of P-PRP treated, L-PRP treated and non-treated TSCs were examined by adipogenic, chondrogenic, and osteogenic induction for28days. The gene expression of TSCs affected by leukocytes in PRP were analysed by real-time Quantitative RT-PCR (qRT-PCR). The cell force of TSCs affected by P-PRP and L-PRP were teseted by CTFM. The TSCs were cultured in growth medium only (10%FBS+DMEM), or treated with10%PRP (PRP group), or10%buffy coat (WBC group), or5%buffy coat+5%PRP (WBC+PRP group). After4days in culture, the expression of COX-1and COX-2genes, collagen type Ⅰ gene, and MMP-13gene was analyzed using real time qRT-PCR.After centrifugation (400g,30min), the rabbit fresh whole blood was separated5levels and we got high concentration of PRP (P-PRP and L-PRP). The platelets in the PRP was1.44×108±0.84×108/mL (P<0.05), compared with whole blood, the platelet concentrate was3.25±0.73. The leukocytes in L-PRP were significant different from in P-PRP (0.66×105±0.36×105/mL in P-PRP and3.28×105±1.22×105/mL in L-PRP, P=0.019, n=8). The concentration of these growth factors in P-PRP group was much higher than in control group and L-PRP group especially the concentration of EGF, TGF-β1, VEGF in L-PRP group was lower than in control group indicated that leukocytes might inhibit PRP to release growth factors (P-PRP and the comparison with the L-PRP medium, P<0.05, n=3); Unattached TSCs increased in L-PRP group; The stem cell surface markers of CD44and CD90increased in P-PRP group while decreased in L-PRP group (P<0.05, n=3); After counting the TSCs cultured in the complete media containing different concentration of P-PRP, L-PRP (0%,2%,5%,10%) at37℃in5%CO2for4days, we got the PDT of different groups. We found that PDT was PRP and leukocyte dosage sensitive. When increased the concentration of P-PRP or decreased the concentration of L-PRP, the PDT shortened (within the group, and comparison between groups, P<0.05, n=3); After21days induction, all TSCs in P-PRP, L-PRP and control groups had been successfully inducted. However, the positive staining cells were much more in L-PRP group than in P-PRP group and control group. Significant difference of adipogenic, chondrogenic and osteogenic induction was seen in L-PRP group compared with P-PRP and control groups. While when TSCs were cultured in chondrogenic induction media, the positive staining cells in P-PRP group were much more than in control group, however, no significant difference of chondrogenic induction in P-PRP group compared with control groups (in groups, P<0.05, n=3); All of the TSCs of control group, P-PRP group and L-PRP group at day4and day14were tested by qRT-PCR. All of the genes were closely related with the concentration of P-PRP and L-PRP. Both on d4and d14, the higher concentration of L-PRP, the lower gene expression of Nanog and Oct4, on the contrary, the higher concentration of L-PRP, the higher gene expression of PPARy, SOX-9, Runx-2, Osteocalscin and mPGEs. Significant difference was seen in L-PRP group and between P-PRP and L-PRP groups (P<0.05, n=3); the cell traction force (CTF) of TSCs treated with P-PRP or L-PRP and control were analyzed. We found that the CTF in L-PRP group was much smaller than both in P-PRP group and control group. Significant difference was seen between L-PRP group and P-PRP group or control group (P<0.05, respectively). However, although CTF of TSCs in P-PRP group was much stronger than in control, there was no significant difference between the two groups (P>0.05)(n=5); both COX-1and COX-2gene expression were markedly increased in WBC treatment group, and this high level of gene expression was significantly reduced by PRP treatment. Additionally, while the expression of collagen type Ⅰ (Col-Ⅰ) was up-regulated in PRP treated cells, its expression levels were completely suppressed by WBC treatment. Finally, WBC treatment significantly increased MMP-13expression, and this increase was significantly reduced by addition of PRP to cell cultures.In short, we can obtain high concentration of PRP (P-PRP and L-PRP) by one time centrifugation and the leukocytes in L-PRP were significant different from that in P-PRP. L-PRP, a kind of PRP with leukocytes, has no benefits to tendon healing since L-PRP inhibits TSCs proliferation, accelerates TSCs differentiation, and induces TSCs to become fragile. WBC treatment also markedly increased both COX-1and COX-2gene expression, and this high level of gene expression was significantly reduced by PRP treatment. Additionally, while the expression of collagen type Ⅰ (Col-Ⅰ) was up-regulated in PRP treated cells, its expression levels were completely suppressed by WBC treatment. Finally, WBC treatment significantly increased MMP-13expression, and this increase was significantly reduced by addition of PRP to cell cultures. These findings support the clinical findings of tendinopathy, and therefore, we can reasonably infer that the P-PRP can promote the healing of tendon and ligament while L-PRP may inhibit the healing of tendon and ligament. Therefore, we should use the P-PRP instead of the leukocyte-containing L-PRP to treat tendinopathy. Part three Construction of P-PRP gel-TSCs compdsite and tracking TSCs in vitro and in vivoThe aim of this study was to determine the effects labeling by super-paramagnetic iron oxide (SPIO) nano-particles on TSCs’functions and the role of P-PRP gel comining with TSCs in repair of tendon defects.Rabbit TSCs were labeled by incubation with50μg/ml of SPIO. Labeling efficiency, cell viability, and proliferation were then measured, and the sternness of TSCs was tested by quantitative real time RT-PCR (qRT-PCR) and immunocytochemistry.We found that the efficiency for labeling TSCs reached as high as98%, and that labeling at50μg/ml of SPIO did not alter cell viability and cell proliferation compared to control cells without labeling. Moreover, the expression levels of stem cell markers (Nucleostemin, Nanog, and Oct-4) did not change in SPIO-labeled TSCs compared to non-labeled cells. Both labeled and non-labeled cells also exhibited similar differentiation potential, as evidenced by the expression of PPAR, Sox9, and Runx2, which are markers of adipogenesis, chondrogenesis, and osteogenesis, respectively. Both in vitro and in vivo tests demonstrated that labeled TSCs could be detected by MRI. Positive Prussian blue staining and collagen I expression of TSCs was found in vivo3weeks after repair.The findings of this study show that labeling TSCs with SPIO particles is a feasible approach to track TSCs in vivo by MRI so that the cells’ role in the repair of injured tendons can be monitored noninvasively. Additionally, MRI tracking in our study showed that labeled TSCs remained in the tendon defect site without migrating into surrounding host tissues two and three weeks after implantation. The finding suggests that PRP is an excellent scaffold; it is known to have chemoattractant property and, as a result, can keep implanted cells in the right place. Therefore, PRP, as an autologous blood product, may be used as an excellent scaffold in TSC therapy or tendon engineering for effective repair of injured tendons.