Objective To explore the effect of the platelet-rich plasma (PRP) on proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) in China goat in vitro. Methods MSCs from the bone marrow of China goat were cultured. The third passage of MSCs were treated with PRP in the PRP group (the experimental group), but the cells were cultured with only the fetal calf serum (FCS) in the FCS group (the control group). The morphology and proliferation of the cells were observed by an inverted phase contrast microscope. The effect of PRP on proliferation of MSCs was examined by the MTT assay at 2,4,6 and 8 days. Furthermore, MSCs were cultured withdexamethasone（DEX）or PRP; alkaline phosphatase (ALP) and the calcium stainingwere used to evaluate the effect of DEX or PRP on osteogenic differatiation of MSCs at 18 days. The results from the PRP group were compared with those from the FCS group. Results The time for the MSCs confluence in the PRP group was earlier than that in the FCS group when observed under the inverted phase contrast microscope. The MTT assay showed that at 2, 4, 6 and 8 days the mean absorbance values were 0.252±0.026, 0.747±0.042, 1.173±0.067, and 1.242±0.056 in the PRP group, but 0.137±0.019, 0.436±0.052, 0.939±0.036, and 1.105±0.070 in the FCS group. The mean absorbance value was significantly higher in the PRP group than in the FCS group at each observation time (P＜0.01). Compared with the FCS group, the positive-ALP cells and the calcium deposition were decreased in the PRP group; however, DEX could increase boththe number of the positiveALP cells and the calcium deposition. Conclusion The PRP can promote proliferation of the MSCs of China goats in vitro but inhibit osteogenic differentiation.
Objective To study the mechanism of compound of calcium phosphate(TCP) and platelet-rich plasma(PRP) in the treatment of femoral head necrosis.Methods The left femoral heads of 48 New Zealand white rabbits were frozen by liquid nitrogen as to make themodel of femoral head necrosis.Twenty-four rabbits were randomly chosen as theexperimental group and their femoral heads were filled with TCP/PRP. The other 24 rabbits were used as the control group and their femoral heads were filled only with TCP. They were sacrificed at 2, 4,8,12 weeks after operation. The specimens were examined with X-ray and histological study.Results At 2 weeks after operation,there was no significant difference in femoral headdensity between the two groups. Four weeks after operation, femoral head density decreased in both groups, while it decreased more in the control group. At 8,12 weeks after operation, the density of the femoral heads in both groups increased, and it was higher in the experimental group. Histology examination showed thatthere was no difference between the two groups 2 weeks after operation. The head became flat at 4 weeks. Control group had more defects. At 4,8,12 weeks, more repairs were observed in the experimental group than that in the control group. The amount and maturity of osteogenesis in experimental group were much more greaterthan those in control group．Bone histomorphometry showed that the volum of thetrabecular was larger in the experimental group (36.65%±7.22%,38.29%±4.28%，39.24%±3.42%) than that of control group(P＜0.05). Conclusion TCP/PRP does not only provide osteoblasts scaffold, butalso promotes bone formation and the head repair. TCP/PRP is a good biomaterialfor the treatment of femur head necrosis.
ObjectiveTo explore the clinical efficacy of the ultrasound-guided intra-articular injection of platelet-rich plasma (PRP) in the treatment of patients with different stages of knee osteoarthritis.MethodsWe retrospectively analyzed the clinical characteristics and X-ray data of patients with knee osteoarthritis who received ultrasound-guided intra-articular injection of PRP in the Department of Rehabilitation Medicine at Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University between May 2018 and June 2019. The patients were grouped according to the Kellgren & Lawrence Classification (K&L 0, Ⅰ, Ⅱ, Ⅲ, and Ⅳ). All the patients received four injections with a one-week interval. The Visual Analogue Scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) were used to evaluate the clinical efficacy before the injection, and 3 and 6 months after the injection. Adverse reactions were recorded.ResultsA total of 102 patients were included without any grade 0 cases. There were 20 patients in K&L Ⅰ group, 37 in Ⅱ group, 31 in Ⅲ group, and 14 in Ⅳ group. No adverse event was reported. Significant differences of VAS scores and WOMAC index were observed in Ⅰ, Ⅱ and Ⅲ groups at the 3rd and 6th month follow-up (P<0.05). VAS and WOMAC scores of the three groups at the 3rd and 6th month after the treatment were significantly improved compared with those before the treatment (P<0.05). There was no significant difference in VAS score at the 3rd or 6th month after the treatment three groups (P>0.05). For K&L Ⅰ group, there was no statistically significant difference in WOMAC score at the 3rd or 6th month after the treatment (P>0.05). However, the WOMAC scores at the 3rd month after the treatment were better than those at the 6th month in K&L Ⅱ and Ⅲ groups (P<0.05). There was no significant time-depended changes in VAS score or WOMAC score in K&L Ⅳ group (P>0.05).ConclusionThe ultrasound-guided intra-articular PRP injection is safe and effective for pain relief and function improvement in patients with knee osteoarthritis at the early and middle stage.
Objective To calculate the recovery rate and enrichment factor and to analyse the correlation by measuring the concentrations of platelets, leukocyte, and growth factors in platelet-rich plasma (PRP) so as to evaluate the feasibil ity and stabil ity of a set of PRP preparation. Methods The peripheral blood (40 mL) was collected from 30 volunteers accorded with the inclusion criteria, and then 4 mL PRP was prepared using the package produced by Shandong Weigao Group Medical Polymer Company Limited. Automatic hematology analyzer was used to count the concentrations of platelets and leukocyte in whole blood and PRP. The enrichment factor and recovery rate of platelets or leukocyte were calculated; the platelet and leukocyte concentrations of male and female volunteers were measured, respectively. The concentrations of platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and vascular endothel ial growth factor (VEGF) were assayed by ELISA. Results The platelet concentrations of whole blood and PRP were (131.40 ± 29.44) × 109/L and (819.47 ± 136.32) × 109/L, respectively, showing significant difference (t=—27.020, P=0.000). The recovery rate of platelets was 60.85% ± 8.97%, and the enrichment factor was 6.40 ± 1.06. The leukocyte concentrations of whole blood and PRP were (5.57 ± 1.91) × 1012/L and (32.20 ± 10.42) × 1012/L, respectively, showing significant difference (t=—13.780, P=0.000). The recovery rate of leukocyte was 58.30% ± 19.24%, and the enrichment factor was 6.10 ± 1.93. The concentrations of platelets and leukocyte in PRP were positively correlated with the platelet concentration (r=0.652, P=0.000) and leukocyte concentration (r=0.460, P=0.011) in whole blood. The concentrations of platelet and leukocyte in PRP between male and female were not significantly different (P gt; 0.05). The concentrations of PDGF, TGF-β, and VEGF in PRP were (698.15 ± 64.48), (681.36 ± 65.90), and (1 071.55 ± 106.04) ng/ mL,which were (5.67 ± 1.18), (6.99 ± 0.61), and (5.74 ± 0.83) times higher than those in the whole blood, respectively. PDGF concentration (r=0.832, P=0.020), TGF-β concentration (r=0.835, P=0.019), and VEGF concentration (r=0.824, P=0.023) in PRP were positively correlated with platelet concentration of PRP. Conclusion PRP with high concentrations of platelets, white blood cells and growth factors can be prepared stably by this package.
ObjectiveTo explore the clinical efficacy and safety of ultrasound-guided intra-articular injection of platelet-rich plasma (PRP) in the treatment of avascular necrosis of the femoral head.MethodsWe retrospectively collected and analyzed the clinical characteristics, imaging data, and clinical outcomes of patients with femoral head necrosis who received ultrasound-guided intra-articular PRP injection in the Department of Rehabilitation Medicine of Sun Yat-sen Memorial Hospital, Sun Yat-sen University between June 2019 and June 2020. All the patients received 4 injections at one-week intervals. The Visual Analogue Scale (VAS), Western Ontario and McMaster University Osteoarthritis Index (WOMAC), and Harris Hip Joint Function Scale (HHS) were evaluated before treatment and 1 month, 3 months, and 6 months after the first injections. Adverse events were recorded. The normally distributed data were presented as mean±standard deviation, and analyzed by one-way repeated measures analysis of variance; the non-normally distributed data were presented as median (lower quartile, upper quartile), and analyzed by Friedman test.ResultsA total of 29 patients were included. According to the Association Research Circulation Osseous classification standard, 2 patients were classified as stageⅠ, 11 as stageⅡ, 11 as stage Ⅲ, and 5 as stage Ⅳ. Before treatment and 1 month, 3 months, and 6 months after treatment, the VAS scores were 7.0 (5.5, 8.0), 4.0 (3.0, 5.0), 3.0 (2.0, 3.0), and 3.0 (2.0, 5.0), respectively, the WOMAC scores were 39.27±11.70, 28.34±8.08, 22.82±6.09, and 24.13±7.55, respectively, and the HHS were 46.0 (40.0, 64.0), 71.0 (57.5, 75.0), 78.0 (68.0, 80.5), and 78.0 (64.0, 80.0), respectively. The time effects in VAS (χ2=65.423, P<0.001), WOMAC (F=46.710, P<0.001), and HHS (χ2=66.347, P<0.001) were all statistically significant. There were significant differences in each index between the values 1 month, 3 months, and 6 months after treatment and those before treatment respectively, and there was also a significant difference in each index between the value 1 month after treatment and that 3 months after treatment (P<0.05). There was no significant difference in any indicator between the value 6 months after treatment and that 3 months after treatment (P>0.05). Significant difference was shown between the value 6 months after treatment and that 1 month after treatment in WOMAC (P=0.016), but not in VAS or HHS (P>0.05). No obvious adverse event was reported during the follow-up period.ConclusionsUltrasound-guided intra-articular PRP injection can effectively alleviate the pain and improve the hip joint function of patients with femoral head necrosis for at least 6 months. However, randomized controlled studies with a larger sample size and longer-term follow-up are needed in the future to confirm the efficacy and safety of PRP injection in femoral head necrosis.
Objective To summarize the research progress of platelet-rich plasma (PRP) for the treatment of discogenic low back pain (DLBP). Methods The literature on the treatment of DLBP with PRP was extensively reviewed, and the classification, treatment mechanism, in vitro and in vivo experiments and clinical trial progress of PRP were summarized. Results According to the PRP composition, preparation methods, and physicochemical properties, there are five commonly used PRP classification systems at present. PRP is involved in delaying or reversing the progress of disc degeneration and pain control by promoting the regeneration of nucleus pulposus cells, increasing the synthesis of extracellular matrix, and regulating the internal microenvironment of degenerative intervertebral disc. Although several in vitro and in vivo studies have confirmed that PRP can promote disc regeneration and repair, significantly relieve pain, and even improve the mobility of DLBP patients. But the contrary conclusion has been reached in a few studies, and there are limitations to the application of PRP. ConclusionCurrent studies have confirmed the effectiveness and safety of PRP in the treatment of DLBP and intervertebral disc degeneration, as well as the advantages of PRP in terms of ease of extraction and preparation, low immunological rejection, high regenerative and repair capacity, and the ability to compensate for the shortcomings of traditional treatment modalities. However, relevant studies are still needed to further optimize PRP preparation methods, unify systematic classification guidelines, and clarify its long-term effectiveness.
Objective To study the effects of platelet-rich plasma(PRP) on the proliferation and osteogenetic activity of the marrow mesenchymal stem cells(MSCs) cultured in vitro to elucidate the cellular and molecularmechanism by which PRP accelerates bone repair.Methods The human MSCs were cultured in vitro and randomly divided into the experimental group(n=9) and control group(n=9). In the experimental group, the MSCs were interfused with PRP(10 μl/ml culture media). The proliferation ability of the cells was tested by flow cytometry and MTT, the osteogenetic activity by alkaline phosphatase(ALP) measuring and tetracycline fluorometry, and cbfal mRNA expression by reverse transcriptPCR.Results PRP could stimulate the MSCs proliferation. The flow cytometry assay showed that the MSCs proportion of S period of the experimental group significantly increased 14.5±0.4 in comparison with that of the control group 7.2±0.5 (P＜0.01) after 24 hours. MTT value showed that MSCs proliferatedto platform period earlier in the experimental group than in the control group. There was a significant increase in ALP activity of the experimental group 7.79±1.98,9.51±2.31and 14.03±3.02 when compared with that of the control group 2.06±0.77,2.84±0.82 and 2.58±0.84 after 3, 6 and 9 days(P＜0.05). The number of mineral nodes increased. Reverse transcript-PCR showed that the expression of cbfal mRNA were elevated gradually at 2,4 and 8 hours after interfused with PRP.Conclusion The effect of PRP on accelerating bone repair is related to its effects on stimulating the proliferation of MSCs, increasing the cbfal expression and promoting the osteogenetic activity.
Objective To investigate the effects of combined platelet-rich plasma (PRP) and arterial supercharging technique on the survival rate and functional restoration of cross-body region skin flaps in rabbits. MethodsTwelve healthy 6-month-old New Zealand White rabbits were randomly assigned to 4 groups (n=3): sham group, PRP group, anastomosis group, and combined treatment group. An axial skin flap with an area of 12 cm×6 cm on the inner side of the hind limbs of all animals were prepared, with the saphenous artery as the main blood supply. Following the ligation of both the proximal and distal ends of the saphenous artery across all groups, the sham group received no further intervention, the PRP group was subjected to PRP injection, the anastomosis group underwent in situ end-to-end anastomosis of the distal saphenous artery, and the combined treatment group received both in situ distal saphenous artery anastomosis and PRP administration. Flap survival was evaluated and recorded on postoperative days 1, 3, and 7, with survival rates calculated accordingly. On day 7, flap tissue samples were harvested for HE staining to assess basal tissue morphology. Additionally, immunohistochemical staining was conducted to detect the expression of α-smooth muscle actin (α-SMA), vascular endothelial growth factor (VEGF), and CD31 in the flap tissues. Results At postoperative day 1, no significant difference in flap survival rates were observed among the 4 groups (P>0.05). At day 3, the PRP group showed no significant difference compared to the sham group (P>0.05); however, both the anastomosis and combined treatment groups exhibited significantly higher survival rates than the sham group (P<0.05), the combined treatment group further demonstrated superior survival rates compared to both the PRP and anastomosis groups (P<0.05). At day 7, the combined treatment group maintained significantly higher survival rates than all other groups (P<0.05), while both the PRP and anastomosis groups exceeded the sham group (P<0.05). HE staining at day 7 revealed persistent inflammatory cell infiltration, sheet-like erythrocyte deposition, and disordered collagen fibers in the sham group. The PRP group showed nascent microvessel formation and early collagen reorganization, whereas the anastomosis group displayed mature microvasculature with resolved interstitial edema. The combined treatment group exhibited differentiated microvessels with densely packed collagen bundles. Immunohistochemical analysis at day 7 demonstrated significantly larger relative area percentages of α-SMA, VEGF, and CD31 positive cells in the combined treatment group compared to all other groups (P<0.05). Both the PRP and anastomosis groups also showed significantly higher values than the sham group (P<0.05). Conclusion The combination of PRP and arterial supercharging techniques significantly enhances flap healing, potentially through mechanisms involving augmented angiogenesis and improved blood supply.
【Abstract】 Objective To find out the best method to prepare platelet-rich plasma (PRP) and to evaluate the effect of PRP gel on skin flap survival and its mechanism. Methods Totally, 72 Wistar rats (aged 12 weeks, weighing 250-300 g) were used for the experiment. The arterial blood (8-10 mL) were collected from the hearts of 24 rats to prepare PRP with three kinds of centrifuge methods: in group A, 200 × g centrifuge for 15 minutes, and 500 × g centrifuge for 10 minutes；in group B, 312 × g centrifuge for 10 minutes, and 1 248 × g centrifuge for 10 minutes；and in group C, 200 × g centrifuge for 15 minutes, and 200 × g centrifuge for 10 minutes. The platelet was counted in the whole blood, PRP, and platelet-poor plasma (PPP) to determine an ideal centrifuge. PRP, PPP, and the serum after first centrifuge were collected. The concentrations of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor β1 (TGF-β1) were measured in the PRP, PPP, and serum using the enzyme-linked immunosorbent assay method, and PRP and PPP gels were prepared. The flaps of 11 cm × 3 cm in size were elevated on the back of 48 rats, which were divided into 3 groups: PRP gel (PRP group, n=16) and PPP gel (PPP group, n=16) were injected, no treatment was given in the control group (n=16). The flap survival rate was measured at 7 days. Histological and real-time PCR were used to count the inflammatory cells and blood vessel density, and to detect the expressions of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), PDGF-AA, and PDGF-BB mRNA at 8 hours, 24 hours, 3 days, and 7 days. Results Platelet counting showed platelet in group A was the highest. ELISA evaluation showed that the concentrations of TGF-β1 and PDGF-BB were significantly higher in PRP than in PPP and serum (P lt; 0.05). The flap survival rate was 61.2% ± 9.1% in PRP group, showing significant differences (P lt; 0.05) when compared with that in PPP group (35.8% ± 11.3%) and control group (28.0% ± 5.4%). The inflammatory cells were significantly lower and the blood vessel density was significantly higher in PRP group than in PPP group and control group (P lt; 0.05). When compared with PPP group and control group, the expressions of VEGF and PDGF-BB increased at all time after operation in PRP group; the expression of EGF increased within 24 hours; and the expression of PDGF-AA increased after 3 days. There were significant differences in PDGF-AA mRNA at 3 days and 7 days, PDGF-BB mRNA at 8 hours, VEGF mRNA at 24 hours and 3 days, and EGF mRNA at 24 hours between PRP group and PPP and control groups (P lt; 0.05). Conclusion 200 × g centrifuge for 15 minutes and 500 × g centrifuge for 10 minutes is the best PRP preparation method. PRP can improve the skin flap survival by regulating the genes involved in angiogenesis.
Objective Platelet-rich plasma (PRP) can promote wound heal ing. To observe the effect of PRP injection on the early heal ing of rat’s Achilles tendon rupture so as to provide the experimental basis for cl inical practice. Methods Forty-six Sprague Dawley rats were included in this experiment, female or male and weighing 190-240 g. PRP and platelet-poor plasma (PPP) were prepared from the heart arterial blood of 10 rats; other 36 rats were made the models of Achilles tendon rupture, and were randomly divided into 3 groups (control group, PPP group, and PRP group), 12 rats for each group. In PPP and PRP groups, PPP and PRP of 100 μL were injected around the tendons once a week, respectively; in the control group, nothing was injected. The tendon tissue sample was harvested at 1, 2, 3, and 4 weeks after operation for morphology, histology, and immunohistochemistry observations. The content of collagen type I fibers also was measured. Specimens of each group were obtained for biomechanical test at 4 weeks. Results All the animals survived till the end of the experiment. Tendon edema gradually decreased and sliding improved with time. The tendon adhesion increased steadily from 1 week to 3 weeks postoperatively, and it was relieved at 4 weeks in 3 groups. There was no significant ifference in the grading of tendon adhesion among 3 groups at 1 week and at 4 weeks (P gt; 0.05), respectively. The inflammatory cell infiltration, angiogenesis, and collagen fibers were more in PRP group than in PPP group and control group at 1 week; with time, inflammatory cell infiltration and angiogenesis gradually decreased. Positive staining of collagen type I fibers was observed at 1-4 weeks postoperatively in 3 groups. The positive density of collagen type I fibers in group PRP was significantly higher than that in control group and PPP group at 1, 2, and 3 weeks (P lt; 0.05), but no significant difference was found among 3 groups at 4 weeks (P gt; 0.05). The biomechanical tests showed that there was no significant difference in the maximal gl iding excursion among 3 groups at 4 weeks postoperatively (P gt; 0.05); the elasticity modulus and the ultimate tensile strength of PRP group were significantly higher than those of control group and PPP group at 4 weeks (P lt; 0.05). Conclusion PRP injection can improve the healing of Achilles tendon in early repair of rat’s Achilles tendon rupture.