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 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.
Objective To systematically evaluate the clinical effectiveness of platelet-rich plasma (PRP) combined with grafting material for the treatment of periodontal intrabony defects. Methods The following databases such as PubMed, The Cochrane Library, EMbase, CNKI, CBM and WanFang Data were searched on computer from inception to August, 2012 to collect the relevant randomized controlled trials (RCTs) on PRP combined with grafting material versus grafting material alone for periodontal intrabony defects. Two reviewers independently screened the literature according to the inclusion and exclusion criteria, extracted the data, and assessed the methodological quality of the included studies. RevMan 5.2 software was applied for meta-analysis. Results A total of 11 RCTs involving 342 patients were included. The pooled analysis on 7 RCTs showed that there was a significant difference in lower increase of clinical attachment loss (WMD=0.70, 95%CI 0.51 to 0.90, Plt;0.000 01) between the PRP combined with grafting material group and the grafting material alone group. But there was no significant difference in the gingival recession (WMD= −0.01, 95%CI −0.15 to 0.13, P=0.86). The pooled analysis on 9 RCTs showed that there was no significant difference in the reduction of plaque index (WMD= −0.04, 95%CI −0.09 to 0.02, P=0.20) between the two groups. Conclusion PRP combined with grafting material is superior to grafting material alone in the clinical attachment loss. But, there are no significant differences in gingival recession and plaque index. However, given the limited sample size and incomplete measure indexes of included studies, this conclusion still needs to be further proved by conducting more high-quality and large-scale RCTs.
Objective To investigate the clinical outcomes of autologous platelet rich plasma (PRP) for anterior cruciate ligament (ACL) reconstruction. Methods Between August 2014 and August 2016, 42 patients with ACL ruptures who underwent arthroscopic ACL reconstruction were randomly divided into 2 groups: 21 patients received graft soaked with PRP (trial group) and 21 patients received routine graft in ACL reconstruction (control group). Because 6 patients failed to be followed up, 17 patients of trial group and 19 of control group were enrolled in the study. There was no significant difference in gender, age, body mass index, side, injury reason, disease duration, Kellgren-Lawrence grade, and preoperative visual analogue scale (VAS), Lysholm score, and International Knee Documentation Committee (IKDC) activity scores between 2 groups (P>0.05). VAS score, Lysholm score, and IKDC activity scores were used to evaluate pain and function at 3 and 12 months postoperatively. Further, second arthroscopy and MRI examination were performed at 12 months postoperatively. Results The patients in both groups were followed up 3 to 12 months with an average of 9.83 months. The VAS score, Lysholm score, and IKDC activity scores were significantly improved at 3 and 12 months after operation in 2 groups (P<0.05), and the scores of trial group were significantly better than those of control group at 3 months (P<0.05), but no significant difference was found between 2 groups at 12 months (P>0.05). No complications of effusion, infection, and allergy were observed in 2 groups during follow-up. MRI showed good position of ACL grafts and good signal quality of the graft in the majority of the cases. However, mixed hyperintense and presence of synovial fluid at the femoral bone-tendon graft interface were found in 3 patients of trial group and 4 patients of control group, indicating poor remodeling ligamentation. MRI score was 3.53±1.13 in trial group and was 3.21±0.92 in control group, showing no significant difference (t=0.936,P=0.356). The second arthroscopy examination showed ligament remodeling score was higher in trial group than control group (t=3.248,P=0.014), but no significant difference was found in synovial coverage score and the incidence of cartilage repair (t=2.190,P=0.064;χ2=0.090,P=0.764). Conclusion PRP application in allograft ACL reconstruction can improve knee function and relieve pain after operation, which may also accelerate graft remodeling.
Objective The tendon-bone heal ing is the key point to ensure the success of the anterior cruciate l igament (ACL) reconstruction. To observe the histological change in the tendon-bone heal ing after ACL reconstruction by different concentrations of platelet-rich plasma (PRP) combined with deproteinized bone (DPB) of calf as bone tunnel infill ing and to investigate the active effect of the complex on tendon-bone heal ing and to define the optimal concentration of PRP. Methods Eight mL blood was drawn from central artery of New Zealand rabbit ears; PRP was prepared by Landesbergmethod, and l iquid supernatant was used as thinner to prepare different concentrations of PRP (30%, 60%, and 100%). Fresh osteoepiphysis spongy bone was harvested from lower end of femur of newborn calf to prepare DPB by way of 30% H2O2 and ether alternating soaking for 24 hours continuous 6 times. DPB was soaked in different concentrations of PRP and mixed with activator to prepare the PRP/DPB complex. A total of 54 New Zealand white rabbits, aging 8-12 months, weighing (2.5 ± 0.4) kg, were divided randomly into 3 groups: group A (30%PRP/DPB complex, n=18), group B (60%PRP/DPB complex, n=18), and group C (100%PRP/DPB complex, n=18). The legs of the rabbits were randomly divided into experimental side and the control side; ACL was reconstructed by semitendinosus and PRP/DPB complex in bone tunnel in the experimental side, and only by semitendinosus in the control side. The general conditions of the rabbits were observed postoperatively and HE staining was used to observe the tendon-bone heal ing, then I-IV levels of semi-quantitative analysis of the tendon-bone heal ing were evaluated according to Demirag standard at 3, 6, and 12 weeks. Results General observation: Synovial fluid sl ightly increased in the specimens and no bony tissue was found in inner of femoral tunnel at 3 weeks; there was no synovial fluid in all the specimens and scar tissue was discovered in inner of femoral tunnel at 6 weeks; and there was no synovial fluid and the tendons became tighter with fibrous tissue at 12 weeks. Histological observation: New granulation tissue formed in the tendon-bone interface of group A experimental sides at 3 weeks; there was various widths of Sharpey type textile fiber in the tendon-bone interface at 6 weeks; Sharpey type textile fiber arranged regularly, which formed an irregular and blur “tidal l ine” at 12 weeks. Group B experimental sides were better than any other group at 3, 6, and 12 weeks; chondrocyte-l ike arranged regularly in the tendonboneinterface at 3 weeks; the number of chondrocyte-l ike per unit area was more than that of the other groups at 6 weeks;and chondrocyte-l ike prol iferated and matured in the tendon-bone interface, Sharpey type textile fiber became tighter andordered. Group C experimental sides were similar to both sides of group A at 3 weeks, however, the prol iferation of relatively mature dense connective tissue was worse than that of other groups at 6 and 12 weeks. According to Demirag grading, there were significant differences in tendon-bone heal ing between the experimental sides and the control sides of group B at 3 and 6 weeks, and between group B experimental sides and group C experimental sides at 12 weeks (P lt; 0.05). Conclusion The mixture of PRP/PRP has good biocompatibil ity and bone induction, so it can enhance tendon-bone heal ing after ACL reconstruction when the concentration of PRP is 60%.
Objective To investigate the factors that affect platelet-rich plasma (PRP) in promoting bone regeneration and repairing. Methods Recent l iterature was reviewed, concerning the preparations of PRP, physiological mechanism and the latest appl ications in orthopedic field. Results PRP, the concentrated body of autologous platelet, was rich in platelets and was the source of autologous growth factors. Many studies had shown that PRP played an important role in promoting bone regeneration and repairing. However, a few experimental results contradicted this point. The reason might be that the biological properties of PRP were influenced by various factors, such as workmanship, vector, activation schemes, working concentration, individual difference. Conclusion The concentration and qual ity of platelet and other related factorsof PRP affect the rel iabil ity of the results and conclusions. So an efficient and stable production method of PRP should beestabl ished.
【Abstract】 Objective To explore the interventional effect of platelet lysate (PL) on osteogenic differentiation ofBMSCs by induction in rats in vitro. Methods Twenty-four clean-grade adult Wistar rats, weighing from 250 g to 300 g, maleor female, were included in this study. PL was obtained through three times of centrifugation and repeated freeze-thaw for the blood aspirated from cardiac cavities in 16 Wistar rats. ELISA assay was conducted to detect the concentration of growth factors PDGF, TGF-β1, IGF-1 and VEGF in PL. The BMSCs harvested by flushing femurs of 8 adult Wistar rats were isolated, cultivated and expanded in vitro. The cells at the 4 passage were performed for osteogenic differentiation by induction in three groups of A (5% PL of final concentration in basic induction medium), B (1% PL of final concentration in basic induction medium), and C (no presence of PL in basic induction medium as a control). The morphological changes of the cells were dynamically observed with inverted phase contrast microscope during the whole period. At different time-points, ALP staining (7 days) and ALP/TP (2, 8, 12 days) of the cells were detected to evaluate ALP activity, and the mineral formation in extracellular martrix was examined with Al izarin red staining which provided quantitative analysis of mineral deposits. Results ELISA assay showed that the content of PDGF, TGF-β1, IGF-1 and VEGF in PL reached (300 ± 30), (140 ± 25), (80 ± 35), (70 ± 20) pg/mL, respectively. Morphological observation displayed BMSCs in group A or B gradually turned from spindle-shape to square- or polygon-shape as the morphorlogical type of osteoblast-l ike cells at 7 days. The cells in group A showed slower shape changesbut higher prol iferation than that in group B or C. Moreover, at the 20 days, the cells in group A still displayed dense gro wth and produced obviously decreased amount of mineral deposits in ECM when compared with group B or C. At the 7 days, the cells ofgroup A showed smaller amount of granules positive for ALP staining in cytoplasm when compared with groups B and C, and displayed marked reduction in ALP activity assay at the 2, 8, and 10 days compared with that of groups B and C (P lt; 0.05). At the 20 days, Al izarin red staining showed the number of mineral deposits in groups A, B and C were 7.67 ± 1.10, 12.87 ± 0.81 and 15.59 ± 0.25, respectively, while the area of mineral deposits were (161 778.70 ± 44 550.80), (337 349.70 ± 56 083.24), and (415 921.70 ± 71 725.39) pixels, respectively. The number of mineral deposits and the area of mineral deposits in group A were smaller than those in groups B and C (P lt;0.05). But there was no statistically significant difference between groups B and C (P gt; 0.05). Conclusion PL is a kind of system carrying various growth factors. Exposure of PL inhibits both ALP activity and mineral formation of BMCs in a dose-dependent way under the osteogenic induction environment.
【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 To investigate the effectiveness of autogenous platelet-rich plasma (PRP) gel with acellular xenogeneic dermal matrix in the treatment of deep II degree burns. Methods From January 2007 to December 2009, 30 cases of deep II degree burns were treated. There were 19 males and 11 females with an average age of 42.5 years (range, 32-57 years).The burn area was 10% to 48% of total body surface area. The time from burn to hospitalization was 30 minutes to 8 hours. All patients were treated with tangential excision surgery, one side of the wounds were covered with autogenous PRP gel and acellular xenogeneic dermal matrix (PRP group), the other side of the wounds were covered with acellular xenogeneic dermal matrix only (control group). The heal ing rate, heal ing time, infection condition, and scar formation were observed. Results At 7 days after operation, the infection rate in PRP group (6.7%, 2/30) was significantly lower than that in control group (16.7%, 5/30, P lt; 0.05). The healing times were (18 ± 4) days and (22 ± 4) days respectively in PRP group and control group, showing significant difference (P lt; 0.05). The healing rates at 14 days and 21 days were 75% ± 7% and 88% ± 5% in PRP group, were 62% ± 15% and 73% ± 7% in control group, showing significant difference (P lt; 0.05). RPR group was superior to control group in elasticity, color, appearance, softness, scar formation, and heal ing qual ity. Conclusion Autogenous PRP gel with acellular xenogeneic dermal matrix can accelerate the wound healing of deep II degree burns as well as alleviate the scar proliferation.
OBJECTIVE: To explore the molecular mechanisms involved in the increased collagen synthesis by platelet-derived wound healing factors (PDWHF) during wound healing in alloxan-induced diabetic rats. METHODS: Thirty-three male SD rats were divided into two groups, the normal (n = 9) (group A) and the diabetic group (n = 24). Two pieces of full-thickness skin with diameter of 1.8 cm were removed from the dorsal site of diabetic rats. PDWHF (100 micrograms/wound) was topically applied to one side of the diabetic wounds (group B) on the operation day and then once a day in the next successive 6 days. Meanwhile, bovine serum albumin (100 micrograms/wound) was applied to the other side of diabetic wound as control group (group C) in the same way. Levels of transforming growth factor-beta 1 (TGF-beta 1) and procollagen I mRNA in wound tissue were inspected by dot blotting. RESULTS: TGF-beta 1 mRNA levels in group B were 4 folds and 5.6 folds compared with those in group C after 5 and 7 days (P lt; 0.01), however, still significantly lower than those of group A (P lt; 0.05). There was no significance difference among three groups on the 10th day after wounding. The levels for procollagen I mRNA in group B amounted to 2.1, 1.8 and 2.3 folds of those in group C after 5, 7, and 10 days (P lt; 0.01), respectively. Compared with those in the group A, procollagen I mRNA levels in the group B were significantly lower after 5 and 7 days (P lt; 0.05), and no significant difference was observed between group B and A after 10 days. CONCLUSION: One important way for PDWHF to enhance the collagen synthesis in diabetic wound healing is to increase the gene expression of endogenous TGF-beta 1.