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 interleukin-17 (IL-17) levels changes in both synovial fluid and venous plasma of patients with primary knee osteoarthritis (OA) after intra-articular injection of platelet-rich plasma (PRP). Methods Between January 2015 and January 2016, 30 patients with primary knee OA were treated by intra-articular injection of PRP once a week for 3 weeks (trial group). Thirty healthy individuals were recruited into the study as control. There was no significant difference in gender, age, and body mass index between 2 groups (P>0.05). Visual analogue scale (VAS) score and Knee Society Score (KSS) were used to evaluate pain level and function of the knee for patients with OA. The IL-17 levels in both venous plasma and synovial fluid were measured before injection and at 1, 3, 6, and 12 months after injection in trial group and the IL-17 levels in venous plasma were measured in control group. The levels were determined using ELISA method. Results There was no knee joint swelling, fever, local infection, or other uncomfortable symptoms for all patients in process of PRP injection. All patients were followed up 13.5 months on average (range, 12-15 months). In trial group, the VAS scores at different time points after injection were significantly lower than that before injection (P<0.05). And the KSS scores at different time points after injection were significantly higher than that before injection (P<0.05). There was no significant difference in VAS and KSS scores between different time points after injection (P>0.05). The IL-17 levels in venous plasma before and after injection in trial group were significantly higher than that in control group (P<0.05). The IL-17 levels in venous plasma at each time point after injection were significantly lower than that before injection (P<0.05). There was no significant difference in IL-17 levels in both venous plasma and synovial fluid between different time points after injection (P>0.05). Conclusion Intra-articular injection of PRP can significantly release the pain symptoms, improve joint function, and reduce IL-17 levels in both synovial fluid and venous plasma of the patients with knee OA, but IL-17 levels can not reduce to normal level.
Objective To introduce the effect of platelet-rich plasma (PRP) in the treatment of chronic tissue lesions. Methods The latest research papers concerning the relevant subject were reviewed and analyzed. Results In PRP, the platelets and cytokines, the interaction of them in the same proportion as they were in the body fluid, played promoting role in repairing the chronic damage of tissues. However, there was still way to go before PRP was appl ied for cl inical use in a widerrange. Conclusion PRP has quite a bright outlook in the treatment of chronic tissue lesions.
Objective To study the effect of platelet-rich plasma (PRP) on repairing chronic wounds of lower l imbs. Methods From May 2007 to November 2007, 47 patients suffering from chronic wounds of lower l imbs were treated. There were 41 males and 6 females, aged from 15 to 68 years (43.2 years on average). The disease was caused by tibiofibulafracture in 20 cases, calcaneus fracture in 4 cases, metatarsal fracture in 1 case, multiple open fracture of lower l imbs in 3 cases, tibia osteomyel itis in 10 cases, femur osteomyel itis in 1 case, soft tissue injury of ankle in 4 cases, infection after amputation in 2 cases, infection after foot orthomorphia in 1 case, and infection after calcaneus tendon neoplasty in 1 case. Their chronic wounds did not healed after 2 to 4 months of therapy. Among them, chronic wounds compl icated with fracture nonunion in 23 cases and positive bacterial culture result in 38 cases. Debridement and autogenous PRP gel injection were appl ied every 2 months and for twice. Results The patients were followed up for 4 months after the first PRP injection. Two months after the first PRP injection, chronic wounds contracted significantly in 34 patients with purulence and necrosis tissue cleaned up, circulation of soft tissue improved and exposed bone or muscle tissue covered by neogenetic granulation. No patient was completely cured. Two months after the second PRP injection, the average coverage rate was 79.3% ± 18.0%, the total cure rate was 29.8%. The volume of the chronic wounds decreased by (9.3 ± 4.9) mL after PRP therapy (2.5 ± 2.7) mL when compared with (11.8 ± 5.6) mL of before therapy, showing significant difference (P lt; 0.05). X-ray photograph showed that among the 23 cases of fracture nonunion, fracture healed completely in 9 cases; bony callus formation increased obviously in 12 cases; no significant change was observed in 2 cases. No aggravated sign of osteomyel itis was notified. Positive results of bacterial culture reduced to 15 cases. Conclusion PRP efficiently enhances the recovery of soft tissue defect and speeds up the chronic wounds heal ing oflower l imbs.
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 effect of various concentration of platelet-rich plasma (PRP) on osteogenic differentiation of rabbit skeletal muscle-derived stem cells (SMSCs) cultured in vitro. Methods Blood drawn from the central ear arteries of 9 one-year-old New Zealand white rabbits weighing 2.5-3.0 kg (male and female) was used to prepare PRP (Landesberg method). Full blood count and platelet count in PRP were tested. Soleus muscle of right hindl imb in rabbit was obtained and used to culture SMSCs in vitro. The cells at passage 3 were randomly divided into different groups: the experimental groups in which the cells were treated by conditioned culture media with various concentrations of autologousPRP (6.25%, 12.50%, 25.00%, 50.00%), and the control group in which the cells were treated with the media without PRP. At different time points after intervention, osteogenetic activity of the cells was detected by ALP staining observation, ALP activity detection was conducted, al izarin red staining for calcium nodules and immunofluorescence staining for osteocalcin were performed, and core binding factor α1 (Cbfα1) of osteogenic gene expression was tested by RT-PCR. Results The full blood PRP count and the platelet count in PRP was (3.06 ± 0.46) × 105/μL and (18.08 ± 2.10) × 105/μL, respectively. ALP staining: the cells in all the experimental groups were positive for the staining with many black sediment particles in cytoplasm; the cells in the control group were negative staining. ALP activity: all the experimental groups were higher than the control group (P lt; 0.05), the experimental group at 12.50% was superior to other experimental groups at each time point (P lt; 0.05). Al izarin red staining: at 14 days after culture, orange-red calcium nodules were evident in all the experimental groups; no orange-red calcium nodules were observed in the control group with a mineral ization rate of zero; there were significant difference between the experimental groups and the control group in terms of mineral ization rate (P lt; 0.05), the experimental group at 12.50% had a higher mineral ization rate than other experimental groups (P lt; 0.05). Immunofluorescence staining for osteocalcin: at 7 days after culture, the experimental groups were positive for the staining with yellow fluorescence in cytoplasm, and the result of the control group was negative. RT-PCR detection: no obvious changes of the gene expression were noted at 4, 12, and 24 hoursafter culture in the control group; the gene expression in all the experimental groups was significant superior to that of control group, especially at 12 hours, and the expression in the experimental group at 12.50% was the highest. Conclusion PRP can obviously promote the osteogenic differentiation of SMSCs cultured in vitro in a concentration-dependent manner, and the 12.50% is proved to be the ideal concentration.
Objective Platelet-rich plasma (PRP) contains high concentrations of platelets and leucocytes, which play a key role in antimicrobial host defense system. To evaluate the antimicrobial efficacy of autologous PRP in vitro and in vivo and to explore the mechanism of action so as to provide the experimental basis for the prevention and treatment of bone infection. Methods PRP was prepared with the method of two centrifugation from 15 health volunteers. Platelet-leukocytegel (PLG) was obtained after activation of PRP with bovine thrombin. Next, PLG was incubated with Staphylococcus aureus (1 × 106 cfu/mL) in vitro compared with PRP, platelet-poor plasma (PPP) and PBS. Samples were taken out after 2, 4, 6, 8, 12, and 24 hours for bacterial culture and colony count. Thirty-six New Zealand adult rabbits, weighing (2.85 ± 0.11) kg, were divided into 4 groups: PLG (n=10), antibiotic (n=10), infection (n=10), and PBS (n=6) groups. The osteomyel itis models were made by injecting 0.1 mL Staphylococcus aureus suspension (1 × 106 cfu/mL) into the tibial canal in PLG group, antibiotic group, and infection group; equal volumes of PBS was injected in PBS group as a control. Autologous PLG was injected immediately after operation in PLG group. Cefazol in (30 mg/kg) was injected through the auricular vein from 1 hour before operation to 72 hours after operation in antibiotic group, once per 8 hours. No treatment was given in infection and PBS groups. The efficacy of PLG for osteomyel itis prophylaxis was evaluated by microbiological, X-ray and histological observation within 28 days. Results The contents of leucocyte and platelet of PRP were 6.2 times and 5.5 times of whole blood, showing signficant differences ((P lt; 0.05); the contents of leucocyte and platelet of PPP were significantly lower than those of whole blood and PRP ((P lt; 0.05). In vitro test showed that PLG had the most obvious bacteriostasis effect. The bacterial count reached a minimum value at 4 hours after incubation in PLG and at 6 hours after incubation in PRP. PPP had slow and no obvious bacteriostasis effect and PBS had no bacteriostasis effect. At 2, 4, 6, 8, 12, and 24 hours of incubation, the bacterial count reduced significantly when compared PLG with PRP and PPP (P lt; 0.05), when compared PRP with PPP (P lt; 0.05). In PLG group and antibiotic group, 1 rabbit died, respectively; 34 rabbits survived to the end of the experiment. There was no significant difference (P gt; 0.05) in temperature, body weight, erythrocyte sedimentation rate and content of leucocyte between 28 days after operation andbefore operation in 4 groups. After 28 days, the X-ray scores were 2.78 ± 1.39, 1.55 ± 1.48, 4.17 ± 1.25, and 0 in PLG, antibiotic,infection, and PBS groups, respectively, which was significantly higher in infection group than in other 3 groups ((P lt; 0.05). Also, the histological scores were 5.89 ± 3.92, 3.00 ± 2.31, 10.33 ± 4.03, and 0, respectively, which was significantly higher in infection group than in other 3 groups (P lt; 0.05), and was significantly lower in antibiotic group than in PLG group ((P lt; 0.05). The results of bacterial culture showed that the infection rates of PLG group (44.4%) and antibiotic group (20.0%) were significantly lower ((P lt; 0.05) than that of infection group (88.9%). The quantitative analysis of bacteria showed that the number of bacteria was signifcantly lower ((P lt; 0.05) in PLG and antibiotic groups than in infection group. Conclusion PRP forms into PLG after activating, it can inhibit Staphylococcus aureus reproduction in vitro and can effectively prevent bone infection in vivo.
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.