ObjectiveTo identify a more popularized preparation protocol of leukocytes-rich platelet-rich plasma (L-PRP) for higher tolerance rate.MethodsThe peripheral blood samples of 76 volunteers (45.0 mL/case) were mixed with 5 mL sodium citrate injection for blood transfusion, and L-PRP was prepared by twice centrifugations. All blood samples were divided into three groups according to the parameters of twice centrifugation: experimental group A (12 cases, 400×g, 10 minutes for the first time and 1 100×g, 10 minutes for the second time), experimental group B (27 cases, 800×g, 10 minutes for the first time and 1 100×g, 10 minutes for the second time), and control group (37 cases, 1 360×g, 10 minutes for the first time and 1 360×g, 10 minutes for the second time). The platelet recovery rate and platelet and leukocyte enrichment coefficient of L-PRP in each group were calculated and compared.ResultsAfter removal of abnormal blood samples (platelet recovery rate was more than 100% or white thrombus), the remaining 55 cases were included in the statistical analysis, including 10 cases in experimental group A, 21 cases in experimental group B, and 24 cases in control group. The platelet enrichment coefficient and platelet recovery rate of experimental group B were significantly higher than those of experimental group A and control group (P<0.05); there was no significant difference between experimental group A and control group (P>0.05). There was no significant difference in leukocyte enrichment coefficient between experimental groups A, B, and control group (P>0.05).ConclusionThe preparation quality of PRP is affected by various factors, including centrifugal force, centrifugal time, temperature, and operation process, etc. Twice centrifugation (800×g, 10 minutes for the first time and 1 100×g, 10 minutes for the second time) is an ideal and feasible centrifugation scheme, which can obtain satisfactory platelet recovery rate and enrichment coefficient with thicker buffy coat, which can reduce the fine operation requirements for operators, improve the fault tolerance rate and generalization.
Objective To investigate the optimal mixing ratio of recombinant human bone morphogenetic protein 2 (rhBMP-2) with porous calcium phosphate cement (PCPC) and autologous bone as bone grafting material for the repair of large bone defects using Masquelet technique. The effect of platelet-rich plasma (PRP) on the healing of bone defects was evaluated under the optimal ratio of mixed bone. Methods Fifty-four New Zealand White rabbits were taken to establish a 2 cm long bone defect model of the ulna and treated using the Masquelet technique. Two parts of the experiment were performed in the second phase of the Masquelet technique. First, 36 modeled experimental animals were randomly divided into 4 groups (n=9) according to the mass ratio of autologous bone and rhBMP-2/PCPC. Group A: autologous bone (100%); group B: 25% autologous bone+75% rhBMP-2/PCPC; group C: 50% autologous bone+50% rhBMP-2/PCPC; group D: 75% autologous bone+25% rhBMP-2/PCPC. The animals were executed at 4, 8, and 12 weeks postoperatively for general observation, imaging observation, histological observation (HE staining), alkaline phosphatase (ALP) activity assay, and biomechanical assay (three-point bending test) were performed to assess the osteogenic ability and to determine the optimal mixing ratio. Then, 18 modeled experimental animals were randomly divided into 2 groups (n=9). The control group was implanted with the optimal mixture ratio of autologous bone+rhBMP-2/PCPC, and the experimental group was implanted with the optimal mixture ratio of autologous bone+rhBMP-2/PCPC+autologous PRP. The same method was used to observe the above indexes at 4, 8, and 12 weeks postoperatively. Results The bone healing process from callus formation to the cortical connection at the defected gap could be observed in each group after operation; new bone formation, bridging with the host bone, and bone remodeling to normal bone density were observed on imaging observation; new woven bone, new capillaries, bone marrow cavity, and other structures were observed on histological observation. The ALP activity of each group gradually increased with time (P<0.05); the ALP activity of group A was significantly higher than that of the other 3 groups at each time point after operation, and of groups C and D than group B (P<0.05); there was no significant difference between groups C and D (P>0.05). Biomechanical assay showed that the maximum load in three-point bending test of each group increased gradually with time (P<0.05), and the maximum loads of groups A and D were significantly higher than that of groups B and C at each time point after operation (P<0.05), but there was no significant difference between groups A and D (P>0.05). According to the above tests, the optimal mixing ratio was 75% autogenous bone+25% rhBMP-2/PCPC. The process of new bone formation in the experimental group and the control group was observed by gross observation, imaging examination, and histological observation, and the ability of bone formation in the experimental group was better than that in the control group. The ALP activity and maximum load increased gradually with time in both groups (P<0.05); the ALP activity and maximum load in the experimental group were significantly higher than those in the control group at each time point after operation (P<0.05), and the maximum load in the experimental group was also significantly higher than that in group A at 12 weeks after operation (P<0.05). ConclusionIn the second phase of Masquelet technique, rhBMP-2/PCPC mixed with autologous bone to fill the bone defect can treat large bone defect of rabbit ulna, and it has the best osteogenic ability when the mixing ratio is 75% autologous bone+25% rhBMP-2/PCPC. The combination of PRP can improve the osteogenic ability of rhBMP-2/PCPC and autologous bone mixture.
ObjectiveTo investigate the effect of human adipose-derived stem cells (hADSCs) on pressure ulcers in mouse.MethodsThe subcutaneous adipose tissue from voluntary donation was harvested. Then the hADSCs were isolated and cultured by mechanical isolation combined with typeⅠcollagenase digestion. The 3rd generation cells were identified by osteogenic, adipogenic, chondrogenic differentiations and flow cytometry. The platelet rich plasma (PRP) from peripheral blood donated by healthy volunteers was prepared by centrifugation. The pressure ulcer model was established in 45 C57BL/6 mice by two magnets pressurized the back skin, and randomly divided into 3 groups (n=15). The wounds were injected with 100 μL of hADSCs (1×106 cells) transfected with a green fluorescent protein (GFP)-carrying virus, 100 μL human PRP, and 100 μL PBS in hADSCs group, PRP group, and control group, respectively. The wound healing was observed after injection. The wound healing rate was calculated on the 5th, 9th, and 13th days. On the 5th, 11th, and 21st day, the specimens were stained with HE staing, Masson staining, and CD31 and S100 immunohistochemical staining to observe the vascular and nerve regeneration of the wound. In hADSCs group, fluorescence tracer method was used to observe the colonization and survival of the cells on the 11th day.ResultsThe cultured cells were identified as hADSCs by induced differentiation and flow cytometry. The platelet counting was significantly higher in PRP group than in normal peripheral blood group (t=5.781, P=0.029). General observation showed that the wound healing in hADSCs group was superior to those in PRP group and control group after injection. On the 5th, 9th, and 13th days, the wound healing rate in hADSCs group was significantly higher than those in PRP group and control group (P<0.05). Histological observation showed that compared with PRP group and control group, inflammatory cell infiltration and inflammatory reaction were significantly reduced in hADSCs group, collagen deposition was significantly increased, and skin appendage regeneration was seen on the 21st day; at each time point, the expression of collagen was significantly higher in hADSCs group than in PRP group and control group (P<0.05). Immunohistochemical staining showed that the number of neovascularization and the percentage of S100-positive cells in hADSCs group were significantly better than those in PRP group and control group on the 5th, 9th, and 13th days (P<0.05). Fluorescent tracer method showed that the hADSCs could colonize the wound and survive during 11 days after injection.ConclusionLocal transplantation of hADSCs can accelerate healing of pressure ulcer wounds in mice and improve healing quality by promoting revascularization and nerve regeneration.
Objective To explore the best centrifuge condition for preparing rabbit leukocyte-poor platelet-rich plasma (LP-PRP) by using single centrifugation method. Methods Sixteen healthy New Zealand rabbits, aged 3-4 months, were utilized in the investigation. A total of 15 mL anticoagulated blood was extracted from the central ear artery of each rabbit, with a repeat of the blood collection procedure after 1 and 2 months. The obtained blood specimens were individually subjected to centrifugation at a radius of 16.7 cm and speeds of 1 200, 1 300, 1 400, and 1 500 r/min (equivalent to centrifugal forces of 269×g, 315×g, 365×g, and 420×g) for durations of 2, 3, 4, and 5 minutes, resulting in a total of 16 groups. Following centrifugation, collect plasma from each group to a distance of 1.5 mL from the separation plane. The volumes, platelet enrichment coefficient, and platelet recovery rates of LP-PRP in each group, under varying centrifugation conditions, were methodically computed and subsequently compared. Results The volume of LP-PRP obtained under all centrifugation conditions ranged from 1.8 to 7.6 mL. At a consistent centrifugal speed, an extension of centrifugation time leaded to a significant increase in the volume of LP-PRP, accompanied by a declining trend in the platelet enrichment coefficient of LP-PRP. When centrifuged for 2 minutes, the volume of LP-PRP at speeds of 1 200 and 1 300 r/min was less than 2.0 mL, while the volume of LP-PRP obtained under other conditions was more than 2.0 mL. When centrifuged for 4 and 5 minutes, the volume of LP-PRP obtained at each speed was more than 4 mL. LP-PRP with a platelet enrichment coefficient more than 2.0 could be prepared by centrifuging at 1 200 r/min for each time group and 1 300 r/min for 2 and 3 minutes, and the highest LP-PRP platelet enrichment coefficient could be obtained by centrifugation for 2 minutes at a speed of 1 200 r/min. The platelet recovery rates of LP-PRP obtained by centrifugation at 1 200 r/min for 4 and 5 minutes, as well as centrifugation at 1 400 r/min for 5 minutes, were both greater than 60%. There was no significant difference between the groups when centrifuged at 1 200 r/min for 4 and 5 minutes (P>0.05). Conclusion In the process of preparing rabbit LP-PRP using a single centrifugation method, collecting 15 mL of blood and centrifuging at a radius of 16.7 cm and speed of 1 200 r/min for 4 minutes can prepare LP-PRP with a volume exceeding 2.0 mL, platelet enrichment coefficient exceeding 2.0, and platelet recovery rate exceeding 60%. This centrifugal condition can achieve the optimal LP-PRP action parameters in the shortest possible time.
Objective To compare effectiveness of injecting platelet-rich plasma (PRP) with different concentrations of leukocytes under ultrasound in treatment of supraspinatus tendon injury. Methods A clinical data of 30 patients with supraspinatus tendon injury, who met the selection criteria and were admitted between December 2022 and December 2023, was retrospectively analyzed. Thirty patients were treated with 4 injections of leukocyte-poor PRP (LP-PRP, n=10), leukocyte-rich PRP (LR-PRP, n=11), and triamcinolone (n=9), with an interval of 7-10 days between each injection. There was no significant difference between groups (P>0.05) in the age, gender, disease duration, affected shoulder side, Ellman classification, preoperative visual analogue scale (VAS) score, Constant score, Disabilities of the Arm, Shoulder, and Hand (DASH) score, and American Shoulder and Elbow Surgeons (ASES) score. At 1, 3, and 6 months after injection, the shoulder pain and function were evaluated by using the VAS score, Constant score, DASH score, and ASES score. MRI was conducted to observe supraspinatus tendon healing. Results No severe adverse reactions was observed in all groups. All patients were followed up 6-7 months, with an average of 6.5 months. After injection, the ASES score and Constant score gradually increased in the LR-PRP group and LP-PRP group, while the VAS score and DASH score decreased, with significant differences compared to before injection (P<0.05). Except for no significant difference between 3 and 6 months after injection in LR-PRP group (P>0.05), the above scores showed significant differences between different time points (P<0.05). At 1 month after injection, the Constant score in triamcinolone group significantly increased compared to before injection, while the VAS score significantly decreased (P<0.05). There was no significant difference in all scores between other time points in the triamcinolone group (P>0.05). Except for 1 month after injection, there was no significant difference in Constant score and VAS score between groups (P>0.05). At all other time points, the LR-PRP group and LP-PRP group had better scores than the triamcinolone group (P<0.05). There was no significant difference between the LR-PRP group and the LP-PRP group (P>0.05). MRI showed that only 4 patients in the LP-PRP group had signs of repair at the supraspinatus tendon injury site at 6 months after injection, while no significant tendon repair sign was observed in the other patients. Conclusion Compared with triamcinolone treatment, multiple injections of LP-PRP and LR-PRP under ultrasound can promote the recovery of shoulder joint function and significantly relieve pain in patients with supraspinatus tendon injury, and imaging improvement can be seen after LP-PRP treatment.
In recent years, regenerative medical technology and modern rehabilitation technology complement each other and develop rapidly. Regenerative rehabilitation with tissue regeneration and functional recovery as the core concept arises at the historic moment. Regenerative rehabilitation can quickly repair damaged or diseased tissues and organs, and restore or improve the function of patients to the greatest extent. This paper introduces the origin and development of regenerative rehabilitation, discusses the research progress and significance of related strategies from three aspects of neurological, motor and circulatory diseases, and stress the importance of regenerative rehabilitation in helping patients to obtain the best curative effect.
ObjectiveTo investigate the effect of vacuum sealing drainage (VSD) combined with autologous platelet-rich plasma gel (PRP) on postoperative wound infection and chronic poor wound healing, so as to provide more economical and safe treatment in clinic.MethodsThe patients with postoperative wound infection and chronic poor wound healing in the Second Affiliated Hospital of Nanchang University and Sixth Affiliated Hospital of Sun Yat-sen University from September 2018 to July 2019 were collected, then were divide into PRP+VSD group and VSD group according to treatment methods. The patients in the PRP+VSD group were filled with PRP and activator calcium thrombin following debridement, then covered with silver ion dressing and continuous VSD; in the VSD group were directly covered with silver ion dressing and then continuous VSD. The general situations of patients in the two groups during the process of replacing the VSD and the wound condition during dressing replacing were observed.ResultsThere were 100 patients in this study, 50 in the PRP+VSD group and 50 in the VSD group. There were no significant differences in the age, gender, body mass index, wound area before treatment, and wound infection type between the two groups (P>0.05). Compared with the VSD group, the PRP+VSD group had higher score of fresh granulation tissue coverage area (P<0.05), shorter wound closure time (P<0.05), shorter wound healing time (P<0.05), lower pain score (P<0.05), and less hospitalization expenses (P<0.05), lower rates of second debridement (P<0.05) and recurrent infection (P<0.05).ConclusionAutologous PRP combined with VSD in treatment of postoperative wound infection and chronic poor wound healing could shorten growth time of wound granulation tissue, promote rapid wound healing, reduce cost, and provide an economic, safe, and effective treatment method for clinical practice.
ObjectiveTo analyze the effectiveness of percutaneous injection of autologous concentrated bone marrow aspirate (cBMA) combined with platelet-rich plasma (PRP) in the treatment of delayed fracture healing.MethodsA prospective, randomized, controlled, single-blind case study was conducted. Between March 2016 and July 2018, 66 patients who met the inclusion and exclusion criteria for delayed fracture healing but had solid internal fixation of the fracture end were randomly divided into control group (31 cases, treated with percutaneous autogenous bone marrow blood injection) and study group (35 cases, treated with percutaneous autogenous cBMA+PRP injection). General data such as gender, age, body mass index, site of delayed fracture healing, length of bone defect at fracture end, and preoperative radiographic union score for tibia (RUST) showed no significant difference between the two groups (P>0.05). Before injection, Kirschner wire was used in both groups to stimulate the fracture end and cause minor injury. The fracture healing time, treatment cost, and adverse reactions were recorded and compared between the two groups. Visual analogue scale (VAS) score was used to evaluate pain improvement. The tibial RUST score was extended to the tubular bone healing evaluation.ResultsNo infection of bone marrow puncture needle eyes occurred in both groups. In the control group, local swelling was obvious in 5 cases and pain was aggravated at 1 day after operation in 11 cases. In the study group, postoperative swelling and pain were not obvious, but 2 cases presented local swelling and pain. All of them relieved after symptomatic treatment. Patients in both groups were followed up, the follow-up time of the control group was 16-36 months (mean, 21.8 months), and the study group lasted 14-33 months (mean, 23.2 months). The amount of bone marrow blood was significantly lower in the study group than in the control group (t=4.610, P=0.000). The degree of postoperative pain in the study group was less than that in the control group, and the treatment cost was higher than that in the control group. But the differences between the two groups in VAS score at 1 day after operation and treatment cost were not significant (P>0.05). Fracture healing was achieved in 19 cases (61.3%) in the control group and 30 cases (85.7%) in the study group. The difference in fracture healing rate between the two groups was significant (χ2=5.128, P=0.024). Fracture healing time and RUST score at last follow-up were significantly better in the study group than in the control group (P<0.05). At last follow-up, RUST scores in both groups were significantly improved when compared with those before operation (P<0.05).ConclusionAutogenous cBMA combined with PRP percutaneous injection can provide high concentration of BMSCs and growth factors, and can improve the fracture healing rate and shorten the fracture healing time better than autogenous bone marrow blood injection.
Objective To explore the effectiveness and mechanism of pure platelet-rich plasma (P-PRP) on osteochondral injury of talus. Methods Thirty-six patients with osteochondral injury of talus selected between January 2014 and October 2017 according to criteria were randomly divided into control group (group A), leukocyte PRP (L-PRP) group (group B), and P-PRP group (group C), with 12 cases in each group. There was no significant difference in gender, age, disease duration, and Hepple classification among the three groups (P>0.05). Patients in the groups B and C were injected with 2.5 mL L-PRP or P-PRP at the bone graft site, respectively. Patients in the group A were not injected with any drugs. The American Orthopaedic Foot and Ankle Society (AOFAS) score and visual analogue scale (VAS) score were used to evaluate the effectiveness before operation and at 3, 6, and 12 months after operation. Study on the therapeutic mechanism of P-PRP: MC3T3-E1 cells were randomly divided into control group (group A), L-PRP group (group B), and P-PRP group (group C). Groups B and C were cultured with culture medium containing 5% L-PRP or P-PRP respectively. Group A was cultured with PBS of the same content. MTT assay was used to detect cell proliferation; ELISA was used to detect the content of matrix metalloprotein 9 (MMP-9) protein in supernatant; alkaline phosphatase (ALP) activity was measured; and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of osteopontin (OPN), collagen type Ⅰ, and MMP-9 in cells. Western blot was used to detect the expression of MMP-9 in supernatant and phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (pAKT), and phosphorylated c-Jun (p-c-Jun) in cells. ResultsAll patients were followed up 13-25 months, with an average of 18 months. No complication such as wound infection and internal fixation failure occurred. MRI showed that the degree of injury was similar between the three groups before operation, and patients in the three groups all recovered at 6 months after operation. Moreover, group C was superior to groups A and B. Compared with preoperation, AOFAS scores and VAS scores in the three groups were all significantly improved at each time point after operation (P<0.05). AOFAS score of group C was significantly higher than that of groups A and B at 3, 6, and 12 months after operation (P<0.05); there was no significant difference in VAS score between the three groups (P>0.05). Study on the therapeutic mechanism of P-PRP: The absorbance (A) value, ALP activity, the relative mRNA expression of OPN and collagen type Ⅰ in group C were significantly higher than those in groups A and B (P<0.05), and those in group B were significantly higher than those in group A (P<0.05). The relative expression of MMP-9 protein and mRNA and the content of MMP-9 protein detected by ELISA in group B were significantly higher than those in groups A and C, while those in group C were significantly lower than those in group A (P<0.05). Western blot detection showed that the relative expression of PI3K, pAKT, and p-c-Jun protein in group B was significantly higher than those in groups A and C (P<0.05), but there was no significant difference between groups A and C (P>0.05). Conclusion P-PRP is superior to L-PRP for osteochondral injury of talus, which may be related to the inhibition of PI3K/AKT/AP-1 signaling pathway in the osteoblast, thereby reducing the secretion of MMP-9.
ObjectiveTo summary the standardized management in research and applications of platelet derivatives for tissue regeneration.MethodsThe related literature about bottlenecks and standardized management of platelet derivatives in recent years was reviewed and analyzed.ResultsAlthough the platelet derivatives are increasingly used to accelerate the regenerative processes of injured joint, skin, nerve, ligament/tendon, and alveolar bone, etc., the large variation in preparation methods, diverse nomenclature, incomplete reporting system, and lack of quantitative and standardized management of the preparation process have caused uncertainty and incomparability of research and application results. In recent years, there has been a trend towards standardized research and management of platelet derivatives.ConclusionThe implementation of standardized research and quality management will contribute to promote the research and application of platelet derivatives in the field of tissue regeneration.