ObjectiveTo evaluate the effect of leucocyte- and platelet-rich plasma (L-PRP) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in treating avascular necrosis of the femoral head (ANFH) in rabbits. MethodsTwenty-four New Zealand white rabbits (4-6 months old, both genders, weighing 2.0-3.0 kg) were used for the establishment of bilateral ANFH models and divided into 4 groups (n=6). BMSCs were isolated from the bone marrow of iliac crest, cultured and identified. L-PRP was prepared by Landesberg method. Core decompression only (group A), core decompression and L-PRP implantation (group B), core decompression and BMSCs implantation (group C), and core decompression and implantation of BMSCs and L-PRP were performed in 4 groups. To evaluate bone formation and remodeling of the defects, X-ray photography was taken at 2, 4, and 8 weeks postoperatively. The modified Lane-Sandhu scoring system was used to evaluate the bone formation. Two rabbits were sacrificed at 2, 4, 8 weeks after operation to harvest the specimens for histological observation, new blood vessel count and new bone area ratio. ResultsThe observations of radiology and histology displayed different degrees of bone regeneration at bone defect sites in each group. At 2, 4, and 8 weeks postoperatively, the results of Lane-Sandhu X-ray photography scoring, new blood vessel count, and new bone area ratio showed that groups C and D were significantly better than groups A and B, group D was significantly better than group C. and group B was significantly better than group A (P<0.05). ConclusionThese findings demonstrate that L-PRP can promote osteogenic differentiation of BMSCs in treating ANFH in rabbits, and core decompression associated with BMSCs and L-PRP is an effective and feasible method to treat ANFH.
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.
ObjectiveTo investigate the effectiveness of micro-fracture therapy combined with intra-articular injection of platelet-rich plasma (PRP) in the treatment of small sized osteochondral lesion of the talus (OLT).MethodsBetween September 2014 and October 2017, 43 patients with small sized OLT met the inclusive criteria were admitted and randomly divided into micro-fracture group (21 cases) and combined group (22 cases). Patients in the micro-fracture group were treated with micro-fracture therapy, and patients in the combined group were treated with micro-fracture therapy combined with intra-articular injection of PRP. There was no significant difference in gender, age, disease duration, side of OLT, injured position, lesion area, Mintz classification, and preoperative American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hind foot score and visual analogue scale (VAS) score between the two groups (P>0.05). After treatment, MRI, VAS score, and AOFAS ankle-hind foot score were used to evaluate the recovery of OLT and the ankle function.ResultsAll incisions healed by first intention, and no complications such as venous thrombosis and ankle joint infection occurred. All patients were followed up 12-18 months after operation, with an average of 15.6 months. The VAS scores and the AOFAS ankle-hind foot scores were significantly improved at 6 and 12 months after operation in the two groups (P<0.05), and the scores at 12 months were significantly improved when compared with postoperative scores at 6 months (P<0.05). Compared with the micro-fracture group, the VAS score and the AOFAS ankle-hind foot score were significantly improved in the combined group at 6 and 12 months after operation (P<0.05). MRI showed that OLT was well filled in both groups at 12 months after operation.ConclusionCompared with micro- fracture therapy, micro-fracture therapy combined with intra-articular injection of PRP can effectively reduce pain, improve ankle function, and has a good effectiveness in the treatment of small sized OLT.
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 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.
ObjectiveTo investigate the effectiveness of extracorporeal shock wave therapy combined with platelet-rich plasma (PRP) injection in treatment of knee osteoarthritis (KOA) by prospective clinical study.MethodsBetween June 2015 and June 2018, 180 patients with KOA met the inclusion criteria were included in study and randomly allocated to group A (n=60), group B (n=60), and group C (n=60). The patients were treated with autologous PRP intra-articular injection in group A, extracorporeal shock wave therapy in group B, and extracorporeal shock wave therapy combined with autologous PRP intra-articular injection in group C, once a week and 5 times a duration of treatment. There was no significant difference in age, gender, disease duration, side of KOA, and Kellgren-Lawrence grading between groups (P>0.05). The pain and function of knee joint were assessed by visual analogue scale (VAS) score, Lequesne Index score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, and knee joint activity before treatment and at 1, 3, and 5 weeks after the first treatment.ResultsThere were significant differences in VAS score, Lequesne Index score, WOMAC score, and knee joint activity between pre- and post-treatment in all groups (P<0.05). VAS score, Lequesne Index score, and WOMAC score gradually decreased with the prolongation of treatment time (P<0.05); but there was no significant difference in knee joint activity between different time points (P>0.05). There was no significant difference in VAS score, Lequesne Index score, WOMAC score, and knee joint activity between groups before treatment (P>0.05); the scores of group C were superior to groups A and B (P< 0.05) at different time points after treatment; while the knee joint activities of 3 groups were similar (P>0.05).ConclusionThe extracorporeal shock wave therapy combined with PRP injection can relieve the pain synergistically for KOA.
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.
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.
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 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.