Objective To investigate the effect of human platelet-rich plasma-derived exosomes(PRP-exos)on the proliferation of Schwann cell(SC)cultured in vitro. Methods PRP-exos were extracted by polymerization-precipitation combined with ultracentrifugation.The morphology of PRP-exos was observed by transmission electron microscopy,and the concentration and particle size distribution of PRP-exos were determined by nanoparticle tracking analysis.Western blotting was employed to determine the expression of the marker proteins CD63,CD81,and CD9 on exosome surface and the platelet membrane glycoprotein CD41.The SCs of rats were isolated and cultured,and the expression of the SC marker S100β was detected by immunofluorescence staining.The fluorescently labeled PRP-exos were co-cultured with SCs in vitro for observation of their interaction.EdU assay was employed to detect the effect of PRP-exos on SC proliferation,and CCK-8 assay to detect the effects of PRP-exos at different concentrations(0,10,20,40,80,and 160 μg/ml)on SC proliferation. Results The extracted PRP-exos appeared as uniform saucer-shaped vesicles with the average particle size of(122.8±38.7)nm and the concentration of 3.5×10¹² particles/ml.CD63,CD81,CD9,and CD41 were highly expressed on PRP-exos surface(P<0.001,P=0.025,P=0.004,and P=0.032).The isolated SCs expressed S100β,and PRP-exos could be taken up by SCs.PRP-exos of 40,80,and 160 μg/ml promoted the proliferation of SCs,and that of 40 μg/ml showed the best performance(all P<0.01). Conclusions High concentrations of PRP-exos can be extracted from PRP.PRP-exos can be taken up by SCs and promote the proliferation of SCs cultured in vitro.
Humans ; Rats ; Animals ; Exosomes/metabolism* ; Platelet-Rich Plasma ; Schwann Cells ; Coculture Techniques ; Cell Proliferation ; Cells, Cultured

Tissue regeneration is an important engineering method for the treatment of oral soft and hard tissue defects.Growth factors,as one of the three elements of tissue regeneration,are a necessary condition for tissue regeneration.Concentrated growth factor(CGF)is a new generation of blood extract prepared by changing the centrifugal speed on the basis of the preparation of platelet-rich plasma(PRP)and platelet-rich fibrin(PRF).It contains abundant growth factors and a fibrin matrix with a three-dimensional network structure,being capable of activating angiogenesis and promoting tissue regeneration and healing.CGF has been widely used in the repair and regeneration of oral soft and hard tissues.This paper introduces the preparation and composition of CGF and reviews the application of CGF in oral implantation and the regeneration of oral bone tissue,periodontal tissue,and dental pulp tissue.
Platelet-Rich Plasma/metabolism* ; Platelet-Rich Fibrin ; Cell Proliferation ; Bone and Bones ; Intercellular Signaling Peptides and Proteins/metabolism* ; Bone Regeneration

OBJECTIVE: To investigate the role of platelet-rich plasma (PRP) in inducing the M2 macrophage polarization via regulating AMPK singling pathway. METHODS: The expressions of M1 marker CD11c and M2 marker CD206 in macrophages of blank control group, LPS group, LPS+PRP group, and LPS+PRP+Compound C group were detected by flow cytometry. Western blot was used to observe the effects of PRP on the expression of AMPK-mTOR signaling pathway-related proteins at different times (12 h, 18 h and 24 h) after LPS treatment. RNA interference technology was used to silence the expression of AMPK in macrophages, and the expression of TGF-β protein was subsequently examined by Western blot. RESULTS: LPS significantly reduced the expression of CD206 and increased the expression of CD11c (P <0.05). After the addition of PRP, the expression of CD206 was significantly increased (P <0.05), while the expression of CD11c was significantly decreased (P <0.05). Compared with LPS group, PRP treatment significantly increased the expressions of p-AMPK and p-ULK1 proteins at 12 h, 18 h and 24 h, while significantly decreased the expression of p-mTOR protein (P <0.05). After the addition of AMPK inhibitor Compound C, the expression of CD206 was significantly reduced (P <0.05) and the expression of CD11c was significantly increased compared with LPS+PRP group (P <0.05). After silencing the expression of AMPK in macrophages, the promotion effect of PRP on TGF-β was significantly reduced (P <0.05). CONCLUSION PRP can stimulate the transformation of macrophages to M2 type via AMPK signalling pathway.
Humans ; AMP-Activated Protein Kinases/pharmacology* ; Lipopolysaccharides/pharmacology* ; Macrophages/metabolism* ; Transforming Growth Factor beta/metabolism* ; Platelet-Rich Plasma/metabolism*

Epithelial attachment via the basal lamina on the tooth surface provides an important structural defence mechanism against bacterial invasion in combating periodontal disease. However, when considering dental implants, strong epithelial attachment does not exist throughout the titanium-soft tissue interface, making soft tissues more susceptible to peri-implant disease. This study introduced a novel synthetic peptide (A10) to enhance epithelial attachment. A10 was identified from a bacterial peptide display library and synthesized. A10 and protease-activated receptor 4-activating peptide (PAR4-AP, positive control) were immobilized on commercially pure titanium. The peptide-treated titanium showed high epithelial cell migration ability during incubation in platelet-rich plasma. We confirmed the development of dense and expanded BL (stained by Ln5) with pericellular junctions (stained by ZO1) on the peptide-treated titanium surface. In an adhesion assay of epithelial cells on A10-treated titanium, PAR4-AP-treated titanium, bovine root and non-treated titanium, A10-treated titanium and PAR4-AP-treated titanium showed significantly stronger adhesion than non-treated titanium. PAR4-AP-treated titanium showed significantly higher inflammatory cytokine release than non-treated titanium. There was no significant difference in inflammatory cytokine release between A10-treated and non-treated titanium. These results indicated that A10 could induce the adhesion and migration of epithelial cells with low inflammatory cytokine release. This novel peptide has a potentially useful application that could improve clinical outcomes with titanium implants and abutments by reducing or preventing peri-implant disease.
Amino Acid Sequence ; Animals ; Benzeneacetamides ; chemical synthesis ; pharmacology ; Cattle ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Cytokines ; metabolism ; Dental Implants ; Enzyme-Linked Immunosorbent Assay ; Epithelial Attachment ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Piperidones ; chemical synthesis ; pharmacology ; Platelet-Rich Plasma ; Receptors, Thrombin ; Surface Properties ; Titanium ; chemistry

STUDY DESIGN: Preliminary clinical trial. PURPOSE: To determine the safety and initial efficacy of intradiscal injection of autologous platelet-rich plasma (PRP) releasate in patients with discogenic low back pain. OVERVIEW OF LITERATURE: PRP, which is comprised of autologous growth factors and cytokines, has been widely used in the clinical setting for tissue regeneration and repair. PRP has been shown in vitro and in vivo to potentially stimulate intervertebral disc matrix metabolism. METHODS: Inclusion criteria for this study included chronic low back pain without leg pain for more than 3 months; one or more lumbar discs (L3/L4 to L5/S1) with evidence of degeneration, as indicated via magnetic resonance imaging (MRI); and at least one symptomatic disc, confirmed using standardized provocative discography. PRP releasate, isolated from clotted PRP, was injected into the center of the nucleus pulposus. Outcome measures included the use of a visual analog scale (VAS) and the Roland-Morris Disability Questionnaire (RDQ), as well as X-ray and MRI (T2-quantification). RESULTS: Data were analyzed from 14 patients (8 men and 6 women; mean age, 33.8 years). The average follow-up period was 10 months. Following treatment, no patient experienced adverse events or significant narrowing of disc height. The mean pain scores before treatment (VAS, 7.5±1.3; RDQ, 12.6±4.1) were significantly decreased at one month, and this was generally sustained throughout the observation period (6 months after treatment: VAS, 3.2±2.4, RDQ; 3.6±4.5 and 12 months: VAS, 2.9±2.8; RDQ, 2.8±3.9; p<0.01, respectively). The mean T2 values did not significantly change after treatment. CONCLUSIONS: We demonstrated that intradiscal injection of autologous PRP releasate in patients with low back pain was safe, with no adverse events observed during follow-up. Future randomized controlled clinical studies should be performed to systematically evaluate the effects of this therapy.
Cytokines ; Female ; Follow-Up Studies ; Humans ; In Vitro Techniques ; Intercellular Signaling Peptides and Proteins ; Intervertebral Disc ; Intervertebral Disc Degeneration ; Leg ; Low Back Pain* ; Magnetic Resonance Imaging ; Male ; Metabolism ; Outcome Assessment (Health Care) ; Platelet-Rich Plasma* ; Regeneration ; Visual Analog Scale

OBJECTIVEWe explored the expressions of the Notch and Wnt signaling pathways and their significance in the repair process of alveolar bone defects by establishing animal models with a composite of autologous bone marrow mesenchymal stem cells (BMSCs) and platelet-rich fibrin (PRF) to repair bone defects in the extraction sockets of rabbits.

METHODSA total of 36 two-month-old male New Zealand white rabbits were randomly divided into four groups, and the left mandibular incisors of all the rabbits were subjected to minimally invasive removalunder general anesthesia. BMSC-PRF compounds, single PRF, and single BMSC were implanted in Groups A, B, and C. No material was implanted in Group D (blank control). The animals were sacrificed at 4, 8 and 12 weeks after surgery, the bone defect was immediately drawn, and the bone specimens underwent surgery after four, eight, and twelve weeks, with three rabbits per time point. The expressions of Notch1 and Wnt3a in the repair process of the bone defect were measured via immunohistochemical and immunofluorescence detection.

RESULTSImmunohistochemistry showed that the expressions of Notch1 and Wnt3a in Groups A, B, and C were higher than that in Group D at the fourth and eighth week after operation (P<0.05). By contrast, the expressions of Notch1 and Wnt3a in Group D were higher than those in Groups A, B, and C at the twelfth week (P<0.05). Immunofluorescence showed that the expressions of both Notch1 and Wnt3a reached their peaks in the new bone cells of the bone defect after four weeks following surgery and gradually disappeared when the bone was repaired completely.

CONCLUSIONNotch1 and Wnt3a signaling molecules are expressed in the process of repairing bone defects using BMSC-PRF composites and can accelerate the healing by regulating the proliferation and differentiation of BMSCs. Moreover, the expressions of Notch and Wnt are similar, and a crosstalk between them may exist it.

Alveolar Bone Grafting ; methods ; Animals ; Blood Platelets ; Bone Marrow Cells ; cytology ; Bone Transplantation ; methods ; Bone and Bones ; abnormalities ; Cell Differentiation ; Fibrin ; administration & dosage ; Male ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; Platelet-Rich Plasma ; Rabbits ; Random Allocation ; Receptor, Notch1 ; metabolism ; Tissue Engineering ; Wnt Signaling Pathway ; Wnt3A Protein ; metabolism ; Wound Healing

This study was conducted to identify the effectiveness of platelet-rich plasma (PRP) and efficacy of intralesional injection as a method of application to acute cutaneous wounds in dogs. Healthy adult beagles (n = 3) were used in this study. Autologous PRP was separated from anticoagulant treated whole blood in three dogs. Cutaneous wounds were created and then treated by intralesional injection of PRP in the experimental group, while they were treated with saline in the control group on days 0, 2 and 4. The healing process was evaluated by gross examination throughout the experimental period and histologic examination on day 7, 14 and 21. In PRP treated wounds, the mean diameter was smaller and the wound closure rate was higher than in the control. Histological study revealed that PRP treated wounds showed more granulation formation and angiogenesis on day 7, and faster epithelialization, more granulation formation and collagen deposition were observed on day 14 than in control wounds. On day 21, collagen deposition and epithelialization were enhanced in PRP treated groups. Overall, PRP application showed beneficial effects in wound healing, and intralesional injection was useful for application of PRP and could be a good therapeutic option for wound management in dogs.
Animals ; Collagen/metabolism ; Dermis/cytology/injuries/physiology ; Dogs ; Epidermis/cytology/injuries/*physiology ; Female ; Granulation Tissue/cytology ; Injections, Intralesional/veterinary ; Male ; Neovascularization, Physiologic ; *Platelet-Rich Plasma ; Regeneration ; Treatment Outcome ; *Wound Healing ; Wounds and Injuries/therapy/*veterinary

Platelet-enriched plasma (PRP) contains high concentration of platelets and abundant growth factors, which is made by centrifuging of blood and separating of blood elements. PRP promotes tendon repair by releasing various cytokines to enhance cell proliferation, tenogenic differentiation, formation and secretion of matrix; meantime, it can reduce pain by inhibiting the expression of pain-associated molecules. A number of clinical studies demonstrated that PRP was effective in treatment of tendinopathy, including patellar tendinopathy, lateral epicondylitis and plantar fasciopathy. However, some studies did not support this conclusion, because of disparity of PRP types, therapeutic courses and injections protocols in clinical application. Based on its safety, PRP can be a choice of treatment for tendinopathy, in case other non-surgical therapies are of no effect.
Blood Platelets ; cytology ; Cytokines ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Platelet-Rich Plasma ; Tendinopathy ; therapy

OBJECTIVETo explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on LPS-induced abnormal activation of platelets in peripheral blood of healthy human donors and its possible molecular mechanism.

METHODSVenous blood samples were collected from a healthy volunteer, and platelet-rich plasma (PRP) from the blood were isolated by differential centrifugation. The PRP was subpackaged into siliconized test tubes and then divided into control group, LPS group, inactive CORM-2 (iCORM-2) group, 10 µmol/L CORM-2 group, and 50 µmol/L CORM-2 group according to the random number table, with 3 tubes in each group. The PRP in control group did not receive any treatment. The PRP in LPS group received LPS (20 mL, 10 µg/mL) stimulation, and the PRP in iCORM-2 group, 10 µmol/L CORM-2 group, and 50 µmol/L CORM-2 group underwent the same LPS stimulation and treatment of 50 µmol/L iCORM-2, 10 µmol/L CORM-2, and 50 µmol/L CORM-2, respectively, with the dosage of 20 mL. After being cultured for 30 min, the platelet adhesion rate was determined by glass bottle method, the number of platelet spreading on fibrinogen was determined with immunofluorescent method, and the platelet aggregation rate was measured by turbidimetric method. The platelet poor plasma (PPP) was prepared from PRP, the levels of ATP in PPP and platelets were determined by chemical fluorescein method. The expressions of platelet glycoprotein I bα (GPIbα) and GPVI were analyzed by flow cytometer. The expressions of glycogen synthase kinase 3β (GSK-3β) and phosphorylated GSK-3β were determined by Western blotting and immunoprecipitation, respectively. Measurement of the above indices was repeated for 3 times. Data were processed with one-way analysis of variance and SNK test.

RESULTSCompared with those in control group, the platelet adhesion rates, numbers of platelets spreading on fibrinogen, platelet aggregation rates, expressions of GPIbα and GPVI in PRP, levels of ATP in PPP in LPS and iCORM-2 groups were significantly increased, while levels of ATP in platelets were significantly decreased (with P values below 0.05). Compared with those in LPS group, the former 7 indices in iCORM-2 group showed no significant differences (with P values above 0.05), while the levels of ATP in platelets in the 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly increased, and the other 6 indices in 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly decreased (with P values below 0.05). The expression levels of GSK-3β of the platelets in PRP in control, LPS, iCORM-2, 10 µmol/L CORM-2, and 50 µmol/L CORM-2 groups were 0.550 ± 0.060, 1.437 ± 0.214, 1.210 ± 0.108, 0.720 ± 0.010, and 0.670 ± 0.010, respectively, and the expression levels of the phosphorylated GSK-3β of the platelets in PRP in the above 5 groups were 0.950 ± 0.070, 1.607 ± 0.121, 1.420 ± 0.040, 1.167 ± 0.015, and 0.513 ± 0.122, respectively. Compared with those in control group, both the expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in LPS and iCORM-2 groups were significantly increased (with P values below 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP between LPS group and iCORM-2 group were similar (with P values above 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly decreased compared with those in LPS group (with P values below 0.05).

CONCLUSIONSLPS stimulation can abnormally activate the platelets in peripheral blood of healthy human, but the abnormal activation can be inhibited by CORM-2 intervention, and the mechanism of the latter may involve the phosphorylation of GSK-3β mediated by GP.

Blood Platelets ; drug effects ; metabolism ; Carbon Monoxide ; metabolism ; Glycogen Synthase Kinase 3 ; Glycogen Synthase Kinase 3 beta ; Humans ; Lipopolysaccharides ; pharmacology ; Organometallic Compounds ; pharmacology ; Phosphorylation ; drug effects ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Platelet-Rich Plasma

Canine mesenchymal cells (MSCs) derived from Wharton's jelly were co-cultured, then supplemented or not supplemented with platelet rich plasma (PRP) and demineralized bone matrix (DBM) to verify osteogenic differentiation. Osteoblastic differentiation followed by mineralized bone matrix production was found to be significantly higher (p < 0.05) when MSCs were associated with PRP/DBM in culture after 14-21-days of induction. Osteopontin and osteocalcin gene expression were significantly superior (p < 0.05) under the same culture conditions after 21 days of observation. In conclusion, addition of PRP to DBM co-cultured with MSCs successfully induced osteogenesis in vitro.
Animals ; Bone Demineralization Technique/veterinary ; Bone Matrix/*metabolism ; Cell Differentiation ; Cells, Cultured ; Coculture Techniques/veterinary ; Dogs ; Mesenchymal Stromal Cells/*metabolism ; *Osteogenesis ; Platelet-Rich Plasma/*metabolism ; Umbilical Cord/metabolism