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1.  Synergistic Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Platelet-Rich Plasma in Streptozotocin-Induced Diabetic Rats 
Annals of Dermatology  2014;26(1):1-10.
Background
Diabetic wounds are a major clinical challenge, because minor skin wounds can lead to chronic, unhealed ulcers and ultimately result in infection, gangrene, or even amputation. Studies on bone marrow derived mesenchymal stem cells (BMSCs) and a series of growth factors have revealed their many benefits for wound healing and regeneration. Platelet-rich plasma (PRP) may improve the environment for BMSC development and differentiation. However, whether combined use of BMSCs and PRP may be more effective for accelerating diabetic ulcer healing remains unclear.
Objective
We investigated the efficacy of BMSCs and PRP for the repair of refractory wound healing in a diabetic rat model.
Methods
Forty-eight rats with diabetes mellitus induced by streptozotocin were divided into four groups: treatment with BMSCs plus PRP, BMSCs alone, PRP alone, phosphate buffered saline. The rate of wound closure was quantified. A histopathological study was conducted regarding wound depth and the skin edge at 7, 14, and 28 days after surgery.
Results
Wound healing rates were significantly higher in the BMSC plus PRP group than in the other groups. The immunohistochemistry results showed that the expression of platelet/endothelial cell adhesion molecule 1, proliferating cell nuclear antigen, and transforming growth factor-β1 increased significantly in the BMSC plus PRP group compared to the other treatment groups. On day 7, CD68 expression increased significantly in the wounds of the BMSC plus PRP group, but decreased markedly at day 14 compared to the controls.
Conclusion
The combination of BMSCs and PRP aids diabetic wound repair and regeneration.
doi:10.5021/ad.2014.26.1.1
PMCID: PMC3956772  PMID: 24648680
Bone marrow-derived mesenchymal stem cell; Diabetes mellitus; Platelet-rich plasma; Wounds
2.  Platelet-rich plasma (PRP) in dental and oral surgery: from the wound healing to bone regeneration 
Platelet-rich plasma (PRP) is a new approach to tissue regeneration and it is becoming a valuable adjunct to promote healing in many procedures in dental and oral surgery, especially in aging patients. PRP derives from the centrifugation of the patient's own blood and it contains growth factors that influence wound healing, thereby playing an important role in tissue repairing mechanisms. The use of PRP in surgical practice could have beneficial outcomes, reducing bleeding and enhancing soft tissue healing and bone regeneration. Studies conducted on humans have yielded promising results regarding the application of PRP to many dental and oral surgical procedures (i.e. tooth extractions, periodontal surgery, implant surgery). The use of PRP has also been proposed in the management of bisphosphonate-related osteonecrosis of the jaw (BRONJ) with the aim of enhancing wound healing and bone maturation. The aims of this narrative review are: i) to describe the different uses of PRP in dental surgery (tooth extractions and periodontal surgery) and oral surgery (soft tissues and bone tissue surgery, implant surgery and BRONJ surgery); and ii) to discuss its efficacy, efficiency and risk/benefit ratio. This review suggests that the use of PRP in the alveolar socket after tooth extractions is certainly capable of improving soft tissue healing and positively influencing bone regeneration but the latter effect seems to decrease a few days after the extraction. PRP has produced better results in periodontal therapy in association with other materials than when it is used alone. Promising results have also been obtained in implant surgery, when PRP was used in isolation as a coating material. The combination of necrotic bone curettage and PRP application seem to be encouraging for the treatment of refractory BRONJ, as it has proven successful outcomes with minimal invasivity. Since PRP is free from potential risks for patients, not difficult to obtain and use, it can be employed as a valid adjunct in many procedures in oral and dental surgery. However, further RCTs are required to support this evidence.
doi:10.1186/1742-4933-10-23
PMCID: PMC3683340  PMID: 23763951
PRP; Wound healing; Bone regeneration; Dental surgery; Oral surgery; Tooth extraction; Periodontal surgery; Implant surgery; BRONJ
3.  Platlet Rich Plasma (PRP) Improves Fat Grafting Outcomes 
Autologous fat transfer offers many qualities of a ideal soft tissue filler. Main advantages of fat grafting ensue from the fact that the lipoaspirate tissue is an abundant source of regenerative pluripotential cells. However, the reported rates of fat cell survival vary greatly in the medical literature (10-90%). Different techniques of harvesting, processing, and reinjecting the fat cells are so claimed to be responsible for these differences, without any agreement concerning the best way to process. To address this important disadvantage, we propose the addition of autologous platelet rich plasma (PRP) which is known as a natural reservoir of growth factors stimulating tissue repair and regeneration. This approach is completely autologous and immediately employed without any type of preconditioning. Platelets rich plasma (PRP) preparation included bleeding of 8 ml of blood from patient’s peripheral vein in Regen Lab© tubes containing sodium citrate anticoagulant. The whole blood was centrifugated at 1500 g during 3 min. As Regen-tubes contained a special gel separator, 99 % of red blood cells were discarded from the plasma at the bottom of the gel, and >90% of platelets were harvested in 4 ml of plasma on the top of the gel, called the platelet-rich plasma (PRP). The purified fat prepared by Coleman technique was mixed with different amount of PRP for in vitro, in vivo (mice) and clinical experiments: >50% of PRP for skin rejuvenation, superficial scars correction, infraorbital region, ..., and for 20% of PRP with 80% of purified fat for deep filler indication (nasolabial folds, lips, or soft tissue defect). In vitro studies demonstrated that PRP increased fat cells survival rate and stem cells differentiation. Animal models showed that fat graft survival rate was significantly increased by addition of PRP. Several clinical cases confirmed the improvement of wound healing and fat grafting survival in facial reconstruction and aesthetic cases by association of fat grafting with PRP. The addition of PRP to fat grafts represented many advantages with a simple, cost-effective and safe method. In addition to its booster effect on fat grafts, PRP had a rejuvenation capacity per se. It is also used on nappage technique, on mask and as a temporary regenerative filler in combination with thrombin. So we consider the addition of 20% PRP to fat grafts offers a better fat grafting survival, a less bruising and inflammation reaction, and easier application of fat grafts due to liquefaction effect of PRP.
PMCID: PMC4238337  PMID: 25489498
Platlet rich plasma; PRP; Fat; Graft; Outcome
4.  Enhanced skin wound healing by a sustained release of growth factors contained in platelet-rich plasma 
Experimental & Molecular Medicine  2011;43(11):622-629.
Platelet-rich plasma (PRP) contains growth factors that promote tissue regeneration. Previously, we showed that heparin-conjugated fibrin (HCF) exerts the sustained release of growth factors with affinity for heparin. Here, we hypothesize that treatment of skin wound with a mixture of PRP and HCF exerts sustained release of several growth factors contained in PRP and promotes skin wound healing. The release of fibroblast growth factor 2, platelet-derived growth factor-BB, and vascular endothelial growth factor contained in PRP from HCF was sustained for a longer period than those from PRP, calcium-activated PRP (C-PRP), or a mixture of fibrin and PRP (F-PRP). Treatment of full-thickness skin wounds in mice with HCF-PRP resulted in much faster wound closure as well as dermal and epidermal regeneration at day 12 compared to treatment with either C-PRP or F-PRP. Enhanced skin regeneration observed in HCF-PRP group may have been at least partially due to enhanced angiogenesis in the wound beds. Therefore, this method could be useful for skin wound treatment.
doi:10.3858/emm.2011.43.11.070
PMCID: PMC3249588  PMID: 21847007
angiogenesis inducing agents; endothelial growth factors; fibrin; fibroblast growth factor 2; heparin; neovascularization, physiologic; platelet-derived growth factor; platelet-rich plasma; wound healing
5.  A Prospective Study Involving the Use of Platelet Rich Plasma in Enhancing the Uptake of Bone Grafts in the Oral and Maxillofacial Region 
Abstract
Platelet-rich plasma (PRP) is an autologous product that contains highly concentrated number of platelets in a small volume of plasma, derived from whole blood by gradient density centrifugation. It has been speculated that local growth factors in human platelets (insulin-like growth factor, IGF; transforming growth factor, TGF-β; platelet derived growth factor, PDGF) would enhance healing of grafts and also counteract resorption. The aim of this study was to evaluate efficacy of PRP on early healing after autogenous bone grafting. Of the twenty patients selected ten were treated with autogenous bone graft and PRP (PRP group) and other ten with autogenous bone graft alone (non-PRP group). PRP group consisted of two benign tumor of mandible, one post surgical defect, two unilateral alveolar cleft, one bilateral alveolar cleft with skeletal class III malocclusion, one maxillary hypoplasia, one oronasal fistula, one recurrent tumor of mandible, one multiple impacted mandibular teeth. Non-PRP group consisted of seven benign tumor of jaw, one keratocyst odontogenic tumor, one orbital blow out fracture, one residual traumatic defect. Biopsies were taken in the native bone, PRP treated grafted bone, grafted bone without PRP at 3 months to assess the maturity of bone. Radiographic imaging was performed by panoramic radiography at 3 and 6 months to evaluate bone opacity of grafted bone on comparison with native bone and computerized tomography at 6 months to evaluate grafted bone morphologically and to measure bone density in Hounsfield units. Microscopic results showed that significantly more matured bone was formed at PRP treated sites as that of native bone and immature bone in controls after 3 months of healing. Bone opacity of PRP treated bone grafts was close to that of native bone than that of non-PRP treated bone grafts on panoramic radiograph at 3 and 6 months. There was graft loss in three cases and graft resorption in one case of non-PRP treated bone grafts at 6 months. In PRP group the compact bone was clearly differentiated from cancellous bone as in native bone and thick in five cases, thin in five cases. In non-PRP group the compact bone was thin as a whole. Comparing native bone group and PRP group the CT value of PRP treated bone graft was more or less close to native bone group and comparing native bone group and non-PRP group CT value was low in non-PRP treated bone graft. Whereas when comparing PRP and non-PRP group CT value was higher in PRP group. Autologous PRP was a safe, biocompatible, effective, source for growth factors and carries no risk of transmissible diseases. It enhances and accelerates bone regeneration of autogenous bone grafts.
doi:10.1007/s12663-012-0466-3
PMCID: PMC3847021  PMID: 24431876
Platelet-rich plasma; Mandibular reconstruction; Nonvascularised bone grafts; Alveolar bone grafting; Hounsfield unit
6.  Platelet-Rich Plasma and Adipose-Derived Mesenchymal Stem Cells for Regenerative Medicine-Associated Treatments in Bottlenose Dolphins (Tursiops truncatus) 
PLoS ONE  2014;9(9):e108439.
Dolphins exhibit an extraordinary capacity to heal deep soft tissue injuries. Nevertheless, accelerated wound healing in wild or captive dolphins would minimize infection and other side effects associated with open wounds in marine animals. Here, we propose the use of a biological-based therapy for wound healing in dolphins by the application of platelet-rich plasma (PRP). Blood samples were collected from 9 different dolphins and a specific and simple protocol which concentrates platelets greater than two times that of whole blood was developed. As opposed to a commonly employed human protocol for PRP preparation, a single centrifugation for 3 minutes at 900 rpm resulted in the best condition for the concentration of dolphin platelets. By FACS analysis, dolphin platelets showed reactivity to platelet cell-surface marker CD41. Analysis by electron microscopy revealed that dolphin platelets were larger in size than human platelets. These findings may explain the need to reduce the duration and speed of centrifugation of whole blood from dolphins to obtain a 2-fold increase and maintain proper morphology of the platelets. For the first time, levels of several growth factors from activated dolphin platelets were quantified. Compared to humans, concentrations of PDGF-BB were not different, while TGFβ and VEGF-A were significantly lower in dolphins. Additionally, adipose tissue was obtained from cadaveric dolphins found along the Spanish Mediterranean coast, and adipose-derived mesenchymal stem cells (ASCs) were successfully isolated, amplified, and characterized. When dolphin ASCs were treated with 2.5 or 5% dolphin PRP they exhibited significant increased proliferation and improved phagocytotic activity, indicating that in culture, PRP may improve the regenerative capacity of ASCs. Taken together, we show an effective and well-defined protocol for efficient PRP isolation. This protocol alone or in combination with ASCs, may constitute the basis of a biological treatment for wound-healing and tissue regeneration in dolphins.
doi:10.1371/journal.pone.0108439
PMCID: PMC4177220  PMID: 25251412
7.  The Effect of Platelet-rich Plasma on Wounds of OLETF Rats Using Expression of Matrix Metalloproteinase-2 and -9 mRNA 
Archives of Plastic Surgery  2012;39(2):106-112.
Background
Complicated diabetic patients show impaired, delayed wound healing caused by multiple factors. A study on wound healing showed that platelet-rich plasma (PRP) was effective in normal tissue regeneration. Nonetheless, there is no evidence that when plateletrich plasma is applied to diabetic wounds, it normalizes the diabetic wound healing process. In this study, we have analyzed matrix metalloproteinase (MMP)-2, MMP-9 expression to investigate the effect of PRP on diabetic wounds.
Methods
Twenty-four-week-old male Otsuka Long-Evans Tokushima Fatty rats were provided by the Tokushima Research Institute. At 50 weeks, wounds were arranged in two sites on the lateral paraspinal areas. Each wound was treated with PRP gel and physiologic saline gauze. To determine the expression of MMP-2, MMP-9, which was chosen as a marker of wound healing, reverse transcription polymerase chain reaction (RT-PCR) was performed and local distribution and expression of MMP-2, MMP-9 was also observed throughout the immunohistochemical staining.
Results
RT-PCR and the immunohistochemical study showed that the levels of MMP-2, MMP-9 mRNA expression in PRP applied tissues were higher than MMP-2, MMP-9 mRNA expression in saline-applied tissues. MMP-9 mRNA expression in wounds of diabetic rats decreased after healing began to occur. But no statistical differences were detected on the basis of body weight or fasting blood glucose levels.
Conclusions
This study could indicate the extracellular matrix-regulating effect observed with PRP. Our results of the acceleration of wound healing events by PRP under hyperglycemic conditions might be a useful clue for future clinical treatment for diabetic wounds.
doi:10.5999/aps.2012.39.2.106
PMCID: PMC3385326  PMID: 22783508
Platelet-rich plasma; Rats, OLETF; Matrix mtalloproteinase-2; Matrix metalloproteinase-9
8.  Characterization of the cytokine profile of platelet rich plasma (PRP) and PRP-induced cell proliferation and migration: Upregulation of matrix metalloproteinase-1 and -9 in HaCaT cells 
The Korean Journal of Hematology  2011;46(4):265-273.
Background
The underlying rationale of platelet rich plasma (PRP) therapy is that an injection of concentrated PRP at the site of injury may promote tissue repair via cytokine release from platelets. The molecular mechanisms of PRP therapy in the skin wound healing process are not well understood at present, and would benefit from clarification.
Methods
PRP was stimulated with angonists for 5 min, and cytokine profile analysis was performed. To investigate the wound healing activity of PRP, cell proliferation and migration analyses were performed in skin cells. The effects of PRP were analyzed on the expression and activity of matrix metalloproteinase (MMP)-1, -2, -9, and the activation of transcription factors.
Results
Thrombin was found to be a strong stimulator of PRP activation to release growth factors and chemokines. PRP induced cell proliferation and migration in HUVECs, HaCaT cells, and HDFs, as well as MMP-1and MMP-9 expression in HaCaT cells, but PRP did not have a significant effect on the expression or activity of MMPs in HDFs. The transcription factors, including signal transducer and activator of transcription-3 (STAT-3) were found to be phosphorylated following PRP treatment in HaCaT cells.
Conclusion
In this study, we have identified the cytokine profile of activated PRP after agonist stimulation. We have shown that PRP plays an active role in promoting the proliferation and migration of skin cells via the regulation of MMPs, and this may be applicable to the future development of PRP therapeutics to enhance skin wound healing.
doi:10.5045/kjh.2011.46.4.265
PMCID: PMC3259519  PMID: 22259633
Platelet rich plasma (PRP); Wound healing; Cytokine profile; Cell proliferation; Cell migration; Matrix metalloproteinase
9.  A new simplified technique for producing platelet-rich plasma: a short technical note 
European Spine Journal  2004;13(Suppl 1):S102-S106.
A possible strategy to promote the wound-healing cascade in both soft and hard tissues is the preparation of an autologous platelet-rich plasma (PRP) to encourage the release of growth factors from activated platelets. In this process, PRP combines the advantage of an autologous fibrin clot that will aid in hemostasis as well as provide growth factors in high concentrations to the site of a tissue defect. The PRP preparation can be used as a biological enhancer in the healing of fractures and lumbar fusions. The local application of growth factors seems to promote initiation and early maturation of bone formation. Autologous bone or bone substitutes can be added to this mixture to increase the volume of grafting material. A simplified technique utilizing a commercially available separation system (GPS—Gravitational Platelet Separation System) is described. This system provides a less costly alternative to other previously described augmentation techniques and also presents a patient-friendly and operator-safe alternative. Further experimental studies of the actual concentrations of the growth factors in the PRP samples are necessary in order to validate the platelet concentration and growth-factor activation by laboratory evidence. In further prospective clinical trials, the safety and efficacy of PRP, in combination with autologous bone or bone graft substitutes, must be evaluated.
doi:10.1007/s00586-004-0715-3
PMCID: PMC3592190  PMID: 15221571
Platelet concentrate; Growth factors; Bone healing; Platelet-rich plasma; GPS
10.  Evaluation of the Effects of Platelet-Rich Plasma (PRP) Therapy Involved in the Healing of Sports-Related Soft Tissue Injuries 
The Iowa Orthopaedic Journal  2012;32:150-163.
Abstract
Musculoskeletal injuries are the most common cause of severe long-term pain and physical disability, and affect hundreds of millions of people around the world. One of the most popular methods used to biologically enhance healing in the fields of orthopaedic surgery and sports medicine includes the use of autologous blood products, namely, platelet rich plasma (PRP). PRP is an autologous concentration of human platelets to supra-physiologic levels. At baseline levels, platelets function as a natural reservoir for growth factors including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-β1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF-I). PRP is commonly used in orthopaedic practice to augment healing in sports-related injuries of skeletal muscle, tendons, and ligaments. Despite its pervasive use, the clinical efficacy of PrP therapy and varying mechanisms of action have yet to be established. Basic science research has revealed that PRP exerts is effects through many downstream events secondary to release of growth factors and other bioactive factors from its alpha granules. These effects may vary depending on the location of injury and the concentration of important growth factors involved in various soft tissue healing responses. This review focuses on the effects of PrP and its associated bioactive factors as elucidated in basic science research. Current findings in PRP basic science research, which have shed light on its proposed mechanisms of action, have opened doors for future areas of PrP research.
PMCID: PMC3565396  PMID: 23576936
11.  Quiescent platelets stimulate angiogenesis and diabetic wound repair 
The Journal of surgical research  2008;160(1):169-177.
Introduction
Platelets partake in hemostasis, wound healing and tumor growth. Although platelet-rich-plasma (PRP) has been used in surgery for several years, its mechanism of action and application methods are still poorly characterized.
Materials and Methods
A single unit of human platelets obtained by plateletpheresis was diluted in plasma and divided into three equal volumes. One volume was stored at room temperature as fresh platelets (RT), another volume frozen by storage at −80 °C (FZ) and the third volume frozen at −80 °C with 6% DMSO (FZ6). Plasma (PL) was used as control. Using flow cytometry, platelets were tested for platelet glycoprotein GPIb and annexin V binding, as survival and activation markers, respectively. Hemostatic function was assessed by thromboelastometry.
In vivo, platelets were topically applied on 1 cm2, full-thickness wounds on db/db mice (n=10/group) and healing was staged microscopically and macroscopically.
Results
All platelet preparations showed hemostatic ability. RT platelets were GPIb positive (nonactivated-quiescent platelets) and stimulated angiogenesis by 3-fold, and cell proliferation by 4-fold in vivo. FZ platelets were positive for annexin V, indicating activated platelets and, in vivo, increased only wound granulation. FZ6 platelets contained 30% nonactivated-quiescent and 50% activated platelets and stimulated granulation, angiogenesis, cell proliferation and promoted re-epithelialization in vivo.
Conclusions
Platelets showed distinct mechanisms to induce hemostasis and wound healing. Quiescent platelets are required to induced angiogenesis in vivo. Platelets stored at room temperature and frozen with 6% DMSO and stored at −80 °C achieved optimal wound healing in diabetic mice.
doi:10.1016/j.jss.2008.09.010
PMCID: PMC2881478  PMID: 19482315
12.  PARot – assessing platelet-rich plasma plus arthroscopic subacromial decompression in the treatment of rotator cuff tendinopathy: study protocol for a randomized controlled trial 
Trials  2013;14:167.
Background
Platelet-rich plasma (PRP) is an autologous platelet concentrate. It is prepared by separating the platelet fraction of whole blood from patients and mixing it with an agent to activate the platelets. In a clinical setting, PRP may be reapplied to the patient to improve and hasten the healing of tissue. The therapeutic effect is based on the presence of growth factors stored in the platelets. Current evidence in orthopedics shows that PRP applications can be used to accelerate bone and soft tissue regeneration following tendon injuries and arthroplasty. Outcomes include decreased inflammation, reduced blood loss and post-treatment pain relief. Recent shoulder research indicates there is poor vascularization present in the area around tendinopathies and this possibly prevents full healing capacity post surgery (Am J Sports Med36(6):1171–1178, 2008). Although it is becoming popular in other areas of orthopedics there is little evidence regarding the use of PRP for shoulder pathologies. The application of PRP may help to revascularize the area and consequently promote tendon healing. Such evidence highlights an opportunity to explore the efficacy of PRP use during arthroscopic shoulder surgery for rotator cuff pathologies.
Methods/Design
PARot is a single center, blinded superiority-type randomized controlled trial assessing the clinical outcomes of PRP applications in patients who undergo shoulder surgery for rotator cuff disease. Patients will be randomized to one of the following treatment groups: arthroscopic subacromial decompression surgery or arthroscopic subacromial decompression surgery with application of PRP.
The study will run for 3 years and aims to randomize 40 patients. Recruitment will be for 24 months with final follow-up at 1 year post surgery. The third year will also involve collation and analysis of the data. This study will be funded through the NIHR Biomedical Research Unit at the Oxford University Hospitals NHS Trust.
Trial registration
Current Controlled Trials: ISRCTN10464365
doi:10.1186/1745-6215-14-167
PMCID: PMC3686643  PMID: 23758981
Platelet-rich-plasma; Rotator cuff; Tendinopathies; Surgery; Growth factors
13.  Platelet Activation by Collagen Provides Sustained Release of Anabolic Cytokines 
Background
Platelet-rich plasma (PRP) has been increasingly used in sports medicine applications. Platelets are thought to release growth factors important in wound healing, including transforming growth factor (TGF-β1), platelet-derived growth factor (PDGF-AB), and vascular endothelial growth factor (VEGF). However, little is known about the effect of platelet activator choice on growth factor release kinetics.
Hypothesis
The choice of platelet activator would affect the timing and level of growth factor release from PRP.
Study Design
Controlled laboratory study.
Methods
Platelet-rich plasma aliquots were activated with either thrombin or collagen. A control group of whole blood aliquots was clotted with thrombin. Supernatant containing the released growth factors was collected daily for 1 week. Levels of TGF-β1, PDGF-AB, and VEGF were measured using enzyme-linked immunosorbent assay (ELISA).
Results
The use of thrombin as an activator resulted in immediate release of TGF-β1 and PDGF-AB, while the collagen-activated PRP clots released similar amounts each day for 5 days. The use of collagen as an activator resulted in an 80% greater cumulative release of TGF-β1 from the PRP aliquots over 7 days (P < .001). Concentrating platelets to 3 times the systemic blood level resulted in a 3-fold higher release of TGF-β1, 2.5-fold greater release of PDGF, and 5-fold greater release of VEGF (all P < .0001) when compared with whole blood control clots, but no significant differences in the timing of release were noted.
Conclusion
These experiments demonstrated that the choice of platelet activator can significantly influence the release kinetics of cytokines from PRP, with thrombin resulting in an immediate release and collagen having a more sustained release pattern.
Clinical Relevance
The level and rate of growth factor release depends on the selected platelet activator, a factor that should be considered when selecting a PRP system for a given application.
doi:10.1177/0363546511401576
PMCID: PMC3176726  PMID: 21398575
blood clot; growth factor; platelet activation; release kinetics
14.  Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors 
Introduction
Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed.
Methods
PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure.
Results
Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only <0.3% from the initial red blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF.
Conclusions
Our study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool from different donors with high reproducibility. These findings support the use of PRP in therapies aiming for tissue regeneration, and its content characterization will allow us to understand and improve the clinical outcomes.
doi:10.1186/scrt218
PMCID: PMC3706762  PMID: 23759113
Platelet-rich plasma; Centrifugation; Growth factors; Cytokine; Activation
15.  Comparison between Conventional Mechanical Fixation and Use of Autologous Platelet Rich Plasma (PRP) in Wound Beds Prior to Resurfacing with Split Thickness Skin Graft 
BACKGROUND
Platelet rich plasma is known for its hemostatic, adhesive and healing properties in view of the multiple growth factors released from the platelets to the site of wound. The primary objective of this study was to use autologous platelet rich plasma (PRP) in wound beds for anchorage of skin grafts instead of conventional methods like sutures, staplers or glue.
METHODS
In a single center based randomized controlled prospective study of nine months duration, 200 patients with wounds were divided into two equal groups. Autologous PRP was applied on wound beds in PRP group and conventional methods like staples/sutures used to anchor the skin grafts in a control group.
RESULTS
Instant graft adherence to wound bed was statistically significant in the PRP group. Time of first post-graft inspection was delayed, and hematoma, graft edema, discharge from graft site, frequency of dressings and duration of stay in plastic surgery unit were significantly less in the PRP group.
CONCLUSION
Autologous PRP ensured instant skin graft adherence to wound bed in comparison to conventional methods of anchorage. Hence, we recommend the use of autologous PRP routinely on wounds prior to resurfacing to ensure the benefits of early healing.
PMCID: PMC4298865  PMID: 25606477
Platelet rich plasma; Hemostasis; Skin graft; Edema
16.  Double-application of platelet-rich plasma on bone healing in rabbits 
Objective: Platelet-rich plasma (PRP) is considered to enhance bone formation especially at early stages of wound healing, depending on the limited and short life-span of platelets and growth factors. The aim of this study was to evaluate efficacy of double-application of PRP (DA-PRP) on bone healing in a rabbit calvarial defect model. Study design: Twenty-eight rabbits, each had two surgically prepared calvarial bone defects (10mm diameter), were included in this study and randomly divided into six groups. Defects (n=56) were treated with single-application of PRP (SA-PRP)(n=10), SA-PRP and beta-tricalciumphosphate (SA-PRP+TCP)(n=10), DA-PRP (n=8), DA-PRP and beta-tricalciumphosphate (DA-PRP+TCP)(n=8), beta-tricalciumphosphate (TCP)(n=10) or left empty (Control)(n=10). Animals were sacrificed at 30 days postoperatively. Results: The new bone (NB%) and defect fill (DF%) percentages were calculated from histological slides by image-analyzer software and statistically analysed. All test groups showed higher NB% than control, but differences among all groups were insignificant. The TCP treated groups had significantly higher DF% than groups treated without TCP, however the DF% differences between control, SA-PRP and DA-PRP or TCP, SA-PRP+TCP or DA-PRP+TCP were insignificant. Conclusion: Although new bone formation was histomorphologically remarkable at double-application PRP groups, statistical analyses of the histomorphometric data revealed no significant difference.
Key words: Platelet-Rich Plasma, double application, bone formation, wound healing.
doi:10.4317/medoral.17336
PMCID: PMC3448207  PMID: 22157673
17.  Bone Marrow Aspiration Concentrate and Platelet Rich Plasma for Osteochondral Repair in a Porcine Osteochondral Defect Model 
PLoS ONE  2013;8(8):e71602.
Background
Bone marrow aspiration concentrate (BMAC) may possess a high potency for cartilage and osseous defect healing because it contains stem cells and multiple growth factors. Alternatively, platelet rich plasma (PRP), which contains a cocktail of multiple growth factors released from enriched activated thrombocytes may potentially stimulate the mesenchymal stem cells (MSCs) in bone marrow to proliferate and differentiate.
Methods
A critical size osteochondral defect (10×6 mm) in both medial femoral condyles was created in 14 Goettinger mini-pigs. All animals were randomized into the following four groups: biphasic scaffold alone (TRUFIT BGS, Smith & Nephew, USA), scaffold with PRP, scaffold with BMAC and scaffold in combination with BMAC and PRP. After 26 weeks all animals were euthanized and histological slides were cut, stained and evaluated using a histological score and immunohistochemistry.
Results
The thrombocyte number was significantly increased (p = 0.049) in PRP compared to whole blood. In addition the concentration of the measured growth factors in PRP such as BMP-2, BMP-7, VEGF, TGF-β1 and PDGF were significantly increased when compared to whole blood (p<0.05). In the defects of the therapy groups areas of chondrogenic tissue were present, which stained blue with toluidine blue and positively for collagen type II. Adding BMAC or PRP in a biphasic scaffold led to a significant improvement of the histological score compared to the control group, but the combination of BMAC and PRP did not further enhance the histological score.
Conclusions
The clinical application of BMAC or PRP in osteochondral defect healing is attractive because of their autologous origin and cost-effectiveness. Adding either PRP or BMAC to a biphasic scaffold led to a significantly better healing of osteochondral defects compared with the control group. However, the combination of both therapies did not further enhance healing.
doi:10.1371/journal.pone.0071602
PMCID: PMC3741121  PMID: 23951201
18.  Optimized Preparation Method of Platelet-Concentrated Plasma and Noncoagulating Platelet-Derived Factor Concentrates: Maximization of Platelet Concentration and Removal of Fibrinogen 
Abstract
Platelet-rich plasma (PRP) has been clinically used as an easily prepared growth factor cocktail that can promote wound healing, angiogenesis, and tissue remodeling. However, the therapeutic effects of PRP are still controversial, due partly to the lack of optimized and standardized preparation protocols. We used whole blood (WB) samples to optimize the preparation protocols for PRP, white blood cell-containing (W-PRP), platelet-concentrated plasma (PCP), and noncoagulating platelet-derived factor concentrate (PFC). PRP and W-PRP were most efficiently collected by 10 min centrifugation in a 15-mL conical tube at 230–270 g and 70 g, respectively. To prepare PCP, platelets were precipitated by centrifugation of PRP at >2300 g, 90% of supernatant plasma was removed, and the platelets were resuspended. For preparation of noncoagulating PFC, the supernatant was replaced with one-tenth volume of saline, followed by platelet activation with thrombin. Platelet (before activation) and platelet-derived growth factor (PDGF)-BB (after activation) concentrations in PCP were approximately 20 times greater than those in WB, whereas PFC contained a 20-times greater concentration of platelets before platelet activation and a 50-times greater concentration of PDGF-BB without formation of a fibrin gel after platelet activation than WB. Surprisingly, total PDGF-BB content in the PFC was twice that of activated WB, which suggested that a substantial portion of the PDGF-BB became trapped in the fibrin glue, and replacement of plasma with saline is crucial for maximization of platelet-derived factors. As an anticoagulant, ethylene di-amine tetra-acetic acid disodium inhibited platelet aggregation more efficiently than acid citrate dextrose solution, resulting in higher nonaggregated platelet yield and final PDGF-BB content. These results increase our understanding of how to optimize and standardize preparation of platelet-derived factors at maximum concentrations.
doi:10.1089/ten.tec.2011.0308
PMCID: PMC3285602  PMID: 21951067
19.  Cascade® Autologous System Platelet-Rich Fibrin Matrix in the Treatment of Chronic Leg Ulcers 
Advances in Wound Care  2012;1(1):52-55.
Problem
Lower extremity ulcers (venous, diabetic) are often unresponsive to standard treatment. Various systemic and local cellular, vascular, and anatomical factors can result in nonhealing wounds that are refractory to normal healing processes and standard care.
Solution
Several published wound care guidelines strongly suggest that if an ulcer does not respond to standard good wound care within 4 weeks, then advanced wound therapies should be considered. These advanced therapies include wound bed preparation agents (negative wound pressure therapy, hyperbaric oxygen), recombinant growth factors, or bioengineered cell therapies.
New Technology
The Cascade® system produces platelet-rich fibrin matrix (PRFM), a novel autologous sterile biologic, produced at the bedside from a small volume (18 mL) of the patient's own blood by using Vacutainer® separation technology optimized for fibrin and platelet isolation. Prepared as an easy to apply, suturable membrane, without the use of exogenous thrombin, PRFM consists of a dense cross-linked fibrin lattice containing intact, viable platelets with their full complement of platelet-derived growth factors.
Indications for Use
From the FDA 510(k) clearance: The Cascade system “is designed to be used for the safe and rapid preparation of autologous platelet-rich plasma from a small sample of blood at the patient point of care.” PRFM has been used to successfully treat severe venous leg ulcer (VLU), neuropathic diabetic foot ulcer (DFU), mixed arterial and Charcot-deformity associated foot ulcers.
Cautions
When treating venous or DFUs, the Cascade system should be used together with standard wound care practice (therapeutic compression for VLU and weight off-loading, debridement, and infection control for DFU) in patients with an adequate blood supply to the lower limb.
doi:10.1089/wound.2011.0290
PMCID: PMC3839008  PMID: 24527280
20.  Platelet Rich Fibrin Matrix Improves Wound Angiogenesis via Inducing Endothelial Cell Proliferation 
The economic, social and public health burden of chronic ulcers and other compromised wounds are enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis and tissue sealing. In this study we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely, platelet rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study for the first-time shows the kinetics of the viability of platelet embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The VEGF released from PRFM was primarily responsible for endothelial mitogenic response via ERK activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers.
doi:10.1111/j.1524-475X.2011.00740.x
PMCID: PMC3623798  PMID: 22092846
platelet rich plasma (PRP); wound healing; angiogenesis; autologous platelet gels; ischemic wounds
21.  Anterior lumbar interbody fusion with carbon fiber cage loaded with bioceramics and platelet-rich plasma. An experimental study on pigs 
European Spine Journal  2004;13(4):354-358.
Platelet-rich plasma (PRP) is an autogenous source of growth factor and has been shown to enhance bone healing both in clinical and experimental studies. PRP in combination with porous hydroxyapatite has been shown to increase the bone ingrowth in a bone chamber rat model. The present study investigated whether the combination of beta tricalcium phosphate (β-TCP) and PRP may enhance spinal fusion in a controlled animal study. Ten Danish Landrace pigs were used as a spinal fusion model. Immediately prior to the surgery, 55 ml blood was collected from each pig for processing PRP. Three-level anterior lumbar interbody fusion was performed with carbon fiber cages and staples on each pig. Autogenous bone graft, β-TCP, and β-TCP loaded with PRP were randomly assigned to each level. Pigs were killed at the end of the third month. Fusion was evaluated by radiographs, CT scanning, and histomorphometric analysis. All ten pigs survived the surgery. Platelet concentration increased 4.4-fold after processing. Radiograph examination showed 70% (7/10) fusion rate in the autograft level. All the levels with β-TCP+PRP showed partial fusion, while β-TCP alone levels had six partial fusions and four non-fusions (P=0.08). CT evaluation of fusion rate demonstrated fusion in 50% (5/10) of the autograft levels. Only partial fusion was seen at β-TCP levels and β-TCP+PRP levels. Histomorphometric evaluation found no difference between β-TCP and β-TCP+PRP levels on new bone volume, remaining β-TCP particles, and bone marrow and fibrous tissue volume, while the same parameters differ significantly when compared with autogenous bone graft levels. We concluded from our results in pigs that the PRP of the concentration we used did not improve the bone-forming capacity of β-TCP biomaterial in anterior spine fusion. Both β-TCP and β-TCP+PRP had poorer radiological and histological outcomes than that of autograft after 3 months.
doi:10.1007/s00586-003-0647-3
PMCID: PMC3468048  PMID: 14730438
Spinal fusion; Bone substitute; Platelet-rich plasma; Tricalcium phosphate; Pig
22.  ACTIVATION OF PLATELET-RICH PLASMA USING SOLUBLE TYPE I COLLAGEN 
PURPOSE
Platelet-rich plasma (PRP) has recently been found to be a useful delivery system for growth factors important in oral tissue healing. However, application of PRP in a liquid form to a wound site within the oral cavity can be complicated by significant loss of the PRP into the surrounding oral space unless gelation via the clotting mechanism is accomplished. Gelation is currently accomplished using bovine thrombin; however, rare but serious complications of this method have led to the search for alternative clotting mechanisms, including the use of soluble collagen as a clotting activator. In this paper, our hypothesis was that soluble Type I collagen would be as effective as bovine thrombin in causing clotting of the PRP and of stimulating growth factor release from the platelets and granulocytes.
MATERIALS AND METHODS
PRP from human donors was clotted using Type I collagen or bovine thrombin. Clot retraction was determined by measuring clot diameters over time. The release of PDGF-AB, TGF-β1 and VEGF from both types of clots was measured over 10 days using ELISA.
RESULTS
Clots formed using Type I collagen had far less retraction than those formed with bovine thrombin. Bovine thrombin and Type I collagen stimulated similar release of PDGF-AB and VEGF between 1 and 10 days; however, thrombin activation resulted in a greater release of TGF-β1 during the first five days after activation.
CONCLUSIONS
The use of Type I collagen to activate clotting of PRP may be a safe and effective alternative to bovine thrombin. The use of collagen results in less clot retraction and equal release of PDGF-AB and VEGF when compared to currently available methods of clot activation.
doi:10.1016/j.joms.2007.06.635
PMCID: PMC2405953  PMID: 18355591
Platelet-rich Plasma; collagen; thrombin; PDGF-AB; TGF-β1; VEGF
23.  Rapidly In Situ Forming Platelet-Rich Plasma Gel Enhances Angiogenic Responses and Augments Early Wound Healing after Open Abdomen 
Objective. The purposes of our present study were to evaluate the potential of platelet-rich plasma gel to enhance granulation tissue formation after open abdomen and to examine whether the effect was attributable to stimulating rapid neovascularization. Methods. Twenty-four rats underwent colon ascendens stent peritonitis surgery to induce sepsis, followed by intraperitoneal injection of nitrogen to create intra-abdominal hypertension. Four hours later, laparotomies were performed. The rats were randomized into three groups (n = 8 for each group): control, platelet-poor plasma (PPP), and platelet-rich plasma (PRP) groups. One week after the treatment, granulation tissue formation and angiogenesis were evaluated by histological and laser Doppler analysis. Results. The resultant platelet count in platelet-rich plasma was higher than that of PPP. The concentrations of platelet-derived growth factor BB, transforming growth factor β-1, and vascular endothelial growth factor in PRP were significantly higher when compared with that of PPP. Myofibroblast count, granulation tissue thickness, vessel numbers, and blood perfusion were increased in PRP group, followed by PPP group, with control being the least. Conclusion. Rapidly in situ forming platelet-rich plasma gel promoted remarkable neovascularization and early wound healing after open abdomen and may lead to novel and effective treatments for open abdominal wounds.
doi:10.1155/2013/926764
PMCID: PMC3870633  PMID: 24382956
24.  Unique Antimicrobial Effects of Platelet-Rich Plasma and Its Efficacy as a Prophylaxis to Prevent Implant-Associated Spinal Infection 
Advanced healthcare materials  2013;2(9):1277-1284.
Platelet-rich-plasma (PRP) has attracted great attention and has been increasingly used for a variety of clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, and sports medicine. However, very little is known about the antimicrobial activities of PRP. In this study, PRP is found to have antimicrobial properties both in vitro and in vivo. In vitro, the antimicrobial properties of PRP have been found to be bacterial strain specific and time specific: PRP has significantly (80–100 fold reduction in colony forming units) inhibited the growth of methicillin sensitive and methicillin resistant Staphylococcus aureus, Group A streptococcus, and Neisseria gonorrhoeae within the first few hours but it has no significant antimicrobial properties against E. coli and Pseudomonas. The antimicrobial properties of PRP also depend on the concentration of thrombin. In vivo, an implant-associated spinal infection rabbit model has been established and used to evaluate the antimicrobial and wound healing properties of PRP. Compared to the infection controls, PRP treatment has resulted in significant reduction in bacterial colonies in bone samples at all time points studied (i.e. 1, 2, and 3 weeks) and significant increase in mineralized tissues (thereby better bone healing) at post-operative weeks 2 and 3. PRP therefore may be a useful adjunct strategy against post-operative implant-associated infections.
doi:10.1002/adhm.201200465
PMCID: PMC3774283  PMID: 23447088
antimicrobial; implant infection; platelet-rich plasma; rabbit spine infection model; Staphylococcus aureus
25.  Platelet-Rich Plasma and Skeletal Muscle Healing: A Molecular Analysis of the Early Phases of the Regeneration Process in an Experimental Animal Model 
PLoS ONE  2014;9(7):e102993.
Platelet-rich plasma (PRP) has received increasing interest in applied medicine, being widely used in clinical practice with the aim of stimulating tissue healing. Despite the reported clinical success, there is still a lack of knowledge when considering the biological mechanisms at the base of the activity of PRP during the process of muscle healing. The aim of the present study was to verify whether the local delivery of PRP modulates specific molecular events involved in the early stages of the muscle regeneration process. The right flexor sublimis muscle of anesthetized Wistar rats was mechanically injured and either treated with PRP or received no treatment. At day 2 and 5 after surgery, the animals were sacrificed and the muscle samples evaluated at molecular levels. PRP treatment increased significantly the mRNA level of the pro-inflammatory cytokines IL-1β, and TGF-β1. This phenomenon induced an increased expression at mRNA and/or protein levels of several myogenic regulatory factors such as MyoD1, Myf5 and Pax7, as well as the muscular isoform of insulin-like growth factor1 (IGF-1Eb). No effect was detected with respect to VEGF-A expression. In addition, PRP application modulated the expression of miR-133a together with its known target serum response factor (SRF); increased the phosphorylation of αB-cristallin, with a significant improvement in several apoptotic parameters (NF-κB-p65 and caspase 3), indexes of augmented cell survival. The results of the present study indicates that the effect of PRP in skeletal muscle injury repair is due both to the modulation of the molecular mediators of the inflammatory and myogenic pathways, and to the control of secondary pathways such as those regulated by myomiRNAs and heat shock proteins, which contribute to proper and effective tissue regeneration.
doi:10.1371/journal.pone.0102993
PMCID: PMC4108405  PMID: 25054279

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