Concentrations Of Platelet-rich Plasma Research Articles

A Randomized Controlled Trial of Lumbar Posterolateral Fusion in Combination with Platelet-Rich Plasma

Introduction Platelet-rich plasma (PRP) has been reported to be a source of autologous growth factors (PDGF and TGF-β) that enhance bone formation. However, PRP has failed to improve fusion rates, perhaps due to suboptimal levels of growth factors. We began a Phase 1 study in July 2009, the purposes of which were to produce highly concentrated PRP enriched in growth factors, to monitor side effects, and to determine subsequent fusion rates. This is the first randomized controlled trial to investigate the potential advantages of using PRP in posterolateral fusion. Platelet-rich plasma (PRP) has been reported to be a source of autologous growth factors (PDGF and TGF-β) that enhance bone formation. However, PRP has failed to improve fusion rates, perhaps due to suboptimal levels of growth factors. We began a Phase 1 study in July 2009, the purposes of which were to produce highly concentrated PRP enriched in growth factors, to monitor side effects, and to determine subsequent fusion rates. This is the first randomized controlled trial to investigate the potential advantages of using PRP in posterolateral fusion. Materials and Methods Thirty-nine patients diagnosed with spinal canal stenosis were divided equally into “PRP” and “control” groups. The PRP patients underwent posterolateral fusion with instrumentation using autograft bone plus PRP, while control patients underwent the same procedure minus PRP. In the PRP group, one unit of whole blood (400 mL) was drawn from each patient at the beginning of surgery and centrifuged to extract platelets. Twenty mL of PRP was obtained, to which thrombin and CaCl2 were added to form a platelet gel for application to the surgical field. Bone fusion was periodically assessed using X-ray and CT. Results The average platelet concentration was 7.7-fold higher in the PRP than plasma. Growth factors released from the platelet gel were more than 50-fold concentrated compared with blood levels. No significant adverse events were observed. Bone union was observed in all patients. The average period was 7.8 months in the PRP group, and 9.8 months in the control group. The average platelet concentration was 7.7-fold higher in the PRP than plasma. Growth factors released from the platelet gel were more than 50-fold concentrated compared with blood levels. No significant adverse events were observed. Bone union was observed in all patients. The average period was 7.8 months in the PRP group, and 9.8 months in the control group. Conclusion In the present study, bone union was achieved more rapidly in the PRP group, which suggests that enhanced bone formation was achieved by providing an elevated concentration of PRP. However, further research is required. In the present study, bone union was achieved more rapidly in the PRP group, which suggests that enhanced bone formation was achieved by providing an elevated concentration of PRP. However, further research is required. Disclosure of Interest None declared

Effect of Autologous Platelet-Rich Plasma in Combination With Bovine Porous Bone Mineral and Bio-Guide Membrane on Bone Regeneration in Mandible Bicortical Bony Defects

Growth factors contained in platelet-rich plasma (PRP) can induce osteoblast differentiation in certain studies, whereas in others, osteogenesis of PRP on mandible bone defects has not been proved clinically. The aim of the study was to investigate the effect of autologous PRP on the osteogenic potential of combining bovine porous bone mineral (BPBM) and bio-guide membrane (BGM) in promoting mandible bicortical bony defects in rabbits. One circular mandible bicortical bony defects were created in each of 54 rabbits, which were divided into 3 groups: group 1: 18 of the defects were left unfilled as a negative control; group 2: 18 of the defects were grafted with autologous PRP and BPBM/BGM; group 3: 18 of the defects were grafted with BPBM/BGM without PRP. Animals were killed at 4, 8, and 12 weeks after operation. Harvested tissue and specimens were evaluated histologically and radiographically, and metabolized observation was performed. Histological parameters associated with osteoblast activities, bone trabecula, neovascularization, newly formed mineralized bone, rudimental grafts and connective tissue formation were measured. Densities of the bones at 4, 8, and 12 weeks were studied by radiographic. The bone defect closure ratio was measured at 12 weeks. The bone metabolized parameter alkaline phosphatase was also measured and compared between 4, 8, and 12 weeks. The platelet concentration of PRP is 4.19- to 4.43-fold to that of the whole blood. Histological analysis showed new bone formation at all therapeutic sites including BPBM/BGM grafts with or without PRP. A statistically significant difference in new bone formation between group PRP/BPBM/BGM and group BPBM/BGM was observed. Untreated defects of group control showed the less bone regeneration. There was significant difference of bone density between group PRP/BPBM/BGM and control, and group BPBM/BGM and control, at 4, 8, and 12 weeks postoperative. There were more bone defects filling, and the grafts were absorbed at 12 weeks of group PRP/BPBM/BGM compared with group BPBM/BGM. Defects treated with PRP/BPBM/BGM demonstrated significantly increased activity of osteoblasts, enhanced amount of mitochondria and rough endoplasmic reticulum in osteoblasts, and increased concentration of alkaline phosphatase at 4, 8, and 12 weeks compared with those treated with BPBM/BGM and control group. Complete closure ratio of bone defects treated with PRP/BPBM/BGM (50%) was significantly increased compared with that treated with BPBM/BGM (16.6%). The study suggested that PRP combination of BPBM and BGM had significant therapeutic effects on mandible bicortical bony defects of rabbits. The effects are associated with the high concentration of platelet in PRP and the porous configuration of BPBM. Although we cannot reveal the detailed statistical relationship of PRP on promoting BPBM/GBM osteoinductive effects, PRP demonstrated superior results of bone regeneration.

Effects of platelet rich plasma and platelet lysate on proliferation of rat bone marrow mesenchymal stem cells: a comparative study

Objective To compare the effects of platelet rich plasma(PRP)and platelet lysate(PL)on proliferation and cell cycle of cultured rat bone marrow stem cells(BMSCs).Methods BMSCs were obtained from twenty 4-week-old SD rats using the whole bone marrow isolation and cultivation method and were identified with flow cytometry.Blood samples were taken from the hearts of thirty 12-week-old SD rats and gradient centrifugation was used to prepare PRP,and PL was obtained after three times of centrifugation and repeated freezing and thawing.The third generation of BMSCs with good growth state were divided into seven groups according to different culture media:ordinary complete medium(A group),1% PRPconditioned medium(B group),1% PL-conditioned medium(C group),5% PRP-conditioned medium(D group),5% PLconditioned medium(E group),10% PRP-conditioned medium(F group),and 10% PL-conditioned medium(G group). The proliferation of BMSCs was assessed by MTT assay.The PCNA protein expression was assessed by immunofluorescence method.Cell cycle of BMSCs was tested by flow cytometry.Western blotting analysis was used to analyze CyclinD1and p27 Kip1protein expression of BMSCs.Results The proliferation of BMSCs was significantly promoted by 1%,5%,10% PRPand 1%,5%,10% PL-conditioned media after cultured for 24h,48h,and 72hin a time-and dose-dependent manner(P 0.05),with the effect of PRP and PL being similar at the same concentration(P0.05).Immunofluorescence assay showed that PRP and PL both significantly promoted PCNA protein expression in a dose-dependent manner(P0.01).Flow cytometry showed that different concentrations of PRP- and PL-conditioned media,compared with the ordinary complete medium,resulted in gradually reduced cells in G0/G1 phase,gradually increased cells in S,G2 /M phase,and significantly increased PI(P0.05),with the changes in a dose-dependent manner.However,the effects of the same concentration of PRP and PL on cell cycle were not significantly different(P0.05).Western blotting analysis showed that 5% PRP and 5% PL significantly up-regulated CyclinD1and down-regulated the expression of p27Kip1.Conclusion Different concentrations of PL and PRP can accelerate cell cycle progression of cultured rat BMSCs by up-regulation CyclinD1and inhibiting p27Kip1, promoting the proliferation of BMSCs in a time-and dose-dependent manner.PL and PRP at the same concentration have the same proliferation-promoting effect,indicating that PL may be used as an alternative of PRP to promote the repair of bone defects.

Effect of PRP and PPP on proliferation and migration of human chondrocytes and synoviocytes in vitro

Abstract Cartilage tissue engineering can provide substantial relief to people suffering from degenerative cartilage disease, such as osteoarthritis. The autologous platelet-rich plasma (PRP) application appears to improve cartilage healing due to its ability to positively influence cellular mechanisms, mainly in cells from synovium and cartilage. Primary cultures of human synovial fluid stem cells (synoviocytes, SCs) and chondrocytes (CCs) were exposed to various concentrations of non-activated PRP and plateletpoor plasma (PPP) prepared by apheresis. Cell proliferation and migration were evaluated in real-time with the non-invasive xCELLigence System. It was found that PRP had a similar effect on the growth of cells as fetal bovine serum (FBS). Surprisingly, our proliferation assay results indicated that 50% PPP had the largest effect on both cell types, with a statistically significant increase in cell number (P<0.001) compared to the (0% FBS) in vitro control. The migratory ability of SCs was significantly enhanced with 10% PRP and 0.8% hyaluronic acid (HA). HA also augmented migration of CCs. In summary, these results demonstrate that directed cell proliferation and migration are inducible in human articular CCs and SCs, and that both platelet-derived fractions may exert a positive effect and modulate several cell responses that are potentially involved in tissue integration during cartilage repair.

The effect of platelet rich plasma on chondrogenic differentiation of human adipose derived stem cells in transwell culture.

Platelet-rich plasma (PRP) has recently emerged as a promising strategy in regenerative medicine due to its multiple endogenous growth factors. Little is known about the role of PRP as a promoter in chondrogenesis of human adipose derived stem cells (hADSCs). The aim of this study was to determine whether PRP may be considered as a natural and easy achievable source of growth factors to promote the chondrogenic differentiation of hADSCs in Transwell culture. Materials and Methods : Biochemical, immunohistological and molecular assays were used to evaluate the effect of different concentrations (5%, 10%, and 15%) of PRP on chondrogenic differentiation of hADSCs in Transwell culture. Results : The cells in the presence of 10% PRP produced markedly higher amounts of GAG and DNA, in comparison to the control group. PRP also increased chondrogenic markers in these cells, such as sox-9, aggrecan and collagen type II. A high expression level of collagen type X as a hypertrophic marker was observed in cartilage produced by using either PRP or TGF-β1. Conclusion : Our findings indicate that autologous PRP at an optimum concentration had beneficial effects on differentiation of hADSCs in Transwell culture. Further, in vivo studies are necessary to fully define the clinical implications of PRP.

Negative Effect of Platelet Rich Plasma on the Differentiation of Synovium-Derived Mesenchymal Stem Cells

Negative Effect of Platelet Rich Plasma on the Differentiation of Synovium-Derived Mesenchymal Stem Cells

A comparative study of effects of autologous versus allogeneic platelet-rich plasma on the proliferation and differentiation of bone marrow stromal stem cells in rabbits

Objective To investigate whether autologous platelet-rich plasma (PRP) can be replaced by allogeneic PRP to promote the in vitro proliferation and differentiation of bone marrow stromal stem cells (BMSCs) in rabbits and to find an optimal concentration at which allogeneic PRP can best promote the proliferation and differentiation of BMSCs.Methods Three New Zealand rabbits were used for conventional culture of BMSCs and preparation of PRP.Experiments were conducted in 4 groups with 4 kinds of culture medium:blank group (DMEM only),conventional group [DMEM containing 10％ fetal bovine serum (FBS)],autologous group (DMEM containing 10％ FBS plus autologous PRP extract) and allogeneic group (DMEM containing 10％ FBS plus allogeneic PRP extract).The allogeneic group was further divided into five subgroups according to the concentrations of PRP extract in the medium:10％,20％,30％,40％ and 50％ respectively.After the best concentration was determined in the allogeneic group,it was used in the autologous group to compare the effects of the 2 kinds of PRP.On day 4,8,12,16 and 20 after intervention,optical density (OD) value in MTT assay and alkaline phosphatase (ALP) activity were tested in all groups and the cell apoptosis rate at the best concentration of PRP extract was also assessed in each group.Results The effect of PRP on the proliferation and differentiation of BMSCs was negatively related to the concentration of PRP.The optimal concentration of PRP for promotion of the proliferation of BMSCs was 30％.There was no significant difference between the autologous and allogeneic PRP (30％) regarding their effects on proliferation and differentiation of BMSCs on day 4,8,12,16 or 20 (P ＞ 0.05).On day 20,the cell apoptosis rates in blank group,conventional group,autologous group (30％) and allogeneic group (30％)were respectively 38.7％ ± 2.0％,24.1％ ± 1.7％,9.7％ ±0.8％ and 10.3 ± l.2％,with no significant difference between autologous and allogeneic groups (P ＞ 0.05).Conclusions Autologous PRP could be replaced by allogeneic PRP in vitro culture of BMSCs; the optimal concentration of PRP for promotion of the proliferation of BMSCs is about 30％. Key words: Platelet-rich plasma; Cell proliferation; Cell differentiation; Alkaline phosphatase

Activated platelet-rich plasma improves adipose-derived stem cell transplantation efficiency in injured articular cartilage

IntroductionAdipose-derived stem cells (ADSCs) have been isolated, expanded, and applied in the treatment of many diseases. ADSCs have also been used to treat injured articular cartilage. However, there is controversy regarding the treatment efficiency. We considered that ADSC transplantation with activated platelet-rich plasma (PRP) may improve injured articular cartilage compared with that of ADSC transplantation alone. In this study, we determined the role of PRP in ADSC transplantation to improve the treatment efficiency.MethodsADSCs were isolated and expanded from human adipose tissue. PRP was collected and activated from human peripheral blood. The effects of PRP were evaluated in vitro and in ADSC transplantation in vivo. In vitro, the effects of PRP on ADSC proliferation, differentiation into chondrogenic cells, and inhibition of angiogenic factors were investigated at three concentrations of PRP (10%, 15% and 20%). In vivo, ADSCs pretreated with or without PRP were transplanted into murine models of injured articular cartilage.ResultsPRP promoted ADSC proliferation and differentiation into chondrogenic cells that strongly expressed collagen II, Sox9 and aggrecan. Moreover, PRP inhibited expression of the angiogenic factor vascular endothelial growth factor. As a result, PRP-pretreated ADSCs improved healing of injured articular cartilage in murine models compared with that of untreated ADSCs.ConclusionPretreatment of ADSCs with PRP is a simple method to efficiently apply ADSCs in cartilage regeneration. This study provides an important step toward the use of autologous ADSCs in the treatment of injured articular cartilage.

The influence of platelet-rich plasma on myogenic differentiation

The ability to expand and direct both precursor and stem cells towards a differential fate is considered extremely advantageous in tissue engineering. Platelet-rich plasma (PRP) possesses a milieu of growth factors and cytokines, which have the potential to have either a differentiative or proliferative influence on the cell type tested. Here, we investigated the effect of PRP on C2C12 myoblasts. A range of PRP concentrations in differentiation media was used to determine whether a concentration dependence existed, while PRP embedded in fibres of aligned electrospun polydioxanone and polycaprolactone was used to determine whether this presence of fibres would cause any differences in response. In both cases, it was found that late myogenic markers were suppressed after 7 days in culture. However, an early differentiation marker, MyoD, was upregulated during this same time period. The results from this study represent the ability of PRP to have an influence over both myogenic proliferation and differentiation, a factor which could prove useful in future studies involved with skeletal muscle tissue engineering.

Platelet-rich Plasma Promotes Healing of Osteoporotic Fractures

With an aging population worldwide, the frequency of osteoporotic fractures is increasing. Therefore, biological methods to enhance the internal fixation of osteoporotic fractures becomes more important to reduce the societal burden of care. The purposes of this study were to evaluate the role of platelet-rich plasma (PRP) in the treatment of osteoporotic fractures and to clarify the best concentration of PRP. Bone marrow mesenchymal stem cells isolated from osteoporotic rats were cultured in high- (8.21±0.4×10(9)/mL), medium-(2.65±0.2×10(9)/mL), and low-concentration (0.85±0.16×10(9)/mL) PRP and in platelet-poor plasma (8±0.5×10(6) platelet/mL). The capacities of cell proliferation and osteogenic and adipogenic differentiation were compared. A transverse osteotomy was performed in the middle of the left femoral diaphysis followed by K-wire fixation, and various concentrations of PRP were transplanted into the fracture zone. Radiologic, mechanical, and histologic evaluations were performed at 2, 4, and 8 weeks, respectively. The results indicated that PRP could inhibit adipogenic differentiation and that medium-concentration PRP was effective in inducing the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells derived from osteoporotic bone marrow and in promoting fracture healing, whereas high-concentration PRP inhibited osteogenic differentiation and callus remodeling. Certain concentrations of PRP can effectively enhance the healing of osteoporotic fractures. Medium-concentration PRP is a suitable concentration to use in practice.

Effect of platelet-rich plasma (PRP) concentration on proliferation, neurotrophic function and migration of Schwann cellsin vitro

Platelet-rich plasma (PRP) contains various growth factors and appears to have the potential to promote peripheral nerve regeneration, but evidence is lacking regarding its biological effect on Schwann cells (SCs). The present study was designed to investigate the effect of PRP concentration on SCs in order to determine the plausibility of using this plasma-derived therapy for peripheral nerve injury. PRP was obtained from rats by double-step centrifugation and was characterized by determining platelet numbers and growth factor concentrations. Primary cultures of rat SCs were exposed to various concentrations of PRP (40%, 20%, 10%, 5% and 2.5%). Cell proliferation assays and flow cytometry were performed to study to assess SC proliferation. Quantitative real-time PCR and ELISA analysis were performed to determine the ability of PRP to induce SCs to produce nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). Microchemotaxis assay was used to analyse the cell migration capacity. The results obtained indicated that the platelet concentration and growth factors in our PRP preparations were significantly higher than in whole blood. Cell culture experiments showed that 2.5-20% PRP significantly stimulated SC proliferation and migration compared to untreated controls in a dose-dependent manner. In addition, the expression and secretion of NGF and GDNF were significantly increased. However, the above effects of SCs were suppressed by high PRP concentrations (40%). In conclusion, the appropriate concentration of PRP had the potency to stimulate cell proliferation, induced the synthesis of neurotrophic factors and significantly increased migration of SCs dose-dependently. Copyright © 2013 John Wiley & Sons, Ltd.

PRP For the Treatment of Cartilage Pathology

In recent years biological strategies are being more widely used to treat cartilage lesions. One of the most exploited novel treatments is Platelet-rich Plasma (PRP), whose high content of growth factors is supposed to determine a regenerative stimulus to cartilaginous tissue. Despite many promising in vitro and in vivo studies, when discussing clinical application a clear indication for the use of PRP cannot be assessed. There are initial encouraging clinical data, but only a few randomized controlled trials have been published, so it is not possible to fully endorse this kind of approach for the treatment of cartilage pathology. Furthermore, study comparison is very difficult due to the great variability in PRP preparation methods, cell content and concentration, storage modalities, activation methods and even application protocols. These factors partially explain the lack of high quality controlled trials up to now. This paper discusses the main aspects concerning the basic biology of PRP, the principal sources of variability, and summarizes the available literature on PRP use, both in surgical and conservative treatments. Based on current evidence, PRP treatment should only be indicated for low-grade cartilage degeneration and in case of failure of more traditional conservative approaches.

Effect of platelet-rich plasma on the biologic activity of the human rotator-cuff fibroblasts: A controlled in vitro study

To assess the in vitro effect of platelet-rich plasma (PRP) on biological activity of the human rotator cuff fibroblasts and to describe the optimal dose-response to maximize cellular stimulation while reducing potential risk. Rotator cuff (RC) fibroblasts of n = 6 patients (mean age of 65.2 years) undergoing arthroscopic cuff tear reconstruction were cultured in vitro for 21 days and stimulated with PRP in three different concentrations (1-, 5-, and 10-fold). Samples were obtained for DNA and GAG measurement at 1, 7, 14, and 21 days. The biological outcomes were regressed on the PRP concentration. The application of PRP significantly influenced the fibroblast proliferation and activity of the human rotator cuff with elevated glycosaminoglycan (GAG) and DNA levels. The dosage of PRP had the significantly highest impact on this proliferation using a onefold or fivefold application. PRP has a significant effect on fibroblast proliferation of the human rotator cuff in vitro with an optimal benefit using a onefold or fivefold PRP concentration. This study justifies further in vivo investigations using PRP at the human rotator cuff.

Effect of platelet-rich plasma on cell proliferation and osteogenic activity of pulp stem cells

To evaluate the effects of different concentrations of platelet-rich plasma (PRP) on human dental pulp stem cells (hDPSC) proliferation and osteogenic differentiation activity so as to provide basis for future application of dental pulp stem cells and PRP in tissue engineering and bone repair therapy. hDPSC were isolated and cultivated in vitro. Flow cytometric analysis was carried out to test the expression of STRO-1.hDPSC were cultured in various concentrations of PRP (1%, 5%, 10%, 20%). At the 2nd and 6th day 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was tested. Osteogenic differentiation of hDPSC was assessed using alkaline phosphatase (ALP) staining and Alizarin Red staining at the 7th and 14th day. Flow cytometric analysis demonstrated that 14.82% of hDPSC were STRO-1 positive. One percent to 20% PRP showed significant effect of promoting hDPSC proliferation. One percent to 10% PRP showed significant effect of promoting hDPSC osteogenic differentiation. Certain concentrations of PRP can promote hDPSC proliferate and osteogenic differentiate, and this finding suggests future application of dental pulp stem cells and PRP in bone tissue engineering.

Effect of PRP on the proliferation of dermal papilla cells and hair follicle regeneration in mice

To investigate the effects of platelet-rich plasma (PRP) on the proliferation of dermal papilla cells (DPCs) and hair follicle regeneration. PRP was prepared using the double-spin method and applied to DPCs. The proliferative effect of activated PRP on DPCs was measured using MTT assay. To understand the influence of activated PRP on the hair-inductive capacity of DPCs, freshly isolated epidermal cells and DPCs of passage 4 were resuspended, mixed with various concentrations of a PRP (0%, 5% or 10%) and were then transferred to a grafting chamber, which was implanted onto the dorsal skin of nude mice. The chambers were removed 1 week after grafting and HF formation was monitored for 4 weeks; the graft site was harvested and processed for histological examination. Activated PRP increased the proliferation benefited the aggregative growth of DPCs. There are significant difference in the yield of hair follicles compared with 10% PRP (344 +/- 27) with 0% PRP (288 +/- 35) in the area of reconstituted skin (P < 0.05). The areas treated with PRP demonstrated an increase in hair follicles density of 19.4%. Ten percent PRP (18 +/- 1) d also can significantly shorten the time of hair formation, compared with 0% PRP (20 +/- 1) d (P < 0.05). There is a considerable effect of PRP on the time of hair formation and the yield of hair follicles reconstitution.

Can platelet-rich plasma (PRP) improve bone healing? A comparison between the theory and experimental outcomes

The increased concentration of platelets within platelet-rich plasma (PRP) provides a vehicle to deliver supra-physiologic concentrations of growth factors to an injury site, possibly accelerating or otherwise improving connective tissue regeneration. This potential benefit has led to the application of PRP in several applications; however, inconsistent results have limited widespread adoption in bone healing. This review provides a core understanding of the bone healing mechanisms, and corresponds this to the factors present in PRP. In addition, the current state of the art of PRP preparation, the key aspects that may influence its effectiveness, and treatment outcomes as they relate specifically to bone defect healing are presented. Although PRP does have a sound scientific basis, its use for bone healing appears only beneficial when used in combination with osteoconductive scaffolds; however, neither allograft nor autograft appear to be appropriate carriers. Aggressive processing techniques and very high concentrations of PRP may not improve healing outcomes. Moreover, many other variables exist in PRP preparation and use that influence its efficacy; the effect of these variables should be understood when considering PRP use. This review includes the essentials of what has been established, what is currently missing in the literature, and recommendations for future directions.

Optimization of Leukocyte Concentration in Platelet-Rich Plasma for the Treatment of Tendinopathy

Numerous methods are available for platelet-rich plasma (PRP) generation, but evidence defining the optimum composition is lacking. We hypothesized that leukocyte-reduced PRP would result in lower inflammatory cytokine expression compared with concentrated-leukocyte PRP and that maintaining the platelet:white blood cell (WBC) ratio would compensate for the effect of increased WBC concentration. Blood and flexor digitorum superficialis tendons were collected from young adult horses. Three PRP groups were generated with the same platelet concentration but different WBC concentrations: intermediate-concentration standard PRP, leukocyte-reduced PRP, and concentrated-leukocyte PRP. An additional high-concentration PRP group was generated with the same WBC concentration as the concentrated-leukocyte PRP group and the same platelet:WBC ratio as the standard PRP group. The PRP groups were used as media for flexor digitorum superficialis tendon explants in culture for seventy-two hours with 10% plasma in Dulbecco modified Eagle medium (DMEM) serving as control. Tendon gene expression for collagen types I (COL1A1) and III (COL3A1), cartilage oligomeric matrix protein (COMP), matrix metalloproteinase (MMP-13), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) was performed. The desired PRP groups were successfully generated. The expression of COMP, the COL1A1:COL3A1 ratio, and the expression of MMP-13 in flexor digitorum superficialis tendon explants was not different between PRP groups. The expression of COMP (p = 0.0027) and the COL1A1:COL3A1 ratio (p &lt; 0.0001) were increased in the PRP groups as compared with the control group, and the expression of MMP-13 was decreased in the PRP groups as compared with the control group (p &lt; 0.0001). The expression of IL-1β was lowest in leukocyte-reduced PRP and highest in concentrated-leukocyte PRP (p = 0.0001). The leukocyte-reduced PRP group and the control group had the lowest TNF-α expression, whereas the high-concentration PRP and concentrated-leukocyte PRP groups had the highest expression (p = 0.0224). A high absolute WBC concentration in PRP contributes to the expression of inflammatory cytokines in flexor digitorum superficialis tendon explants, and maintenance of the platelet:WBC ratio is not able to counteract this effect. The optimum composition of PRP for the treatment of tendinopathy has not been directly investigated. Persistent inflammation results in inferior repair with scar tissue. The present study indicates that in an animal model, WBC in PRP contributes to inflammatory cytokine production. Therefore, leukocyte-reduced PRP may be the optimum preparation to stimulate superior healing without scar tissue formation.

Effect of autologous and allogenic platelet‐rich plasma on human gingival fibroblast function

Platelet-rich plasma (PRP) has been proposed as a method of delivering growth factors to enhance regeneration. The aim of this study was to investigate the use of autogenous and allogenic PRP and platelet-poor plasma (PPP) on migration and proliferation of human gingival fibroblasts in vitro. Various concentrations of PRP, as well as PPP, were prepared from autologous and allogenic sources and applied to primary gingival fibroblasts. Migration was determined by assessing the fibroblast response to a concentration gradient. (3)H-thymidine incorporation and crystal violet colorimetric assays were utilized to assess DNA synthesis and proliferation. Platelet-rich plasma provides a significant migratory stimulus to gingival fibroblasts. Furthermore, the various concentrations of PRP (50%, 20% and 10%) do not promote DNA synthesis in the short term (24 h), but over the longer term (5 days) they stimulate an increase in cell proliferation. Compared with PPP, PRP was superior in terms of encouraging migration, but was inferior in terms of promoting DNA synthesis and cell proliferation. No difference was noted between the autologous and allogenic PRP preparations on cell function. Both PPP and PRP promote gingival fibroblast migration and proliferation in vitro, without differences between preparations obtained from autologous and allogenic sources.

Adult and umbilical cord blood-derived platelet-rich plasma for mesenchymal stem cell proliferation, chemotaxis, and cryo-preservation

Platelet-rich plasma (PRP) was prepared from human adult peripheral blood and from human umbilical cord (uc) blood and the properties were compared in a series of in vitro bioassays. Quantification of growth factors in PRP and platelet-poor plasma (PPP) fractions revealed increased levels of mitogenic growth factors PDGF-AB, PDGF-BB, and FGF-2, the angiogenic agent VEGF and the chemokine RANTES in ucPRP compared to adult PRP (aPRP) and PPP. To compare the ability of the various PRP products to stimulate proliferation of human bone marrow (BM), rat BM and compact bone (CB)-derived mesenchymal stem cells (MSC), cells were cultured in serum-free media for 4 and 7 days with varying concentrations of PRP, PPP, or combinations of recombinant mitogens. It was found that while all forms of PRP and PPP were more mitogenic than fetal bovine serum, ucPRP resulted in significantly higher proliferation by 7 days than adult PRP and PPP. We observed that addition of as little as 0.1% ucPRP caused greater proliferation of MSC effects than the most potent combination of recombinant growth factors tested, namely PDGF-AB + PDGF-BB + FGF-2, each at 10 ng/mL. Similarly, in chemotaxis assays, ucPRP showed greater potency than adult PRP, PPP from either source, or indeed than combinations of either recombinant growth factors (PDGF, FGF, and TGF-β1) or chemokines previously shown to stimulate chemotactic migration of MSC. Lastly, we successfully demonstrated that PRP and PPP represented a viable alternative to FBS containing media for the cryo-preservation of MSC from human and rat BM.

Effects of Platelet-Rich Plasma Composition on Anabolic and Catabolic Activities in Equine Cartilage and Meniscal Explants.

Objective:To evaluate the effects of single- and double-spin preparations of platelet-rich plasma (PRP) on anabolic and catabolic activities of cartilage and meniscal explants in vitro.Methods:Single- and double-spin PRP was prepared using laboratory processing or commercial kits. The cellular contents were quantified, and each PRP was mixed in equal quantities with cell culture medium and added to cartilage or meniscus explant cultures, with or without interleukin 1 β (IL-1β). Extracellular matrix synthesis was quantified over 24 hours via 35S-sulfate and 3H-proline incorporation, while gene expression of catabolic enzymes was evaluated using real-time PCR.Results:The platelet concentration in single-spin laboratory PRP was 59% higher than blood. Platelet and white blood cell concentrations in single-spin laboratory and kit PRP were not significantly different, while the double-spin kit resulted in approximately 2.5-fold higher platelet and approximately 400-fold higher white blood cell concentrations. In cartilage cultures without IL-1β, radiolabel incorporation in single-spin PRP cultures was significantly higher than in double-spin cultures. Similar results were obtained for 35S-sulfate incorporation in meniscus cultures without IL-1β. In IL-1β, radiolabel incorporation was largely similar among all PRPs. After 24 hours of culture, ADAMTS-4 gene expression in cartilage was lowest for single-spin PRP, while expression in the double-spin kit was not significantly different from double-spin laboratory PRP in which platelets were concentrated 6-fold.ConclusionsThis study suggests that single-spin PRP preparations may be the most advantageous for intra-articular applications and that double-spin systems should be considered with caution.