Presence Of Platelet-rich Plasma Research Articles

Fibrochondrogenic Differentiation Potential of Human Adiposederived Mesenchymal Stem Cells in a Type I Collagen-based Meniscus Scaffold with Activated Platelet-Rich Plasma Stimulation In-vitro

Introduction: Despite efforts to use scaffolds to treat meniscus tears, minimal progress has been made in facilitating meniscus regeneration and return of function. Our research objective was to develop a meniscus repair and regeneration implant by applying a resorbable scaffold in combination with cells and growth factors. We report here the results of using Platelet-Rich Plasma (PRP) as a source of growth factors to induce fibrochondrogenic differentiation of human Adipose- Derived Mesenchymal Stem Cells (hADSC) in a three-dimensional (3D) Type I collagen-based scaffold in-vitro. Methods: Scaffold Preparation: Type I collagen scaffolds were prepared following a protocol previously published. Two different densities of scaffolds, High Density (HD) and Low Density (LD), were produced for in-vitro study. hADSC and PRP Preparation. hADSCs were cultured to the fifth passage to reach the desired number for experimentation. PRP was collected from human blood and activated. Cell Culture Procedure: Effects of PRP on hADSC proliferation and differentiation into fibrochondrogenic cells were examined in four scaffold groups: LD, HD, LD+PRP and HD+PRP. hADSCs were seeded onto scaffolds (n=5) at a concentration of 2 × 106 cells/scaffold. 1% of PRP was added to the experimental media. Cellular proliferation was assessed at 1, 7, 14 and 21 days. Differentiation was measured using qRT-PCR on Days 14 and 21. qRT- PCR analysis of gene expression was completed with primers for COLLAGEN 1 and AGGRECAN. Data Analysis: ANOVAs were conducted (two-tailed tests) at the .05 significance level. Results: Cellular proliferation of hADSCs seeded on each scaffold increased over time. Similar trend was observed for cells seeded on HD scaffolds with and without PRP. hADSC showed significant increase in cellular proliferation on the LD scaffolds at Days 1 and 7. At Day 21, PRP treatment and LD scaffold had a synergistic positive effect on Type I collagen gene expression. PRP did not elevate type I collagen gene in the HD group, the HD scaffold alone had the same level of type I collagen gene expression as LD+PRP. Aggrecan expression was elevated in the presence of PRP in both the HD and LD scaffold groups, indicating enhanced fibrochondrogenic differentiation of hADSCs. Effective cell infiltration was observed across both HD and LD scaffolds with and without PRP treatment. HD scaffolds displayed larger cell clusters and more extensive cell migration over time compared to LD scaffolds. However, LD scaffolds resulted a more uniform cellular distribution than HD scaffolds. Conclusion: Our study demonstrates that PRP can play an important role in directing hADSCs towards fibrochondrogenic differentiation in Type I collagen-based scaffolds in-vitro. Additionally, our study shows that collagen scaffold density can influence the spatial distribution and cellular behavior of infiltrated cells.

REACTIVATION OF THE NUCLEUS PULPOUS CELLS BY PLATELET DERIVATIVES AS A NEW TOOL IN INTERVERTEBRAL DISC REGENERATION

Degenerative disc disease, associated to low back pain, afflicts more than 50% of humans, and represents a major healthcare problem, especially for the pathology initiation. Current treatments range from conservative strategies to more invasive surgical techniques, such as disc removal and vertebral fusion. In the Intervertebral Disease (IVD) the nucleus pulposus (NP) degeneration is a key factor for the pathology initiation. Several tissue engineering approaches aiming to restore the appropriate NP cell (NPCs) and matrix content, were attempted by using adult stromal cells either from bone marrow or adipose tissue, chondrocytes, notochordal cells and more recently also pluripotent stem cells. However, none was fully satisfactory since the NP acid and a-vascularized environment appeared averse to the implanted heterologous cells. Several studies demonstrated the efficacy of platelet derivatives such as platelet rich plasma (PRP) in promoting the regeneration of connective tissues. We investigated the efficacy of PRP on NPCs proliferation and differentiation with the goal to propose the direct stimulation of resident cells (stimulation of endogenous cells – less invasive surgical procedure) or the implantation of NPCs expanded in vitro in the presence of PRP as therapeutic agents in IVD degeneration.NPCs were isolated from small fragments of NP explants, cultivated in medium supplemented with PRP or FCS (standard condition control) and characterized by FACS analysis for the expression of the typical mesenchymal stem cells markers CD34, CD44, CD45, CD73, CD90 and CD105. NPCs cultured in PL showed a phenotypic profile like the cells cultured in FCS. However, compared to NPCs expanded in the presence of FCS, NPCs expanded in PRP showed a much better proliferation and differentiation capacity. NPCs differentiation was evaluated by the cell ability to produce an organized metachromatic cartilaginous matrix, confirmed by the positive immunohistochemical staining for chondrogenic markers.

BIOFUNCTIONALIZED 3D-BIOPRINTED VASCULAR-LIKE STRUCTURES: AN ADVANCED APPROACH TO PROMOTE ENDOTHELIAL TISSUE REGENERATION

Biofabrication is a popular technique to produce personalized constructs for tissue engineering. In this study we combined laponite (Lap), gellan gum (GG) with platelet-rich plasma (PRP) aiming to enhance the endothelial regeneration through the synergistic effects of their individual properties. Laponite has the ability to form porous three-dimensional networks mimicking the extracellular matrix structure, and PRP delivery of growth factors stimulates the endothelial cell proliferation and migration, offering a composite bioink for cell growth and support. The sustained release of these growth factors from the GG-laponite-PRP composite material over time provides a continuous source of stimulation for the cells, leading to more effective tissue engineering strategies for endothelial tissue regeneration. Four blend compositions comprising 1% w/v GG and 0.5 or 1% w/v Lap and 25% v/v PRP were combined with Wharton jelly mesenchymal stem cells (WJ-MSCs) and bioprinted into vessel-like structures with an inner diameter of 3 mm and a wall thickness of 1 mm. Stress/strain analysis revealed the elastomeric properties of the hydrogels with Young modulus values of 10 MPa. Increasing the Lap concentration led to a non-significant decrease of swelling ratio from 93 to 91%. Live/dead assay revealed cell viability of at least 76%, with the 0.5%Lap-GG viability exceeding 99% on day 21. Gradual increase of glycosaminoglycans accumulation and collagen production indicate promotion of ECM formation. The expression and membranous localization of PECAM-1 from day 7 and the granular intracellular localization of vWF after 2 weeks demonstrate in vitro endothelial functionality. In vivo subcutaneous implantation indicated the absence of any adverse immunological reactions. The results reveal the expression of both vWF and PECAM-1 by WJ-MSCs entrapped in all four construct compositions with significantly higher expression of vWF in the presence of PRP.

Enzymatically cross-linked hyaluronic acid hydrogels as in situ forming carriers of platelet-rich plasma: Mechanical properties and bioactivity levels evaluation

New studies have shown the great potential of the combination of in situ enzymatically cross-linked hydrogels based on tyramine derivative of hyaluronic acid (HA-TA) with platelet-rich plasma (PRP) and platelet lysate in regenerative medicine. This study describes how the presence of PRP and platelet lysate affects the kinetics of gelation, viscoelastic properties, swelling ratio, and the network structure of HA-TA hydrogels and how the encapsulation of PRP in hydrogels affects the bioactivity of released PRP determined as the ability to induce cell proliferation. The properties of hydrogels were tuned by a degree of substitution and concentration of HA-TA derivatives. The addition of platelet derivatives to the reaction mixture slowed down the cross-linking reaction and reduced elastic modulus (G′) and thus cross-linking efficiency. However, low-swellable hydrogels (7–190%) suitable for soft tissue engineering with G’ 200–1800 Pa were prepared with a gelation time within 1 min. It was confirmed that tested cross-linking reaction conditions are suitable for PRP incorporation because the total bioactivity level of PRP released from HA-TA hydrogels was ≥87% and HA-TA content in the hydrogels and thus mesh size (285–482 nm) has no significant effect on the bioactivity level of released PRP.

In vitro and in vivo biocompatibility of decellularized cellulose scaffolds functionalized with chitosan and platelet rich plasma for tissue engineering applications

This study describes the fabrication of cellulose scaffold (CS) and cellulose-chitosan (CS/CHI) scaffolds from the immature endosperm of Borassus flabellifer (Linn.) (BF) loaded with platelet rich plasma (PRP). Thus, developed scaffolds were evaluated for their physicochemical and mechanical behavior, growth factor release and biological performance. Additionally, in vivo response was assessed in a sub cutaneous rat model to study vascularization, host inflammatory response and macrophage polarization. The results of this study demonstrated that CS and CS/CHI scaffolds with PRP demonstrated favorable physiochemical and morphogical properties. The scaffold groups CS-PRP and CS/CHI-PRP were able to release growth factors in a well sustained manner under physiological conditions. The presence of PRP in cellulosic scaffolds did show significant differences in their behavior when investigated under in vitro studies, where the release of diverse cytokines improved the cellular proliferation and differentiation of osteoblasts. Finally, the PRP enriched scaffolds when studied under in vivo conditions showed increased angiogenesis and re-epithelialization with adequate collagen deposition and tissue remodeling. Our results suggest that besides the conventional carrier systems, this new-generation of plant-based cellulosic scaffolds with/without any modification can serve as a suitable carrier for PRP encapsulation and release, which can be used in numerous tissue regenerative therapies.

Evaluation of in vitro biocompatibility of scaffolds for the repair of bone defects

The use of bone scaffolds in traumatology and orthopedics is an extremely important issue. The growing number of cases of significant bone defects, in particular after revision arthroplasty, combat trauma and due to the introduction of new methods of reconstructive surgery of bones and joints, requires more detailed studies of the using different osteoplastic materials. Materials and methods. As scaffolds used 4 types of materials that are most often used in the clinic for the correction of bone defects - ceramic hydroxylapatite, beta-tricalcium phosphate, allogeneic bone matrix treated with gamma irradiation, allogeneic bone matrix scaffold. The effect of matrices on the viability of normal human fibroblasts (M19 cell line) in cell culture in vitro was studied. The viability of cells after their co-cultivation with scaffolds was determined by colorimetric method by staining with crystal violet. To obtain an osteoinductive effect used platelet-rich plasma (PRP), standardized by the method of Araki with some modifications. The proliferative activity of fibroblasts was assessed by the level of expression of the proliferation marker Ki-67 by immunocytochemical analysis. Results. It was found that the least pronounced antiproliferative effect is shown by allogeneic bone matrix treated with gamma irradiation. Data on the complex effect of co-cultivation of fibroblasts with scaffolds in the presence of PRP on cell viability and proliferative activity were obtained. It was found that PRP improves the survival of fibroblasts by 15-30 % and increases their proliferative activity by 35-75 %. Delipidization of scaffold from allogeneic bone matrix, heat-treated by local bone bank technology, increased its biocompatibility with human fibroblasts. Conclusions. According to the results of a comparative analysis of the impact of different scaffolds on the viability of normal human fibroblasts, it was found that scaffolds from allogeneic bone matrix have the least pronounced antiproliferative effect. Platelet-rich plasma has been shown to improve fibroblast survival and increase their proliferative activity. Treatment with 70 % ethyl alcohol scaffold from allogeneic bone matrix, heat-treated by local bone bank technology, increased its biocompatibility with human fibroblasts.

Use of Platelet-Rich Plasma Plus Suramin, an Antifibrotic Agent, to Improve Muscle Healing After Injuries

Background: The increasing use of platelet-rich plasma (PRP) to treat muscle injuries raises concerns because transforming growth factor–beta (TGF-β) in PRP may promote fibrosis in the injured muscle and thus impair muscle regeneration. Purpose: To investigate whether suramin (a TGF-β inhibitor) can reduce muscle fibrosis to improve healing of the injured muscle after PRP treatment and identify the underlying molecular mechanism. Study Design: Controlled laboratory study. Methods: Myoblasts isolated from the gastrocnemius muscle of Sprague Dawley rats were treated with PRP or PRP plus suramin. MTT assays were performed to evaluate cell viability. The expression of fibrosis-associated proteins (such as type I collagen and fibronectin), Smad2, and phosphorylated Smad2 was determined using Western blot analysis and immunofluorescent staining. An anti–TGF-β antibody was employed to verify the role of TGF-β in fibronectin expression. Gastrocnemius muscles were injured through a partial transverse incision and then treated using PRP or PRP plus suramin. Hematoxylin and eosin staining was conducted to evaluate the healing process 7 days after the injury. Immunofluorescent staining was performed to evaluate fibronectin expression. Muscle contractile properties—fast-twitch and tetanic strength—were evaluated through electric stimulation. Results: PRP plus 25 μg/mL of suramin promoted myoblast proliferation. PRP induced fibronectin expression in myoblasts, but suramin reduced this upregulation. The anti–TGF-β antibody also reduced the upregulation of fibronectin expression in the presence of PRP. The upregulation of phosphorylated Smad2 by PRP was reduced by either the anti–TGF-β antibody or suramin. In the animal study, no significant difference was discovered in muscle healing between the PRP versus PRP plus suramin groups. However, the PRP plus suramin group had reduced fibronectin expression at the injury site. Fast-twitch strength and tetanic strength were significantly higher in the injured muscle treated using PRP or PRP plus suramin. Conclusion: Simultaneous PRP and suramin use reduced fibrosis in the injured muscle and promoted healing without negatively affecting the muscle’s contractile properties. The underlying molecular mechanism may be associated with the phosphorylated Smad2 pathway. Clinical Relevance: Simultaneous PRP and suramin use may reduce muscle fibrosis without compromising muscle contractile properties and thus improve muscle healing.

Platelet-rich plasma-hyaluronic acid/chondrotin sulfate/carboxymethyl chitosan hydrogel for cartilage regeneration.

In this study, the chondrogenic potential of hyaluronic acid/chondrotin sulfate/carboxymethyl chitosan hydrogels with adipose-derived mesenchymal stem cells (ADMSCs) was evaluated. Here, hyaluronic acid, chondrotin sulfate, and carboxymethyl chitosan were used as the substrate for cartilage tissue engineering in which the hydrogel is formed due to electrostatic and hydrogen bonds through mixing the polymers. Because of the instability of this hydrogel in the biological environment, 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride/N-hydroxy-succinimide was used as a crosslinker to increase the hydrogel stability. The hydrogels showed reasonable stability due to the combined effect of self-crosslinking and chemical crosslinking. The cells were treated with the prepared hydrogel samples for 14 and 21 days in nondifferentiation medium for evaluation of the cellular behavior of ADMSCs. Gene expression evaluation was performed, and expression of specific genes involved in differentiation was shown in the crosslinked hydrogel with platelet-rich plasma (PRP) (H-EN-P) had increased the gene expression levels. Quantification of immunofluorescence intensity indicated the high level of expression of SOX9 in H-EN-P hydrogel. Based on the results, we confirmed that the presence of PRP and the similarity of the hydrogel constituents to the cartilage extracellular matrix could have positive effects on the differentiation of the cells, which is favorable for cartilage tissue engineering approaches.

Evaluating the Interactions between Endothelial and Smooth Muscle Cells in the Presence of Drug-Eluting Stents and Platelets: Is Interleukin6 a New Possible Therapeutic Target for In-Stent Restenosis?

The success of percutaneous coronary intervention (PCI) procedures is limited by in-stent restenosis (ISR). The use of drug-eluting stents (DES) decreased the incidence of ISR; however, ISR continues to affect a percentage of patients with ischemic cardiac events. Therefore, there is room for improvement considering not only the effectiveness of DES but also the adjunct pharmacological interventions for patients after PCI. Interleukin 6 (IL-6) can stimulate the growth of vascular smooth muscle cells (SMCs) in a platelet-derived growth factor (PDGF)-dependent manner and that antibodies against the IL-6 receptor reduced intimal hyperplasia in mice. In this study, our aim is to develop an in vitro study, based on co-cultures of endothelial cells (EC) and SMCs in the presence of platelets and stents, to analyze the cytokine responses (IL-6 and other inflammation markers) associated with ISR, in the quest for new therapeutic targets. Overall, in the presence of platelet-rich plasma (PRP), there was an increase in the production of reactive oxygen species in the groups that received the stents, probably because platelets potentiate stent-associated oxidative stress. Moreover, the analysis of the expression of inflammatory markers showed a difference in the expression of IL-6 only in the groups that received stents, which further increased in the presence of platelets. Altogether, our findings suggest that the down regulation of IL-6 expression using pharmacologic inhibitors is a possible new therapeutic strategy in the context of ISR.

Repair of rat cranial bone defect by using amniotic fluid-derived mesenchymal stem cells in polycaprolactone fibrous scaffolds and platelet-rich plasma

Introduction: Tissue regenerative medicine strategies, as a promising alternative has become of major interest to the reconstruction of critical size bone defects. This study evaluated the effects of the simultaneous application of polycaprolactone (PCL), amniotic fluid mesenchymal stem cells (AF-MSCs) and platelet-rich plasma (PRP) on the repair of rat cranial bone defects. Methods: The AF-MSCs were isolated at the end of the second week of pregnancy in rats. PRP obtained from rat blood and the random PCL fibrous scaffolds were prepared using the electrospinning method. Circular full thickness (5 mm) bone defects were developed on both sides of the parietal bones (animal number=24) and the scaffolds containing AF-MSCs and PRP were implanted in the right lesions. Thereafter, after eight weeks the histological and immunohistochemistry studies were performed to evaluate the bone formation and collagen type I expression. Results: The spindle-shaped mesenchymal stem cells were isolated and the electron microscope images indicated the preparation of a random PCL scaffold. Immunohistochemical findings showed that collagen type I was expressed by AF-MSCs cultured on the scaffold. The results of hematoxylin and eosin (H&E) staining indicated the formation of blood vessels in the presence of PRP. Additionally, immunofluorescence findings suggested that PRP had a positive effect on collagen type I expression. Conclusion: The simultaneous application of fibrous scaffold + AF-MSCs + PRP has positive effects on bone regeneration. This study showed that PRP can affect the formation of new blood vessels in the scaffold transplanted in the bone defect.

Comparative lyophilized platelet-rich plasma wafer and powder for wound-healing enhancement: formulation, in vitro and in vivo studies

Platelet-rich plasma (PRP) accelerates wound healing, as it is an excellent source of growth factors. PRP was separated from whole human blood by centrifugation. PRP powder and wafers were prepared by lyophilization, with the wafers prepared using sodium carboxymethylcellulose (Na CMC). The PRP wafers showed porous structures, as indicated by scanning electron microscopy (SEM) images, and the ability of the wafer to absorb exudates and thus promote wound healing was tested with the hydration capacity test. The platelet count was tested and indicated that the presence of PRP in the wafers had no effect on the platelet count. An antimicrobial activity test was carried out, showing that PRP had antibacterial activity against Gram-negative bacteria. Compared with lyophilized PRP powder and PRP-free wafers, PRP wafers showed the highest percent of wound size reduction on induced wounds in rats. Histopathological examination of rat skin showed that the PRP wafers achieved the shortest healing time, followed by the lyophilized PRP powder and finally the PRP-free wafers. The present study revealed that PRP can be formulated as a wafer, which is a promising pharmaceutical delivery system that can be used for enhanced wound-healing activity and improved the ease of application compared to lyophilized PRP powder.

In Vitro Evaluation of Proliferation and Migration Behaviour of Human Bone Marrow-Derived Mesenchymal Stem Cells in Presence of Platelet-Rich Plasma.

Objective To access the effects of platelet-rich plasma (PRP) on the behaviour of human bone marrow-derived mesenchymal stem cells (hBMSCs), including proliferation and migration. Methods PRP was diluted with DMEM/F12, resulting in concentrations of 1%, 2%, and 5%. The proliferation of hBMSCs was examined by 2 methods: cell-number counting with the haemocytometer method and the colony-forming unit-fibroblast (CFU-F) assay. Cell migration was evaluated using the scratch wound healing (SWH) assay; after that, the recorded digital images were analysed by the Image-Analysis J 1.51j8 software to compare the cell-free areas between groups after 0, 24, and 48 hours. Results hBMSCs cultured in DMEM/F12 at PRP concentrations of 1%, 2%, and 5% were all able to proliferate and migrate. In the 5% PRP group, hBMSCs proliferated greatly with a significantly higher cell number than reported for all other groups on days 5, 7, and 9. CFU-Fs were observed in all groups, except for the negative control group. The SWH assay demonstrated that hBMSCs cultured in 2% and 5% PRP almost filled the artificial wound scratch and significantly migrated more than those of all other groups at both 24 h and 48 h. Conclusion This study indicated that, due to the significant enhancement of cell proliferation and migration, 5% PRP might be the optimal concentration that should be used to promote the potential of hBMSCs in wound healing.

Platelet-rich plasma shows beneficial effects for patients with knee osteoarthritis by suppressing inflammatory factors.

Knee osteoarthritis is a degenerative disease that may develop due ageing, obesity, strain, congenital abnormal joints, joint deformity or trauma. It is caused by many factors, such as degradation of articular cartilage injury, joint edge and subchondral bone hyperplasia of reactivity. Platelet-rich plasma (PRP) is an autologous blood sample that contains highly concentrated platelets and multiple cell growth factors. PRP promotes synovial cell proliferation and differentiation and may recover cartilage morphology. In the present study, the clinical efficacy of PRP was investigated in patients with knee osteoarthritis aged between 18 and 30 years in a phase-III clinical study. Following an 8-week baseline, patients with knee osteoarthritis were randomized into once-weekly, double-blind treatment with PRP (2-14 ml) or placebo groups. The results indicated that patients with osteoarthritis treated with PRP had modulated plasma concentrations of inflammatory factors and pro-angiogenic factors compared with the placebo group. Treatment responses were assessed by median percent reduction in inflammatory and pro-angiogenic factors and these improved with PRP treatment compared with the placebo. Clinical data indicated that PRP alleviated knee osteoarthritis and reduced humoral and cellular immune responses that led to beneficial effects on histological parameters. Inflammation was significantly alleviated in patients receiving PRP compared with the placebo group. The most common treatment-emergent adverse events in the presence of PRP were hypertension and proteinuria. In conclusion, treatment with PRP for patients with knee osteoarthritis presented beneficial effects in alleviating joint inflammation, cartilage destruction and bone damage, and repairing joint tissue. These results suggested that PRP may be a potential therapeutic agent for knee osteoarthritis.

Using Electrospun Scaffolds to Promote Macrophage Phenotypic Modulation and Support Wound Healing

AbstractThe development of pressure ulcers in spinal cord injury patients is extremely common, often requiring extensive surgical procedures. Macrophages (MACs) play a crucial role in the innate immune system, contributing to wound healing and overall regeneration. MACs have been found to possess the potential to be activated by external factors from their M0 inactive state to an M1 proinflammatory or M2 regenerative state. This study conducted a comprehensive evaluation of MAC phenotype in response to electrospun scaffolds of varying material fiber/pore diameter, fiber stiffness, and +/− inclusion of platelet-rich plasma (PRP). Generally, itwas found that the addition of PRP resulted in decreased pore size, where 5 silk fibroin (SF) had the stiffest fibers. Furthermore, PRP scaffolds demonstrated an increased production of VEGF and chemotaxis. The polycaprolactone (PCL) and SF scaffolds had the largest cell infiltration and proliferation. Overall, it was found that 5% SF had both ideal fiber and pore structure, allowing for cell infiltration further enhanced by the presence of PRP. Additionally, this scaffold led to a reasonable production of VEGF while still allowing fibroblast proliferation to occur. These results suggest that such a scaffold could provide an off-the-shelf product capable of modifying the local MAC response.

Platelet-rich plasma (PRP) and adipose-derived mesenchymal stem cells: stimulatory effects on proliferation and migration of fibroblasts and keratinocytes in vitro.

The clinical use of tissue engineering associated with cell therapy is considered a new alternative therapy for the repair of chronic lesions with potential application in different medical areas, mostly in orthopedic and dermatological diseases. Platelet-rich plasma (PRP) is a rich source of growth factors and cytokines important for wound healing. Adipose-derived mesenchymal stem cells (ADSCs) have shown potential to accelerate the resolution of ulcers, to stimulate cell proliferation, and to benefit the quality of skin repair. This study aims to determine the effect of PRP and conditioned medium (CM) from ADSC on fibroblast and keratinocyte proliferation in vitro. Migration and proliferation assays were performed to evaluate the growth of fibroblasts and keratinocytes in the presence of PRP, CM, and CM+PRP. Significant proliferative stimulation was observed after 48h of culture (p<0.05) on mean absorbance of fibroblasts cultured with 10 and 25% PRP, 100% CM, and 25% PRP+25% CM, if compared with control. Keratinocyte proliferation was stimulated after 48h in cultures with 25, 50, and 100% CM, and growth was compared with controls. The migration assay detected a significant migratory stimulus in fibroblasts cultured with 10% PRP+10% CM after 48h. These in vitro results suggest that PRP and ADSC have therapeutic potential for healing and re-epithelialization of chronic wounds in vivo.

Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway

Platelet-rich plasma (PRP) contains a high concentration of several growth factors and contributes to soft-tissue engineering and wound healing. However, the effect of PRP on human dermal fibroblast proliferation and responses is unknown. This was investigated in the present study using PRP prepared from the whole human blood using the double-spin method. Human dermal fibroblast cultures were established from skin samples collected during plastic surgery. Platelet concentration and growth factor levels in PRP were estimated, and a cell proliferation assay was carried out after PRP treatment. The role of Ras-dependent extracellular signal-regulated kinase (ERK)1/2 in the effects of PRP was investigated in human dermal fibroblasts by suppressing ERK1/2 expression with an inhibitor or by short interfering (si)RNA-mediated knockdown, and assessing ERK1/2 phosphorylation by western blotting as well as proliferation in PRP-treated cells. We found that PRP stimulated human dermal fibroblast proliferation, which was suppressed by ERK1/2 inhibitor treatment (P<0.01). ERK1/2 phosphorylation was increased in the presence of PRP, while siRNA-mediated knockdown of ERK1/2 blocked cell proliferation normally induced by PRP treatment (P<0.01). These results demonstrate that PRP induces human dermal fibroblast proliferation via activation of ERK1/2 signaling. Our findings provide a basis for the development of agents that can promote wound healing and can be applied to soft-tissue engineering.

Platelet-Rich Plasma Enhances the Cellular Function of Equine Bone Marrow-Derived Mesenchymal Stem Cells

Rationale: Equine mesenchymal stem cells (eMSCs) and platelet-rich plasma (PRP) are cell-based therapies being used clinically to repair damaged tissue of horses. Numerous reports including data from our laboratory show that there are variations in the biological properties of MSCs and platelet – rich plasma, which can impact their biological functions. A single study describes the use of the eMSCs and PRP in tendon healing, with minimal success. The exact mechanism of action using the combination of the two therapies is unknown. This study was performed to evaluate and understand the effects of PRP, if any on the cellular performance of eBMMSCs in culture. Objective: To assess the effects of PRP in vitro on the rate of proliferation, expression of protein markers and osteogenic and chondrogenic differentiation of the primary cultures of eBMMSCs. Methods and Results: A commercially available stall side, portable kit was used to isolate PRP. To investigate the effect of PRP on eBMMSCs, the rate of proliferation of eBMMSCs was measured using the MTS assay, and subsequently the viability and the stemness of eBMMSCs was assessed using fluorescent staining and the expression of CD90. Finally, the osteogenic and chondrogenic differentiation of eBMMSCs was assessed by lineage-specific staining and the expressions of lineage – specific mRNAs. All assays were performed at a concentration of 50 million platelets/mL. Significant increase in proliferation and the differentiation profiles were observed in presence of PRP. Most importantly, the stem cell characteristics of inferior eBMMSCs showed a marked improvement in presence of PRP. Conclusions: The addition of PRP improves the in vitro function of eBMMSCs by enhancing the proliferation and osteo - and chondro - genesis. The presence of an optimal dose of platelets may enhance the in vivo performance of eBMMSCs and may be indicated when using autologous eBMMSCs therapy in the clinic.

Human Fibroblast–Like Cultures in the Presence of Platelet-Rich Plasma as a Single Growth Factor Source

The purpose of this study was to compare the proliferation, morphology, and antigenic expression of human fibroblast-like cells between primary cultures treated with platelet-rich plasma (PRP) or fetal bovine serum (FBS) as the growth factor source. Cells from human gingival tissue samples obtained from healthy volunteers during oral surgery were studied. Isolated cells were cultured in media supplemented with 10% PRP or FBS. Platelet-rich plasma was prepared from the venous blood of each patient. The authors studied short- and long-term cell cultures in the presence of PRP or FBS as the sole growth factor source in order to determine (a) cell growth rate, by MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay; (b) cell morphology, by electronic microscopy; and (c) antigenic expression, by flow cytometry and confocal microscopy. In short-term cultures, the cell growth rate was higher with PRP versus FBS treatment. No differences in morphology or expression of vimentin, fibronectin, or α-actin antigens were observed between PRP and FBS cultures. In long-term cultures, PRP and FBS did not significantly differ in cell growth rate but differed in morphology and in the expression of vimentin, fibronectin, and α-actin. The PRP enhances cell proliferation over the short term and induces cell differentiation of fibroblast-like cells to myofibroblast-like cells over the long term, suggesting that fibroblast differentiation to myofibroblasts may underlie the action mechanism of PRP in soft tissue regeneration.

Establishment of a Promising Human Nucleus Pulposus Cell Line for Intervertebral Disc Tissue Engineering

Low-back pain caused by intervertebral disc degeneration could be recovered by the regeneration of the nucleus pulposus (NP). This study aimed to establish a chondrogenic recovery model with promising a human NP (hNP) cell line, an immortalized hNP (ihNP), which could be a screening platform to identify regenerative drugs. The ihNP cells were created from primary human NP cells transfected with a retroviral vector-driven HPV16 E6/E7. Growth properties and characteristics of ihNP were evaluated by comparing with parental NP cells. Successful immortalization of ihNP cells stably expressed HPV 16 E6/E7 mRNA. The doubling time of ihNP was shortened to 53.16±2.63 h compared with parental hNP-P1. Cell cycle regulators, including p53, p21, and pRB were downregulated compared to parental hNP-P1. The in vivo neoplastic forming assay also demonstrated that the ihNP was nontumorigenic. After 25 generations of cell cultures, the ihNP cells, yet stably expressed chondrogenic genes, including (SOX9), type II collagen (Col II), aggrecan, decorin, biglycan, and versican. Higher expressions of chondrogenic proteins, including Col II, phosphorylated SOX9 (p-SOX9), and CD44 were also determined. Under the stressful inflammatory conditions induced by lipopolysaccharides (LPS), the regenerative and anti-inflammatory potentials of ihNP in two-dimensional culture with the presence of platelet-rich plasma (PRP) were evaluated by reverse transcriptase polymerase chain reaction. PRP showed significant effects on restoring diminished chondrogenic markers and deleterious inflammatory responses induced by LPS in ihNP. The therapeutic potentials of ihNP in three-dimensional neocartilage model could also be exerted by PRP using histological evaluation and immunological staining. Hence, the established ihNP cells can provide a chondrogenic recovery model as a regenerative drug screening tool for further regenerative drug discovery and development.

Supplementation With Platelet-Rich Plasma Improves the In Vitro Formation of Tissue-Engineered Cartilage With Enhanced Mechanical Properties

This study aimed to determine the effects of platelet-rich plasma (PRP) on the histologic, biochemical, and biomechanical properties of tissue-engineered cartilage. Chondrocytes isolated from bovine metacarpal-phalangeal articular cartilage were seeded on top of a porous ceramic substrate (calcium polyphosphate [CPP]). Cultures were supplemented with fetal bovine serum (FBS), PRP, or platelet-poor plasma (PPP) at 5%. On day 5, the concentration was increased to 20%. PRP and PPP were obtained through centrifugation of whole blood withdrawn from a mature cow. After 2 weeks, samples (n = 8) were analyzed histologically, biochemically, and biomechanically. Data were analyzed using the Wilcoxon test (significance, P < .05). Chondrocytes cultured in 20% PRP formed thicker cartilage tissue (1.6 ± 0.2 mm) than did cells grown in 20% FBS (0.7 ± 0.008 mm; P = .002) and 20% PPP (0.8 ± 0.2 mm; P = .03). Cartilage tissue generated in the presence of 20% PRP had a greater equilibrium modulus of 38.1 ± 3.6 kPa versus 15.6 ± 1.5 kPa (P = .0002) for 20% PPP and 20.4 ± 3.5 kPa (P = .007) for 20% FBS. Glycosaminoglycan (GAG) content was increased in tissues formed in 20% PRP (176 ± 18.8 μg GAG/mg) compared with those grown in 20% FBS (112 ± 10.6 μg GAG/mg; P = .01) or 20% PPP (131.5 ± 14.8 μg GAG/mg; P = .11). Hydroxyproline content was similar whether the media was supplemented with 20% PRP (8.7 ± 0.9 μg/mg), 20% FBS (7.6 ± 0.9 μg/mg; P = .37), or 20% PPP (6.4 ± 1 μg/mg; P = .28). DNA content was similar in all tissues whether formed in 20% PRP (11.9 ± 3.5 μg/mg), 20% FBS (9.3 ± 2.5 μg/mg; P = .99), or 20% PPP (7.2 ± 1.3 μg/mg; P = .78). Immunostained samples showed prevalence of type II collagen in tissues formed in the presence of 20% PRP. The presence of PRP in the culture media enhances the in vitro formation of cartilage, with increased GAG content and greater compressive mechanical properties, while maintaining characteristics of hyaline phenotype. Understanding the in vitro effects of PRP on tissue-engineered cartilage may lead to the creation of engineered cartilage tissue with enhanced properties suitable for cartilage repair.