Platelet-Rich Plasma, Adipose Tissue, and Scar Modulation

Platelet-Rich Plasma, Adipose Tissue, and Scar Modulation Abstract In developed countries, over 100 million people per year form scars, of which 30% become hypertrophic. These scars are defined histologically by the presence of persistently contractile myofibroblasts. Recent in vitro research suggests that certain bone morphogenic proteins (BMP) can induce scar myofibroblast dedifferentiation and reprogramming into adipocytes. Since platelets contain BMPs within their granules, it is possible that platelet-rich plasma (PRP) can act as a vehicle to deliver BMP to sites of scarring or potential scarring. Additionally, when PRP is mixed with fat graft tissue, synergistic adipogenic growth factors (including BMPs) are released which can help complete myofibroblast transformation and adipogenesis. The aim of this article is to corroborate these findings by systematically reviewing articles demonstrating the following concepts: (1) the effect of PRP on scar modulation; (2) the extraction of BMP from PRP; (3) BMP-induced myofibroblastic dedifferentiation; and (4) the effect of PRP on adipose-derived stem cells using the MEDLINE database. This search yielded 2830 articles, of which 38 met the inclusion criteria for this literature review. Level of Evidence: 4 [AU: Please provide some keywords] Platelet-rich plasma (PRP) is a personalized therapeutic procedure that involves centrifuging a patient’s blood and removing certain components in order to concentrate the number of platelets.1 In most cases, PRP is activated to release of growth factors from the intramembranous granules of the platelets and can then be utilized for a variety of therapeutic purposes.1 Platelet activation occurs when the intracellular granule fuses to the platelet membrane, and the contained growth factors undergo a final modification into an active state and are released.2 Under physiologic conditions, the content of these granules work as mediators in wound healing and hemostasis.2 Platelets contain both alpha and dense granules. Alpha granules store growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), and other members from the TGF-β superfamily. Dense granules contain bioactive molecules, such as serotonin, histamine, and adenosine, which modulate membrane permeability and local inflammation (Figure 1).3,4 Figure 1. View largeDownload slide Platelet containing alpha and dense granules and some of their bioactive contents. Figure 1. View largeDownload slide Platelet containing alpha and dense granules and some of their bioactive contents. PRP therapy is currently being used for many dermatological and nondermatological purposes, in tandem with other treatments, to enhance overall results.1 Cosmetically, PRP shows efficacy in treating alopecia, acne, traumatic scars, contractile scars, wrinkles, stretch marks, chronic ulcers, as well as enhancing laser resurfacing and postsurgical wound healing.1,3,5-7 For orthopedic purposes, PRP is used for spine fusions, osteoarthritis, cartilage, bone and tendon injuries, and various types of fractures.8 One recent study found that mixing PRP with percutaneous needle tenotomy relieved pain in a patient with De Quervain’s tenosynovitis for whom other treatments failed.9 Bone morphogenic proteins (BMPs), a subset of the TGF-β superfamily, are a group of growth factors found in PRP that were originally discovered and named for their role in cartilage and bone development.10 In addition, BMPs are implicated in a variety of embryological and physiological regulatory roles related to adipose tissue, such as adipocyte development and regulating brown/white adipocyte differentiation.10-12 There are at least 15 different BMPs, each uniquely effective at regulating the type and rate of stem cell differentiation into either adipogenic, chondrogenic, or osteogenic precursors.13 Their roles and effectiveness are based not only on the type of BMP and BMP receptor, but also on the local environment, including the presence of other signaling factors.13 BMP-4, BMP-7, and to a lesser degree BMP-2, are all potent adipogenic inducers of stem cells under the right conditions and are even necessary for proper adipocyte differentiation.14-16 Our laboratory, The Center for Tissue Engineering (CTE) at UC Irvine, collaborates with the Plikus lab at UC Irvine to study BMP. In a murine model of wound healing, it was demonstrated that actively growing hair follicles induce surrounding adipogenesis initiated by the dedifferentiation of myofibroblasts into adipocytes under BMP influence.17 Other studies show that BMPs, namely BMP-4 and 7, stall and reverse myofibroblast differentiation by antagonizing TGF-β promyofibroblastic signaling and inducing greater PPARγ expression.18,19 These findings support a possible role for BMPs in preventing and even reducing scarring by inducing adipogeneic transformation of myofibroblasts in scar tissue. Since platelets contain BMPs within their granules, it is possible that PRP acts as a vehicle to deliver BMP to sites of scarring or potential scarring. PRP may be particularly effective when mixed with fat graft tissue, which would provide the other synergistic adipogenic growth factors that BMP-4 and 7 lack to complete myofibroblast-induced adipogenesis. Here, we systematically review literature from studies indicating the following: (1) the effect of PRP on scar modulation; (2) the extraction of BMP from PRP; (3) BMP-induced myofibroblastic dedifferentiation; and (4) the effect of PRP on adipose-derived stem cells (ADSCs) (Tables 1-4). Finally, we propose a potentially new therapeutic approach for hypertrophic scarring. Table 1. Studies Evaluating Effects of PRP on Scar Modulation Study Study design Study method No. of patients Results Cervelli et al20 Clinical: traumatic scars (1) Fat graft + PRP + laser (2) Fat graft + PRP (3) Laser alone 60 patients split into 3 groups Scar management: Triple therapy > Fat graft + PRP > Laser alone Eichler et al21 Retrospective questionnaire Port area scars treated with or without PRP 20 of 120 patients received PRP PRP patients > Non-PRP treated patients in reduced scar dissatisfaction score Klosova et al22 Clinical: burn scars PRP + split thickness skin graft 23 patients with 38 scars Viscoelastic properties: STSG + PRP > STSG alone Nofal et al5 Clinical study: acne scars (1) PRP injections (2) Skin needling (3) PRP + skin needling 45 patients split into 3 groups No single treatment significantly better than the other Lee et al23 Clinical: acne scars After receiving CO2 fractional resurfacing patients underwent split face trial: (1) ½ face PRP injection (2) ½ face Saline injection 14 patients PRP enhanced recovery and scar appearance when compared to saline Na et al24 Clinical: postfractional CO2 recovery (FcxCR) Bilateral inner arm FcxCR followed by either PRP or saline injection 25 patients PRP after FxCR enhanced wound healing and recovery Jones et al7 Retrospective case review Patients with keloids received intraoperative PRP combined with postoperative superficial radiation therapy 49 patients with 50 ear keloids 94% nonrecurrence after 2 years Study Study design Study method No. of patients Results Cervelli et al20 Clinical: traumatic scars (1) Fat graft + PRP + laser (2) Fat graft + PRP (3) Laser alone 60 patients split into 3 groups Scar management: Triple therapy > Fat graft + PRP > Laser alone Eichler et al21 Retrospective questionnaire Port area scars treated with or without PRP 20 of 120 patients received PRP PRP patients > Non-PRP treated patients in reduced scar dissatisfaction score Klosova et al22 Clinical: burn scars PRP + split thickness skin graft 23 patients with 38 scars Viscoelastic properties: STSG + PRP > STSG alone Nofal et al5 Clinical study: acne scars (1) PRP injections (2) Skin needling (3) PRP + skin needling 45 patients split into 3 groups No single treatment significantly better than the other Lee et al23 Clinical: acne scars After receiving CO2 fractional resurfacing patients underwent split face trial: (1) ½ face PRP injection (2) ½ face Saline injection 14 patients PRP enhanced recovery and scar appearance when compared to saline Na et al24 Clinical: postfractional CO2 recovery (FcxCR) Bilateral inner arm FcxCR followed by either PRP or saline injection 25 patients PRP after FxCR enhanced wound healing and recovery Jones et al7 Retrospective case review Patients with keloids received intraoperative PRP combined with postoperative superficial radiation therapy 49 patients with 50 ear keloids 94% nonrecurrence after 2 years PRP, platelet rich plasma; STSG, split thickness skin grafts. View Large Table 1. Studies Evaluating Effects of PRP on Scar Modulation Study Study design Study method No. of patients Results Cervelli et al20 Clinical: traumatic scars (1) Fat graft + PRP + laser (2) Fat graft + PRP (3) Laser alone 60 patients split into 3 groups Scar management: Triple therapy > Fat graft + PRP > Laser alone Eichler et al21 Retrospective questionnaire Port area scars treated with or without PRP 20 of 120 patients received PRP PRP patients > Non-PRP treated patients in reduced scar dissatisfaction score Klosova et al22 Clinical: burn scars PRP + split thickness skin graft 23 patients with 38 scars Viscoelastic properties: STSG + PRP > STSG alone Nofal et al5 Clinical study: acne scars (1) PRP injections (2) Skin needling (3) PRP + skin needling 45 patients split into 3 groups No single treatment significantly better than the other Lee et al23 Clinical: acne scars After receiving CO2 fractional resurfacing patients underwent split face trial: (1) ½ face PRP injection (2) ½ face Saline injection 14 patients PRP enhanced recovery and scar appearance when compared to saline Na et al24 Clinical: postfractional CO2 recovery (FcxCR) Bilateral inner arm FcxCR followed by either PRP or saline injection 25 patients PRP after FxCR enhanced wound healing and recovery Jones et al7 Retrospective case review Patients with keloids received intraoperative PRP combined with postoperative superficial radiation therapy 49 patients with 50 ear keloids 94% nonrecurrence after 2 years Study Study design Study method No. of patients Results Cervelli et al20 Clinical: traumatic scars (1) Fat graft + PRP + laser (2) Fat graft + PRP (3) Laser alone 60 patients split into 3 groups Scar management: Triple therapy > Fat graft + PRP > Laser alone Eichler et al21 Retrospective questionnaire Port area scars treated with or without PRP 20 of 120 patients received PRP PRP patients > Non-PRP treated patients in reduced scar dissatisfaction score Klosova et al22 Clinical: burn scars PRP + split thickness skin graft 23 patients with 38 scars Viscoelastic properties: STSG + PRP > STSG alone Nofal et al5 Clinical study: acne scars (1) PRP injections (2) Skin needling (3) PRP + skin needling 45 patients split into 3 groups No single treatment significantly better than the other Lee et al23 Clinical: acne scars After receiving CO2 fractional resurfacing patients underwent split face trial: (1) ½ face PRP injection (2) ½ face Saline injection 14 patients PRP enhanced recovery and scar appearance when compared to saline Na et al24 Clinical: postfractional CO2 recovery (FcxCR) Bilateral inner arm FcxCR followed by either PRP or saline injection 25 patients PRP after FxCR enhanced wound healing and recovery Jones et al7 Retrospective case review Patients with keloids received intraoperative PRP combined with postoperative superficial radiation therapy 49 patients with 50 ear keloids 94% nonrecurrence after 2 years PRP, platelet rich plasma; STSG, split thickness skin grafts. View Large Table 2. Studies Demonstrating and Supporting PRP/BMP’s Ability to Induce Myofibroblastic Dedifferentiation Study Study type Result Plikus et al17 Basic science BMP-2 and BMP-4 strongly expressed and induced myofibroblast dedifferentiation and redifferentiation into adipocytes. BMP-4 converted keloid scar to lipid containing adipocytes Anitua et al25 Basic science Gingival fibroblasts exposed to plasma rich in growth factors showed enhanced growth and expression of VEGF, hepatocyte growth factor, hyaluronic acid. Myofibroblast phenotype was inhibited and reverted with plasma rich in growth factors Anitua et al26 Basic science Platelets obtained from young and middle-aged donors reduced myofibroblast differentiation rate and decreased myofibroblast levels Anitua et al28 Basic science Plasma rich in growth factors stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-β1 induced myodifferentiation Anitua et al27 Basic science Plasma rich in growth factors significantly reduced the myofibroblastic phenotype and accelerated corneal wound healing compared to controls Izumi et al29 Basic science BMP-7 decreased TGF-β dependent fibrogenic activity in mouse pulmonary myofibroblastic cells Liang et al30 Basic science BMP-7 antagonized TGF-β signaling pathways and suppressed silica-induced pulmonary fibrosis Bin et al31 Basic science BMP-7 antagonized TGF-β signaling and inhibited fibroblast morphology in dermal papilla cells Midgley et al19 Basic science BMP-7 exposure reversed myofibroblast differentiation Yano et al32 Basic science BMP-6 participates in progressive renal fibrosis through the development of myofibroblasts in relation with TGF-β Sharma et al33 Basic science Treated with a combination of BMP-6, BMP-7 and hormone cocktail stimulated skeletal muscle precursor cells to express genes found in brown preadipocytes Krause et al34 Basic science BMP-6 reduced TGF-β expression in Dupuytren’s fibroblasts. Treatment with TGF-β receptor kinase inhibitor and BMP-6 led to decreased contractility of Dupuytren’s fibroblasts. Kim et al35 Basic science Scar tissue fibroblasts expressed GREM1 a competitive antagonist of BMP Study Study type Result Plikus et al17 Basic science BMP-2 and BMP-4 strongly expressed and induced myofibroblast dedifferentiation and redifferentiation into adipocytes. BMP-4 converted keloid scar to lipid containing adipocytes Anitua et al25 Basic science Gingival fibroblasts exposed to plasma rich in growth factors showed enhanced growth and expression of VEGF, hepatocyte growth factor, hyaluronic acid. Myofibroblast phenotype was inhibited and reverted with plasma rich in growth factors Anitua et al26 Basic science Platelets obtained from young and middle-aged donors reduced myofibroblast differentiation rate and decreased myofibroblast levels Anitua et al28 Basic science Plasma rich in growth factors stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-β1 induced myodifferentiation Anitua et al27 Basic science Plasma rich in growth factors significantly reduced the myofibroblastic phenotype and accelerated corneal wound healing compared to controls Izumi et al29 Basic science BMP-7 decreased TGF-β dependent fibrogenic activity in mouse pulmonary myofibroblastic cells Liang et al30 Basic science BMP-7 antagonized TGF-β signaling pathways and suppressed silica-induced pulmonary fibrosis Bin et al31 Basic science BMP-7 antagonized TGF-β signaling and inhibited fibroblast morphology in dermal papilla cells Midgley et al19 Basic science BMP-7 exposure reversed myofibroblast differentiation Yano et al32 Basic science BMP-6 participates in progressive renal fibrosis through the development of myofibroblasts in relation with TGF-β Sharma et al33 Basic science Treated with a combination of BMP-6, BMP-7 and hormone cocktail stimulated skeletal muscle precursor cells to express genes found in brown preadipocytes Krause et al34 Basic science BMP-6 reduced TGF-β expression in Dupuytren’s fibroblasts. Treatment with TGF-β receptor kinase inhibitor and BMP-6 led to decreased contractility of Dupuytren’s fibroblasts. Kim et al35 Basic science Scar tissue fibroblasts expressed GREM1 a competitive antagonist of BMP BMP, bone morphogenic proteins; PRP, platelet rich plasma; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. View Large Table 2. Studies Demonstrating and Supporting PRP/BMP’s Ability to Induce Myofibroblastic Dedifferentiation Study Study type Result Plikus et al17 Basic science BMP-2 and BMP-4 strongly expressed and induced myofibroblast dedifferentiation and redifferentiation into adipocytes. BMP-4 converted keloid scar to lipid containing adipocytes Anitua et al25 Basic science Gingival fibroblasts exposed to plasma rich in growth factors showed enhanced growth and expression of VEGF, hepatocyte growth factor, hyaluronic acid. Myofibroblast phenotype was inhibited and reverted with plasma rich in growth factors Anitua et al26 Basic science Platelets obtained from young and middle-aged donors reduced myofibroblast differentiation rate and decreased myofibroblast levels Anitua et al28 Basic science Plasma rich in growth factors stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-β1 induced myodifferentiation Anitua et al27 Basic science Plasma rich in growth factors significantly reduced the myofibroblastic phenotype and accelerated corneal wound healing compared to controls Izumi et al29 Basic science BMP-7 decreased TGF-β dependent fibrogenic activity in mouse pulmonary myofibroblastic cells Liang et al30 Basic science BMP-7 antagonized TGF-β signaling pathways and suppressed silica-induced pulmonary fibrosis Bin et al31 Basic science BMP-7 antagonized TGF-β signaling and inhibited fibroblast morphology in dermal papilla cells Midgley et al19 Basic science BMP-7 exposure reversed myofibroblast differentiation Yano et al32 Basic science BMP-6 participates in progressive renal fibrosis through the development of myofibroblasts in relation with TGF-β Sharma et al33 Basic science Treated with a combination of BMP-6, BMP-7 and hormone cocktail stimulated skeletal muscle precursor cells to express genes found in brown preadipocytes Krause et al34 Basic science BMP-6 reduced TGF-β expression in Dupuytren’s fibroblasts. Treatment with TGF-β receptor kinase inhibitor and BMP-6 led to decreased contractility of Dupuytren’s fibroblasts. Kim et al35 Basic science Scar tissue fibroblasts expressed GREM1 a competitive antagonist of BMP Study Study type Result Plikus et al17 Basic science BMP-2 and BMP-4 strongly expressed and induced myofibroblast dedifferentiation and redifferentiation into adipocytes. BMP-4 converted keloid scar to lipid containing adipocytes Anitua et al25 Basic science Gingival fibroblasts exposed to plasma rich in growth factors showed enhanced growth and expression of VEGF, hepatocyte growth factor, hyaluronic acid. Myofibroblast phenotype was inhibited and reverted with plasma rich in growth factors Anitua et al26 Basic science Platelets obtained from young and middle-aged donors reduced myofibroblast differentiation rate and decreased myofibroblast levels Anitua et al28 Basic science Plasma rich in growth factors stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-β1 induced myodifferentiation Anitua et al27 Basic science Plasma rich in growth factors significantly reduced the myofibroblastic phenotype and accelerated corneal wound healing compared to controls Izumi et al29 Basic science BMP-7 decreased TGF-β dependent fibrogenic activity in mouse pulmonary myofibroblastic cells Liang et al30 Basic science BMP-7 antagonized TGF-β signaling pathways and suppressed silica-induced pulmonary fibrosis Bin et al31 Basic science BMP-7 antagonized TGF-β signaling and inhibited fibroblast morphology in dermal papilla cells Midgley et al19 Basic science BMP-7 exposure reversed myofibroblast differentiation Yano et al32 Basic science BMP-6 participates in progressive renal fibrosis through the development of myofibroblasts in relation with TGF-β Sharma et al33 Basic science Treated with a combination of BMP-6, BMP-7 and hormone cocktail stimulated skeletal muscle precursor cells to express genes found in brown preadipocytes Krause et al34 Basic science BMP-6 reduced TGF-β expression in Dupuytren’s fibroblasts. Treatment with TGF-β receptor kinase inhibitor and BMP-6 led to decreased contractility of Dupuytren’s fibroblasts. Kim et al35 Basic science Scar tissue fibroblasts expressed GREM1 a competitive antagonist of BMP BMP, bone morphogenic proteins; PRP, platelet rich plasma; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. View Large Table 3. Studies Demonstrating That BMP Can Be Extracted From PRP Study PRP type Results Schmidmaier et al37 Activated PRP vs PPP (platelet poor plasma) Levels of growth factors in PRP were significantly higher than in PPP Betsch et al38 Activated PRP vs plasma Thrombin activated PRP contained significantly higher levels of BMP-2 and BMP-7 than plasma alone Jungbluth et al39 Activated PRP vs plasma Thrombin activated PRP had a 17.6-fold increase in BMP-2 and 1.5-fold increase in BMP-7 compared to plasma alone Osada et al40 Activated PRP Podoplanin and C-type lectin-like receptor 2 activated PRP released BMP-9 Arslan et al41 Activated PRP PRP activated by a commercial PRP kit had high levels of BMP-2,4 and 7 Wahlstrom et al42 PRP activated in acidic buffer PRP activated in acidic buffers had significantly greater levels of PDGF, TGF-β, VEGF, and BMP-2 in a pH dependent manner and compared to those exposed to neutral buffers. Additionally, BMP-2 was only detected at pH 4.3. Kalen et al43 PRP activated in acidic buffer Acidic preparation (pH 4.3) of PRP released significantly more BMP-2 and BMP-4 compared to neutral buffer (pH 7.4) Kruger et al44 Activated PRP Range of BMP-2,4,6, and 7 levels detected amongst different PRP donors Strandberg et al45 PRP exposed to freeze-thaw cycles BMP-2 levels did not significantly change with multiple freeze-thaw cycles of platelet lysate Sellberg et al46 PRP exposed to freeze-thaw cycles BMP-2 levels were not significantly different between fresh platelets and platelets that underwent freeze-thaw and subsequent storage at room temperature Study PRP type Results Schmidmaier et al37 Activated PRP vs PPP (platelet poor plasma) Levels of growth factors in PRP were significantly higher than in PPP Betsch et al38 Activated PRP vs plasma Thrombin activated PRP contained significantly higher levels of BMP-2 and BMP-7 than plasma alone Jungbluth et al39 Activated PRP vs plasma Thrombin activated PRP had a 17.6-fold increase in BMP-2 and 1.5-fold increase in BMP-7 compared to plasma alone Osada et al40 Activated PRP Podoplanin and C-type lectin-like receptor 2 activated PRP released BMP-9 Arslan et al41 Activated PRP PRP activated by a commercial PRP kit had high levels of BMP-2,4 and 7 Wahlstrom et al42 PRP activated in acidic buffer PRP activated in acidic buffers had significantly greater levels of PDGF, TGF-β, VEGF, and BMP-2 in a pH dependent manner and compared to those exposed to neutral buffers. Additionally, BMP-2 was only detected at pH 4.3. Kalen et al43 PRP activated in acidic buffer Acidic preparation (pH 4.3) of PRP released significantly more BMP-2 and BMP-4 compared to neutral buffer (pH 7.4) Kruger et al44 Activated PRP Range of BMP-2,4,6, and 7 levels detected amongst different PRP donors Strandberg et al45 PRP exposed to freeze-thaw cycles BMP-2 levels did not significantly change with multiple freeze-thaw cycles of platelet lysate Sellberg et al46 PRP exposed to freeze-thaw cycles BMP-2 levels were not significantly different between fresh platelets and platelets that underwent freeze-thaw and subsequent storage at room temperature BMP, bone morphogenic proteins; PDGF, platelet-derived growth factor; PPP, platelet poor plasma; PRP, platelet rich plasma; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. View Large Table 3. Studies Demonstrating That BMP Can Be Extracted From PRP Study PRP type Results Schmidmaier et al37 Activated PRP vs PPP (platelet poor plasma) Levels of growth factors in PRP were significantly higher than in PPP Betsch et al38 Activated PRP vs plasma Thrombin activated PRP contained significantly higher levels of BMP-2 and BMP-7 than plasma alone Jungbluth et al39 Activated PRP vs plasma Thrombin activated PRP had a 17.6-fold increase in BMP-2 and 1.5-fold increase in BMP-7 compared to plasma alone Osada et al40 Activated PRP Podoplanin and C-type lectin-like receptor 2 activated PRP released BMP-9 Arslan et al41 Activated PRP PRP activated by a commercial PRP kit had high levels of BMP-2,4 and 7 Wahlstrom et al42 PRP activated in acidic buffer PRP activated in acidic buffers had significantly greater levels of PDGF, TGF-β, VEGF, and BMP-2 in a pH dependent manner and compared to those exposed to neutral buffers. Additionally, BMP-2 was only detected at pH 4.3. Kalen et al43 PRP activated in acidic buffer Acidic preparation (pH 4.3) of PRP released significantly more BMP-2 and BMP-4 compared to neutral buffer (pH 7.4) Kruger et al44 Activated PRP Range of BMP-2,4,6, and 7 levels detected amongst different PRP donors Strandberg et al45 PRP exposed to freeze-thaw cycles BMP-2 levels did not significantly change with multiple freeze-thaw cycles of platelet lysate Sellberg et al46 PRP exposed to freeze-thaw cycles BMP-2 levels were not significantly different between fresh platelets and platelets that underwent freeze-thaw and subsequent storage at room temperature Study PRP type Results Schmidmaier et al37 Activated PRP vs PPP (platelet poor plasma) Levels of growth factors in PRP were significantly higher than in PPP Betsch et al38 Activated PRP vs plasma Thrombin activated PRP contained significantly higher levels of BMP-2 and BMP-7 than plasma alone Jungbluth et al39 Activated PRP vs plasma Thrombin activated PRP had a 17.6-fold increase in BMP-2 and 1.5-fold increase in BMP-7 compared to plasma alone Osada et al40 Activated PRP Podoplanin and C-type lectin-like receptor 2 activated PRP released BMP-9 Arslan et al41 Activated PRP PRP activated by a commercial PRP kit had high levels of BMP-2,4 and 7 Wahlstrom et al42 PRP activated in acidic buffer PRP activated in acidic buffers had significantly greater levels of PDGF, TGF-β, VEGF, and BMP-2 in a pH dependent manner and compared to those exposed to neutral buffers. Additionally, BMP-2 was only detected at pH 4.3. Kalen et al43 PRP activated in acidic buffer Acidic preparation (pH 4.3) of PRP released significantly more BMP-2 and BMP-4 compared to neutral buffer (pH 7.4) Kruger et al44 Activated PRP Range of BMP-2,4,6, and 7 levels detected amongst different PRP donors Strandberg et al45 PRP exposed to freeze-thaw cycles BMP-2 levels did not significantly change with multiple freeze-thaw cycles of platelet lysate Sellberg et al46 PRP exposed to freeze-thaw cycles BMP-2 levels were not significantly different between fresh platelets and platelets that underwent freeze-thaw and subsequent storage at room temperature BMP, bone morphogenic proteins; PDGF, platelet-derived growth factor; PPP, platelet poor plasma; PRP, platelet rich plasma; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. View Large Table 4. Studies Showing Effects of PRP on Adipose Derived Stem Cells (ADSC) and Preadipocytes Study Study model Assay Result McLaughlin et al47 Basic science Growth of ADSCs + PRP vs ADSCs + 10% fetal bovine serum ADSCs + PRP combination showed increased ADSC proliferation, expression of BMP-4 and BMP-2, and reduced expression of PDGF-B and bFGF D’Esposito et al48 Basic science Studied ADSCs + PRP vs adipocytes + PRP Adding PRP increased ADSC viability and proliferation. PRP also enhanced secretion of pro-angiogenic factors from mature adipocytes facilitating tissue regeneration Liao et al49 Basic science ADSCs grown in general media vs adipogenic media supplemented with and without PRP PRP enhanced the proliferation of ADSCs even when grown in anti-proliferative, pro-adipogenic media. PRP inhibited adipogenic differentiation through downregulation of BMP Receptor 1A and FGF receptor 1 Seyhan et al50 Basic science Fat grafts combined with either PRP, ADSCs, or both Fat graft retention was significantly improved in rats who received grafts with PRP and ADSCs combined. Li et al51 Basic science Growth of ADSCs in PRPLipoinjection of granular fat, ADSCs and PRP into nude mice PRP enhanced ADSC proliferation and expression of adipogenic-related genes such as PPARγ, lipoprotein lipase and adipophilin. Granular fat grafts with 20% PRP supplementation had significantly improved residual volumes Li et al52 Basic science Fat grafts prepared with ADSCs at different densities with and without PRP Residual fat volume of grafts containing 105 /mL ADSC + PRP was significantly higher than other treatment conditions after 90 days. Increased adipocyte area and capillary formation was observed Fukaya et al53 Basic science Growth of preadipocytes in either PRP, fetal bovine serum (FBS) or platelet-poor plasma (PPP) PRP stimulated proliferation of preadipocytes in a dose-dependent fashion. Growth in 2% PRP was significantly higher than in 2% FBS or 2% PPP Chignon-Sicard et al54 Basic science Combined PRP + ADSCs PRP reduced the potential for ADSC to differentiate and led to generation of myofibroblast-like cells through TGF-β signaling Study Study model Assay Result McLaughlin et al47 Basic science Growth of ADSCs + PRP vs ADSCs + 10% fetal bovine serum ADSCs + PRP combination showed increased ADSC proliferation, expression of BMP-4 and BMP-2, and reduced expression of PDGF-B and bFGF D’Esposito et al48 Basic science Studied ADSCs + PRP vs adipocytes + PRP Adding PRP increased ADSC viability and proliferation. PRP also enhanced secretion of pro-angiogenic factors from mature adipocytes facilitating tissue regeneration Liao et al49 Basic science ADSCs grown in general media vs adipogenic media supplemented with and without PRP PRP enhanced the proliferation of ADSCs even when grown in anti-proliferative, pro-adipogenic media. PRP inhibited adipogenic differentiation through downregulation of BMP Receptor 1A and FGF receptor 1 Seyhan et al50 Basic science Fat grafts combined with either PRP, ADSCs, or both Fat graft retention was significantly improved in rats who received grafts with PRP and ADSCs combined. Li et al51 Basic science Growth of ADSCs in PRPLipoinjection of granular fat, ADSCs and PRP into nude mice PRP enhanced ADSC proliferation and expression of adipogenic-related genes such as PPARγ, lipoprotein lipase and adipophilin. Granular fat grafts with 20% PRP supplementation had significantly improved residual volumes Li et al52 Basic science Fat grafts prepared with ADSCs at different densities with and without PRP Residual fat volume of grafts containing 105 /mL ADSC + PRP was significantly higher than other treatment conditions after 90 days. Increased adipocyte area and capillary formation was observed Fukaya et al53 Basic science Growth of preadipocytes in either PRP, fetal bovine serum (FBS) or platelet-poor plasma (PPP) PRP stimulated proliferation of preadipocytes in a dose-dependent fashion. Growth in 2% PRP was significantly higher than in 2% FBS or 2% PPP Chignon-Sicard et al54 Basic science Combined PRP + ADSCs PRP reduced the potential for ADSC to differentiate and led to generation of myofibroblast-like cells through TGF-β signaling ADSC, adipose-derived stem cells; BMP, bone morphogenic proteins; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; PRP, platelet rich plasma; TGF, transforming growth factor. View Large Table 4. Studies Showing Effects of PRP on Adipose Derived Stem Cells (ADSC) and Preadipocytes Study Study model Assay Result McLaughlin et al47 Basic science Growth of ADSCs + PRP vs ADSCs + 10% fetal bovine serum ADSCs + PRP combination showed increased ADSC proliferation, expression of BMP-4 and BMP-2, and reduced expression of PDGF-B and bFGF D’Esposito et al48 Basic science Studied ADSCs + PRP vs adipocytes + PRP Adding PRP increased ADSC viability and proliferation. PRP also enhanced secretion of pro-angiogenic factors from mature adipocytes facilitating tissue regeneration Liao et al49 Basic science ADSCs grown in general media vs adipogenic media supplemented with and without PRP PRP enhanced the proliferation of ADSCs even when grown in anti-proliferative, pro-adipogenic media. PRP inhibited adipogenic differentiation through downregulation of BMP Receptor 1A and FGF receptor 1 Seyhan et al50 Basic science Fat grafts combined with either PRP, ADSCs, or both Fat graft retention was significantly improved in rats who received grafts with PRP and ADSCs combined. Li et al51 Basic science Growth of ADSCs in PRPLipoinjection of granular fat, ADSCs and PRP into nude mice PRP enhanced ADSC proliferation and expression of adipogenic-related genes such as PPARγ, lipoprotein lipase and adipophilin. Granular fat grafts with 20% PRP supplementation had significantly improved residual volumes Li et al52 Basic science Fat grafts prepared with ADSCs at different densities with and without PRP Residual fat volume of grafts containing 105 /mL ADSC + PRP was significantly higher than other treatment conditions after 90 days. Increased adipocyte area and capillary formation was observed Fukaya et al53 Basic science Growth of preadipocytes in either PRP, fetal bovine serum (FBS) or platelet-poor plasma (PPP) PRP stimulated proliferation of preadipocytes in a dose-dependent fashion. Growth in 2% PRP was significantly higher than in 2% FBS or 2% PPP Chignon-Sicard et al54 Basic science Combined PRP + ADSCs PRP reduced the potential for ADSC to differentiate and led to generation of myofibroblast-like cells through TGF-β signaling Study Study model Assay Result McLaughlin et al47 Basic science Growth of ADSCs + PRP vs ADSCs + 10% fetal bovine serum ADSCs + PRP combination showed increased ADSC proliferation, expression of BMP-4 and BMP-2, and reduced expression of PDGF-B and bFGF D’Esposito et al48 Basic science Studied ADSCs + PRP vs adipocytes + PRP Adding PRP increased ADSC viability and proliferation. PRP also enhanced secretion of pro-angiogenic factors from mature adipocytes facilitating tissue regeneration Liao et al49 Basic science ADSCs grown in general media vs adipogenic media supplemented with and without PRP PRP enhanced the proliferation of ADSCs even when grown in anti-proliferative, pro-adipogenic media. PRP inhibited adipogenic differentiation through downregulation of BMP Receptor 1A and FGF receptor 1 Seyhan et al50 Basic science Fat grafts combined with either PRP, ADSCs, or both Fat graft retention was significantly improved in rats who received grafts with PRP and ADSCs combined. Li et al51 Basic science Growth of ADSCs in PRPLipoinjection of granular fat, ADSCs and PRP into nude mice PRP enhanced ADSC proliferation and expression of adipogenic-related genes such as PPARγ, lipoprotein lipase and adipophilin. Granular fat grafts with 20% PRP supplementation had significantly improved residual volumes Li et al52 Basic science Fat grafts prepared with ADSCs at different densities with and without PRP Residual fat volume of grafts containing 105 /mL ADSC + PRP was significantly higher than other treatment conditions after 90 days. Increased adipocyte area and capillary formation was observed Fukaya et al53 Basic science Growth of preadipocytes in either PRP, fetal bovine serum (FBS) or platelet-poor plasma (PPP) PRP stimulated proliferation of preadipocytes in a dose-dependent fashion. Growth in 2% PRP was significantly higher than in 2% FBS or 2% PPP Chignon-Sicard et al54 Basic science Combined PRP + ADSCs PRP reduced the potential for ADSC to differentiate and led to generation of myofibroblast-like cells through TGF-β signaling ADSC, adipose-derived stem cells; BMP, bone morphogenic proteins; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; PRP, platelet rich plasma; TGF, transforming growth factor. View Large Study Design A PubMed literature search was conducted on July 5, 2017 for the following keywords after review and approval by an independent librarian (S.C): “BMP,” “BMPs,” “Bone Morphogenic Protein,” “Bone Morphogenic Protein 2,” “Bone Morphogenic Protein 4,” “Bone Morphogenic Protein 7,” “BMP 4,” “BMP 2,” and “BMP 7.” Keywords for the PRP search included: “PRP,” “Platelet Enriched Plasma,” “Platelet Rich Plasma,” “Platelets,” and “Platelet.” Key words for the scar modulation included: “Wound Healing,” “Skin Wound Healing,” “Myofibroblast,” “Adipogenesis,” “Adipocyte differentiation,” “Adipose derived stem cell,” “ADSC,” “Keloid,”, “Hypertrophic Scar,” and “Scar.” Four search cohorts were created using PRP and BMP terms (542 articles), PRP and scar modulation terms (165 articles), BMP and scar modulation terms (2090 articles), and PRP, BMP, and scar modulation combined (33 articles). In total 2830 abstracts where reviewed for inclusion criteria. For additional information see PRISMA diagram for compiled search groups (Figure 2). Figure 2. View largeDownload slide Flow diagram: 2830 articles were obtained from search terms. After reviewing all 2830 abstracts only 231 articles met the inclusion criteria for complete full-text assessment. After full-text assessment only 38 articles were found that met selection criteria. Figure 2. View largeDownload slide Flow diagram: 2830 articles were obtained from search terms. After reviewing all 2830 abstracts only 231 articles met the inclusion criteria for complete full-text assessment. After full-text assessment only 38 articles were found that met selection criteria. Selection Criteria and Data Collection All 2830 abstracts were assessed and selected if they demonstrated the following inclusion criteria: studies showing PRP contains BMP or evaluating the effects of PRP and BMP on either scar modulation, adipogenesis, or myofibroblast dedifferentiation. Studies were excluded if they described PRP or BMP use for orthopedic purposes. The full text of each article was read critically by a research assistant (Z.O., J.P., L.T.) to ensure each article met the selection criteria. Each summary and article were then read and cross-referenced for completeness (R.S). In total, 38 articles met the inclusion criteria and were included in the review. The Effect of PRP on Scar Modulation A few studies have been published evaluating the use of PRP for the treatment of scars. However, PRP activation and application has yet to be standardized, leading to a multitude of different treatment protocols with varying levels of efficacies. Additionally, PRP is often paired with other scar treatment modalities. Here, we review papers showing the positive effect of PRP on scar modulation. Cervelli et al randomly assigned 60 patients with traumatic scars to one of three groups: fat graft combined with PRP, nonablative laser resurfacing, or all three modalities combined.20 They found that the most effective scar treatment was fat grafting with PRP plus nonablative laser resurfacing (P < 0.05).20 The improvement in scar healing for treatments using the three modalities together was 22% greater than the fat graft and PRP group and was 11% greater than nonablative laser resurfacing alone.20 To test patient satisfaction with PRP-treated scars, Eichler et al administered a retrospective questionnaire to 120 patients who had their venous access device removed within six months and received either PRP or no treatment for their port area scar.21 The 20 patients who received PRP treatment showed a significant reduction in their desire to improve port area scarring and had reduced scar dissatisfaction (PRP: 10% vs control: 40.2% dissatisfaction, P < 0.05).21 Clinicians have also experimented with using PRP for skin grafts as an adjunctive therapy for the treatment of burns. For example, Klosova et al combined PRP with split thickness skin grafts (STSG) in patients who originally presented with scars from deep burns.22 They found that the viscoelastic properties of scars treated with STSG and PRP returned more rapidly than areas treated with STSG alone.22 Nofal et al treated 45 patients with either intradermal PRP injections, skin needling, or both treatments every two weeks for a six week period.5 All the groups showed a statistically significant improvement in the degree of acne scars when compared to control (P < 0.001), yet no single treatment was superior to the other.5 To study the effects of combining PRP and CO2 fractional resurfacing for acne scars, Lee et al subjected half of a patient’s face to PRP and the other half to saline injections post-CO2 fractional resurfacing.23 Erythema, edema, and posttreatment crusting all improved more rapidly with PRP treatment (P < 0.05).23 Similarly, Na et al subjected patients’ bilateral inner arms to PRP or a saline treatment postfractional CO2 laser resurfacing.24 They found that the treatment with PRP conferred several beneficial effects including a thicker epidermis, more organized stratum corneum and collagen fibers, and a higher collagen density compared to the control (P < 0.05).24 PRP has also been applied for keloid therapy. Jones et al conducted a retrospective analysis of 49 patients who received ear keloid surgery and were then treated postoperatively with PRP addition over the surgical site and superficial radiation therapy (SRT).7 They reported a 94% success rate, supporting the role of PRP in this combination therapy for the management of keloids.7 In summary, a few studies show that PRP treatment produces dermatological benefits when used alone or in combination with other therapeutic modalities. Many of the studies presented above are clinical assessments, with little attention placed on the components of PRP that may be producing these positive effects. Recent research indicates that BMP may be a significant contributor to the positive effects of PRP on scar modulation. BMP/PRP-Induced Myofibroblastic Dedifferentiation While clinical studies show that PRP may have an effect on scar modulation, very little attention has centered on the components in PRP that may be producing these effects. Recent investigations show that certain BMPs induce myofibroblastic dedifferentiation into preadipocytes, which is a potential explanation for the positive effect of PRP on scars. In a landmark study, Plikus et al utilized a murine model and demonstrated that myofibroblasts have the capacity to dedifferentiate into adipocytes during wound healing.17 Myofibroblast reprogramming was found to require neogenic hair follicles, which trigger BMP signaling and the activation of adipocyte transcription factors.17 Furthermore, the overexpression of a BMP antagonist (Noggin) in hair follicles, or the deletion of the BMP receptor in myofibroblasts, prevented adipocyte formation.17 The expression of BMP-2 and BMP-4 were found to be critical for myofibroblast reprogramming and transcriptomic data showed that BMP-2 and BMP-7 expression were upregulated, while the BMP antagonists, GREM1 and Bambi, were downregulated in this process.17 The upregulation of pSmad 1/5/8 in dermal cells adjacent to the regenerated hair follicles served as an indicator that BMP signaling was occurring.17 Additionally, when human-derived keloid cells were pretreated with BMP-4 (20 ng/mL) and exposed to adipogenic culture conditions, they consistently began adipogenic conversion.17 Thus, the possibility of fatty conversion of established scar tissue appears to be a real possibility. In 2012, Anitua et al studied the effects of activated platelets in gingival tissue regeneration and periodontal wound healing.25 In their preparation, PRP was anticoagulated with sodium citrate, activated with calcium chloride, and the leukocytes were removed.25 They found that activated PRP significantly increased gingival fibroblast proliferation, migration, and cell adhesion on a type I collagen matrix (P < 0.05).25 However, the myofibroblast phenotype, which severely impairs tissue function when extracellular matrix protein secretion becomes excessive, was inhibited and reverted with activated PRP treatment.25 Citing other studies, the authors hypothesized that the hepatocyte growth factor (HGF) found in the platelets might inhibit TGF-β1-induced myofibroblast differentiation.25 Similarly, in 2016, the same group studied the effects of TGF-β1-induced myofibroblast differentiation in dermal fibroblasts.26 Dermal fibroblasts isolated from healthy human skin were exposed to activated platelets obtained from four healthy, young patients and four healthy, middle-aged patients.26 Both the young and middle-aged donor platelets, even in the presence of TGF-β1, had reduced myofibroblast differentiation rates.26 Furthermore, the young and middle-aged PRP donor samples resulted in a, respectively, 23% ± 4% and 20% ± 1% decrease in myofibroblast levels (P < 0.05).26 In this study, TGF-β1, PDGF, epidermal growth factor (EGF), HGF, insulin growth factor (IGF), and basic fibroblast growth factor (bFGF) levels in the activated PRP from the two age groups were not significantly different.26 Finally, in two other studies, Anitua et al also showed that activated platelets reversed the myofibroblast phenotype in corneal fibroblast exposed to TGF-β1 (P < 0.05).27,28 Several other studies also examined the role of BMPs and their effects on the myofibroblast phenotype. Izumi et al studied both BMP-7 and TGF-β1 and showed that BMP-7 antagonizes the TGF-β1-dependent fibrogenic activity of mouse pulmonary myofibroblasts through the induction of the inhibitor of differentiation (Id) proteins Id2 and Id3 (P < 0.05).29 Through immunocytochemistry, they demonstrated that the ectopic expression of BMP-7 led to the nuclear localization of Phospho-Smad1/5/8 and the suppression of Smad3.29 Furthermore, BMP-7 suppressed the mRNA expression of COL1A2 (the TGF-β response element) and tiMMP2.29 Lastly, they found that BMP-7 increased Id 2 and 3, which suppresses COLIA2 promoter activity.29 In a similar study, Liang et al showed that BMP-7 inhibits silica-induced pulmonary fibrosis via the suppression of the TGF-β/Smad pathway (P < 0.05).11,29,30 Furthermore, Bin et al exposed dermal papilla cells (DPC) to 10 ng/mL TGF-β1 for 48 hours and found that they differentiated into cells with a fibroblast-like morphology (P < 0.05).31 However, all the changes were completely inhibited by BMP-7 treatment (P < 0.05).31 Bin et al concluded that hair follicle DPCs might play a role in wound healing by converting into fibroblasts via the TGF-β1 pathway.31 In addition, BMP-7, which is prominently expressed in the epidermis and papillary dermis and undergoes hair-cycle related changes in expression, halts fibrotic disease progression and promotes recovery by antagonizing TGF-β1-stimulated epithelial-mesenchymal transition.31 Finally, these authors hypothesized that the antagonistic effects of BMP-7 on the TGF-β1 pathway might suppress hypertrophic scar formation.31 In addition, Midgley et al tested the effect of recombinant BMP-7 on myofibroblasts.19 After exposing human lung fibroblast to TGF-β1 and confirming their differentiation into myofibroblasts using α-SMA, they demonstrated that 400 ng/mL of BMP-7 was required to reverse myofibroblast differentiation (P < 0.05).19 Several studies involving BMP signaling present contrasting results, and therefore, further understanding of the signaling pathway is necessary. Yano et al studied the role of BMP-6 in rat renal fibrosis. Treatment with BMP-6 increased α-SMA expression, which was analogous to the effects of TGF-β1 (P < 0.05).32 Collectively, BMP-6 was shown to participate in progressive renal fibrosis through the development of myofibroblasts in relation with TGF-β1.32 In contrast, Sharma et al found that when human skeletal muscle precursor cells were stimulated for two days with BMP-6 (205 ng/mL), followed by three days of an induction hormone cocktail, this was sufficient to induce a gene expression that was characteristic of brown preadipocytes (P < 0.05).33 A similar yet less pronounced effect was demonstrated using BMP-7 (P < 0.05).33 Moreover, Krause et al showed an increased TGF-β1 expression in Dupuytren’s disease fibroblasts compared to normal fibroblasts.34 Additionally, treatment with a TGF-β1 type I receptor inhibitor (SB-431542) and BMP-6 inhibited Smad and ERK1/2/MAP kinase signaling and decreased the contractility of Dupuytren’s fibroblasts (P < 0.05).34 Cotreatment of Dupuytren’s fibroblasts with a TGF-β1 inhibitor (SB-431542) and a MAP kinase 1 inhibitor (PD98059) attenuated the proliferation and contraction of Dupuytren’s-associated fibroblasts (P < 0.05).34 Given these findings, the researchers concluded that the TGF-β and ERK1/2 MAP kinase pathways are prime targets for nonsurgical intervention of Dupuytren’s disease.34 Finally, Kim et al found that scar tissue fibroblasts express GREM1, a competitive antagonist of BMP, and this expression was not observed in the dermis of normal skin (P < 0.05).35 The researchers concluded that GREM1 might serve as a marker for activated myofibroblasts in scars.35 In summary, convincing evidence exists demonstrating that BMP-2, 4, and 7, released by neogenic hair follicles, stimulate the dedifferentiation of myofibroblasts to preadipocytes. In addition, studies show that the TGF-β1-dependent conversion of fibroblasts to myofibroblast is reversed by BMP-7. This effect of BMP is thought to be produced by the intracellular signals SMAD 6 and 7 which inhibit the TGF-β1 pathway. In addition, the use of TGF-β1 inhibitors or BMP-6 alone is sufficient to reduce the contractility of Dupuytren’s-associated fibroblasts, creating a new avenue for nonsurgical treatment of the disease. Finally, GREM1, a competitive BMP antagonist, is expressed by fibroblasts in the dermis of scar tissue and may serve as a marker for persistent myofibroblasts PRP Contains Extractible BMP High concentrations of BMP (BMP-4: 20 ng/mL) induce the dedifferentiation of myofibroblasts, which raises the question of whether PRP alone contains enough extractable BMP to produce the desired effects. Whole blood, on its own, has detectable levels of BMP-1, BMP-2, BMP-3, BMP-4, BMP-6, BMP-7, and BMP-8.36 Here, we review studies that indicate the presence of BMP in PRP, how the preparation of PRP affects BMP release, and how levels of BMP are impacted by storage and free-thaw methods. Comparing PRP to platelet-poor plasma (PPP), some investigators show that PRP has levels of detectable growth factors, other than BMP-4, which are not detected in PPP.37 For example, BMP-2 concentrations are 1.9-fold higher in PRP than in PPP (0.03 ng/ml in PRP vs 0.015 ng/mL in PPP, P < 0.05).37 Studies investigating BMP levels in activated PRP are more common than those looking at BMP levels in nonactivated PRP. For example, Betsch et al demonstrated that the activation of PRP with thrombin yielded more BMP-2 and BMP-7 than in plasma alone ([BMP-2]: 167.7 ± 83.42 pg/mL in PRP vs 12.87 + 62.06 pg/mL in plasma; [BMP-7]: 178.86 + 128.98 pg/mL in PRP vs 95.856 + 37.28 pg/mL in plasma; P < 0.05).38 To further support these findings, Jungbluth et al found similar concentrations of BMP-2 and BMP-7 in thrombin-activated PRP from minipigs ([BMP-2]: 204.9 ± 180.4 pg/mL; [BMP-7]:125.2 ± 83.7 pg/mL; P < 0.05).39 This amounted to a 17.6- and 1.5-fold increase in BMP-2 and BMP-7, respectively, compared to plasma levels. However, they did note variations in the BMP concentrations between different minipig donors.39 Furthermore, Osada et al showed that platelets are activated via podoplanin and C-type lectin-like receptor 2 (CLEC-2) interactions which results in the release of BMP-9 (P < 0.05).40 Using the commercial advanced tissue regeneration kit (ATR) to activate PRP, Arslan et al obtained high concentrations of BMP-2, BMP-4, and BMP-7 from PRP (77.7 ± 9.58 pg/mL, 230 ± 48.9 pg/mL, and 3,059 ± 272 pg/mL, respectively; P < 0.05).41 Other studies also show that acidic preparations of PRP increase the BMP levels obtained from PRP.42,43 For example, Wahlstrom et al tested human PRP in buffers with pH values that ranged from 4.3 to 8.6 and found that only PRP incubated at pH 4.3 released BMP-2 (57 ng/mL, P < 0.05).42 In addition, Kalen et al found that PRP samples released significantly more BMP-2 (20 ng/L) and BMP-4 when they were lysed in an acidic buffer (pH 4.3) rather than in a neutral buffer (pH 7.4) (BMP-2, 32% vs 3%; BMP-4, 87% vs 52% released; P < 0.05).43 This group also found detectable levels of BMP-7 (1,536 ng/L) and BMP-6 (414-14,144 ng/L). However, no statistically significant difference was observed in the concentration of BMP-6 and BMP-7 in acidic vs neutral preparations.43 Similar to other studies, Kalen et al observed a considerable variation in the levels of the different BMPs released from different donors. For instance, only two of the 31 donors had detectable levels of all the four studied BMPs (P < 0.05).43 The heterogeneity among donors, with regard to the BMP levels in PRP, was replicated by Kruger et al who detected a range of BMP-2, BMP-4, BMP-6, and BMP-7 concentrations in 6 different donors.44 Interestingly, BMP-2 was present in only five of the six donors (average BMP-2, 0.31 ng/mL).44 Consequently, PRP activation methods as well as heterogeneity in BMPs and their levels amongst donors must be considered when using PRP alone as a therapeutic. Other studies investigated different preparation techniques in order to lengthen the storage time of PRP without affecting the relative concentrations of the growth factors. Strandberg et al found that after 1, 3, 5, 10, and 30 freeze-thaw cycles of the platelet lysate, the concentration of BMP-2 was not significantly decreased (196.0 ± 11.9 pg/mL at cycle 0 and 177.5 ± 8.7 pg/mL at cycle 30).45 In addition, Sellberg et al showed that the concentration of BMP-2 in fresh platelets was equivalent to those that underwent three freeze-thaw cycles and were subsequently stored at room temperature for five days (1250 pg/mL).46 In summary, the studies above clearly indicate that PRP contains variable concentrations of the different BMPs across various donors. Yet, the activation of PRP through different methods leads to a considerable increase in BMPs. For example, several studies indicate that acid may increase the amount of BMP-2 and BMP-4 released from PRP. Studies looking at the effects of freeze-thaw cycles for the purpose of PRP storage show no difference in BMP concentrations when compared to fresh isolates. Finally, although different levels of BMPs can be extracted to some degree from PRP, the levels may not be sufficient to produce scar myofibroblastic dedifferentiation, which requires nanograms of BMP per milliliter. Effect of PRP on Adipose Derived Stem Cells The levels of BMP extracted from PRP may not be enough alone to dedifferentiate myofibroblast into preadipocytes. However, several studies indicate that combining fat grafts containing adipose-derived stem cells (ADSCs) with PRP increases local BMP levels, affects ASDC commitment to the adipocyte lineage, and improves graft retention rates. Consequently, here, we review articles describing the effects of PRP on ADSCs. Only one study has investigated the expression of BMP-2 and 4 after adding activated PRP (platelet relesate) to ADSCs. McLaughlin et al demonstrated that the addition of PRP increased the proliferation and expression of BMP-4 (5.7 ± 0.97 fold increase) and BMP-2 (4.7 ± 1.3 fold increase) and reduced the expression of PDGF-β and bFGF in the ADSCs compared to ADSCs grown in 10% fetal bovine serum (P < 0.05).47 Furthermore, they found that the ADSCs retained their potential to differentiate into osteogenic, chondrogenic, and adipogenic cell lines.47 Consequently, activated PRP is thought to stimulate BMP-2 and 4 production from ADSCs, thereby increasing the concentration of these factors locally. Several groups have used PRP in combination with ADSCs, investigating its utility in soft tissue reconstruction. D’Esposito et al combined PRP with ADSCs to investigate its potential use for tissue regeneration.48 They found that PRP increased the viability, proliferation rate, and G1-S cell cycle progression of ADSCs, with higher PRP concentrations having larger effects (P < 0.05).48 In mature adipocytes, they found no change in PPARγ expression, cell viability and differentiation upon exposure to PRP.48 However, they did find an increase in the expression of leptin, angiogenesis, and the release of IL-6, 8, 10, Interferon-γ, and vascular endothelial growth factor (VEGF) in these mature adipocytes (P < 0.05).48 This was consistent with the stimulatory effect of the platelets’ contents on adipose tissue.48 Consequently, the researchers concluded that PRP may facilitate tissue regeneration because it stimulates the recruitment and proliferation of ADSCs without changing cell viability.48 The ability of PRP to stimulate ADSC proliferation is hypothesized to play a role in fat graft survival. Liao et al found that PRP significantly enhanced ADSC proliferation even when the ADSCs were grown in antiproliferative, proadipogenic media (P < 0.05).49 Interestingly, they found that PRP inhibited the adipogenic differentiation of ADSCs in this media across all the PRP concentrations tested.49 This effect was attributed to the downregulation of the major adipogenic mediating receptors bone morphogenetic protein receptor IA (BMPRIA) and fibroblast growth factor receptor 1 (FGFR1).49 Thus, when PRP is added to fat grafts, the ADSCs respond by undergoing proliferation rather than differentiation.49 As a result, the larger pool of ADSCs participate in hormone production and the subsequent differentiation into adipocytes after the PRP is reabsorbed.49 Consequently, one can hypothesize that when PRP is added to fat grafts, the resultant proliferation of the ADSCs and their production of hormones may increase the concentration of BMP-2 and 4 to the levels needed for myofibroblastic dedifferentiation. Seyhan et al studied fat graft retention in rats and found that those treated with both PRP and ADSCs had higher graft retention rates, as well as increased growth factors levels (VEGF, TGF-β, FGF), after 12 weeks when compared to the controls (P < 0.05).50 Similarly, Li et al tested cell viability and graft take after adding PRP at different concentrations (0%, 10%, 20%, and 30%) to the ADSC media.51 Here, PRP, ADSCs, and granular fat were combined and were grafted subcutaneously into nude mice and examined histologically at different time periods.51 They found that PRP improved ADSC proliferation and that the grafts with PRP added at concentrations of 20% and 30% showed significantly improved residual volumes (P < 0.05). However, when comparing the 20% and 30% PRP groups, no difference was observed in the residual volumes and histology.51 Considering the cost of PRP, the researchers suggested using 20% PRP with ADSCs for improving fat graft survival.51 In a separate study, Li et al found that grafts containing 105 ADSCs/mL with PRP were optimal for adipogenesis, and the residual fat volume was significantly higher than in the other treatment conditions (eg, 107, 106, 104, and 0 cells/mL ± PRP) after 90 days (P < 0.05).52 Similarly, Fukaya et al found that adding PRP to preadipocytes stimulated the proliferation of preadipocytes in a dose-dependent manner (P < 0.05).53 Furthermore, in a study performed by Chignon-Sicard et al, the effects of freeze-activated PRP on antagonizing ADSC proliferation was assessed.54 Here, PRP reduced the potential of ADSCs to undergo differentiation into adipocytes and, in fact, led to the generation of myofibroblast-like cells (P < 0.05).54 The researchers concluded that the TGF-β found in PRP played a critical role in the promyofibroblastic and antiadipogenic fate of the ADSCs.54 Here, again, emphasis must be placed on the PRP preparation technique (activated vs nonactivated, method of activation, and the presence or removal of leukocytes) in order to understand its effects on ADSCs. In summary, the studies above indicate that when PRP is combined with fat grafts, ADSCs are stimulated, showing an increased expression of BMP-2 and 4 by roughly 5-fold. In addition, the rapid proliferation of ADSCs and an improved fat graft retention is also observed with the addition of PRP to ADSCs. Additional studies are needed to quantify the levels of BMP-2 and 4 produced by PRP-stimulated ADSCs to assess whether the concentrations needed to produce myofibroblastic dedifferentiation can be achieved. CONCLUSION In developed countries, over 100 million people per year form scars, of which 30% become hypertrophic or keloid scars due to abnormal wound healing.55 Patients who suffer from these abnormalities not only have aesthetic, social, and psychological impairments but can also have a reduced quality of life.55 Hypertrophic scars, which are visible, elevated, and do not spread into surrounding tissues, contain an increased amount of myofibroblasts that fail to undergo normal apoptosis. In addition, they have a reduced or complete lack of cutaneous fat and growing hair follicles, which may contribute to their pathophysiology.55 Research demonstrates that actively growing hair follicles locally secrete BMPs, which induce myofibroblast dedifferentiation and reprogramming into adipocytes.17,30,31,56 Furthermore, BMPs induce adipogenesis from local ADSC sources, promote angiogenesis, reduce inflammation, and stimulate proper wound healing.18,57-59 However, the lack of hair follicles and, thus, the reduced local BMP levels in hypertrophic scar tissue prevents many of these beneficial events from occurring.55 To address the lack of local BMP in hypertrophic scars, we sought to investigate the literature for natural sources of BMP-2 and 4, which are implicated in myofibroblast dedifferentiation, and would bypass the necessity to produce them via recombinant techniques. Through our systematic review, we found studies showing that BMPs can be obtained from PRP following various activation techniques, yet evidence of whether the released amounts are high enough to produce myofibroblastic dedifferentiation (BMP-4, 20ng/ml) is yet to be achieved. While PRP alone may not provide a sufficient source of BMP for therapeutic applications, PRP stimulates fat grafts containing ADSCs to produce a five-fold increase in expression of BMP-2 and 4. Taken together, we hypothesize that PRP, which serves as both a natural reservoir for BMP and as a stimulus for ADSC-derived BMP production, combined with fat grafts, may offer a new treatment modality for hypertrophic scars and contractures (Figure 3). The beneficial effects of this treatment on hypertrophic scars is postulated to be achieved through the ability of BMP-2 and BMP-4 to dedifferentiate and reprogram persistently contracted myofibroblasts into adipocytes. Figure 3. View largeDownload slide Activated PRP when combined with adipose derived stem cells in fat grafts may synergistically increase BMP-2, 4, and 7 levels to the thresholds shown to dedifferentiate hypertrophic scar myofibroblasts into preadipocytes. Figure 3. View largeDownload slide Activated PRP when combined with adipose derived stem cells in fat grafts may synergistically increase BMP-2, 4, and 7 levels to the thresholds shown to dedifferentiate hypertrophic scar myofibroblasts into preadipocytes. Since we propose using activated PRP as source of BMP, several important limitations may exist which include, but are not limited to, unwanted osteogenesis, chondrogenesis, neurogenesis, or neoplasm formation.60-63 These adverse effects might occur based on many factors, including the type of BMP produced, the target cell and its expressed receptors, and the local environment including other growth factors in addition to the delivered BMP.11,13,64 However, as the studies in our review show, PRP added to a fat graft mixture should preferentially stimulate ADSCs to undergo proliferation and adipogenesis, avoiding these unwanted effects. Another important consideration is the lack of standardization in the PRP preparation methods, with many different techniques producing quite variable results.5 Using PRP kits with large platelet multipliers may prove most beneficial as they maximize the platelet numbers and, thus, the released growth factors. Many authors suggest PRP protocols aiming to set a standard, but unfortunately there is still no consensus on the best technique.2 There is also considerable heterogeneity in the concentration of platelets in the patients’ blood. Consequently, a range of platelet concentrations are often obtained in PRP between donors prepared with the same protocol.2 To make matters more complicated, not every patient’s platelets contain the same amount or type of BMPs. One study on a cohort of 31 donors demonstrated that only two patients contained all four studied BMPs (2, 4, 6, and 7) in their platelets, and another two patients contained no detectable BMPs.43 This issue may contribute to the variable results seen in previous PRP studies and serve as significant limitation. To avoid these issues and ineffective therapy, researchers can screen a patient’s platelet-derived BMPs and levels prior to treatment. Additionally, fat grafts with ADSCs plus PRP provide a mechanism for overcoming BMP deficiencies via de novo ADSC BMP production. Unfortunately, nonactivated and leukocyte-rich PRP stimulate the formation of the myofibroblast phenotype. To address these issues, protocols for preparing pure PRP (P-PRP) instead of leukocyte-rich PRP (L-PRP) can be utilized to minimize the release of the antiadipogenic, proinflammatory molecules, such as TGF-β and TNF-α, found in leukocytes.65,66 In addition, activating PRP in an acidic environment maximizes the BMP yield and serves as the only way to release a detectable amount of BMP-2.42,43 Finally, it is desirable to examine different methods of activating PRP to assess whether even higher BMP levels can be achieved. The pathophysiology of hypertrophic scarring lies in the existence of persistently present and contracted myofibroblasts. The management of hypertrophic scarring through surgical and nonsurgical techniques have yet to fully address this underlying pathology. Our laboratory, The Center for Tissue Engineering at UC Irvine, is currently working on studying the effects of combining PRP and fat grafts as a novel injectable for dedifferentiating myofibroblasts in hypertrophic scars. Disclosures The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding The authors received no financial support for the research, authorship, and publication of this article. REFERENCES 1. Lynch MD , Bashir S . Applications of platelet-rich plasma in dermatology: a critical appraisal of the literature . J Dermatolog Treat . 2016 ; 27 ( 3 ): 285 - 289 . Google Scholar Crossref Search ADS PubMed 2. Dhurat R , Sukesh M . Principles and methods of preparation of platelet-rich plasma: a review and author’s perspective . 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Hyaluronan regulates bone morphogenetic protein-7-dependent prevention and reversal of myofibroblast phenotype . J Biol Chem . 2015 ; 290 ( 18 ): 11218 - 11234 . Google Scholar Crossref Search ADS PubMed 20. Cervelli V , Nicoli F , Spallone D , et al. Treatment of traumatic scars using fat grafts mixed with platelet-rich plasma, and resurfacing of skin with the 1540 nm nonablative laser . Clin Exp Dermatol . 2012 ; 37 ( 1 ): 55 - 61 . Google Scholar Crossref Search ADS PubMed 21. Eichler C , Najafpour M , Sauerwald A , Puppe J , Warm M . Platelet-rich plasma in the treatment of subcutaneous venous access device scars: a head-to-head patient survey . Biomed Res Int . 2015 ; 2015 : 630601 . Google Scholar Crossref Search ADS PubMed 22. Klosová H , Stětinský J , Bryjová I , Hledík S , Klein L . Objective evaluation of the effect of autologous platelet concentrate on post-operative scarring in deep burns . Burns . 2013 ; 39 ( 6 ): 1263 - 1276 . 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Plasma rich in growth factors enhances wound healing and protects from photo-oxidative stress in dermal fibroblasts and 3D skin models . Curr Pharm Biotechnol . 2016 ; 17 ( 6 ): 556 - 570 . Google Scholar Crossref Search ADS PubMed 27. Anitua E , Muruzabal F , Alcalde I , Merayo-Lloves J , Orive G . Plasma rich in growth factors (PRGF-Endoret) stimulates corneal wound healing and reduces haze formation after PRK surgery . Exp Eye Res . 2013 ; 115 : 153 - 161 . Google Scholar Crossref Search ADS PubMed 28. Anitua E , Sanchez M , Merayo-Lloves J , De la Fuente M , Muruzabal F , Orive G . Plasma rich in growth factors (PRGF-Endoret) stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-beta1-Induced myodifferentiation . Invest Ophthalmol Vis Sci . 2011 ; 52 ( 9 ): 6066 - 6073 . Google Scholar Crossref Search ADS PubMed 29. Izumi N , Mizuguchi S , Inagaki Y , et al. BMP-7 opposes TGF-beta1-mediated collagen induction in mouse pulmonary myofibroblasts through Id2 . Am J Physiol Lung Cell Mol Physiol . 2006 ; 290 ( 1 ): L120 - L126 . Google Scholar Crossref Search ADS PubMed 30. Liang D , Wang Y , Zhu Z , et al. BMP-7 attenuated silica-induced pulmonary fibrosis through modulation of the balance between TGF-β/Smad and BMP-7/Smad signaling pathway . Chem Biol Interact . 2016 ; 243 : 72 - 81 . Google Scholar Crossref Search ADS PubMed 31. Bin S , Li HD , Xu YB , et al. BMP-7 attenuates TGF-β1-induced fibroblast-like differentiation of rat dermal papilla cells . Wound Repair Regen . 2013 ; 21 ( 2 ): 275 - 281 . Google Scholar Crossref Search ADS PubMed 32. Yano R , Golbar HM , Izawa T , Sawamoto O , Kuwamura M , Yamate J . Participation of bone morphogenetic protein (BMP)-6 and osteopontin in cisplatin (CDDP)-induced rat renal fibrosis . Exp Toxicol Pathol . 2015 ; 67 ( 2 ): 99 - 107 . Google Scholar Crossref Search ADS PubMed 33. Sharma A , Huard C , Vernochet C , et al. Brown fat determination and development from muscle precursor cells by novel action of bone morphogenetic protein 6 . PLoS One . 2014 ; 9 ( 3 ): e92608 . Google Scholar Crossref Search ADS PubMed 34. Krause C , Kloen P , Ten Dijke P . Elevated transforming growth factor β and mitogen-activated protein kinase pathways mediate fibrotic traits of Dupuytren’s disease fibroblasts . Fibrogenesis Tissue Repair . 2011 ; 4 ( 1 ): 14 . Google Scholar Crossref Search ADS PubMed 35. Kim HS , Shin MS , Cheon MS , et al. GREM1 is expressed in the cancer-associated myofibroblasts of basal cell carcinomas . PLoS One . 2017 ; 12 ( 3 ): e0174565 . Google Scholar Crossref Search ADS PubMed 36. Smiler DG , Soltan M , Soltan C , Matthews C . Growth factors and gene expression of stem cells: bone marrow compared with peripheral blood . Implant Dent . 2010 ; 19 ( 3 ): 229 - 240 . Google Scholar Crossref Search ADS PubMed 37. 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Strandberg G , Sellberg F , Sommar P , et al. Standardizing the freeze-thaw preparation of growth factors from platelet lysate . Transfusion . 2017 ; 57 ( 4 ): 1058 - 1065 . Google Scholar Crossref Search ADS PubMed 46. Sellberg F , Berglund E , Ronaghi M , et al. Composition of growth factors and cytokines in lysates obtained from fresh versus stored pathogen-inactivated platelet units . Transfus Apher Sci . 2016 ; 55 ( 3 ): 333 - 337 . Google Scholar Crossref Search ADS PubMed 47. McLaughlin M , Gagnet P , Cunningham E , et al. Allogeneic platelet releasate preparations derived via a novel rapid thrombin activation process promote rapid growth and increased BMP-2 and BMP-4 expression in human adipose-derived stem cells . Stem Cells Int . 2016 ; 2016 : 7183734 . Google Scholar Crossref Search ADS PubMed 48. D’Esposito V , Passaretti F , Perruolo G , et al. Platelet-rich plasma increases growth and motility of adipose tissue-derived mesenchymal stem cells and controls adipocyte secretory function . J Cell Biochem . 2015 ; 116 ( 10 ): 2408 - 2418 . Google Scholar Crossref Search ADS PubMed 49. Liao HT , James IB , Marra KG , Rubin JP . The effects of platelet-rich plasma on cell proliferation and adipogenic potential of adipose-derived stem cells . Tissue Eng Part A . 2015 ; 21 ( 21-22 ): 2714 - 2722 . Google Scholar Crossref Search ADS PubMed 50. Seyhan N , Alhan D , Ural AU , Gunal A , Avunduk MC , Savaci N . The effect of combined use of platelet-rich plasma and adipose-derived stem cells on fat graft survival . Ann Plast Surg . 2015 ; 74 ( 5 ): 615 - 620 . Google Scholar Crossref Search ADS PubMed 51. Li F , Guo W , Li K , et al. Improved fat graft survival by different volume fractions of platelet-rich plasma and adipose-derived stem cells . Aesthet Surg J . 2015 ; 35 ( 3 ): 319 - 333 . Google Scholar Crossref Search ADS PubMed 52. Li K , Li F , Li J , et al. Increased survival of human free fat grafts with varying densities of human adipose-derived stem cells and platelet-rich plasma . J Tissue Eng Regen Med . 2017 ; 11 ( 1 ): 209 - 219 . Google Scholar Crossref Search ADS PubMed 53. Fukaya Y , Kuroda M , Aoyagi Y , et al. Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system . Exp Mol Med . 2012 ; 44 ( 5 ): 330 - 339 . Google Scholar Crossref Search ADS PubMed 54. Chignon-Sicard B , Kouidhi M , Yao X , et al. Platelet-rich plasma respectively reduces and promotes adipogenic and myofibroblastic differentiation of human adipose-derived stromal cells via the TGFβ signalling pathway . Sci Rep . 2017 ; 7 ( 1 ): 2954 . Google Scholar Crossref Search ADS PubMed 55. Mokos ZB , Jović A , Grgurević L , et al. Current therapeutic approach to hypertrophic scars . Front Med (Lausanne) . 2017 ; 4 : 83 . Google Scholar Crossref Search ADS PubMed 56. Botchkarev VA . Bone morphogenetic proteins and their antagonists in skin and hair follicle biology . J Invest Dermatol . 2003 ; 120 ( 1 ): 36 - 47 . Google Scholar Crossref Search ADS PubMed 57. Wei X , Li G , Yang X , et al. Effects of bone morphogenetic protein-4 (BMP-4) on adipocyte differentiation from mouse adipose-derived stem cells . Cell Prolif . 2013 ; 46 ( 4 ): 416 - 424 . Google Scholar Crossref Search ADS PubMed 58. Muir AM , Massoudi D , Nguyen N , et al. BMP1-like proteinases are essential to the structure and wound healing of skin . Matrix Biol . 2016 ; 56 : 114 - 131 . Google Scholar Crossref Search ADS PubMed 59. François S , Eder VV , Belmokhtar K , et al. Synergistic effect of human Bone Morphogenic Protein-2 and Mesenchymal Stromal Cells on chronic wounds through hypoxia-inducible factor-1 α induction . Sci Rep . 2017 ; 7 ( 1 ): 4272 . Google Scholar Crossref Search ADS PubMed 60. Berner A , Boerckel JD , Saifzadeh S , et al. Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration . Cell Tissue Res . 2012 ; 347 ( 3 ): 603 - 612 . Google Scholar Crossref Search ADS PubMed 61. Li H , Han Z , Liu D , Zhao P , Liang S , Xu K . Autologous platelet-rich plasma promotes neurogenic differentiation of human adipose-derived stem cells in vitro . Int J Neurosci . 2013 ; 123 ( 3 ): 184 - 190 . Google Scholar Crossref Search ADS PubMed 62. Pountos I , Panteli M , Georgouli T , Giannoudis PV . Neoplasia following use of BMPs: is there an increased risk ? Expert Opin Drug Saf . 2014 ; 13 ( 11 ): 1525 - 1534 . Google Scholar Crossref Search ADS PubMed 63. Tang HC , Chen WC , Chiang CW , Chen LY , Chang YC , Chen CH . Differentiation effects of platelet-rich plasma concentrations on synovial fluid mesenchymal stem cells from pigs cultivated in alginate complex hydrogel . Int J Mol Sci . 2015 ; 16 ( 8 ): 18507 - 18521 . Google Scholar Crossref Search ADS PubMed 64. Lee SY , Lee JH , Kim JY , Bae YC , Suh KT , Jung JS . BMP2 increases adipogenic differentiation in the presence of dexamethasone, which is inhibited by the treatment of TNF-α in human adipose tissue-derived stromal cells . Cell Physiol Biochem . 2014 ; 34 ( 4 ): 1339 - 1350 . Google Scholar Crossref Search ADS PubMed 65. Zhou Y , Zhang J , Wu H , Hogan MV , Wang JH . The differential effects of leukocyte-containing and pure platelet-rich plasma (PRP) on tendon stem/progenitor cells - implications of PRP application for the clinical treatment of tendon injuries . Stem Cell Res Ther . 2015 ; 6 : 173 . Google Scholar Crossref Search ADS PubMed 66. Anitua E , Prado R , Orive G . Platelet-rich plasma and myofibroblasts: is the composition the key to success ? Adv Skin Wound Care . 2015 ; 28 ( 5 ): 198 - 199 . Google Scholar Crossref Search ADS PubMed © 2018 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Aesthetic Surgery Journal Oxford University Press

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Oxford University Press
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© 2018 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com
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Abstract

Abstract In developed countries, over 100 million people per year form scars, of which 30% become hypertrophic. These scars are defined histologically by the presence of persistently contractile myofibroblasts. Recent in vitro research suggests that certain bone morphogenic proteins (BMP) can induce scar myofibroblast dedifferentiation and reprogramming into adipocytes. Since platelets contain BMPs within their granules, it is possible that platelet-rich plasma (PRP) can act as a vehicle to deliver BMP to sites of scarring or potential scarring. Additionally, when PRP is mixed with fat graft tissue, synergistic adipogenic growth factors (including BMPs) are released which can help complete myofibroblast transformation and adipogenesis. The aim of this article is to corroborate these findings by systematically reviewing articles demonstrating the following concepts: (1) the effect of PRP on scar modulation; (2) the extraction of BMP from PRP; (3) BMP-induced myofibroblastic dedifferentiation; and (4) the effect of PRP on adipose-derived stem cells using the MEDLINE database. This search yielded 2830 articles, of which 38 met the inclusion criteria for this literature review. Level of Evidence: 4 [AU: Please provide some keywords] Platelet-rich plasma (PRP) is a personalized therapeutic procedure that involves centrifuging a patient’s blood and removing certain components in order to concentrate the number of platelets.1 In most cases, PRP is activated to release of growth factors from the intramembranous granules of the platelets and can then be utilized for a variety of therapeutic purposes.1 Platelet activation occurs when the intracellular granule fuses to the platelet membrane, and the contained growth factors undergo a final modification into an active state and are released.2 Under physiologic conditions, the content of these granules work as mediators in wound healing and hemostasis.2 Platelets contain both alpha and dense granules. Alpha granules store growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), and other members from the TGF-β superfamily. Dense granules contain bioactive molecules, such as serotonin, histamine, and adenosine, which modulate membrane permeability and local inflammation (Figure 1).3,4 Figure 1. View largeDownload slide Platelet containing alpha and dense granules and some of their bioactive contents. Figure 1. View largeDownload slide Platelet containing alpha and dense granules and some of their bioactive contents. PRP therapy is currently being used for many dermatological and nondermatological purposes, in tandem with other treatments, to enhance overall results.1 Cosmetically, PRP shows efficacy in treating alopecia, acne, traumatic scars, contractile scars, wrinkles, stretch marks, chronic ulcers, as well as enhancing laser resurfacing and postsurgical wound healing.1,3,5-7 For orthopedic purposes, PRP is used for spine fusions, osteoarthritis, cartilage, bone and tendon injuries, and various types of fractures.8 One recent study found that mixing PRP with percutaneous needle tenotomy relieved pain in a patient with De Quervain’s tenosynovitis for whom other treatments failed.9 Bone morphogenic proteins (BMPs), a subset of the TGF-β superfamily, are a group of growth factors found in PRP that were originally discovered and named for their role in cartilage and bone development.10 In addition, BMPs are implicated in a variety of embryological and physiological regulatory roles related to adipose tissue, such as adipocyte development and regulating brown/white adipocyte differentiation.10-12 There are at least 15 different BMPs, each uniquely effective at regulating the type and rate of stem cell differentiation into either adipogenic, chondrogenic, or osteogenic precursors.13 Their roles and effectiveness are based not only on the type of BMP and BMP receptor, but also on the local environment, including the presence of other signaling factors.13 BMP-4, BMP-7, and to a lesser degree BMP-2, are all potent adipogenic inducers of stem cells under the right conditions and are even necessary for proper adipocyte differentiation.14-16 Our laboratory, The Center for Tissue Engineering (CTE) at UC Irvine, collaborates with the Plikus lab at UC Irvine to study BMP. In a murine model of wound healing, it was demonstrated that actively growing hair follicles induce surrounding adipogenesis initiated by the dedifferentiation of myofibroblasts into adipocytes under BMP influence.17 Other studies show that BMPs, namely BMP-4 and 7, stall and reverse myofibroblast differentiation by antagonizing TGF-β promyofibroblastic signaling and inducing greater PPARγ expression.18,19 These findings support a possible role for BMPs in preventing and even reducing scarring by inducing adipogeneic transformation of myofibroblasts in scar tissue. Since platelets contain BMPs within their granules, it is possible that PRP acts as a vehicle to deliver BMP to sites of scarring or potential scarring. PRP may be particularly effective when mixed with fat graft tissue, which would provide the other synergistic adipogenic growth factors that BMP-4 and 7 lack to complete myofibroblast-induced adipogenesis. Here, we systematically review literature from studies indicating the following: (1) the effect of PRP on scar modulation; (2) the extraction of BMP from PRP; (3) BMP-induced myofibroblastic dedifferentiation; and (4) the effect of PRP on adipose-derived stem cells (ADSCs) (Tables 1-4). Finally, we propose a potentially new therapeutic approach for hypertrophic scarring. Table 1. Studies Evaluating Effects of PRP on Scar Modulation Study Study design Study method No. of patients Results Cervelli et al20 Clinical: traumatic scars (1) Fat graft + PRP + laser (2) Fat graft + PRP (3) Laser alone 60 patients split into 3 groups Scar management: Triple therapy > Fat graft + PRP > Laser alone Eichler et al21 Retrospective questionnaire Port area scars treated with or without PRP 20 of 120 patients received PRP PRP patients > Non-PRP treated patients in reduced scar dissatisfaction score Klosova et al22 Clinical: burn scars PRP + split thickness skin graft 23 patients with 38 scars Viscoelastic properties: STSG + PRP > STSG alone Nofal et al5 Clinical study: acne scars (1) PRP injections (2) Skin needling (3) PRP + skin needling 45 patients split into 3 groups No single treatment significantly better than the other Lee et al23 Clinical: acne scars After receiving CO2 fractional resurfacing patients underwent split face trial: (1) ½ face PRP injection (2) ½ face Saline injection 14 patients PRP enhanced recovery and scar appearance when compared to saline Na et al24 Clinical: postfractional CO2 recovery (FcxCR) Bilateral inner arm FcxCR followed by either PRP or saline injection 25 patients PRP after FxCR enhanced wound healing and recovery Jones et al7 Retrospective case review Patients with keloids received intraoperative PRP combined with postoperative superficial radiation therapy 49 patients with 50 ear keloids 94% nonrecurrence after 2 years Study Study design Study method No. of patients Results Cervelli et al20 Clinical: traumatic scars (1) Fat graft + PRP + laser (2) Fat graft + PRP (3) Laser alone 60 patients split into 3 groups Scar management: Triple therapy > Fat graft + PRP > Laser alone Eichler et al21 Retrospective questionnaire Port area scars treated with or without PRP 20 of 120 patients received PRP PRP patients > Non-PRP treated patients in reduced scar dissatisfaction score Klosova et al22 Clinical: burn scars PRP + split thickness skin graft 23 patients with 38 scars Viscoelastic properties: STSG + PRP > STSG alone Nofal et al5 Clinical study: acne scars (1) PRP injections (2) Skin needling (3) PRP + skin needling 45 patients split into 3 groups No single treatment significantly better than the other Lee et al23 Clinical: acne scars After receiving CO2 fractional resurfacing patients underwent split face trial: (1) ½ face PRP injection (2) ½ face Saline injection 14 patients PRP enhanced recovery and scar appearance when compared to saline Na et al24 Clinical: postfractional CO2 recovery (FcxCR) Bilateral inner arm FcxCR followed by either PRP or saline injection 25 patients PRP after FxCR enhanced wound healing and recovery Jones et al7 Retrospective case review Patients with keloids received intraoperative PRP combined with postoperative superficial radiation therapy 49 patients with 50 ear keloids 94% nonrecurrence after 2 years PRP, platelet rich plasma; STSG, split thickness skin grafts. View Large Table 1. Studies Evaluating Effects of PRP on Scar Modulation Study Study design Study method No. of patients Results Cervelli et al20 Clinical: traumatic scars (1) Fat graft + PRP + laser (2) Fat graft + PRP (3) Laser alone 60 patients split into 3 groups Scar management: Triple therapy > Fat graft + PRP > Laser alone Eichler et al21 Retrospective questionnaire Port area scars treated with or without PRP 20 of 120 patients received PRP PRP patients > Non-PRP treated patients in reduced scar dissatisfaction score Klosova et al22 Clinical: burn scars PRP + split thickness skin graft 23 patients with 38 scars Viscoelastic properties: STSG + PRP > STSG alone Nofal et al5 Clinical study: acne scars (1) PRP injections (2) Skin needling (3) PRP + skin needling 45 patients split into 3 groups No single treatment significantly better than the other Lee et al23 Clinical: acne scars After receiving CO2 fractional resurfacing patients underwent split face trial: (1) ½ face PRP injection (2) ½ face Saline injection 14 patients PRP enhanced recovery and scar appearance when compared to saline Na et al24 Clinical: postfractional CO2 recovery (FcxCR) Bilateral inner arm FcxCR followed by either PRP or saline injection 25 patients PRP after FxCR enhanced wound healing and recovery Jones et al7 Retrospective case review Patients with keloids received intraoperative PRP combined with postoperative superficial radiation therapy 49 patients with 50 ear keloids 94% nonrecurrence after 2 years Study Study design Study method No. of patients Results Cervelli et al20 Clinical: traumatic scars (1) Fat graft + PRP + laser (2) Fat graft + PRP (3) Laser alone 60 patients split into 3 groups Scar management: Triple therapy > Fat graft + PRP > Laser alone Eichler et al21 Retrospective questionnaire Port area scars treated with or without PRP 20 of 120 patients received PRP PRP patients > Non-PRP treated patients in reduced scar dissatisfaction score Klosova et al22 Clinical: burn scars PRP + split thickness skin graft 23 patients with 38 scars Viscoelastic properties: STSG + PRP > STSG alone Nofal et al5 Clinical study: acne scars (1) PRP injections (2) Skin needling (3) PRP + skin needling 45 patients split into 3 groups No single treatment significantly better than the other Lee et al23 Clinical: acne scars After receiving CO2 fractional resurfacing patients underwent split face trial: (1) ½ face PRP injection (2) ½ face Saline injection 14 patients PRP enhanced recovery and scar appearance when compared to saline Na et al24 Clinical: postfractional CO2 recovery (FcxCR) Bilateral inner arm FcxCR followed by either PRP or saline injection 25 patients PRP after FxCR enhanced wound healing and recovery Jones et al7 Retrospective case review Patients with keloids received intraoperative PRP combined with postoperative superficial radiation therapy 49 patients with 50 ear keloids 94% nonrecurrence after 2 years PRP, platelet rich plasma; STSG, split thickness skin grafts. View Large Table 2. Studies Demonstrating and Supporting PRP/BMP’s Ability to Induce Myofibroblastic Dedifferentiation Study Study type Result Plikus et al17 Basic science BMP-2 and BMP-4 strongly expressed and induced myofibroblast dedifferentiation and redifferentiation into adipocytes. BMP-4 converted keloid scar to lipid containing adipocytes Anitua et al25 Basic science Gingival fibroblasts exposed to plasma rich in growth factors showed enhanced growth and expression of VEGF, hepatocyte growth factor, hyaluronic acid. Myofibroblast phenotype was inhibited and reverted with plasma rich in growth factors Anitua et al26 Basic science Platelets obtained from young and middle-aged donors reduced myofibroblast differentiation rate and decreased myofibroblast levels Anitua et al28 Basic science Plasma rich in growth factors stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-β1 induced myodifferentiation Anitua et al27 Basic science Plasma rich in growth factors significantly reduced the myofibroblastic phenotype and accelerated corneal wound healing compared to controls Izumi et al29 Basic science BMP-7 decreased TGF-β dependent fibrogenic activity in mouse pulmonary myofibroblastic cells Liang et al30 Basic science BMP-7 antagonized TGF-β signaling pathways and suppressed silica-induced pulmonary fibrosis Bin et al31 Basic science BMP-7 antagonized TGF-β signaling and inhibited fibroblast morphology in dermal papilla cells Midgley et al19 Basic science BMP-7 exposure reversed myofibroblast differentiation Yano et al32 Basic science BMP-6 participates in progressive renal fibrosis through the development of myofibroblasts in relation with TGF-β Sharma et al33 Basic science Treated with a combination of BMP-6, BMP-7 and hormone cocktail stimulated skeletal muscle precursor cells to express genes found in brown preadipocytes Krause et al34 Basic science BMP-6 reduced TGF-β expression in Dupuytren’s fibroblasts. Treatment with TGF-β receptor kinase inhibitor and BMP-6 led to decreased contractility of Dupuytren’s fibroblasts. Kim et al35 Basic science Scar tissue fibroblasts expressed GREM1 a competitive antagonist of BMP Study Study type Result Plikus et al17 Basic science BMP-2 and BMP-4 strongly expressed and induced myofibroblast dedifferentiation and redifferentiation into adipocytes. BMP-4 converted keloid scar to lipid containing adipocytes Anitua et al25 Basic science Gingival fibroblasts exposed to plasma rich in growth factors showed enhanced growth and expression of VEGF, hepatocyte growth factor, hyaluronic acid. Myofibroblast phenotype was inhibited and reverted with plasma rich in growth factors Anitua et al26 Basic science Platelets obtained from young and middle-aged donors reduced myofibroblast differentiation rate and decreased myofibroblast levels Anitua et al28 Basic science Plasma rich in growth factors stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-β1 induced myodifferentiation Anitua et al27 Basic science Plasma rich in growth factors significantly reduced the myofibroblastic phenotype and accelerated corneal wound healing compared to controls Izumi et al29 Basic science BMP-7 decreased TGF-β dependent fibrogenic activity in mouse pulmonary myofibroblastic cells Liang et al30 Basic science BMP-7 antagonized TGF-β signaling pathways and suppressed silica-induced pulmonary fibrosis Bin et al31 Basic science BMP-7 antagonized TGF-β signaling and inhibited fibroblast morphology in dermal papilla cells Midgley et al19 Basic science BMP-7 exposure reversed myofibroblast differentiation Yano et al32 Basic science BMP-6 participates in progressive renal fibrosis through the development of myofibroblasts in relation with TGF-β Sharma et al33 Basic science Treated with a combination of BMP-6, BMP-7 and hormone cocktail stimulated skeletal muscle precursor cells to express genes found in brown preadipocytes Krause et al34 Basic science BMP-6 reduced TGF-β expression in Dupuytren’s fibroblasts. Treatment with TGF-β receptor kinase inhibitor and BMP-6 led to decreased contractility of Dupuytren’s fibroblasts. Kim et al35 Basic science Scar tissue fibroblasts expressed GREM1 a competitive antagonist of BMP BMP, bone morphogenic proteins; PRP, platelet rich plasma; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. View Large Table 2. Studies Demonstrating and Supporting PRP/BMP’s Ability to Induce Myofibroblastic Dedifferentiation Study Study type Result Plikus et al17 Basic science BMP-2 and BMP-4 strongly expressed and induced myofibroblast dedifferentiation and redifferentiation into adipocytes. BMP-4 converted keloid scar to lipid containing adipocytes Anitua et al25 Basic science Gingival fibroblasts exposed to plasma rich in growth factors showed enhanced growth and expression of VEGF, hepatocyte growth factor, hyaluronic acid. Myofibroblast phenotype was inhibited and reverted with plasma rich in growth factors Anitua et al26 Basic science Platelets obtained from young and middle-aged donors reduced myofibroblast differentiation rate and decreased myofibroblast levels Anitua et al28 Basic science Plasma rich in growth factors stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-β1 induced myodifferentiation Anitua et al27 Basic science Plasma rich in growth factors significantly reduced the myofibroblastic phenotype and accelerated corneal wound healing compared to controls Izumi et al29 Basic science BMP-7 decreased TGF-β dependent fibrogenic activity in mouse pulmonary myofibroblastic cells Liang et al30 Basic science BMP-7 antagonized TGF-β signaling pathways and suppressed silica-induced pulmonary fibrosis Bin et al31 Basic science BMP-7 antagonized TGF-β signaling and inhibited fibroblast morphology in dermal papilla cells Midgley et al19 Basic science BMP-7 exposure reversed myofibroblast differentiation Yano et al32 Basic science BMP-6 participates in progressive renal fibrosis through the development of myofibroblasts in relation with TGF-β Sharma et al33 Basic science Treated with a combination of BMP-6, BMP-7 and hormone cocktail stimulated skeletal muscle precursor cells to express genes found in brown preadipocytes Krause et al34 Basic science BMP-6 reduced TGF-β expression in Dupuytren’s fibroblasts. Treatment with TGF-β receptor kinase inhibitor and BMP-6 led to decreased contractility of Dupuytren’s fibroblasts. Kim et al35 Basic science Scar tissue fibroblasts expressed GREM1 a competitive antagonist of BMP Study Study type Result Plikus et al17 Basic science BMP-2 and BMP-4 strongly expressed and induced myofibroblast dedifferentiation and redifferentiation into adipocytes. BMP-4 converted keloid scar to lipid containing adipocytes Anitua et al25 Basic science Gingival fibroblasts exposed to plasma rich in growth factors showed enhanced growth and expression of VEGF, hepatocyte growth factor, hyaluronic acid. Myofibroblast phenotype was inhibited and reverted with plasma rich in growth factors Anitua et al26 Basic science Platelets obtained from young and middle-aged donors reduced myofibroblast differentiation rate and decreased myofibroblast levels Anitua et al28 Basic science Plasma rich in growth factors stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-β1 induced myodifferentiation Anitua et al27 Basic science Plasma rich in growth factors significantly reduced the myofibroblastic phenotype and accelerated corneal wound healing compared to controls Izumi et al29 Basic science BMP-7 decreased TGF-β dependent fibrogenic activity in mouse pulmonary myofibroblastic cells Liang et al30 Basic science BMP-7 antagonized TGF-β signaling pathways and suppressed silica-induced pulmonary fibrosis Bin et al31 Basic science BMP-7 antagonized TGF-β signaling and inhibited fibroblast morphology in dermal papilla cells Midgley et al19 Basic science BMP-7 exposure reversed myofibroblast differentiation Yano et al32 Basic science BMP-6 participates in progressive renal fibrosis through the development of myofibroblasts in relation with TGF-β Sharma et al33 Basic science Treated with a combination of BMP-6, BMP-7 and hormone cocktail stimulated skeletal muscle precursor cells to express genes found in brown preadipocytes Krause et al34 Basic science BMP-6 reduced TGF-β expression in Dupuytren’s fibroblasts. Treatment with TGF-β receptor kinase inhibitor and BMP-6 led to decreased contractility of Dupuytren’s fibroblasts. Kim et al35 Basic science Scar tissue fibroblasts expressed GREM1 a competitive antagonist of BMP BMP, bone morphogenic proteins; PRP, platelet rich plasma; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. View Large Table 3. Studies Demonstrating That BMP Can Be Extracted From PRP Study PRP type Results Schmidmaier et al37 Activated PRP vs PPP (platelet poor plasma) Levels of growth factors in PRP were significantly higher than in PPP Betsch et al38 Activated PRP vs plasma Thrombin activated PRP contained significantly higher levels of BMP-2 and BMP-7 than plasma alone Jungbluth et al39 Activated PRP vs plasma Thrombin activated PRP had a 17.6-fold increase in BMP-2 and 1.5-fold increase in BMP-7 compared to plasma alone Osada et al40 Activated PRP Podoplanin and C-type lectin-like receptor 2 activated PRP released BMP-9 Arslan et al41 Activated PRP PRP activated by a commercial PRP kit had high levels of BMP-2,4 and 7 Wahlstrom et al42 PRP activated in acidic buffer PRP activated in acidic buffers had significantly greater levels of PDGF, TGF-β, VEGF, and BMP-2 in a pH dependent manner and compared to those exposed to neutral buffers. Additionally, BMP-2 was only detected at pH 4.3. Kalen et al43 PRP activated in acidic buffer Acidic preparation (pH 4.3) of PRP released significantly more BMP-2 and BMP-4 compared to neutral buffer (pH 7.4) Kruger et al44 Activated PRP Range of BMP-2,4,6, and 7 levels detected amongst different PRP donors Strandberg et al45 PRP exposed to freeze-thaw cycles BMP-2 levels did not significantly change with multiple freeze-thaw cycles of platelet lysate Sellberg et al46 PRP exposed to freeze-thaw cycles BMP-2 levels were not significantly different between fresh platelets and platelets that underwent freeze-thaw and subsequent storage at room temperature Study PRP type Results Schmidmaier et al37 Activated PRP vs PPP (platelet poor plasma) Levels of growth factors in PRP were significantly higher than in PPP Betsch et al38 Activated PRP vs plasma Thrombin activated PRP contained significantly higher levels of BMP-2 and BMP-7 than plasma alone Jungbluth et al39 Activated PRP vs plasma Thrombin activated PRP had a 17.6-fold increase in BMP-2 and 1.5-fold increase in BMP-7 compared to plasma alone Osada et al40 Activated PRP Podoplanin and C-type lectin-like receptor 2 activated PRP released BMP-9 Arslan et al41 Activated PRP PRP activated by a commercial PRP kit had high levels of BMP-2,4 and 7 Wahlstrom et al42 PRP activated in acidic buffer PRP activated in acidic buffers had significantly greater levels of PDGF, TGF-β, VEGF, and BMP-2 in a pH dependent manner and compared to those exposed to neutral buffers. Additionally, BMP-2 was only detected at pH 4.3. Kalen et al43 PRP activated in acidic buffer Acidic preparation (pH 4.3) of PRP released significantly more BMP-2 and BMP-4 compared to neutral buffer (pH 7.4) Kruger et al44 Activated PRP Range of BMP-2,4,6, and 7 levels detected amongst different PRP donors Strandberg et al45 PRP exposed to freeze-thaw cycles BMP-2 levels did not significantly change with multiple freeze-thaw cycles of platelet lysate Sellberg et al46 PRP exposed to freeze-thaw cycles BMP-2 levels were not significantly different between fresh platelets and platelets that underwent freeze-thaw and subsequent storage at room temperature BMP, bone morphogenic proteins; PDGF, platelet-derived growth factor; PPP, platelet poor plasma; PRP, platelet rich plasma; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. View Large Table 3. Studies Demonstrating That BMP Can Be Extracted From PRP Study PRP type Results Schmidmaier et al37 Activated PRP vs PPP (platelet poor plasma) Levels of growth factors in PRP were significantly higher than in PPP Betsch et al38 Activated PRP vs plasma Thrombin activated PRP contained significantly higher levels of BMP-2 and BMP-7 than plasma alone Jungbluth et al39 Activated PRP vs plasma Thrombin activated PRP had a 17.6-fold increase in BMP-2 and 1.5-fold increase in BMP-7 compared to plasma alone Osada et al40 Activated PRP Podoplanin and C-type lectin-like receptor 2 activated PRP released BMP-9 Arslan et al41 Activated PRP PRP activated by a commercial PRP kit had high levels of BMP-2,4 and 7 Wahlstrom et al42 PRP activated in acidic buffer PRP activated in acidic buffers had significantly greater levels of PDGF, TGF-β, VEGF, and BMP-2 in a pH dependent manner and compared to those exposed to neutral buffers. Additionally, BMP-2 was only detected at pH 4.3. Kalen et al43 PRP activated in acidic buffer Acidic preparation (pH 4.3) of PRP released significantly more BMP-2 and BMP-4 compared to neutral buffer (pH 7.4) Kruger et al44 Activated PRP Range of BMP-2,4,6, and 7 levels detected amongst different PRP donors Strandberg et al45 PRP exposed to freeze-thaw cycles BMP-2 levels did not significantly change with multiple freeze-thaw cycles of platelet lysate Sellberg et al46 PRP exposed to freeze-thaw cycles BMP-2 levels were not significantly different between fresh platelets and platelets that underwent freeze-thaw and subsequent storage at room temperature Study PRP type Results Schmidmaier et al37 Activated PRP vs PPP (platelet poor plasma) Levels of growth factors in PRP were significantly higher than in PPP Betsch et al38 Activated PRP vs plasma Thrombin activated PRP contained significantly higher levels of BMP-2 and BMP-7 than plasma alone Jungbluth et al39 Activated PRP vs plasma Thrombin activated PRP had a 17.6-fold increase in BMP-2 and 1.5-fold increase in BMP-7 compared to plasma alone Osada et al40 Activated PRP Podoplanin and C-type lectin-like receptor 2 activated PRP released BMP-9 Arslan et al41 Activated PRP PRP activated by a commercial PRP kit had high levels of BMP-2,4 and 7 Wahlstrom et al42 PRP activated in acidic buffer PRP activated in acidic buffers had significantly greater levels of PDGF, TGF-β, VEGF, and BMP-2 in a pH dependent manner and compared to those exposed to neutral buffers. Additionally, BMP-2 was only detected at pH 4.3. Kalen et al43 PRP activated in acidic buffer Acidic preparation (pH 4.3) of PRP released significantly more BMP-2 and BMP-4 compared to neutral buffer (pH 7.4) Kruger et al44 Activated PRP Range of BMP-2,4,6, and 7 levels detected amongst different PRP donors Strandberg et al45 PRP exposed to freeze-thaw cycles BMP-2 levels did not significantly change with multiple freeze-thaw cycles of platelet lysate Sellberg et al46 PRP exposed to freeze-thaw cycles BMP-2 levels were not significantly different between fresh platelets and platelets that underwent freeze-thaw and subsequent storage at room temperature BMP, bone morphogenic proteins; PDGF, platelet-derived growth factor; PPP, platelet poor plasma; PRP, platelet rich plasma; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. View Large Table 4. Studies Showing Effects of PRP on Adipose Derived Stem Cells (ADSC) and Preadipocytes Study Study model Assay Result McLaughlin et al47 Basic science Growth of ADSCs + PRP vs ADSCs + 10% fetal bovine serum ADSCs + PRP combination showed increased ADSC proliferation, expression of BMP-4 and BMP-2, and reduced expression of PDGF-B and bFGF D’Esposito et al48 Basic science Studied ADSCs + PRP vs adipocytes + PRP Adding PRP increased ADSC viability and proliferation. PRP also enhanced secretion of pro-angiogenic factors from mature adipocytes facilitating tissue regeneration Liao et al49 Basic science ADSCs grown in general media vs adipogenic media supplemented with and without PRP PRP enhanced the proliferation of ADSCs even when grown in anti-proliferative, pro-adipogenic media. PRP inhibited adipogenic differentiation through downregulation of BMP Receptor 1A and FGF receptor 1 Seyhan et al50 Basic science Fat grafts combined with either PRP, ADSCs, or both Fat graft retention was significantly improved in rats who received grafts with PRP and ADSCs combined. Li et al51 Basic science Growth of ADSCs in PRPLipoinjection of granular fat, ADSCs and PRP into nude mice PRP enhanced ADSC proliferation and expression of adipogenic-related genes such as PPARγ, lipoprotein lipase and adipophilin. Granular fat grafts with 20% PRP supplementation had significantly improved residual volumes Li et al52 Basic science Fat grafts prepared with ADSCs at different densities with and without PRP Residual fat volume of grafts containing 105 /mL ADSC + PRP was significantly higher than other treatment conditions after 90 days. Increased adipocyte area and capillary formation was observed Fukaya et al53 Basic science Growth of preadipocytes in either PRP, fetal bovine serum (FBS) or platelet-poor plasma (PPP) PRP stimulated proliferation of preadipocytes in a dose-dependent fashion. Growth in 2% PRP was significantly higher than in 2% FBS or 2% PPP Chignon-Sicard et al54 Basic science Combined PRP + ADSCs PRP reduced the potential for ADSC to differentiate and led to generation of myofibroblast-like cells through TGF-β signaling Study Study model Assay Result McLaughlin et al47 Basic science Growth of ADSCs + PRP vs ADSCs + 10% fetal bovine serum ADSCs + PRP combination showed increased ADSC proliferation, expression of BMP-4 and BMP-2, and reduced expression of PDGF-B and bFGF D’Esposito et al48 Basic science Studied ADSCs + PRP vs adipocytes + PRP Adding PRP increased ADSC viability and proliferation. PRP also enhanced secretion of pro-angiogenic factors from mature adipocytes facilitating tissue regeneration Liao et al49 Basic science ADSCs grown in general media vs adipogenic media supplemented with and without PRP PRP enhanced the proliferation of ADSCs even when grown in anti-proliferative, pro-adipogenic media. PRP inhibited adipogenic differentiation through downregulation of BMP Receptor 1A and FGF receptor 1 Seyhan et al50 Basic science Fat grafts combined with either PRP, ADSCs, or both Fat graft retention was significantly improved in rats who received grafts with PRP and ADSCs combined. Li et al51 Basic science Growth of ADSCs in PRPLipoinjection of granular fat, ADSCs and PRP into nude mice PRP enhanced ADSC proliferation and expression of adipogenic-related genes such as PPARγ, lipoprotein lipase and adipophilin. Granular fat grafts with 20% PRP supplementation had significantly improved residual volumes Li et al52 Basic science Fat grafts prepared with ADSCs at different densities with and without PRP Residual fat volume of grafts containing 105 /mL ADSC + PRP was significantly higher than other treatment conditions after 90 days. Increased adipocyte area and capillary formation was observed Fukaya et al53 Basic science Growth of preadipocytes in either PRP, fetal bovine serum (FBS) or platelet-poor plasma (PPP) PRP stimulated proliferation of preadipocytes in a dose-dependent fashion. Growth in 2% PRP was significantly higher than in 2% FBS or 2% PPP Chignon-Sicard et al54 Basic science Combined PRP + ADSCs PRP reduced the potential for ADSC to differentiate and led to generation of myofibroblast-like cells through TGF-β signaling ADSC, adipose-derived stem cells; BMP, bone morphogenic proteins; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; PRP, platelet rich plasma; TGF, transforming growth factor. View Large Table 4. Studies Showing Effects of PRP on Adipose Derived Stem Cells (ADSC) and Preadipocytes Study Study model Assay Result McLaughlin et al47 Basic science Growth of ADSCs + PRP vs ADSCs + 10% fetal bovine serum ADSCs + PRP combination showed increased ADSC proliferation, expression of BMP-4 and BMP-2, and reduced expression of PDGF-B and bFGF D’Esposito et al48 Basic science Studied ADSCs + PRP vs adipocytes + PRP Adding PRP increased ADSC viability and proliferation. PRP also enhanced secretion of pro-angiogenic factors from mature adipocytes facilitating tissue regeneration Liao et al49 Basic science ADSCs grown in general media vs adipogenic media supplemented with and without PRP PRP enhanced the proliferation of ADSCs even when grown in anti-proliferative, pro-adipogenic media. PRP inhibited adipogenic differentiation through downregulation of BMP Receptor 1A and FGF receptor 1 Seyhan et al50 Basic science Fat grafts combined with either PRP, ADSCs, or both Fat graft retention was significantly improved in rats who received grafts with PRP and ADSCs combined. Li et al51 Basic science Growth of ADSCs in PRPLipoinjection of granular fat, ADSCs and PRP into nude mice PRP enhanced ADSC proliferation and expression of adipogenic-related genes such as PPARγ, lipoprotein lipase and adipophilin. Granular fat grafts with 20% PRP supplementation had significantly improved residual volumes Li et al52 Basic science Fat grafts prepared with ADSCs at different densities with and without PRP Residual fat volume of grafts containing 105 /mL ADSC + PRP was significantly higher than other treatment conditions after 90 days. Increased adipocyte area and capillary formation was observed Fukaya et al53 Basic science Growth of preadipocytes in either PRP, fetal bovine serum (FBS) or platelet-poor plasma (PPP) PRP stimulated proliferation of preadipocytes in a dose-dependent fashion. Growth in 2% PRP was significantly higher than in 2% FBS or 2% PPP Chignon-Sicard et al54 Basic science Combined PRP + ADSCs PRP reduced the potential for ADSC to differentiate and led to generation of myofibroblast-like cells through TGF-β signaling Study Study model Assay Result McLaughlin et al47 Basic science Growth of ADSCs + PRP vs ADSCs + 10% fetal bovine serum ADSCs + PRP combination showed increased ADSC proliferation, expression of BMP-4 and BMP-2, and reduced expression of PDGF-B and bFGF D’Esposito et al48 Basic science Studied ADSCs + PRP vs adipocytes + PRP Adding PRP increased ADSC viability and proliferation. PRP also enhanced secretion of pro-angiogenic factors from mature adipocytes facilitating tissue regeneration Liao et al49 Basic science ADSCs grown in general media vs adipogenic media supplemented with and without PRP PRP enhanced the proliferation of ADSCs even when grown in anti-proliferative, pro-adipogenic media. PRP inhibited adipogenic differentiation through downregulation of BMP Receptor 1A and FGF receptor 1 Seyhan et al50 Basic science Fat grafts combined with either PRP, ADSCs, or both Fat graft retention was significantly improved in rats who received grafts with PRP and ADSCs combined. Li et al51 Basic science Growth of ADSCs in PRPLipoinjection of granular fat, ADSCs and PRP into nude mice PRP enhanced ADSC proliferation and expression of adipogenic-related genes such as PPARγ, lipoprotein lipase and adipophilin. Granular fat grafts with 20% PRP supplementation had significantly improved residual volumes Li et al52 Basic science Fat grafts prepared with ADSCs at different densities with and without PRP Residual fat volume of grafts containing 105 /mL ADSC + PRP was significantly higher than other treatment conditions after 90 days. Increased adipocyte area and capillary formation was observed Fukaya et al53 Basic science Growth of preadipocytes in either PRP, fetal bovine serum (FBS) or platelet-poor plasma (PPP) PRP stimulated proliferation of preadipocytes in a dose-dependent fashion. Growth in 2% PRP was significantly higher than in 2% FBS or 2% PPP Chignon-Sicard et al54 Basic science Combined PRP + ADSCs PRP reduced the potential for ADSC to differentiate and led to generation of myofibroblast-like cells through TGF-β signaling ADSC, adipose-derived stem cells; BMP, bone morphogenic proteins; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; PRP, platelet rich plasma; TGF, transforming growth factor. View Large Study Design A PubMed literature search was conducted on July 5, 2017 for the following keywords after review and approval by an independent librarian (S.C): “BMP,” “BMPs,” “Bone Morphogenic Protein,” “Bone Morphogenic Protein 2,” “Bone Morphogenic Protein 4,” “Bone Morphogenic Protein 7,” “BMP 4,” “BMP 2,” and “BMP 7.” Keywords for the PRP search included: “PRP,” “Platelet Enriched Plasma,” “Platelet Rich Plasma,” “Platelets,” and “Platelet.” Key words for the scar modulation included: “Wound Healing,” “Skin Wound Healing,” “Myofibroblast,” “Adipogenesis,” “Adipocyte differentiation,” “Adipose derived stem cell,” “ADSC,” “Keloid,”, “Hypertrophic Scar,” and “Scar.” Four search cohorts were created using PRP and BMP terms (542 articles), PRP and scar modulation terms (165 articles), BMP and scar modulation terms (2090 articles), and PRP, BMP, and scar modulation combined (33 articles). In total 2830 abstracts where reviewed for inclusion criteria. For additional information see PRISMA diagram for compiled search groups (Figure 2). Figure 2. View largeDownload slide Flow diagram: 2830 articles were obtained from search terms. After reviewing all 2830 abstracts only 231 articles met the inclusion criteria for complete full-text assessment. After full-text assessment only 38 articles were found that met selection criteria. Figure 2. View largeDownload slide Flow diagram: 2830 articles were obtained from search terms. After reviewing all 2830 abstracts only 231 articles met the inclusion criteria for complete full-text assessment. After full-text assessment only 38 articles were found that met selection criteria. Selection Criteria and Data Collection All 2830 abstracts were assessed and selected if they demonstrated the following inclusion criteria: studies showing PRP contains BMP or evaluating the effects of PRP and BMP on either scar modulation, adipogenesis, or myofibroblast dedifferentiation. Studies were excluded if they described PRP or BMP use for orthopedic purposes. The full text of each article was read critically by a research assistant (Z.O., J.P., L.T.) to ensure each article met the selection criteria. Each summary and article were then read and cross-referenced for completeness (R.S). In total, 38 articles met the inclusion criteria and were included in the review. The Effect of PRP on Scar Modulation A few studies have been published evaluating the use of PRP for the treatment of scars. However, PRP activation and application has yet to be standardized, leading to a multitude of different treatment protocols with varying levels of efficacies. Additionally, PRP is often paired with other scar treatment modalities. Here, we review papers showing the positive effect of PRP on scar modulation. Cervelli et al randomly assigned 60 patients with traumatic scars to one of three groups: fat graft combined with PRP, nonablative laser resurfacing, or all three modalities combined.20 They found that the most effective scar treatment was fat grafting with PRP plus nonablative laser resurfacing (P < 0.05).20 The improvement in scar healing for treatments using the three modalities together was 22% greater than the fat graft and PRP group and was 11% greater than nonablative laser resurfacing alone.20 To test patient satisfaction with PRP-treated scars, Eichler et al administered a retrospective questionnaire to 120 patients who had their venous access device removed within six months and received either PRP or no treatment for their port area scar.21 The 20 patients who received PRP treatment showed a significant reduction in their desire to improve port area scarring and had reduced scar dissatisfaction (PRP: 10% vs control: 40.2% dissatisfaction, P < 0.05).21 Clinicians have also experimented with using PRP for skin grafts as an adjunctive therapy for the treatment of burns. For example, Klosova et al combined PRP with split thickness skin grafts (STSG) in patients who originally presented with scars from deep burns.22 They found that the viscoelastic properties of scars treated with STSG and PRP returned more rapidly than areas treated with STSG alone.22 Nofal et al treated 45 patients with either intradermal PRP injections, skin needling, or both treatments every two weeks for a six week period.5 All the groups showed a statistically significant improvement in the degree of acne scars when compared to control (P < 0.001), yet no single treatment was superior to the other.5 To study the effects of combining PRP and CO2 fractional resurfacing for acne scars, Lee et al subjected half of a patient’s face to PRP and the other half to saline injections post-CO2 fractional resurfacing.23 Erythema, edema, and posttreatment crusting all improved more rapidly with PRP treatment (P < 0.05).23 Similarly, Na et al subjected patients’ bilateral inner arms to PRP or a saline treatment postfractional CO2 laser resurfacing.24 They found that the treatment with PRP conferred several beneficial effects including a thicker epidermis, more organized stratum corneum and collagen fibers, and a higher collagen density compared to the control (P < 0.05).24 PRP has also been applied for keloid therapy. Jones et al conducted a retrospective analysis of 49 patients who received ear keloid surgery and were then treated postoperatively with PRP addition over the surgical site and superficial radiation therapy (SRT).7 They reported a 94% success rate, supporting the role of PRP in this combination therapy for the management of keloids.7 In summary, a few studies show that PRP treatment produces dermatological benefits when used alone or in combination with other therapeutic modalities. Many of the studies presented above are clinical assessments, with little attention placed on the components of PRP that may be producing these positive effects. Recent research indicates that BMP may be a significant contributor to the positive effects of PRP on scar modulation. BMP/PRP-Induced Myofibroblastic Dedifferentiation While clinical studies show that PRP may have an effect on scar modulation, very little attention has centered on the components in PRP that may be producing these effects. Recent investigations show that certain BMPs induce myofibroblastic dedifferentiation into preadipocytes, which is a potential explanation for the positive effect of PRP on scars. In a landmark study, Plikus et al utilized a murine model and demonstrated that myofibroblasts have the capacity to dedifferentiate into adipocytes during wound healing.17 Myofibroblast reprogramming was found to require neogenic hair follicles, which trigger BMP signaling and the activation of adipocyte transcription factors.17 Furthermore, the overexpression of a BMP antagonist (Noggin) in hair follicles, or the deletion of the BMP receptor in myofibroblasts, prevented adipocyte formation.17 The expression of BMP-2 and BMP-4 were found to be critical for myofibroblast reprogramming and transcriptomic data showed that BMP-2 and BMP-7 expression were upregulated, while the BMP antagonists, GREM1 and Bambi, were downregulated in this process.17 The upregulation of pSmad 1/5/8 in dermal cells adjacent to the regenerated hair follicles served as an indicator that BMP signaling was occurring.17 Additionally, when human-derived keloid cells were pretreated with BMP-4 (20 ng/mL) and exposed to adipogenic culture conditions, they consistently began adipogenic conversion.17 Thus, the possibility of fatty conversion of established scar tissue appears to be a real possibility. In 2012, Anitua et al studied the effects of activated platelets in gingival tissue regeneration and periodontal wound healing.25 In their preparation, PRP was anticoagulated with sodium citrate, activated with calcium chloride, and the leukocytes were removed.25 They found that activated PRP significantly increased gingival fibroblast proliferation, migration, and cell adhesion on a type I collagen matrix (P < 0.05).25 However, the myofibroblast phenotype, which severely impairs tissue function when extracellular matrix protein secretion becomes excessive, was inhibited and reverted with activated PRP treatment.25 Citing other studies, the authors hypothesized that the hepatocyte growth factor (HGF) found in the platelets might inhibit TGF-β1-induced myofibroblast differentiation.25 Similarly, in 2016, the same group studied the effects of TGF-β1-induced myofibroblast differentiation in dermal fibroblasts.26 Dermal fibroblasts isolated from healthy human skin were exposed to activated platelets obtained from four healthy, young patients and four healthy, middle-aged patients.26 Both the young and middle-aged donor platelets, even in the presence of TGF-β1, had reduced myofibroblast differentiation rates.26 Furthermore, the young and middle-aged PRP donor samples resulted in a, respectively, 23% ± 4% and 20% ± 1% decrease in myofibroblast levels (P < 0.05).26 In this study, TGF-β1, PDGF, epidermal growth factor (EGF), HGF, insulin growth factor (IGF), and basic fibroblast growth factor (bFGF) levels in the activated PRP from the two age groups were not significantly different.26 Finally, in two other studies, Anitua et al also showed that activated platelets reversed the myofibroblast phenotype in corneal fibroblast exposed to TGF-β1 (P < 0.05).27,28 Several other studies also examined the role of BMPs and their effects on the myofibroblast phenotype. Izumi et al studied both BMP-7 and TGF-β1 and showed that BMP-7 antagonizes the TGF-β1-dependent fibrogenic activity of mouse pulmonary myofibroblasts through the induction of the inhibitor of differentiation (Id) proteins Id2 and Id3 (P < 0.05).29 Through immunocytochemistry, they demonstrated that the ectopic expression of BMP-7 led to the nuclear localization of Phospho-Smad1/5/8 and the suppression of Smad3.29 Furthermore, BMP-7 suppressed the mRNA expression of COL1A2 (the TGF-β response element) and tiMMP2.29 Lastly, they found that BMP-7 increased Id 2 and 3, which suppresses COLIA2 promoter activity.29 In a similar study, Liang et al showed that BMP-7 inhibits silica-induced pulmonary fibrosis via the suppression of the TGF-β/Smad pathway (P < 0.05).11,29,30 Furthermore, Bin et al exposed dermal papilla cells (DPC) to 10 ng/mL TGF-β1 for 48 hours and found that they differentiated into cells with a fibroblast-like morphology (P < 0.05).31 However, all the changes were completely inhibited by BMP-7 treatment (P < 0.05).31 Bin et al concluded that hair follicle DPCs might play a role in wound healing by converting into fibroblasts via the TGF-β1 pathway.31 In addition, BMP-7, which is prominently expressed in the epidermis and papillary dermis and undergoes hair-cycle related changes in expression, halts fibrotic disease progression and promotes recovery by antagonizing TGF-β1-stimulated epithelial-mesenchymal transition.31 Finally, these authors hypothesized that the antagonistic effects of BMP-7 on the TGF-β1 pathway might suppress hypertrophic scar formation.31 In addition, Midgley et al tested the effect of recombinant BMP-7 on myofibroblasts.19 After exposing human lung fibroblast to TGF-β1 and confirming their differentiation into myofibroblasts using α-SMA, they demonstrated that 400 ng/mL of BMP-7 was required to reverse myofibroblast differentiation (P < 0.05).19 Several studies involving BMP signaling present contrasting results, and therefore, further understanding of the signaling pathway is necessary. Yano et al studied the role of BMP-6 in rat renal fibrosis. Treatment with BMP-6 increased α-SMA expression, which was analogous to the effects of TGF-β1 (P < 0.05).32 Collectively, BMP-6 was shown to participate in progressive renal fibrosis through the development of myofibroblasts in relation with TGF-β1.32 In contrast, Sharma et al found that when human skeletal muscle precursor cells were stimulated for two days with BMP-6 (205 ng/mL), followed by three days of an induction hormone cocktail, this was sufficient to induce a gene expression that was characteristic of brown preadipocytes (P < 0.05).33 A similar yet less pronounced effect was demonstrated using BMP-7 (P < 0.05).33 Moreover, Krause et al showed an increased TGF-β1 expression in Dupuytren’s disease fibroblasts compared to normal fibroblasts.34 Additionally, treatment with a TGF-β1 type I receptor inhibitor (SB-431542) and BMP-6 inhibited Smad and ERK1/2/MAP kinase signaling and decreased the contractility of Dupuytren’s fibroblasts (P < 0.05).34 Cotreatment of Dupuytren’s fibroblasts with a TGF-β1 inhibitor (SB-431542) and a MAP kinase 1 inhibitor (PD98059) attenuated the proliferation and contraction of Dupuytren’s-associated fibroblasts (P < 0.05).34 Given these findings, the researchers concluded that the TGF-β and ERK1/2 MAP kinase pathways are prime targets for nonsurgical intervention of Dupuytren’s disease.34 Finally, Kim et al found that scar tissue fibroblasts express GREM1, a competitive antagonist of BMP, and this expression was not observed in the dermis of normal skin (P < 0.05).35 The researchers concluded that GREM1 might serve as a marker for activated myofibroblasts in scars.35 In summary, convincing evidence exists demonstrating that BMP-2, 4, and 7, released by neogenic hair follicles, stimulate the dedifferentiation of myofibroblasts to preadipocytes. In addition, studies show that the TGF-β1-dependent conversion of fibroblasts to myofibroblast is reversed by BMP-7. This effect of BMP is thought to be produced by the intracellular signals SMAD 6 and 7 which inhibit the TGF-β1 pathway. In addition, the use of TGF-β1 inhibitors or BMP-6 alone is sufficient to reduce the contractility of Dupuytren’s-associated fibroblasts, creating a new avenue for nonsurgical treatment of the disease. Finally, GREM1, a competitive BMP antagonist, is expressed by fibroblasts in the dermis of scar tissue and may serve as a marker for persistent myofibroblasts PRP Contains Extractible BMP High concentrations of BMP (BMP-4: 20 ng/mL) induce the dedifferentiation of myofibroblasts, which raises the question of whether PRP alone contains enough extractable BMP to produce the desired effects. Whole blood, on its own, has detectable levels of BMP-1, BMP-2, BMP-3, BMP-4, BMP-6, BMP-7, and BMP-8.36 Here, we review studies that indicate the presence of BMP in PRP, how the preparation of PRP affects BMP release, and how levels of BMP are impacted by storage and free-thaw methods. Comparing PRP to platelet-poor plasma (PPP), some investigators show that PRP has levels of detectable growth factors, other than BMP-4, which are not detected in PPP.37 For example, BMP-2 concentrations are 1.9-fold higher in PRP than in PPP (0.03 ng/ml in PRP vs 0.015 ng/mL in PPP, P < 0.05).37 Studies investigating BMP levels in activated PRP are more common than those looking at BMP levels in nonactivated PRP. For example, Betsch et al demonstrated that the activation of PRP with thrombin yielded more BMP-2 and BMP-7 than in plasma alone ([BMP-2]: 167.7 ± 83.42 pg/mL in PRP vs 12.87 + 62.06 pg/mL in plasma; [BMP-7]: 178.86 + 128.98 pg/mL in PRP vs 95.856 + 37.28 pg/mL in plasma; P < 0.05).38 To further support these findings, Jungbluth et al found similar concentrations of BMP-2 and BMP-7 in thrombin-activated PRP from minipigs ([BMP-2]: 204.9 ± 180.4 pg/mL; [BMP-7]:125.2 ± 83.7 pg/mL; P < 0.05).39 This amounted to a 17.6- and 1.5-fold increase in BMP-2 and BMP-7, respectively, compared to plasma levels. However, they did note variations in the BMP concentrations between different minipig donors.39 Furthermore, Osada et al showed that platelets are activated via podoplanin and C-type lectin-like receptor 2 (CLEC-2) interactions which results in the release of BMP-9 (P < 0.05).40 Using the commercial advanced tissue regeneration kit (ATR) to activate PRP, Arslan et al obtained high concentrations of BMP-2, BMP-4, and BMP-7 from PRP (77.7 ± 9.58 pg/mL, 230 ± 48.9 pg/mL, and 3,059 ± 272 pg/mL, respectively; P < 0.05).41 Other studies also show that acidic preparations of PRP increase the BMP levels obtained from PRP.42,43 For example, Wahlstrom et al tested human PRP in buffers with pH values that ranged from 4.3 to 8.6 and found that only PRP incubated at pH 4.3 released BMP-2 (57 ng/mL, P < 0.05).42 In addition, Kalen et al found that PRP samples released significantly more BMP-2 (20 ng/L) and BMP-4 when they were lysed in an acidic buffer (pH 4.3) rather than in a neutral buffer (pH 7.4) (BMP-2, 32% vs 3%; BMP-4, 87% vs 52% released; P < 0.05).43 This group also found detectable levels of BMP-7 (1,536 ng/L) and BMP-6 (414-14,144 ng/L). However, no statistically significant difference was observed in the concentration of BMP-6 and BMP-7 in acidic vs neutral preparations.43 Similar to other studies, Kalen et al observed a considerable variation in the levels of the different BMPs released from different donors. For instance, only two of the 31 donors had detectable levels of all the four studied BMPs (P < 0.05).43 The heterogeneity among donors, with regard to the BMP levels in PRP, was replicated by Kruger et al who detected a range of BMP-2, BMP-4, BMP-6, and BMP-7 concentrations in 6 different donors.44 Interestingly, BMP-2 was present in only five of the six donors (average BMP-2, 0.31 ng/mL).44 Consequently, PRP activation methods as well as heterogeneity in BMPs and their levels amongst donors must be considered when using PRP alone as a therapeutic. Other studies investigated different preparation techniques in order to lengthen the storage time of PRP without affecting the relative concentrations of the growth factors. Strandberg et al found that after 1, 3, 5, 10, and 30 freeze-thaw cycles of the platelet lysate, the concentration of BMP-2 was not significantly decreased (196.0 ± 11.9 pg/mL at cycle 0 and 177.5 ± 8.7 pg/mL at cycle 30).45 In addition, Sellberg et al showed that the concentration of BMP-2 in fresh platelets was equivalent to those that underwent three freeze-thaw cycles and were subsequently stored at room temperature for five days (1250 pg/mL).46 In summary, the studies above clearly indicate that PRP contains variable concentrations of the different BMPs across various donors. Yet, the activation of PRP through different methods leads to a considerable increase in BMPs. For example, several studies indicate that acid may increase the amount of BMP-2 and BMP-4 released from PRP. Studies looking at the effects of freeze-thaw cycles for the purpose of PRP storage show no difference in BMP concentrations when compared to fresh isolates. Finally, although different levels of BMPs can be extracted to some degree from PRP, the levels may not be sufficient to produce scar myofibroblastic dedifferentiation, which requires nanograms of BMP per milliliter. Effect of PRP on Adipose Derived Stem Cells The levels of BMP extracted from PRP may not be enough alone to dedifferentiate myofibroblast into preadipocytes. However, several studies indicate that combining fat grafts containing adipose-derived stem cells (ADSCs) with PRP increases local BMP levels, affects ASDC commitment to the adipocyte lineage, and improves graft retention rates. Consequently, here, we review articles describing the effects of PRP on ADSCs. Only one study has investigated the expression of BMP-2 and 4 after adding activated PRP (platelet relesate) to ADSCs. McLaughlin et al demonstrated that the addition of PRP increased the proliferation and expression of BMP-4 (5.7 ± 0.97 fold increase) and BMP-2 (4.7 ± 1.3 fold increase) and reduced the expression of PDGF-β and bFGF in the ADSCs compared to ADSCs grown in 10% fetal bovine serum (P < 0.05).47 Furthermore, they found that the ADSCs retained their potential to differentiate into osteogenic, chondrogenic, and adipogenic cell lines.47 Consequently, activated PRP is thought to stimulate BMP-2 and 4 production from ADSCs, thereby increasing the concentration of these factors locally. Several groups have used PRP in combination with ADSCs, investigating its utility in soft tissue reconstruction. D’Esposito et al combined PRP with ADSCs to investigate its potential use for tissue regeneration.48 They found that PRP increased the viability, proliferation rate, and G1-S cell cycle progression of ADSCs, with higher PRP concentrations having larger effects (P < 0.05).48 In mature adipocytes, they found no change in PPARγ expression, cell viability and differentiation upon exposure to PRP.48 However, they did find an increase in the expression of leptin, angiogenesis, and the release of IL-6, 8, 10, Interferon-γ, and vascular endothelial growth factor (VEGF) in these mature adipocytes (P < 0.05).48 This was consistent with the stimulatory effect of the platelets’ contents on adipose tissue.48 Consequently, the researchers concluded that PRP may facilitate tissue regeneration because it stimulates the recruitment and proliferation of ADSCs without changing cell viability.48 The ability of PRP to stimulate ADSC proliferation is hypothesized to play a role in fat graft survival. Liao et al found that PRP significantly enhanced ADSC proliferation even when the ADSCs were grown in antiproliferative, proadipogenic media (P < 0.05).49 Interestingly, they found that PRP inhibited the adipogenic differentiation of ADSCs in this media across all the PRP concentrations tested.49 This effect was attributed to the downregulation of the major adipogenic mediating receptors bone morphogenetic protein receptor IA (BMPRIA) and fibroblast growth factor receptor 1 (FGFR1).49 Thus, when PRP is added to fat grafts, the ADSCs respond by undergoing proliferation rather than differentiation.49 As a result, the larger pool of ADSCs participate in hormone production and the subsequent differentiation into adipocytes after the PRP is reabsorbed.49 Consequently, one can hypothesize that when PRP is added to fat grafts, the resultant proliferation of the ADSCs and their production of hormones may increase the concentration of BMP-2 and 4 to the levels needed for myofibroblastic dedifferentiation. Seyhan et al studied fat graft retention in rats and found that those treated with both PRP and ADSCs had higher graft retention rates, as well as increased growth factors levels (VEGF, TGF-β, FGF), after 12 weeks when compared to the controls (P < 0.05).50 Similarly, Li et al tested cell viability and graft take after adding PRP at different concentrations (0%, 10%, 20%, and 30%) to the ADSC media.51 Here, PRP, ADSCs, and granular fat were combined and were grafted subcutaneously into nude mice and examined histologically at different time periods.51 They found that PRP improved ADSC proliferation and that the grafts with PRP added at concentrations of 20% and 30% showed significantly improved residual volumes (P < 0.05). However, when comparing the 20% and 30% PRP groups, no difference was observed in the residual volumes and histology.51 Considering the cost of PRP, the researchers suggested using 20% PRP with ADSCs for improving fat graft survival.51 In a separate study, Li et al found that grafts containing 105 ADSCs/mL with PRP were optimal for adipogenesis, and the residual fat volume was significantly higher than in the other treatment conditions (eg, 107, 106, 104, and 0 cells/mL ± PRP) after 90 days (P < 0.05).52 Similarly, Fukaya et al found that adding PRP to preadipocytes stimulated the proliferation of preadipocytes in a dose-dependent manner (P < 0.05).53 Furthermore, in a study performed by Chignon-Sicard et al, the effects of freeze-activated PRP on antagonizing ADSC proliferation was assessed.54 Here, PRP reduced the potential of ADSCs to undergo differentiation into adipocytes and, in fact, led to the generation of myofibroblast-like cells (P < 0.05).54 The researchers concluded that the TGF-β found in PRP played a critical role in the promyofibroblastic and antiadipogenic fate of the ADSCs.54 Here, again, emphasis must be placed on the PRP preparation technique (activated vs nonactivated, method of activation, and the presence or removal of leukocytes) in order to understand its effects on ADSCs. In summary, the studies above indicate that when PRP is combined with fat grafts, ADSCs are stimulated, showing an increased expression of BMP-2 and 4 by roughly 5-fold. In addition, the rapid proliferation of ADSCs and an improved fat graft retention is also observed with the addition of PRP to ADSCs. Additional studies are needed to quantify the levels of BMP-2 and 4 produced by PRP-stimulated ADSCs to assess whether the concentrations needed to produce myofibroblastic dedifferentiation can be achieved. CONCLUSION In developed countries, over 100 million people per year form scars, of which 30% become hypertrophic or keloid scars due to abnormal wound healing.55 Patients who suffer from these abnormalities not only have aesthetic, social, and psychological impairments but can also have a reduced quality of life.55 Hypertrophic scars, which are visible, elevated, and do not spread into surrounding tissues, contain an increased amount of myofibroblasts that fail to undergo normal apoptosis. In addition, they have a reduced or complete lack of cutaneous fat and growing hair follicles, which may contribute to their pathophysiology.55 Research demonstrates that actively growing hair follicles locally secrete BMPs, which induce myofibroblast dedifferentiation and reprogramming into adipocytes.17,30,31,56 Furthermore, BMPs induce adipogenesis from local ADSC sources, promote angiogenesis, reduce inflammation, and stimulate proper wound healing.18,57-59 However, the lack of hair follicles and, thus, the reduced local BMP levels in hypertrophic scar tissue prevents many of these beneficial events from occurring.55 To address the lack of local BMP in hypertrophic scars, we sought to investigate the literature for natural sources of BMP-2 and 4, which are implicated in myofibroblast dedifferentiation, and would bypass the necessity to produce them via recombinant techniques. Through our systematic review, we found studies showing that BMPs can be obtained from PRP following various activation techniques, yet evidence of whether the released amounts are high enough to produce myofibroblastic dedifferentiation (BMP-4, 20ng/ml) is yet to be achieved. While PRP alone may not provide a sufficient source of BMP for therapeutic applications, PRP stimulates fat grafts containing ADSCs to produce a five-fold increase in expression of BMP-2 and 4. Taken together, we hypothesize that PRP, which serves as both a natural reservoir for BMP and as a stimulus for ADSC-derived BMP production, combined with fat grafts, may offer a new treatment modality for hypertrophic scars and contractures (Figure 3). The beneficial effects of this treatment on hypertrophic scars is postulated to be achieved through the ability of BMP-2 and BMP-4 to dedifferentiate and reprogram persistently contracted myofibroblasts into adipocytes. Figure 3. View largeDownload slide Activated PRP when combined with adipose derived stem cells in fat grafts may synergistically increase BMP-2, 4, and 7 levels to the thresholds shown to dedifferentiate hypertrophic scar myofibroblasts into preadipocytes. Figure 3. View largeDownload slide Activated PRP when combined with adipose derived stem cells in fat grafts may synergistically increase BMP-2, 4, and 7 levels to the thresholds shown to dedifferentiate hypertrophic scar myofibroblasts into preadipocytes. Since we propose using activated PRP as source of BMP, several important limitations may exist which include, but are not limited to, unwanted osteogenesis, chondrogenesis, neurogenesis, or neoplasm formation.60-63 These adverse effects might occur based on many factors, including the type of BMP produced, the target cell and its expressed receptors, and the local environment including other growth factors in addition to the delivered BMP.11,13,64 However, as the studies in our review show, PRP added to a fat graft mixture should preferentially stimulate ADSCs to undergo proliferation and adipogenesis, avoiding these unwanted effects. Another important consideration is the lack of standardization in the PRP preparation methods, with many different techniques producing quite variable results.5 Using PRP kits with large platelet multipliers may prove most beneficial as they maximize the platelet numbers and, thus, the released growth factors. Many authors suggest PRP protocols aiming to set a standard, but unfortunately there is still no consensus on the best technique.2 There is also considerable heterogeneity in the concentration of platelets in the patients’ blood. Consequently, a range of platelet concentrations are often obtained in PRP between donors prepared with the same protocol.2 To make matters more complicated, not every patient’s platelets contain the same amount or type of BMPs. One study on a cohort of 31 donors demonstrated that only two patients contained all four studied BMPs (2, 4, 6, and 7) in their platelets, and another two patients contained no detectable BMPs.43 This issue may contribute to the variable results seen in previous PRP studies and serve as significant limitation. To avoid these issues and ineffective therapy, researchers can screen a patient’s platelet-derived BMPs and levels prior to treatment. Additionally, fat grafts with ADSCs plus PRP provide a mechanism for overcoming BMP deficiencies via de novo ADSC BMP production. Unfortunately, nonactivated and leukocyte-rich PRP stimulate the formation of the myofibroblast phenotype. To address these issues, protocols for preparing pure PRP (P-PRP) instead of leukocyte-rich PRP (L-PRP) can be utilized to minimize the release of the antiadipogenic, proinflammatory molecules, such as TGF-β and TNF-α, found in leukocytes.65,66 In addition, activating PRP in an acidic environment maximizes the BMP yield and serves as the only way to release a detectable amount of BMP-2.42,43 Finally, it is desirable to examine different methods of activating PRP to assess whether even higher BMP levels can be achieved. The pathophysiology of hypertrophic scarring lies in the existence of persistently present and contracted myofibroblasts. The management of hypertrophic scarring through surgical and nonsurgical techniques have yet to fully address this underlying pathology. Our laboratory, The Center for Tissue Engineering at UC Irvine, is currently working on studying the effects of combining PRP and fat grafts as a novel injectable for dedifferentiating myofibroblasts in hypertrophic scars. Disclosures The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding The authors received no financial support for the research, authorship, and publication of this article. REFERENCES 1. Lynch MD , Bashir S . Applications of platelet-rich plasma in dermatology: a critical appraisal of the literature . J Dermatolog Treat . 2016 ; 27 ( 3 ): 285 - 289 . Google Scholar Crossref Search ADS PubMed 2. Dhurat R , Sukesh M . Principles and methods of preparation of platelet-rich plasma: a review and author’s perspective . 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Reprints and permission: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Aesthetic Surgery JournalOxford University Press

Published: Nov 12, 2018

References

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