Natsuko Kakudo

Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

Background: This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-&bgr;1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose–derived stem cells and human dermal fibroblasts. Methods: Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-&bgr;1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco’s Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco’s Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose–derived stem cells and human dermal fibroblasts were assayed for proliferation. Results: Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-&bgr;1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose–derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Conclusions: Platelet-rich plasma can enhance the proliferation of human adipose–derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

Bone regeneration with BMP-2 and hydroxyapatite in critical-size calvarial defects in rats

BACKGROUND This study describes the effect of bone formation by BMP-2 (bone morphogenetic protein-2), a bone formation inducer, with or without hydroxyapatite (HAP) application to critical-size defects in rat calvarial bone. MATERIAL AND METHODS Twenty male Wistar rats were divided into four groups of 5 animals each: control, HAP, BMP, and mixed BMP/HAP. A Critical-size defect of 4mm was made using a trephine in the calvarial bone and, after that, BMP and/or HAP was applied to the defect according to the grouping. Defects were evaluated radiographically and histologically using ImageJ color analyzer software at 4 weeks postoperatively. RESULTS The histological data were more precise than the radiologic data due to the white color of the porous-type HAP material. The highest radiopacity was noted in the mixed BMP/HAP group (162.07±9.06), followed by the HAP group (133.15±21.8), then the BMP group (100.79±8.27), and, lastly, the control group (54.45±8.39). After subtracting the white background and using ImageJ for histological analysis, the highest rate of osteochondrogenesis was in the mixed BMP/HAP group (85.29%±8.21), and then the BMP group (77.34%±7.39), followed by the HAP group (59.82%±11.23), and, lastly, the control group (40.27%±7.44). Differences in the values between groups were then analyzed using confidence intervals (CI) of 95 and 99%. CONCLUSION Within 4 weeks, the mixed BMP/HAP group showed the highest level of bone induction, especially compared to the BMP group, but this was non-significant; even with a 95% CI, the result was negative. This reveals that BMP alone can be applied, with a final result the same as that seen in the mixed BMP/HAP group. BMP and HAP, both being osteoinducting agents, even though they differ from a material classification point of view, have a positive effect on osteogenesis.

Effects of platelet-rich plasma on proliferation and myofibroblastic differentiation in human dermal fibroblasts.

AbstractPlatelet-rich plasma (PRP) is plasma that is produced from autologous blood, and its usefulness in plastic surgery and dermal wound healing has garnered attention in recent years. The aim of this study was to investigate the effects of PRP and platelet-poor plasma on the proliferation and differentiation of skin fibroblasts into myofibroblasts and on wound contraction using Western blotting, immunofluorescence staining, and collagen gels containing an embedded fibroblast model. PRP promotes proliferation of human dermal fibroblasts. PRP addition enhanced the expression of alpha-smooth muscle actin protein, a myofibroblast marker, as shown by immunofluorescence staining and Western blotting. PRP-treated groups demonstrated more marked contraction in the collagen gel model than the platelet-poor plasma and vehicle groups. PRP promotes proliferation, causes the differentiation of human dermal fibroblasts into myofibroblasts and promotes wound contraction, thus providing a potential therapeutic agent for skin wound healing.

Platelet-rich plasma releasate promotes angiogenesis in vitro and in vivo

Platelet-rich plasma (PRP) is a plasma fraction in which several growth factors are concentrated at high levels. In recent years, the biological effects on various cells of the active soluble releasate that is isolated following platelet activation of PRP [PRP-releasate (PRPr)] have been reported. The purpose of this study was to determine the angiogenic effects of human PRPr in vitro and in vivo. PRPr was prepared from human whole blood using the double spin method and was activated with CaCl2 and autologous thrombin. PRPr stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) and in vivo angiogenesis-inducing ability in nude mice. PRPr led to the phosphorylation of Erk1/2 and Akt in HUVECs, and the induction of proliferation and migration by PRPr was suppressed by PRPr inhibitors PD98059 and LY294002. PRPr induces angiogenesis in vitro and in vivo, and the present findings suggest that the mechanism for this is activation of the ERK and phosphatidylinositol-3-kinase–Akt pathways. Our results demonstrate that PRPr is a promising autologous source for therapeutic angiogenesis in treating cardiovascular disease.

Platelet and growth factor concentrations in activated platelet-rich plasma: a comparison of seven commercial separation systems

Platelet-rich plasma (PRP) is blood plasma that has been enriched with platelets. It holds promise for clinical use in areas such as wound healing and regenerative medicine, including bone regeneration. This study characterized the composition of PRP produced by seven commercially available separation systems (JP200, GLO PRP, Magellan Autologous Platelet Separator System, KYOCERA Medical PRP Kit, SELPHYL, MyCells, and Dr. Shin’s System THROMBO KIT) to evaluate the platelet, white blood cell, red blood cell, and growth factor concentrations, as well as platelet-derived growth factor-AB (PDGF-AB), transforming growth factor beta-1 (TGF-β1), and vascular endothelial growth factor (VEGF) concentrations. PRP prepared using the Magellan Autologous Platelet Separator System and the KYOCERA Medical PRP Kit contained the highest platelet concentrations. The mean PDGF-AB concentration of activated PRP was the highest from JP200, followed by the KYOCERA Medical PRP Kit, Magellan Autologous Platelet Separator System, MyCells, and GLO PRP. TGF-β1 and VEGF concentrations varied greatly among individual samples, and there was almost no significant difference among the different systems, unlike for PDGF. The SELPHYL system produced PRP with low concentrations of both platelets and growth factors. Commercial PRP separation systems vary widely, and familiarity with their individual advantages is important to extend their clinical application to a wide variety of conditions.

Platelet-rich plasma promotes epithelialization and angiogenesis in a splitthickness skin graft donor site.

Autologous platelet-rich plasma contains multiple growth factors. We performed a side-by-side (half-side) test between the platelet-rich plasma (PRP)-treated and control (untreated) sides of a split-thickness skin graft donor site, and compared the number of days until epithelialization and pain during gauze change. On day 13 after surgery, we performed punch biopsy on the two sides and for adjacent normal skin tissue and compared the epidermal thickness and numbers of collagen fibers and newly formed vessels in the dermis by H&E staining, elastica van Gieson staining, and α-smooth muscle actin (α-SMA) immunostaining. Epithelialization progressed more rapidly, pain during gauze change was milder, and the epidermal thickness and number of newly formed vessels in the dermis were significantly greater on the PRP-treated side. This study revealed that PRP promotes epithelialization and angiogenesis of split-thickness skin graft donor sites.

Hypoxia Enhances Proliferation of Human Adipose-Derived Stem Cells via HIF-1ɑ Activation

Background Adipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined. Methods Normal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF–1α-binding hypoxia responsive element in FGF–2 was performed. HIF–1α was knocked down by siRNA, and FGF–2 expression was investigated. Results ASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5–15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF–1α expression. The levels of VEGF and FGF–2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF–2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF–1α to FGF–2 in hypoxia. HIF–1α knockdown by siRNA partially inhibited the FGF–2 expression of ASCs induced by hypoxia. Conclusion ASC proliferation was enhanced by hypoxia. HIF–1α activation, FGF–2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.

Effect of recombinant human fibroblast growth factor-2 on intramuscular ectopic osteoinduction by recombinant human bone morphogenetic protein-2 in rats

To clarify the effects of recombinant human fibroblast growth factor‐2 (rhFGF‐2) on the osteoconduction ability of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) in vivo, rhBMP‐2 (2 μg) was mixed with different doses of rhFGF‐2 (0, 16, 80, 400, or 2,000 ng), and implanted into the lower leg muscle of rats using type I collagen as a carrier. Twenty‐one days later, ectopic neoplastic bones had bone mineral content, bone area, and bone mineral density measured by means of dual energy X‐ray absorptiometry and imaged by soft X‐ray. The values for alkaline phosphatase activity and calcium content were determined, and histology obtained. In the group treated with rhFGF‐2 at 16 ng, alkaline phosphatase activity, calcium content, bone mineral content, bone area, and bone mineral density were the greatest of all treatment groups, and the richest trabeculae were histologically observed in this group. In the groups treated with rhFGF‐2 at 80, 400, or 2,000 ng, bone formation was suppressed in a dose‐dependent manner. These results indicate that rhFGF‐2 promotes ectopic rhBMP‐2‐related osteoinduction at a low concentration (16 ng) in vivo, and that it suppresses osteoinduction at a higher amount (>80 ng).

Adipose-derived regenerative cell (ADRC)-enriched fat grafting: optimal cell concentration and effects on grafted fat characteristics

BackgroundTo overcome the absorption of traditional fat grafting, techniques for adipose-derived regenerative cell (ADRC)-enriched fat grafting are currently being adapted for practical application. The Celution®800/CRS (Cytori Therapeutics, San Diego, CA) has enabled rapid grafting of the patient’s own freshly harvested ADRCs without requiring a culturing step. However, the optimal cell concentration and the effects of ADRCs on the characteristics of grafted fat after free fat grafting remain unclear.MethodsADRCs were isolated and purified from human fat tissue using the Celution®800/CRS. Animals that received fat grafting without the addition of ADRCs were designated the control group (group A). The number of ADRCs per grafted fat volume (mL) was adjusted to 3 × 105, 1.5 × 106, and 3 × 106 cells/mL (groups B, C, and D, respectively), mixed with free fat, and transplanted as ADRC-enriched fat grafting. These mixtures were transplanted subcutaneously into BALB/C Jcl-nu/nu mice. The volume of grafted fat was determined 5 months after transplantation, and histological assessments were performed.ResultsADRC-enriched fat grafting resulted in decreased fat absorption and the formation of greater numbers of new blood vessels in the grafted fat. The optimal ADRC concentration in this study was found to be 3 × 105 cells/mL (group B), with higher concentrations resulting in increased cyst and fibril formation in the grafted fat.ConclusionsThis study used the Celution®800/CRS for free fat grafting and demonstrated that the concentration of transplanted ADRCs affected the engraftment and quality of the grafted fat.

Effects of transforming growth factor-beta1 on cell motility, collagen gel contraction, myofibroblastic differentiation, and extracellular matrix expression of human adipose-derived stem cell.

Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, and their usefulness in plastic surgery has garnered attention in recent years. Although, there have been expectations that ASCs might function in wound repair and regeneration, no studies to date have examined the role of ASCs in the mechanism that promotes wound-healing. Transforming growth factor-beta1 (TGF-β1) is a strong candidate cytokine for the triggering of mesenchymal stem cell migration, construction of extracellular matrices, and differentiation of ASCs into myofibroblasts. Cell proliferation, motility, and differentiation, as well as extracellular matrix production, play an important role in wound-healing. We have evaluated the capacity of ASCs to proliferate and their potential to differentiate into phenotypic myofibroblasts, as well as their cell motility and collagen gel contraction ability, when cultured with TGF-β1. Cell motility was analyzed using a wound-healing assay. ASCs that differentiated into myofibroblasts expressed the gene for alpha-smooth muscle actin, and its protein expression was detected immunohistochemically. The extracellular matrix expression in ASCs was evaluated using real-time RT-PCR. Based on the results, we conclude that human ASCs have the potential for cell motility, extracellular matrix gene expression, gel contraction, and differentiation into myofibroblasts and, therefore, may play an important role in the wound-healing process.