Naoto Yamamoto

Accelerated wound healing in healing-impaired db/db mice by autologous adipose tissue-derived stromal cells combined with atelocollagen matrix.

Adipose tissue-derived stromal cells (ATSCs) have recently gained widespread attention as a potential alternate source to bone marrow–derived mesenchymal stem cells with a proliferative capacity and a similar ability to undergo multilineage differentiation. In this study, we evaluated the effectiveness of freshly isolated autologous ATSCs-containing atelocollagen matrix with silicon membrane (ACMS) on wound healing of diabetic (db/db) mice. Cultured ATSCs from (db/db) mice secreted significant amounts of growth factors and cytokines, which are suitable for wound repair. Two full thickness round skin defects were made on the backs of healing-impaired db/db mice. Freshly isolated autologous ATSCs-containing ACMS or ACMS alone were applied to the wounds. Twelve mice were treated and then killed at 1 or 2 weeks (n = 6 each). Histologic sections of the wounds were prepared at each time period after treatment. Histologic examination demonstrated significantly advanced granulation tissue formation, capillary formation, and epithelialization in diabetic healing-impaired wounds treated with autologous ATSCs-containing ACMS, compared with mice treated with ACMS alone. These results suggested that transplantation of autologous ATSCs-containing ACMS significantly accelerated wound healing in diabetic healing-impaired db/db mice.

Enhanced Effect of Platelet-Rich Plasma Containing a New Carrier on Hair Growth

BACKGROUND Treatments for alopecia are in high demand, but not all are safe and reliable. Dalteparin and protamine microparticles (D/P MPs) can effectively carry growth factors (GFs) in platelet‐rich plasma (PRP). OBJECTIVE To identify the effects of PRP‐containing D/P MPs (PRP&D/P MPs) on hair growth. METHODS & MATERIALS Participants were 26 volunteers with thin hair who received five local treatments of 3 mL of PRP&D/P MPs (13 participants) or PRP and saline (control, 13 participants) at 2‐ to 3‐week intervals and were evaluated for 12 weeks. Injected areas comprised frontal or parietal sites with lanugo‐like hair. Experimental and control areas were photographed. Consenting participants underwent biopsies for histologic examination. RESULTS D/P MPs bind to various GFs contained in PRP. Significant differences were seen in hair cross‐section but not in hair numbers in PRP and PRP&D/P MP injections. The addition of D/P MPs to PRP resulted in significant stimulation in hair cross‐section. Microscopic findings showed thickened epithelium, proliferation of collagen fibers and fibroblasts, and increased vessels around follicles. CONCLUSION PRP&D/P MPs and PRP facilitated hair growth but D/P MPs provided additional hair growth. The authors have indicated no significant interest with commercial supporters.

Enhanced healing of mitomycin C-treated wounds in rats using inbred adipose tissue-derived stromal cells within an atelocollagen matrix.

The aim of this study was to evaluate the potential accelerating effects of an adipose tissue‐derived stromal cells (ATSC)‐containing atelocollagen matrix with silicone membrane (ACMS) for repairing mitomycin C‐treated healing‐impaired wounds. Mitomycin C was applied to full‐thickness skin incisions in this study to create a healing‐impaired wound model in rat. After thoroughly washing out the mitomycin C from the wound, ACMS alone or ATSC‐containing ACMS was applied to the wounds. Histological sections of the wounds were then prepared at indicated time periods after the treatments. These results indicated significantly advanced granulation tissue and capillary formations in the healing‐impaired wounds treated with ATSC‐containing ACMS compared with those treated with ACMS alone. Thus, this study suggested that transplantation of inbred ATSC‐containing ACMS is effective for repairing healing‐impaired wounds.

Platelet-rich Plasma (prp) Promotes Survival of Fat-grafts in Rats

This study evaluated the effects of platelet-rich plasma (PRP) on resorption and adipocyte survival in autologous fat-graft of rats prepared with isogenous PRP. Fat grafts prepared without PRP (control group) became united to the tissue adjacent to the implantation site and were significantly resorbed from 30 days. On the other hand, fat grafts prepared with PRP (PRP group) demonstrated little resorption from 30 to 120 days and appeared pink, had a soft, supple feel, and were easily compressible. Histologic sections of grafts in the control and PRP groups at 10 days exhibited similar consolidation of the grafted tissue, which contained morphologically normal adipocytes with different degrees of granulation and capillary formation. From 20 days normal adipocytes were obviously decreased in the control group, while the PRP group demonstrated increased granulation tissue and capillary formation and good maintenance of normal adipocytes for at least 120 days.

Detection of skin perforators by indocyanine green fluorescence nearly infrared angiography.

Perforator-based island flaps are widely used to reconstruct skin defects. For this procedure to succeed, a perforator with adequate blood flow must be selected, and precise preoperative prediction of the location of the perforators is required. To identify perforators, a variety of methods have bee

Enhancement of vascularization and granulation tissue formation by growth factors in human platelet‐rich plasma‐containing fragmin/protamine microparticles

The purpose of this study was to evaluate effects of human platelet-rich plasma (PRP)-containing fragmin/protamine microparticles (F/P MPs) as a protein carrier on neovascularization and granulation tissue formation. Frozen and thawed PRP contains high concentrations of various growth factors (GFs) and F/P MPs effectively adsorb those GFs. Human microvascular endothelial cells (MVECs) and dermal fibroblast cells (DFCs) were optimally grown in medium containing 4% PRP and the addition of F/P MPs significantly maintained and protected the proliferative activity of PRP incubated at 37°C for more than 10 days. When PRP-containing F/P MPs were subcutaneously injected into the back of mice, significant neovascularization was induced near the injected site with enhanced filtration of inflammatory cells from day 3 to day 30, compared with controls (injections of PRP, F/P MPs, and saline). Both PRP-containing F/P MPs and PRP alone induced significant formation of granulation tissue at the injected site. However, thickness of induced granulation tissues was well maintained for 30 days only in PRP-containing F/P MP-injected group. Those bound GFs may be gradually diffused and released from F/P MPs in vitro and in vivo. Thereby, PRP-containing F/P MPs offer significantly higher inductions of vascularization and fibrous tissue formation in vivo than PRP alone.

PRP&F/P MPs Improved Survival of Dorsal Paired Pedicle Skin Flaps in Rats

BACKGROUND Skin flap necrosis is a problem encountered postoperatively. The purpose of this study was to evaluate the effects of platelet-rich plasma containing fragmin/protamine microparticles (PRP&F/P MPs) on viability in a rat dorsal paired pedicle skin (DPPS) flap. MATERIALS AND METHODS Two symmetrical adjoining rectangular flaps (8 × 2 cm each) were drawn on the rat dorsum. Two days after PRP&F/P MPs-, PRP-, F/P MPs-, and saline (control)-injections (n = 8 each), flaps were elevated as a random pattern flap without the lateral thoracic, posterior intercostal, and deep circumflex iliac vessels. The flaps were immediately sutured back and the flap survival area was measured 7 d after flap elevation. RESULTS The flap survival rate in PRP&F/P MPs-injected groups (73.1% ± 4.2%) was significantly higher than those in PRP (64.9% ± 4.0%), F/P MPs (59.4 ± 4.5%), and control (61.2% ± 4.2%) groups. Histologic observation of the flaps showed survived thick granulation tissue and neovascularization in PRP&F/P MPs-injected groups. CONCLUSIONS When PRP&F/P MPs are administered 2 d before the flap elevation, the improved flap survivals are observed. The pre-injection of PRP&F/P MPs may thus represent a promising treatment to prevent skin flap necrosis in reconstructive surgery.

The role of collagen arrangement change during tendon healing demonstrated by scanning electron microscopy

The dry weight of tendon tissue is accounted for mainly by collagen fibers. Accordingly, the tendon-healing process primarily involves repair of collagen fibers. During the remodeling phase of tendon healing, newly proliferating collagen fibers are transformed into a mature repaired tendon. Despite the importance of this phenomenon, the details of fibrous rebuilding have not been reported previously. The aim of this study was to visualize the ultrastructural changes and to obtain a clear understanding of the reorganization of the collagen fibers in the tendon repair site, using rat Achilles tendons. We used scanning electron microscopy (SEM) with cell maceration as the main method of analysis. Pretreatment with cell maceration removed the cellular components successfully. This allowed precise visualization of each collagen fiber and the three-dimensional network of the fibers. This study was the first to apply the cell-maceration/SEM method to observe tendon tissue. Seven days after surgery, new collagen fibers grew extensively in the repair site in a random arrangement. Fourteen days after surgery, the collagen fibers began to form an axial arrangement. Near the tendon stump, this change progressed from the outer layer to the core region. On the other hand, in the middle of the repair site, it progressed from the core to the outer layer. Change in the axial arrangement of collagen fibers contributes to the connection between the repair site and the tendon stump and to the separation of the repair site from the paratenon.

Stimulatory Effect of Autologous Adipose Tissue-Derived Stromal Cells in an Atelocollagen Matrix on Wound Healing in Diabetic db/db Mice

We aimed to evaluate the effectiveness of the application of an atelocollagen matrix containing autologous adipose tissue-derived stromal cells (ASCs) on wound healing in diabetic (db/db) mice. Cultured ASCs from db/db mice and from db/+ mice secreted identical amounts of growth factors, cytokines, and type I collagen. ASCs from db/db mice proliferated at the same rate as those from db/+ mice. When DiI-labeled ASCs were applied to full-thickness round skin wounds on the backs of diabetic db/db mice, histological observation at 2 weeks showed that red fluorescent-labeled tissues were formed in the epidermis, dermis, and capillaries. Twelve db/db mice were treated with either matrix alone or matrix containing ASCs and then sacrificed at 1 or 2 weeks. A histological examination demonstrated significantly advanced granulation tissue formation, capillary formation, and epithelialization in those wounds treated with atelocollagen matrix containing ASCs, compared with wounds treated with matrix alone.

Increased survival of free fat grafts and vascularization in rats with local delivery of fragmin/protamine microparticles containing FGF-2 (F/P MP-F)

We evaluated the effects of fragmin/protamine micro-particles (F/P MPs) containing FGF-2 (F/P MP-F) as carriers for the controlled release of FGF-2 for adipocyte-survival and capillary formation in inbred rats with subdivided free fat grafts. F/P MPs could immobilize FGF-2, thereafter gradually releasing the bound FGF-2. Inbred Fisher 344 rats weighing around 150 g were anesthetized and implanted with paste comprising harvested fat combined with F/P MP-F. The effect of F/P MP-F on the survival, granulation, and capillary formation in fat grafts was histologically compared with control grafts containing either FGF-2, F/P MPs or PBS. The control fat grafts became attached to tissues adjacent to the implantation site and were significantly resorbed after 30 days. In contrast, pink, soft, supple grafts were compressible and were little resorbed in the group given F/P FP MP-F at 30-120 days. Normal adipocytes were obviously decreased in the control groups with increased granulation tissues, whereas normal adipocytes with capillary formations were maintained in the F/P MP-F group. Thus, adding F/P MP-F to subdivided fat grafts helps to improve graft volume retention and survival in soft-tissue reconstruction through accelerating adipocyte-survival rates and angiogenesis.