Sadanori Akita

Human mesenchymal stem cells successfully improve skin-substitute wound healing.

Background  Large or deteriorated skin defects are sometimes life threatening. There is increasing evidence that adult stem cells are useful for tissue regeneration. Human mesenchymal stem cells (hMSCs) are self‐renewing and are potent in differentiating into multiple cells and tissues.

Cranial bone defect healing is accelerated by mesenchymal stem cells induced by coadministration of bone morphogenetic protein-2 and basic fibroblast growth factor

To facilitate bone healing in difficult circumstances, and to replace conventional therapeutic modalities, highly purified bone marrow‐derived human mesenchymal stem cells (hMSCs) were investigated for induction of their osteogenic lineage upon provision of cytokine cues in vitro and in the cranial defect model in vivo. Alkaline phosphatase‐expressing cells were most frequently observed when the hMSCs were treated with 2.5 ng/ml of basic fibroblast growth factor (bFGF) and 50 ng/ml of bone morphogenetic protein (BMP)‐2 for 4 days in culture after a 6‐day incubation in osteogenic medium containing dexamethasone, ascorbic acid‐2‐phosphate, and β‐glycerophosphate. Four‐millimeter full‐thickness cranial defect wounds were made in male nude rats (F344/NJCl‐rnu), whose deficit in the T cell compartment prevented T‐cell–mediated cellular rejection. The animals were treated for 4 weeks with hMSCs and application of 10 µg each of bFGF and BMP‐2 that had been soaked into a gelatin sponge carrier. Significant bone mineral density was observed by dual X‐ray absorptiometry and this treatment also produced histologically mature osteocytes surrounded by both osteoblasts and osteoclasts expressing alkaline phosphatase and osteocalcin. The bone mineral densities and histological structures were matched at 8 weeks post‐transplantation. Therefore, human bone marrow‐derived mesenchymal stem cells are able to differentiate into an osteogenic lineage upon cytokine stimulation and accelerate healing in a nude rat cranial bone healing model.

Basic fibroblast growth factor accelerates and improves second-degree burn wound healing.

Second‐degree burns are sometimes a concern for shortening patient suffering time as well as the therapeutic choice. Thus, adult second‐degree burn patients (average 57.8 ± 13.9 years old), mainly with deep dermal burns, were included. Patients receiving topical basic fibroblast growth factor (bFGF) or no bFGF were compared for clinical scar extent, passive scar hardness and elasticity using a Cutometer, direct scar hardness using a durometer, and moisture analysis of the stratum corneum at 1 year after complete wound healing. There was significantly faster wound healing with bFGF, as early as 2.2 ± 0.9 days from the burn injury, compared with non‐bFGF use (12.0 ± 2.2 vs. 15.0 ± 2.7 days, p<0.01). Clinical evaluation of Vancouver scale scores showed significant differences between bFGF‐treated and non‐bFGF–treated scars (p<0.01). Both maximal scar extension and the ratio of scar retraction to maximal scar extension, elasticity, by Cutometer were significantly greater in bFGF‐treated scars than non‐bFGF–treated scars (0.23 ± 0.10 vs. 0.14 ± 0.06 mm, 0.59 ± 0.20 vs. 0.49 ± 0.15 mm: scar extension, scar elasticity, bFGF vs. non‐bFGF, p<0.01). The durometer reading was significantly lower in bFGF‐treated scars than in non‐bFGF–treated scars (16.2 ± 3.8 vs. 29.3 ± 5.1, p<0.01). Transepidermal water loss, water content, and corneal thickness were significantly less in bFGF‐treated than in non‐bFGF–treated scars (p<0.01).

Mesenchymal stem cell therapy for cutaneous radiation syndrome.

Systemic and local radiation injuries caused by nuclear power reactor accidents, therapeutic irradiation, or nuclear terrorism should be prevented or properly treated in order to improve wound management and save lives. Currently, regenerative surgical modalities should be attempted with temporal artificial dermis impregnated and sprayed with a local angiogenic factor such as basic fibroblast growth factor, and secondary reconstruction can be a candidate for demarcation and saving the donor morbidity. Human mesenchymal stem cells and adipose-derived stem cells, together with angiogenic and mitogenic factor of basic fibroblast growth factor and an artificial dermis, were applied over the excised irradiated skin defect and were tested for differentiation and local stimulation effects in the radiation-exposed wounds. The perforator flap and artificial dermal template with growth factor were successful for reconstruction in patients who were suffering from complex underlying disease. Patients were uneventfully treated with minimal morbidities. In the experiments, the hMSCs are strongly proliferative even after 20 Gy irradiation in vitro. In vivo, 4 Gy rat whole body irradiation demonstrated that sustained marrow stromal (mesenchymal stem) cells survived in the bone marrow. Immediate artificial dermis application impregnated with cells and the cytokine over the 20 Gy irradiated skin and soft tissues demonstrated the significantly improved fat angiogenesis, architected dermal reconstitution, and less inflammatory epidermal recovery. Detailed understanding of underlying diseases and rational reconstructive procedures brings about good outcomes for difficult irradiated wound healing. Adipose-derived stem cells are also implicated in the limited local injuries for short cell harvesting and processing time in the same subject.

The quality of pediatric burn scars is improved by early administration of basic fibroblast growth factor.

Pediatric burn wounds can be problematic because an accurate evaluation is difficult as the result of anatomically immature vasculature or immobilization failure, especially in patients with second-degree burns, and because the burn surface areas and the burn depth tend to worsen over the course of time. Delayed wound healing results in unsightly scarring, such as hypertrophic scars, which are problematic both esthetically and functionally. Among cytokines and growth factors, basic fibroblast growth factor (bFGF) is clinically proven, having demonstrated accelerated acute and chronic wound healing. Accelerated wound healing may lead to improved scarring. To elucidate the effects of bFGF on second-degree pediatric burn wounds, a comparative study was performed. A total of 20 pediatric patients ranging from 8 month to 3 years (average 1 year, 3 months ± 6 months) who suffered from the burns by various causes were divided into two groups, conventional (n = 10) and treatment with bFGF (n = 10). A moisture meter, used to objectively measure the stratum corneum and epithelial—mesenchymal functions, was used to assess scars at least 1 year after wound healing. Clinical evaluation of pigmentation, pliability, height, and vascularity demonstrated significant differences between conventional and bFGF-treated scars (1.7 ± 0.55 vs 0.7 ± 0.58, 2.4 ± 0.82 vs 1.1 ± 0.69, 1.8 ± 0.66 vs 0.5 ± 0.57, 1.9 ± 0.63 vs 0.8 ± 0.68; conventional vs bFGF-treated, pigmentation, pliability, height, and vascularity, respectively, P < .01). The effective contact coefficient was significantly greater in conventional wounds than bFGF-treated wounds (14.6 ± 1.68 % vs 8.7 ± 2.82 %; conventional vs bFGF, P < .01) and bFGF-treated wounds demonstrated significantly less transepidermal water loss values than conventional treatment (8.3 ± 1.90 g/m2/h vs 5.7 ± 1.85 g/m2/hr; conventional vs bFGF, P < .01). Pediatric burn patients treated with bFGF showed less damaging function of the stratum corneum after healing both in clinical assessment and moisture meter analysis.

Noncultured autologous adipose-derived stem cells therapy for chronic radiation injury.

Increasing concern on chronic radiation injuries should be treated properly for life-saving improvement of wound management and quality of life. Recently, regenerative surgical modalities should be attempted with the use of noncultured autologous adipose-derived stem cells (ADSCs) with temporal artificial dermis impregnated and sprayed with local angiogenic factor such as basic fibroblast growth factor, and secondary reconstruction can be a candidate for demarcation and saving the donor morbidity. Autologous adipose-derived stem cells, together with angiogenic and mitogenic factor of basic fibroblast growth factor and an artificial dermis, were applied over the excised irradiated skin defect and tested for Patients who were uneventfully healed with minimal donor-site morbidity, which lasts more than 1.5 years.

PAX9 and TGFB3 are linked to susceptibility to nonsyndromic cleft lip with or without cleft palate in the Japanese: population-based and family-based candidate gene analyses

AbstractThe prevalence of nonsyndromic cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO) are believed to be higher in the Japanese than in Americans, Europeans or Africans. The purpose of this study was to investigate, in a Japanese population, relationships between CL/P or CPO and seven candidate genes (TGFB3, DLX3, PAX9, CLPTM1, TBX10, PVRL1, TBX22) that showed positive associations in other populations and are expressed in the oral/lip region in developing mice. We first searched for mutations in these genes among 112 CL/P and 16 CPO patients, and found a heterozygous missense mutation (640A>G, S214G) in exon 3 of PAX9 in two sibs with CL/P and their phenotypically normal mother from a Japanese family. A population-based case-control analysis and a family-based transmission disequilibrium test (TDT), using single nucleotide polymorphisms (SNPs), and two-SNP haplotypes of the genes, between the 112 CL/P cases with their parents and 192 controls indicated a significant association at one SNP site, IVS1+5321, in TGFB3 with a P-value of 0.0016. Population-based haplotyping revealed that the association was most significant for haplotype “A/A” consisting of IVS1+5321 and IVS1−1572; TDT also gave a P-value of 0.0252 in this haplotype.

Bone morphogenetic protein-2 regulates proliferation of human mesenchymal stem cells

Human mesenchymal stem cells obtained from the iliac crest of a single donor were investigated for cell proliferation, cell cycle profile, gene expression, and ultrastructural changes using electron microscopy. The human mesenchymal stem cells significantly increased their cell number by day 2 after treatment with bone morphogenetic protein‐2 alone, or basic fibroblast growth factor alone or combinations of both proteins under serum‐free conditions (p < 0.01). The human mesenchymal stem cells showed marked expression of cell nuclear antigen, notably at day 1, and pituitary tumor transforming gene throughout the experiment, suggesting cell cycle progression by bone morphogenetic protein‐2 treatment. In addition, strong cellular nuclear bromodeoxyuridine incorporation was seen by immunocytochemistry. Fluorescence‐activated cell sorting also showed a similar pattern of cell cycle progression with bone morphogenetic protein‐2 treatment in serum‐free medium and 10% fetal bovine serum treatment. The bone morphogenetic protein‐2–treated human mesenchymal stem cells showed heterochromatin in the nucleus, suggesting cell differentiation, and well‐developed granular endoplasmic reticulum, indicative of protein production. Overall, the human mesenchymal stem cells successfully proliferated with appropriate cell cycle progression and the cell ultrastructural morphology suggested marked nuclear and granular endoplasmic reticulum induction by bone morphogenetic protein‐2 treatment in serum‐free medium. (WOUND REP REG 2003;11:354–360)

Leukemia inhibitory factor gene improves skin allograft survival in the mouse model

BACKGROUND Leukemia inhibitory factor (LIF) is a widely expressed cytokine involved in both local and systemic immune response. Furthermore, it has been implicated in various immunological processes including thymic T cell maturation and embryo implantation. We investigated implication of various modalities in the application of prolonged and viable allograft to the wound, using cytokines and growth factors. MATERIALS BALB/c and B6D2F1 strains of mice were used either as a skin graft donor or host. LIF cDNA inserted in plasmid vector or the vector alone was injected intradermally in graft skin and observed up to 21 days. LIF, LIF-receptor, gp130, as well as type 1 and 2 T helper cytokine expressions were investigated by reverse transcription polymerasse chain reaction, in situ hybridization, and histological studies. RESULTS LIF cDNA-treated groups showed significantly improved graft survival compared to the vector-treated control in 21 days postoperatively for grafting from B6D2F1 to BALB/c and BALB/c to B6D2F1. LIF and LIF receptor mRNA expressions were observed 24 hr and 21 days posttransplantation. The gp130 expression was only observed in LIF-treated B6D2F1 to BALB/c allografting on day 21 posttransplantation. LIF transcripts were strongly present in the epidermal, dermal, and subdermal tissues as determined by an in situ hybridization of LIF-treated grafting. CONCLUSIONS These results suggest that LIF cDNA treatment is an effective and beneficial adjuvant for the skin allograft survival. Improved skin allograft modulation by cytokine gene transfer is a potentially promising therapy for temporary large skin coverage.

A Basic Fibroblast Growth Factor Improves Lower Extremity Wound Healing With a Porcine-Derived Skin Substitute

BACKGROUND Although a number of cytokine or growth factor therapies for wound acceleration have been reported, few mentioned the quality of the outcome. The lower extremity is important in esthetics as well as in function, because it is exposed. Recently, a growth factor, namely basic growth factor (bFGF) is widely used for difficult wound healing with a porcine-derived bilayered artificial dermis for better wound closure. Thus, their combination use was tested clinically. METHODS Sequential lower extremity reconstruction by an artificial dermis with or without bFGF administration and secondary split-thickness skin grafting was measured for hardness using a durometer, and the moisture parameters assessed such as effective contact coefficient, transepidermal water loss (TEWL), water content and thickness using a moisture meter for at least 6 months after the final procedure and compared with normal skin control. RESULTS There was significantly less skin hardness using a durometer in bFGF treatment compared with non-bFGF treatment (16.2 +/- 3.83 vs. 29.2 +/- 4.94, p < 0.01). Effective contact coefficient, TEWL, water content, and thickness in non-bFGF treatment were all significantly greater than those in bFGF treatment, whereas water content and thickness in bFGF treatment were comparable with those of the control. CONCLUSION The use of bFGF as artificial dermis for extensive and deeper tissue loss coverage demonstrated better reconstruction quality in terms of hardness using a durometer and the function of the stratum corneum by moisture analysis.