吉村 浩太郎

BACKGROUND: Clinical sequelae of irradiation result in tissue devitalization (e.g., ischemia, fibrosis, and atrophy) where wound healing capacity is impaired. Fat-derived products may work to treat such pathology. METHODS: Nonlethal irradiation at various doses (5, 10, and 15 Gy) and frequencies (one to three times on sequential days) was delivered to dorsal skin of nude mice, and subsequent gross and microscopic changes were evaluated for up to 4 weeks. Cutaneous punch wounds were then created to compare wound healing in irradiated and nonirradiated states. Wounds were also locally injected with vehicle, cultured adipose-derived stem cells, centrifuged fat tissue, or micronized cellular adipose matrix, and the therapeutic impact was monitored for up to 15 days. RESULTS: Nude mice given total doses greater than 15 Gy spontaneously developed skin ulcers, and radiation damage was dose-dependent; however, a fractionated irradiation protocol was able to reduce the damage. Histologic assessment revealed dose-dependent dermal fibrosis/thickening and subcutaneous atrophy. Dose-dependent (5 to 15 Gy) impairment of wound healing was also evident. At the highest dosage (15 Gy three times), open wounds persisted on day 15. However, wounds injected with cultured adipose-derived stem cells were nearly healed on day 12, and those treated with injection of centrifuged fat or micronized tissue healed faster than untreated controls (p < 0.05). There was no significant differences between treated groups. CONCLUSIONS: Tissue devitalization by irradiation was dose-dependent, although fractionated protocols helped to reduce it. Adipose-derived stem cells and other fat-derived products harboring adipose-derived stem cells successfully revitalized irradiated tissues and accelerated wound healing.

Chronic changes following radiotherapy include alterations in tissue-resident stem cells and vasculatures, which can lead to impaired wound healing. In this study, novel recombinant human collagen peptide (rhCP) scaffolds were evaluated as a biomaterial carrier for cellular regenerative therapy. Human adipose-derived stem cells (hASCs) were successfully cultured on rhCP scaffolds. By hASC culture on rhCP, microarray assay indicated that expression of genes related to cell proliferation and extracellular matrix production was upregulated. Pathway analyses revealed that signaling pathways related to inflammatory suppression and cell growth promotion were activated as well as signaling pathways consistent with some growth factors including vascular endothelial growth factor, hepatocyte growth factor, and transforming growth factor beta, although gene expression of these growth factors was not upregulated. These findings suggest the rhCP scaffold showed similar biological actions to cytokines regulating cell growth and immunity. In subsequent impaired wound healing experiments using a locally irradiated (20 Gray) mouse, wound treatment with rhCP sponges combined with cultured hASCs and human umbilical vein endothelial cells accelerated wound closure compared with wounds treated with rhCP with hASCs alone, rhCP only, and control (dressing alone), with better healing observed according to this order. These results indicating the therapeutic value of rhCP scaffolds as a topical biomaterial dressing and a biocarrier of stem cells and vascular endothelial cells for regenerating therapies. The combination of rhCP and functional cells was suggested to be a potential tool for revitalizing stem cell-depleted conditions such as radiation tissue damage.

BACKGROUND: Fat grafting frequently requires multiple treatments and thus repeated liposuction to achieve treatment goals. The purpose of this study was to evaluate whether cryopreservation of adipose tissue may facilitate future fat grafting. METHODS: Lipoaspirates were harvested from six women and preserved using two cryopreservation methods: (1) simple cooling to -80°C (cryo-1); or (2) programmed cooling to -196°C (cryo-2). Fresh fat, cryo-1 fat, and cryo-2 fat were analyzed both in vitro and in vivo. RESULTS: Immunohistochemistry of both types of cryopreserved adipose tissue revealed that most adipocytes were necrotic. The cell number and viability of stromal vascular fraction cells were significantly decreased in cryo-1 fat (1.7 × 10 cells, 42.6 percent viable) and cryo-2 fat (2.0 × 10 cells, 55.4 percent viable), compared with fresh fat (3.9 × 10 cells, 90.6 percent viable). Although adipose-derived stem cells were cultured successfully from all fats, functional adipose-derived stem cells from cryopreserved fats were much fewer, with comparable multilineage differentiating capacity. In vivo studies using human fat grafted into immunocompromised mice revealed that, 3 months after transplantation, all of the cryopreserved fats maintained their volume to some extent; however, the cryopreserved fats were mostly filled with dead tissue and produced significantly lower engraftment scores than fresh fat. CONCLUSIONS: Most adipocytes were killed in the process of cryopreservation and thawing. Adipose-derived stem cells were isolated from cryopreserved fat, but the number of functional adipose-derived stem cells was very limited in both cryopreservation methods. After grafting, cryopreserved fat was retained as dead and fibrous tissue, suggesting a risk of clinical complications such as oil cysts.