Nobuyuki Shioya

Stimulation of glucose utilization and lactate production in cultured human fibroblasts by thyroid hormone

Human dermal fibroblasts were obtained by harvesting outgrowing cells from the dermal tissue explants and cultured in Dulbeccos modified Eagle medium containing 10% fetal calf serum. After the cells reached confluency, culture was continued in the medium containing calf serum which was deprived of thyroid hormone by the treatment with activated charcoal. These fibroblasts were responsive to exogeneously added thyroid hormone (triiodothyronine) at physiological concentrations, resulting in enhanced utilization of glucose and production of lactate. This timulation by thyroid hormone was dependent upon the length of exposure to the hormone and its concentration. The hormone did not show any effect on cellular DNA and protein content. The experimental system described above seems to be easy to reconstitute and should be useful for the elucidation of the mechanism of thyroid hormone action.

A Cultured Skin Substitute Composed of Fibroblasts and Keratinocytes with a Collagen Matrix: Preliminary Results of Clinical Trials

The cultured skin substitute was created through successive cultivation of fibroblasts and keratinocytes that were combined within a collagen matrix. This collagen matrix was composed of a collagen spongy sheet and a collagen gel. The collagen spongy sheet was designed to produce a honeycomb structure having many holes in which all holes through the sheet were filled with collagen gel. This specific structure thereby allows for the nourishment of the cultured keratinocytes on the surface of the matrix when placed on the graft bed. In this study, autologous cultured skin substitute was applied to a 51-year-old man who had sustained a burn injury. Three sheets of the cultured skin substitute (6 × 9.5 cm) were grafted onto the full-thickness excised wound in the right anterior chest wall. One week after grafting most of the matrix disappeared and stratified keratinocytes were seen to have firmly attached to the underlying tissue. Five weeks after grafting a cornified epidermal layer was seen. Ten months after grafting a mature epidermis and a well-differentiated papillary and reticular dermis replacement were observed. The physical properties and color of this grafted area resemble those of normal skin. In the second test case, autologous cultured skin substitute was applied to a 30-year-old man with a scar remaining after tattoo removal. Eight sheets of the cultured skin substitute (10 × 18 cm) were applied on an excised wound (thickness, 0.02–0.025 in.) of both the fore- and upper arms. The histological appearance of a biopsied skin specimen from the grafted area at 3 months after grafting showed a mature epidermis and a well-differentiated reticular dermis replacement. The regenerated skin at 14 months after grafting showed an excellent result.

Effect of a collagen matrix containing epidermal growth factor on wound contraction

Excessive wound contraction is known to lead to pathological wound contracture. Using a rabbit model, we applied a bovine type I collagen matrix sponge as a dermal substitute and human epidermal growth factor to full‐thickness excisional wounds. Wound contraction was assessed 14 and 28 days after wounding. It was found that both collagen matrix and epidermal growth factor significantly inhibited wound contraction (p < 0.001) in all wounds treated with collagen matrix alone or treated with 0.1 and 1 µg of epidermal growth factor 28 days after wounding. Interestingly, the combination of collagen matrix with epidermal growth factor strongly inhibited wound contraction over matrix alone (p < 0.01 on day 28). Histological analyses showed a regular horizontal arrangement of collagen fibers in the dermis under wounds treated with these substances but not under untreated wounds. Furthermore, using a fibroblast‐populated collagen gel, the direct inhibitory effect of epidermal growth factor on gel contraction by fibroblasts was also observed. Collagen gels without stimulation contracted to 29.5 ± 0.6% of their original size, as determined 6 days after culturing. At 3 days or more, epidermal growth factor inhibited collagen gel contraction by fibroblasts (after 6 days: 34.2 ± 1.8%, p > 0.05; 36.5 ± 2.8%, p < 0.05; and 39.8 ± 2.1%, p < 0.001 at 1, 10, and 100 ng/ml of epidermal growth factor, respectively). In conclusion, collagen matrix and epidermal growth factor, particularly in combination, may be useful in the prevention of wound contracture.

Improved in vitro angiogenesis model by collagen density reduction and the use of type III collagen.

Angiogenesis was examined by a three-dimensional model in vitro, using human umbilical vein endothelial cells (HUVECs) cultured in a collagen gel. An abundant capillary-like network with a lumen structure was identified histologically and shown to have formed at a collagen density of 0.05% or 0.10% instead of 0.15%, for either type I or type III collagen. At the same density, type III collagen induced a capillary-like network with HUVECs at an earlier stage of culture than type I collagen. In a two-dimensional culture, HUVECs were viable and proliferated to a great extent on the culture dish coated with collagen. This was particularly so in the case of type III collagen at lower density (5 ng/cm2). Type III collagen at a sufficiently low density is thus shown useful for studying angiogenesis in vitro. The capillary-like network that formed in the three-dimensional culture appeared somewhat labile, but became stable with the continuous addition of endothelial cell growth supplement and increase in the plating number of HUVECs.

Development of a new wound dressing with antimicrobial delivery capability

A bilaminar wound dressing composed of an outer membrane and an inner three‐dimensional matrix of a fabric or a sponge may be considered to constitute an ideal structure that promotes wound healing: the outer membrane prevents body fluid loss, controls water evaporation, and protects the wound surface from bacterial invasion, and the inner matrix encourages adherence by tissue growth into the matrix. Using this concept, we developed a biosynthetic wound dressing with a drug delivery capability. This medicated wound dressing is composed of a spongy sheet of a chitosane derivative and collagen mixture that is laminated to an antimicrobial‐impregnated polyurethane membrane. In this study, a gentamycin sulfate—impregnated wound dressing was prepared and evaluated. The antimicrobial efficacy of this wound dressing was examined on an agar plate seeded with Pseudomonas aeruginosa. Also, the cytotoxicity of an antimicrobial released from this wound dressing was examined in an in vitro system with cultured skin substitutes. Both in vitro tests have shown that this wound dressing is capable of suppressing bacterial growth and minimizing cellular damage. In addition, in the treatment of wounds inflicted on rats and rabbits, this wound dressing was shown to be efficacious in covering full‐thickness and split‐thickness skin defects. Finally, the efficacy of this wound dressing was evaluated in a nonrandomized open‐label study of 31 clinical cases. In 31 cases treated with this wound dressing, good or excellent wound healing was achieved.

Cryosurgery for hemangiomas of the body surface and oral cavity.

The indications for and limitations of cryosurgery for various types of hemangiomas on the body surface or in the intraoral region are reported based on our clinical experience. In our opinion, cryosurgery is not the method of choice in the management of hemangiomas but is useful under certain conditions, as with a localized cavernous under certain conditions, as with a localized cavernous hemangioma in the facial or intraoral region. It is also valuable for strawberry marks with bleeding or infection and may be acceptable for small port-wine stains; in the treatment of extensive port-wine stains, a resulting hypertrophic scar or depigmentation may present difficulties.

Evaluation of the Median Forehead Flap and the Nasolabial Flap in Nasal Reconstruction

Abstract. Basal cell carcinoma, which accounts for 70%–80% of all cutaneous malignancies in the United States, has increased recently in Japan. We compared methods for reconstruction after surgery for basal cell carcinoma, which is expected to increase further in the future. Thus patients who underwent reconstruction after surgery for basal cell carcinoma of the nose using medial forehead flaps and nasolabial flaps were selected, and the effectiveness of these flaps was compared by taking the size and location of the tissue defect into consideration. As a result, possibly because of anatomical and histological differences of the face between Caucasians and Asians, better results were obtained with nasolabial flaps than with median forehead flaps.

Development of new wound dressing composed of spongy collagen sheet containing dibutyryl cyclic AMP

Although cyclic AMP has been considered to regulate cell proliferation, the mechanism of this function is largely unknown. Recent studies suggest that cyclic AMP promotes the proliferation of skin cells in a dose-dependent manner. An ointment containing dibutyryl cyclic AMP has been used in the treatment of skin ulcers and found to be effective in promoting tissue repair. To search more efficacious wound management, the authors developed a new wound dressing composed of a spongy atelo-collagen sheet containing dibutyryl cyclic AMP. This wound dressing was evaluated in two types of animal tests. One is the application of the wound dressing to a full-thickness skin defect in order to evaluate the granulation tissue formation and the wound size reduction. The wound dressing was found to promote the granulation tissue formation and naturally reduce the wound size. The other test was the application of the wound dressing to the full-thickness skin defect, leaving behind a skin island in a central portion, in order to evaluate the epithelialization. This skin island left in a full-thickness skin defect was extremely enlarged. The enlargement of the skin island seems to be related to the epithelialization from the margin of the skin island as well as by the expansion of a skin island induced by contraction of the developed granulation tissue in the surrounding wound area. These results suggest that an atelo-collagen spongy sheet containing dibutyryl cyclic AMP is effective in promoting the granulation tissue formation and epithelialization.

Formation of hair follicles from a single-cell suspension of embryonic rat skin by a two-step procedure in vitro

SummaryA technique for culturing skin was devised whereby hair follicles in a normal state were generated from a single-cell suspension of embryonic rat skin. Dissociated cells obtained by trypsinization of the day-15 embryonic lip were cultured by a two-step procedure in vitro. Reorganization of hair-follicle rudiments was accompanied by reaggregation of the cells during a 24-hour initial culture with rotation, and the rudiments differentiated into hair follicles within a week during subsequent subculture of the cell aggregates by floatation. The light-microscopic features and the size of the follicles were similar to those of day-18 vibrissa follicles during normal development in vivo. Furthermore, the stratification of cells, including subcellular differentiation, and the ultrastructure of the hair follicles generated in vitro were similar to those of normal hair follicles with well-keratinized hair shafts. The present system appears to be a useful model for analytical studies in vitro on the formation of hair follicles and for studies designed to facilitate the transplantation of human hair.

Modified Limberg flap for lumbosacral meningomyelocele defects

We have recently developed a technique for closing round or wide oval lumbosacral meningomyelocele defects by modifying the Limberg flap. Four cases of repair of lumbosacral meningomyelocele were treated, with good skin closure in 3. The indications for and advantages of our method are discussed.