Eiju Uchinuma

CLINICAL EVALUATION OF AN ALLOGENEIC CULTURED DERMAL SUBSTITUTE COMPOSED OF FIBROBLASTS WITHIN A SPONGY COLLAGEN MATRIX

We have developed an allogeneic cultured dermal substitute (CDS) that was prepared by plating fibroblasts on to a spongy collagen matrix and culturing them for 7 to 10 days. The matrix was freeze-dried from a 1% aqueous solution of bovine-hide atelocollagen. This study was designed to evaluate the efficacy of promoting epithelialisation clinically on 26 donor-site wounds for split-thickness skin grafts. One half of a wound was covered with an allogeneic CDS and the other half side was covered with a commercially-available freeze-dried porcine dermis (FPD). Both macroscopically and histologically the epithelialisation on the area of the donor site that was covered with allogeneic CDS was more rapid than that covered with FPD. In a representative donor-site wound covered with allogeneic CDS, there was a stratified structure of epithelial cells on the underlying connective tissue on day 5, and the epithelium had matured by day 12. When covered with FPD a stratified structure of epithelial cells was noted on day 8, and the epithelium had matured by day 15. We conclude that allogeneic CDS provides a good environment for epithelialisation.

An anatomical study of the medial canthus using a three-dimensional model.

Abstract. Opinions on the direction and insertion of the muscle and tendon of the medial canthus not only differ depending on the reporter, but, to date, have lacked objectivity. The direction and insertion of the muscle and tendon of the medial canthus have, therefore, not been clear to surgeons operating on the medial canthus. In order to fully grasp the anatomy of this construct three-dimensionality, we constructed a 3D model of successive sections of the medial canthus in a frontal direction using five cadavers, and then studied this model. The pretarsal part of the orbicularis oculi muscle is formed from a single muscle bundle of both the upper and lower eyelids, and runs into the medial palpebral tendon. This muscle bundle further branches off along the outside of the lacrimal sac, internally. It surrounds the back of the lacrimal sac without entering it. The preseptal part of the orbicularis oculi muscle consists of a single muscle bundle for both the upper and lower eyelids. The muscle fibers on the side of the skin run into the medial palpebral tendon. The muscle fibers posterior to this muscle bundle run into tendinous fibers, and, in all of the upper eyelids examined, they stop at the lacrimal fornix. In three out of the five lower eyelids examined the muscle fibers stop at the anterior surface of the lacrimal sac, while in the remaining cases they run into the medial palpebral tendon, as with the muscle fibers on the side of the skin. The medial palpebral tendon traverses the anterior surface of the lacrimal sac in an internal direction without branching off anteroposteriorly.

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.

Role of COX-2 in lymphangiogenesis and restoration of lymphatic flow in secondary lymphedema

The pathophysiology of secondary lymphedema remains poorly understood. To clarify the roles of cyclooxygenase (COX)-2 in enhancement of lymphangiogenesis during secondary lymphedema, we tested a mouse tail model and evaluated the recurrence of lymph flow. To induce lymphedema, a circumferential incision was made in the tail of anesthetized mice to sever the dermal lymphatic vessels. The maximum diameters of the tails were measured weekly. We found that the diameters of the tails around the wounds were markedly increased after surgery, and reached maximum size 2 weeks after wounding in mice without a COX-2 inhibitor, celecoxib (Celecoxib–). Expression of COX-2 in wound granulation tissues was markedly increased 1 week after surgery compared with unwounded naive control mice. In Celecoxib–, recurrence of lymphatic flow in the wound granulation tissues was detected 3 weeks after surgical treatment. In contrast, lymphatic flow was markedly suppressed in mice treated with celecoxib (Celecoxib+). Newly formed lymphatic structures were identified in the granulation tissues formed at wounded lesions in Celecoxib–, whereas those were markedly suppressed in Celecoxib+. Interstitial tissue pressures in the distal areas of the tail wounds were markedly increased in Celecoxib+ with reduced expression of vascular endothelial cell growth factor (VEGF)-C. F4/80-positive cells were accumulated to the wound granulation tissues in Celecoxib–, and the accumulation of these cells was suppressed in Celecoxib+. Prostaglandin E2 (PGE2) upregulated the expressions of VEGF-A and VEGF-C in cultured macrophages, but not human lymphatic microvascular endothelial cells. The present study therefore suggests that lymphangiogenesis, together with recurrence of lymph flow after surgical induction of lymphedema, is upregulated by COX-2 possibly via generation of PGs.

A novel cell‐adhesive scaffold material for delivering keratinocytes reduces granulation tissue in dermal wounds

Novel peptide‐conjugated chitosan membranes were fabricated and used to deliver keratinocytes to dermal wounds in mice. Three active peptides of 12 or 13 amino acids each, RLVSYNGIIFFLK (A5G27), ASKAIQVFLLAG (A5G33), and AGTFALRGDNPQG (A99) were selected from a cell‐adhesive peptide library of laminin, a major constituent of basement membrane. The peptides were synthesized and coupled to chitosan membranes, and the resulting peptide–chitosan membranes were tested for keratinocyte attachment. Two of the peptides that bind to cell surface heparin‐like receptors (A5G27 and A5G33) were found to promote strong keratinocyte attachment, whereas the one that binds to integrin (A99) was inactive. Subsequently, A5G27– and A5G33–chitosan membranes were tested as vehicles for keratinocyte delivery in a wound model. We found that keratinocytes were delivered into the full‐thickness wound with either membrane. Using the A5G33–chitosan membrane, we further evaluated the activity of the delivered keratinocytes in wound healing. Immunohistochemistry for granulation tissue markers, including tenascin and α‐smooth muscle actin, showed that keratinocyte delivery by the present peptide–chitosan membranes in the wound bed provided a favorable condition for keratinocyte migration along the wound surface and reduced granulation tissue formation.

Hes1 regulates formations of the hypophyseal pars tuberalis and the hypothalamus

The hypophyseal pars tuberalis surrounds the median eminence and infundibular stalk of the hypothalamus as thin layers of cells. The pars tuberalis expresses MT1 melatonin receptor and participates in mediating the photoperiodic secretion of pituitary hormones. Both the rostral tip of Rathke’s pouch (pars tuberalis primordium) and the pars tuberalis expressed αGSU mRNA, and were immunoreactive for LH, chromogranin A, and TSHβ in mice. Hes genes control progenitor cell differentiation in many embryonic tissues and play a crucial role for neurulation in the central nervous system. We investigated the Hes1 function in outgrowth and differentiation of the pars tuberalis by using the markers for the pars tuberalis. In homozygous Hes1 null mutant embryos, the rostral tip was formed in the basal-ventral part of Rathke’s pouch at embryonic day (E)11.5 as well as in wild-type embryos. In contrast to the wild-type, the rostral tip of null mutants could not extend rostrally with age; it remained in the low extremity of Rathke’s pouch during E12.5–E13.5 and disappeared at E14.5, resulting in lack of the pars tuberalis. Development of the ventral diencephalon was impaired in the null mutants at early stages. Rathke’s pouch, therefore, could not link with the nervous tissue and failed to receive inductive signals from the diencephalon. In a very few mutant mice in which the ventral diencephalon was partially sustained, some pars tuberalis cells were distributed around the hypoplastic infundibulum. Thus, Hes1 is required for development of the pars tuberalis and its growth is dependent on the ventral diencephalon.

Reconstruction of the Umbilicus Using a Reverse Fan-Shaped Flap

Abstract The goal of the reconstruction for umbilical absence is to obtain a natural three-dimensional appearance of the umbilicus with minimal operative scarring. This paper presents two cases of umbilical reconstruction using a reverse fan-shaped flap. In both cases, the umbilicus was lost during surgical procedures on the abdominal wall when the patients were newborns. We performed this technique in both cases. This technique is simple and safe. With this technique, a permanent umbilical depth and ring can be obtained without any complications.

Anterofrontal superficial temporal artery island flap for full-thickness eyelid reconstruction

The development of movable skin flaps appears to be a popular aspiration of contemporary plastic surgeons, although it seems to us that the classic skin flap, devised by pioneers a century ago, still remains worthy of application.

Effect of EGF and bFGF on fibroblast proliferation and angiogenic cytokine production from cultured dermal substitutes.

Abstract Growth factors accelerate wound healing but the underlying mechanisms remain poorly understood. The aim of this study was to investigate the effect of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on fibroblast proliferation and production of angiogenic factors from cultured dermal substitutes (CDS). In the first experiment, fibroblasts were seeded into a flask at a density of 1 × 104 cells/cm2.Cell proliferation was assessed after culturing in media containing EGF or bFGF at concentrations ranging from 2 to 50 μg. The number of fibroblasts increased significantly in the presence of EGF or bFGF, but fibroblasts detached from the flasks in the presence of 50 μg bFGF. In the second experiment, CDS were prepared by incorporating fibroblasts into collagen gels. To make a wound surface model, the CDS was elevated to the air–liquid interface, on which a spongy sheet of hyaluronic acid (HA) containing EGF or bFGF was placed. The amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released from the CDS after 1 week of cultivation was measured by ELISA. When the CDS was covered with a HA sponge containing EGF (Group 1), fibroblasts released 3.5-times more VEGF compared with a HA-alone sponge (control group). When covered with a HA sponge containing bFGF (Group 2), 8.7-times more VEGF was released compared with the control group. Fibroblasts in Groups 1 and 2 released 9.6- and 9.3-times more HGF, respectively, compared with the control group. Thus, EGF stimulates fibroblasts to produce VEGF and HGF, in addition to its ability to enhance epidermal cell proliferation.

Clinical trial of allogeneic cultured dermal substitutes for intractable skin ulcers of the lower leg

The efficacy of allogeneic cultured dermal substitute (CDS) on wound healing was evaluated in six patients with intractable skin ulcers on the lower extremities. Allogeneic CDS was repeatedly applied to wounds at intervals of 4–7 days to prepare a wound bed acceptable for skin grafting or to induce resurfacing through the granulation tissue formation associated with epithelialization. In one patient with a leg ulcer, the wound size decreased to 32% of the original size within 10 weeks and skin grafting was conducted. In the other five patients with leg, ankle, or foot ulcers, the wound size decreased to 9%–25% of the original size within 6 weeks.