Yukio Sumi

Peri-implant soft tissue management through use of cultured mucosal epithelium

OBJECTIVE In implant therapy, peri-implant soft tissue management through use of mucosal grafting or skin grafting is necessary in some patients who do not have enough attached gingiva around the abutment. However, limitation of donor site size is a problem for the mucosal graft, and the different characteristics of skin, such as hair growth, are disadvantages in treatment that involves the use of skin graft. On the other hand, cultured epithelium fabricated with living mucosal cells has proved to be a good grafting material for any kind of mucosal defect. In this study, we used cultured mucosal epithelium for soft tissue management in implant therapy. STUDY DESIGN In the first surgical procedure of the implant therapy, a small segment of oral mucosa was sampled from a patient. The cultured epithelium was fabricated and then stored until it was grafted in the second surgery. RESULTS Twelve cases in which patients underwent peri-implant soft tissue management through use of cultured mucosal epithelium for implant therapy are presented, and the usefulness of this technique in the making of attached gingiva is analyzed. CONCLUSIONS From this study it was concluded that cultured mucosal epithelium can serve as a proper material for peri-implant soft tissue management.

Thrombospondin-1 promotes fibroblast-mediated collagen gel contraction caused by activation of latent transforming growth factor β-1

BACKGROUND Grafting of cultured epithelium has become a useful technique for the treatment of epithelial defects, since grafted epithelial cells secrete factors promoting wound healing. We identified one such factor produced by cultured oral epithelial cells as thrombospondin-1 (TSP-1). Recently, TSP-1 was reported to act as an activator of transforming growth factor-beta1 (TGF-beta1). OBJECTIVE The role of TSP-1 in wound healing and its mechanism were investigated in vitro and in vivo. METHODS The cultured oral epithelial cell-conditioned medium was harvested and applied to Heparin-Sepharose affinity chromatography. Proteins were analyzed by N-Terminal sequencer. TSP-1 and the other factors were applied to fibroblasts-mediated collagen gel contraction assay. The amount of TGF-beta1 (latent TGF-beta1 (LTGF) and active TGF-beta1) in collagen gels was quantified by ELISA and Western blotting analysis. Collagen sponges were soaked with TSP-1 and implanted subcutaneously into rats. RESULTS A 38 kDa protein secreted from cultured oral epithelial cells was found to be human TSP-1. TSP-1 promoted collagen gel contraction activity, and anti-human TSP-1 and TGF-beta1 antibody inhibited the activity. The diameters of the gels treated with LTGF and TSP-1 were reduced to a greater extent than those of gels treated with either factor alone. Although there were no significant differences in the amounts of total TGF-beta1, which include LTGF, the amount of 25 kDa TGF-beta1 was 3.30-fold greater in TSP-1-treated samples than controls. In vivo, 7 days after implantation, increased numbers of fibroblasts were observed in the sponges treated with TSP-1. CONCLUSION These findings suggested that TSP-1 causes collagen gel contraction by activation of LTGF. TSP-1 is expected to be especially suitable for regulating wound healing.

Successful Culture and Sustainability in Vivo of Gene-Modified Human Oral Mucosal Epithelium

Human oral mucosal cells are an attractive site for tissue engineering because they are the most accessible cells in the body and easy to manipulate in vitro. They thus have possibilities for targeting by somatic gene therapy. We examined the efficiency of retrovirus-mediated gene transfer and the construction of mucosal epithelium in vivo. Human oral mucosal cells were transduced with a retroviral vector carrying the lacZ gene at high efficiency and constructed epithelium after G418 selection with 3T3 cells in vitro. The cultured oral mucosal epithelium membrane was then grafted onto immunodeficient mice. Beta-Gal expression was detected histochemically in vivo 5 weeks after grafting. Furthermore, we transduced factor IX cDNA into the mucosal epithelium membrane, and it was then transplanted into nude mice. Between 0.6 and 1.8 ng of human factor IX per milliliter was found in mouse plasma, and the production was continued for 23 days in vivo. These results confirmed that the oral mucosal epithelium is an ideal target tissue for gene therapy or tissue engineering.

In vitro analysis of distraction osteogenesis.

Distraction osteogenesis has been widely used for lengthening craniomaxillofacial bone. The healing process of distracted bone has been studied mainly by histological observation in vivo. To analyze the cellular response to the mechanical stress of distraction, we have established and evaluated an in vitro model of distraction osteogenesis using an organ culture technique. Five-week-old male Wistar rats were used for the experiments. The tibial bone was fractured by hand and fixed for 1 week by acrylic resin. After the initial healing period, the tibial bone was harvested and used for this study. A distraction instrument was devised to control the strain on the cultured bone using a micrometer. After distraction, the samples were histologically evaluated for ossification by hematoxylin and eosin stain and Alcian blue stain. As a result, the histological finding for the bone region at a slow rate of distraction (0.5 mm/day) was different from that at a rapid rate of distraction (1.0 mm/day). The proliferation of cartilage was inhibited at the rapid distraction rate. Thus, we hypothesized that mechanical stress regulated cartilaginous growth in tissue cultivation. Judging from this experiment, the model was useful for investigation of the mechanism of bone formation in distraction osteogenesis, because it was simple and served to isolate many factors.

Cultured oral epithelium as an effective biological dressing using for palatal wounds after palatoplasty

Abstract Cultured allografts derived from mucosal tissue provide a potent stimulus to wound healing in a wide variety of wounds. In the field of oral surgery, mucoperiosteal defect of the hard palate after palatoplasty causes scar contraction, leading to poor growth of the maxilla. The promotion of wound healing in these cases through cultured epithelial allografting has been reported. Cultured epithelial allografting was done using a strangers cultured cells. We grafted cultured oral epithelium in the hope of improving growth of maxilla. Clefts of the soft and hard palate (seven patients), and a cleft of the soft palate (two patients) were present. Average patient age was 1 year 4 months. Palatoplasty was done by a conventional pushback operation. Oral epithelial cells in healthy adults were cultured using 3T3 cells as the feeder layer. After 3 weeks, cultured oral mucosal epithelium was grafted on a raw surface following palatoplasty. In all patients, the grafted areas underwent re-epithelialization after about 1 week and did not exhibit any side effects of graft rejection. Grafted areas healed completely after 2–3 weeks in all cases. Cultured epithelial allografts serve as a temporary biological dressing, and accelerates epithelialization and wound healing. Allografting by cultured oral epithelium has proved to be a very useful therapeutic modality in palatoplasty, as well as effective augmentation materials in cases of oral mucosal defects.

An ex vivo model for gene therapy of hemophilia B using cultured human oral mucosal epithelium

Abstract Human oral mucosal cells are important target tissues for regenetic engineering and are sources of epithelial sheets. In this study, we examine the possibility of mucosal epithelial sheet as a target tissue for gene therapy. Human oral mucosal cells were transduced with a retroviral vector carrying human factor IX gene and constructed epithelium after G418 selection with 3T3 cells in vitro. The cultured oral mucosal epithelium membrane was then grafted onto immunodeficient mice. Between 0.6 and 1.8 ng of human factor IX per milliliter was found in mouse plasma, and the production was continued for 23 days in vivo. These results demonstrate a model of ex vivo gene therapy for hemophilia B using gene-modified oral mucosal epithelium.