Yukihiko Kinoshita

Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma

Double‐stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21–23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi downregulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis‐dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS‐1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF‐transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin‐1 by pericytes. These data suggest that cationized‐gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS‐1 squamous cell carcinoma xenograft model. (Cancer Sci 2006; 97: 313 – 321)

Soft tissue reaction to collagen-immobilized porous polyethylene: subcutaneous implantation in rats for 20 wk.

Collagen-immobilized porous polyethylene, in which the immobilization was through covalent bonding, and virgin porous polyethylene were implanted subcutaneously in rats from 1 to 20 wk. The results were the ingrowth of the connective tissue into collagen-immobilized porous polyethylene was rich and contained a low level of inflammatory cellular infiltration compared with that of virgin porous polyethylene. The material-tissue interface showed that the living body-originated collagen fibres were firmly anchored into the immobilized collagen layer. These results suggested that covalent immobilization of collagen on to the biomaterial surface is useful in promoting the ingrowth of soft tissue and the tissue adhesion.

Recent Developments of Functional Scaffolds for Craniomaxillofacial Bone Tissue Engineering Applications

Autogenous bone grafting remains a gold standard for the reconstruction critical-sized bone defects in the craniomaxillofacial region. Nevertheless, this graft procedure has several disadvantages such as restricted availability, donor-site morbidity, and limitations in regard to fully restoring the complicated three-dimensional structures in the craniomaxillofacial bone. The ultimate goal of craniomaxillofacial bone reconstruction is the regeneration of the physiological bone that simultaneously fulfills both morphological and functional restorations. Developments of tissue engineering in the last two decades have brought such a goal closer to reality. In bone tissue engineering, the scaffolds are fundamental, elemental and mesenchymal stem cells/osteoprogenitor cells and bioactive factors. A variety of scaffolds have been developed and used as spacemakers, biodegradable bone substitutes for transplanting to the new bone, matrices of drug delivery system, or supporting structures enhancing adhesion, proliferation, and matrix production of seeded cells according to the circumstances of the bone defects. However, scaffolds to be clinically completely satisfied have not been developed yet. Development of more functional scaffolds is required to be applied widely to cranio-maxillofacial bone defects. This paper reviews recent trends of scaffolds for crania-maxillofacial bone tissue engineering, including our studies.

Reconstruction of mandibular continuity defects in dogs using poly (L-lactide) mesh and autogenic particulate cancellous bone and marrow: preliminary report.

PURPOSE This study evaluated the reconstruction of continuity defects in the canine mandible using a poly [L-lactide] (PLLA) mesh tray and particulate cancellous bone and marrow (PCBM). MATERIALS AND METHODS Eight adult dogs were divided into two groups of four dogs each. In group A, each dog had a tray fixed with stainless steel wires on each side of the mandibular stumps with the concave surface of the tray attached to the inferior border of the mandible (U-fixation). In group B, the concave surface was attached to the superior border (inverted U-fixation). Each tray was filled with PCBM from the ilium. After the operation, the dogs were radiographed, and specimens were examined histologically at 3-, 6-, and 12-month intervals. RESULTS All of group A showed good clinical healing and the continuity of the mandibular bone was regained within 3 months postoperatively. However, fibrous tissue had invaded through the area above the tray, resulting in a poorly shaped alveolar ridges. In group B, the dogs showed good bony regeneration with well-shaped alveolar ridges. However, two animals in this group had partial exposure of the PLLA mesh tray into the oral cavity. CONCLUSION It is suggested that a combination of the PLLA mesh and PCBM grafts might be a useful technique for functional reconstruction of the jaw bone, specifically using method A (U-fixation) as a technique to reconstruct continuity defects of the mandible, and method B (inverted, U-fixation) as a promising method for alveolar reconstruction to make wearing dentures possible.

Alveolar bone regeneration using absorbable poly(L-lactide-co-ɛ-caprolactone)/β-tricalcium phosphate membrane and gelatin sponge incorporating basic fibroblast growth factor

The aim of this study was to evaluate the effects of combining a porous poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and gelatin sponge incorporating basic fibroblastic growth factor (bFGF) on bone regeneration in mandibular ridges. Four full-thickness saddle-type defects (10 mm long x 5 mm deep) were symmetrically created in both edentulous mandibular alveolar ridges of 6 beagles. The dome-shaped membrane was secured to each defect site, and a gelatin sponge containing 200 microg bFGF was implanted on the left side of each defect (experimental group). Only the membranes (control group) were secured to the defect sites on the right. Three and 6 months later, 3 animals were killed. Bone regeneration was analyzed by soft X-ray photographs, micro-computed tomography (CT) images, and peripheral quantitative CT (pQCT), and then examined histologically. Soft X-ray examination revealed an increase in new bone volume in the experimental group 6 months postoperatively. pQCT showed that immature bone density was higher in the experimental group. Micro-CT images revealed well formed new bone along the original contour of the dome-shaped membrane in the experimental group. Histologically, inflammatory infiltration of tissue surrounding the membranes was slight. These results suggest that combining the poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and bFGF-gelatin sponge is promising for alveolar ridge reconstruction.

Study on the efficacy of biodegradable poly (L-lactide) mesh for supporting transplanted particulate cancellous bone and marrow : Experiment involving subcutaneous implantation in dogs

Poly(L-lactide) (PLLA) mesh sheets, monofilaments and mesh cylinders filled with fresh autogenic particulate cancellous bone and marrow (PCBM) were implanted subcutaneously into the back of 22 adult dogs for 1 yr. Polypropylene (PP) was used as a control. The inflammatory response to PLLA mesh sheets was slight, similar to PP mesh sheets. However, 3 months after implantation, histiocytes and multinucleate giant cells appeared on the surface of the monofilaments of PLLA mesh sheets and gradually increased in number as the monofilaments were degraded and absorbed. Almost no inflammatory cellular infiltration was seen in the tissue around PLLA mesh sheets or between the monofilaments. When a PLLA mesh cylinder filled with PCBM was implanted subcutaneously, the bone formation reached its peak 1 month after implantation, as in the case of a similar PP mesh cylinder, and bone formation was observed along the inner wall of the cylinder. In addition, a vascular net was abundant within the cylinder. The mechanical strength of the test material (PLLA monofilaments) did not change for almost 2 months after implantation, and 80% of its initial strength was retained for 3 months. These observations suggest that a PCBM graft supported by PLLA mesh would be very effective in the reconstruction of damaged maxillofacial bones, since histological tissue reactions associated with biodegradation of the PLLA mesh are mild and the PLLA mesh does not interfere with bone formation of PCBM. It was also found that the PLLA mesh retains its mechanical strength long enough to allow the PCBM transplant to develop sufficient strength to sustain the damaged region.

Enhanced Regeneration of Critical Bone Defects Using a Biodegradable Gelatin Sponge and β-Tricalcium Phosphate with Bone Morphogenetic Protein-2

We examine the osteogenicity of a sponge biomaterial consisting of a biodegradable mixture of gelatin and β-tricalcium phosphate (βTCP) that bound bone morphogenetic protein 2 (BMP-2) in critical-sized bone defects in rats. Gelatin-βTCP sponges containing either phosphate buffered saline or incorporating BMP-2 are implanted into 5 mm diameter bone defects created in rat mandibles. We assess the defects biweekly for 8 weeks following implantation. There is significantly higher osteoinductive activity and significantly more Gla-osteocalcin content at bone-defect healing sites treated with gelatin-βTCP sponges incorporating BMP-2 than there is in those treated with sponges that did not contain BMP-2. Histologically, new bone that contains bone marrow and that is connected to the original bone almost entirely replaces the regenerated bone. These results show that biodegradable gelatin-βTCP incorporating BMP-2 is osteogenic enough to promote healing in large bone defects.

Reduction in tumour formation on porous polyethylene by collagen immobilization

After surface modification with collagen immobilization through covalent bonding, porous polyethylene pieces with an average pore size of 400 microns were implanted subcutaneously into the back of rats for 1 yr. It was found that connective tissues with abundant blood vessels were formed clearly, filling more than 90% of the pore volume and bound firmly to the pore walls. A tumour was found in only one of 24 implanted pieces (4.2%). On the other hand, the virgin porous polyethylene pieces without collagen immobilization exhibited inflammatory reactions within the pores and the connective tissues produced filled only 15% of the pore volume. Formation of a malignant histiocytoma was observed in 11 of the 24 pieces which had been implanted (45.8%). Thus, immobilization of collagen on the surface of an artificial material through covalent bonding proved to be very effective not only for firm bonding with soft connective tissues but also for a reduction of tumour formation.

Functional Reconstruction of the Jaw Bones Using Poly(L-Lactide) Mesh and Autogenic Particulate Cancellous Bone and Marrow

The goal of the reconstruction of jaw bone defects should be not only to recover deformities, but also to reconstruct the jaw with which the patient can perform normal functions. To achieve this purpose, it is essential that a method of promoting regeneration of physiological bone is developed so that dentures can be accommodated. In this report a new method of functional reconstruction using fresh autogenous particulate cancellous bone and marrow and a poly(L-lactide) mesh tray is presented. This method will make physiological reconstruction of the jaw possible. In the future this method is also expected to assist in the development of simultaneous functional reconstruction of the jaw bone and oral rehabilitation when dental implants are included in the tray.

Gastrointestinal mucosal cyst of the oral cavity: report of case and review of literature.

The presence of a gastrointestinal cyst or heterotopic mucosa in the oral cavity is a very rare condition. Gorlin and Jirasek reviewed the literature in 1970 and reported 12 cases of cysts containing gastrointestinal mucosa, including one of their own, and two cases of masses of heterotopic gastrointestinal tissue in the oral cavity.’ Following their report, 15 additional cases of gastrointestinal cyst2-12 and three cases of masses of heterotopic gastrointestinal tissues”-” in the oral cavity have been described. This report describes an additional case of this rare condition.