Bone regeneration is accompanied by a cascade of events. Migration and proliferation of osteogenic cells occurred at the early stage of bone regeneration, and these osteogenic cells are involved in the synthesis of the bone matrix and its mineralization depending on their maturation stages. The remodeling of fracture callus is a crucial event that restores mechanical integrity and integrates the newly formed bone into the preexisting skeletal structure. Various molecules are involved in these processes to achieve complete bone healing, but the molecular mechanism underlying these processes has not been fully understood. To develop biology-based regenerative medicine/dentistry, we conduct BONE REGENERATION PROJECT by investigating molecular mechanism of osteoblast differentiation and bone regeneration. I will present some results from this project.
Bone grafts are frequently applied to patients with severe bone defects as part of implant surgery. Normally, bone particles collected during the drilling process and other fragments obtained from the donor site are temporarily stored in saline. However, it is doubtful whether saline is suitable for implant surgery. To address this issue, we examined the effects of three different solutions on cells of osteoblastic lineage. Rat bone marrow cells were preserved in normal saline, α minimum essential medium, or Hanks' balanced salt solution (HBSS) for 1 hour, after which the cells were inoculated to culture plates. At Days 7, 10 and 14, alkaline phosphatase activity and DNA amount were measured and cell viability was assessed. These results indicate that HBSS has the potential to provide superior storage conditions than saline for osteoblast differentiation. Currently, saline is conventionally used in dental implant surgery; however, HBSS is more suited to maintaining osteogenic cells for bone grafting.
Gingivectomy is a periodontal surgery which favors a reformation of the gingival-tooth attachment. Information on the regeneration process of gingival nerve fibers is meager although that of axotomy has been well-documented. The regeneration process of rat gingival nerve fibers in gingivectomy was therefore examined by immunocytochemistry for PGP 9.5, CGRP, and p75-NGFR in comparison with that in axotomy. In the control group without any surgical treatment, the junctional and gingival sulcular epithelia received a dense supply of nerve fibers with each immunoreaction. In the axotomy group at postoperative day 7 (PO 7), the penetration of regenerating nerve fibers was recognizable in the gingival epithelium after the disappearance of any immunoreaction at PO 1. The gingivectomy group 1 also had no neural staining in healing tissues at PO 1. At PO 3, when the gingival epithelium had begun to regenerate, a few thin nerve fibers sprouted from the damaged nerve fibers in the connective tissue. Healed gingival connective tissue was reinnervated by PO 7, but intraepithelial nerve fibers were not found in the regenerated gingival epithelium until PO 14, indicating that neural regeneration occurred more quickly in axotomy groups than in gingivectomy groups. Since the gingival tissue has been suggested to have a potential for the production of neurotrophins such as nerve growth factor (NGF), it is considered that this time lag in neural regeneration between the two experimental groups is caused by the pres-ence/absence of the surrounding tissue which contains rich Schwann cells and fibroblasts.
The cell-therapy for the tooth root tissue regeneration combined with tissue-stem cells and tissue-engineering technology would be a very powerful tool in the future, but there is not enough accumulation of basic knowledge about tooth root development. In fact, several signalling molecules in the Shh, FGF, BMP and Wnt families appear to regulate the developmantal steps of tooth morphogenesis via sequential and reciprocal interactions between the oral epithelium and cranial neural crest cells (CNCCs). Recent findings that non-odontogenic mesenchyme, bone marrow derived mesenchymal stem cells (MSCs), can construct tooth structure with the help of odontogenic epithelium. We improved the tissue culture techniques to isolate the cell populations of CNCCs and MSCs. Next, we examined CNCC culture of Wnt1-Cre/R26R to confirm the specificity of the cultured CNCCs by lacZ staining. For understanding the molecular basis of the odontogenic poten-tiality of CNCCs and MSCs, we did the DNA microarray analysis with a small amount of cultured CNCCs and MSCs to compare the genome-wide expression pattern between CNCCs and MSCs. In addition, we examined the biological effects for the root development of typical signaling molecules, SHH, BMP, FGF family and found that FGFs were most stimulative for root development. It is expected that the further studies involved in mechanisms of tooth root development and tissue-stem cell biology could lead to the innovation of tooth regenerative therapy based on the biological evidence.
Tissue engineering is the three-dimensional assembly over time of vital tissues and organs by a process involving cells, signals and extracellular matrix. Use of endogenous stem/progenitor cells combined into designed scaffolds with cytokines or growth factors for local delivery has been demonstrated as a discipline of more effective tissue engineering. However, several barriers to effective and safe local delivery of proteins in vivo have been identified because of their short life, high costing, difficulty to maintain full bioactivity and high-dose local delivery associated with both local and systemic toxicity. To overcome these barriers, gene therapy has emerged as an effective approach by which therapeutic proteins could be carried as genes. The preliminary studies introduced here are to explore the feasibility of our designed tissue engineering constructs using our modified gene activated matrix (GAM) with calcium phosphate and transgene for BMP-2 for in vivo, and periosteal cell culture with a collagen sponge and transgene for BMP-2/VEGF for ex vivo. Our modified GAM tremendously bridged the bone defect with 12µg plasmid encoding for BMP-2 after 6 weeks, which was followed by mechanically strong bone replacement. And the second study implicated that the implanted collagen sponges harboring periosteal cells with BMP-2/VEGF contained the most alkaline phosphatase activity, enhancement of many of osteogenic gene expressions and prominent angiogenesis.
Periodontal fibroblasts and pulpal fibroblasts have been considered as the major cells responsible for new alveolar bone and dentin formation, respectively. Bone morphogenic protein-2 (BMP-2) has great clinical potential for inducing bone and dentin repair and regeneration. The objective of this study was to investigate the effect of acemannan, polysaccharide fraction of aloe vera gel on BMP-2 expression in primary human periodontal fibroblasts and pulpal fibroblasts. Cells were treated with increasing concentration of acemannan for 24 and 48 hours. The reverse transcriptase-PCR and ELISA assays were used to investigate the effects of acemannan on the levels of BMP-2 mRNA and protein expression, respectively. The results from RT-PCR and ELISA revealed that acemannan induced BMP-2 mRNA level and protein synthesis in both cells. The effective concentrations of acemannan were 0.25-1 mg/ml. Collectively, these results suggest that acemannan is a potent plant polysaccharide for stimulating BMP-2 expression in periodontal fibroblasts and pulpal fibroblasts.
The features of cementum and periodontal ligament (PDL) remain poorly defined and a specific marker for cementum or PDL has not been identified. We therefore aimed to isolate RNA from cementoblasts and PDL cells of mouse mandibular molars using laser capture microdissection (LCM). Murine mandibular frozen sections were prepared using Tape Transfer System. Over 500 cementoblasts and PDL cells were separately captured. The quality of total RNA extracted from specimens and isolated RNAs was determined using an Agilent bioanalyzer, which detected 18s and 28s peaks in the dissected tissue, suggesting good RNA preservation. We then amplified the RNA, synthesized antisense RNA and performed RT-PCR analyses. The mRNA expression of GAPDH and bone-associated markers was detected. These results suggest that LCM is useful for comparing mRNA expression in adjacent tissues.
Soft lining material is a soft elastic and resilient material which helps alleviate stresses from the denture bases to the underlying supporting tissues in the pa-tients. The purpose of this study was to develop the new soft lining material from natural rubber to replace commercial soft lining materials which have several disadvantages and limitations. In this study, the high-ammonium natural rubber latex was vulcanized and grafted with ethyl methacrylate by γ-irradiation to improve its quality. The grafted natural rubber latex, socalled grafted copolymer, was then studied for its ultrastructure and prepared for evaluation of its bonding ability to the denture base material and cytotoxicity by using light and electron microscopies and human gingival fibroblast co-culture method. The results from electron microscopic study showed a complete junction between the grafted copolymer and the denture base material. Microscopic investigations in cell culture, the human gingival fibroblasts demonstrated well proliferation and well attachment around and on the surface of the grafted copolymer. In conclusion, the grafted copolymer has a good bonding to the denture base material and a good cellular biocompatibility. Therefore, it would be worth to modify the grafted copolymer for using as denture soft lining materials.
Chitin is the principle component of the exoskeleton of crustaceans which are abound in Thailand. Chitin as well as its derivative, chitosan, have recently been used widely as biomedical materials for different treatment purposes and in different products. The objective of this study was to develop chitosan to be used as dental biomaterials. In this study, chitosan powder was prepared into 4 prepara-tions using different protocols. All products were then studied for their cellular biocompatibility and biodegradability in vitro and in vivo. The results from in vitro study showed that all 4 products were biocompatible to human gingival fibroblasts while the results from in vivo study were different among preparations. The chitosan sheet was neither biodegradable nor induce any tissue reaction within the experimental period while the sheet and sponge prepareded from carboxy-methylchitosan demonstrated different degrees of biodegradation with very little tissue response. The sponge prepared from chitosan/collagen revealed partial biodegradation with foreign body reaction. These results suggest that some of these preparations might be worth to develop to be used as dental biomaterials.