To understand the molecular basis mechanisms of β-TCP in accelerating of bone formation, β-TCP was implanted into bone defects of mandible in beagle dogs, and gene expression profiles were examined using GeneChip. Significant higher gene mRNA levels of fibronectin (FN) and focal adhesion kinase (FAK) were observed in β-TCP implanted mandible when compared with the controls. These enhancements of gene expressions were successfully confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. FN involved in the early stages of osteogenesis and in mineralization processes. FN linked through integrin clustering to activate FAK and FAK is an important signaling molecule to lead the cell proliferation and activate the transcription of bone-specific genes. Thus, the stimulation of the FN and FAK gene expressions by β-TCP may be one of molecular mechanisms for accelerating bone formation.
Recent studies demonstrated that hydroxyl radical (OH⋅;) and various extracellular matrix degrading enzymes coexisted in osteoarthritic TMJ (OA-TMJ), however exact roles of these molecular species are not fully understood. We tried to elucidate whether OH⋅; could modify pro-form of matrix metalloproteinase-9 (MMP-9) to acquire collagenolytic activity. OH⋅; was generated in vitro by Fenton reaction where the reaction was originated by mixing hydrogen peroxide and FeSO4-7H2O. When exposed to OH⋅;, the intensity of the pro-form of MMP-9 (92kDa) was decreased but intermediately- (83kDa) and fully-activated (67kDa) MMP-9 were increased in their intensities. The activated MMP-9 could cleave type IV collagen. It was thus demonstrated that MMP-9 was partially- or fully- activated in vitro by OH⋅; without losing its collagenolytic function. These results suggest that among various pathways of MMP activation, free radical-prone pathway is really existed, and this could be feasible in the OA-TMJ.
We combined the use of bone morphogenetic protein-2(BMP-2)-loaded collagen scaffold and cultured bone marrow stromal cell (BMSC) sheet in order for developing new bone tissue engineering. Rat BMSC sheets were generated in the presence of dexamethasone, β-glycerophosphate and ascorbic acid. Four types of the grafts were implanted in the intermuscular region of rat femurs: control group, collagen sponge; cell group, BMSC sheet in collagen sponge; BMP group, BMP-2-loaded collagen sponge; and BMP-cell group, BMSC sheet in BMP-2-loaded collagen sponge. Four weeks after surgery, histological and histomorphometric analysis were performed. The BMP-cell group demonstrated abundant new bone formation compared to all the other groups (p<0.05) and marked resorption of the implanted collagen when compared to the control and cell groups (p<0.05). The combination of BMP-2-loaded collagen sponge and BMSC sheet appeared to successfully promote an osteoinduction.
Higher propagation of Candida biofilms may aggravate intraoral health such as periodontium, leading to unfavorable prognoses for tissue regeneration. We examined protamine and its hydrolysates to determine whether they offer the possibility of being used for antifungal activity for PMMA dental prosthesis. Candida albicans (C. albicans), a pathogenic microorganism causing oral candidiasis, was used as the target strain to evaluate antifungal activity. We also investigated surface attachment of the protamine onto PMMA and analyzed surface characterization of treated PMMA using XPS analysis. After oxygen (O2)-plasma treatment on a PMMA surface (PMMA+O2), XPS analysis showed that the intensity ratios of O1s/C1s and C = O/C-C both became higher in that of the PMMA+O2 compared to that of untreated PMMA. On an adsorption assay using the quartz crystal microbalance with dissipation (QCM-D) technique, protamine and its hydrolysates were demonstrably more able to adhere to the PMMA+O2 sensors than to the untreated PMMA sensors. The amounts of C. albicans that initially attached to and the fungi that colonized on the PMMA and PMMA+O2 specimens were lower than on untreated PMMA (control). A tendency to decrease was noted for protamine hydrolysates compared with native protamine. These results indicated that protamine and its hydrolysates could be immobilized on PMMA surfaces, providing antifungal activity on these surfaces.
The purpose of this study was to evaluate the effect of implanted collagen hydrogel-sponge composite on periodontal wound healing. One-wall infrabony defects (depth: 5 mm; width: 3 mm) were surgically created in three beagle dogs. The exposed root surface was planed and demineralized with EDTA. In the experimental group, the defects were filled with collagen hydrogel-sponge composite. Conversely, no collagen hydrogel-sponge composite was applied to defects in the control group. Histomorphometric parameters were evaluated four weeks after surgery. In the experimental group, regeneration of alveolar bone and cementum was frequently observed. Periodontal ligament tissue was reestablished between the alveolar bone and cementum. New bone height, new bone area, new cementum and new periodontal ligament in the experimental group were significantly greater than those in the control group (p < 0.05). These findings suggest that implanted collagen hydrogel-sponge composite facilitates periodontal wound healing in one-wall infrabony defects in beagle dogs.
Low-intensity pulsed ultrasound (LIPUS) is therapeutic instrument of improving bone healing, and Bone Morphogenetic Protein-2 (BMP-2) has been demonstrated to enhance bone formation. The present study was designed to evaluate the effects of LIPUS on bone formation induced by BMP-2 at palatal subperiosteal sites in Wister rats. Alginated gel (AG) contained BMP-2 (1.0µg/µL) was implanted into the palatal left site (BMP site), and AG alone was the right site (AG site). The rats were divided into two groups; LIPUS group, ultrasound (1MHz, 160mW, 5 min) was performed every three days, and non-LIPUS group. At 3 weeks after surgery, thickness of new bone of BMP site was significantly higher than that of AG site (P<0.05). Newly formed bone of BMP site was continuous with the original bone at non-LIPUS group, whereas it was almost integrated with the original bone at LIPUS group. These results suggest that LIPUS has the ability to enhance the maturation of newly formed bone induced by BMP-2.
In the present study, 5-MHz and 27-MHz titanium (Ti) quartz crystal microbalance (QCM) sensors were produced by a thin film magnetron that sputtered Ti onto gold-plated quartz. Scanning probe microscope (SPM) analysis and X-ray photoelectron spectroscopy (XPS) analysis were used to characterize the nanostructure of the Ti films. The respective amounts of adsorption of albumin for the 5-MHz and 27-MHz Ti QCM sensors were compared. SPM analysis showed that the surface roughness of the film was slightly increased by the sputtering process. However, it was covered with an almost uniform particle layer. Furthermore, XPS analysis showed that the film consisted mainly of TiO2. The 27-MHz Ti QCM sensor offers an approximately 24-fold increase in sensitivity over 5-MHz Ti QCM, which determines its attractiveness for application in biomolecular sensing with Ti.
In the present in vitro study, we evaluated peri-implant (IP) osteogenesis generated by 3-D culture of osteogeneic JCRB1119:KUSA/A1 cells with a collagen gel scaffold on Ti and Ti alloy IPs, which have osteoconductive and osteoinductive surface texture and substrates (i.e., IP-AO, IP-HA, IP-SPI) that are commercially available. In the histological study, we identified bone elaboration associated with formation of cement line (interfacial matrix) on the different substrate surfaces; the integrated mineralizing layer provided a biomimetic scaffold which favored static osteogenesis at the IP surfaces. Meanwhile, we elucidated contact osteogenesis showing different distribution and morphology at the cell-substratum interface. Furthermore, matrix mineralization showing the histology that mimicked distant osteogenesis in vivo was evident in the growing peri-IP opaque matrix; the IP-HA have acquired more opaque matrix in the day 21 specimens. The present GBR method engineered KUSA/A1 cells in a 3-D collagen scaffold to initiate an osteogeneic process of new (de novo) bone formation without intervening soft tissue in the peri-IP osteogenesis; this peri-IP osteoid tissue might be employed to a biocompatible and biodegradable tissue, which could be remodeled and modeled for functional osseointegration.
We evaluated the influences of the component ions (Ag, Au, Cu, In, Ni, Pd, Ti, and Zn) of dental alloys on the in vitro capillary formation of new blood vessels using a human angiogenesis kit (Kurabo). The percent area of new capillary in the presence of Zn ions was the highest, being 106.1% of that in the control group. The percent area decreased in the order of Ag, In, Ti, Ni, Cu, Pd, Au and Zn ions. The new capillary length was also the greatest for Zn ions and the smallest for Ag ions, but the order of the other types of metal ions differed from that of the percent area. Based on these results, if the release of a small amount of Zn ions can be controlled, the active rate of capillary formation may increase, resulting in earlier tissue regeneration.
Embryotoxic risk factors in dental biomaterials can be examined at the normal differentiation level of mouse embryonic stem cells. On the other hand, cell differentiation is promoted in regenerative medicine. Therefore, mechanisms involved in embryonic stem cell differentiation may have application to regenerative medicine using the embryonic stem cell test (EST). I explain about the EST in the dental field. To evaluate the embryotoxicity of dental biomaterials, animal experiments are widely performed. However, such experiments are inappropriate for evaluation of the serious influences of biomaterials in the long term. There is a possibility that models for the prediction of long-term embryotoxicity can be produced by in vitro tests. In 1997, Spielmann et al. proposed the first in vitro embryotoxicity test method using ES and 3T3 cells. The EST holds promise as a screening assay based on the most important mechanisms in embryotoxicity, cytotoxicity and differentiation, as well on differences in sensitivity between adult and embryonic tissues.