To obtain the basic information concerning time-sequential development of new bone in the same mouse, micro X-ray CT system was adopted. A cone beam type CT system, for small animals, enabled to extract three-dimensional (3D) images from the time point of bone morphogenetic protein (BMP) implantation through 3–4 weeks postoperatively when usually new ossification could be observed histologically. The results obtained showed the process of new bone formation by BMP from 3D viewpoint. BMP initial contact area to tissue reflected the final shape of new bone, and this observation suggested the technical method to prepare the composite of BMP and scaffold.
Adiponectin, an adipocyte-derived biologically active molecule, is a ∼30-kDa polypeptide containing an N-terminal signal sequence, variable domain, collagen-like domain, and C-terminal globular domain. Previously we demonstrated that full-length adiponectin up-regulated the expression of genes related to osteoblastic differentiation, although the free globular domain down-regulated them. It was hypothesized that the collagen-like domain may be involved in osteoblastic differentiation, so we investigated the effect of novel synthesized collagen-like peptide on osteoblastic differentiation of murine pro-osteoblastic cells.Microarray analysis showed that the collagen-like peptide up-regulated the genes related to various signaling pathways involved in osteoblastic differentiation. In contrast, the globular domain suppressed the above genes and induced the expression of genes related to cell morphogenesis and neurological system processes. Real-time RT-PCR for osteocalcin clarified the microarray observation. Therefore, the novel synthesized collagen-like peptide had positive action on osteoblastic differentiation.
It remains unclear whether p38 mitogen activated protein kinase (p38 MAPK) is involved in anesthetic preconditioning (APC). We investigated the effect of inhibiting p38 MAPK activation on sevoflurane-induced APC. Isolated perfused guinea pig hearts underwent 30 min ischemia and 120 min reperfusion. APC was elicited with 2% sevoflurane administration. Inhibition of p38 MAPK was achieved using SB203580. Contractile recovery was monitored by left ventricular developed (LVDP) and end-diastolic (LVEDP) pressure. Infarct size was determined by triphenyltetrazolium chloride stain. Expression of p38 MAPK was determined by Western blot. After ischemia/reperfusion, APC hearts had higher LVDP and lower LVEDP compared to CTL. Infarct size was significantly reduced in APC. SB203580 administration failed to abolish APC. Western blot analysis demonstrated that phosphorylation of p38 MAPK was increased by APC, which was enhanced by SB 203580.In conclusion, sevoflurane increases p38 MAPK phosphorylation but this is not required for APC.
Previously we reported a successful three-dimensional cartilage regeneration using an RWV (rotation wall vessel) bioreactor from human mesenchymal stem cells (hMSCs)(Sakai et al. J Orthop Res. 27, 2009), which is useful for better understanding the initiation mechanism of chondrogenic differentiation of hMSCs and discovering its associated proteins. Entire proteome of hMSCs and spherical cartilaginous constructs by RWV culture was comparatively analyzed using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization/ time-of-flight/mass spectrometry (MALDI-TOF-MS/MS) to determine candidate proteins associated with early chondrogenesis. Among them, there found a greatly up-regulation of COL6A1 and COL6A2 proteins on day 10, which encode matrix proteins with great changes at early stage of chondrogenesis in parallel with the increase of SOX9. A specific antibody against to the extracellular protein COL6 effectively postponed the progress of early chondrogenesis, followed by the successive reduction of extracellular COL6 in spite of a temporary increase in genes expression and up-regulation of SOX9 phosphorylation on the early stage of antibody neutralization. It suggested that the extracellular matrix protein COL6 might be a potential key molecule to involve in early events of chondrogenesis via regulating or influencing the expression of SOX9.
In bone tissue engineering, scaffolds play a pivotal role for bone regeneration. Recent research has focused on producing scaffolds by 3D modeling for bone tissue engineering. This study evaluates the biocompatibility of selectively laser-sintered nylon-12 (SLS Nylon12) as a scaffold material. We perform cytotoxicity tests and an implantation test in subcutaneous tissue according to ISO standards. The SLS Nylon12 has good biocompatibility, comparable to the negative control. SLS Nylon12 can therefore be used as a scaffold material for clinical applications.
The teratogenicity of thalidomide was demonstrated by a modified Embryonic Stem Cell (EST) method using a hybrid culture with human hepatocytes. Six kinds of primary cells derived from the mouse uterus or oviduct were used as feeder layers. As a result, the beating rate due to EL-M3 cell differentiation was slightly lower for some feeder layers after the addition of thalidomide. However, there was no significant difference due to the addition of thalidomide. This may be explained by the presence of atypical cells in the feeder cells. The results will be further analyzed.
In this study, mesenchymal stem cells (MSC) were combined with platelet-rich plasma (PRP) and platelet-poor plasma (PPP), to determine their effects on bone regeneration in mini-pig calvarial defects, which have similar bone regeneration rates as humans. MSCs were harvested from Clawn mini-pigs and cultured for 2 weeks using osseo-induction medium. MSCs were mixed with PRP and PPP as graft materials. Graft materials were transplanted into mini-pig calvarial defects created for 8, 12 and 18 weeks. By micro CT analysis, the volumetric density of new bone in the PRP + MSC and PPP + MSC groups were larger than that of the control group after 12 and 18 weeks transplantation, with the PRP + MSC group being the highest after 18 weeks. Histological analysis showed and new bone formation that was continuous along the original bone was observed in the PRP + MSC and PPP + MSC groups. This study demonstrated that the combination of PRP, PPP and MSCs were sufficiently adaptable for treating bone defects in mini-pigs.
Previously, the authors have reported the acceleration of tooth movement and osteoclastogenesis on the pressure site in an experimental tooth movement model by low-energy laser irradiation (LELI). Osteopontin (OPN) is an essential and sufficient for osteoclastogenesis. However, the effect of LELI on osteogenesis on the tension site is not known clearly. Therefore, the present study was designed to examine the effects of LELI on the expression of OPN during experimental tooth movement. To induce experimental tooth movement in rats, 10 g force was applied to the upper right first molar with a Nickel titanium closed-coil spring. Next, a gallium-aluminum-arsenide (Ga-Al-As) diode laser was used to irradiate the area around the moved tooth, and the amount of tooth movement was measured for 21 days. Histopathological examination was also performed. Furthermore, the effect of LELI on OPN release from stretched human periodontal ligament (hPDL) cells was evaluated by Enzyme-linked immunosorbent assay (ELISA) in vitro. The amount of tooth movement in the LELI group was significantly greater than in the non-irradiation group by the end of the experimental period. Cells positively stained with OPN were found to be significantly increased in the LELI group on days 3 as compared with the non-irradiation group. Furthermore, the OPN releases from stretched hPDL cells were significantly higher concentration in the LELI group than in the non-irradiation group in vitro.These findings suggest that LELI induces the expression of OPN in tension side during orthodontic tooth movement.
Prosthetic alloys corrode in the oral cavity over a prolonged period, eluting trace amounts of constituent ions into the saliva. The adverse effects of the eluted ions on biological safety have become a major problem in the dental field. Thus, various alloy compositions resistant to corrosion in the oral cavity have been intensively investigated.We hypothesized that small amounts of such metal ions would act on cells to promote capillary angiogenesis. Then, the effects of Ag, Cu, In, Pd, Sn, and Zn, possibly eluted from silver alloys that are used widely in the dental field, on human angiogenesis were investigated using an angiogenesis kit (Kurabo), demonstrating that capillary angiogenesis was slightly promoted by exposure to a trace amount of Zn ions. Furthermore, promotion of capillary angiogenesis was not observed in the case of mixing all of the metal ions.Moreover, capillary angiogenesis is observed within a narrow Zn concentration range. Thus, a method to stabilize and maintain Zn ion elution should be developed for application to dental regenerative medicine.