The purpose of this study is to evaluate osteogenic differentiation of dedifferentiated fat cells (DFATs) in 3D culture with RADA16, a self-assembling peptide. DFATs derived from rabbit subcutaneous fat were cultured in RADA16 in either a control medium (CM) or an osteogenic medium (OM) containing dexamethasone, L-ascorbic acid 2-phosphate, and β-glycerophosphate for 21 days. The cells cultured in the OM showed significantly higher DNA content on days 7 and 21, significantly higher osteocalcin content on days 7 and 14, and significantly higher calcium content on days 7, 14, and 21, compared to the cells cultured in CM. Von Kossa staining revealed the formation of a mineralized extracellular matrix with calcium deposition on days 14 and 21 of culture. These findings demonstrate that DFATs cultured in RADA16 differentiate into osteoblasts as early as day 7 of culture to form cultured bone in vitro. RADA16 promotes proliferation and differentiation of DFATs and thus might be a useful scaffold in bone tissue engineering.
Tissue engineering involves the morphogenesis of new tissues formed from biocompatible polymers and mesenchymal stem cells (MSCs). We evaluated bone production after combining MSCs derived from mini-pig posterior iliac crest and polypeptides consisting of a Pro-Hyp-Gly sequence [poly(PHG)] sponge. A cell group using poly(PHG) sponge as a scaffold, a poly(PHG) sponge only group, and a bone defect only (control) group were created for transplantation experiments using a mini-pig calvarial defect model. The percentage of new bone in the cell group was significantly greater than that in the control group at weeks 12 and 18, as determined using microradiography. At week 18, the poly(PHG) scaffolds were almost fully degraded, and remodeling of mature compact bone was observed in the cell group. Bone formation materials that combine poly(PHG) sponge as a scaffold with MSCs have good osteogenic ability, and should be studied for clinical application.
Bone morphogenetic protein-2 (BMP-2) may play a major role in osteoinduction and osteoclast differentiation. The influence of BMP-2 conditioning in graded doses to root dentin surface on periodontal wound healing was examined. Twenty-four periodontal defects were surgically created on the buccal roots of four beagle dogs. The denuded root dentin surfaces were demineralized with 24% EDTA, 0.1 and 1.0 μg/μL BMP-2 solution (15 μL) was applied, and the groups were labeled as low-dose BMP (BL) and high-dose BMP (BH), respectively. In the control roots, phosphate-buffered saline was applied to the root surface. Specimens were histologically analyzed 16 weeks after surgery. Periodontal wound healing was stimulated by conditioning with BMP-2. Formation of alveolar bone and cementum-like tissue was observed; however, in BH group, ankylosis and root resorption were accelerated in the defect area, and there was little evidence of periodontal ligament formation between alveolar bone and root surface.
The aim of this study was to investigate the local osteogenic effect of fluvastatin incorporated in biodegradable gelatin-hydrogel (Flu-GH) as a carrier. Two bone defects were created in the calvarial bone of rats: a left defect in the experimental Flu-GH group and a right defect in the untreated control group. New bone formation was quantitatively analyzed over 21 days using a new micro-computed tomography system, the R-mCT®. Histological observation was also performed. The R-mCT® system enabled continuous observation of the dynamics of bone formation in the same animal over 21 days, revealing clear differences between the two groups. No significant differences in new bone formation were observed, however, between the two groups, except outside the bone defects. Further investigation is necessary to establish a suitable drug delivery system for fluvastatin.
The DNA/protamine complex was prepared from the reaction of high molecule weight DNA and protamine. The obtained high molecule weight DNA/protamine complex was water-insoluble white powder. The yield was 75%. The molar binding ratio (molar of protamine/ 1 base pair of DNA) DNA to protamine in was 0.129. SEM observation revealed that prepared complex had a porous structure. The porous disk could be easily prepared. In cell culture tests using MC 3T3-E1 mouse osteoblast cells, cell viabilities for the DNA/protamine complex were from 87.1% to 99.0%. In vivo test, complex showed a mild tissue response after the implantation into the back of skin of rats. The complex was gradually biodegraded during implantation, and a part of complex resided 30 days after implantation. These results suggested that high molecule weight DNA do not influence cytotoxicity and tissue response of DNA/protamine complex.
It is important to investigate the influence of neuropeptides on pulpitis and apical periodontitis to understand the pathogenic mechanism of these inflammations at the cellular level. Evaluating the effects of neuropeptides on cells is also useful for establishing, new treatments to prevent the spread of inflammation. Then, neuropeptide is related to these mechanisms of tissue regeneration. The present study investigated the influence of stimulating human dental pulp cells (HDPC) and human periodontal ligament fibroblasts (HPdLF) with neuropeptides alone on the expression of proinflammatory cytokine mRNA. HDPC collected from the extracted first premolar for orthodontic treatment and commercially available healthy HPdLF were used for the study. Substance P (SP) and calcitonin gene-related peptide (CGRP) were used as neuropeptides. The mRNA expression of the proinflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α were quantified at 6 and 24 hours. Significant mRNA expression of only IL-8 among the proinflammatory cytokines was observed 6 hours after stimulation with both SP and CGRP in HDPC (P < 0.05). In HPdLF, significant IL-8 mRNA expression was observed after SP stimulation alone. Although a significant increase in IL-8 mRNA expression was observed in both cells by SP+CGRP stimulation (P < 0.05), there was no synergetic effect. The IL-8 mRNA expression level was significantly higher in HDPC than HPdLF both when the cells were stimulated with SP and when they were stimulated with CGRP. These results suggest that local neutrophil infiltration is induced by IL-8 expression, and that HDPC is more sensitive than HPdLF to neuropeptides when pulp and periodontal membrane cells were stimulated with neuropeptides alone.
The embryonic stem cell test (EST) is an in vitro assay that has been developed to assess the embryotoxic potential of chemicals and biomaterials. An attempt for improvement in that ES cells used in the EST protocol are restricted to ES-D3 cells. If other kinds of ES cell become available, its experimental application will be further usefulness. We compared the incidence of pulsation between ES-D3 cells requiring no feeder cells for cultivation and EL M3 cells or ES-R1-EGFP B2/EGFP cells requiring feeder cells, to explore the experimental possibility of using ES cells requiring feeder cells. As the present results with ES-D3 and EL M3 cells were similar to those obtained under the two-dimensional condition, these two kinds of cell are thought to be equally available under the present three-dimensional conditions. On the other hand, because ES-R1-EGFP B2/EGFP cells did not show any pulsation at all in the three-dimensional culture, other experimental conditions for the three-dimensional culture method need to be established with those cells. Besides, it was suggested that similar results could be obtained with EL M3 cells requiring feeder cells for cultivation compared to those with ES-D3 cells.
A woven-fabric scaffold, composed of biodegradable poly-L-lactic acid resin fibers and injected with marrow-derived mesenchymal cells mixed with platelet-rich plasma, was developed to evaluate the in vivo performance of a cell-scaffold graft material in experimental bone defects created in the dog mandible. Defects of regular shape and significant size were created at furcation sites and on the mesial aspects of lower premolar roots and then treated with graft material and subjected to histological analysis after 4 and 8 weeks of healing. In the experimental group, bone regeneration in defects on the tooth root mesial aspect occurred at 8 weeks, accompanied by the regeneration of periodontal tissues, such as cementum and periodontal ligaments. In contrast, delayed and immature healing was observed in the control group defects with no graft material. The findings here indicated that a three-dimensional, woven-fabric, composite scaffold combined with mesenchymal cells and platelet-rich plasma could accelerate the regeneration of alveolar bone and periodontal tissues.