Effect of collagenase concentration on the isolation of small adipocytes from human buccal fat pad.

Dedifferentiated fat (DFAT) cells were isolated from mature adipocytes using the ceiling culture method. Recently, we successfully isolated DFAT cells from adipocytes with a relatively small size (<40 µm). DFAT cells have a higher osteogenic potential than that of medium adipocytes. Therefore, the objective of this study was to determine the optimal concentration of collagenase solution for isolating small adipocytes from human buccal fat pads (BFPs). Four concentrations of collagenase solution (0.01%, 0.02%, 0.1%, and 0.5%) were used, and their effectiveness was assessed by the number of small adipocytes and DFAT cells isolated. The total number of floating adipocytes that dissociated with 0.02% collagenase was 2.5 times of that dissociated with 0.1% collagenase. The number of floating adipocytes with a diameter of ≤29 µm that dissociated with 0.02% collagenase was thrice of those dissociated with 0.1% and 0.5% collagenase. The number of DFAT cells that dissociated with 0.02% collagenase was 1.5 times of that dissociated with 0.1% collagenase. In addition, DFAT cells that dissociated with 0.02% collagenase had a higher osteogenic differentiation potential than those that dissociated with 0.1% collagenase. These results suggest that 0.02% is the optimal collagenase concentration for isolating small adipocytes from BFPs.

Transplantation of mature adipocyte-derived dedifferentiated fat cells into three-wall defects in the rat periodontium induces tissue regeneration.

The transplantation of dedifferentiated fat (DFAT) cells in combination with poly(d,l-lactic-co-glycolic acid) (PLGA) scaffolds has previously been proven as an effective approach in promoting periodontal tissue regeneration in a rat fenestration defect model. The aim of this study was to assess the regenerative potential of DFAT cells in a rat model of three-wall periodontal bone defect. Three-wall bone defects were created bilaterally on the mesial side of rat maxillary first molars and were either left untreated or treated by implantation of PLGA scaffolds with DFAT cells or PLGA alone. Four weeks after surgery, the tissues were processed for micro-computed tomography (micro-CT) and histomorphometric examination. Micro-CT revealed that the PLGA/DFAT group had significantly higher rates of bone regeneration than the other groups, while histomorphometric analysis showed that the PLGA/DFAT group had significantly higher densities of collagen fiber bundles in acellular and cellular cementum than the PLGA group. Moreover, the results indicate that the placement of the PLGA scaffold prevented the downgrowth of the junctional epithelium. These findings suggest that DFAT cells contribute to tissue regeneration in three-wall periodontal defects, while PLGA provides space necessary for periodontal tissue restoration.

Use of Rat Mature Adipocyte-Derived Dedifferentiated Fat Cells as a Cell Source for Periodontal Tissue Regeneration.

Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cells. We obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid) on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA) and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3, and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration.

Small Buccal Fat Pad Cells Have High Osteogenic Differentiation Potential.

Dedifferentiated fat (DFAT) cells derived from mature adipocytes have mesenchymal stem cells' (MSCs) characteristics. Generally, mature adipocytes are 60-110 µm in diameter; however, association between adipocyte size and dedifferentiation efficiency is still unknown. This study, therefore, investigated the dedifferentiation efficiency of adipocytes based on cell diameter. Buccal fat pad was harvested from five human donors and dissociated by collagenase digestion. After exclusion of unwanted stromal cells by centrifugation, floating adipocytes were collected and their size distribution was analyzed. The floating adipocytes were then separated into two groups depending on cell size using 40- and 100-µm nylon mesh filters: cell diameters less than 40 µm (small adipocytes: S-adipocytes) and cell diameters of 40-100 µm (large adipocytes: L-adipocytes). Finally, we evaluated the efficiency of adipocyte dedifferentiation and then characterized the resultant DFAT cells. The S-adipocytes showed a higher capacity to dedifferentiate into DFAT cells (S-DFAT cells) compared to the L-adipocytes (L-DFAT cells). The S-DFAT cells also showed a relatively higher proportion of CD146-positive cells than L-DFAT cells, and exhibited more osteogenic differentiation ability based on the alkaline phosphatase activity and amount of calcium deposition. These results suggested that the S- and L-DFAT cells had distinct characteristics, and that the higher dedifferentiation potential of S-adipocytes compared to L-adipocytes gives the former group an advantage in yielding DFAT cells.

High-magnitude mechanical strain inhibits the differentiation of bone-forming rat calvarial progenitor cells.

PURPOSE: Orthodontic tooth movement occurs during the bone remodeling induced by therapeutic mechanical strain. It is important to investigate the relation between the strength of mechanical stress and bone formation activity. The aim of this study was to determine the effect of high-magnitude mechanical strain on bone formation in detail. MATERIALS AND METHODS: Osteoblast-like cells isolated from fetal rat calvariae were loaded with 18% cyclic tension force (TF) for 48 h. To phenotypically investigate the effect of TF, we measured the number and the size of bone nodules stained by von Kossa technique on day 21 after cell seeding and determined the calcium content of bone nodules on day 14. Furthermore, we examined the gene expression of BMP-2, Runx2 and Msx2, which are important factors for bone nodule formation, on days 1, 4 and 7 after TF loading. RESULTS: The maximum bone nodule size in the control group was 1620 and 719 µm in the TF group. Furthermore, the mean number of bone nodules sized over 360 µm in the TF group was significantly decreased compared to the control group. The calcium content was also significantly decreased to 42% by TF loading. The mRNA expression of BMP-2, Runx2 and Msx2 was decreased 1 and 4 days after TF loading. CONCLUSION: The differentiation of bone forming progenitor cells into bone nodule forming cells was inhibited by TF due to the decreased expression of bone formation related factors such as BMP-2, Runx2 and Msx2.

Effects of palate depth, modified arm shape, and anchor screw on rapid maxillary expansion: a finite element analysis.

OBJECTIVES: This study examined the effects of palate depth, modifications of the arm shape, and anchor screw placement in the mid-palatal area on rapid maxillary expansion (RME) using finite element (FE) analysis. MATERIALS AND METHODS: Three-dimensional FE models were constructed that included the maxilla (cortical and cancellous bone), maxillary sinus, maxillary first molar and first premolar, periodontal membrane, and an RME appliance with arms, bands, and anchor screws. The expansion screws were activated 0.2mm transversely. RESULTS: The deepest palate model had the smallest lateral displacement of the tooth and expansion of the mid-palatal suture and the greatest strain of the arm among the models with different palate heights. The model with a larger diameter arm had the smallest arm strain among the models with various arm shapes. The model with an anchor screw had the greatest lateral displacement of the tooth and expansion of the mid-palatal suture among all models. CONCLUSIONS: For a deeper palate, the arm strain increased and the effect of RME decreased. Modified arm shapes such as a larger diameter arm, arms connected by a diagonal wire, a straight arm, and a shorter arm efficiently expanded the maxillary dental arch. Anchor screws increased the effect of RME, generated more and closer bodily movement of the tooth, and parallel expansion of the mid-palatal suture. The model with an anchor screw without arms decreased the displacement of the teeth compared to the models with arms, so the arms are necessary for effective RME.