In the present study, the author prepared surface hydrolyzed poly(lactic acid) (PLA) films (PLA-COOH), i.e., carboxylic acid introduction, by NaOH hydrolysis and investigated apatite formation on PLA-COOH films after immersion in Hanks' balanced salt solution (HBSS, pH 7.4) without organic species. In addition, the degradation behavior was monitored in buffered solution with or without surface carboxylic acid groups. HBSS was used as simulated body fluid for evaluating in vitro biocompatibility. PLA-COOH was easily prepared by NaOH hydrolysis for 3h. PLA-COOH showed a greater degree of degradation after immersion in phosphate buffered saline solution (pH 7.4) compared with native PLA. The weight loss of PLA and PLA-COOH films was approximately 3.2% and 4.9% after 115 days immersion, respectively. Scanning electron microscopy observation revealed that PLA-COOH film showed a quicker and greater amount of precipitation on the film compared with original unmodified PLA after immersion in HBSS. Analysis of the precipitates by X-ray diffraction and Fourier-transformed infrared spectroscopy confirmed that their main-component was carbonate-containing apatite. In conclusion, it is expected that PLA-COOH is a good candidate for a scaffold material to enhance bone formation in vivo and will have a potential for dental clinical use.
In vitro differentiation induction of normal human periodontal ligament fibroblasts, HpdLF, into an osteoblast lineage was attempted and the changes in gene expression were examined. The cells changed to an osteoblast-like cuboid morphology at 3 weeks after induction. Moreover, calcified nodules were detected and osteocalcin expression was confirmed at 3 weeks after induction, suggesting that the differentiation was progressing toward an osteoblast lineage. The expression of bone differentiation markers such as alkaline phosphatase, type 1 collagen, insulin-like growth factor (IGF) 1, IGF2, bone morphogenic protein 2 (BMP2) and osteocalcin at 3 weeks after induction were increased compared with pre-induction. The up-regulation of IGF1 or IGF2 may be due to activation of pathways for the induction of alkaline phosphatase and type 1 collagen, and the differentiation of HpdLF into an osteoblast lineage. The increased expression of BMP2 may be due to the production of osteocalcin and maturation of osteoblasts.
Complexes of daunorubicin hydrochloride (DH)-intercalated DNA and artificial lipids were synthesized. These DH-pre-intercalated DNA/lipid complexes were blended with PLGA (poly (D, L-lactide-co-glycolide)) to prepare DH-releasable films as a carrier material for a drug delivery system (DDS). The films could release DH in PBS solution. The amount of DH released was dependent on the chemical structure of the lipid and initial concentration of DH in the pre-intercalated DNA. Pre-intercalation slowed the release of DH better than post-intercalation. The DH released caused a reduction in cell viability during the culture of L-929 mouse fibroblasts. DH-pre-intercalated DNA/lipid/PLGA films have potential for the controlled-release of DH.
We evaluated the transplant of cultivated beagle dog periodontal ligament (PDL) cells grown on human amniotic membrane (AM) to promote periodontal regeneration in artificial fenestration defects in jaw bones. PDL cells were obtained from the extracted mandibular lateral incisors of three beagle dogs. PDL-derived cells were grown on an AM substrate. Artificial fenestration in periodontal defects was made bilaterally in the maxillary canines, PDL-derived cell sheets on AM were transplanted in the experimental group, and mucoperiosteal flaps were returned. The other sides were applied AM only as the control group. Four weeks after surgery, the dogs were sacrificed and specimens were examined histologically. In the experimental group, new cementum and bone were observed in the periodontal defect, whereas in the controls, connective tissue could only be observed. The results of the in vivo experimental study suggested that PDL-derived cell sheets on AM were useful biomaterial for periodontal regeneration.
In a previous in vivo transplantation study, we demonstrated hard tissue formation related with human dental pulp-derived (HDPD) cells in alginate scaffold. In the present in vitro study, we investigated the histology of the same HDPD cells with a collagen type I gel scaffold cultured in conditioned medium. Immunohistochemical, fine structure transmission electron microscopy (TEM), and molecular cell biological studies of HDPD cells-and-collagen mixture (cell clusters) demonstrated differentiation of odontoblast-like cells, which secreted dentin sialoprotein, dentin phosphoprotein and collagen types I and III in the extracellular matrix. The TEM study also revealed initial matrix vesicle-related and subsequent collagen-related mineralization. Results of this study elucidate that odontogenic HDPD cells have same properties as the odontoblast-like cells observed in our previous in vivo transplantation study. We concluded that HDPD cells differentiate and initiate dentin formation in the either collagen type I gel or alginate scaffolds, but they showed different morphology in in vivo and in vitro environments.
Dedifferentiated fat cells (DFATs) possess osteogenic potential making them a promising cell source for bone regeneration. PuraMatrixTM (PM), a self-assembling peptide scaffold, produces a nanoscale environment for cells. We examined radiologically the osteogenic potential and application of DFATs with PM for bone regeneration in rabbit calvarial defect models. DFATs were obtained from ceiling culture, and subsequently cultured in normal media (NM) or osteogenic media (OM). Their in vitro osteogenic differentiation potential was assessed using alizarin red staining, and by measuring osteocalcin expression and calcium concentration. Eighteen 6-mm calvarial defects were randomly treated in vivo with osteo-induced DFATs + PM, DFATs + PM, and PM scaffold, or no treatment. After 8 weeks, calvaria were harvested and the degree of healing between treatment groups was radiologically compared. Osteocalcin expression in OM significantly increased at 7 and 14 days, as well as calcium concentration at 21 days. There was no difference in healing among the four groups radiologically. Rabbit DFATs efficiently differentiate into osteoblasts when cultured in OM. To demonstrate DFAT usefulness in vivo, progressively more sophisticated animal models must be developed.