The in vivo origin of bone-producing osteoblasts is not fully defined. Skeletal stem cells, a population of mesenchymal stem cells resident in the bone marrow compartment, are thought to act as osteoprogenitors during growth and adulthood. Quiescent bone lining cells (BLCs) have been suggested as a population capable of activation into mature osteoblasts. These cells were defined by location and their morphology and studies addressing their significance have been hampered by their inaccessibility, and lack of markers that would allow for their identification and tracing. Using lineage tracing models, we have observed labeled osteoblasts at time points extending beyond the reported lifespan for this cell type, suggesting continuous reactivation of BLCs. BLCs also make a major contribution to bone formation after osteoblast ablation, which includes the ability to proliferate. In contrast, mesenchymal progenitors labeled by Gremlin1 or alpha smooth muscle actin do not contribute to bone formation in this setting. BLC activation is inhibited by glucocorticoids, which represent a well-established cause of osteoporosis. BLCs express cell surface markers characteristic of mesenchymal stem/progenitors that are largely absent in osteoblasts including Sca1 and Leptin Receptor. BLCs also show different gene expression profiles to osteoblasts, including elevated expression of Mmp13, and osteoclast regulators RANKL and macrophage colony stimulating factor, and retain osteogenic potential upon transplantation. Our findings provide evidence that bone lining cells represent a major source of osteoblasts during adulthood.

Heterotopic ossification (HO) is a pathological process where bone forms in connective tissues such as skeletal muscle. Previous studies have suggested that muscle-resident non-myogenic mesenchymal progenitors are the likely source of osteoblasts and chondrocytes in HO. However, the previously identified markers of muscle-resident osteoprogenitors label up to half the osteoblasts within heterotopic lesions, suggesting other cell populations are involved. We have identified alpha smooth muscle actin (αSMA) as a marker of osteoprogenitor cells in bone and periodontium, and of osteo-chondro progenitors in the periosteum during fracture healing. We therefore utilized a lineage tracing approach to evaluate whether αSMACreERT2 identifies osteoprogenitors in the muscle. We show that in the muscle, αSMACreERT2 labels both perivascular cells, and satellite cells. αSMACre-labeled cells undergo osteogenic differentiation in vitro and form osteoblasts and chondrocytes in BMP2-induced HO in vivo. In contrast, Pax7CreERT2-labeled muscle satellite cells were restricted to myogenic differentiation in vitro, and rarely contributed to HO in vivo. Our data indicate that αSMACreERT2 labels a large proportion of osteoprogenitors in skeletal muscle, and therefore represents another marker of muscle-resident cells with osteogenic potential under HO-inducing stimulus. In contrast, muscle satellite cells make minimal contribution to bone formation in vivo.

The development of bioactive injectable system as cell carrier with minimal impact on viability of encapsulated cells represents a great challenge. In the present work, we propose a new pH-responsive chitosan-hydroxyapatite-based hydrogel with sodium bicarbonate (NaHCO) as the gelling agent. The in situ synthesis of hydroxyapatite phase has resulted in stable composite suspension and final homogeneous hydrogel. The application of sodium bicarbonate has allowed non-cytotoxic fast gelation of chitosan-hydroxyapatite within 4min, and without excess of sodium ions concentration. Rheological properties of crosslinked hydrogel have demonstrated possible behaviour as 'strong physical hydrogel'. The live dead staining has confirmed good viability and dispersion, as well as proliferation of encapsulated cells by the culture time. Presented preliminary results show good potential of chitosan-hydroxyapatite/NaHCO as a cell carrier, whose impact on the cell differentiation need to be confirmed by encapsulation of other cell phenotypes.

AbstractObjective:To determine the difference in the levels of Streptococcus mutans and S sobrinus in stimulated saliva in orthodontic patients with different bracket types (stainless steel and esthetic brackets) using polymerase chain reaction and cultivation method.Materials and Methods:Thirty-two patients, aged 13 to 30 years, were selected following these criteria: 1) orthodontic treatment indication, 2) systemic health, and 3) no tobacco and antibiotic consummation for three months prior to the commencement of the study. Patients were divided into two groups according to the bracket type; 16 patients formed the conventional bracket group (stainless steel brackets), and 16 patients formed the esthetic bracket group (plastic brackets). The levels of S mutans and S sobrinus in stimulated whole saliva samples were collected prior to fixed orthodontic appliance placement (T1) and 12 weeks after placement (T2), as were the Decayed, Missing, and Filled Surface Index (DMFS) and Oral Hygiene Index-Simplified (OHI-S). Mann-Whitney, Wilcoxon, and chi-square tests were used for statistical analysis.Results:Statistical analysis (chi-square test) showed no difference in S mutans and S sobrinus counts among patients with different brackets at either T1 or T2. There was no difference in total bacteria counts after fixed orthodontic appliance placement.Conclusion:The number of colony-forming units of S mutans and S sobrinus in stimulated saliva samples does not seem to be significantly different between patients with stainless steel brackets and patients with plastic brackets.

Objective The purpose of this study was to assess differences in gene expression between cementoblasts and osteoblasts using gene profiling of cell populations isolated directly from osteocalcin-GFP transgenic mice. Background Cementum and bone are similar mineralized tissues, but cementum accumulates much more slowly, does not have vasculature or innervation or undergo remodeling. Despite these differences, there are no well-established markers to distinguish cementoblasts from other mature mineralizing cells such as osteoblasts and odontoblasts. Methods Osteocalcin-GFP (OC-GFP) reporter mice were used as they show labeling of cementoblasts, osteoblasts and odontoblasts, but not periodontal ligament fibroblasts, within the periodontium. We sorted cells digested from the molar root surface to isolate OC-GFP+ cementoblasts. Osteoblasts were isolated from calvarial digests. Microarray analysis was performed, and selected results were confirmed by real time PCR and immunostaining or in situ hybridization. Results Microarray analysis identified 95 genes that were expressed at least 2-fold higher in cementoblasts than osteoblasts. Our analysis indicated that the Wnt signaling pathway was differentially regulated, as were genes related to skeletal development. Real time PCR confirmed that expression of Wnt inhibitors Wif1 and Sfrp1 was elevated in cementoblasts compared to osteoblasts, and Wif1 expression was localized to the apical root region. In addition, the transcription factor Barx1 was expressed at higher levels in cementoblasts, and immunohistochemistry indicated that BARX1 expression was detectable in apical cementoblasts and cementocytes, but was not present in osteoblasts or odontoblasts. Conclusion The OC-GFP mouse provides a good model for selectively isolating cementoblasts, and allowed for identification of differentially expressed genes between cementoblasts and osteoblasts.

Presently there is no clear evidence for the ability of mature osteogenic lineage cells to dedifferentiate. In order to identify and trace mature osteogenic lineage cells, we have utilized transgenic mouse models in which the dentin matrix protein 1 (Dmp1) promoter drives expression of GFP (active marker) or Cre recombinase (historic label) in preosteocytes/osteocytes. In long bone chip outgrowth cultures, in which cells on the bone surface were enzymatically removed, cells with previous activity of the Dmp1 promoter migrated onto plastic and down-regulated Dmp1-GFP expression. Dmp1Cre-labeled cells from these cultures had the potential to re-differentiate into the osteogenic lineage, while the negative population showed evidence of adipogenesis. We observed numerous Dmp1Cre-labeled osteoblasts on the surface of bone chips following their in vivo transplantation. Our data indicate that cells embedded in bone matrix are motile, and once given access to the extra bony milieu will migrate out of their lacunae. This population of cells is phenotypically and functionally heterogeneous in vitro. Once the preosteocytes/osteocytes leave lacunae, they can dedifferentiate, potentially providing an additional source of functional osteoblasts.

AIM: Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs).METHODS: Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers – alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry.RESULTS: In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein.CONCLUSION: Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage

Purpose Osteogenesis imperfecta is a serious genetic disorder that results from improper type I collagen production. We aimed to evaluate whether bone marrow stromal cells (BMSC) delivered locally into femurs were able to engraft, differentiate into osteoblasts, and contribute to formation of normal bone matrix in the osteogenesis imperfect murine (oim) model. Methods Donor BMSCs from bone-specific reporter mice (Col2.3GFP) were expanded in vitro and transplanted into the femoral intramedullary cavity of oim mice. Engraftment was evaluated after 4 weeks. Results We detected differentiation of donor BMSCs into Col2.3GFP+ osteoblasts and osteocytes in cortical and trabecular bone of transplanted oim femurs. New bone formation was detected by deposition of dynamic label in the proximity to the Col2.3GFP+ osteoblasts, and new bone showed more organized collagen structure and expression of type I α2 collagen. Col2.3GFP cells were not found in the contralateral femur indicating that transplanted osteogenic cells did not disseminate by circulation. No osteogenic engraftment was observed following intravenous transplantation of BMSCs. BMSC cultures derived from transplanted femurs showed numerous Col2.3GFP+ colonies, indicating the presence of donor progenitor cells. Secondary transplantation of cells recovered from recipient femurs and expanded in vitro also showed Col2.3GFP+ osteoblasts and osteocytes confirming the persistence of donor stem/progenitor cells. Conclusion We show that BMSCs delivered locally in oim femurs are able to engraft, differentiate into osteoblasts and osteocytes and maintain their progenitor potential in vivo. This suggests that local delivery is a promising approach for introduction of autologous MSC in which mutations have been corrected.