Development and Characterization of Alkaline Phosphatase-Positive Human Umbilical Cord Perivascular Cells.

Human umbilical cord perivascular cells (HUCPVCs), harvested from human umbilical cord perivascular tissue, show potential for future use as an alternative to mesenchymal stromal cells. Here, we present the results for the characterization of the properties alkaline phosphatase-positive HUCPVCs (ALP(+)-HUCPVCs). These ALP(+)-HUCPVCs were created from HUCPVCs in this study by culturing in the presence of activated vitamin D3, an inhibitor of bone morphogenetic protein signaling and transforming growth factor-beta1 (TGF-ß1). The morphological characteristics, cell proliferation, gene expression, and mineralization-inducing ability of ALP(+)-HUCPVCs were investigated at the morphological, biological, and genetic levels. ALP(+)-HUCPVCs possess high ALP gene expression and activity in cells and a slow rate of cell growth. The morphology of ALP(+)-HUCPVCs is fibroblast-like, with an increase in actin filaments containing alpha-smooth muscle actin. In addition to ALP expression, the gene expression levels of type I collagen, osteopontin, elastin, fibrillin-1, and cluster of differentiation 90 are increased in ALP(+)-HUCPVCs. ALP(+)-HUCPVCs do not have the ability to induce mineralization nodules, which may be due to the restriction of phosphate uptake into matrix vesicles. Moreover, ALP(+)-HUCPVCs may produce anti-mineralization substances. We conclude that ALP(+)-HUCPVCs induced from HUCPVCs by a TGF-ß1 stimulation possess myofibroblast-like properties that have little mineralization-inducing ability.

Effects of chronic Porphyromonas gingivalis lipopolysaccharide infusion on skeletal muscles in mice.

Periodontitis, which is caused by various oral organisms, predominantly affects adults, and is one of the main causes of tooth loss, as well as leading to progression of numerous systemic diseases. However, its relationship to sarcopenia (aging-associated degenerative loss of skeletal muscle mass and function) remains unclear. The aim of this study was to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) on skeletal muscle in mice, and to establish the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8 mg/kg/day) for 4 weeks. This treatment significantly decreased the weight of fast-twitch skeletal muscles (masseter and tibialis anterior muscles), but not that of slow-twitch skeletal muscle (soleus muscle). The area of fibrosis was significantly increased in masseter muscle, but remained unchanged in the other two muscles. The number of apoptotic myocytes was significantly increased (approximately eightfold) in masseter muscle. These data suggest that persistent subclinical exposure to PG-LPS might reduce the size of fast-twitch skeletal muscle, but not slow-twitch skeletal muscle. Masseter muscle appears to be especially susceptible to the adverse effects of PG-LPS, because muscle remodeling (muscle fibrosis and myocyte apoptosis) was induced solely in masseter muscle. Thus, periodontitis might be one of the major causes of oral sarcopenia.

Interleukin-4 released from human gingival fibroblasts reduces osteoclastogenesis.

OBJECTIVE: Human gingival epithelium is continuously exposed to bacteria and acts as the first line of defense in periodontal tissues. It is crucial to maintain healthy, non-inflamed gingival tissue to avoid gingivitis and periodontitis. The purpose of this study was to investigate the influence of IL-4 in human gingival fibroblasts (hGF) on the activation of osteoclasts. DESIGN: Two hGF samples were obtained from two healthy patients, and one was collected from a commercially available resource. The hGFs were cultured, and conditioned medium of hGF (hGF-CM) was stocked at -80°C. The mRNA was isolated from the hGF cultures and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of suppressive osteoclastogenetic mediators, such as interleukin (IL)-4, osteoprotegerin (OPG), IL-10, IL-27, and IL-33. The hGF-CM was used to investigate the inhibitory function of mouse macrophages supplemented with either glutathione S-transferase-Receptor activator of NF-kB ligand (GST-RANKL), human recombinant (rh)IL-4, or rhOPG but not a combination. Differentiation of osteoclasts was examined by tartrate resistant acid phosphatase (TRAP) staining and TRAP assay. The suppressive role of IL-4 was assessed by neutralizing IL-4 antibody in the TRAP assay. RESULTS: The hGF-CM reduced both TRAP positive staining and activity in a dose-dependent manner. IL-4 and OPG mRNA expressions were expressed in hGF-CM from three different donors but that of IL-10, IL-27, or IL-33 was not detected. In the RAW264 culture, rhIL-4 and rhOPG reduced TRAP positive staining as well as activity in a dose-dependent manner. Moreover, addition of neutralizing antibodies for IL-4 reduced the inhibitory effect of conditioned medium from gingival fibroblasts in the RAW264 culture. CONCLUSION: We concluded that hGF potentially contained suppressive mediators, such as IL-4 and OPG, for osteoclastogenesis. Moreover, we confirmed that the differential inhibition of osteoclast is caused by OPG as well as IL-4 in hGF-CM.

Bone formation by human umbilical cord perivascular cells.

We investigated the possibility of employing human umbilical perivascular cells (HUCPVCs) within the context of finding an alternative source of mesenchymal stromal cells (MSC) for bone tissue engineering. Since it has previously been reported that conditioned medium (CM) from osteogenic bone marrow (BM) MSCs can potentiate osteogenic differentiation in a secondary cell population, we also employed BM-MSCs to generate CM to stimulate osteogenesis in the HUCPVCs. The BM-MSCs were a commercially available immortalized human cell line. In vitro assays showed negligible levels of osteogenic gene expression in HUCPVCs compared to BM-MSC, but alkaline phosphatase was detected when HUCPVC were cultured in osteogenic medium in the presence of CM from BM-MSC. An in vivo assay employing a rat calvarial osteotomy defect, together with a collagen sponge scaffold, showed that HUCPVCs provided statistically significant bony repair compared to controls. BM-MSC loaded scaffolds were not statistically different from either controls or HUCPVCs. The addition of BM-MSC CM to HUCPVCs also produced no statistically significant difference to the bone formed by HUCPVCs alone. Our results demonstrate that the in vitro assays employed did not predict in vivo outcomes, and that the BM-MSC cell line employed, or CM from such cells, provided no osteogenic advantage over the use of HUCPVCs alone.

Peri-implant crestal bone loss: a putative mechanism.

Purpose. The immunological mechanisms of peri-implant crestal bone loss have, hitherto, not been elucidated. We hypothesized that bacterial products from the microgap cause upregulation of cytokines in otherwise healthy peri-implant cells, which results in osteoclast formation and, ultimately, in bone resorption. Materials and Methods. We used RT-PCR and ELISA to assay mediators of osteoclastogenesis in rat and human macrophages (r-and hMO); bone marrow derived stromal cells (r-and hBMCs); and human gingival fibroblasts (hGF)-with or without stimulation by LPS. TRAP positive multinucleate cells were assessed for their resorptive ability. Results. We show that IL-1α, IL-1ß, and IL-6 were expressed by all examined cell types, and TNF-α was upregulated in hGF. Secretion of IL-1α and IL-1ß proteins was stimulated in hMO by LPS, and IL-6 protein secretion was highly stimulated in hBMCs and hGF. Both LPS and RANKL stimulated macrophages to form osteoclast-like TRAP positive cells, which resorbed calcium phosphate substrates. Conclusion. Taken together, the results of our study support the hypothesis that bacterial endotoxins upregulate enhanced mediators of osteoclastogenesis in resident cells found in the healthy peri-implant compartment and that the local synergistic action of cytokines secreted by such cells results in the genesis of resorptively active osteoclasts.