Fotinos S. Panagakos

Insulin-like growth factors-I and -II stimulate chemotaxis of osteoblasts isolated from fetal rat calvaria

Repair and regeneration of damaged bone is believed to be regulated in part by growth factors stored in the bone matrix. These growth factors are synthesized and secreted by osteoblasts and are incorporated into the developing bone. This pool of stored growth factors is then released into the immediate area following resorption of the matrix. One of the initial steps in bone repair is the recruitment of osteoblasts to the repair site. Growth factors, such as TGF-beta and PDGF, which are present in bone matrix, have been shown to be chemotactic for osteoblasts. In this study, primary cultures of osteoblasts isolated from fetal rat calvaria were examined for chemotaxis in response to IGF-I and IGF-II. IGF-I stimulated a dose-dependent increase in osteoblast chemotaxis, while IGF-II stimulated chemotaxis maximally at the lowest concentration studied (0.1 ng/ml), and had no effect at the highest concentration studied (100 ng/ml). IGF-I and -II had no effect on osteoblast proliferation at any of the concentrations examined. These results indicate that IGFs may be playing an important role in the early stages of bone repair by stimulating osteoblast chemotaxis to the repair site.

Formation and mineralization of extracellular matrix secreted by an immortal human osteoblastic cell line: modulation by tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF -ga), a 17-kDa cytokine produced by stimulated macrophages/monocytes, modulates the functions of a variety of cells and has been shown to induce bone resorption in vitro. However, the effects that TNF-α may have on the process of bone formation are not completely understood. In order to study the effects of TNF-α on matrix development and mineralization, we utilized a human osteoblastic cell line, HOS TE85. Our results show that HOS TE85, which has been shown to be responsive to hormones active on normal osteoblasts, forms an extensive extracellular matrix (ECM) that mineralizes during extended culture. Treatment during the development of the matrix with TNF-α has little effect on cell number and DNA synthesis, showing thereby that TNF-α is not cytotoxic to the cells. However, TNF-α inhibits the formation of alkaline phosphatase (AP) -positive foci in a dose-dependent manner at concentrations of 0.1–10 ng/ml. TNF-α treatment caused a significant decrease in the incorporation of collagen into the developing matrix. In addition, TNF-α treatment resulted in a significant decrease in the synthesis of AP by HOS TE85 cells during the process of ECM formation and resulted in a pronounced lack of mineralization of the ECM. These results indicate that TNF-α may be acting as an uncoupler by decreasing the synthesis and incorporation of proteins required for bone formation, and inhibiting matrix formation and mineralization in vitro.

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: Effect of tumor necrosis factor-alpha

Recent work by a number of investigators has demonstrated that the process of bone matrix formation and mineralization is under the influence of growth factors and cytokines present in the local environment. Utilizing primary and established osteoblast cell culture systems, these studies have examined the regulation of bone matrix protein synthesis and deposition into the extracellular matrix (ECM) and subsequent mineralization. In previous studies, we have utilized the human osteoblastic cell line, HOS TE85, to study the effects of Tumor Necrosis Factor - alpha (TNF-α) on the regulation of matrix proteins and proteolytic function in monolayer cultures as well as during the development and calcification of ECM formed by HOS TE85 cells during extended culture. Our studies demonstrate that TNF-α inhibited formation and mineralization of nodules. In the study reported here, we evaluated the ultrastructural morphology of the cell-matrix complex formed by HOS TE85 cells in the presence and absence of TNF-α at selected time points during the matrix development process utilizing both transmission electron microscopy and light microscopy. In the presence of TNF-α, the cell-matrix complex does not develop normally, with a lack of organization and mineralization, when compared to untreated cells. The lack of mineralization appears to result from the lack of normal collagen fibril deposition and formation of an appropriate ECM essential for the mineralization process. These results support our previous observations that TNF-α inhibits HOS TE85 cells from forming a mineralizing ECM by inhibiting incorporation of collagen into the ECM and inducing the synthesis of proteolytic enzymes capable of degrading collagen in the ECM.

Differentiation of human osteoblastic cells in culture

We have followed the synthesis and secretion of urokinase-type plasminogen activator (u-PA) and its inhibitor, PAI-1, and matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP-1) during differentiation of a human osteoblastic cell line, HOS TE85, and the effect of TNF-α on this process. Our results show that the ratio of u-PA/PAI-1 associated with the cell-matrix components increases during differentiation of these cells over a 14-day period. Although TNF-α suppresses the induced increase in steady-state mRNA levels of u-PA and PAI-1 during maturation of extracellular matrix (ECM), the u-PA/PAI-1 ratio is altered in such a way that PA activity associated with the ECM is higher than control cells. The expression of MMP-1 is low and remains essentially invariant over a culture period of 14 days. TNF-α enhances MMP-1 transcription nearly 12-fold initially, after which mRNA levels drop off but remain significantly higher than the controls. Activities and steady-state mRNA levels of MMP-2 and MMP-9 increase nearly 15-fold during maturation of the ECM, but the level of TIMP-1 mRNA is not appreciably altered. The presence of TNF-α suppresses maturation-induced transcription of MMP-2, enhances TIMP-1 transcription, but has little effect on MMP-9 mRNA levels. The data show that chronic exposure to TNF-α alters the balance between u-PA/PAI- 1 and MMPs/TIMP-1, which favors higher activity of proteinases. Accordingly, the presence of TNF-α in chronic inflammatory episodes would be expected to alter bone remodeling by inhibiting maturation of ECM and formation of bone.

The effect of gallium nitrate on synoviocyte MMP activity

Gallium, a group IIIa metal salt, has been demonstrated to be an effective immunosuppressive agent. Gallium has also been shown to inhibit the production of inflammatory cytokines, such as IL-1beta, produced by macrophage-like cells in vitro. To further characterize the effects of gallium on the inflammatory process, we examined the effects of gallium nitrate on matrix metalloproteinase (MMP) activity utilizing the rabbit synoviocyte cell line HIG-82. HIG-82 cells were incubated with IL-1beta and TPA, with and without increasing concentrations of gallium nitrate. Conditioned medium was collected and assayed for MMP activity using a synthetic substrate and substrate gel zymography. IL-1beta and TPA alone induced MMP activity in HIG-82 cells. A dose-dependent inhibition of IL-1beta and TPA stimulated MMP activity by gallium nitrate at increasing concentrations was observed. This study demonstrates that gallium nitrate can inhibit the activity of MMPs and may be useful as a modulator of inflammation in arthritis.

Incidence of percutaneous injuries at a dental school: a 4-year retrospective study.

BACKGROUND Infection control training for predoctoral dental students, dental hygiene students, and dental assistant students has assumed an important role in the educational process at our institution. As part of an ongoing review of the curriculum at our school, we conducted a retrospective analysis of reported percutaneous injuries during the years 1991 through 1994 to determine whether the increase in infection control training introduced at the school in 1990 has had an effect on our rate of percutaneous injuries. METHODS The population examined in this retrospective study consisted of predoctoral and postdoctoral dental students, dental hygiene students, dental assistant students, and staff. The data for this retrospective study were obtained from annual reports of occupational exposures incurred by students and staff. These annual reports were generated by compiling and summarizing all percutaneous injury incident reports that were prepared for that year. RESULTS Our results indicate, that except for an increase in 1992, the total number and incidence of reported percutaneous injuries decreased from 1991 to 1994. Statistically significant decreases were seen in the total number of reported percutaneous injuries for all students, staff, and all groups combined. On the basis of data available for 1993 and 1994, the incidence of reported percutaneous injuries per 1000 procedures was fairly constant over these 2 years. Distribution of percutaneous injuries by source varied during the 4-year period. CONCLUSIONS As part of the outcomes assessment program at our institution, we conducted a retrospective study of reported percutaneous injuries from 1991 to 1994. This study demonstrated that, although the total number of injuries decreased significantly, the rates within certain individual groups remained unchanged. On the basis of this observation, increased emphasis in the prevention of percutaneous injuries through additional training is indicated for these groups.

Heparin fails to potentiate the effects of IL-1β-mediated bone resorption of fetal rat long bones iin vitro

Heparin has been identified as a potent modulator of bone resorption. Heparin induces osteoporosis during long-term administration and has been shown in vitro to enhance the effects of other bone resorbing factors, including parathyroid hormone. In this study, we examined the effects of heparin on the bone-resorbing activity of the inflammatory cytokine IL-1 beta. Resorption was determined by measuring release of previously incorporated 45Ca from fetal rat long bones cultured in medium supplemented with either 0.1% bovine serum albumin or 10% heat-inactivated fetal calf serum. Heparin, in the absence of serum, decreased basal resorption at 4 and 10 units/ml, and slightly increased resorption at 30 units/ml. Heparin had no effect on IL-1 beta-stimulated resorption. In the presence of serum, heparin induced a two-fold increase in resorption alone, however, when cocultured with IL-1 beta, heparin failed to further enhance IL-1 beta-stimulated resorption.