Osteoblast-like Line Research Articles

The Effect and the Potential Use of Magnetic–Dam Barrier in Guided Bone Regeneration: A Laboratory Study

Guided bone regeneration (GBR) has been shown to be an optimal technique to accelerate the bone regeneration process thanks to the action of membrane barriers that promote tissue healing through the process of osteogenesis, inducing the repopulation with osteoprogenitor cells that prevent the invasion of non-osteogenic tissue. However, current membranes, such as expanded polytetrafluoroethylene or rubber dam, have some disadvantages that could potentially reduce the effectiveness of GBR. Recently, some scaffolds with magnetic properties have been tested to promote rapid osteogenesis. The aim of this laboratory study was to evaluate the intensity of the magnetic field generated by a custom-made rubber dam magnetised with neodymium-iron-boron (Nd2F14B) (three layers of latex filled with Nd2F14B powder on the inner surface) and to understand the effects of such a membrane on cell viability. A magnetic field of 750 G, 400 G, and 900 G was generated on the surface and on the long and wide sides of 3 and 2 cm in contact with the rubber dam. At a distance of 1 mm from the magnetic dam, a magnetic field of 300 G, 150 G, and 400 G was measured on the surface and on the long and wide sides of the rubber dam, respectively. After 72 h, the MG-63 osteoblast-like line showed a slight decrease in cell proliferation (85 ± 10) compared with the unmodified dam (95 ± 6) and the cell control population. According to our findings, this magnetic cofferdam is able to generate a static magnetic field and significantly affect cell proliferation in contrast to other nonabsorbable membranes. Further laboratory studies and subsequent clinical trials are needed to evaluate the significant improvements that can be achieved by using this type of magnetic rubber dam in GBR.

Cytobiocompatibility of collagen and ePTFE membranes on osteoblast‐like cells in vitro

In guided bone regeneration (GBR), a semipermeable membrane is placed over an osseous defect to create a secluded environment in which bone formation can proceed without ingrowth of connective tissue cells from the overlaying soft tissue. Although the cell-occlusive property of GBR membranes appear to be essential to new bone formation, the role of transmembrane tissue fluid diffusion is not known. The objective of this study was to evaluate the degree to which diffusion across commonly used GBR membranes can support functional properties of osteoblast-like cells in vitro. Cells from an established osteoblast-like line (SAOS-2) were cultured on membranes of cross-linked collagen, noncross linked collagen, and ePTFE. The membranes rested on metal grids which allowed the membranes to lightly contact the surface of the culture medium. As a control, cells were directly plated and cultured in control wells. At days 7 and 21, cells were harvested by scraping the membranes or culture wells and analyzed for expression of alkaline phosphatase (ALP), core binding factor 1 (cbfa-1), bone sialoprotein-2 (BSP-2), and osteocalcin (OC). Expression was determined by quantitative real-time PCR. Glucose-6-phosphate dehydrogenase (G6PD) served as a reference gene. The membranes were examined by transmission light microscopy. RT-PCR revealed up-regulation of ALP of up to 60-fold and of cbfa-1 and BSP of up to threefold relative to G6PDH. Expression of OC was less then onefold. The expression profile for each of the four genes tested demonstrated small variations among cells grown on different membranes. Microscopic observations revealed remnants of undisrupted osteoblast-like cells attached to both collagen membranes. Cell morphology and spatial arrangement indicated that vitality was maintained. Diffusion through the three membranes evaluated in this study was sufficient to support osteoblast-like cell differentiation.

Clodronate stimulates osteoblast differentiation in ST2 and MC3T3-E1 cells and rat organ cultures

We investigated the direct effects of various bisphosphonates on osteoblasts. At 10 −5 M, clodronate increased alkaline phosphatase activity in cultured MC3T3-E1 (osteoblast-like line) and ST2 (pluripotent mesenchymal line) cells. Etidronate significantly increased alkaline phosphatase activity at 10 −5 M only in MC3T3-E1 cells. These effects were due to an increase in alkaline phosphatase-positive cell numbers, and the differentiation-enhanced cells were capable of mineralization (von Kossa stain). Other bisphosphonates (pamidronate, alendronate, and incadronate) did not increase alkaline phosphatase activity in either cell line. In cultured rat calvariae, clodronate stimulated the expression of genes for alkaline phosphatase and osteocalcin (osteoblast-differentiation markers), but decreased the expression of the gene for tartrate-resistant acid phosphatase (osteoclast marker). Clodronate, etidronate, and incadronate inhibited protein Tyr phosphatase and Ser/Thr phosphatase activities in MC3T3-E1 cells. These data suggest that clodronate acts directly on mesenchymal cells to enhance osteoblast differentiation, and this effect may be partly expressed through inhibition of protein Tyr phosphatase and/or Ser/Thr phosphatase activity.

Further analysis of spontaneous membrane potential activity and the hyperpolarizing response to parathyroid hormone in osteoblastlike cells.

Whole cell voltage clamp measurements using the patch technique on well-attached and well-spread cells of an osteoblastlike line (ROS 17/2.8) show the same spontaneous membrane potential activity as measurements with inserted microelectrodes. Furthermore, membrane potential measurements during the first 80 milliseconds (ms) following microelectrode penetration of the cell membrane usually show no decay. There is also good agreement between values of cell membrane resistance obtained by the microelectrode technique, the whole cell patch clamp technique, and the single channel patch clamp technique. These results indicate that our microelectrode measurements are not dominated by leak-induced artifacts, and that the spontaneous membrane potential activity is not induced by Ca2+ leakage around the microelectrode. The spontaneous membrane potential activity is eliminated in the presence of the Ca2+ ionophore A23187, also in serum-free medium, and by K+ and Ca2+ channel blockers, but it is not affected by the hyperpolarizing responses to parathyroid hormone (PTH) and dibutyryl cAMP, which persist under all of these conditions. These results support the hypothesis that the spontaneous membrane potential activity is related to repeated fluctuations of internal [Ca2+] and that such fluctuations result from a feedback loop involving Ca2+ channels or Ca2+ pumps in the cell membrane.

Electrophysiological responses of osteoclasts to hormones.

Electrophysiological measurements were carried out on osteoclasts in vitro. Such isolated osteoclasts are able to resorb bone in vitro and contract in response to calcitonin (CT). Our measurements show that individual osteoclasts respond to CT with a significant transient hyperpolarization of membrane potential. Application of parathyroid hormone (PTH) and dibutyryl cAMP produced a transient hyperpolarization in some osteoclasts. Measurements on an osteoblastlike line (ROS 17/2.8) showed a sustained hyperpolarizing response to CT, which is similar to but smaller than the hyperpolarizing response to PTH and dibutyryl cAMP in this and some other osteoblastlike lines. In contrast to osteoblastlike cells, the osteoclasts have no long term membrane potential response to CT, to PTH, or to dibutyryl cAMP. These results show that there are distinct differences between osteoclasts and osteoblasts in their ion transport responses to hormones.