Neonatal Rat Calvarial Osteoblasts Research Articles

Quantification of Adhesiveness of Osteoblasts to Titanium Surfacesin Vitroby the Micropipette Aspiration Technique

For osseointegration to occur at a biomaterial surface implanted in tissue, osteoblasts must first adhere to the surface and subsequently lay down a framework of submatrix proteins and mineral. Thus the adhesion force of an osteoblast to a surface can serve as a measure of its biocompatibility during the first hours after implantation. This study used the micropipette aspiration technique to quantify the adhesion force of individual Ost-6 neonatal rat calvarial osteoblasts interacting with smooth, rough, fibronectin (FN)-coated, and hydroxyapatite (HA)-coated commercially pure titanium surfaces. Individual cellular adhesion forces ranged from 2 to 320 mdyn and were proportional to the time allowed to adhere (seeding time, 15-120 min) and adsorbed FN coating concentration (5-20 microg/ml). For the 60 min seeding time, osteoblasts adhered 80% more strongly to 5 microg/ml FN-coated titanium than to smooth uncoated titanium, due to specific affinity of the alpha(v)beta(1) transmembrane integrin for the FN molecule. Average adhesion force on plasma sprayed HA-coated titanium was only 40% as high as the uncoated surface, indicating that the osteoconductive effects of HA were not evident in the first 60 min of adhesion. There was no difference in adhesion force between the uncoated smooth and rough titanium surfaces for the 60 min seeding time. The uncoated and FN-coated titanium surfaces had the same average cell-surface contact angle used to assess the degree of cell flattening on the titanium surface. This indicated that osteoblasts formed equal quantities of cell-substrate bonds to the two surfaces but with very different adhesion forces, thus osteoblast affinity for the titanium surfaces was expressed in higher individual cell-substrate bond forces.

Analysis of osteoblast mineral deposits on orthopaedic/dental implant metals

Neonatal rat calvarial osteoblasts were cultured on Ti-6Al-4V, Co-Cr-Mo alloy, 316L stainless steel and polystyrene (reference substrate) in the presence of ascorbic acid and 10 m m β-glycerophosphate for 16, 17, 18, 19, 20, 21, 24 and 28 d. Scanning electron microscopy examination revealed that osteoblasts cultured on these orthopaedic/dental implant metals synthesized and deposited an extracellular matrix containing collagenous and non-collagenous components, as well as mineral nodules of various morphologies. Energy dispersive spectrometry revealed that the mineral deposits consisted of three distinct chemical compositions: calcium phosphate, calcium-sulphur-phosphorus, and calcium only. Backscattered electron imaging demonstrated that both the calcium phosphate and calcium-only deposits were electron dense, while the calcium-sulphur-phosphorus deposits were electron translucent. X-ray diffraction analysis indicated that the bulk of the osteoblast mineral deposits was amorphous hydroxyapatite; in addition, electron diffraction analysis revealed small regions of crystalline hydroxyapatite.

Examination of osteoblastorthopaedic biomaterial interactions using molecular techniques

Molecular techniques can be used to elucidate the effects of extended periods of cellbiomaterial interactions on the time-course and level of expression of particular genes which determine cellular phenotype. We used the polymerase chain reaction to demonstrate the expression of genes for the bone-related proteins osteocalcin, osteonectin and osteopontin by neonatal rat calvarial osteoblasts. In addition, Northern blotting was subsequently used to show that messenger RNAs encoding osteonectin and osteopontin were consistently expressed during a 5 wk period of interaction of osteoblasts with Ti-6AI-4V, a commercial brand of hydroxyapatite, and tissue culture polystyrene.

Osteoblast responses to orthopedic implant materials in vitro

Responses of neonatal rat calvarial osteoblasts to a variety of orthopedic implant materials were examined in vitro. Attachment, proliferation, and collagen synthesis of a well-characterized line of osteoblasts with 316L stainless steel, Ti-6Al-4V, Co-Cr-Mo, PMMA, hydroxyapatite, borosilicate glass, and tissue culture polystyrene were studied. Cell adhesion and growth were similar on nonapatitic materials. In contrast, attachment and growth of osteoblasts were significantly lower and slower, respectively, on hydroxyapatite. Collagen synthesis per cell and relative collagen synthesis, however, were comparable on all the materials tested.

Contribution of marrow stromal cells to the regulation of osteoblast proliferation in rats: evidence for the involvement of insulin-like growth factors

Fibroblast-like marrow stromal cells are believed to play a role in the maintenance of osteoblast populations at the marrow-bone interface. We now report that this interaction may be very specific. Stromal cell conditioned medium (SC-CM) stimulated DNA synthesis and proliferation in culture of neonatal rat calvarial osteoblasts at low concentrations (1.25–5%), but was inhibitory at 10%. At growth promoting effective concentrations, the activity of osteoblast alkaline phosphatase was decreased. This action was selective since SC-CM failed to promote the growth of rat calvarial fibroblasts. Characterization of the SC-CM indicated the cells produced IGF-I and -II and a wide range of molecular weight fractions with putative stimulatory action (FPLC analysis using Superose 12 and 6 gel permeation columns). HPAEPAD analysis showed that some elements were glycosylated, and the composition suggested the presence of N- and O-linked oligosaccharide chains. Because rat marrow stromal fibroblast-like cells produce a number of osteotropic factors which affect calvarial osteoblast growth, these interactions may be important to considerations about the etiology of the osteoporoses.

Partial characterization of rat marrow stromal cells.

Fibroblast-like rat marrow stromal cell (CFU-F) cultures have been characterized in terms of their responsiveness to calciotropic hormones, metal ions, the nonsteroidal antiinflammatory drug, and by their putative paracrine role in the maintenance of active populations of osteoblasts at the marrow-bone interface. These studies indicate that CFU-Fs lack a complete osteoblast signature. Subconfluent CFU-Fs grown in the presence or absence of 10(-7) M dexamethasone lack receptors for PTH and calcitonin, and fail to show enhanced cAMP or cGMP responses to 10(-7) M 1-34 PTH (rat), or any evidence of osteocalcin production [+/- 10(-9) M 1,25-(OH)2D3]. Low concentrations of fluoride [10(-12) and 10(-9) M] stimulated CFU-F grown in vitro in serum-free media, though higher levels (10(-7) and 10(-6) M), inhibited growth in vivo and in vitro. Aluminum (10(-12)-10(-7) M) and ibuprofen (10(-7) M) did not alter normal growth patterns, indicating an action on bone cells more differentiated than CFU-Fs. Serum-free conditioned medium (CM) from control and ovariectomized (OVX)/OVX+ dihydrotachysterol-Rx rat CFU-F cultures was mitogenic for neonatal rat calvarial osteoblasts in vitro, but not for ROS 17/2.8 cells. The studies affirm the mesenchymal-like character of CFU-Fs and project their significant role in sustaining functional endosteal osteogenic cell populations.

RGDS tetrapeptide binds to osteoblasts and inhibits fibronectin-mediated adhesion

The mechanisms of osteoblast attachment to surfaces were probed using the adhesive tetrapeptide RGDS (Arg-Gly-Asp-Ser) and the related but non-adhesive RGES (Arg-Gly-Glu-Ser). Specifically, RGDS and RGES were investigated for their ability both to bind to a suspension of well-characterized neonatal rat calvarial osteoblasts and to inhibit cell attachment to fibronectin-coated microtiter plates. RGDS bound to the cells with an average K d ~ 9.4 × 10 − 4 M, and RGES bound with an average K d ~ 3.0 × 10 −4 M; at saturation, the osteoblasts bound almost twice as much RGDS as RGES. RGDS partially inhibited cell adhesion (55% to 60%) in a competitive, dose-dependent manner. In contrast, RGES had minimal effect on cell attachment. Since complete inhibition of attachment was not observed, it is likely that a synergistic adhesion site in the fibronectin molecule and/or cell surface molecules such as proteoglycans are active in mediating osteoblast/substrate adhesion.