D. M. Brunette

Titanium in Medicine : material science, surface science, engineering, biological responses and medical applications

This comprehensive book provides state-of-the-art scientific and technical information in a clear format and consistent structure making it suitable for formal course work or self-instruction. T ...

A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system.

SummaryA dextran-polythylene glycol aqueous two-phase system has been used to separate cell surface membranes from other cellular organelles. The surface membranes have been identified on the basis of morphology, content of Na+, K+-ATPase, and presence of surface antigen as detected by a51Cr release method. Contamination of the surface membrane preparations by smooth endoplasmic reticulum, mitochondria, and nuclei has been found to be minimal. An average of 6.5% of the total protein was found in the membrane fraction. Less than two hours is required to isolate the membrane fraction after preparation of a Dounce homogenate. Fractionation by aqueous two-phase polymer systems appears to be a rapid and effective method for the isolation of surface membranes.

Ouabain-resistant mutants of mouse and hamster cells in culture

Abstract Somatic cell mutants resistant to ouabain, which inhibits the plasma membrane Na/K ATPase, have been isolated from mouse L and Chinese hamster ovary (CHO) cells. Ouabain at concentrations ≥ 1 mM with 5–6 mM K + , or ≥ 0.1 mM with 0.5 mM K + , inhibits the growth of the wild-type cells and is ultimately cytotoxic. Clones 2- to 100-fold more resistant than wild type in terms of dose can be obtained by single-step selection from a wild-type population in the presence of ouabain. The phenotypes of ouabain-resistant (OUA R ) clones are reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Wild-type and OUA R L cell clones were compared with respect to their susceptibility to ouabain inhibition of 42 K uptake by whole cells and of Na/K ATPase activity in isolated plasma membranes. In both respects the OUA R cells are less sensitive to the drug than are wild-type cells. Conditions for optimal ATPase activity in the absence of ouabain were indistinguishable for the wild-type and one OUA R clone examined in detail and were comparable to requirements reported for ATPase preparations from other source materials. The frequency of OUA R cells in a wild-type population can be substantially increased, to approximately 10 −4 per viable cell, by exposure to the chemical mutagen EMS. The spontaneous mutation rate to 10-fold increase in ouabain-resistance is estimated by Luria-Delbruck fluctuation analyses to be 5–6 × 10 −8 per cell per generation for both L and CHO cells. Cell-cell hybridization experiments utilizing OUA R and wild-type CHO cells indicate that resistance to ouabain behaves as a codominant trait, and that this marker can be useful for selection of somatic cell hybrids.

The effects of the surface topography of micromachined titanium substrata on cell behavior in vitro and in vivo.

Surface properties, including topography and chemistry, are of prime importance in establishing the response of tissues to biomaterials. Microfabrication techniques have enabled the production of precisely controlled surface topographies that have been used as substrata for cells in culture and on devices implanted in vivo. This article reviews aspects of cell behavior involved in tissue response to implants with an emphasis on the effects of topography. Microfabricated grooved surfaces produce orientation and directed locomotion of epithelial cells in vitro and can inhibit epithelial downgrowth on implants. The effects depend on the groove dimensions and they are modified by epithelial cell-cell interactions. Fibroblasts similarly exhibit contact guidance on grooved surfaces, but fibroblast shape in vitro differs markedly from that found in vivo. Surface topography is important in establishing tissue organization adjacent to implants, with smooth surfaces generally being associated with fibrous tissue encapsulation. Grooved topographies appear to have promise in reducing encapsulation in the short term, but additional studies employing three-dimensional reconstruction and diverse topographies are needed to understand better the process of connective-tissue organization adjacent to implants. Microfabricated surfaces can increase the frequency of mineralized bone-like tissue nodules adjacent to subcutaneously implanted surfaces in rats. Orientation of these nodules with grooves occurs both in culture and on implants. Detailed comparisons of cell behavior on micromachined substrata in vitro and in vivo are difficult because of the number and complexity of factors, such as population density and micromotion, that can differ between these conditions.

Fibroblasts on micromachined substrata orient hierarchically to grooves of different dimensions

Contact guidance was studied by light, scanning (SEM) and transmission electron microscopy (TEM) in cultures of human gingival fibroblasts cultured on grooved surfaces. The grooves were originally produced in silicon wafers by micromachining, a process which is based on the methods used to fabricate microelectronic components, and the grooved surfaces were then replicated in Epon. Micromachining enables precise control of groove depth, groove spacing, and groove shape to be obtained. In silicon wafers with appropriate crystal orientation, a second smaller set of grooves, called the minor grooves, is found on the floor of the major grooves. The minor grooves are oriented at a 54 degree angle to the major grooves, so that cells cultured on such surfaces are concurrently exposed to grooves of different dimensions which direct cell migration in different directions. Marked fibroblast alignment with the major grooves was observed both within the grooves and in the intervening flat ridges between the grooves. In addition, shallow and closely spaced grooves in epon or titanium-coated polymer or silicon were also capable of orienting fibroblasts. Although the minor grooves were able to orient fibroblasts in the absence of any other orienting influence, when fibroblasts were concurrently exposed to major and minor grooves the cells aligned themselves with the major grooves. TEM showed that the cellular filamentous cytoskeletal elements reflected the orientation of the cell as a whole. Fibroblasts on grooved substrata appeared to have more filopodia and to round up more frequently than fibroblasts cultured on flat substrata. It is suggested that both the mechanical properties of the cytoskeleton as well as the durability of the cellular attachment to groove edges may play a role in the contact guidance effected by grooved surfaces produced by micromachining.

Mechanical stretching increases the number of cultured bone cells synthesizing DNA and alters their pattern of protein synthesis.

SummaryA simple method was devised for applying mechanical stretching to bone cell cultures. Bone cells cultured on the flexible plastic membrane of a Petriperm dish are placed over a template with a convex surface. A lead weight is then placed on top of the dish which causes the membrane and the tightly attached cells to be stretched. Mechanical stretching, applied either intermittently or continuously for a 2-hour period resulted in a 64% increase in the number of cells synthesizing DNA. Stretching the cells also significantly increased incorporation of tritiated proline and tritiated leucine. To assay the ratio of collagenous to noncollagenous protein, medium and cell layers of cultures labeled with tritiated leucine were incubated with collagenase and the digests chromatographed on PD 10 columns. The amount of collagen synthesized by stretched and unstretched cultures did not differ; but an increased synthesis of noncollagenous proteins was observed in the stretched cultures.

Spreading and orientation of epithelial cells on grooved substrata

The spreading and orientation of epithelial (E) cells was studied on titanium-coated grooved substrata by light, transmission (TEM) and scanning electron microscopy (SEM). Vertical-walled grooves and V-shaped grooves, 3-60 microns deep, were produced in silicon wafers by micromachining, a process which was developed for the fabrication of micro-electronic components, and the grooved substrata were replicated in Epon. Photolithography was used to prepare photoresist-based and silicon dioxide-silicon substrata with grooves of approximately 2 and approximately 0.5 micron deep, respectively. Cell clusters were markedly oriented by all the grooved substrata examined, with the orientation index being highest for substrata with grooves of the smallest repeat spacing. Time-lapse cinemicrography showed that the grooves directed the migration of E cells, but the control was not absolute, as some cells crossed over the ridges and descended into the grooves. The 0.5 micron grooves appeared less effective than the deeper grooves in directing cell locomotion. SEM and TEM of E cells spreading on the grooved substrata demonstrated that cell processes, including lamellae and filopodia, were capable of bending around and closely adapting to groove edges. E cells did not flatten as extensively on a substratum with 22 microns deep V-shaped grooves as on a smooth surface, although some cells were markedly elongated. One mechanism proposed to explain contact guidance of fibroblasts is that linear elements of the locomotory system, such as microfilament bundles, are unable to operate when bent. The observed flexibility of epithelial cell processes and the ability of substrata with shallow grooves to orient E cells indicate that contact guidance of E cells on micromachined substrata cannot be explained by the mechanical stiffness of long linear cytoskeletal elements.

Culture and origin of epithelium-like and fibroblast-like cells from porcine periodontal ligament explants and cell suspensions

Abstract A method was developed for preparing cell suspensions from the periodontal ligament of porcine primary molars. Culture of these isolated cells resulted in the growth of two morphologically distinct cell populations; one epithelium-like and one fibroblast-like. The plating efficiency of the cell suspension was estimated at about 0.02 per cent. Similar cell populations were also obtained using a simple explant technique. Autoradiographic examination of explants labelled with [H 3 ]-thymidine indicated that DNA-synthesizing cells were first detectable between 18–30 hr after explantation. The most likely origin of the epithelium-like cells was the epithelial rests of Malassez. Some of the cells of fibroblast-like morphology that incorporated the label at the earliest times appeared to be located in the immediate vicinity of blood vessels. These findings are novel in that growth of cells from dissociated periodontal ligament, elucidation of the origin of monolayers derived from connective tissue cells of periodontal ligament, and cell culture of epithelium derived from this source have not previously been reported.

Grooved Titanium Surfaces Orient Growth and Migration of Cells from Human Gingival Explants

A silicon mask-etching technique was used to prepare grooved surfaces that control the direction of outgrowths of human gingival explants. The method used to produce the grooves is excellent in terms of both the uniformity of the grooves and the control with which surfaces of the desired specifications can be obtained.

Properties and Biological Significance of Natural Oxide Films on Titanium and Its Alloys

This chapter covers information on the composition, microstructure and physico-chemical properties of thin oxide films on titanium and titanium alloys. The focus is on thin layers in the sense of ‘natural’ oxide films grown at ambient or higher temperatures with emphasis on titanium oxide, with some selected additional information on oxides related to metals commonly used as alloying elements in titanium alloys for biomedical applications. This chapter does not, however, include thicker oxide films such as those produced by electrochemical or plasma techniques, which are covered in Chap. 8.