Cell adhesion to textured silicone surfaces: the influence of time of adhesion and texture on focal contact and fibronectin fibril formation.

Cell adhesion and spreading on biomaterials is a key issue in the study of cell-biomaterial interactions. With the development of new disciplines within biomaterials research such as tissue engineering and cellular therapy, information at molecular and structural levels is needed in order to conceive and design biomaterials that elicit specific, functional cell responses. In this study we determined the formation of focal adhesions and fibronectin fibrillar structures by human fibroblasts and human umbilical vein endothelial cells adhered to fibronectin-precoated, smooth, and textured silicones as a function of time. Textures consisted of parallel ridges and 0.5 mm deep grooves with a width of 2, 5, and 10 mm. In addition, pillar and well constructs were used. Cells assembled focal adhesions within the first 24 h of adhesion. Fibronectin production and assembly resulted in a dense fibrillar network at day 6. Initial focal adhesion density and size were dictated by the presence of the texture. Topography also influenced initial fibronectin deposition, although the differences did not result in apparent differences in fibronectin networks after 6 days of incubation. Without fibronectin preadsorption, cells did not proliferate on the silicone surfaces. Cells adhered to glass removed all the preabsorbed fibronectin, whereas on silicone they did not. The present study shows that different textures initially give rise to differences in focal contact and fibronectin fibril assembly. The effects of the small, initial in vitro differences on in vivo tissue biocompatibility remains to be studied.

Orientation of ECM protein deposition, fibroblast cytoskeleton, and attachment complex components on silicone microgrooved surfaces.

The microfilaments and vinculin-containing attachment complexes of rat dermal fibroblasts (RDF) incubated on microtextured surfaces were investigated with confocal laser scanning microscopy (CLSM) and digital image analysis (DIA). In addition, depositions of bovine and endogenous fibronectin and vitronectin were studied. Smooth and microtextured silicone substrata were produced that possessed parallel surface grooves with a groove and ridge width of 2.0, 5.0, and 10.0 microns. The groove depth was approximately 0.5 micron. CLSM and DIA make it possible to visualize and analyze intracellular and extracellular proteins and the underlying surface simultaneously. It was observed that the microfilaments and vinculin aggregates of the RDFs on the 2.0 microns grooved substrata were oriented along the surface grooves after 1, 3, 5, and 7 days of incubation while these proteins were significantly less oriented on the 5.0 and 10.0 microns grooved surfaces. Vinculin was located mainly on the surface ridges on all textured surfaces. In contrast, bovine and endogenous fibronectin and vitronectin were oriented along the surface grooves on all textured surfaces. These proteins did not seem to be hindered by the surface grooves since many groove-spanning filaments were found on all the microgrooved surfaces. In conclusion, it can be said that microtextured surfaces influence the orientation of intracellular and extracellular proteins. Although results corroborate three earlier published hypotheses, they do not justify a specific choice of any one of these hypotheses.

Influence of silicone (PDMS) surface texture on human skin fibroblast proliferation as determined by cell cycle analysis.

In vivo biocompatibility of soft-tissue implants is often hampered by development of capsules that eventually might contract and impair implant function. It has been shown that capsule formation can be significantly reduced by using materials with textured surface elements in the micron range. In this study the interaction of human fibroblasts with silicone surfaces was analyzed using cell cycle analysis. Silicone was textured with 2, 5, and 10 microns wide grooves (2MU, 5MU, 10MU, respectively) or kept smooth (SMT). Cell cycle analysis was performed after staining of cells with propidium iodide. Cells proliferated on the fibronectin-preadsorbed silicone, as demonstrated by increased coverage and occurrence of subpopulations in the S and G2/M phase of the cell cycle. Cells on SMT went faster into the S phase than cells on textured silicones. Cells on 10MU showed less proliferation than cells on 2MU and 5MU. Besides the basic percentages of cells in the different cycle phases, DNA profiles were also influenced by incubation time and texture, especially with respect to the presence of hypodiploid populations and asymmetry of the G0/G1 peak. Finally scatter characteristics were influenced. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay data did not reveal significant differences among the different samples. Fibronectin preadsorption of silicone only resulted in slightly higher MTT conversion. Cell cycle analysis proved to be a sensitive screening method for proliferation on the silicone surfaces and provided information beyond the normal G0/G1, S and G2/M subpopulations.

Types I and III collagen in the tissue capsules of titanium and stainless-steel implants.

Collagen plays an important role in wound healing and as such is present in connective tissue capsules around implanted materials. The proportion of type I collagen to type III collagen is lower during wound healing than that found in normal dermis, but the amount of type I collagen gradually increases and type III decreases as healing continues. The intent of this research was to investigate a possible correlation between implant metals and type I and type III collagen formation in the ensuing soft-tissue capsules. Smooth implants of grade 4 commercially pure titanium and 316L stainless steel were placed via injection needle into the subcutaneous tissue of 12 rats for 6 weeks. The soft-tissue capsule that formed around each implant was evaluated histologically and by immunofluorescence. Although the two metal implant types differ in many respects, they were chosen for their differences in surface conductivity. Titanium oxide is semiconducting, whereas the oxide of stainless steel is conducting. Impedance spectroscopy was used for conductivity analysis of the surface. No qualitative differences were found in amount of type I or type III collagen in the capsules of the two metals. However, the capsules around titanium stained for type I collagen were thicker than capsules around stainless steel. In addition, type I collagen showed evidence of a greater association with densely packed, highly orientated collagen fibers.

Intraocular lens implants: a biocompatibility review.

The natural aging process of the eye inevitably leads to the formation of a cataract, resulting in an increasing loss of vision. A cataract is the clouding of the natural lens in the eye and represents a major physical impairment. Modern surgical techniques allow for removal of the clouded lens and replacement with a prosthetic intraocular lens. This article reviews the intraocular tissue response to the implant, which frequently leads to postoperative complications for the patient.

Quantitative analysis of fibroblast morphology on microgrooved surfaces with various groove and ridge dimensions.

Fibroblasts have been shown to respond to substratum surface roughness. The change in cell size, shape and orientation of rat dermal fibroblasts (RDF) was therefore studied using smooth and microtextured silicone rubber substrata. The microtextured substrata possessed parallel surface microgrooves that ranged in width from 1.0 to 10.0 microns, and were separated by ridges of 1.0 to 10.0 microns. The grooves were either 0.45 or 1.00 microns deep. Prior to incubation, the substrata were cleaned and given a radio frequency glow discharge treatment. After surface evaluation with scanning electron microscopy and confocal laser scanning microscopy, RDF were incubated on these substrata for 5 days. During this period of incubation, the RDF were photographed on days 1, 2, 3, 4, and 5, using phase contrast microscopy. Digital image analysis of these images revealed that on surfaces with a ridge width < or = 4.0 microns, cells were highly orientated (< 10 degrees) and elongated along the surface grooves. Protrusions contacting the ridges specifically could be seen. If the ridge width was larger than 4.0 microns, cellular orientation was random (approximately 45 degrees) and the shape of the RDF became more circular. Furthermore, results showed that the ridge width is the most important parameter, since varying the groove width and groove depth did not affect the RDF size, shape, nor the angle of cellular orientation.

Quantitative analysis of cell proliferation and orientation on substrata with uniform parallel surface micro-grooves.

In order to quantify the effect of the substrata surface topography on cellular behaviour, planar and micro-textured silicon substrata were produced and made suitable for cell culture by radio frequency glow discharge treatment. These substrata possessed parallel surface grooves with a groove and ridge width of 2.0 (SilD02), 5.0 (SilD05) and 10 microns (SilD10). Groove depth was approximately 0.5 micron. Rat dermal fibroblasts (RDFs) were cultured on these substrata and a tissue culture polystyrene control surface for 1, 2, 3, 5 and 7 days. After incubation the cell proliferation was quantified with a Coulter Counter, and RDF size, shape and orientation with digital image analysis. Cell counts proved that neither the presence of the surface grooves nor the dimension of these grooves had an effect on the cell proliferation. However, RDFs on SilD02, and to a lesser extent on SilD05 substrata, were elongated and aligned parallel to the surface grooves. Orientation of the RDFs on SilD10 substrata proved to be almost comparable to the SilD00 substrata. Finally, it was observed that the cells on the micro-textured substrata were capable of spanning the surface grooves.

Breast implants: facts, controversy, and speculations for future research.

The breast implant crisis has been widely publicized. Beyond its immediate problems for the patients, the crisis has also discredited the use of silicone rubber as one of the most widely used biomaterials. Silicone elastomer or gel, the primary material in mammary prostheses, may be exposed to the body's tissues via envelope rupture, gel bleed, or elastomer fragmentation. Local responses to silicone include granulomatous inflammation, capsular contraction, and infection, all in varying degrees depending on ill-defined factors, which may include patient condition, peri- and postoperative complications, and hereditary predisposition, as well as material properties such as surface texture. The theory that silicone breast implants cause immunological disorders has not been proven. However, further study is necessary because some patients report autoimmune-like disorders (human adjuvant disease) following implant placement. Like autoimmune disease, human adjuvant disease is characterized by abnormalities of the immune response, autoantibody formation, and chronic inflammation. Silicone has been shown to play the role of an adjuvant, providing constant nonspecific stimulation of the immune system. Some researchers have hypothesized the role of silicone in specific immune reactions, including immunoglobulin formation and T-cell activation. This report examines the role of silicone as an agent of disease, focusing on material surface-tissue interactions.

Effect of parallel surface microgrooves and surface energy on cell growth.

To evaluate the effect of surface treatment and surface microtexture on cellular behavior, smooth and microtextured silicone substrata were produced. The microtextured substrata possessed parallel surface grooves with a width and spacing of 2.0 (SilD02), 5.0 (SilD05), and 10 microns (SilD10). The groove depth was approximately 0.5 microns. Subsequently, these substrata were either left untreated (NT) or treated by ultraviolet irradiation (UV), radiofrequency glow discharge treatment (RFGD), or both (UVRFGD). After characterization of the substrata, rat dermal fibroblasts (RDF) were cultured on the UV, RFGD, and UVRFGD treated surfaces for 1, 3, 5, and 7 days. Comparison between the NT and UV substrata revealed that UV treatment did not influence the contact angles and surface energies of surfaces with a similar surface topography. However, the contact angles of the RFGD and UVRFGD substrata were significantly smaller than those of the UV and NT substrata. The dimension of the surface microevents did not influence the wettability characteristics. Cell culture experiments revealed that RDF cell growth on UV-treated surfaces was lower than on the RFGD and UVRFGD substrata. SEM examination demonstrated that the parallel surface grooves on the SilD02 and SilD05 substrata were able to induce stronger cell orientation and alignment than the events on SilD10 surfaces. By combining all of our findings, the most important conclusion was that physicochemical parameters such as wettability and surface free energy influence cell growth but play no measurable role in the shape and orientation of cells on microtextured surfaces.

Effectiveness of cleaning surgical implants: quantitative analysis of contaminant removal.

Surgical implants need to be free from contaminants before implantation. The effectiveness of a presently used Clemson bioengineering cleaning (CBC) protocol was evaluated for cleaning three different biomaterials (titanium, aluminum oxide, and polyethylene terephthalate, PET) contaminated with three different contaminants (calcium chloride, zinc chloride, and hexadecane). Radiolabeled tracer analysis (RTA), with the use of liquid scintillation, was used as the surface analytical technique to quantitatively determine the percent contaminant removed from the biomaterial surface. On average, the ultrasonic cleaning step removed 99.96% of all three contaminants from both titanium and aluminum oxide. The CBC protocol did not sufficiently clean PET fabric contaminated with hexadecane leaving 11.76% of the contaminant after the ultrasonic step. With the use of isopropyl alcohol in series with 1% Liquinox, the ultrasonic step cleaned the fabric soiled with hexadecane within 30 min, removing 99.85% of the hexadecane initially on the surface. RTA proved to be an excellent method of quantifying surface contamination on implant materials, and for assessing the effectiveness of cleaning protocols in question.

Educational goals for biomaterials science and engineering: prospective view.

The research field of biomaterials and surgical implants has matured to a point suggesting that a formal and comprehensive education is now required to handle all professional issues related to biomaterials and implant development. A professional curriculum is proposed for a discipline of biomaterials science and engineering on a graduate level. The curriculum includes the definition of an essential knowledge base and describes two track options for a study period of 3 years. Lists of prerequisites as well as required and suggested courses are presented and discussed. Continuing education courses are presented as examples. A quick vision of the immediate future of the field enforces the need for biomaterials professionals to take the lead in bringing the field into the next century.

The influence of micro-topography on cellular response and the implications for silicone implants.

Tissue attachment to substratum surfaces is of central importance to the in vivo performance of prosthetic implant materials. It is not yet understood why connective tissue does not attach to the surface of silicone or any other polymeric material. Recently the authors have conclusively demonstrated that micro-range surface roughness modifies cellular responses in cell culture and modifies biocompatibility and tissue attachment in vivo significantly. In order to better understand the basic interactions between living cells or tissues on one hand and man-made substratum surfaces on the other hand, the germane literature is reviewed here. Cells adhere to substratum surfaces mainly through focal adhesions which are a complex of intracellular transmembrane and extracellular proteins. Adhesion is facilitated and modified by proteins adsorbed to the substratum surface. Protein adsorption in turn is modified by the underlying substratum surface properties including surface chemistry, charge, and free energy. When silicone and other polymeric implants having well-defined surface topographic features including pores, pillars, or grooves were implanted, the tissue response to these implants was strongly influenced by the dimensions of these features as well as by other geometric details. Highest biocompatibility along with tissue attachment was seen when topographic features had dimensions of 1-3 microns and a uniform distribution. Cell culture studies revealed that topographic features affect cellular alignment, direction of proliferation, cellular attachment, growth rate, metabolism, and cytoskeletal arrangement. Since discontinuities or curvatures associated with topographic features may represent local changes in surface free energy, it is hypothesized that these discontinuities trigger changes in protein adsorption, protein configuration, and cellular response.

Fibroblast response to microtextured silicone surfaces: texture orientation into or out of the surface.

Previous studies suggested that surface topographic configurations of 1-3 microns influence cellular behavior and tissue response. They did not address which specific aspect of the configurations elicits the cellular response. We therefore investigated the effect of the orientation of several surface configurations. Seven different textures on polydimethyl siloxane (silicone; Dow Corning Silastic) specimens were used to test the question of whether orientation into (down) or out of the surface (up) affected cellular response to a material. The textures were smooth and photoetched configurations of 2 microns up, 2 microns down, 5 microns up, 5 microns down, 10 microns up, and 10 microns down. The response of cultured fibroblasts on these surfaces was compared with that of a standard tissue culture material, polyethylene terepthalate (Thermanox). The cell density was measured over a 12-day period with the use of a colorimetric assay. The uptake of methylene blue was measured daily and compared as an absorbance in a destaining agent. Cells on the 2 and 5 microns up arrays showed increased rates of proliferation and cell density as compared with their down counterparts. This would indicate that textures of 2 and 5 microns have a significant influence on cell growth, and that the surface with hills has a greater effect than the surface with wells. In contrast, the 10 microns up and 10 microns down arrays did not prove to be statistically different from smooth ones. This indicates that the orientation effect is related to the configuration size and that this configuration size is not viewed differently from smooth silicone by the cells. The presented data are in agreement with results of this laboratory and others that fibroblasts recognize the dimensions of surface configurations and react accordingly. Specifically, they appear to react to the uppermost surface area presented to them, but conclusive data can only be obtained from a study of the focal adhesions.

Semi-quantitative and qualitative histologic analysis method for the evaluation of implant biocompatibility.

An adequate histologic evaluation is a prerequisite for a good understanding of the behavior of tissue to implant materials. However, despite improvements in histologic sectioning techniques, few studies have used histomorphometric methods for the quantification of the tissue response. This paper discusses new simple histologic grading scales, which can be used for the fast standardized light microscopic analysis of the biocompatibility of hard and soft tissue implants. Two examples of the application of the grading scales are demonstrated.

Fibroblast anchorage to microtextured surfaces.

The contact between tissue and the implanted biomaterial is influenced by the micromorphology of the implant surface as well as biomechanical reactions. This effect is mediated by subcellular morphological structures and can affect the anchorage of the material inside the body of the host. The aim of the present study was to ascertain by transmission electron microscopy how human gingival fibroblasts interact with surface events. A special replica technique was used to produce a line pattern of 1 micron pitch with a depth of 1 micron. It was demonstrated, by transmission electron microscopy, that cells seeded on this surface extended cellular processes into the grooves, leading to an intensive contact and probably to mechanical interlocking. The typical morphological structures at several points indicated the presence of focal adhesion sites.

Collagen types I and III at the implant/tissue interface.

Collagen composition in tissue capsules around implants has been reported to differ histologically from collagen in subcutaneous connective tissue. In the present study, an immune histochemical analysis of collagen types I and III was undertaken in tissue capsules of various implant materials. The materials included polyvinyl chloride/polyacrylonitrile copolymer, poly(ethylene terephthalate), polysiloxane, titanium, and hydroxyapatite, which had been implanted into the dorsal subcutaneous space of rabbits for various time periods from 28 and to 90 days. The results indicate that collagen type III stained in all capsules independent of the evaluated materials, implantation periods, and material surface roughness. Collagen type I stained only in titanium implant capsules and dominated there over collagen type III. The staining sensitivity was highly specific and reproducible. The presence of collagen type III can be expected because it is the collagen of connective tissue healing. Collagen type I appears to be a response to chemical or electrochemical titanium surface properties but not to surface roughness. The quantitative relationship between the two collagen types may indicate capsule tissue stability and therefore serve as another biocompatibility measure.

Surface micromorphology and cellular interactions.

Contact guidance induced by the topographical properties of the underlying substratum is of great importance in morphogenesis and also influences the interaction of tissue cells with implanted material. A large body of evidence has accumulated since the first detection of this phenomenon in 1910. Several major hypotheses have been developed to explain the observed cell behaviour. The technological progress enabled researchers to produce pure substrata with a defined and controlled surface microgeometry. Based on these specimens, it could be demonstrated that cytoskeletal structures and receptors forming focal adhesions most likely are involved in contact guidance. In a study using human gingival fibroblasts, the reaction of these cells to a regular surface microstructure of 1 micron pitch and 1 micron depth was tested. After two days on the microstructured samples, all the cells showed a strong alignment to the topography of the surface. Transmission electron microscopy revealed that the cells either bridged the grooves or conformed to the surface structures. The latter confirms earlier investigations with porous subcutaneous implants, where the inflammatory reaction and the formation of a fibrous tissue capsule was reduced due to enhanced tissue adhesion.

On the use of primary reference grade polydimethylsiloxane.

There has been an increase in the use of primary reference material as a standard for identifying the cellular response to biomaterials. One such material is NHLBI-DTB polydimethylsiloxane (PDMS). The PDMS was developed for blood contacting studies and is composed of PDMS backed on one side with mylar. The results of implantation studies of two different publications are discussed in light of the different materials and different surface topographies of each of the materials. The appropriateness of in vivo studies using this reference material is questioned.

Macrophage response to microtextured silicone.

Seven different silicone surface textures were tested for effect on macrophage spreading and metabolic activity in vitro. Variables of the textured arrays that could modify spreading were determined to be the size, spacing between, depth, density, and orientation of the individual surface events and the roughness of the surfaces. Cells were influenced by the size of the events and the roughness of the surfaces more than any other variables. Cell morphology data, surface area and perimeter, could be divided into discrete regions that correlated well with the size of the events. Cell dimensions on 5 microns textures were smallest while those on smooth silicone and glass surfaces were the largest. Surface texture events may be modifying contact guidance of the cells or interacting with specific transmembrane proteins to alter cell shape and function. The mitochondrial activity of cells attached to the textured silicones was determined by measuring the amount of reduced MTT directly through live cells. Cells on polystyrene (PS), 5VP and 8VP textures were metabolically more active than cells on the other textures. PMA was used to stimulate cells on the various textures. PMA-stimulated cells, on the smaller textures, 2VP, 5VP and 5CP, were less active than test cells that were not stimulated. The inability of PMA to stimulate these cells may be due to a structural alteration of protein kinase C. An hypothesis is introduced that includes a possible mechanism of how a micrometre-sized surface texture could modify cell function.