Cell surface interactions in the study of biocompatibility.

Implantation of materials in the body can lead to to adverse local and systematic reactions. knowledge of basic mechanisms of cell material interaction and better understanding of the ongoing processes at the cellular and intracellular level during interaction of anchorage dependent cells with biomaterials will help in the development of new biocompatible materials. As fibroblasts are the predominant tissue cell coming in contact with most of the material in the body, mamalian fibroblast cells were used for the study. Cell attachment and adhesion pattern of cells on different materials were studied using microscopic techniques. Fluorscent labelling of actin and vinculin of osteoblast cells demonstrated a typical sequence of events of cells as rounded, attachment with attachment structures, microfilaments etc.

INTRODUCTION

Biocompatibility and biosafety are considered to imply that the clinical application of a biomaterial should neither cause any adverse reaction nor endanger the life of a patient. More precise definitions have already been coined as the ability of a material to perform with an appropriate host response in a specific application (1) Animal testing is an inherent component of biocompatibility testing. The use of in vitro methods can reduce the extend of animal testing and significantly reduce time and cost of testing. Knowledge of basic mechanisms of cell-material interaction and better understanding of ongoing processes at the cellular level during interaction of anchorage dependent cells can aid in the development of new biomaterials. The recent development of tissue engineering in the field of orthopedic research makes it possible to envisage the association of autologous cells and proteins that promote cell adhesion with osteoconductive material to create osteoinductive materials (2). Nature of interaction between osteoblast cells and their substrate can influence the ability of these cells to produce an osteoid matrix around an implant which in turn will determine the fate of the implant (3).

Attachment of anchorage dependent cells is the first step in the process of cell-surface interactions which in turn can affect subsequent cellular and tissue responses. Cells attach to substrates through contact sites which are classified as focal contacts, close contacts and extracellular contacts depending on the distance of cell from the substrate and the presence of certain proteins. It is important to understand the nature of contact of cells interacting with biomaterials. Changes in cell morphology can be studied using different microscopic techniques like phase contrast and electron microscopy. Morphological changes in cells can also be assessed by labeling specific cellular structures such as acting cytoskeleton (4). In case of implants intended for orthopedic application where close apposition with bone cells is required for better osteointegration, cell attachment and adhesivity play an important role.

In the present study initial attachment characteristics were assessed by Scanning Electron microscopy, cytoskeletal organization by actin staining and focal contact by vinculin staining followed by fluorescence microscopy, enabling to examine microfilaments and focal contacts.

MATERIALS & METHODS

Materials

Metals, polymers and ceramics are routinely used biomaterials. Representatives of these materials. Titanium, non tissue culture grade polymer, Hydroxy apatite and glass) were cleaned, sterilized by steam and used. Copper was used as positive control which had proved to give a chronic response (4).

Cell culture systems

L-929 (Mammalian fibroblasts) obtained from NCCS, Pune were maintained in Minimum Essential Medium supplemented These Cells were subcultured and approximately 1X[10.sup.3] cells were seeded onto different materials to study initial attachment pattern. After 30minutes & 90minutes, cells were fixed in Glutaraldehyde and processed for Scanning Electron Microscopy (Hitachi).

MG-63 osteoblast cells (obtained from NCCS, Pune) were maintained in RPMI media supplemented with foetal calf serum. These cells were seeded onto different materials for 6h and cell seeded materials were stained for actin & vinculin. For this, cells were fixed in buffered formalin and washed with phosphate buffered saline. For actin, fixed cells were incubated with FITC conjugated phalloidin. For Vinculin, indirect immunofluorescent staining was employed using primary antibody of mouse anti vinculin and secondary antibody of FITC conjugated rabbit anti mouse immunoglobulin. Stained preparations were viewed under Fluorescent microscope (Nikon). Fluorescent images were captured using Optimas image acquiring system.

RESULTS & DISCUSSION

Attachment of anchorage dependent cells to substrates is the initial process in the process of cell-surface interactions. Attachment of cells to substrate is followed by translational and transcriptional cellular events. Attachment phase of cell adhesion occurs rapidly and involves physicochemical linkages between cells and materials including ionic forces. Initial fibroblast attachment on different substrates are depicted in figures1-3. Cells seeded on control glass showed normal morphology during the initial steps of attachment (Fig1d&2d). Attached cells on polymer (Fig.1c&2c) showed less attachment compared to other substrates. Attached cells were spherical with rough texture. More number of cells were attached on Titanium & Hydroxy apatite after30minutes (Fig.1a&b).

[FIGURES 1-3 OMITTED]

Attached cells were more widespread after 90 minutes of contact. Some of the attached cells spread radially from the centre and developed filopodia. (Fig 3a&b). Spread cells generally showed a comparatively smooth surface. The surface of cells not yet spread, were convoluted into microappendages and ridges (Fig.3c). Neighbouring cells maintained physical contact with one another through multiple extensions (Fig3d). Cell spreading is an essential function of a cell which has adhered to any surface and precedes the function of cell proliferation to finally provide a cell covered surface. Diminished cell adhesion is used as a measure of toxicity, if initial attachment of cells is first investigated. Impaired cell adhesion of suspended cells to different substrata measures the adhesivity of test surface depending on its physicochemical properties (5)

Adhesion phase occurring at long term contact involves various biological molecules like extracellular matrix proteins, cell membrane proteins and cytoskeletal proteins which interact together to induce signal transduction, promoting the action of transcription factors and consequently regulating gene expression (2). Cell spreading and adhesion are phenomena involving complex cytoskeletal reorganization. F-actin containing microfilaments demonstrated attachment of initially rounded cells (Fig.4a) to substrate with subsequent spreading of the cells (Fig.4b). Actin was generally distributed with circumferential banding near the edge of the cell and a cytoplasmic meshwork was becoming apparent in certain cells (Fig.4a). Diffused actin meshwork overlayed the entire cell image. Lack of nuclear image sparing showed that the localization is not deep in the cytoplasm but is just beneath the plasma membrane on the top and bottom of the image of the nucleus. Cells were more spread on Titanium and nuclear sparing was more evident (fig.4b). Fewer prominent transcellular filaments were observed on well spread cell on ceramics (Fig.4c). However ceramic Hydroxy apatite showed high background fluorescence, due to nonspecific adsorption of fluorescent labels on the substrate. Bright fluorescence seen on Titanium after 36 hours represented a folded lamellar ruffle, projecting upward from the substratum (Fig.4d)

[FIGURE 4 OMITTED]

Nature of contacts formed between cells and surface influence cellular response to substrates. Contact sites on materials were visualized by fluorescent microscopy in conjunction with a specific antibody against vinculin, a protein which is found on the cytoplasmic face of focal contacts. Vinculin staining of cells seeded on coverslip showed diffused cytoplasmic distribution with circumfrential labeling at the borders of the cells (Fig5a&b).

[FIGURE 5 OMITTED]

Cells on Copper demonstrated necrotic cells evidenced by the changes in cell size and nuclear damage (Fig.5c). In case of hydroxy apatite, cells started spreading, favouring cell adhesion characteristics (Fig.5d). In case of Titanium, cells became polygonal, spread and showed diffused cytoplasmic labeling (Fig.5e). Fluorescent images were captured using Optimas Image analysis system. Image intensifiers coupled to the fluorescent microscopes allowed recording of weakly fluorescent images without significant loss in resolution (6). During cell adhesion, integrins gather in focal adhesions whereby they link the extracellular matrix with intracellular cytoskeleton. Various signaling molecules transmit signals resulting in the reorganization of the cytoskeleton and regulation of gene activi ty in the nucleus (7). Transfer of signals from the substratum into the cells depends on the physicochemical nature of the material. Difference in these signals play an important role in actin polymerization and regulation of genes for protein synthesis in the cells (8).

In the present study the nature of initial contact, attachment of cells, cytoskeletal organization, focal contacts and spreading of cells were evaluated by SEM & fluorescent labeling. Diminished adhesion and spreading of cells is indicated by diffuse presence of actin and absence of focal contacts. As these findings are dependent on the physicochemical nature of the substrate, will indicate the cytocompatibility of the material. Cell surface interaction and cell adhesion are complex processes involving cytoskeletal reorganization of actin and vinculin. Better knowledge of cell-surface interaction is useful in assessing biocompatibility thereby improving material characteristics for developing new materials and modification of existing materials.

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Kumari. T.V *, Usha Vasudev, Anil Kumar & Bindu Menon

Division of Implant Biology

Sree Chitra Tirunal Institute For Medical Sciences & Technology

Thiruvananthapuram 695 012

* MAHE Award for best presentation--Second Prize