Model Biomaterial Surfaces Research Articles

Cellular responses to chemical and morphologic aspects of biomaterial surfaces. I. A novel in vitro model system.

The clinical success of any implant is directly dependent upon the cellular behavior in the immediate vicinity of the interface established between the host tissue and the biomaterial(s) used to fabricate the device. All biomaterials have morphologic, chemical, and electrical surface characteristics that influence the cellular response to the implant. Quantitative measurement of specific aspects of this local host response to different but well-characterized biomaterial surfaces provides a crucial link in the understanding of the overall phenomenon of implant biocompatibility. A system has been devised for in vitro examination of responses of cells to controlled but independent changes in both the chemistry and morphology of polystyrene (PS) tissue culture surfaces. Micromachined silicon wafers were used as templates to solvent-cast PS replicas [using 0, 1, or 2 wt % styrene (S) monomer additions] with either none, 0.5- or 5.0-microns-deep surface grooves arranged in a radial array. When all possible morphologies were combined with all possible polymers, nine model biomaterial surfaces (MBSs) were produced. The chemical characteristics of the MBSs were determined using electron spectroscopy for chemical analysis, secondary ion mass spectroscopy, and contact angle techniques and were found to be distinct. The types and amount of proteins that adsorb onto these surfaces from serum containing media were examined and found to consist of multiple molecular layers of relatively uniform composition. Self-contained tissue culture vessels formed from the MBSs were capable of supporting the growth of confluent cultures of rat calvarial cells. The model biomaterial system described here can be used to examine how simultaneous stimuli resulting from the chemical and morphological characteristics of a test material may influence biologic responses. Such multifactorial biocompatibility research is needed to properly document material-host interactions.

Attachment of human bone cells to tissue culture polystyrene and to unmodified polystyrene: the effect of surface chemistry upon initial cell attachment.

Cell culture studies have often been used in the determination of the suitability of biomaterials as surfaces for the attachment and growth of cells. For such studies of surfaces for potential use in bone implants, cells derived from bone may be maintained in culture on tissue culture polystyrene (TCPS). We have determined the contribution that serum fibronectin (FN) or vitronectin (VN) make to the attachment and spreading of cells cultured from explanted human bone (bone-derived cells) during the first 90 min following seeding on culture surfaces. The attachment of bone-derived cells to TCPS was simulated two-fold by the addition of 10% (v/v) fetal bovine serum (FBS) to the seeding culture medium. The roles of FN and VN were determined by selective removal of the FN or VN from the FBS prior to addition to the culture medium. FBS from which the VN had been removed did not have this stimulatory activity. In contrast, the attachment of bone-derived cells onto TCPS from medium containing FN-depleted serum (which contained VN) was the same as when intact FBS was used. There was incomplete attachment of bone-derived cells (27% of cells) when seeded in medium containing FBS depleted of both VN and FN. Our results show that for human bone-derived cells, the attachment onto TCPS of cells planted in medium containing FBS during the first 90 min of culture is principally as a result of adsorption onto the surface of serum VN. As unmodified polystyrene (PS) has also been used previously as a model biomaterial surface, PS was compared to TCPS for attachment of the bone-derived cells. Attachment of bone-derived cells to TCPS was twice that onto PS, both when the medium was serum-free and when it contained FBS. Bone-derived cells attached to TCPS or PS onto which purified VN or FN had been precoated, with VN adsorbed onto PS being as effective as was VN adsorbed onto TCPS. With FN, there was an effect of the polystyrene surface chemistry which was evident in that suboptimal concentrations of FN had a slightly higher potency when adsorbed onto TCPS than did the same concentrations of FN coated onto PS. When preadsorbed onto TCPS, the potency of FN for attachment of bone-derived cells was at least equal to that of VN.

Peptide, protein, and cellular interactions with self‐assembled monolayer model surfaces

Model biomaterial surfaces with well defined chemistry were prepared from close-packed alkyltrichlorosilane monolayers on polished silicon and glass. The outermost molecular groups which come in direct contact with the biological environment were varied across a wide range of oxidation states by employing -CF3, -CH3, -CO2CH3, and -CH2OH terminal functionalities. Characterization by contact angles, surface spectroscopy, and ellipsometry verified that these model surfaces could be repeatedly prepared with good consistency for routine use to study biomolecule adsorption onto model surfaces. Adhesion of canine endothelial cells and the adsorption of proteins (human serum albumin and human fibrinogen) as well as series of synthetic defined oligopeptides to these model surfaces have been studied. Endothelial cells attachment and growth were in the rank order of: -CH2OH > -CO2Me > -CH3 > -CF3. The peptides were comprised of different alternating sequences of lysine, leucine, and tryptophan residues. These structural differences imparted different amphiphilic characters that led to measurable differences in the adsorption of these peptides to liquid-vapor interfaces. The adsorption to model surfaces was studied using ESCA, radiometry, and concentration-dependent contact angles. ESCA and radiometry measured irreversible biomolecules adsorption whereas the contact angle method measured steady-state adsorption. Radiometric results were inconsistent with ESCA, possibly due to artifacts associated with protein radiolabeling.

Plasma protein adsorption on model biomaterial surfaces

The study of plasma protein adsorption onto biomaterial surfaces using a quantitative, high-resolution, two-dimensional polyacrylamide gel electrophoresis technique (2-D PAGE) is reported. Surfaces of three different types of biomaterial were examined: calcium hydroxyapatite, low-temperature isotropic carbon particles, and polyetherurethane particles. All three biomaterials were found to bind, simultaneously and in a kinetic dependent way, a number of plasma proteins. In the case of CaHA, the experimental titration of diluted plasma with the adsorbent allowed calculation of the adsorption isotherms and permitted estimation of the affinity of individual proteins for the surface. In the case of an LTI carbon surface, the kinetics of binding were different for different proteins; fibrinogen was completely removed in 10 min from diluted plasma, while the adsorption kinetics of other proteins were slower and resulted only in their partial removal from solution. The surface of carbon was also found to be morphologically heterogeneous due to the different orientation of carbon crystallites. This might have an effect on the binding of some larger plasma proteins, like fibrinogen. The polyurethane surface might also have been heterogeneous; it is thought to consist of separate microdomains of hard and soft segments. The present results indicated that some proteins, like hemopexin, might have an extraordinary affinity for PU surfaces. Advantages and disadvantages of the 2-D PAGE technique applied to the study of biomaterial-protein interactions are discussed.

Role of serum vitronectin and fibronectin in adhesion of fibroblasts following seeding onto tissue culture polystyrene.

The suitability of polymeric biomaterials as surfaces for the attachment and growth of cells has often been investigated in tissue culture. In this study the contribution that adsorption of serum fibronectin (Fn) or vitronectin (Vn) make to the attachment and spreading of fibroblast cells during the first 90 min following seeding was determined for two modified tissue culture polystyrenes, as model biomaterial surfaces. The amount of serum Vn and Fn which adsorbed onto tissue culture grade polystyrene (TCP) from different serum concentrations over the range of 0.1-30% (v/v) were determined and compared to attachment of cells of the BHK-21 and HT1080 fibroblast lines. There was no simple correlation between the amount of Fn or the amount of Vn adsorbed and cell attachment and spreading. The requirement for Fn or Vn for attachment and spreading of BHK-21 or HT1080 cells onto modified polystyrene (either TCP or to Primaria) during the first 90 min of cell culture was directly tested by selective removal of Fn or Vn from the serum prior to addition to the culture medium. Attachment and spreading of BHK-21 or HT1080 cells onto TCP or Primaria surfaces were reduced in a concentration-dependent manner when the cells were seeded in medium containing 2% (v/v) or higher concentrations of Vn-depleted serum. BHK-21 cells or HT1080 cells seeded in medium containing Fn-depleted serum (which contained Vn) attached and spread onto TCP or Primaria. Both BHK-21 cells and HT1080 cells failed to attach to TCP or Primaria when seeded in medium containing serum depleted of both Vn and Fn. The requirement for serum Vn or Fn for fibroblast attachment to TCP was also tested using cells of a human dermal fibroblast strain. The attachment of the dermal fibroblasts to TCP during the first 90 min of culture was not decreased by depletion of Vn from the 15% (v/v) serum, but there was a reduction in the proportion of the attached cells which had spread. Selective depletion of serum Fn did not have any effect on either cell attachment or spreading. Our results show that for fibroblast cells, particularly with cell lines such as BHK-21 or HT1080 but also with cell strains, the first binding of cells onto tissue culture polystyrene when plated in medium containing serum is a result of adsorption onto the surface of serum Vn. The adsorption of serum Vn onto the surface overcomes the effect of serum components which tend to decrease cell attachment.