Plasma-polymerized surfaces for culture of human keratinocytes and transfer of cells to an in vitro wound-bed model

The aim of this study was to develop plasma-polymerized surfaces suitable for the attachment and culture of human keratinocytes and that would allow their subsequent transfer to a wound-bed model. Keratinocyte attachment has been assessed on a carrier polymer, either untreated or treated with a hydrocarbon plasma polymer, collagen I, or carboxylic-acid-containing plasma copolymers. Cell attachment was poor on the "bare" carrier polymer and hydrocarbon plasma polymer (PP) surfaces. Cell attachment was good and comparable on collagen I-coated carrier polymer and carrier polymer plasma coated with carboxylic acid functionalities. After 24 h of cell culture, surfaces were inverted so that cells were adjacent to a de-epidermalized dermis (DED) for 4 days. After 4 days in contact with DED, the surfaces were removed and the level of residual cells and cells transferred to DED were assessed using a cell viability assay. Cell transfer from the collagen I-coated surface was on the order of 90%. Transfer from the carrier polymer surface and the hydrocarbon-coated surface was poor while cells cultured on acid-containing surfaces showed high levels of transfer. Cell transfer was greatest from those surfaces containing the highest level of acid functionality (ca. 21%). Cell transfer was not significantly affected by the choice of carrier polymer material although some sample-to-sample variation was seen. To determine that plasma-polymerized surfaces could be used clinically, selected samples were sterilized with ethylene oxide. Subsequent analysis and cell culture indicated that the surface chemistry and cell-transfer capability of these plasma-polymerized surfaces were unaffected by the sterilization procedure. Plasma-polymerized carboxylic-acid-containing surfaces show great promise in the field of wound healing, encouraging keratinocyte attachment and permitting keratinocyte transfer to a wound bed.     

Development of a stable chemically defined surface for the culture of human keratinocytes under serum-free conditions for clinical use

Within the field of tissue engineering there is a need to develop new approaches to achieve effective wound closure in patients with extensive skin loss or chronic ulcers. This article exploits the well-known interdependency of epithelial keratinocytes and stromal fibroblasts in conjunction with plasma surface technology. The aim was to produce a chemically defined surface, which with the aid of a feeder layer of lethally irradiated dermal fibroblasts would improve the attachment and proliferation of the keratinocyte cell from which subconfluent cells can be transferred to wound bed models. Plasma copolymers of acrylic acid/octa-1,7-diene have been prepared and characterized by X-ray photoelectron spectroscopy. The fibroblasts and keratinocytes were cultured on plasma polymer-coated 24-well plates. Cell attachment and proliferation were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-eluted stain assay (MTT-ESTA) and DNA assay. Attachment and proliferation of both cell types on plasma polymer surfaces were compared with tissue culture plastic and collagen I, plus a negative control of a pure hydrocarbon layer. A pure acrylic acid surface, fabricated at a power of 10 W and containing 9.2% carboxylate groups, was found to promote both fibroblast and keratinocyte attachment and proliferation and permit the serum-free coculture of keratinocytes and irradiated fibroblasts.     

Adhesion and proliferation of corneal epithelial cells on self-assembled monolayers

The effect of surface chemistry on the proliferation and adhesion of SV-40 human corneal epithelial cells was investigated. The surface chemistry of substrates was controlled by the deposition of self-assembled monolayers (SAMs) terminated with the following functional groups: -CF3, -CH3, -CO(2)H, and -NH(2). SAMs of alkanethiols on gold and of alkylsiloxanes on SiOx were included in the study. Comparisons are made between different types and functionalities of SAMs and between SAM-covered substrates and tissue culture polystyrene. Adhesion assays were performed after incubation of the cells for 1 h in 10% fetal bovine serum and in serum-free conditions. The cellular response was found to be a function of surface chemistry and the presence of exogenous proteins. The number of cells that adhered to most of the SAMs in 10% serum and in serum-free conditions was not significantly different from the number of cells that adhered to TCPS. Proliferation assays were carried out in 10% serum and in 0.5% serum. Cell behavior was influenced by surface chemistry but did not deviate significantly from the behavior on TCPS for most of the SAMs. Serum level did not play a major role in cell proliferation. Our data establish the expected behaviors for a corneal epithelial cell line under defined conditions on specific surfaces.     

Small-diameter porous poly (epsilon-caprolactone) films enhance adhesion and growth of human cultured epidermal keratinocyte and dermal fibroblast cells

Autologous keratinocyte grafts provide clinical benefit by rapidly covering wounded areas, but they are fragile. We therefore developed biocompatible hexagonal-packed porous films with uniform, circular pore sizes to support human keratinocytes and fibroblasts. Cells were cultured on these porous poly (epsilon-calprolactone) films with pore sizes ranging from novel ultra-small 3 microm to 20 microm. These were compared with flat (pore-less) films. Cell growth rates, adhesion, migration, and ultrastructural morphology were examined. Human keratinocytes and fibroblasts attached to all films. Furthermore, small-pore (3-5 microm) films showed the highest levels of cell adhesion and survival and prevented migration into the pores and opposing film surface. Keratinocyte migration over small-pore film surface was inhibited. Keratinocytes optimally attached to 3-microm-pore films due to a combination of greater pore numbers (porosity), a greater circumference of the pore edge per unit surface area, and greater frequency of flat surface areas for attachment, allowing better cell-substrate and cell-cell attachment and growth. The 3-microm pore size allowed cell-cell communication, together with diffusion of soluble nutrients and factors from the culture medium or wound substrate. These characteristics are considered important in developing grafts for use in the treatment of human skin wounds.     

Protein adsorption and human osteoblast-like cell attachment and growth on alkylthiol on gold self-assembled monolayers.

Protein adsorption and growth of primary human osteoblasts on self-assembled monolayers of alkylthiols on gold (SAMs) with carboxylic acid and hydroxyl and methyl termini were investigated. Single-component SAMs and SAMs patterned by photolithographic techniques were used. Cell growth on patterned SAMs demonstrated preferences for one pattern region in all combinations of alkylthiols, with the hierarchical preference COOH > OH > CH(3). Patterned SAMs and immunochemistry were used to investigate adsorption of fibronectin and albumin with respect to different alkylthiol termini. Fibronectin adsorption from both pure solution and serum containing cell culture medium (SDMEM) followed the sequence COOH > OH > CH(3). Albumin adsorption from pure solution followed the sequence OH > COOH > CH(3); from SDMEM the sequence was CH(3) > OH > COOH. Cell attachment to SAMs with the above termini, after preadsorption with fibronectin, albumin, or mixtures of fibronectin and albumin, was measured. Attachment was maximal on COOH-terminated SAMs precoated with fibronectin. Attachment to COOH was significantly reduced only when fibronectin was omitted from the protein preadsorption solution. On OH and CH(3) SAMs increasing the proportion of albumin in the solution was sufficient to significantly reduce cell attachment. The distribution vinculin and the integrins alpha(5)beta(1) and alpha(v)beta(3) indicated that focal contact formation by cells varied with alkylthiol termini in the following sequence: COOH > OH > CH(3).     

Development of a plasma-polymerized surface suitable for the transplantation of keratinocyte-melanocyte cocultures for patients with vitiligo

The purpose of this study was to develop a convenient methodology for the coculture of autologous melanocytes and keratinocytes for grafting of patients with vitiligo. While grafting of pure melanocytes may achieve repigmentation, the inclusion of keratinocytes ensures rapid reepithelialization. Previously we have used confluent sheets of keratinocytes (with melanocytes present) to transfer cells. However, we found that as the keratinocyte density increased, melanocyte number and function were downregulated. Accordingly in this study we explored combinations of three culture surfaces and three media, seeking to achieve subconfluent culture of primary keratinocytes with a reasonable density of melanocytes, using cells immediately after isolation from skin. For this in vitro study, the surfaces studied were uncoated glass coverslips, and glass coverslips coated with collagen I or a nitrogen-containing plasma polymer. The results show that both the substrate surface and the medium composition influence the proliferation and survival of melanocytes. Keratinocytes and melanocytes could be successfully cocultured on a chemically defined plasma polymer substrate using a serum-free medium.     

A denatured collagen microfiber scaffold seeded with human fibroblasts and keratinocytes for skin grafting

Biomaterial scaffolds are categorized into artificial or natural polymers, or combinations of the two. Artificial polymers often undergo serum protein adsorption, elicit foreign body and encapsulation immune responses post-implantation. Large pore bovine electrospun collagen I was therefore screened as a candidate for human keratinocyte and fibroblast cell scaffolds. Human HaCaT keratinocyte and dermal fibroblasts were seeded on electrospun denatured collagen I microfiber (DCM) scaffolds and after 72 h Livedead(?) assays performed to determine adhesive cell, survival and scaffold penetration. Both keratinocytes and fibroblasts attached to and survived on DCM scaffolds, however only fibroblasts migrated over and into this biomaterial. HaCaT keratinocytes remained largely stationary on the scaffold surface in discrete islands of monolayered cells. For this reason, normal human epidermal keratinocyte (NHEK) scaffold interactions were assessed using scanning and transmission electron microscopy (EM) that demonstrated DCM scaffolds comprised networks of interlocking and protruding collagen fibers with a mean diameter of 2-5 μm, with a mean inter-fiber pore size of 6.7 μm (range 3-10 μm) and scaffold thickness 50-70 μm. After 72 h the keratinocytes and fibroblasts on DCM scaffolds had attached, flattened and spread over the entire scaffold with assembly of lamellapodia and focal adhesion (FA)-like junctions. Using transmission EM, NHEKs and HaCaT keratinocytes assembled desmosomes, lamellapodia and FA junctions, however, neither hemidesmosomes nor basal lamina were present. In long term (21 day) co-culture fibroblasts migrated throughout the scaffold and primary keratinocytes (and to a lesser extend HaCaTs) stratified on the scaffold surface forming a human skin equivalent (HSE). In vivo testing of these HSEs on immunocompetent (BalbC) and immunodeficient (SCID) excisionally wounded model mice demonstrated scaffold wound biocompatibility and ability to deliver human cells after scaffold biodegradation.     

Cultured human keratinocytes on type I collagen membranes to reconstitute the epidermis

The development of new techniques and modifications to overcome some of the disadvantages in cultured keratinocyte grafting has been motivated by several well-known drawbacks in the use of cultured epithelial autografts such as long culture periods, lack of adherence, difficulty in handling, lack of dermal substrates, and high costs. Two recent insights have influenced further research. On the one hand, it has been shown that the use of undifferentiated proliferative cells in fibrin glue suspensions is effective in epithelial reconstitution. On the other hand, the enzymatic release of cells from the culture surfaces is a critical step leading to at least temporary destruction of anchoring structures of the cultured cells. In this study, we tried to combine these two aspects in an attempt to modify common modalities of keratinocyte transplantation. To avoid dispase dissolving of the cultured cells, keratinocytes were seeded onto bovine collagen type I membranes without feeder layers and under serum-free culture conditions. Subconfluent monolayers of cultured human keratinocytes were transplanted as an upside-down graft on collagen membranes (keratinocyte collagen membrane grafts [KCMG], n = 12) after 3 days of culture or as membrane grafts alone (n = 12) onto standard nude mice full-thickness wounds. Fully differentiated epidermis was found at 21 days after grafting KCMG with persistence of human keratinocytes. This study demonstrates that upside-down grafts of undifferentiated monolayers of keratinocytes on non-cross-linked bovine type I collagen membranes do lead to an early reconstitution of multilayered squamous epithelium with enhanced wound healing compared to the control group. The upside down KCMG grafting technique is able to transfer actively proliferative keratinocytes and simplifies the application compared to conventional epithelial sheet grafting.     

Controlling the orientation of bone osteopontin via its specific binding with collagen I to modulate osteoblast adhesion

Osteopontin (OPN) is an important matricellular protein that modulates cell functions. It is potentially an excellent surface-coating component for engineered biomaterials. It is believed that in its preferred orientation and conformation on a surface, the functional domains of OPN such as the arginine-glycine-aspartic acid (RGD) motif will be presented to cells to the greatest extent. Previously, the authors demonstrated that OPN orientation could be modulated by surface charge. In this work, the authors attempt to control the orientation/conformation of bone OPN via its specific interactions with type I collagen. Surface plasmon resonance was used to confirm the specific binding between bone OPN and collagen I. A radiolabeled OPN adsorption assay was used to determine the amount of adsorbed OPN on tissue culture polystyrene (TCPS) surfaces with or without collagen I as an interlayer. An in vitro cell adhesion assay using osteoblast MC3T3-E1 was performed to compare the functionality of collagen-bound OPN and adsorbed OPN on TCPS. With the same amount of OPN on the surfaces, the number of cells adhered to collagen-bound OPN is significantly higher than to OPN alone on TCPS. A cell inhibition assay using soluble GRGDSP peptides showed that a higher GRGDSP concentration was needed to completely block osteoblast adhesion to collagen-bound OPN than to OPN directly on TCPS. Enhanced cell adhesion and higher blocking peptide concentration suggest that collagen-bound bone OPN has a preferable orientation/conformation for cell adhesion compared with OPN alone on TCPS. Thus, the specific binding of OPN to collagen I may naturally orient OPN, thus influencing osteoblast adhesion.     

Behavior of osteoblasts on a type I atelocollagen grafted ozone oxidized poly L-lactic acid membrane

With oxidizing poly-L-lactic acid (PLLA) surface by ozone, peroxide groups are easily generated on the surface. Those peroxides are broken down by redox-coupling reaction, and provide active species that initiate grafting by reaction with the collagen molecules. The surface density of generated peroxide on a PLLA surface was determined by an iodide method. The maximum concentration of peroxide was about 2.87 x 10(-8) mol/cm2 when ozone oxidation was performed at 60 V for 60 min. After the surface oxidation, type I atelocollagen was grafted onto PLLA surface. All physical measurements on the collagen-grafted surface indicated that the PLLA surface was effectively grafted with type I atelocollagen. Behavior of rat calvaria osteoblasts on type I atelocollagen grafted PLLA (PLLA + COL) surface was observed. Initial attachment of osteoblasts on the surface was significantly enhanced, and it is assumed that the atelocollagen matrix supported the initial attachment and growth of cells. Collagenous protein synthesis of osteoblasts was maintained at relatively low level in the early stage of proliferation due to the primarily existing grafted type I atelocollagen, and then increased in 7 days as the osteoblast differentiated. After 7 days, collagenous protein synthesis in osteoblasts was activated. Alkaline phosphatase (ALPase) activity and mineralization by osteoblasts were promoted on PLLA + COL surface. In comparison with PLLA + COL, non-treated PLLA and tissue culture plate (TCPS) did not show any feature expressed in osteoblasts' maturation up to 9 days in this experiment. The grafted type I atelocollagen provided a favorable matrix for cell migration in relation with collagenase expression. Ozone oxidation might be a favorable method for surface modification of PLLA membranes by collagen grafting, and cell behavior could be modulated by the grafted collagen.     

Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment

The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.     

Human skin cell cultures onto PLA50 (PDLLA) bioresorbable polymers: influence of chemical and morphological surface modifications

Poly(alpha-hydroxy acid)s derived from lactic and glycolic acid are bioresorbable polymers which can cover a large range of thermal, physical, mechanical, and biological properties. Human keratinocytes have been shown as able to grow on a poly(DL-lactic acid) film. However the keratinocyte growth was delayed with respect to culture on standard tissue culture polystyrene, even though the same plateau level was observed after 2 weeks. In order to improve the performance of poly(DL-lactic acid) films as skin culture support, their surface was modified by creating tiny cavities using a method based on the leaching out of poly(ethylene oxide) from poly(lactic acid)-poly(ethylene oxide) heterogeneous blends. The surface of the films was also chemically modified by alkaline attack with sodium hydroxide and by type-I collagen coating. Murine fibroblast cell line and primary cultures of human fibroblasts and of two types of keratinocytes were allowed to adhere and to grow comparatively on the different films. The presence of cavities affected neither the adhesion of dermal fibroblasts nor that of keratinocytes. Only keratinocyte proliferation was significantly reduced by the presence of cavities. Collagen coating improved skin cell adhesion and proliferation as well, except in the case of murine fibroblasts. In the case of the NaOH treatments, similar trends were observed but their extent depended on the treatment time. In the case of chemical modifications, fluorescence microscopy bore out adhesion and proliferation tendencies deduced from MTT tests.     

Surface modification by complexes of vitronectin and growth factors for serum-free culture of human osteoblasts

Cell attachment, expansion, and migration in three-dimensional biomaterials are crucial steps for effective delivery of osteogenic cells into bone defects. Complexes composed of vitronectin (VN), insulin-like growth factors (IGFs), and insulin growth factor-binding proteins (IGFBPs) have been reported to enhance cell attachment, proliferation, and migration in a variety of cell lines in vitro. The aim of this study was to examine whether prebound complexes of VN and IGFs +/- IGFBPs could facilitate human osteoblast serum-free expansion in vitro and enhance cell attachment, proliferation, and migration in three-dimensional biomaterial constructs. Human osteoblasts derived from alveolar bone chips and the established human osteoblast cell line Saos-2 were used. These cells were seeded on tissue culture plates and porous scaffolds of type I collagen sponges and polyglycolic acid (PGA), which had been coated with VN +/- IGFBP-5 +/- IGF-I. Cell attachment, proliferation, and migration were evaluated by cell counting, confocal microscopy, and scanning electron microscopy. The number of attached human osteoblasts was significantly higher in VN-coated polystyrene culture dishes. Furthermore, significant increases in cell proliferation were observed when growth factors were bound to these surfaces in the presence of VN. In the two scaffold materials examined, greater cell attachment was found in type I collagen sponges compared with PGA scaffolds. However, coating the scaffolds with complexes composed of VN + IGF-I or VN + IGFBP-5 + IGF-I enhanced cell attachment on PGA. Moreover, the presence of VN + IGFBP-5 + IGF-I resulted in significantly greater osteoblast migration into deep pore areas as compared with untreated scaffolds or scaffolds treated with fetal calf serum. These results demonstrated that complexes of VN + IGFBP-5 + IGF-I can be used to expand osteoblasts in vitro under serum-free conditions and enhance the attachment and migration of human osteoblasts in three-dimensional culture. This in turn suggests a potential application in surface modification of biomaterials for tissue reconstruction.     

Self-assembled monolayers of alkanethiols on gold modulate electrophysiological parameters and cellular morphology of cultured neurons

Self-assembled monolayers (SAMs) of omega-substituted alkanethiols on gold have been explored as well defined in vitro model surfaces for the investigation of neuronal growth and function. When used as cell culture substrates, surfaces with monolayers functionalized with terminal -COOH groups support neuron attachment and growth even without an intermediate protein layer. Addition of a poly-L-lysine layer (PLL) to the -COOH terminated monolayers significantly increases total neurite outgrowth. Mixed monolayers containing -COOH and -CH3 terminal groups in 1:10 and 1:100 ratios poorly support neuron adhesion and preclude neurite extension. A layer of PLL improves the ability of mixed monolayer surfaces to support neuronal growth in culture. The morphology of cultured neurons depends on the chemical composition of SAMs on the support surface. Using glass microelectrode intracellular recording, the properties of cell culture substrates modulate the dynamic properties of action potentials of cultured neurons. These findings provide insight into the cellular responses of excitable cells to the chemical details of a surface and, thus, may help direct the rational design of biologically active materials.     

Type V collagen represses the attachment, spread, and growth of porcine vascular smooth muscle cells in vitro.

We attempted to clarify the effects of various purified extracellular components, including types I, III, IV, and V collagen and fibronectin on attachment, spread, growth, and DNA synthesis of porcine aortic smooth muscle cells (SMCs) in vitro. The number, area and shape index (SI = 4 pi S/L2) of cells attached to different substrates were determined at various intervals of incubation. The cell number and [3H]thymidine incorporation into DNA were measured on the 1st and 6th days of culture. SMCs showed the largest number of attached cells on fibronectin, but the smallest number of attached cells on type V collagen. There was no evidence of effects of the serum in media on the attachment of SMCs to the substrates. The areas of attached SMCs were the largest on fibronectin and the smallest on type V collagen. The shape index of SMCs on fibronectin decreased relative to those on other substrates. On the 6th day in culture, the number and population doubling of SMCs on type V collagen were significantly fewer than those on other substrates. Both the incorporation rate of [3H]thymidine into DNA and the percentage of nuclei labeled with [3H]thymidine were significantly less in the SMCs on type V collagen on the 1st day than those on other substrates. SMCs on types I, III, and IV collagen showed intermediate levels of cell attachment, spread, and growth. These results suggest that attachment, spread, and growth of SMCs are affected mainly by solid phase purified extracellular components and are most strongly suppressed by type V collagen. When DNA synthesis of growth-arrested SMCs was reinitiated by the addition of serum, type V collagen most intensively inhibited the rate and amount of [3H]thymidine incorporation. Flow cytometric analysis demonstrated an increased in the proportion of cells in G0/G1 phase on type V collagen in comparison with that on other substrates. Thus, the antiproliferative effect of type V collagen may relate to inhibition of transition of SMCs from the G0/G1 into the S phase.     

Correlating fibronectin adsorption with endothelial cell adhesion and signaling on polymer substrates

Fibronectin (Fn) adsorption was studied on different commercial polymer surface chemistries, including tissue culture polystyrene (TCPS), bacteriologic polystyrene (BPS), fluoropolymer Teflon AF, and poly-L-lactide (PLLA). Antibody probes detected the availability of Fn's cell binding domain on adsorbed Fn in the competitive presence and absence of bovine serum albumin (BSA). Domain availability was highest for Fn adsorbed on TCPS, especially in the presence of either serum albumin or dilute serum. Attachment and growth efficiencies for human umbilical venous endothelial cells (HUVECs) cultured on surfaces preadsorbed with Fn in serum and serum-free media correlated with antibody cell-binding domain availability: TCPS > BPS, Teflon AF > PLLA. Intracellular signaling from the GTPase, RhoA, was highest (RhoA:RhoGDI inhibitor ratio) in cells cultured on the Teflon AF surfaces, indicating that despite lower attached cell numbers on Teflon AF compared to TCPS, cell signaling remained activated after 24 h of growth. Up-regulated cellular Fn mRNA messages, assessed using RT-PCR techniques, supported HUVECs' producing the endogenous extracellular matrix (ECM) protein Fn in order to attach and survive on the suboptimal Teflon AF culture surfaces.     

The titanium surface texture effects adherence and growth of human gingival keratinocytes and human maxillar osteoblast-like cells in vitro.

The adhesion, orientation and proliferation of human gingival epithelial cells and human maxillar osteoblast-like cells in primary and secondary culture were studied on glossy polished, sandblasted and plasma-sprayed titanium surfaces by scanning electron microscopy and in thin sections. The primary cultured explants of human gingival epithelial cells attached, spread and proliferated on all titanium surfaces with the greatest extension on the polished and the smallest extension on plasma-sprayed surfaces. In secondary suspension cultures of gingival keratinocytes, attachment spreading and growth was only observed on polished and plasma-sprayed surfaces, but not on sandblasted surfaces. Moreover, the attachment of these cells depended on the seeding concentration as well as on the coating with fetal calf serum. Cells on polished surfaces developed an extremely flat cell shape, but on sandblasted and plasma-sprayed surfaces a more cuboidal shape. In contrast human maxillar osteoblasts seeded as secondary suspension cultures attached very well to all three differently textured titanium surfaces and showed identical growth patterns independent of the titanium surface structure. These findings suggest that cell morphology, orientation, proliferation and adhesion of human gingival epithelial cells in primary or secondary culture are dependent on the texture of the titanium surface whereas no such differences were observed for maxillar osteoblast-like cells. In conclusion, the soft tissue integration and response is more influenced by the surface texture than the process of osseointegration.     

The response of osteoblast-like cells towards collagen type I coating immobilized by p-nitrophenylchloroformate to titanium

The scaffold surface composition can be altered by the use of surface coatings. The use of thin coatings will give special surface properties, while the bulk properties of the scaffold are preserved. Collagen type I is known to play an important role during cell adhesion as well as osteoblast differentiation. A common way to coat surfaces is the adsorption method. An alternative way is the use of a protein immobilization method like p-nitrophenyl chloroformate. In this study, we investigated the effect of a collagen type I coating and p-nitrophenyl chloroformate as a protein immobilization method on osteoblast adhesion, proliferation, and differentiation. Titanium fiber meshes were treated with sodium hydroxide (NaOH), followed by p-nitrophenyl chloroformate, and coated with collagen type I. Osteoblast-like cells were seeded into the meshes and cultured for 24 days. The cell attachment, proliferation, and differentiation were measured by using Live and Dead assay, cell counting, DNA analysis, alkaline phosphatase activity assay, calcium content measurement, Real Time PCR (QPCR), and scanning electron microscopy (SEM). Results demonstrated that initially less cells were attached to the covalently bounded collagen meshes (NPC-Col) compared with titanium as control (Ti) and adsorbed collagen meshes (ABS-Col). Further, a decreased growth curve of cells cultured on the NPC-Col meshes was observed in comparison with Ti and ABS-Col meshes. The calcium measurements and SEM pictures revealed that all three surfaces showed differentiation of osteoblast-like cells after 8-24 days. On the basis of our results, we conclude that initially less cells were attached to the NPC-Col meshes and that they had a decreased proliferation rate. Further, we conclude that an adsorbed collagen type I coating stimulated the osteoblastic differentiation of rat bone marrow cells.     

Glycosaminoglycans and the substrate attachment of murine myeloma, 3T3, and cutaneous fibrosarcoma cells

The attachment of murine myeloma, 3T3, and cutaneous fibrosarcoma cells to substrates of either fibronectin, type I collagen, or two types of tissue culture plastic was examined in the presence and absence of specific exogenous glycosaminoglycans. Fibrosarcoma and 3T3 cells were found to be nondiscriminatory with respect to their avidity of attachment to substrates of either of the proteins or of conventional tissue culture plastic, whereas the myeloma cells attached significantly less well to a substrate of collagen than to the other two matrices. On tissue culture plastic and collagen the fibrosarcoma cells attached more rapidly than did the other two cell types. Selective and partial inhibition of cell attachment to type I collagen, and, to a lesser extent, fibronectin, occurred upon preincubating these substrates with the sulfated glycosaminoglycans, heparin and heparan sulfate, at concentrations of 1 to 100 micrograms/ml; for 3T3 cells heparin was significantly more inhibitory (mean maximal inhibition of approximately 40%) than were two heparan sulfate fractions. Attachment of fibrosarcoma and 3T3 cells to a nitrogenated tissue culture plastic surface with a net positive charge (Primaria) was nearly 50% inhibited by heparin at the higher concentration and to a lesser extent by the two heparin sulfate fractions. Myeloma cell attachment to this same substrate was inhibited, to a lesser degree, by all three sulfated glycosaminoglycans. Hyaluronic acid, dermatan sulfate, and chondroitin 6-sulfate were inactive in our attachment assays. We suggest that the functional role of glycosaminoglycans in substrate attachment may vary depending on the cell type and the matrix involved in the specific interaction. In particular, the net charge of the substrate appears to be an important factor, and on positively charged surfaces these substances may serve a greater function. However, since nearly complete abrogation of cell attachment should have been achievable by some of the exogenous preparations if cell surface sulfated glycosaminoglycans were to comprise the major cellular binding sites for matrices, we conclude that it is unlikely that these complex polysaccharides function as the principal determinant of simple cell attachment.     

Enhanced adherence of human adult endothelial cells to plasma discharge modified polyethylene terephthalate

Human adult aortic endothelial cell attachment to polyethyleneterephthalate (PET as mylar film) was examined in vitro. PET was examined in both the unmodified form (PET-) and in a modified form (PET+) that had undergone plasma discharge surface modification (PDSM). These surfaces were compared to unmodified tissue culture polystyrene (PS-). The kinetics of attachment and the force of attachment using the rotating disc were determined as a function of surface and substrate protein applied to the surface. Four proteins--fibronectin, collagen I/III, collagen IV/V, and laminin--were added and compared to saline pretreatment. The most significant variable affecting attachment was the time of incubation. When corrected for time, PET+ demonstrated significantly superior attachment kinetics when compared to PET- in most cases. These kinetics were similar to those seen on PS-. Fibronectin precoating of the surface greatly enhanced attachment kinetics on PET+ and PS- but to a much lesser degree on PET-. The fibronectin effect was synergistic with PDSM, suggesting that PDSM enhances protein adsorption on the surface. The force of attachment was generally independent of incubation time and surface/substrate combination except for laminin precoating. Taken together, these data indicate that human endothelial cell adherence to PET may be significantly enhanced by PDSM and surface precoating with fibronectin. Attachment occurs rapidly and, once attached, the cells demonstrate a very firm attachment force capable of resisting shear stresses up to 90 dynes/cm2.