Modulus-dependent macrophage adhesion and behavior.

Macrophage attachment and activation to implanted materials is crucial in determining the extent of acute and chronic inflammation, and biomaterials degradation. In an effort to improve implant performance, considerable attention has centered on altering material surface chemistry to modulate macrophage behavior. In this work, the influence of the modulus of a material on the behavior of model macrophages (i.e., human promonocytic THP-1 cells) was investigated. We synthesized interpenetrating polymer network (IPN) coatings with varying moduli to test the hypothesis that lower moduli surfaces attenuate THP-1 cell attachment and activation. The surface chemistry and moduli of the IPN coatings were characterized using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM), respectively. THP-1 cells preferentially attached to stiffer coatings of identical surface chemistry, confirming that fewer macrophages attach to lower moduli surfaces. The secretion of human TNF-alpha, IL-10, IL-8 and IL-1beta from THP-1 cells attached to the IPNs was measured to assess the concentration of both pro- and anti-inflammatory cytokines. The global amount of TNF-alpha released did not vary for IPN surfaces of different moduli; however, the amount of the pro-inflammatory cytokine IL-8 released demonstrated a biphasic response, where lower (approx. 1.4 kPa) and very high (approx. 348 kPa) moduli IPN surfaces attenuated IL-8 secretion. The different trends for TNF-alpha and IL-8 secretion highlight the complexity of the wound healing response, suggesting that there may not be a unique surface chemistry and substratum modulus combination that minimizes the pro-inflammatory cytokines produced by activated macrophages.

Development and characterization of a high-throughput system for assessing cell-surface receptor-ligand engagement.

A nonfouling interfacial interpenetrating polymer network (IPN) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] was grafted to polystyrene for use as a novel platform for the development of high-throughput assays for screening of specific bimolecular interactions (i.e., receptor-ligand engagement). For the development of the IPN, a water-soluble hydrogen-abstracting photoinitiator was investigated: (4-benzoylbenzyl)trimethylammonium chloride. IPN-modified polystyrene surfaces were characterized using XPS, contact angle goniometry, and protein adsorption analysis. These IPN surfaces minimized fibrinogen adsorption compared to tissue culture polystyrene (>96% reduction), prevented mammalian cell adhesion, and served as nonfouling surfaces to graft biological ligands. For bimolecular interaction studies, a model peptide ligand from bone sialoprotein (Ac-CGGNGEPRGDTYRAY-NH(2)) was grafted to p(AAm-co-EG/AAc) via a 3400 M(w) linear pEG spacer. Ligand density measurements, cell culture, and a centrifugal adhesion assay were used to study cell adhesion to peptide-modified IPNs (i.e., receptor-ligand engagement). Ligand density (Gamma) was controllable from approximately 1 to 20 pmol/cm(2) by modulating the peptide input concentration (0.02-20 microM). Cell adhesion was directly dependent on the ligand density. This technology creates a powerful high-throughput system to simultaneously probe a myriad of cell-surface receptor-ligand interactions.

Saline irrigation does not affect bone formation or fixation strength of hydroxyapatite/tricalcium phosphate-coated implants in a rat model.

Intramembranous bone regeneration is critical to implant fixation. In cementless joint replacement (as opposed to cemented joint replacement), saline irrigation is not typically performed during surgery so that the osteogenic stimulus provided by the marrow is preserved. Several groups are now using the rat marrow ablation model to study intramembranous bone regeneration and implant fixation. In this model, the marrow contents are mechanically disrupted, and debris is often cleared by saline irrigation, a step that appears inconsistent with the clinical situation. Furthermore, in contrast to conventional wisdom, it has been reported that saline irrigation enhanced bone-implant contact and peri-implant bone formation in the rat model (Ishizaka et al. Bone 1996;19:589-594), although mechanical fixation of the implant was not investigated. Accordingly, the present study was performed to determine if saline irrigation leads to enhanced mechanical fixation of implants in the rat model. Forty-eight 400 to 450 g male rats were divided equally into two groups. The treatment group, in contrast to the control group, received saline irrigation in the ablated medullary canal prior to placement of hydroxyapatite/tricalcium phosphate-coated implants. Eight animals in each group were killed at 2, 4, or 8 weeks after implantation, at which time the specimens were analyzed by micro computed tomography to measure bone formation around the implant, followed by a mechanical pull-out test to measure the strength of fixation of the implant. As expected, there was increased fixation strength over time, but there were no significant differences in peri-implant bone volume, bone-implant contact, or implant fixation strength between the two groups. Thus, we found no effect of saline irrigation on bone formation or implant fixation strength in this study in which the implant had an osteoconductive coating.

Analysis of interpenetrating polymer networks via quartz crystal microbalance with dissipation monitoring.

A quartz crystal microbalance with dissipation monitoring (QCM-D) was used to assess the physical properties of interpenetrating polymer networks (IPNs) through swelling experiments in ambient humidity and in phosphate-buffered saline (PBS), pH 7.4. The IPNs, based on acrylamide (AAm) and poly(ethylene glycol) (pEG), swell from thin, rigid films when dry (16.7 +/- 5.2 nm on Si/SiO(2)) to expanded, viscoelastic films when hydrated (107 +/- 24.2 nm on Si/SiO2). The dry IPNs could be analyzed using the Sauerbrey relationship, but for the hydrated films it was necessary to interpret QCM-D data with a Kelvin-Voigt viscoelastic model. A complex modulus |G| of 116 +/- 38.1 kPa for the swollen IPN surface on Si/SiO2 was defined by the model. The QCM-D was also employed to quantify the adsorption of human fibrinogen, a protein important in thrombus formation, onto the IPNs. Fibrinogen adsorption studies demonstrated the sensitivity of the QCM-D, as well as confirmed the nonfouling nature of the IPN surface, where less than 5 ng/cm2 of fibrinogen was adsorbed.

A low-temperature biomimetic calcium phosphate surface enhances early implant fixation in a rat model.

The present study demonstrates increased early mechanical fixation of titanium implants coated with a new biomimetic apatite surface in a rat model. Male Sprague-Dawley rats received unilateral femoral medullary implants for periods of 1-4 weeks. The strength of fixation of the implant to the host bone increased more rapidly in the group receiving apatite-treated implants compared with the control group as evidenced by the apatite group's 21-fold greater fixation strength at 1 week (p = 0.009), 4-fold greater fixation strength at 2 weeks (p = 0.041), and 2-fold greater fixation strength at 4 weeks (p = 0.093) compared with the control. Fixation strength was correlated with bone-implant contact as determined from micro computed tomography assessment of the specimens (r2 = 0.338, p = 0.011 in the control group and r2 = 0.543, p < 0.001 in the apatite group). Furthermore, for a given amount of bone-implant contact, the fixation strength was higher in the apatite group than in the control group (p = 0.011), suggesting that the bone formed a stronger bond to the apatite coating than to the titanium. This difference in bonding strength accounted for the difference in mechanical behavior.

Endothelial cell function on a poly(acrylamide-co-polyethylene acid) interpenetrating polymer network: cardiovascular applications.

Hydrogel coatings have been widely researched as a nonfouling surface modification of materials for cardiovascular applications. In this study, we examined cell-surface interactions between a poly(acrylamide-copolyethylene glycol/acrylic acid) interpenetrating network (IPN) hydrogel and aortic endothelial cells (ECs). The IPN was covalently attached to polystyrene to form a nanometer scale thick hydrogel, and the IPN layer was activated by conjugation of the cell adhesion peptide Arg-Gly-Asp (RGD). On IPN surfaces lacking the RGD peptide, EC did not adhere and spread even after long-term incubation. The IPN was able to support greater EC adhesion and spreading with increasing RGD surface concentrations. Upon adequate adhesion and spreading, ECs migrated and proliferated at high rates regardless of the RGD surface concentration. These results suggest that this IPN can be used to promote endothelialization of vascular implants made of polymeric and metal materials for cardiovascular applications.

Thermo-responsive peptide-modified hydrogels for tissue regeneration.

Loosely cross-linked hydrogels consisting of N-isopropylacrylamide (NIPAAm) and acrylic acid (AAc) were synthesized, characterized, and used as model scaffolds for studying cell-material interactions in three-dimensions (3D). The AAc groups were functionalized with peptides containing the -RGD- and -FHRRIKA- sequences found in bone sialoprotein. Chemical modification of the hydrogels was verified via solid-state (1)H nuclear magnetic resonance spectroscopy, lower critical solution temperature studies, and volume change studies. The peptide-modified hydrogels were pliable at 22 degrees C and could be injected through a small-diameter aperture. Rat calvarial osteoblasts (RCO) seeded into the peptide-modified hydrogels were viable for at least 21 days of in vitro culture. The RCO spread more and demonstrated significantly greater proliferation when cultured within the peptide-modified hydrogels, as compared to control hydrogels. These peptide-modified P(NIPAAm-co-AAc) hydrogels serve as useful tools for studying cell-material interactions within 3D structures and have the potential to be used as injectable scaffolds for tissue engineering applications.

A system to impose prescribed homogenous strains on cultured cells.

There is presently significant interest in cellular responses to physical forces, and numerous devices have been developed to apply stretch to cultured cells. Many of the early devices were limited by the heterogeneity of deformation of cells in different locations and by the high degree of anisotropy at a particular location. We have therefore developed a system to impose cyclic, large-strain, homogeneous stretch on a multiwell surface-treated silicone elastomer substrate plated with pulmonary epithelial cells. The pneumatically driven mechanism consists of four plates each with a clamp to fix one edge of the cruciform elastomer substrate. Four linear bearings set at predetermined angles between the plates ensure a constant ratio of principal strains throughout the stretch cycle. We present the design of the device and membrane shape, the surface modifications of the membrane to promote cell adhesion, predicted and experimental measurements of the strain field, and new data using cultured airway epithelial cells. We present for the first time the relationship between the magnitude of cyclic mechanical strain and the extent of wound closure and cell spreading.

A biodegradable polymer scaffold for delivery of osteotropic factors.

Despite discoveries and developments in osteotropic factors, therapies exploiting these macromolecules have been limited due to a lack of suitable delivery vehicles and three dimensional (3D) scaffolds that promote bone regeneration. To address this limitation, an emulsion freeze-drying process was developed to fabricate biodegradable scaffolds with controlled microarchitecture, and the ability to incorporate and deliver bioactive macromolecules for bone regeneration. The effect of median pore size and protein loading on protein release kinetics was investigated using scaffolds with different protein loading and median pore sizes ranging from 7 to 70 microm. Graphs of protein release from scaffolds showed an initial burst followed by a slower sustained release. Release kinetics were characterized using an unsteady-state, diffusion-controlled model with an effective diffusivity that took tortuosity (tau) and partition coefficient for protein adsorption (Kp) onto the scaffold walls into account. Tortuosity and partition coefficient significantly reduced the protein diffusivity by a factor of 41 +/- 43 and 105 +/- 51 for 60 and 30-microm median pore-sized scaffolds, respectively. The activity of the protein released from these scaffolds was demonstrated by delivering rhBMP 2 and [A-4] (an amelogenin derived polypeptide) proteins from the scaffold and regenerating bone in a rat ectopic bone induction assay [Whang et al. J Biomed Mater Res 1998;42:491-9, Veis et al. J Bone Mineral Res, Submitted].

The effect of peptide surface density on mineralization of a matrix deposited by osteogenic cells.

The density of Arg-Gly-Asp-containing peptides covalently grafted to solid materials has been shown to affect adhesion, spreading, and focal contact formation. The objective of this study was to examine the effect of ligand density on mineralization of the extracellular matrix deposited by osteoblasts. In particular, RGD-modified quartz surfaces with ligand densities varying over two orders (0.01-3.6 pmol/cm(2)) of magnitude were prepared to assess the long-term function of osteoblasts on peptide-derivatized surfaces. After 3 weeks in culture, surfaces modified with a 15 amino acid peptide (Ac-Cys-Gly-Gly-Asn-Gly-Glu-Pro-Arg-Gly-Asp-Thr-Tyr-Arg-Ala-Tyr-NH(2) ) at a density > or =0.62 pmol/cm(2) significantly (p<0.05) enhanced mineralization compared with a RGD surface density of 0.01 pmol/cm(2), RGE surfaces, or clean surfaces adsorbed with serum proteins. These results suggest that regulation of the surface density of adhesive ligands on biomaterial surfaces is a critical determinant in a strategy to alter the degree of extracellular matrix maturation in contact with solid surfaces (e.g., implants). Further studies are required to elucidate the intracellular signal transduction pathways that mediate long-term matrix mineralization through the initial engagement of these adhesive ligands.

Surface modification of poly(ethylene terephthalate) angioplasty balloons with a hydrophilic poly(acrylamide-co-ethylene glycol) interpenetrating polymer network coating.

An interpenetrating polymer network (IPN) of poly(acrylamide-co-ethylene glycol) (p(AAm-co-EG)) hydrogel was covalently grafted to polyethylene terephthalate (PET) angioplasty balloons to increase surface hydrophilicity and improve lubricity. A 2-step graft polymerization protocol was followed to first polymerize and cross-link acrylamide onto the substrate with a photosensitizer and/or oxygen plasma pretreatment. The effects of varying photo-initiation and plasma exposure times were investigated separately and conjunctively using water contact angles to obtain optimal coating deposition parameters. A poly(ethylene glycol) network was then grafted by swelling the preexisting polyacrylamide network to allow inter-diffusion of the monomer and cross-linker, which were then polymerized by photo-initiation. When the photo-initiation time was long enough to reach near gelation, pretreatment of PET with oxygen plasma did not offer significant benefit. X-ray photoelectron spectroscopy confirmed the presence of both polymer layers, and composition depth profiles supported the assessment that an interpenetrating network was formed. Tensile testing and application of Weibull statistics on unmodified and modified films indicated that the surface modification approach did not significantly alter the mechanical integrity of the material. These findings indicate that a p(AAm-co-EG) coating can be effectively deposited on PET surfaces without compromising the structural integrity of the substrate.

Specific amelogenin gene splice products have signaling effects on cells in culture and in implants in vivo.

Low molecular mass amelogenin-related polypeptides extracted from mineralized dentin have the ability to affect the differentiation pathway of embryonic muscle fibroblasts in culture and lead to the formation of mineralized matrix in in vivo implants. The objective of the present study was to determine whether the bioactive peptides could have been amelogenin protein degradation products or specific amelogenin gene splice products. Thus, the splice products were prepared, and their activities were determined in vitro and in vivo. A rat incisor tooth odontoblast pulp cDNA library was screened using probes based on the peptide amino acid sequencing data. Two specific cDNAs comprised from amelogenin gene exons 2,3,4,5,6d,7 and 2,3,5,6d, 7 were identified. The corresponding recombinant proteins, designated r[A+4] (8.1 kDa) and r[A-4] (6.9 kDa), were produced. Both peptides enhanced in vitro sulfate incorporation into proteoglycan, the induction of type II collagen, and Sox9 or Cbfa1 mRNA expression. In vivo implant assays demonstrated implant mineralization accompanied by vascularization and the presence of the bone matrix proteins, BSP and BAG-75. We postulate that during tooth development these specific amelogenin gene splice products, [A+4] and [A-4], may have a role in preodontoblast maturation. The [A+4] and [A-4] may thus be tissue-specific epithelial mesenchymal signaling molecules.

Quantification of the surface density of a fluorescent label with the optical microscope.

Fluorescence microscopy can offer unique advantages for biomaterials characterization. Like spectroscopy or radioactivity, it can be used to quantify specific binding to surfaces, but it can also assess surface homogeneity at the micron scale or detect protein aggregation. To fully utilize the potential of this technique, there must be a way to calibrate the microscope in terms of the moles of a fluorophore per unit area. The method we propose involves the following steps: fluorescent labeling of erythrocytes and quantification of the label by flow cytometry; flattening of fluorescent erythrocytes for microscopic observation; imaging and digital analysis to relate the gray level intensities to the fluorophore density; and using this procedure to characterize a different, more easily obtainable, standard. The latter can be a 50% solution of Na fluorescein that yields a highly reproducible and uniform fluorescence. Concentrated fluorescein solution can also be used to correct images for the spatial nonuniformity of illumination and detection (shading correction). By applying this method to study the binding of IgG and fibrinogen to glass or amidated glass, we showed that protein adsorption to glass may result in protein aggregation that may affect the biological activity of the adsorbed protein.

Protein adsorption and cell attachment to patterned surfaces.

To better understand the events involved in the generation of defined tissue architectures on biomaterials, we have examined the mechanism of attachment of human bone-derived cells (HBDC) to surfaces with patterned surface chemistry in vitro. Photolithography was used to generate alternating domains of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) and dimethyldichlorosilane (DMS). At 90 min after seeding, HBDC were localized preferentially to the EDS regions of the pattern. Using sera specifically depleted of adhesive glycoproteins, this spatial organization was found to be mediated by adsorption of vitronectin (Vn) from serum onto the EDS domains. In contrast, fibronectin (Fn) was unable to adsorb in the face of competition from other serum components. These results were confirmed by immunostaining, which also revealed that both Vn and Fn were able to adsorb to EDS and DMS regions when coated from pure solution, i.e., in the absence of competition. In this situation, each protein was able to mediate cell adhesion across a range of surface densities. Cell spreading was constrained on the EDS domains, as indicated by cell morphology and the lack of integrin receptor clustering and focal adhesion formation. This spatial constraint may have implications for the subsequent expression of differentiated function.

Integrin subunits responsible for adhesion of human osteoblast-like cells to biomimetic peptide surfaces.

We have identified the integrin subunits responsible for the initial adhesion of human osteoblast-like cells to peptide-modified surfaces. Biomimetic peptide surfaces containing homogenous RGD (Arg-Gly-Asp), homogenous FHRRIKA (Phe-His-Arg-Arg-Ile-Lys-Ala), and a mixed ratio of FHRRIKA:RGD (25:75) were used to assess integrin-mediated adhesion. The RGD and FHRRIKA peptides were selected from the cell-binding and putative heparin-binding domains of bone sialoprotein. A panel of monoclonal antibodies against human alpha1, alpha2, alpha3, alpha4, alpha5, beta1, alpha(v), and alpha(v)beta3 was used to identify the subunits most dominant in mediating short-term (10 or 30 minutes) and long-term (4 hours) cell adhesion to the peptide surfaces. Anti-alpha2, anti-beta1, and anti-alpha(v) significantly (p < 0.05) diminished cell attachment to homogenous RGD surfaces following 30 minutes of incubation. After 4 hours of incubation on RGD-grafted surfaces, immunostaining of these integrin subunits revealed discrete localization of the alpha(v) subunit at the periphery of the cell (similar to focal contact points), whereas the alpha2 and beta1 subunits stained very diffusely throughout the cell. A radial-flow apparatus was used to determine the effect of anti-integrin antibodies on strength of cell detachment following 10 minutes of incubation on peptide-grafted surfaces. The strength of detachment from surfaces containing RGD was significantly reduced (p < 0.05) in the presence of anti-alpha2, anti-alpha(v), or anti-beta1 compared with controls (presence of preimmune mouse IgG). None of the antibodies significantly influenced cell attachment to homogenous FHRRIKA-grafted surfaces. These results demonstrate that initial (30 minutes) attachment of human osteoblast-like cells to homogenous RGD surfaces was mediated by the collagen receptor alpha2beta1 and the vitronectin receptor alpha(v)beta3, whereas only the vitronectin receptor governed longer term (longer than 30 minutes) adhesion (localization to focal contacts). The importance of distinct integrins in mediating the attachment of bone cells to RGD-immobilized surfaces indicates a strategy for engineering orthopaedic implants with a built-in surface specificity for cell adhesion.

Designing biomaterials to direct biological responses.

We have set forth a design strategy for creating biomimetic materials that direct the formation of tissue surrounding implants or regeneration within porous scaffolds. Our studies have established that heterogeneous mimetic peptide surfaces (MPS) containing both the -RGD- (cell-binding) and-FHRRIKA- (putative heparin-binding) peptides, unique to BSP, in the ratio of 75:25 (MPS II) or 50:50 (MPS III) proved to be more biologically relevant and specific for RCO cell function. The initial response of human osteoblast-like cells to these surfaces was mediated by the collagen (alpha 2 beta 1) and vitronectin receptors (alpha v beta 3), whereas the vitronectin receptor alone dominated longer-term events (> 30 min). MPS II and III surfaces enhanced cell spreading and long-term events such as mineralization of the extracellular matrix compared to homogenous peptide surfaces and controls. Furthermore, extensive mineralization of the ECM deposited by RCOs occurred when the peptide was coupled to an interfacial interpenetrating polymer network (IPN) that resisted protein deposition (i.e., non-specific adsorption) and fouling. Work on thermo-reversible P(NIPAAm-co-AAc) hydrogels demonstrated the ability to create materials that can be delivered to the body in a minimally invasive manner and support tissue regeneration. These hydrogels can be modified to incorporate biofunctional components such as the biomimetic peptides, theoretically enhancing their ability to foster tissue regeneration. These results suggest that biomaterials can be engineered to mimic ECM components of bone (e.g., various organs) by grafting peptides in the appropriate ratios of the cell and heparin-binding domains, and ultimately modulate the expression of the osteoblast cell phenotype. Approaches similar to the one presented in this work can be used to design materials for hybrid artificial organs and other tissues.