Ellen S. Gawalt

Cell attachment and spreading on metal implant materials

Abstract Strong bonding and coverage of organic siloxanes or phosphonates on both Ti and Ti–6Al–4V can be obtained using either of two novel metal–organic interfaces. Surface coverages are considerably higher than can be effected by direct silanization of the native oxide surfaces. Furthermore, these interfaces can be used to attach the fibronectin attachment peptide arginine–glycine–aspartic acid (RGD) to Ti–6Al–4V. Essentially, no osteoblast adhesion occurred on unmodified Ti–6Al–4V or on alkyl, ω-hydroxy, ω-carboxylic acid, or ω-carboxylate-terminated alkylphosphonate-modified surfaces. Adhesion and spreading of osteoblasts on RGD-modified surfaces was quite substantial.

Understanding Organic Film Behavior on Alloy and Metal Oxides

Native oxide surfaces of stainless steel 316L and Nitinol alloys and their constituent metal oxides, namely nickel, chromium, molybdenum, manganese, iron, and titanium, were modified with long chain organic acids to better understand organic film formation. The adhesion and stability of films of octadecylphosphonic acid, octadecylhydroxamic acid, octadecylcarboxylic acid, and octadecylsulfonic acid on these substrates were examined in this study. The films formed on these surfaces were analyzed by diffuse reflectance infrared Fourier transform spectroscopy, contact angle goniometry, atomic force microscopy, and matrix-assisted laser desorption ionization mass spectrometry. The effect of the acidity of the organic moiety and substrate composition on the film characteristics and stability is discussed. Interestingly, on the alloy surfaces, the presence of less reactive metal sites does not inhibit film formation.

Antibody-functionalized peptidic membranes for neutralization of allogeneic skin antigen-presenting cells.

We report herein application of an in situ material strategy to attenuate allograft T cell responses in a skin transplant mouse model. Functionalized peptidic membranes were used to impede trafficking of donor antigen-presenting cells (dAPCs) from skin allografts in recipient mice. Membranes formed by self-assembling peptides (SAPs) presenting antibodies were found to remain underneath grafted skins for up to 6 days. At the host-graft interface, dAPCs were targeted by using a monoclonal antibody that binds to a class II major histocompatibility complex (MHC) molecule (I-A(d)) expressed exclusively by donor cells. Using a novel cell labeling near-infrared nanoemulsion, we found more dAPCs remained in allografts treated with membranes loaded with anti-I-A(d) antibodies than without. In vitro, dAPCs released from skin explants were found adsorbed preferentially on anti-I-A(d) antibody-loaded membranes. Recipient T cells from these mice produced lower concentrations of interferon-gamma cultured ex vivo with donor cells. Taken together, the data indicate that the strategy has the potential to alter the natural course of rejection immune mechanisms in allogeneic transplant models.

Coassembly of amphiphilic peptide EAK16-II with histidinylated analogues and implications for functionalization of β-sheet fibrils in vivo

EAK16-II (AEAEAKAKAEAEAKAK) is one of the first building blocks of environmentally responsive materials. This self-assembling peptide undergoes solution-to-gel transition when transferred from a low to high ionic strength environment. Previously we have demonstrated the histidinylated analogue EAKIIH6 (AEAEAKAKAEAEAKAKHHHHHH) coassembles with the parent peptide to render His-tags as a functionalization mechanism in vitro and in vivo. The present study aimed to understand the pathways by which the analogue coassembles with EAK16-II. The results presented herein suggested two competing but not mutually exclusive events in the coassembly. Atomic force microscopic and gel electrophoretic data showed that EAKIIH6 self-sorted to high molecular weight species without EAK16-II. Self-sorting of EAKIIH6 was inhibited by the parent peptide in a concentration dependent manner. Injecting mixtures containing EAKIIH6 subcutaneously rendered His-tags detectable in live mice for at least 312 h, despite diluting the histidinylated analogue by 10-50 folds compared to a previous formulation. The study provided a formulation by which in vivo display of His-tags was attained without excess amphiphilic peptides. By increasing coassembling efficiency, the likelihood of generating immunogenic aggregates outside the main fibrils could be minimized. These findings provide insights for rational functionalization of in situ self-gelling materials.

Polystyrene Formation on Monolayer-Modified Nitinol Effectively Controls Corrosion

A surface-initiated polymerization of styrene on carboxylic acid terminated phosphonic monolayers was utilized to increase the corrosion resistance of nitinol and nickel oxide surfaces. Alkyl chain ordering, organic reactions, wettability, and film quality of the monolayers and polymers were determined by infrared spectroscopy, atomic force microscopy, matrix-assisted laser desorption ionization spectrometry, and water contact angles. The polystyrene film proved to be a better corrosion barrier than phosphonic acid monolayers by analysis with cyclic voltammetry and electrochemical impedance spectroscopy. The protection efficiency of the polystyrene film on nitinol was 99.4% and the monolayer was 42%.

A novel calcium aluminate‐melatonin scaffold enhances bone regeneration within a calvarial defect

Abstract:  Over 500,000 bone graft or bio‐implant procedures are performed annually in the United States. It has been reported that osseous autograft procurement may result in donor site complications and bio‐implant allografts have been associated with disease transmission. Ceramic scaffolds are only osteoconductive, limiting their clinical use. The objective of this study was to create a bone filler substitute with regenerating properties similar to natural bone. Therefore, melatonin and platelet‐rich plasma (PRP) were utilized for their known osteoinductive properties. It was hypothesized that melatonin and/or PRP would enhance the osteoinductive and osteoconductive properties of calcium aluminate (CA) scaffolds to promote bone regeneration in a model of calvarial defects. The biocompatibility of CA and CA‐Mel scaffolds was tested in vitro and in vivo. Data show that CA‐Mel scaffolds, in comparison with CA scaffolds, enhanced the adhesion, viability, and proliferation of normal human osteoblasts cells but not that of NIH3T3 fibroblasts. Data also showed that human adult mesenchymal stem cells grown on CA or CA‐Mel scaffolds showed a time‐dependent induction into osteoblasts over 14 days revealed through scanning electron microscopy and by alkaline phosphatase analyses. Implantation of CA‐Mel scaffolds into critical size calvarial defects in female, ovariectomized rats showed that the CA‐Mel scaffolds were biocompatible, allowed for tissue infiltration, and showed evidence of scaffold biodegradation by 3 and 6 months. Bone regeneration, assessed using fluorochrome labeling at 3 and 6 months, was greatest in animals implanted with the CA‐Mel scaffold. Overall, results from this study show that CA‐Mel scaffolds were osteoconductive and osteoinductive.

Biofilms, biomaterials, and device-related infections

The chapter “History of Biomaterials” at the start of this book documents the development and design of indwelling materials for medical and dental purposes. The initial design criteria in the choice of materials were pragmatic, and based on the necessary mechanical properties required to fashion a functional device. Orthopedic implants require strong materials for weight-bearing, and articulating surfaces such as joints require durability and resistance to wear. Stents and shunts require flexibility and patency, and sutures require a high tensile strength yet also must be flexible enough for intricate manipulation. As the devices became more sophisticated and developments in materials science provided more options for manufacture, implants are being used more frequently and with longer anticipated lifetimes. Concurrently, the design process increasingly incorporated biocompatibility and comfort into the design criteria. However, with longer lifetimes, the more frequent use of invasive surgical procedures involving indwelling devices and biologically-friendly materials, there has been a rise in the number of incidences of device-related infection. Urinary catheters have been estimated to account for 30% of all nosocomial infections (Gould et al., 2010). Between 66 and 88% of these occur after urinary catheterization (Wong, 1983). It is also reported that almost 100% of catheterized patients develop an infection in an openly draining indwelling catheter which has been in place for four days or more (Wong, 1983). For some procedures, such as orthopedic joint arthroplasties, the diagnosed surgical site infection rates are relatively low (between 1% and 2%; Hsieh et al., 2009); however, the increasing number of patients undergoing joint-replacement surgery translates to large numbers of patients afflicted with the consequences of complicating infections per year. Furthermore, infection of artificial joints can be devastating, since oral or IV antibiotic therapy frequently fails to resolve the infection, with the only remaining course of action being surgical debridement or partial or total revision. These two examples, the first with very high numbers of patients but of lesser severity in terms of impact to the individual, and the second, low numbers but severe patient impact, reflect the incentive to pursue a third design criteria – that of infection resistance – into materials and devices (Maki and Tambyah, 2001). In the following sections we will discuss the role of bacterial biofilms in infection, and the growing literature highlighting biofilms as an important cause of device-related infection

Perfluorocarbon Thin Films and Polymer Brushes on Stainless Steel 316 L for the Control of Interfacial Properties

Perfluorocarbon thin films and polymer brushes were formed on stainless steel 316 L (SS316L) to control the surface properties of the metal oxide. Substrates modified with the films were characterized using diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), contact angle analysis, atomic force microscopy (AFM), and cyclic voltammetry (CV). Perfluorooctadecanoic acid (PFOA) was used to form thin films by self-assembly on the surface of SS316L. Polypentafluorostyrene (PFS) polymer brushes were formed by surface-initiated polymerization using SAMs of 16-phosphonohexadecanoic acid (COOH-PA) as the base. PFOA and PFS were effective in significantly reducing the surface energy and thus the interfacial wetting properties of SS316L. The SS316L control exhibited a surface energy of 38 mN/m compared to PFOA and PFS modifications, which had surface energies of 22 and 24 mN/m, respectively. PFOA thin films were more effective in reducing the surface energy of the SS316L compared to PFS polymer brushes. This is attributed to the ordered PFOA film presenting aligned CF(3) terminal groups. However, PFS polymer brushes were more effective in providing corrosion protection. These low-energy surfaces could be used to provide a hydrophobic barrier that inhibits the corrosion of the SS316L metal oxide surface.

Increased osteoblast adhesion on physically optimized KRSR modified calcium aluminate

Calcium aluminate (CA) is a porous biocompatible material easily cast at room temperature. Through this casting process, the average surface pore size of CA was varied from an average of 100 to 290 microns. The optimal surface pore size of the hydrated CA for cell viability was determined to be 100 microns. Further, a three step-solution deposition technique was developed to covalently immobilize cell adhesion peptides, RGD, and KRSR to the CA surface. Cell adhesion for 1-, 4-, and 7-day time periods was tested with primary osteoblasts and NIH 3T3 fibroblasts. Both peptides were found to increase fibroblast adhesion to the CA surface. However, only KRSR increased osteoblast adhesion to the surface of the CA, which may aid in bone formation after implantation.

Calcium Aluminate, RGD-Modified Calcium Aluminate, and β-Tricalcium Phosphate Implants in a Calvarial Defect

Calcium aluminate (CaAl), arginine-glycine-aspartic acid-modified CaAl, and &bgr;-tricalcium phosphate (TCP) implants were studied in a rat calvarial critical-sized defect model. The rates of newly formed bone and osteointegration were measured using 3 different methods: radiography, micro-computed tomography, and histologic examination. After 4 weeks, there was no new bone formed and no signs of osteointegration into the skull bone in the CaAl or arginine-glycine-aspartic acid-modified CaAl groups, and thick fibrous capsules were visible around the whole circumference of the implants in both groups. In the &bgr;-TCP group, neovascularization of the implant was observed, which is consistent with the early phase of new bone formation. In addition, in the &bgr;-TCP group, signs of implant integration into the host tissue were evident at 4 weeks. There was no soft tissue reaction around the &bgr;-TCP implant. These observations suggest that more specific adhesion peptides may be needed to activate the bioinert CaAl implant and promote bone formation in the craniofacial skeleton.