Abstract:The surface of implantable biomaterials is in direct contact with the host tissue and plays a critical role in determining biocompatibility. In order to improve the integration of implants, it is desirable to control interfacial reactions such that nonspecific adsorption of proteins is minimized and tissue-healing phenomena can be controlled. In this regard, our goal has been do develop a method to functionalize oxidized titanium surfaces by the covalent immobilization of bioactive organic molecules. Titanium first was chemically treated with a mixture of sulfuric acid and hydrogen peroxide to eliminate surface contaminants and to produce a consistent and reproducible titanium oxide surface layer. An intermediary aminoalkylsilane spacer molecule was then covalently linked to the oxide layer, followed by the covalent binding of either alkaline phosphatase or albumin to the free terminal NH 2 groups using glutaraldehyde as a coupling agent. Surface analyses following coating procedures consisted of X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Enzymatic activity of coupled alkaline phosphatase was assayed colorimetrically, and surface coverage by bound albumin was evaluated by SEM visualization of colloidal gold immunolabeling. Our results indicate that the linkage of the aminoalkylsilane to the oxidized surface is stable and that bound proteins such alkaline phosphatase and albumin retain their enzymatic activity and antigenicity, respectively. The density of immunolabeling for albumin suggests that the binding and surface coverage obtained is in excess of what would be expected for inducing biological activity. In conclusion, this method offers the possibility of covalently linking selected molecules with known biological activity to oxidized titanium surfaces in order to guide and promote the tissue healing that occurs during implant integration in bone and soft tissues.
The surface characteristics of biomaterials can influence protein adsorption, cellular functions, and ultimately tissue formation. Controlled chemical oxidation of titanium-based surfaces with a mixture of H(2)SO(4)/H(2)O(2) creates a nanopatterned surface that has been shown to affect early osteogenic events. The objective of this study was to evaluate the effect over time of this nanopattern on various key parameters of osteogenesis, and determine whether these effects ultimately translate into more mineralized matrix production. Osteogenic cells were obtained by enzymatic digestion of newborn rat calvaria and grown on treated and untreated titanium discs for periods of up to 14 days. Alkaline phosphatase activity peaked earlier and cell number was higher as of day 7 on the nanopatterned discs. Immunofluorescence showed that the treated surface favored early bone sialoprotein and osteopontin secretion, and fibronectin accumulation. Alizarin red staining revealed that, at days 10 and 14, there were significantly more mineralized nodules on treated than on untreated discs. These results demonstrate that simple chemical treatment of titanium with H(2)SO(4)/H(2)O(2) accelerates the in vitro osteogenic potential of calvaria-derived cells. They also suggest that this treatment may represent an advantageous approach for producing "intelligent surfaces" that stimulate bone formation and enhance bone-implant contact.
SUMMARYMineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonamelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for amelogenin, but not for ameloblastin, during the early presecretory and midto late maturation stages, during which mRNA signals were detected but no proteins appeared to be secreted. Extracellular amelogenin immunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 m to reach a level slightly above an amount expected if the protein were being deposited evenly across the enamel layer. Immunolabeling for ameloblastin showed an inverse pattern, with relatively more gold particles near secretory surfaces and much fewer deeper into the enamel layer. Administration of brefeldin A and cycloheximide to stop protein secretion revealed that the immunoblotting pattern of amelogenin was relatively stable, whereas ameloblastin broke down rapidly into lower molecular weight fragments. The distance from the cell surface at which immunolabeling for amelogenin stabilized generally corresponded to the point at which that for ameloblastin started to show a net reduction. These data suggest a correlation between the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elongation. A meloblasts , like all hard tissue-forming cells, release an intricate set of extracellular matrix proteins optimized for promoting the development of a closely associated mineral phase (reviewed in Deutsch et al.
The liver is generally considered negative for the vitamin D nuclear receptor (VDR n ), even though several studies have shown significant effects of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) on liver cell physiology. The low abundance of VDR n in the liver led us to propose that hepatocytes (the largest hepatic cell population) were most likely negative for the receptor, whereas the small hepatic sinusoidal and ductular cell populations that contain cell types known to express VDR n in other tissues should express the receptor. Using freshly isolated cells from normal livers as well as biliary and epithelial hepatic cell lines, our data show that the human, rat, and mouse hepatocytes express very low VDR n messenger RNA (mRNA) and protein levels. I t is generally recognized that the liver is largely negative for the vitamin D nuclear receptor (VDR n ), although estrogens have been shown to induce the appearance of the VDR n in male and female rat livers 1 and Sandgren et al. 2 reported the presence of a very low VDR n density in normal rat livers (1,300-fold lower than in intestine). Segura et al. 3 recently reported that fetal, neonatal, and adult rat hepatocytes show nuclear immunoreactivity for the VDR n , although the specificity of the labeling observed was not entirely clear.Experimental evidence indicates that the vitamin D 3 (D 3 ) hormone 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) has selective activity in the liver. Baran et al. 4,5 have shown that 1,25(OH) 2 D 3 increases intracellular Ca 2ϩ in rat hepatocytes, although the mode of action of the hormone in these studies may be due to a membrane receptor rather than the VDR n . 6 Studies performed in our laboratory 7,8 as well as by Whitfield et al. 9,10 indicate that the liver responds to 1,25(OH) 2 D 3 , as indicated by its control of DNA polymerase ␣ activity as well as cytoplasmic and nuclear protein kinases leading to an accelerated regeneration process following two-thirds partial hepatectomy in the rat. The latter observations suggest that 1,25(OH) 2 D 3 has a significant effect on liver cell physiology during the compensatory growth process. In addition, the hormone has been reported as an inhibitor of the D 3 -25 hydroxylase 11,12 and a significant modulator of the gene encoding the mitochondrial D 3 -25/cholesterol/bile acid hydroxylase (CYP27A). 13 However, the hormone mode of action during these experimental paradigms has not been investigated and the presence of the VDR n in liver cells still remains an open question. The absence or extremely low level of VDR n reported to date in liver led us to propose that hepatocytes, which constitute the largest hepatic cell population, were most
In the field of regenerative medicine, nanoscale physical cuing is clearly becoming a compelling determinant of cell behavior. Developing effective methods for making nanostructured surfaces with well-defined physicochemical properties is thus mandatory for the rational design of functional biomaterials. Here, we demonstrate the versatility of simple chemical oxidative patterning to create unique nanotopographical surfaces that influence the behavior of various cell types, modulate the expression of key determinants of cell activity, and offer the potential of harnessing the power of stem cells. These findings promise to lead to a new generation of improved metal implants with intelligent surfaces that can control biological response at the site of healing.
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