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RESEARCH REPORT |
1 Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033; and
2 Tissue Engineering and Bone Biology Lab, Dows Institute for Dental Research, University of Iowa College of Dentistry, Iowa City, IA, USA;
* corresponding author, joldak{at}usc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: biomimetic coating • apatite • amelogenin • osteoblast • titanium implants.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Amelogenins, the predominant protein components of the secretory-stage tooth enamel, have been used to fabricate biomimetic coatings on titanium and bioglass surfaces (Wen et al., 1999; Wen and Moradian-Oldak, 2003). A recombinant amelogenin affected the morphology of apatite crystals grown on titanium and bioglass, resulting in the modulation of surface microtopography. Amelogenins undergo a self-assembly process into nanosphere structures that play an important role in the formation of highly ordered and uniquely elongated apatite mineral in enamel (Fincham et al., 1999; Iijima and Moradian-Oldak, 2004; Du et al., 2005). Furthermore, amelogenins have potential signal transduction functions during tooth or bone development (Veis et al., 2000; Veis, 2003; Tompkins et al 2005), and can promote the adhesion of several cell types, including normal human periodontal ligament cells and the MG-63 human osteoblast-like osteosarcoma cell line (Hoang et al., 2002). A porcine-derived enamel matrix protein mixture, enriched in amelogenins (enamel matrix derivative, EMD), enhanced bone formation (Boyan et al., 2000; Kawana et al., 2001) and osteoblast cell proliferation (Schwarz et al., 2004), and promoted the regeneration of acellular cementum, periodontal ligament, and alveolar bone in treating periodontal defects (Hammarström et al., 1997; Heijl, 1997).
Based on the reported potential signaling effect of amelogenin, and its ability to promote cell adhesion and control apatite crystal morphology and organization, we hypothesized that a biomimetic apatite/amelogenin coating on titanium surfaces would effectively promote osteoblastic differentiation and extracellular matrix production. We proposed that this biomimetic coating would not only improve the implant osseointegration, but also promote bone tissue engineering. We used Human Embryonic Palatal Mesenchymal (HEPM) pre-osteoblasts as a model cell line. We investigated the adhesion, morphology, and osteogenic gene expression of osteoblast precursor cells cultured on apatite/amelogenin coatings, as compared with pure titanium and apatite coating surfaces.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Calcium Phosphate Coating on Titanium
The biomimetic coating was prepared as reported previously (Wen and Moradian-Oldak, 2003). In brief, titanium samples (10 x 10 x 1.3 mm3) (Titanium Industries, Parsippany, NJ, USA, Grade 2, ASTM B265) were ultrasonically cleaned and chemically treated. They were incubated in a supersaturated calcifying solution (136.8 mM NaCl, 3.10 mM CaCl2•2H2O, 1.86 mM K2HPO4, 50 mM Tris-HCl) at 37°C, pH 7.4, for 4 hrs, rinsed with distilled-de-ionized water, and further incubated in another blank or amelogenin-containing (25 to 200 µg/mL) calcifying solution [142.8 mM KNO3, 1.5 mM Ca(NO3)2•4H2O, 0.9 mM K2HPO4] at 37°C for 3 days. The coatings were characterized by means of x-ray diffraction (XRD, Cu K
radiation, 50 kV/70 mA, Rigaku) and scanning electron microscopy (SEM, 15 kV, Cambridge 360).
Amelogenin Incorporation into the Coatings and its Release into Solution
The distribution of protein on the coating surface was detected by FITC fluorescent labeling (Pierce, Rockford, IL, USA). The titanium samples with a blank (as a control) or rM179-containing coating were incubated in 0.5 µg/mL FITC in 2 mL of 100 mM carbonate/bicarbonate buffer (pH 9.0) at room temperature in the dark for 1 hr. The samples were thoroughly washed with de-ionized water for 10 min and observed under a fluorescent microscope. The protein content in the coating was determined quantitatively by bicinchoninic acid (BCA) protein assay (BCATM Protein Assay Kit, Pierce, Rockford, IL, USA). The coatings with or without rM179 were dissolved in 0.1% trifluoroacetic acid (TFA). We developed the colorimetric reaction at 37°C for 3 hrs by mixing 200 µL of sample solution with 1.8 mL of BCA reagent. The calibration curve was established simultaneously, and a linear response (R2 > 0.99) was obtained over the range of 15.6–250 µg/mL rM179 in 0.1% TFA. To characterize the protein release kinetics, we incubated the titanium samples with a blank or amelogenin-containing coating (prepared at 50 µg/mL rM179) in 1.2 mL of distilled water or phosphate-buffered saline (PBS, pH 7.4) at room temperature. A 200-µL aliquot of eluate was removed for measurement after 2, 5, 24, 72, and 90 hrs, respectively. A like volume of fresh solution was refilled at each time point. The eluate sample was mixed with 50 µL 0.1% TFA and stored at 4°C until the measurement. After 90 hrs, the coating was dissolved in 300 µL of 0.1% TFA for release of the retained protein. The amount of both released and retained amelogenin was determined by analytical HPLC (C4-214TP54 column), with 150-µL sample injections. The calibration was established over 8–125 µg/mL rM179 in 0.1% TFA. We incubated the pre-formed blank coating in 142.8 mM KNO3 solution with 50 µg/mL amelogenin for 1 to 3 days to obtain the control sample with superficially adsorbed protein. The protein content and release kinetics were characterized in the same way.
Cell Culture and Immunofluorescent Staining
Human embryonic palatal mesenchymal pre-osteoblast cells (HEPM 1486, ATCC, Manassas, VA, USA) were cultured in Eagle’s minimum essential medium (EMEM) supplemented with Eagle’s salts, L-glutamine (2 mM), non-essential amino acids (0.1 mM), sodium pyruvate (1 mM), 10% fetal bovine serum (to maintain their growth) (Yoneda and Pratt, 1981), and 25 µg/mL penicillin/streptomycin. Cells were cultured (50,000 cells/mL) in triplicate onto different substrates for 24 hrs (50% confluence), fixed in 3.7% formaldehyde for 10 min, rinsed in Tris-buffered saline (TBS), permeabilized for 8 min in 0.5% Triton X-100 in TBS, and rinsed again in TBS. Cells were dual-labeled with rhodamine-phalloidin (Molecular Probes, Eugene, OR, USA) for F-actin (1:400) and for the focal adhesion protein vinculin with the monoclonal antibody 7F9 (kindly provided by Dr. Keith Burridge) undiluted for 45 min at room temperature, rinsed in TBS for 5 min, then rinsed in de-ionized water for 1 min. We then mounted a glass coverslip onto the sample surface using Fluorosave (Calbiochem Corp., San Diego, CA, USA). Surfaces were viewed with an Olympus BX40 microscope equipped with a 40x fluorescence objective.
Real-time RT-PCR
Cells cultured for 72 hrs were washed, and RNA was extracted for real-time reverse-transcription polymerase chain-reaction analysis. Primers and probes were designed for type I collagen, alkaline phosphatase (ALP), and osteocalcin with Primer Express software (Perkin Elmer, Boston, MA, USA) from the known human sequences (Table
). RNA extracts were analyzed in triplicate by quantitative real-time multiplex RT-PCR, with a TaqMan Gold RT-PCR Kit (Perkin Elmer, Foster City, CA, USA). 18s ribosomal RNA (Applied Biosystems Kit 4310893E) was used as an endogenous control. Detailed descriptions of mRNA transcription, target gene amplification, RT-PCR reaction, and analysis of steady-state mRNA levels have been reported previously (Schneider et al., 2003). Steady-state mRNA levels were normalized to 18s rRNA. The mRNA levels on different substrates were further normalized to that on TCP. Statistical analysis of the Ct value, the threshold cycle value of the multiplex reaction of the control vs. experimental target gene (
Ct), was performed by a one-way analysis of variance (ANOVA) with a Tukey’s Multiple Comparison Test or a paired one-tailed t test to a confidence level of P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The superficial adsorption onto the pre-formed blank coating at 50 µg/mL amelogenin resulted in a protein content of 15.8 ± 14.5 µg protein/titanium piece (N = 3), which was significantly lower than the co-precipitated protein content. In the case of the co-precipitated protein, no detectable release was observed when the samples were incubated in PBS. However, a minor release was observed when the sample was incubated in distilled water (Fig. 1B). Less than 10% of total incorporated protein was released within 5 hrs, and no further release was observed thereafter within a 90-hour period (Fig. 1C). In the case of superficially adsorbed protein, no ‘burst’ release was observed (Fig. 1B). In both cases, the majority of the protein was retained in the coating (Fig. 1B).
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2C). The HEPM cells attached and extensively spread over the glass coverslip surface (Fig. 2A). The cells on a commercially pure titanium (cpTi) surface showed a similar pattern (data not shown). On blank apatite coating, the cells were well-attached but remained coalesced, with minimal spreading (Fig. 2B). Similar cellular behavior was observed on the coating prepared at 25 µg/mL amelogenin (data not shown). In contrast, the cells showed intermediate spreading on the coatings prepared at 50 µg/mL amelogenin and higher concentrations, and nodule formation was frequently observed at a concentration greater than 100 µg/mL amelogenin (square in Fig. 2C). Numerous filopodia were observed along the periphery of cells cultured on cpTi (Fig. 2D) or the coating prepared with 50 µg/mL amelogenin (Fig. 2F), as well as on glass. The cells on the blank coating (Fig. 2E) or the coating prepared with 25 µg/mL amelogenin showed fewer filopodia, although they appeared to be well-attached to the surface.
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). The effects appeared to be dose-dependent. The coatings prepared with 25 µg/mL amelogenin showed an effect similar to that on the blank apatite coatings. A maximal enhancement in the expression of type I collagen (about 7.5-fold) occurred on the coatings produced with 50 µg/mL amelogenin. On these coatings, there was a slight but insignificant increase (average, 1.6-fold, with a large SD = 1.1) in the expression of ALP. The increase in the osteocalcin expression prevailed on the coatings produced with 100 µg/mL (seven- to eight-fold) and 200 µg/mL amelogenin (four-fold). Amelogenin added to the culture media had a similar, but less profound, effect on these gene expressions (Fig. 3B). At 50 ng/mL amelogenin, the expression of type I collagen increased to about three- to four-fold, and that of ALP to about 1.5- to 3.5-fold relative to those from the untreated cells. The expressions of these 2 genes at 1000 ng/mL amelogenin were comparable with those at the untreated levels. In contrast, the expressions of osteocalcin were increased at 1000 ng/mL protein (three- to four-fold). Bovine serum albumin (BSA) did not promote the expression of osteocalcin, but had an effect on the expression of type I collagen and ALP (Fig. 3B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The up-regulation of type I collagen, ALP, and osteocalcin gene expressions by rM179 added to the cell culture media indicated that this recombinant amelogenin can promote the osteoblastic differentiation of HEPM cells at both early and late stages in a dose-dependent manner. Although BSA affected the expression of ALP and type I collagen, it did not affect that of the bone-specific marker osteocalcin. We therefore suggest that the up-regulation of osteocalcin in the HEPM cell line is specific to amelogenin. It is not known, however, whether the presence of 10% fetal bovine serum included in the culture medium would inhibit or promote the effects of amelogenin. Two small alternatively spliced mRNA-translated amelogenins ([A+4] and [A-4]) have been reported to enhance the ALP activity from the treated osteoblast precursor C2C12 cells in a dose-dependent manner (Veis, 2003). The rM179 that contains an amino acid sequence similar to that in [A-4] had an active molar concentration similar to that of [A-4] for its effect on the expression of ALP. The general effect of our current biomimetic coating and rM179 is also qualitatively similar to those observations on BMP-2-containing coatings (Liu et al., 2004).
Similar dose-dependent, but more profound, enhancement effects were observed when the cells were cultured on apatite/amelogenin coatings, in contrast to those on TCP, cpTi, and blank apatite coating. The co-precipitation of amelogenin and apatite resulted in higher incorporation of protein into the coating, in contrast to the superficial adsorption on the pre-formed coating. Analysis of protein release kinetics suggested a stable presence of amelogenin on the coating, even after 90 hrs. The profound effects could thus result from amelogenin either released into the culture medium or retained on the coating. Furthermore, the interaction between amelogenin and mineral components during the co-precipitation process modified the crystal morphology and, subsequently, the microtopography of the surface. The blank apatite and the coating prepared with 25 µg/mL amelogenin did not promote the spreading of cells, and neither showed any enhancement on target gene expression. These two coatings have similar surface morphologies (Wen and Moradian-Oldak, 2003). Coatings prepared with 50 µg/mL protein had plate-like crystals, but of a slightly reduced size. At 100–200 µg/mL amelogenin, the coatings consisted of thickened crystals, which are bundles of apatite crystals organized along their c-axis, with an increased curvature. It was likely that these microtopographic alterations had a synergetic contribution to the enhancement effects. The surface roughness or microtopography of a titanium implant has been shown to affect osteoblastic gene expression (Schneider et al., 2003) and pre-osteoblast differentiation (Schneider et al., 2004), as well as the responsiveness of osteoblast-like cells to extrinsic regulatory factor (Boyan et al., 1998).
In summary, the co-precipitation of amelogenins and apatite resulted in a significantly higher incorporation of protein into the coating, in comparison with superficial adsorption. The majority of the protein for which the dose can be controlled remained stable in/on the coating after a minor release. Amelogenin added to the culture media specifically up-regulated the gene expression of osteocalcin by HEPM cells in a dose-dependent manner. The biomimetic apatite/amelogenin coating on titanium had dose-dependent, but more profound, enhancement effects on the bone-related gene expression. The co-precipitation of amelogenin into biomimetic coatings is a promising approach to benefit implant osseointegration or bone tissue engineering.
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ACKNOWLEDGMENTS |
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Received May 20, 2004; Last revision July 14, 2005; Accepted July 24, 2005
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