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RESEARCH REPORT |
The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, 10833 Le Conte Avenue (B3-081 CHS), Box 951668, Los Angeles, CA 90095-1668, USA;
* corresponding author, tack{at}dent.ucla.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: osseointegration • bone-titanium integration • nano-indentation • dental pulp stem cell • dual-acid-etched surface
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Implant therapy has become a standard tool for dental and maxillofacial restoration and reconstruction. However, the success rates may be affected by various risk factors, including porous host bone and systemic diseases that impair bone metabolism (Stanford, 1999; Fiorellini et al., 2000). Implants are often placed after necessary site development, such as bone augmentation, sinus lifting, and ridge expansion (Rachmiel et al., 2001; Fugazzotto and De, 2002; McCarthy et al., 2003). These bone-grafting procedures require extended healing times for bone to integrate to the titanium surface, and may result in lower implant success rates (Corrente et al., 2000).
Bone marrow stromal stem cells have been selected as an ex vivo bone-engineering tool (Pelled et al., 2002), based on their mineralizing potential and an established in vitro protocol to induce this potential. We postulated that taking advantage of the bone-regenerative potential of dental pulp stem cells (DPSCs), possibly harvested from extracted wisdom teeth or periodontally compromised teeth, could be a novel approach for future implant therapy. Potential applications may include the use of the expanded DPSCs for stem cell transplantation around titanium implants and the development of pre-osseointegrated implants by ex vivo culturing the DPSCs on titanium for accelerated bone anchorage. We hypothesized that dental pulp cells possess the potential to generate mineralized tissue on titanium, and that this potential is modulated by the surface topography of the involved titanium. To test the hypotheses, we investigated the gene expression, elemental composition, tissue surface morphology, and biomechanical properties of tissue cultured from dental pulp cells on titanium with different surface topographies.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture
Dental pulp tissue was extracted from the lower central incisors of eight-week-old male Sprague-Dawley rats. The lower central incisors were carefully pulled out without fracture, to avoid the periodontal tissue flux into the pulp tissue. The periodontal tissue and other soft tissue remnants were removed from the root part of the teeth, and the incisal tip and root parts were then cut to allow for the pulp tissue to be washed out. The extracted tissue was treated with 0.1% collagenase in 0.25% Trypsin-1 mM EDTA-4 Na in 37°C for 15 min. The pellet of released cells centrifuged at 10,000 rpm for 5 min was re-suspended in alpha-modified Eagle’s medium supplemented with 15% fetal bovine serum, 50 µg/mL ascorbic acid, 10 mM Na-ß-glycerophosphate, 10 mM dexamethasone, and antibiotic-antimycotic solution, supplemented with 10,000 units/mL penicillin G sodium, 10,000 mg/mL streptomycin sulfate, and 25 mg/mL amphotericin B antibiotics. The cells were incubated in 100-mm-diameter culture dishes in a humidified atmosphere of 95% air and 5% CO2 at 37°C. When 80% confluent, the cells were detached by treatment with 0.25% Trypsin-1 mM EDTA-4 Na, and seeded in 12-well culture plates at a density of 4 x 104 cells/cm on one of 3 different surfaces: a polystyrene dish, a machined titanium disk, or a DAE titanium disk. The medium was replaced every 3 days. This study protocol was approved by the UCLA Chancellor’s Animal Research Committee. All experiments were performed in accordance with the United States Department of Agriculture guidelines for animal research.
Characterization of Dental Pulp Cells
To characterize the dental-pulp-derived cells, we performed a flow-cytometric analysis for the identification of cell populations, alkaline phosphatase activity assay, and Von Kossa stain for their mineralizing capability. The detailed procedures are described in Appendix 1.
Characterization of Mineralized Tissue Cultured on Titanium
Surface morphology, elemental composition, biomechanical properties, and cellular phenotype of the mineralized tissue cultured on titanium were evaluated by scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), nanoindentation, and reverse-transcriptase polymerase chain-reaction (RT-PCR), respectively. The details are described in Appendix 1.
Statistical Analyses
One-way analysis of variance (ANOVA) was used to evaluate the effects of culture conditions on ALP activity, mineralized nodule area, biomechanical properties, and atomic percentage of the tissue. When appropriate, Bonferroni multiple-comparison testing was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The machined titanium disk showed anisotropic turned ridges (Fig. 1B), whereas the dual-acid-etched (DAE) titanium disk displayed a uniformly rough surface (Fig. 1C). The AFM images exhibited a nano-level roughness of the polystyrene (Fig. 1D), and confirmed a three-dimensionally rougher surface on the DAE titanium surface than on the machined titanium (Figs. 1E, 1F). Average roughness (Ra), maximum peak-to-valley length (Rp-v), and inter-irregularities space (Sm) were 0.002 ± 0.000 µm, 0.027 ± 0.004 µm, and 0.381 ± 0.062 µm, respectively, for the polystyrene, 0.024 ± 0.005 µm, 0.194 ± 0.040 µm, and 0.651 ± 0.201 µm, respectively, for the machined surface, and 0.231 ± 0.051 µm, 1.988 ± 0.454 µm, and 1.163 ± 0.252 µm, respectively, for the DAE surface. All variables were significantly different among the 3 substrates (p < 0.0001, Bonferroni test).
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). No difference was found in the CD44+ percentage with or without dexamethasone in the culture medium. Although some cells were alkaline phosphatase (ALP)-positive in the culture medium without dexamethasone, the ALP-positive cells were significantly greater in number in the medium containing dexamethasone at days 7 and 14 (Fig. 2B). The cells cultured in the absence of dexamethasone exhibited little increase in ALP activity from day 7 to day 14, while the ALP-positive area was increased by 100% in the cells cultured with dexamethasone. Both day 14 cultures with or without dexamethasone were positively stained with Von Kossa (Fig. 2C). However, the mineralized nodule area was appreciably smaller in the medium without dexamethasone than with dexamethasone.
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, 3D, 3G). The tissue on the machined titanium exhibited fibrous structures at day 14 (Fig. 3B), but by day 28, uniform, plate-like structures appeared and predominated (Figs. 3E, 3H), while the appearance of tissue on the DAE titanium was laminar, with few fibrous structures on the surface, from day 14 through day 42 (Figs. 3C, 3F, 3I). The day 42 tissue on DAE titanium displayed fibrous and small globular structures embedded in the lamina (Fig. 3I). Tissue cultures at day 42, regardless of the substrate used, showed elemental peaks of titanium, calcium, and phosphorus (Fig. 3J); the atomic ratio of calcium to phosphorus ranged from 1.39 to 1.41 and did not vary among the 3 cultures (Fig. 3K) (ANOVA, p > 0.05).
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). The day 42 tissue cultured on the machined titanium showed approximately 1.5 times greater hardness than that cultured on the polystyrene dish (Bonferroni, p = 0.0082), while tissue cultured on the DAE titanium showed approximately 3 times greater hardness than that cultured on the polystyrene (p < 0.0001). The elastic modulus of the tissue was also influenced by the culture conditions (ANOVA, p < 0.0001); the tissue on the DAE titanium exhibited the highest value (p < 0.0001) and the polystyrene culture the lowest (p = 0.0027) (Fig. 3M).
Gene Expression
Similarity in the gene expression pattern was observed among collagen I, osteopontin, and osteocalcin (Figs. 4A, 4B). The expression of these genes was up-regulated up to 3 times in the culture on the DAE titanium disks, compared with the polystyrene and machined titanium disks, within the first week of culture through day 28. The culture on the machined titanium exhibited up-regulation of these genes at the later stage of day 42, as compared with the polystyrene and DAE titanium. The highest gene expression of dentin sialoprotein was seen at day 14 in all culture conditions. The expression appeared to be down-regulated in the cultures with titanium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The extracted cell population in the present study was positive for CD44, a surface protein marker for marrow stem cells and DPSCs (Gronthos et al., 2000). We characterized the cells cultured with or without dexamethasone, which is a glucocorticoid class of hormone and is known as an osteoblastic inducer. The DPC phenotype at day 7, showing alkaline phosphatase (ALP) activity even without dexamethasone, may indicate that the cells contained some mature mineralizing cells, suggestively odontoblastic cells. By day 14 of culture, however, the cells exhibited little increase of ALP activity and limited mineralizing capability in the absence of dexamethasone, when the majority of the cells were CD44-positive. With the presence of dexamethasone in the culture medium, the cells showed intense ALP activity and generated robust mineralized nodules. These outcomes indicated that the extracted cell population shows some characteristics of the previously identified dental pulp stem cells, and that the stem cell properties were induced to osteoblastic lineage.
Comparisons of the differentiation mechanisms between DPSCs and bone marrow stromal stem cells (BMSSCs) have been investigated for the identification of their similarities and differences. Although they represent a remarkably similar gene expression profile, DPSC-specific expression of growth factors, such as insulin-like growth factor-2 (IGF-2), suggests that osteogenesis and dentinogenesis mediated by BMSSCs and DPSCs, respectively, may be regulated by distinct mechanisms (Shi et al., 2001; Batouli et al., 2003). In the present study, dentin sialoprotein gene expression, a specific odontoblastic marker (Batouli et al., 2003), diminished in the cultures of dental pulp cells with titanium, while the expression of collagen I, osteopontin, and osteocalcin was increased. Culturing DPCs on titanium may suppress the odontogenic phenotype, but promote the osteogenic phenotype, suggesting that titanium may regulate DPSC differentiation. Titanium and its surface roughness promote the differentiation of bone-marrow-derived osteoblasts and osteoblastic cell lines, and the osteoblastic phenotype in in vivo bone healing (Kieswetter et al., 1996; Mustafa et al., 2001; Ogawa and Nishimura, 2003; Schneider et al., 2003). This study demonstrates that this modulation also occurs in dental pulp cells.
Candidate triggering factors to promote osteogenic differentiation are surface chemistry and surface topography of the substrates. Our previous study (Ogawa et al., 2000) showed that there is no sulfur remnant resulting from the DAE treatment, and there is no elemental difference between the machined and DAE surfaces. However, the electrochemical potential, the degree of oxidation, and the surface energy caused by topographical differences and the atomic direction of titanium, as well as other physical properties, may potentially affect the cellular response (Boyan et al., 1996). Another issue that needs to be addressed in the future is the effect of surface chemistry between the polystyrene and titanium. It has been impossible for titanium culture models to isolate the genuine effect of titanium, because even machined or polished titanium disks have their own topographies that are different from those of the polystyrene dish. The comparison of such cultures between the polystyrene dish and titanium disk involves the cross-effects of titanium, as a material, and its surface topography. The machined and dual-acid-etched surfaces used in this study showed over 10 times and 100 times, respectively, greater average roughness than the polystyrene. It is possible that greater surface roughness of titanium induced the DPC gene modulation, rather than titanium as a material alone. The development of a titanium culture model having a surface topography equivalent to that of the polystyrene dish is needed for better evaluation of the osteogenic potential of titanium per se.
The hardness and elastic modulus of the mineralized tissue generated by dental pulp cells increased when cultured on the titanium surface compared with the polystyrene surface. The DAE titanium surface fostered even higher hardness and elastic modulus values. A previous report of tests on human femoral bone showed that the hardness is 0.25–0.4 GPa, 0.45–0.6 GPa, and 0.55–0.65 GPa for the trabecular, osteon, and interstitial bone tissues, respectively (Hoffler et al., 2000). Normal dentin measures approximately 0.8 GPa, 0.6 GPa, and 0.4 GPa by 200-mN indentation for the normal area, the highly mineralized area close to pulp, and the less-mineralized area in the mid-coronal area, respectively (Kinney et al., 2003). Intertubular and peritubular dentin properties differ: Hardness of peritubular dentin is 2–2.5 GPa, and that of intertubular dentin, 0.1–0.5 GPa (Kinney et al., 1996). Our hardness data of mineralized tissue on the DAE titanium by rat dental pulp cells were consistent with reported human trabecular bone hardness, indicating that the hardness of in vitro-cultured mineralized tissue by dental pulp cells can be enhanced by titanium to the level equivalent to that of in vivo bone tissue.
The greater hardness values in the titanium cultures may be attributed to its enhanced mineralization (Roy et al., 2001). Morphological characterization of the tissue showed the clear distinction with or without titanium in the cultures. Tissue cultured on the polystyrene was characterized by the exposed fibrous and small globular extracellular matrix (ECM) structure observed in SEM images, while tissue cultured on titanium exhibited plate-like morphology, indicating the formation of laminar mineralized tissue. An increased production of calcium-binding matrix, osteopontin, and osteocalcin, resulting from the up-regulated expression, may have contributed to the advancement of mineralized tissue formation. Involvement of collagen architecture and density in determining mechanical properties of bone and teeth has been demonstrated (Hoffler et al., 2000; Kinney et al., 2003). Although collagen synthesis at the protein level and its localization were not studied here, increased collagen deposition by up-regulated collagen I gene expression may have induced the synergetic effect on biomechanical enhancement (Wassen et al., 2000). The calcium-to-phosphorus molar ratio (Ca/P ratio) seemed to be independent of the substrate surface. The Ca/P ratio is 1.55–1.70 and 1.40–1.60 for the vertebrate bone and dentin, respectively (Bloebaum et al., 1997; Mishima and Kozawa, 1998). Little information was available for the Ca/P ratio of the cultured mineralized tissue. Ca/P ratios obtained from the cultured dental pulp cells were approximately 1.40.
We conclude that dental pulp cells have the potential to generate mineralized tissue on titanium, which can be enhanced by titanium surface roughness. This study provides the first step in pursuing tissue-engineered osseointegration using stem cell properties of dental pulp cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received February 18, 2004; Last revision February 22, 2005; Accepted March 22, 2005
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