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Molecular and Biomechanical Characterization of Mineralized Tissue by Dental Pulp Cells on TitaniumThe 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; Correspondence: * corresponding author, tack{at}dent.ucla.edu
The application of implant therapy is still limited, because of various risk factors and the long healing time required for bone-titanium integration. This study explores the potential for osseointegration engineering with dental pulp cells (DPCs) by testing a hypothesis that DPCs generate mineralized tissue on titanium. DPCs extracted from rat incisors positive for CD44, alkaline phosphatase activity, and mineralizing capability were cultured on polystyrene and on machined and dual-acid-etched (DAE) titanium. Tissue cultured on titanium with a Ca/P ratio of 1.4 exhibited plate-like morphology, while that on the polystyrene exhibited fibrous and punctate structures. Tissues cultured on titanium were harder than those on polystyrene, 1.5 times on the machined and 3 times on the DAE. Collagen I, osteopontin, and osteocalcin genes were up-regulated on titanium, especially the DAE surface. In conclusion, DPCs showing some characteristics of the previously identified dental pulp stem cells can generate mineralized tissue on titanium via the osteoblastic phenotype, which can be enhanced by titanium surface roughness.
Key Words: osseointegration • bone-titanium integration • nano-indentation • dental pulp stem cell • dual-acid-etched surface
The dental pulp consists of a population of cells that contain lineage-specific progenitor cells, as well as stem cells with multi-lineage differentiation capabilities (Gronthos et al., 2002; Nakashima et al., 2002). Dental pulp cells cultured in osteogenic-inducing media show phenotypes similar to those of osteoblasts, as represented by the production of collagen type I, osteopontin, and bone sialoprotein, as well as mineralizing capability (Kasugai et al., 1993; Shi et al., 2001). Therefore, dental pulp tissue is expected to be a cellular source for bone tissue repair and engineering (Krebsbach and Robey, 2002); the in vivo bone-regenerative ability of pulp tissue extracted from the primary and wisdom teeth has been demonstrated (Miura et al., 2003). 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.
Titanium Disks and Surface Analysis Two types of commercially pure titanium disks (20 mm in diameter and 1.5mm thick) were fabricated for this study: (1) a disk with a machined surface; and (2) a disk dual-acid-etched (DAE) with H2SO4 and HCl (Osseotite®; Implant Innovations, West Palm Beach, FL, USA). Surface morphology was examined by scanning electron microscopy (SEM) (Stereoscan 250, Cambridge Co., Cambridge, MA, USA) and atomic force microscopy (AFM) (SPM-9500J3, Shimadzu, Tokyo, Japan) as described in Appendix 1.
Cell Culture
Characterization of Dental Pulp Cells
Characterization of Mineralized Tissue Cultured on Titanium
Statistical Analyses
Surface Characteristics of Titanium Disks SEM observation showed that the surface of the polystyrene was smooth and flat, without any structural irregularity (Fig. 1A  ). 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).
Characterization of Dental-pulp-derived Cells The flow-cytometric analysis for the extracted cells showed that approximately 67.5% of cells were CD44-positive at day 1, which increased to approximately 90% in the subcultured cells at day 14 post-extraction (Fig. 2A ). 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.
Tissue Surface Morphology and Atomic Composition SEM images of the cultured tissue on the polystyrene dish showed progressive accumulation of fibrous structure and accretion of small globules between day 14 and day 42 (Figs. 3A , 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).
Biomechanical Property of Mineralized Tissue The tissue hardness varied significantly with the culture conditions (ANOVA, p < 0.0001) (Fig. 3L  ). 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
Osteoblastic gene expression, plate-like structure formation, and the presence of calcium and phosphorus elements indicate successful formation of mineralized tissue by dental pulp cells on titanium. Mineralized tissue formation by cells cultured on titanium exhibiting up-regulated osteoblastic gene expression and enhanced biomechanical properties serves as a basis for pursuing a new way of engineering tissue-titanium integration and further characterization of dental pulp stem cells (DPSCs) for their bone-regenerative potential. 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.
This work was partially supported by 3i Implant Innovations, Inc. and by the Nissenken Institute.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication February 18, 2004. Revision received February 22, 2005. Accepted for publication March 22, 2005.
Journal of Dental Research, Vol. 84, No. 6,
515-520 (2005)
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