Skip to main content
NCBI home page
Journal of Dental Research logoLink to Journal of Dental Research
. 2014 Dec;93(12):1290–1295. doi: 10.1177/0022034514547914

Fluorapatite-modified Scaffold on Dental Pulp Stem Cell Mineralization

T Guo 1,2,†, Y Li 1,3,†, G Cao 2, Z Zhang 1, S Chang 1, A Czajka-Jakubowska 4, JE Nör 1, BH Clarkson 1, J Liu 1,*
Editors: Jack L Ferracane, William V Giannobile
PMCID: PMC4462802  PMID: 25139361

Abstract

In previous studies, fluorapatite (FA) crystal-coated surfaces have been shown to stimulate the differentiation and mineralization of human dental pulp stem cells (DPSCs) in two-dimensional cell culture. However, whether the FA surface can recapitulate these properties in three-dimensional culture is still unknown. This study examined the differences in behavior of human DPSCs cultured on electrospun polycaprolactone (PCL) NanoECM nanofibers with or without the FA crystals. Under near-physiologic conditions, the FA crystals were synthesized on the PCL nanofiber scaffolds. The FA crystals were evenly distributed on the scaffolds. DPSCs were cultured on the PCL+FA or the PCL scaffolds for up to 28 days. Scanning electron microscope images showed that DPSCs attached well to both scaffolds after the initial seeding. However, it appeared that more multicellular aggregates formed on the PCL+FA scaffolds. After 14 days, the cell proliferation on the PCL+FA was slower than that on the PCL-only scaffolds. Interestingly, even without any induction of mineralization, from day 7, the upregulation of several pro-osteogenic molecules (dmp1, dspp, runx2, ocn, spp1, col1a1) was detected in cells seeded on the PCL+FA scaffolds. A significant increase in alkaline phosphatase activity was also seen on FA-coated scaffolds compared with the PCL-only scaffolds at days 14 and 21. At the protein level, osteocalcin expression was induced only in the DPSCs on the PCL+FA surfaces at day 21 and then significantly enhanced at day 28. A similar pattern was observed in those specimens stained with Alizarin red and Von Kossa after 21 and 28 days. These data suggest that the incorporation of FA crystals within the three-dimensional PCL nanofiber scaffolds provided a favorable extracellular matrix microenvironment for the growth, differentiation, and mineralization of human DPSCs. This FA-modified PCL nanofiber scaffold shows promising potential for future bone, dental, and orthopedic regenerative applications.

Keywords: odontogenesis, osteogenesis, biomaterial(s), cell differentiation, nanofibers, tissue engineering

Introduction

In dentistry and medicine, regeneration and functional restoration of impaired hard tissues remain a huge challenge. It is widely accepted that stem cells, biomimetic scaffolds, cellular inductive growth, and differentiation factors play crucial roles in these tissue engineering and regeneration processes (Langer and Vacanti, 1993; Ripamonti and Reddi, 1997; Polykandriotis et al., 2010).

Among various mesenchymal stem cell lines, dental pulp stem cells (DPSCs) have been shown to have self-renewal capacity, high proliferative rate, and the multilineage differentiation potential (Gronthos et al., 2000; Zhang et al., 2008). Previous studies have shown that DPSCs exhibited adequate odontogenic/osteogenic differential capabilities on various scaffolds and with different treatments in both in vitro and in vivo studies (d’Aquino et al., 2009; Wang et al., 2010, 2011; Zheng et al., 2011; Qu et al., 2014; Iohara et al., 2014). Thus, DPSCs are attractive candidates for bone and dentin/pulp tissue engineering and regeneration applications. Over the past several years, many three-dimensional scaffolds have been fabricated that have intended to replace the previously used two-dimensional scaffolds. Electrospun polycaprolactone (PCL) nanofiber has enabled the formation of a hierarchical organized mesh structure designed to mimic the extracellular matrix nanotopographic environment (Dong et al., 2009). It is biocompatible and biodegradable and has a high surface-to-volume ratio that has been shown to enhance cell adhesion.

Previously, the inorganic modified PCL fiber scaffolds were created for the growth and differentiation of dental pulp cells (Yang et al., 2010; Kim et al., 2014); however, these scaffolds all require the mineralization inductive supplement for the induction and promotion of the pulp cell differentiation and mineralization. In our previous studies, fluorapatite (FA) crystal surfaces were shown to be biocompatible and biodegradable. Importantly, they are capable of inducing the mineralization of DPSCs, MG-63, and adipose-derived stem cells (ASCs) without adding any inductive promoting supplements (Wang et al., 2012; Liu et al., 2010, 2012). Furthermore, ordered FA was able to enhance the mineralization, in coculture, of ASCs and microvascular endothelial cells (Wang et al., 2014). The odontogenic/osteogenic inducing and promoting effects of these FA crystals strongly support their incorporation as a bioactive component within an appropriate scaffold for hard tissue regeneration applications. Thus, in the present study, we aimed to fabricate a composite scaffold of FA crystal-coated PCL nanofibers and then investigate its effect on human DPSCs proliferation and mineralization.

Materials & Methods

Coating of FA on PCL Nanofiber Surfaces

The NanoECM nanofiber scaffolds were purchased from Nanofiber SOLUTIONS (Columbus, Ohio, USA) and are composed of randomly oriented electrospun PCL nanofibers. These nanofiber scaffolds were fabricated to mimic the three-dimensional nanofibrous extracellular matrix found throughout human tissues. The synthesis and coating of the FA crystals on the PCL nanofiber scaffolds were a modification of the previously described method by Yin et al. (2009). Briefly, for a typical synthesis of FA crystals, the scaffolds were immersed in a solution with 0.10M HEDTA-Ca, 0.06M KH2PO4, and 0.02M KF, incubated at 37°C under ambient pressure condition for 1 day. The final scaffolds with the newly grown FA layer were rinsed with phosphate-buffered saline (GIBCO, Invitrogen, Carlsbad, CA, USA), dried in air, and stored prior to the following experiments.

Cell Culture and Seeding

DPSCs were a gift from Dr. S. Shi (University of Southern California, Los Angeles, CA, USA; Gronthos et al., 2002). The cells were cultured in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO), 100 units/mL of penicillin and 100 mg/mL of streptomycin, under standard culture conditions. The medium was changed every 2 days, and the cells of passages 6 to 9 were used in this investigation.

Before cell seeding, the scaffolds with FA (PCL+FA) or without FA (PCL) were rinsed in phosphate-buffered saline 5 times, then preincubated in the medium at 37°C for 2 hr. DPSCs were seeded on each scaffold at a seeding density of 1 × 105 cells per scaffold. After 2 hr, the medium was replenished with a sufficient volume to cover the scaffolds and changed every 2 days. The cells were cultured for up to 28 days.

Fluorescent Microscopy

After being cultured for 1 day, DPSCs were labeled with CellTracker Green CMFDA (Invitrogen) according to the manufacturer’s protocols and viewed by fluorescent microscopy (Leica DMI3000B).

DNA Quantitation

DNA content of DPSCs growing on PCL and PCL+FA scaffolds for 1, 3, 7, 14, 21, and 28 days was measured with a DNA quantitation kit (Sigma, St. Louis, MO, USA). The DNA quantities were calculated from a standard curve by measuring the fluorescent values emitted at 460 nm after excitation at 360 nm.

Scanning Electron Microscope Observation and Energy Dispersive X-ray Spectroscopy Analysis

After the cell scaffolds were cultured for 7, 14, 21, and 28 days, the samples were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde, and serially dehydrated in 50% to 100% ethanol before placement into desiccators to dry overnight. These specimens were observed under a Philips XL30 FEG scanning electron microscope (SEM; FEI Company) at 10 kV. The elemental contents of the coated FA crystals and the cell-mediated mineralized nodules were analyzed by energy dispersive x-ray spectroscopy (EDX; Phoenix XEDS system).

RNA Isolation and Reverse Transcription

At day 7 and 21, total cellular RNA was extracted from DPSCs grown on PCL and PCL+FA scaffolds through Trizol (Invitrogen) according to the manufacturer’s protocols. Following the SuperScript II Reverse Transcriptase (Invitrogen) manufacturer’s protocols, the total RNA was converted into cDNA.

Real-time Polymerase Chain Reaction

The real-time polymerase chain reaction (PCR) quantitation was performed with a ViiA 7 (Life Tech). The assay-on-demand human gene products were used for the quantitative detection of the gene markers—including dentin matrix protein 1 (dmp1), dentin sialophosphoprotein (dspp), runt-related transcription factor 2 (runx2), osteocalcin (ocn), secreted phosphoprotein 1 (spp1), collagen type I alpha 1 (col1a1), and growth differentiation factor 5 (gdf5). The PCR was performed with the TaqMan Universal PCR Master Mix Kit (Applied Biosystems). The target gene expression was normalized to the housekeeping gene GAPDH. Relative gene expression values were calculated by ΔΔCT-based fold-change calculations.

Alkaline Phosphatase Activity Assay

After culturing for 7, 14, 21, and 28 days, alkaline phosphatase (ALP) activity of the DPSCs was measured with a Senso Lyte pNPP Alkaline Phosphatase Assay Kit (AnaSpec, San Jose, CA, USA) according to the manufacturer’s protocol, and the ALP activity was calculated through the OD405 values obtained from a standard curve and then normalized with its corresponding total protein content.

Alizarin Red Staining and Osteogenesis Quantitative Assay

The DPSC-scaffold constructs cultured for 21 and 28 days were collected for Alizarin red staining according to the manufacturer’s instructions (Osteogenesis Assay Kit, Millipore). The extracted Alizarin red from each stained specimen was then quantified by measuring the OD405 values obtained from a standard curve. The FA-coated scaffolds without cells served as controls.

Von Kossa Staining

The DPSC-scaffold constructs, cultured for 21 and 28 days, were collected for the Von Kossa staining. Briefly, after being rinsed, the specimens were fixed with 95% ethanol for 15 min, serially hydrated into distilled water, stained with 5% silver nitrate for 1 hr in the dark at 37°C, and then exposed to bright light for 30 min for color development. The FA-coated scaffolds without cells served as controls.

Western Blot

Cells cultured at day 21 and 28 were lysed in NP-40 protein lysis buffer, and proteins were separated by 15% SDS-PAGE and transferred electrophoretically to a nitrocellulose membrane. The membrane was incubated with mouse anti-human osteocalcin (OCN) antibody (1:1000) at 4°C overnight and further incubated with secondary antibody anti-mouse IgG (1:10,000) for 2 hr. β-actin was used as a loading control. Relative band densities were measured by determining the ratio of OCN/β-actin according to Image J program (National Institutes of Health).

Statistical Analysis

Statistical analysis of DNA quantification, ALP activities, osteogenesis quantitation, and OCN expression was carried out through one-way analysis of variance and Tukey’s post hoc test with an average of 3 to 5 replicates from each group. Significance was considered at p < .05.

Results

Fabrication of the FA-modified PCL Nanofiber Scaffolds

The SEM images showed that the electrospun PCL nanofiber scaffolds consisted of fibers <700 nm in diameter. The FA crystals were evenly distributed onto the nanofiber surfaces. The FA crystals were approximately 1.6 µm in cross section and 5 µm in length (Fig. 1A).

Figure 1.

Figure 1.

Open in a new tab

Dental pulp stem cell (DPSC) growth on scaffolds. (A) Scanning electron microscope observation of polycaprolactone (PCL) nanofiber scaffold (a) and fluorapatite (FA)–coated PCL scaffold (b-d). The cross-sectional diameter of each FA crystal is approximately 1.6 µm, and the length is approximately 5 µm. (B) Fluorescent microscopy observation of DPSCs grown on PCL and PCL+FA scaffolds for 3 days. At day 3, the DPSCs formed small masses, multicellular aggregates, on the PCL+FA scaffolds but not on the PCL scaffolds. Scale bar: 200 µm. (C) DNA quantitation of DPSCs grown on PCL and PCL+FA scaffolds up to 28 days. Starting from day 14, the DNA amount of DPSCs grown on the PCL+FA scaffolds was significantly lower than that on PCL scaffolds. *p < .05. Triplicate samples from each group were used for the statistical analysis.

DPSC Adhesion and Proliferation on PCL and PCL+FA Scaffolds

The fluorescent images of the DPSCs on day 2 indicated that the cells had attached and spread well on both scaffolds. At day 3, the DPSCs formed small masses, multicellular aggregates, on the PCL+FA scaffolds but not on the PCL scaffolds (Fig. 1B). However, at later time points, the cells covered all scaffolds equally well.

The DNA quantitation data showed continual cell growth on both scaffolds. Starting from day 14, there were significantly fewer cells on the PCL+FA scaffolds than the PCL scaffolds (Fig. 1C).

SEM observation showed that DPSCs attached, flattened, and spread well on both scaffolds. At day 14, the cells grown on the PCL scaffolds covered 90% of the scaffold, whereas the cells grown on the PCL+FA scaffolds were more sparsely distributed (Fig. 2A).

Figure 2.

Figure 2.

Open in a new tab

Observation and analysis of synthesized fluorapatite (FA) crystals and cell-mediated mineral nodules. (A) Scanning electron microscope observation of dental pulp stem cells (DPSCs) grown on polycaprolactone (PCL) and PCL+FA scaffolds for 7, 14, 21 and 28 days. After 21 and 28 days, densely deposited mineral nodules were seen on the PCL+FA scaffolds. (B) Energy dispersive x-ray spectroscopy analysis of synthesized FA crystals (a) and the mineral nodules (b) formed after the DPSCs were grown on PCL+FA scaffolds for 28 d. (C) Ca/P ratio of synthesized FA and the above mineral nodules.

Differentiation and Mineralization of the DPSCs on PCL+FA Scaffolds

After 21 and 28 days, densely deposited mineral nodules were seen on the PCL+FA scaffolds but not on PCL scaffolds. The nodules became more apparent over time (Fig. 2A). The EDX analysis data showed that the Ca/P ratio of the synthesized FA crystals was 1.4 and 1.26 for the cell-mediated mineral nodules formed on PCL+FA scaffolds after 28 days (Fig. 2B, 2C).

At day 7, real-time PCR showed that cells grown on the PCL+FA scaffolds expressed more than twice as much dmp1, dspp, runx2, ocn, spp1, and col1a1, while gdf5 was downregulated more than half compared with cells grown on the PCL scaffolds. Apart from col1a1 expression, similar results of other transcripts were seen on day 21 (Fig. 3).

Figure 3.

Figure 3.

Open in a new tab

Real-time polymerase chain reaction quantification of the expression of odontogenesis/osteogenesis-related molecules of dental pulp stem cells (DPSCs) grown on polycaprolactone (PCL) and PCL + fluorapatite (FA) scaffolds for 7 and 21 days. At day 7, cells grown on the PCL+FA scaffolds expressed more than twice as much dmp1, dspp, runx2, ocn, spp1, and col1a1, while gdf5 was downregulated more than half compared with cells grown on the PCL scaffolds. Apart from col1a1 expression, similar results of other transcripts were seen on day 21.

After culture for 14 and 21 days, ALP activity of the DPSCs grown on PCL+FA scaffolds was significantly higher than that on PCL scaffolds (Fig. 4A).

Figure 4.

Figure 4.

Open in a new tab

Induction and stimulation of dental pulp stem cell (DPSC) differentiation and mineralization by polycaprolactone + fluorapatite (PCL+FA) scaffolds. (A) Alkaline phosphatase (ALP) activity quantitation of DPSCs grown on PCL and PCL+FA scaffolds for 7, 14, 21 and 28 days. ALP activity of the DPSCs grown on PCL+FA scaffolds was significantly higher than that on PCL scaffolds at day 14 and 21. *p < .05. Triplicate samples from each group were used for the statistical analysis. (B) Alizarin red staining of DPSCs grown on PCL and PCL+FA scaffolds for 21 and 28 days. PCL+FA without cells served as control. (C) Von Kossa staining of DPSCs grown on PCL and PCL+FA scaffolds for 21 and 28 days. PCL+FA without cells served as control. (D) Quantitative analysis of Alizarin red staining of DPSCs grown on PCL and PCL+FA scaffolds for 21 and 28 days. The FA-coated scaffolds without cells served as controls. *p < .05. Four scaffolds from each group were tested for statistical analysis. (E) Western blot of osteocalcin (OCN) and β-actin expression of DPSCs grown on PCL and PCL+FA scaffolds for 21 and 28 days. (F) Quantitative analysis of the optical band density of OCN expression by the ratio of OCN/β-actin. *p < .05.

Positive Alizarin red staining and Von Kossa staining were seen only in the DPSCs grown on PCL+FA scaffolds but not on PCL scaffolds. PCL scaffolds and PCL+FA scaffolds without cells showed no Alizarin red staining or Von Kossa staining (Fig. 4B, 4C). The osteogenesis quantitative assay showed that, after 21 and 28 days, the extracted Alizarin red amount was significantly higher when the DPSCs were grown on PCL+FA scaffolds than on PCL scaffolds (Fig. 4D). From day 21 to day 28, cells grown on the PCL+FA showed more obvious OCN expression. No OCN expression was seen in the cells grown on PCL scaffolds at these time points (Fig. 4E, 4F).

Discussion

Previous studies have described the considerable efforts, through tissue engineering techniques, to regenerate bone and the dentin/pulp. These efforts have met with various successes. Therefore, a bioactive scaffold facilitating the odontogenic/osteogenic differentiation and subsequent mineralization would be a promising contribution to the field of hard tissue regeneration.

In this study, the biomimetic and bioactive scaffold has been created by coating the PCL nanofibers with FA crystals, which is odontogenic/osteogenic inductive and mimics the mineralized nanofiber meshwork of the native bone and dentin matrices. It showed the promising potential to treat maxillofacial and alveolar bony defects and, perhaps, to maintain the vitality of the dentin-pulp complex. FA crystals have been shown to be biocompatible and can induce mineralization of several cell lines in two-dimensional culture (Liu et al., 2010, 2012; Wang et al., 2012, 2014). Differences in the conditions for the synthesis of the FA crystals—for example time, temperature, and pressure—will affect their size and shape (Chen et al., 2006; Czajka-Jakubowska et al., 2009; Li et al., 2014).

To mimic the biological, architectural, and mechanical properties of hard tissue matrices, we fabricated a PCL+FA composite scaffold at 37°C under ambient pressure. These novel PCL+FA scaffolds were shown in this study to support the attachment, growth, differentiation, and mineralization of the DPSCs. At day 3, multicellular aggregates formed on the PCL+FA scaffolds but not on the PCL scaffolds. In other studies, these multicellular aggregates were associated with cellular differentiation (Kinney et al., 2014). It has also been reported that once cellular differentiation has started, cellular proliferation slows, which would explain why the cells showed a statistically significantly faster proliferation on the PCL scaffolds, where no multicellular aggregates were seen (Dreesmann et al., 2009). This was further supported by the real-time PCR data, which showed that cells cultured on the PCL+FA scaffolds expressed less gdf5 than the cells cultured on the PCL scaffolds. Similar results were found in previous studies showing that gdf5 was upregulated during cell proliferation but downregulated during differentiation process (Chang et al., 2013).

In the present study, at day 7, the expression of pro-osteogenic/odontogenic molecules (dmp1, dspp, runx2, ocn, spp1, col1a1) was upregulated in the cells grown on the PCL+FA scaffold compared with those on PCL scaffold. This indicates that the PCL+FA scaffold induced the DPSC differentiation toward mineral-forming cells from as early as day 7. Then, at days 14 and 21, without any mineralization induction, significantly increased ALP activity was observed on the PCL+FA scaffolds compared to the PCL scaffolds. This was consistent with the downregulation of gdf5 expression, since gdf5 had been reported to inhibit the ALP activity of dental pulp cells (Chang et al., 2013). At day 21, most of the pro-osteogenic/odontogenic molecules were still upregulated in DPSCs grown on PCL+FA scaffolds. Unsurprising, at the protein level, the OCN expression was also induced from DPSCs grown on the PCL+FA scaffolds but not on PCL scaffolds. Consistently, this elevated pro-osteogenic/odontogenic molecule and protein expression was coordinated with obvious Alizarin red and Von Kossa staining, which was seen in the cells grown on PCL+FA scaffolds but not on the PCL scaffolds or the PCL+FA scaffolds without cells. This was further confirmed by the osteogenesis quantitative assay along with mineralized nodule formation observed under the SEM. These data identified acceleration in mineral formation from that previously reported from our two-dimensional studies, as the mineralized nodules and Alizarin red staining were seen after 4 wk in two-dimensional culture, compared with 3 wk in three-dimensional culture. As expected, the late-stage mineralization marker, OCN, was expressed at 3 wk when the cells were grown on this PCL+FA scaffolds, whereas in our two-dimensional culture studies, OCN was expressed at 4 wk (Liu et al., 2010, 2012; Wang et al., 2012, 2014). The effect of the FA crystals was also seen in the EDX analysis data, which identified the mineral nodules formed on the PCL+FA surfaces as apatite-like structures.

All these data provided important evidence that DPSCs were induced to differentiate into mineral-forming cells and start forming apatite-like structures by the PCL+FA scaffolds, without any differentiating supplement. It has been suggested that the intrinsic properties of FA crystals could have favored the recruitment of pro-osteogenic growth factors from the culture media to create an osteoinductive microenvironment (Lin et al., 2009) for the DPSCs. Yet, the FA could have also interacted with the DPSCs by generating potent inductive factors, which in turn induced the osteogenic differentiation of these cells (Liu et al., 2010). In our most recent studies, the osteogenic differentiation of ASCs stimulated by the FA surfaces has been shown to be mediated through the FGF and VEGF signaling pathways (Clark et al., 2014; Wang et al., 2014). The three-dimensional microenvironment and FA crystals may act synergistically in inducing and promoting this DPSC differentiation and mineralization process. Therefore, compared to other inorganic modified scaffolds (Yang et al., 2010; Antebi et al., 2013; Kim et al., 2014), this FA+PCL composite scaffold provides us a new biomimetic material, which contains similar crystalline structures as dentin and bone and induces and promotes odontogenic/osteogenic differentiation of mesenchymal stem cells.

For the first time, our study shows that an FA-coated PCL nanofiber three-dimensional scaffold supports the growth, differentiation, and mineralization of DPSCs to a greater degree than two-dimensional FA surfaces. This PCL+FA scaffold serves as an excellent in vitro model for studying the mechanisms underlying the stem cell differentiation and mineralization process in a biomimetic three-dimensional microenvironment. Most important, this FA-modified PCL nanofiber construct is a new biomimetic material that has the promising potential to be used as an odontogenic/osteogenic scaffold in tissue engineering regeneration of bone and dentin/pulp.

Acknowledgments

The authors thank Dr. Haifeng Chen (College of Engineering, Peking University, China) for his technical guidance on fluorapatite synthesis at near-physiologic conditions.

Footnotes

This work was supported by the Department of Cariology, Restorative Sciences, and Endodontics at the University of Michigan, School of Dentistry.

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

References

  1. Antebi B, Cheng X, Harris JN, Gower LB, Chen XD, Ling J. (2013). Biomimetic collagen-hydroxyapatite composite fabricated via a novel perfusion-flow mineralization technique. Tissue Eng Part C Methods 19:487-496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chang MC, Lin LD, Tseng HC, Chang BE, Chan CP, Lee SY, et al. (2013). Growth and differentiation factor-5 regulates the growth and differentiation of human dental pulp cells. J Endod 39:1272-1277. [DOI] [PubMed] [Google Scholar]
  3. Chen H, Sun K, Tang Z, Law RV, Mansfield JF, Clarkson BH. (2006). Synthesis of fluorapatite nanorods and nanowires by direct precipitation from solution. Cryst Growth Des 6:1504-1508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clark D, Wang X, Chang S, Czajka-Jakubowska A, Clarkson BH, Liu J. (2014). VEGF promotes osteogenic differentiation of ASCs on ordered fluorapatite surfaces. J Biomed Mater Res A [Epub ahead of print 5/5/2014]. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Czajka-Jakubowska AE, Liu J, Chang SR, Clarkson BH. (2009). The effect of the surface characteristics of various substrates on fluorapatite crystal growth, alignment, and spatial orientation. Med Sci Monit 15:MT84-88. [PubMed] [Google Scholar]
  6. d’Aquino R, De Rosa A, Lanza V, Tirino V, Laino L, Graziano A, et al. (2009). Human mandible bone defect repair by the grafting of dental pulp stem/progenitor cells and collagen sponge biocomplexes. Eur Cell Mater 18:75-83. [DOI] [PubMed] [Google Scholar]
  7. Dong Y, Liao S, Ngiam M, Chan CK, Ramakrishna S. (2009). Degradation behaviors of electrospun resorbable polyester nanofibers. Tissue Eng Part B Rev 15:333-351. [DOI] [PubMed] [Google Scholar]
  8. Dreesmann L, Mittnacht U, Lietz M, Schlosshauer B. (2009). Nerve fibroblast impact on Schwann cell behavior. Eur J Cell Biol 88:285-300. [DOI] [PubMed] [Google Scholar]
  9. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 97:13625-13630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. (2002). Stem cell properties of human dental pulp stem cells. J Dent Res 81:531-535. [DOI] [PubMed] [Google Scholar]
  11. Iohara K, Murakami M, Nakata K, Nakashima M. (2014). Age-dependent decline in dental pulp regeneration after pulpectomy in dogs. Exp Gerontol 52:39-45. [DOI] [PubMed] [Google Scholar]
  12. Kim JJ, Bae WJ, Kim JM, Kim JJ, Lee EJ, Kim HW, et al. (2014). Mineralized polycaprolactone nanofibrous matrix for odontogenesis of human dental pulp cells. J Biomater Appl 28:1069-1078. [DOI] [PubMed] [Google Scholar]
  13. Kinney MA, Hookway TA, Wang Y, McDevitt TC. (2014). Engineering three-dimensional stem cell morphogenesis for the development of tissue models and scalable regenerative therapeutics. Ann Biomed Eng 42:352-367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Langer R, Vacanti JP. (1993). Tissue engineering. Science 260:920-926. [DOI] [PubMed] [Google Scholar]
  15. Li X, Zhu J, Man Z, Ao Y, Chen H. (2014). Investigation on the structure and upconversion fluorescence of Yb (3+)/Ho (3+) co-doped fluorapatite crystals for potential biomedical applications. Sci Rep 4:4446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lin L, Chow KL, Leng Y. (2009). Study of hydroxyapatite osteoinductivity with an osteogenic differentiation of mesenchymal stem cells. J Biomed Mater Res A 89:326-335. [DOI] [PubMed] [Google Scholar]
  17. Liu J, Jin T, Chang S, Czajka-Jakubowska A, Zhang Z, Nör JE, et al. (2010). The effect of novel fluorapatite surfaces on osteoblast-like cell adhesion, growth, and mineralization. Tissue Eng Part A 16:2977-2986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liu J, Wang X, Jin Q, Jin T, Chang S, Zhang Z, et al. (2012). The stimulation of adipose-derived stem cell differentiation and mineralization by ordered rod-like fluorapatite coatings. Biomaterials 33:5036-5046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Polykandriotis E, Popescu LM, Horch RE. (2010). Regenerative medicine: then and now—an update of recent history into future possibilities. J Cell Mol Med 14:2350-2358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Qu T, Jing J, Jiang Y, Taylor RJ, Feng JQ, Geiger B, et al. (2014). Magnesium-containing nanostructured hybrid scaffolds for enhanced dentin regeneration. Tissue Eng Part A [Epub ahead of print 4/3/2014]. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ripamonti U, Reddi AH. (1997). Tissue engineering, morphogenesis, and regeneration of the periodontal tissues by bone morphogenetic proteins. Crit Rev Oral Biol Med 8:154-163. [DOI] [PubMed] [Google Scholar]
  22. Wang J, Liu X, Jin X, Ma H, Hu J, Ni L, et al. (2010). The odontogenic differentiation of human dental pulp stem cells on nanofibrous poly (L-lactic acid) scaffolds in vitro and in vivo. Acta Biomater 6:3856-3863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang J, Ma H, Jin X, Hu J, Liu X, Ni L, et al. (2011). The effect of scaffold architecture on odontogenic differentiation of human dental pulp stem cells. Biomaterials 32:7822-7830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wang X, Jin T, Chang S, Zhang Z, Czajka-Jakubowska A, Nör JE, et al. (2012). In vitro differentiation and mineralization of dental pulp stem cells on enamel-like fluorapatite surfaces. Tissue Eng Part C Methods 18:821-830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wang X, Zhang Z, Chang S, Czajka-Jakubowska A, Nör JE, Clarkson BH, et al. (2014). Fluorapatite enhances mineralization of mesenchymal/endothelial cocultures. Tissue Eng Part A 20:12-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yang X, Yang F, Walboomers XF, Bian Z, Fan M, Jansen JA. (2010). The performance of dental pulp stem cells on nanofibrous PCL/gelatin/nHA scaffolds. J Biomed Mater Res A 93:247-257. [DOI] [PubMed] [Google Scholar]
  27. Yin Y, Yun S, Fang J, Chen H. (2009). Chemical regeneration of human tooth enamel under near-physiological conditions. Chem Commun (Camb) 39:5892-5894. [DOI] [PubMed] [Google Scholar]
  28. Zhang W, Walboomers XF, Van Kuppevelt TH, Daamen WF, Van Damme PA, Bian Z, et al. (2008). In vivo evaluation of human dental pulp stem cells differentiated towards multiple lineages. J Tissue Eng Regen Med 2:117-125. [DOI] [PubMed] [Google Scholar]
  29. Zheng L, Yang F, Shen H, Hu X, Mochizuki C, Sato M, et al. (2011). The effect of composition of calcium phosphate composite scaffolds on the formation of tooth tissue from human dental pulp stem cells. Biomaterials 32:7053-7059. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Dental Research are provided here courtesy of International and American Associations for Dental Research

RESOURCES