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A First Step in De Novo Synthesis of a Living Pulp Tissue Replacement Using Dental Pulp MSCs and Tissue Growth Factors, Encapsulated within a Bioinspired Alginate Hydrogel Manasi Bhoj, BDS,* Chengfei Zhang, DDS, PhD,† and David W. Green, BSc, PhD* Abstract Introduction: A living, self-supporting pulp tissue replacement in vitro and for transplantation is an attractive yet unmet bioengineering challenge. Our aim is to create 3-dimensional alginate-based microenvironments that replicate the shape of gutta-percha and comprise key elements for the proliferation of progenitor cells and the release of growth factors. Methods: An RGD-bearing alginate framework was used to encapsulate dental pulp stem cells and human umbilical vein endothelial cells in a ratio of 1:1. The alginate hydrogel also retained and delivered 2 key growth factors, vascular endothelial growth factor-121 and fibroblast growth factor, in a sufficient amount to induce proliferation. A method was then devised to replicate the shape of gutta-percha using RGD alginate within a custommade mold of thermoresponsive N-isopropylacrylamide. Plugs of alginate containing different permutations of growth factor–based encapsulates were tested and evaluated for viability, proliferation, and release kinetics between 1 and 14 days. Results: According to scanning electron microscopic and confocal microscopic observations, the encapsulated human endothelial cells and dental pulp stem cell distribution were frequent and extensive throughout the length of the construct. There were also high levels of viability in all test environments. Furthermore, cell proliferation was higher in the growth factor–based groups. Growth factor release kinetics also showed significant differences between them. Interestingly, the combination of vascular endothelial growth factor and fibroblast growth factor synergize to significantly up-regulate cell proliferation. Conclusions: RGD-alginate scaffolds can be fabricated into shapes to fill the pulp space by simple templating. The addition of dual growth factors to cocultures of stem cells within RGD-alginate scaffolds led to the creation of microenvironments that significantly enhance the proliferation of
dental pulp stem cell/human umbilical vein endothelial cell combinations. (J Endod 2015;-:1–8)
Key Words Dental pulp stem cells, fibroblastic growth factor, human umbilical vein endothelial cells, RGD alginate, vascular endothelial growth factor
R
oot canal therapy is currently the preferred treatment for irreversible pulpitis wherein pulp is completely excavated and the root canal is prepared, debrided, and later obturated with gutta-percha (1, 2). What makes the prognosis for root canal treatment questionable are complications such as bacterial metabolite leakage (3), failure to achieve a proper seal (4), fracture upon application of force, and loss of vitality (5). Efforts toward endodontic regeneration have involved implementation of the latest tissue engineering strategies, including root revascularization by forced induction of blood clots, use of postnatal stem cell therapy (6), pulp implantation, injectable biochemically active cell-free scaffolds, 3-dimensional (3D) cell printing, and gene therapy (7). The greatest challenge of tissue engineering the ‘‘pulp’’ is to achieve in vivo revascularization from the host blood supply (8). The other barriers to progress in regenerative endodontics are dentinogenesis and maintaining the afferent nerve supply. Emphasis must be placed on replicating the pulp’s intricate zonal structure with their functions, namely mesenchymal stem cell self-renewal, odontogenic repair (formation of secondary dentin), and sensory and blood vessel apparatus (9). Bioengineering of the pulp requires a judicious selection and combination of stem cells and morphogens delivered over time in convoluted spatial patterns within a supporting framework. So far, the optimal permutation of factors has not been determined (10). A variety of dental stem cells have been implemented in pulp bioengineering strategies such as dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth, stem cells from apical papilla, and periodontal ligament stem cells. DPSCs exhibit strong, stable proliferation and self-renewal properties, directly differentiate along the odontoblast lineage, and are the most common dental stem cell used in pulp tissue engineering (11). A key process in early tissue morphogenesis is the interplay between stem cells and vascular endothelium. In a dental context, it has been shown that the coculture of postnatal DPSCs with endothelial cells (ECs) offer superior regenerative potential than when used individually (12). Other than the cellular and
From the *Oral Biosciences and †Comprehensive Dental Care, Faculty of Dentistry, The University of Hong Kong Hospital, Sai Ying Pun, Hong Kong. Address requests for reprints to Dr David W. Green, Oral Biosciences; or Dr Chengfei Zhang, Comprehensive Dental Care, Faculty of Dentistry, The University of Hong Kong Hospital, Floor 3A, 34 Hospital Road, Sai Ying Pun, Hong Kong. E-mail address: [email protected] or [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.03.006
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Basic Research—Biology TABLE 1. Various Test Groups of Pulp Construct Encapsulate
Quantity (mL)
DPSCs DPSCs:HUVECs DPSCs:HUVECs+VEGF DPSCs:HUVECs+VEGF: FGF (1:3)
106 cells 106 cells in ratio of 1:1 106 cells in 1:1 ratio of 50 ng VEGF 106 cells 1:1 ratio in 50 ng VEGF + 150 ng FGF in 1:3 ratio
DPSCs, dental pulp stem cells; FGF, fibroblast growth factor; HUVECs, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor.
structural environments, the biochemical composition is important in driving tissue production by cells. Tissue morphogens are small proteins inducing an array of cellular activities even at low concentrations in picogram quantities (10). Bone morphogenetic protein, vascular endothelial growth factor (VEGF), fibroblastic growth factor (FGF-2), and transforming growth factor are the principle growth factors that are being used frequently in conjunction with dental stem cells to induce various tissue structures (9). Cumulative evidence showed that VEGF and FGF in a pulp context specifically lead to angiogenesis, whereas bone morphogenetic protein is proposed to induce dentinogenesis at the pulp periphery (13–15). All dental cells require a 3D framework to provide safe anchorage and spatial organization and to be used as modules for transplantation to the desired site of reconstruction. Various synthetic and natural scaffold materials are available to build structural environments that mimic natural tissue frameworks, but accurate matching of the biology and biodegradation characteristics to a tissue exists as the major area of difficulty in optimizing scaffold material. Recent documented attempts indicated the formation of dentinlike precursor tissue, with the use of gelatin hydrogels, collagen, peptide-based gels, and polyglycolic acid (PGA) at the boundary within existing dentin (16, 17). Other tissue engineering biomaterials such as alginate have been virtually absent from pulp bioengineering research even though it is suitable for this context. Alginate, which is derived from marine seaweed, is widely available at relatively low costs and is used broadly in experimental tissue engineering as well as by food and drug manufacturers and has Food and Drug Administration approval (18). Continuous efforts are needed to engineer a scaffold that can properly self-organize and support the expansion and differentiation of stem cells into naturally simulated pulp, especially once it is implanted in the root canal. To start tackling these design flaws, we have engineered a morphologically accurate pulp replacement from stiffened alginate, which express internal cellular and molecular environments necessary for beginning pulp tissue reconstruction. The ultimate purpose of such biomimetic designs for pulp microenvironments is to eliminate the shortcomings of current pulp regeneration strategies by building an endothelial tubule network (protovasculature) that is the foundation for tissues. The presence of these structures guarantees a connection with the host blood supply after implantation. Such a strategy is critical to revitalizing and strengthening the existing diseased tooth structures.
Figure 1. A schematic of the procedure used to fabricate cell-seeded alginate pulp replacement frameworks. Step 1: cultivation and expansion of human cells. Steps 2 and 3: mixing of cells and growth factors in RGD-alginate solution. Steps 4, 5, and 6: fabrication of temperature-responsive sockets and molds. Step 7: (A) extruded pulp constructs, (B) confocal image, and (C) SEM. Step 8: prospective clinical end point filling of pulp cavity.
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Materials and Methods Cell Sources and Extraction Procedure Human umbilical vascular endothelial cells (HUVECs) were obtained commercially (ScienCell Research Laboratories, San Diego, CA) and cultured in endothelial cell medium (ECM) (ScienCell Research Laboratories Cat. No. 1001) at a seeding density of 5000
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Basic Research—Biology cells/cm2 at 37 C with 5% CO2. DPSCs were isolated from extracted human premolars by combining elements from 2 established protocols described by Yildirim (19). First, the enzyme digestion method was followed by adding collagenase type I (3 mg/mL) (Gibco-Invitrogen, Carlsbad, CA) and dispase (4 mg/mL) (Gibco-Invitrogen) to the freshly extirpated pulp tissue from sound permanent human tooth (under 18 years) and maintained in the incubator for 30 minutes, vortexed for 30 seconds, and centrifuged for 5 minutes at 1200 rpm. The supernatant fraction was discarded, and fresh medium was added to the remaining pellet, which was then thoroughly vortexed to produce a new
cell suspension. Normally, at this stage, the suspension is passed through a cell strainer. However, the suspension was directly cultured in a 75-cm2; culture flask to facilitate outgrowth of the DPSC population from the emulsified pulpal fragments.
Mold Fabrication for Producing the 3D RGD-alginate Pulp Plug Replacement A mold with specially imprinted sockets was fabricated with a thermoresponsive polymer, poly-N-isopropylacrylamide (pNIPAAm), using
Figure 2. (A) Confocal fluorescence images in the fluorescein isothiocyanate (FITC) channel showing DPSC and HUVEC viability inside the RGD-alginate framework over 14 days at 20 magnification. (B) Confocal fluorescence images in the FITC channel showing DPSC/HUVEC coculture viability inside a whole pulp plug alginate construct at 4 magnification.
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Basic Research—Biology imprints made by gutta-percha points ISO40 (Dentsply, Ballaigues, Switzerland). The entire experiment was performed on ice. In brief, following the procedure of Sasaki et al (20), polyethylene glycol (PEG) dimethacrylate (Sigma-Aldrich 437468) 613 mg and 1.58 g NIPAAm (Sigma-Aldrich 731129) (14 mmol crystal powder) were added into 19 mL deionized water. The reaction bottle was kept on ice, whereas nitrogen gas was aspirated into a monomer mixture. A polymerization starter mixture was prepared by adding ammonium persulfate (APS) (02602-02; Nakalai USA, San Diego, CA) 32 mg in 1 mL distilled water and thoroughly mixed. This starter solution was transferred into NIPAAm solution, and then 20 mL tetramethylethylenediamine (TEMED) (330401-72, Nakalai) was added while constantly stirring and keeping on ice. At the end, this solution was poured into a beaker with gutta-percha suspended upright in the mixture (Fig. 1A–C). After overnight gelation, the gutta-percha points were removed, and the mold was thoroughly washed in deionized water and then soaked in ethanol solution for 30 minutes to remove all unreacted cytotoxic monomers.
Preparation of Plug-shaped Cell-seeded Alginate Matrix Environments Four individual groups of RGD-alginate materials (NovaMatrix, Drammen, Norway) were prepared with different encapsulates and fabricated into pulp shape using the templating method described previously. The individual encapsulates used were DPSCs, HUVECs, VEGF121 (Human VEGF-121/VEGF-A Protein; Sino Biological Inc, Beijing, China), and FGF-2 (Recombinant Human bFGF/FGF-2 Protein, Sino Biological Inc). The first group of plugs consisted of DPSCs; the second group consisted of a combination of DPSCs and HUVECs (coculture 1:1) (12); the third group consisted of DPSCs, HUVECs, and VEGF; and the fourth group was composed of DPSC/HUVEC coculture and VEGF and FGF-2 (1:3) (Table 1). To achieve the reported synergistic effect between the 2 growth factors within the alginate gel, VEGF was added at a concentration of 50 ng/mL and 150 ng/mL for basic fibroblast growth factor (bFGF) (21). Each of the alginate encapsulate compositions were loaded into the prepared pNIPAAM sockets and immersed in alpha-Minimum Essential Media (a-MEM) with 50 mmol/L CaCl2 solution for 20 minutes. During this incubation period, the alginate constructs hardened into a stiff gel and were gently handled with forceps and placed into a 12-well plate containing aMEM, fetal bovine serum (FBS), and penicillin/streptomycin solution for 14 days with a change in media every other day. Cell Viability within RGD-alginate Environments Cell viability in the 4 composition groups of alginate scaffolds were evaluated on days 1, 7, and 14 using the LIVE/DEAD Cell Staining Kit (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions (22, 23). The live cells (green fluorescence) and necrotic cells (red fluorescence) were examined using a confocal fluorescence microscope (FluoView FV1000; Olympus, Tokyo, Japan) and analyzed using bioImageL V2 software (Faculty of Odontology, Malm€o University, Malm€o, Sweden). Depolymerization and Retrieval of Cells Embedded in RGD-alginate Constructs The constructs were suspended in phosphate-buffered saline containing 55 mmol/L trisodium citrate (Sigma-Aldrich, St. Louis, MO) and 150 mmol/L sodium chloride (Sigma-Aldrich) for 30 minutes in an incubator to cause degelling and the release of cells from the alginate scaffold. The cells were recovered by centrifugation at 1200 rpm for 5 minutes (24). 4
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Cell Proliferation Quantification in Growth Factor–infused Scaffolds Cell proliferation was measured using the standard MTT assay kit (M5655 and M8910, Sigma-Aldrich). In the first instance, the cells were released from the scaffold using a specially prepared depolymerization solution as mentioned previously. The experiment was run in triplicates and averaged from 4 different samples and a blank group at 1-, 7-, and 14-day time points as for the viability assays (25). Scanning Electron Microscopy The morphology and internal structure of alginate scaffolds were observed and imaged by scanning electron microscopy (SUI510; Hitachi, Tokyo, Japan). Scanning electron microscopy (SEM) was also used to observe cell morphology. The control (scaffold with no cells) and treatment scaffold groups (combinations of cells and factors) were fixed overnight in 4% v/v formalin containing 100 mmol/L CaCl2 followed by ethanol submersion (26) for 2 hours and then attached to sample stubs with conductive paint and sputter coated with gold and palladium in the MPS-2S Magnetron Ion Sputter Device (IXRF Systems, Austin, TX). The samples were viewed under SEM at an accelerating voltage of 15 kV. VEGF-121 and FGF-2 Release and Retention Kinetics from Alginate Scaffold Each of the 4 plug types by composition (n = 3 for each of 4 study groups) were placed in 6-well plates (each construct having a volume of 150 mL) and incubated in culture medium at 37 C (27). The concentration of VEGF used was 50 ng/mL, and for FGF-2 it was 150 ng/mL. Later, 1.0 mL solution from each well was collected in an Eppendorf tube at time points 1, 3, 5, 7, 9, 11, and 13 days and replaced with fresh medium and preserved at 20 C. The Human VEGF Quantikine ELISA Kit (DVE00) and Human FGF basic Quantikine HS ELISA Kit were used according to the manufacturer’s protocol. Average values below 0 were considered to be nondetectable. A cumulative release profile was assessed by total FGF and VEGF secreted during each time point from within the constructs (sum of FGF and VEGF released over the study until the last time point). The actual release profile was determined by the slope of each cumulative release of each group over the given time period. The detection limit for VEGF was 9 pg/mL and 0.07 pg/mL for bFGF. VEGF and FGF2 in the samples were determined by interpolation from the standard curve (28).
Figure 3. Proliferation levels of DPSCs and HUVECs in different alginate environments on days 1, 7, and 14.
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Basic Research—Biology Statistical Analysis IBM SPSS (Armonk, NY) statistics 20.0 software was used for data analysis with 1-way analysis of variance and Bonferroni multiple comparisons for analysis at a confidence of 95% with P > .05. Data points and error bars in the graphs represent the mean value and standard error calculated from different experiments.
the growth factor–treated alginate environments within 24 hours, but it was not sustained for 14 days. The DPSCs and HUVECs were viable throughout the entire pulp plug construct from the coronal to the apical end (Fig. 2B). Significant increases can be seen in the cell densities on day 7 compared with day 1. However, on day 14, few dead cells were seen within the construct and viable cells clustered together, which is a typical first stage of prototissue development inside alginate gels.
Results Cell Viability in Alginate Environments Encapsulated DPSC and HUVEC cocultures were viable throughout the 14-day culture period assessed by live-dead staining (Fig. 2A). However, there were noticeable differences in viability between the experimental groups. In particular, the number of viable cells increased in all
Cell Proliferation in Alginate Environments Significant differences in cell proliferation were measured between all the experimental groups. Statistical analysis was performed on the quantitative cell counts on 1, 7, and 14 days using 1-way analysis of variance (P < .05). Bonferroni multiple
Figure 4. DPSCs and HUVECs embedded within an RGD-alginate scaffold on day 1. Comparing alginate (left) with no cells and (right) with cells after 24 hours at different magnifications.
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Figure 5. The release properties of VEGF from different alginate environments until day 13. (A) The cumulative profile of VEGF released from alginate plugs. (B) The actual release on 1, 3, 5, 7, 9, 11, and 13 days.
comparisons showed that on day 1 there was no significant difference in cell numbers between the DPSC and DPSC + HUVEC groups (Fig. 3). However, on day 7, there was a significant increase in the DPSC + HUVEC group compared with the DPSC-alone group. This meant that the coculture of DPSCs and HUVECs increased the rate of cell proliferation between days 1 and 7 compared with DPSConly alginate (Fig. 3). The plugs containing growth factors also induced higher multiplication rates compared with the DPSC-only control. However, on day 14, there was no significant difference in proliferation between all the experimental groups. Clearly, the growth factors induced proliferation within the first 7 days, but their effect dissipated by day 14 (Fig. 3).
SEM Under SEM, DPSCs and HUVECs are shown fixed into the scaffold material and are spherical in shape shown early in cultivation. The distribution of cells is uniform along the whole length and depth of the construct, also reaching the apical tip. The homogenous cell distribu-
tion is needed to establish functional living tissue along the whole length of the root canal, which guarantees long-term survival (Fig. 4).
Cumulative Growth Factor Release from Alginate Constructs The temporal pattern of VEGF released from alginate scaffolds was typical of the release curve for small proteins entrapped within a hydrogel (Fig. 5A). On days 1 and 3, there was a large ‘‘burst’’ release of VEGF, but this gradually declined at the end of the study (ie, on day 13). Significantly, there was an increase in VEGF release from environments containing FGF along with VEGF compared with environments with cells and VEGF. Overall, the cumulative increase was highest in the groups having DPSCs/HUVECs/VEGFs and DPSCs/HUVECs/VEGFs/FGFs in alginate environments (Fig. 5A). Using Bonferroni multiple comparisons, it was concluded that at 24 hours there was no significant difference between VEGF release from these 2 environments, and both were significantly higher than the cellular environments without growth factors present (P < .05). By day 3, significant differences were observed among all
Figure 6. The release properties of FGF-2 from different alginate environments until day 13. (A) The cumulative profile of FGF-2 released from alginate plugs. (B) The actual release on 1, 3, 5, 7, 9, 11, and 13 days.
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Basic Research—Biology the environments, with the highest release noted in the all-mix environments (Fig. 5B). The combination of FGF with VEGF caused higher quantities of VEGF release than when VEGF was used alone. The same type of results was measured in the quantities of FGF release. The quantity of FGF was highest from the ‘‘all-mix’’ environments (Fig. 6A and B). The results from the FGF data highlighted a sustained and controlled release profile until day 13. It was concluded that the alginate matrix possesses a higher FGF-2 retention capacity compared with VEGF.
Discussion The successful bioengineering of a living pulp tissue in vitro will have important clinical implications because it provides a permanent strategy for saving the necrotic teeth arising from caries. Studies have shown that stem cell type, matrix identity, and protein adjuncts are crucial, in selected combinations, for successful de novo tissue formation. Still, there is no clinically viable procedure that has been delivered from these approaches in building bridges to dentin and developing and reconnecting a blood vessel system. Galler et al (9) have focused on matrix qualities engineered with enzyme remodeling labile cross-links and nanofibrous architecture, whereas Huang et al (29) centered on achieving the correct cell phenotype to manufacture pulp de novo directly inside the pulp cavity of mice. Rather than injecting the cells inside, they were seeded into a poly-D,L-lactide/glycolide carrier for transplantation. In this study, we implemented the ‘‘classic’’ tissue engineering approach to pulp bioengineering in a new strategy using pulp tissue progenitor cells and growth factors within RGD-alginate matrix material. The pulp tissue possesses an intricate internal structure, which is not easily replicated using biomaterial frameworks and building blocks for now. Simplified designs that create environments with the potential to promote anatomically correct pulp tissues are perhaps viable alternatives. In our simplified design, DPSCs were used as a source of tissue-producing cells, HUVECs as a source of endothelial tubule–generating cells, and growth factors to induce angiogenesis and morphogenesis events. DPSCs used for pulp regeneration have been previously characterized in a number of published studies (30, 31). They possess strong stem cell properties of self-renewal, and the differentiated daughter populations have a strong direction toward odontogenesis (32). The combination of HUVEC cells alongside DPSCs in calibrated proportions of 1:1, 1:3, and 1:5 have been found to increase the degree of angiogenesis and the cell proliferation rate (12). Thus, this type of cell coculture is a new route for the beginnings in dental pulp regeneration. Dissanayaka et al have shown that the coculture of HUVECs and DPSCs when injected into mice leads to vascularization and tubule formation in a scaffold-free approach (33). The piecing together of these factors could define a relevant primordial environment that would initiate the first step in pulp tissue regeneration based on a vascular endothelial tubule network spread throughout the construct. The idea of developing specially tailored progenitor cell microenvironments for tissue morphogenesis is to generate a blood vessel network that signals pulp stem cells for tissue formation and could eventually reconnect and function with the native blood supply. The creation of functional living tissue is built on the foundations of a blood vessel network after implantation; this must continue to provide an effective vascular supply, which, in turn, can effectively supply oxygen and nutrients to the tissue and the removal of waste products. Apart from the coculture of cells in this study, we tested the synergistic effects of combining the 2 growth factor proteins, VEGF and FGF-2, on the coculture of cells in a partial ECM matrix of the RGD-alginate scaffold. This scaffold has already been shown to promote better attachment, spreading, proliferation, and differentiation of various encapsuJOE — Volume -, Number -, - 2015
lated human cells (34). Further cell spreading and migratory activities can be promoted significantly by infusing ECM proteins such as collagen IV and seeding the cells onto the alginate matrix, which is 1 next step in the future development of this system. The same responses have been seen in encapsulated stem cells from apical papilla (SCAPs) and HUVECs in a 1:5 ratio (unpublished data). Among all the available identified factors, VEGF and FGF-2 have been shown to promote effective microcirculation in combination with stem cells in vitro (35, 36). The results show that the combination of VEGF and FGF lead to increased cell proliferation. The alginate microenvironments that we created were compatible with the DPSCs and HUVECs, but they could also retain and locally release functional quantities of active molecules. Our results also showed that the addition of FGF and VEGF together led to an increased combined DPSC and HUVEC proliferation in the first 24 hours compared with the environments with a single morphogen or none at all. Both the MTT and live-dead assay showed that cell proliferation was highest until day 7. However, on day 14, the cells cluttered into spherical balls, and proliferation declined compared with day 7, which possibly could be caused by the growth factors added. Notably, VEGF and FGF-2 quantities released on day 1 may have played a direct role in up-regulating the cell number compared with the cellular environments devoid of morphogens in other groups. Although it was evident from the results that alginate possessed biologically relevant retention capacities for FGF-2, the release of VEGF was almost complete within the first 7 days. We propose extending the system to include an exterior polysaccharide coating around the alginate construct loaded with DPSCs. Dentinogenic-specific conditions can be set within the new DPSC-seeded layer that promote the generation of odontoblastic cells. This outer coating will also function to slower the outward diffusion of growth factors from the alginate. However, further optimization and extension of the time line for culture are required to confirm proper vasculature formation, but this study could be a first step in fabricating a construct with a shape of gutta-percha and establishing a synergistic effect of growth factors in enhancing cell proliferation. In terms of future clinical application, this material could be used as injectable material (37) for posterior teeth or custom-made conical shape construct for anterior teeth. This study may prove to be a new direction in the field of pulp regeneration by creating environments necessary for dental pulp de novo and compartmentalizing them as well.
Acknowledgments Supported by General Research Fund (HKU 784912 M) grant to Chengfei Zhang. The authors deny any conflict of interest related to this study.
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23. Moshaverinia A, Chen C, Akiyama K, et al. Alginate hydrogel as a promising scaffold for dental-derived stem cells: an in vitro study. J Mater Sci Mater Med 2012;23:3041–51. 24. Johnstone B, Yoo J, Stewart M. Cell sources for cartilage tissue engineering. In: Vunja-Novakovic R, Freshny RI, eds. Culture of Cells for Tissue Engineering. Hoboken, NJ: John Wiley & Sons; 2006:83–111. 25. Kanafi MM, Ramesh A, Gupta PK, Bhonde RR. Dental pulp stem cells immobilized in alginate microspheres for applications in bone tissue engineering. Int Endod J 2014; 47:687–97. 26. Wegman F, Bijenhof A, Schuijff L, et al. Osteogenic differentiation as a result of BMP2 plasmid DNA based gene therapy in vitro and in vivo. Eur Cell Mater 2011;21: 230–42. 27. Patel Z, Ueda H, Yamamoto M, et al. In-vitro and in-vivo release of vascular endothelial growth factor from gelatin microparticles and biodegradable composite scaffolds. Pharm Res 2008;25:2370–8. 28. Claesson-Welsh L, Welsh M, Ito N, et al. Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrinbinding motif RGD. Proc Natl Acad Sci U S A 1998;95:5579–83. 29. Huang GT, Yamaza T, Shea LD, et al. Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng Part A 2010;16:605–10. 30. Karamzadeh R, Eslaminejad MB, Aflatoonian R. Isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods. J Vis Exp 2012; http://dx.doi.org/ 10.3791/4372. pii: 4372. 31. Yildirim S. Characterization of DPSC. Dental Pulp Stem Cells. New York: Springer; 2013:53–64. 32. Wang J, Liu X, Jin X, et al. The odontogenic differentiation of human dental pulp stem cells on nanofibrous poly(L-lactic acid) scaffolds in vitro and in vivo. Acta Biomater 2010;6:3856–63. 33. Dissanayaka WL, Zhu L, Hargreaves KM, et al. Scaffold-free prevascularized microtissue spheroids for pulp regeneration. J Dent Res 2014;93:1296–303. 34. Sharma S, Srivastava D, Grover S, Sharma V. Biomaterials in tooth tissue engineering: a review. J Clin Diagn Res 2014;8:309. 35. Lokmic Z, Mitchell GM. Engineering the microcirculation. Tissue Eng Part B Rev 2008;14:87–103. 36. Yuan C, Wang P, Zhu L, et al. Coculture of stem cells from apical papilla and human umbilical vein endothelial cell under hypoxia increases the formation of threedimensional vessel-like structures in vitro. Tissue Eng A 2015;21:1163–72. 37. Marler JJ, Guha A, Rowley J, et al. Soft-Tissue augmentation with injectable alginate and syngeneic fibroblasts. Plast Reconstr Surg 2000;105:2049–58.
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A First Step in De Novo Synthesis of a Living Pulp Tissue Replacement Using Dental Pulp MSCs and Tissue Growth Factors, Encapsulated within a Bioinspired Alginate Hydrogel Manasi Bhoj, BDS,* Chengfei Zhang, DDS, PhD,† and David W. Green, BSc, PhD* Abstract Introduction: A living, self-supporting pulp tissue replacement in vitro and for transplantation is an attractive yet unmet bioengineering challenge. Our aim is to create 3-dimensional alginate-based microenvironments that replicate the shape of gutta-percha and comprise key elements for the proliferation of progenitor cells and the release of growth factors. Methods: An RGD-bearing alginate framework was used to encapsulate dental pulp stem cells and human umbilical vein endothelial cells in a ratio of 1:1. The alginate hydrogel also retained and delivered 2 key growth factors, vascular endothelial growth factor-121 and fibroblast growth factor, in a sufficient amount to induce proliferation. A method was then devised to replicate the shape of gutta-percha using RGD alginate within a custommade mold of thermoresponsive N-isopropylacrylamide. Plugs of alginate containing different permutations of growth factor–based encapsulates were tested and evaluated for viability, proliferation, and release kinetics between 1 and 14 days. Results: According to scanning electron microscopic and confocal microscopic observations, the encapsulated human endothelial cells and dental pulp stem cell distribution were frequent and extensive throughout the length of the construct. There were also high levels of viability in all test environments. Furthermore, cell proliferation was higher in the growth factor–based groups. Growth factor release kinetics also showed significant differences between them. Interestingly, the combination of vascular endothelial growth factor and fibroblast growth factor synergize to significantly up-regulate cell proliferation. Conclusions: RGD-alginate scaffolds can be fabricated into shapes to fill the pulp space by simple templating. The addition of dual growth factors to cocultures of stem cells within RGD-alginate scaffolds led to the creation of microenvironments that significantly enhance the proliferation of
dental pulp stem cell/human umbilical vein endothelial cell combinations. (J Endod 2015;-:1–8)
Key Words Dental pulp stem cells, fibroblastic growth factor, human umbilical vein endothelial cells, RGD alginate, vascular endothelial growth factor
R
oot canal therapy is currently the preferred treatment for irreversible pulpitis wherein pulp is completely excavated and the root canal is prepared, debrided, and later obturated with gutta-percha (1, 2). What makes the prognosis for root canal treatment questionable are complications such as bacterial metabolite leakage (3), failure to achieve a proper seal (4), fracture upon application of force, and loss of vitality (5). Efforts toward endodontic regeneration have involved implementation of the latest tissue engineering strategies, including root revascularization by forced induction of blood clots, use of postnatal stem cell therapy (6), pulp implantation, injectable biochemically active cell-free scaffolds, 3-dimensional (3D) cell printing, and gene therapy (7). The greatest challenge of tissue engineering the ‘‘pulp’’ is to achieve in vivo revascularization from the host blood supply (8). The other barriers to progress in regenerative endodontics are dentinogenesis and maintaining the afferent nerve supply. Emphasis must be placed on replicating the pulp’s intricate zonal structure with their functions, namely mesenchymal stem cell self-renewal, odontogenic repair (formation of secondary dentin), and sensory and blood vessel apparatus (9). Bioengineering of the pulp requires a judicious selection and combination of stem cells and morphogens delivered over time in convoluted spatial patterns within a supporting framework. So far, the optimal permutation of factors has not been determined (10). A variety of dental stem cells have been implemented in pulp bioengineering strategies such as dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth, stem cells from apical papilla, and periodontal ligament stem cells. DPSCs exhibit strong, stable proliferation and self-renewal properties, directly differentiate along the odontoblast lineage, and are the most common dental stem cell used in pulp tissue engineering (11). A key process in early tissue morphogenesis is the interplay between stem cells and vascular endothelium. In a dental context, it has been shown that the coculture of postnatal DPSCs with endothelial cells (ECs) offer superior regenerative potential than when used individually (12). Other than the cellular and
From the *Oral Biosciences and †Comprehensive Dental Care, Faculty of Dentistry, The University of Hong Kong Hospital, Sai Ying Pun, Hong Kong. Address requests for reprints to Dr David W. Green, Oral Biosciences; or Dr Chengfei Zhang, Comprehensive Dental Care, Faculty of Dentistry, The University of Hong Kong Hospital, Floor 3A, 34 Hospital Road, Sai Ying Pun, Hong Kong. E-mail address: [email protected] or [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.03.006
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Basic Research—Biology TABLE 1. Various Test Groups of Pulp Construct Encapsulate
Quantity (mL)
DPSCs DPSCs:HUVECs DPSCs:HUVECs+VEGF DPSCs:HUVECs+VEGF: FGF (1:3)
106 cells 106 cells in ratio of 1:1 106 cells in 1:1 ratio of 50 ng VEGF 106 cells 1:1 ratio in 50 ng VEGF + 150 ng FGF in 1:3 ratio
DPSCs, dental pulp stem cells; FGF, fibroblast growth factor; HUVECs, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor.
structural environments, the biochemical composition is important in driving tissue production by cells. Tissue morphogens are small proteins inducing an array of cellular activities even at low concentrations in picogram quantities (10). Bone morphogenetic protein, vascular endothelial growth factor (VEGF), fibroblastic growth factor (FGF-2), and transforming growth factor are the principle growth factors that are being used frequently in conjunction with dental stem cells to induce various tissue structures (9). Cumulative evidence showed that VEGF and FGF in a pulp context specifically lead to angiogenesis, whereas bone morphogenetic protein is proposed to induce dentinogenesis at the pulp periphery (13–15). All dental cells require a 3D framework to provide safe anchorage and spatial organization and to be used as modules for transplantation to the desired site of reconstruction. Various synthetic and natural scaffold materials are available to build structural environments that mimic natural tissue frameworks, but accurate matching of the biology and biodegradation characteristics to a tissue exists as the major area of difficulty in optimizing scaffold material. Recent documented attempts indicated the formation of dentinlike precursor tissue, with the use of gelatin hydrogels, collagen, peptide-based gels, and polyglycolic acid (PGA) at the boundary within existing dentin (16, 17). Other tissue engineering biomaterials such as alginate have been virtually absent from pulp bioengineering research even though it is suitable for this context. Alginate, which is derived from marine seaweed, is widely available at relatively low costs and is used broadly in experimental tissue engineering as well as by food and drug manufacturers and has Food and Drug Administration approval (18). Continuous efforts are needed to engineer a scaffold that can properly self-organize and support the expansion and differentiation of stem cells into naturally simulated pulp, especially once it is implanted in the root canal. To start tackling these design flaws, we have engineered a morphologically accurate pulp replacement from stiffened alginate, which express internal cellular and molecular environments necessary for beginning pulp tissue reconstruction. The ultimate purpose of such biomimetic designs for pulp microenvironments is to eliminate the shortcomings of current pulp regeneration strategies by building an endothelial tubule network (protovasculature) that is the foundation for tissues. The presence of these structures guarantees a connection with the host blood supply after implantation. Such a strategy is critical to revitalizing and strengthening the existing diseased tooth structures.
Figure 1. A schematic of the procedure used to fabricate cell-seeded alginate pulp replacement frameworks. Step 1: cultivation and expansion of human cells. Steps 2 and 3: mixing of cells and growth factors in RGD-alginate solution. Steps 4, 5, and 6: fabrication of temperature-responsive sockets and molds. Step 7: (A) extruded pulp constructs, (B) confocal image, and (C) SEM. Step 8: prospective clinical end point filling of pulp cavity.
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Materials and Methods Cell Sources and Extraction Procedure Human umbilical vascular endothelial cells (HUVECs) were obtained commercially (ScienCell Research Laboratories, San Diego, CA) and cultured in endothelial cell medium (ECM) (ScienCell Research Laboratories Cat. No. 1001) at a seeding density of 5000
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Basic Research—Biology cells/cm2 at 37 C with 5% CO2. DPSCs were isolated from extracted human premolars by combining elements from 2 established protocols described by Yildirim (19). First, the enzyme digestion method was followed by adding collagenase type I (3 mg/mL) (Gibco-Invitrogen, Carlsbad, CA) and dispase (4 mg/mL) (Gibco-Invitrogen) to the freshly extirpated pulp tissue from sound permanent human tooth (under 18 years) and maintained in the incubator for 30 minutes, vortexed for 30 seconds, and centrifuged for 5 minutes at 1200 rpm. The supernatant fraction was discarded, and fresh medium was added to the remaining pellet, which was then thoroughly vortexed to produce a new
cell suspension. Normally, at this stage, the suspension is passed through a cell strainer. However, the suspension was directly cultured in a 75-cm2; culture flask to facilitate outgrowth of the DPSC population from the emulsified pulpal fragments.
Mold Fabrication for Producing the 3D RGD-alginate Pulp Plug Replacement A mold with specially imprinted sockets was fabricated with a thermoresponsive polymer, poly-N-isopropylacrylamide (pNIPAAm), using
Figure 2. (A) Confocal fluorescence images in the fluorescein isothiocyanate (FITC) channel showing DPSC and HUVEC viability inside the RGD-alginate framework over 14 days at 20 magnification. (B) Confocal fluorescence images in the FITC channel showing DPSC/HUVEC coculture viability inside a whole pulp plug alginate construct at 4 magnification.
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Basic Research—Biology imprints made by gutta-percha points ISO40 (Dentsply, Ballaigues, Switzerland). The entire experiment was performed on ice. In brief, following the procedure of Sasaki et al (20), polyethylene glycol (PEG) dimethacrylate (Sigma-Aldrich 437468) 613 mg and 1.58 g NIPAAm (Sigma-Aldrich 731129) (14 mmol crystal powder) were added into 19 mL deionized water. The reaction bottle was kept on ice, whereas nitrogen gas was aspirated into a monomer mixture. A polymerization starter mixture was prepared by adding ammonium persulfate (APS) (02602-02; Nakalai USA, San Diego, CA) 32 mg in 1 mL distilled water and thoroughly mixed. This starter solution was transferred into NIPAAm solution, and then 20 mL tetramethylethylenediamine (TEMED) (330401-72, Nakalai) was added while constantly stirring and keeping on ice. At the end, this solution was poured into a beaker with gutta-percha suspended upright in the mixture (Fig. 1A–C). After overnight gelation, the gutta-percha points were removed, and the mold was thoroughly washed in deionized water and then soaked in ethanol solution for 30 minutes to remove all unreacted cytotoxic monomers.
Preparation of Plug-shaped Cell-seeded Alginate Matrix Environments Four individual groups of RGD-alginate materials (NovaMatrix, Drammen, Norway) were prepared with different encapsulates and fabricated into pulp shape using the templating method described previously. The individual encapsulates used were DPSCs, HUVECs, VEGF121 (Human VEGF-121/VEGF-A Protein; Sino Biological Inc, Beijing, China), and FGF-2 (Recombinant Human bFGF/FGF-2 Protein, Sino Biological Inc). The first group of plugs consisted of DPSCs; the second group consisted of a combination of DPSCs and HUVECs (coculture 1:1) (12); the third group consisted of DPSCs, HUVECs, and VEGF; and the fourth group was composed of DPSC/HUVEC coculture and VEGF and FGF-2 (1:3) (Table 1). To achieve the reported synergistic effect between the 2 growth factors within the alginate gel, VEGF was added at a concentration of 50 ng/mL and 150 ng/mL for basic fibroblast growth factor (bFGF) (21). Each of the alginate encapsulate compositions were loaded into the prepared pNIPAAM sockets and immersed in alpha-Minimum Essential Media (a-MEM) with 50 mmol/L CaCl2 solution for 20 minutes. During this incubation period, the alginate constructs hardened into a stiff gel and were gently handled with forceps and placed into a 12-well plate containing aMEM, fetal bovine serum (FBS), and penicillin/streptomycin solution for 14 days with a change in media every other day. Cell Viability within RGD-alginate Environments Cell viability in the 4 composition groups of alginate scaffolds were evaluated on days 1, 7, and 14 using the LIVE/DEAD Cell Staining Kit (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions (22, 23). The live cells (green fluorescence) and necrotic cells (red fluorescence) were examined using a confocal fluorescence microscope (FluoView FV1000; Olympus, Tokyo, Japan) and analyzed using bioImageL V2 software (Faculty of Odontology, Malm€o University, Malm€o, Sweden). Depolymerization and Retrieval of Cells Embedded in RGD-alginate Constructs The constructs were suspended in phosphate-buffered saline containing 55 mmol/L trisodium citrate (Sigma-Aldrich, St. Louis, MO) and 150 mmol/L sodium chloride (Sigma-Aldrich) for 30 minutes in an incubator to cause degelling and the release of cells from the alginate scaffold. The cells were recovered by centrifugation at 1200 rpm for 5 minutes (24). 4
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Cell Proliferation Quantification in Growth Factor–infused Scaffolds Cell proliferation was measured using the standard MTT assay kit (M5655 and M8910, Sigma-Aldrich). In the first instance, the cells were released from the scaffold using a specially prepared depolymerization solution as mentioned previously. The experiment was run in triplicates and averaged from 4 different samples and a blank group at 1-, 7-, and 14-day time points as for the viability assays (25). Scanning Electron Microscopy The morphology and internal structure of alginate scaffolds were observed and imaged by scanning electron microscopy (SUI510; Hitachi, Tokyo, Japan). Scanning electron microscopy (SEM) was also used to observe cell morphology. The control (scaffold with no cells) and treatment scaffold groups (combinations of cells and factors) were fixed overnight in 4% v/v formalin containing 100 mmol/L CaCl2 followed by ethanol submersion (26) for 2 hours and then attached to sample stubs with conductive paint and sputter coated with gold and palladium in the MPS-2S Magnetron Ion Sputter Device (IXRF Systems, Austin, TX). The samples were viewed under SEM at an accelerating voltage of 15 kV. VEGF-121 and FGF-2 Release and Retention Kinetics from Alginate Scaffold Each of the 4 plug types by composition (n = 3 for each of 4 study groups) were placed in 6-well plates (each construct having a volume of 150 mL) and incubated in culture medium at 37 C (27). The concentration of VEGF used was 50 ng/mL, and for FGF-2 it was 150 ng/mL. Later, 1.0 mL solution from each well was collected in an Eppendorf tube at time points 1, 3, 5, 7, 9, 11, and 13 days and replaced with fresh medium and preserved at 20 C. The Human VEGF Quantikine ELISA Kit (DVE00) and Human FGF basic Quantikine HS ELISA Kit were used according to the manufacturer’s protocol. Average values below 0 were considered to be nondetectable. A cumulative release profile was assessed by total FGF and VEGF secreted during each time point from within the constructs (sum of FGF and VEGF released over the study until the last time point). The actual release profile was determined by the slope of each cumulative release of each group over the given time period. The detection limit for VEGF was 9 pg/mL and 0.07 pg/mL for bFGF. VEGF and FGF2 in the samples were determined by interpolation from the standard curve (28).
Figure 3. Proliferation levels of DPSCs and HUVECs in different alginate environments on days 1, 7, and 14.
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Basic Research—Biology Statistical Analysis IBM SPSS (Armonk, NY) statistics 20.0 software was used for data analysis with 1-way analysis of variance and Bonferroni multiple comparisons for analysis at a confidence of 95% with P > .05. Data points and error bars in the graphs represent the mean value and standard error calculated from different experiments.
the growth factor–treated alginate environments within 24 hours, but it was not sustained for 14 days. The DPSCs and HUVECs were viable throughout the entire pulp plug construct from the coronal to the apical end (Fig. 2B). Significant increases can be seen in the cell densities on day 7 compared with day 1. However, on day 14, few dead cells were seen within the construct and viable cells clustered together, which is a typical first stage of prototissue development inside alginate gels.
Results Cell Viability in Alginate Environments Encapsulated DPSC and HUVEC cocultures were viable throughout the 14-day culture period assessed by live-dead staining (Fig. 2A). However, there were noticeable differences in viability between the experimental groups. In particular, the number of viable cells increased in all
Cell Proliferation in Alginate Environments Significant differences in cell proliferation were measured between all the experimental groups. Statistical analysis was performed on the quantitative cell counts on 1, 7, and 14 days using 1-way analysis of variance (P < .05). Bonferroni multiple
Figure 4. DPSCs and HUVECs embedded within an RGD-alginate scaffold on day 1. Comparing alginate (left) with no cells and (right) with cells after 24 hours at different magnifications.
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Figure 5. The release properties of VEGF from different alginate environments until day 13. (A) The cumulative profile of VEGF released from alginate plugs. (B) The actual release on 1, 3, 5, 7, 9, 11, and 13 days.
comparisons showed that on day 1 there was no significant difference in cell numbers between the DPSC and DPSC + HUVEC groups (Fig. 3). However, on day 7, there was a significant increase in the DPSC + HUVEC group compared with the DPSC-alone group. This meant that the coculture of DPSCs and HUVECs increased the rate of cell proliferation between days 1 and 7 compared with DPSConly alginate (Fig. 3). The plugs containing growth factors also induced higher multiplication rates compared with the DPSC-only control. However, on day 14, there was no significant difference in proliferation between all the experimental groups. Clearly, the growth factors induced proliferation within the first 7 days, but their effect dissipated by day 14 (Fig. 3).
SEM Under SEM, DPSCs and HUVECs are shown fixed into the scaffold material and are spherical in shape shown early in cultivation. The distribution of cells is uniform along the whole length and depth of the construct, also reaching the apical tip. The homogenous cell distribu-
tion is needed to establish functional living tissue along the whole length of the root canal, which guarantees long-term survival (Fig. 4).
Cumulative Growth Factor Release from Alginate Constructs The temporal pattern of VEGF released from alginate scaffolds was typical of the release curve for small proteins entrapped within a hydrogel (Fig. 5A). On days 1 and 3, there was a large ‘‘burst’’ release of VEGF, but this gradually declined at the end of the study (ie, on day 13). Significantly, there was an increase in VEGF release from environments containing FGF along with VEGF compared with environments with cells and VEGF. Overall, the cumulative increase was highest in the groups having DPSCs/HUVECs/VEGFs and DPSCs/HUVECs/VEGFs/FGFs in alginate environments (Fig. 5A). Using Bonferroni multiple comparisons, it was concluded that at 24 hours there was no significant difference between VEGF release from these 2 environments, and both were significantly higher than the cellular environments without growth factors present (P < .05). By day 3, significant differences were observed among all
Figure 6. The release properties of FGF-2 from different alginate environments until day 13. (A) The cumulative profile of FGF-2 released from alginate plugs. (B) The actual release on 1, 3, 5, 7, 9, 11, and 13 days.
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Basic Research—Biology the environments, with the highest release noted in the all-mix environments (Fig. 5B). The combination of FGF with VEGF caused higher quantities of VEGF release than when VEGF was used alone. The same type of results was measured in the quantities of FGF release. The quantity of FGF was highest from the ‘‘all-mix’’ environments (Fig. 6A and B). The results from the FGF data highlighted a sustained and controlled release profile until day 13. It was concluded that the alginate matrix possesses a higher FGF-2 retention capacity compared with VEGF.
Discussion The successful bioengineering of a living pulp tissue in vitro will have important clinical implications because it provides a permanent strategy for saving the necrotic teeth arising from caries. Studies have shown that stem cell type, matrix identity, and protein adjuncts are crucial, in selected combinations, for successful de novo tissue formation. Still, there is no clinically viable procedure that has been delivered from these approaches in building bridges to dentin and developing and reconnecting a blood vessel system. Galler et al (9) have focused on matrix qualities engineered with enzyme remodeling labile cross-links and nanofibrous architecture, whereas Huang et al (29) centered on achieving the correct cell phenotype to manufacture pulp de novo directly inside the pulp cavity of mice. Rather than injecting the cells inside, they were seeded into a poly-D,L-lactide/glycolide carrier for transplantation. In this study, we implemented the ‘‘classic’’ tissue engineering approach to pulp bioengineering in a new strategy using pulp tissue progenitor cells and growth factors within RGD-alginate matrix material. The pulp tissue possesses an intricate internal structure, which is not easily replicated using biomaterial frameworks and building blocks for now. Simplified designs that create environments with the potential to promote anatomically correct pulp tissues are perhaps viable alternatives. In our simplified design, DPSCs were used as a source of tissue-producing cells, HUVECs as a source of endothelial tubule–generating cells, and growth factors to induce angiogenesis and morphogenesis events. DPSCs used for pulp regeneration have been previously characterized in a number of published studies (30, 31). They possess strong stem cell properties of self-renewal, and the differentiated daughter populations have a strong direction toward odontogenesis (32). The combination of HUVEC cells alongside DPSCs in calibrated proportions of 1:1, 1:3, and 1:5 have been found to increase the degree of angiogenesis and the cell proliferation rate (12). Thus, this type of cell coculture is a new route for the beginnings in dental pulp regeneration. Dissanayaka et al have shown that the coculture of HUVECs and DPSCs when injected into mice leads to vascularization and tubule formation in a scaffold-free approach (33). The piecing together of these factors could define a relevant primordial environment that would initiate the first step in pulp tissue regeneration based on a vascular endothelial tubule network spread throughout the construct. The idea of developing specially tailored progenitor cell microenvironments for tissue morphogenesis is to generate a blood vessel network that signals pulp stem cells for tissue formation and could eventually reconnect and function with the native blood supply. The creation of functional living tissue is built on the foundations of a blood vessel network after implantation; this must continue to provide an effective vascular supply, which, in turn, can effectively supply oxygen and nutrients to the tissue and the removal of waste products. Apart from the coculture of cells in this study, we tested the synergistic effects of combining the 2 growth factor proteins, VEGF and FGF-2, on the coculture of cells in a partial ECM matrix of the RGD-alginate scaffold. This scaffold has already been shown to promote better attachment, spreading, proliferation, and differentiation of various encapsuJOE — Volume -, Number -, - 2015
lated human cells (34). Further cell spreading and migratory activities can be promoted significantly by infusing ECM proteins such as collagen IV and seeding the cells onto the alginate matrix, which is 1 next step in the future development of this system. The same responses have been seen in encapsulated stem cells from apical papilla (SCAPs) and HUVECs in a 1:5 ratio (unpublished data). Among all the available identified factors, VEGF and FGF-2 have been shown to promote effective microcirculation in combination with stem cells in vitro (35, 36). The results show that the combination of VEGF and FGF lead to increased cell proliferation. The alginate microenvironments that we created were compatible with the DPSCs and HUVECs, but they could also retain and locally release functional quantities of active molecules. Our results also showed that the addition of FGF and VEGF together led to an increased combined DPSC and HUVEC proliferation in the first 24 hours compared with the environments with a single morphogen or none at all. Both the MTT and live-dead assay showed that cell proliferation was highest until day 7. However, on day 14, the cells cluttered into spherical balls, and proliferation declined compared with day 7, which possibly could be caused by the growth factors added. Notably, VEGF and FGF-2 quantities released on day 1 may have played a direct role in up-regulating the cell number compared with the cellular environments devoid of morphogens in other groups. Although it was evident from the results that alginate possessed biologically relevant retention capacities for FGF-2, the release of VEGF was almost complete within the first 7 days. We propose extending the system to include an exterior polysaccharide coating around the alginate construct loaded with DPSCs. Dentinogenic-specific conditions can be set within the new DPSC-seeded layer that promote the generation of odontoblastic cells. This outer coating will also function to slower the outward diffusion of growth factors from the alginate. However, further optimization and extension of the time line for culture are required to confirm proper vasculature formation, but this study could be a first step in fabricating a construct with a shape of gutta-percha and establishing a synergistic effect of growth factors in enhancing cell proliferation. In terms of future clinical application, this material could be used as injectable material (37) for posterior teeth or custom-made conical shape construct for anterior teeth. This study may prove to be a new direction in the field of pulp regeneration by creating environments necessary for dental pulp de novo and compartmentalizing them as well.
Acknowledgments Supported by General Research Fund (HKU 784912 M) grant to Chengfei Zhang. The authors deny any conflict of interest related to this study.
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JOE — Volume -, Number -, - 2015
