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Enhanced chondrogenic differentiation of dental pulp stem cells using nanopatterned PEG-GelMA-HA hydrogels.

ABSTRACT: We have examined the effects of surface nanotopography and hyaluronic acid (HA) on in vitro chondrogenesis of dental pulp stem cells (DPSCs). Ultraviolet-assisted capillary force lithography was employed to fabricate well-defined nanostructured scaffolds of composite PEG-GelMA-HA hydrogels that consist of poly(ethylene glycol) dimethacrylate (PEGDMA), methacrylated gelatin (GelMA), and HA. Using this microengineered platform, we first demonstrated that DPSCs formed three-dimensional spheroids, which provide an appropriate environment for in vitro chondrogenic differentiation. We also found that DPSCs cultured on nanopatterned PEG-GelMA-HA scaffolds showed a significant upregulation of the chondrogenic gene markers (Sox9, Alkaline phosphatase, Aggrecan, Procollagen type II, and Procollagen type X), while downregulating the pluripotent stem cell gene, Nanog, and epithelial-mesenchymal genes (Twist, Snail, Slug) compared with tissue culture polystyrene-cultured DPSCs. Immunocytochemistry showed more extensive deposition of collagen type II in DPSCs cultured on the nanopatterned PEG-GelMA-HA scaffolds. These findings suggest that nanotopography and HA provide important cues for promoting chondrogenic differentiation of DPSCs.

SUBMITTER: Nemeth CL

PROVIDER: S-EPMC4229712 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Enhanced chondrogenic differentiation of dental pulp stem cells using nanopatterned PEG-GelMA-HA hydrogels.

Nemeth Cameron L CL Janebodin Kajohnkiart K Yuan Alex E AE Dennis James E JE Reyes Morayma M Kim Deok-Ho DH

Tissue engineering. Part A 20140630 21-22

We have examined the effects of surface nanotopography and hyaluronic acid (HA) on in vitro chondrogenesis of dental pulp stem cells (DPSCs). Ultraviolet-assisted capillary force lithography was employed to fabricate well-defined nanostructured scaffolds of composite PEG-GelMA-HA hydrogels that consist of poly(ethylene glycol) dimethacrylate (PEGDMA), methacrylated gelatin (GelMA), and HA. Using this microengineered platform, we first demonstrated that DPSCs formed three-dimensional spheroids, w ...[more]

PMID: 24749806

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Project description:Pulpal revascularization is commonly used in the dental clinic to obtain apical closure of immature permanent teeth with thin dentinal walls. Although sometimes successful, stimulating bleeding from the periapical area of the tooth can be challenging and in turn may deleteriously affect tooth root maturation. Our objective here was to define reliable methods to regenerate pulp-like tissues in tooth root segments (RSs). G1 RSs were injected with human dental pulp stem cells (hDPSCs) and human umbilical vein endothelial cells (HUVECs) encapsulated in 5% gelatin methacrylate (GelMA) hydrogel. G2 RSs injected with acellular GelMA alone, and G3 empty RSs were used as controls. White mineral trioxide aggregate was used to seal one end of the tooth root segment, while the other was left open. Samples were cultured in vitro in osteogenic media (OM) for 13 d and then implanted subcutaneously in nude rats for 4 and 8 wk. At least 5 sample replicates were used for each experimental group. Analyses of harvested samples found that robust pulp-like tissues formed in G1, GelMA encapsulated hDPSC/HUVEC-filled RSs, and less cellularized host cell-derived pulp-like tissue was observed in the G2 acellular GelMA and G3 empty RS groups. Of importance, only the G1, hDPSC/HUVEC-encapsulated GelMA constructs formed pulp cells that attached to the inner dentin surface of the RS and infiltrated into the dentin tubules. Immunofluorescent (IF) histochemical analysis showed that GelMA supported hDPSC/HUVEC cell attachment and proliferation and also provided attachment for infiltrating host cells. Human cell-seeded GelMA hydrogels promoted the establishment of well-organized neovasculature formation. In contrast, acellular GelMA and empty RS constructs supported the formation of less organized host-derived vasculature formation. Together, these results identify GelMA hydrogel combined with hDPSC/HUVECs as a promising new clinically relevant pulpal revascularization treatment to regenerate human dental pulp tissues.

Project description:BACKGROUND CONTEXT:Advances in the development of biomaterials and stem cell therapy provide a promising approach to regenerating degenerated discs. The normal nucleus pulposus (NP) cells exhibit similar phenotype to chondrocytes. Because dental pulp stem cells (DPSCs) can be differentiated into chondrogenic cells, the DPSCs and DPSCs-derived chondrogenic cells encapsulated in type I and type II collagen hydrogels can potentially be transplanted into degenerated NP to repair damaged tissue. The motility of transplanted cells is critical because the cells need to migrate away from the hydrogels containing the cells of high density and disperse through the NP tissue after implantation. PURPOSE:The purpose of this study was to determine the motility of DPSC and DPSC-derived chondrogenic cells in type I and type II collagen hydrogels. STUDY DESIGN/SETTING:The time lapse imaging that recorded cell migration was analyzed to quantify the cell migration velocity and distance. METHODS:The cell viability of DPSCs in native or poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG)-crosslinked type I and type II collagen hydrogels was determined using LIVE/DEAD cell viability assay and AlamarBlue assay. DPSCs were differentiated into chondrogenic cells. The migration of DPSCs and DPSC-derived chondrogenic cells in these hydrogels was recorded using a time lapse imaging system. This study was funded by the Regional Institute on Aging and Wichita Medical Research and Education Foundation, and the authors declare no competing interest. RESULT:DPSCs showed high cell viability in non-crosslinked and crosslinked collagen hydrogels. DPSCs migrated in collagen hydrogels, and the cell migration speed was not significantly different in either type I collagen or type II collagen hydrogels. The migration speed of DPSC-derived chondrogenic cells was higher in type I collagen hydrogel than in type II collagen hydrogel. Crosslinking of type I collagen with 4S-StarPEG significantly reduced the cell migration speed of DPSC-derived chondrogenic cells. CONCLUSIONS:After implantation of collagen hydrogels encapsulating DPSCs or DPSC-derived chondrogenic cells, the cells can potentially migrate from the hydrogels and migrate into the NP tissue. This study also explored the differential cell motility of DPSCs and DPSC-derived chondrogenic cells in these collagen hydrogels.

Project description:Mesenchymal stem cells (MSCs) derived from dental and orofacial tissues provide an alternative therapeutic option for craniofacial bone tissue regeneration. However, there is still a need to improve stem cell delivery vehicles to regulate the fate of the encapsulated MSCs for high quality tissue regeneration. Matrix elasticity plays a vital role in MSC fate determination. Here, we have prepared various hydrogel formulations based on alginate and gelatin methacryloyl (GelMA) and have encapsulated gingival mesenchymal stem cells (GMSCs) and human bone marrow MSCs (hBMMSCs) within these fabricated hydrogels. We demonstrate that addition of the GelMA to alginate hydrogel reduces the elasticity of the hydrogel mixture. While presence of GelMA in an alginate-based scaffold significantly increased the viability of encapsulated MSCs, increasing the concentration of GelMA downregulated the osteogenic differentiation of encapsulated MSCs in vitro due to decrease in the stiffness of the hydrogel matrix. The osteogenic suppression was rescued by addition of a potent osteogenic growth factor such as rh-BMP-2. In contrast, MSCs encapsulated in alginate hydrogel without GelMA were successfully osteo-differentiated without the aid of additional growth factors, as confirmed by expression of osteogenic markers (Runx2 and OCN), as well as positive staining using Xylenol orange. Interestingly, after two weeks of osteo-differentiation, hBMMSCs and GMSCs encapsulated in alginate/GelMA hydrogels still expressed CD146, an MSC surface marker, while MSCs encapsulated in alginate hydrogel failed to express any positive staining. Altogether, our findings suggest that it is possible to control the fate of encapsulated MSCs within hydrogels by tuning the mechanical properties of the matrix. We also reconfirmed the important role of the presence of inductive signals in guiding MSC differentiation. These findings may enable the design of new multifunctional scaffolds for spatial and temporal control over the fate and function of stem cells even post-transplantation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2957-2967, 2017.

Project description:Preservation of a vital dental pulp is a central goal of restorative dentistry. Currently, there is significant interest in the development of tissue engineering scaffolds that can serve as biocompatible and bioactive pulp-capping materials, driving dentin bridge formation without causing cytotoxic effects. Our earlier in vitro studies described the biocompatibility of multidomain peptide (MDP) hydrogel scaffolds with dental pulp-derived cells but were limited in their ability to model contact with intact 3-dimensional pulp tissues. Here, we utilize an established ex vivo mandible organ culture model to model these complex interactions. MDP hydrogel scaffolds were injected either at the interface of the odontoblasts and the dentin or into the pulp core of mandible slices and subsequently cultured for up to 10 d. Histology reveals minimal disruption of tissue architecture adjacent to MDP scaffolds injected into the pulp core or odontoblast space. Additionally, the odontoblast layer is structurally preserved in apposition to the MDP scaffold, despite being separated from the dentin. Alizarin red staining suggests mineralization at the periphery of MDP scaffolds injected into the odontoblast space. Immunohistochemistry reveals deposition of dentin sialophosphoprotein by odontoblasts into the adjacent MDP hydrogel, indicating continued functionality. In contrast, no mineralization or dentin sialophosphoprotein deposition is evident around MDP scaffolds injected into the pulp core. Collagen III expression is seen in apposition to gels at all experimental time points. Matrix metalloproteinase 2 expression is observed associated with centrally injected MDP scaffolds at early time points, indicating proteolytic digestion of scaffolds. Thus, MDP scaffolds delivered centrally and peripherally within whole dental pulp tissue are shown to be biocompatible, preserving local tissue architecture. Additionally, odontoblast function and pulp vitality are sustained when MDP scaffolds are intercalated between dentin and the odontoblast region, a finding that has significant implications when considering these materials as pulp-capping agents.

Dataset Information

Enhanced chondrogenic differentiation of dental pulp stem cells using nanopatterned PEG-GelMA-HA hydrogels.

Publications

Enhanced chondrogenic differentiation of dental pulp stem cells using nanopatterned PEG-GelMA-HA hydrogels.

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