Scaffolds play a vital role in tissue engineering. They serve as three-dimensional tissue templates and are intended to provide synthetic ECM microenvironments for cell attachment, proliferation, differentiation, and neo tissue genesis. An advanced scaffold therefore may benefit from mimicking certain advantageous features of the natural ECM [6
]. Collagen type I accounts for 80–90% of the organic substances of demineralized dentin ECM and is correlated closely with the dentin formation [7
]. Previous studies demonstrated that collagen matrix promoted odontogenic differentiation and mineralization and has been used as a scaffold in tooth tissue engineering [8
]; however, there are concerns over the potential pathogen transmission, immune reactions and the poor mechanical properties of collagen matrix. In our laboratory, NF-PLLA scaffolds were fabricated to emulate the nanofibrous architecture of collagen fibers [18
]. Both SW and NF scaffolds with similar interconnected spherical pores were formed by using a paraffin sphere porogen to facilitate cell seeding, migration and growth. The only difference between the two types of scaffolds was in the architecture of the macropore walls of the scaffolds, which was either nanofibrous or solid-walled (smooth). Our previous studies showed that the nanofibrous architecture facilitated osteoblast proliferation, differentiation and mineralization compared to smooth pore wall architecture [21
]. Since dentin is a tissue analogous to bone and shares many extracellular components with bone, we therefore hypothesized that NF scaffolds had better potential in promoting the proliferation and odontogenic differentiation of DPSCs, resulting in enhanced dentin-like tissue formation than SW scaffolds. This study was intended to test this hypothesis.
Histological staining and DNA assay in vitro
demonstrated that NF scaffolds provided a better extracellular microenvironment for the proliferation of DPSCs and their ECM production than SW scaffolds. Since both scaffolds had similar macropore structures, the nanofibrous features seemed to play an important role in cell attachment and odontogenic differentiation. Our previous studies demonstrated that nanofibrous architecture enhanced protein adsorption, including fibronectin and vitronectin, contributing to pre-osteoblast cell attachment [26
]. This was consistent with our observation that more human DPSCs were present throughout the macropores of NF scaffolds compared to those of SW scaffolds. Another possible interpretation of the enhanced cell attachment and growth could be that filopodia played an important role in biological processes [29
] and nanofibrous architecture could have altered the mode of anchorage, allowing filopodia to anchor more tightly. In addition, the local mass transport conditions in the nanofibrous scaffolds is likely better than those in solid-walled architecture. The nanofibrous pore walls might improve nutrient/oxygen supply to and metabolic waste removal from the attached cells, contributing to better extracellular environment for DPSCs growth and ECM production.
To investigate the ability of odontogenic differentiation and mineralization on NF and SW scaffolds, ALP activity quantification, real-time PCR, calcium content assay, SEM and histological analyses were carried out. ALP is regarded as an early marker of osteogenic differentiation and hard tissue formation [31
]. Col I, OCN and DSPP were chosen as gene markers for the odontogenic phenotype. Collagen I is the predominant protein and the basis for dentin repair [7
]. OCN, a vitamin K-dependent noncollagenous ECM protein, is generally regarded as a late marker for osteogenic and odontogenic differentiation [32
]. DSPP is the major dentinal noncollagenous protein and plays a crucial role during dentinogenesis [33
]. From our results, DPSCs cultured on NF scaffolds showed significantly higher ALP activity than those on SW scaffolds. In addition, the expression of the specific markers associated with odontogenic differentiation, especially OCN and DSPP, was upregulated on NF scaffolds compared with those on SW scaffolds. The high levels of OCN and DSPP mRNA in cells on NF scaffolds were consistent with more mineral formation observed in NF scaffolds. These findings suggest that DPSCs grown on NF scaffolds allowed for enhanced expression of the odontoblast phenotype compared to SW scaffolds. In terms of mineralization, von Kossa staining and calcium content assay revealed a greater amount of mineral generated in the NF scaffolds compared with that in the SW scaffolds. This was also confirmed by the SEM and EDX results. Consistent with the in vitro
studies, results from in vivo
studies showed that the newly formed ECM in NF scaffolds were strongly immunostained for DSP, which indicated that the DPSCs differentiated into odontoblast-like cells and regenerated dentin-like hard tissue in the NF scaffolds. Taken together, these outcomes provided strong evidence that NF scaffolds better promoted odontogenic differentiation and mineralization than SW scaffolds both in vitro
and in vivo
Several factors might have contributed to the enhanced odontogenic differentiation and mineralization of human DPSCs on the NF scaffolds. In dentin tissue, type I collagen is considered to provide initiation sites for calcification and have been verified to modulate odontogenic differentiation and mineralization [7
]. These effects were mediated by the interaction of collagen with integrin receptors present on the cell membrane [8
]. NF-PLLA scaffolds with interconnected macropores were fabricated in our laboratory to imitate type I collagen fibers [18
]. Our previous results demonstrated that NF scaffolds adsorbed greater quantities of cell adhesion proteins (such as fibronectin) than SW scaffolds [26
]. In dentin tissue, fibronectin enhances the differentiation of odontoblasts and dentine formation [34
]. A fibronectin-rich matrix may serve as a reservoir of growth factors, which have participated in the differentiation of odontoblasts [36
]. Our findings suggest that nanofibrous architecture of PLLA scaffolds exhibited certain characteristics similar to natural collagen fibers to facilitate the odontogenic differentiation and biomineralization of human DPSCs.
Although DSP-positive staining and hard tissue formation were confirmed, no typical palisade arrangement of the cells and dentin-pulp like tissue were identified in the implants in both of the scaffolds. In this study, we focused on investigating the influences of NF scaffolds on the differentiation of the cells. While not being the focus of this study, signaling molecules also play a vital role in tissue regeneration. More investigation on optimal odontogenic factors, their spatial and temporal application, and integration with scaffolds may lead to further improved microenvironments for high quality dentin regeneration, which will be explored in our future studies.