Regenerative endodontic procedures are becoming more feasible in everyday clinical scenarios. However, optimizing a bioactive scaffold that could recapitulate true dentin-pulp regeneration as a cell-homing approach is still a critical area of research (2). Ideal scaffolds should be biocompatible, preserve native structure, and promote tissue regeneration rather than repair (31).
In this study, a naturally derived biomimetic P-ECM scaffold was prepared from a widely available bovine source., This could offer tissue specific endodontic regeneration as it provides a natural microenvironment for recruited stem cells (32–34). However, being prepared from a bovine source, optimal decellularization must be ensured to avoid possible immunogenicity without altering the structure and bioactive properties of the scaffold (8, 31, 32, 35). In this study, a 5 days decellularization protocol was chosen following the protocol described by Bakhtiar et al. (7). However, modifications were made in the trypsin/EDTA concentration, the use of deionized water and PBS washes based on a decellularization pilot study. In this decellularization protocol, sodium dodecyl sulfate (SDS) was not used as several studies showed that it results in collagen degradation and residual toxicity in the tissues (24, 36). The residual DNA content of the extracellular matrix is thought to be the main source of antigenicity that could provoke a host immunological response (37). However, it has been reported that there is no significant host response to decellularized ECM containing 50 ng/mg of DNA or less (13). Therefore, DNase treatment followed by several washes with deionized water and PBS were made to ensure that residual DNA was below the cut-off point. Indeed, DNA content was found to be significantly less following decellularization in comparison to native tissues, which was consistent with Alqahtani et al.(9), Bakhtiar et al.(24) and Lee et al.(38). In addition, qualitative histological assessment confirmed the absence of nuclei in decellularized pulp, retention of glycosaminoglycans (GAGs), and collagen content which was in accordance with the findings reported by Elwerfelli et al. (8) and Song et al. (39). In fact, it was reported that tissue collagen and GAGs play important roles in the regulation of cell-ECM interaction and subsequent intercellular processes. They also contain matrix-bound adhesion/growth factors, which are crucial for the regeneration process(40).
In this study, a concentration of 3.00 mg/ml of bovine dental pulp-derived ECM, prepared in a hydrogel form, was used according to a study by Bakhtiar et al who reported that this concentration had optimal physical and biological properties (7). Being an injectable hydrogel may provide a clinically applicable ready-made material that conforms to irregular canal shapes (16, 41). It is worth mentioning that ten bovine molar teeth used in this study were enough to prepare 150–170 ml of P-ECM hydrogel. This could offer a cost effective product for REPs in which only approximately 20 µL of hydrogel is needed to fill each canal (15). For the preparation of a novel ECM + HA hydrogel, physical mixing of gelled P-ECM with HA hydrogel was done in a 1:1 ratio. This novel combination was thought to enhance the mechanical properties of the hydrogel by having a sustained release of growth factors (17, 21, 41, 42).
In this study the total protein content in the P-ECM hydrogel was measured. Moreover, both hydrogels showed continuous protein release over a period of 28 days (43–45). P-ECM hydrogel showed higher protein release which continued to gradually decrease over time. While P-ECM + HA showed a greater retention of proteins throughout the timepoints. This might be due to physical mixing with HA which might alter the degree of release of growth factors and proteins having a more sustained release (42). However, it is important to note that P-ECM hydrogel contained 3 mg/ml ECM while P-ECM + HA were prepared with a 50:50 composition to have a final ECM concentration of 1.5 mg/ml.
In REPs, the ideal rate of scaffold degradation is thought to be from 6–8 weeks in order to allow for remodeling and replacement by de-novo tissues and to sustain the turnover of inflammation and regeneration (46, 47). In this study, it was found that P-ECM + HA had more degradation rate percentage than P-ECM. This could be explained by the fact that the ECM in the combination group was crushed during mixing exposing more surface area to be biodegraded. It should also be noted that the addition of HA may have led to increased initial weight of the scaffold. This was in accordance with that described by Bakhtiar et al who reported a similar rate of degradation (7, 46).
Moreover, hydrogel topology revealed the presence of pores, and intermingling of fibers after decellularization which is due to the ECM component which was in accordance with that described by Elwerfelli et al. and Yuanyuan Shi et al. (8, 31, 48). However, P-ECM + HA had a larger fiber diameter, smaller pore area percentage, and more dense structure, which might be due to mixing with HA(46).
In REPs, the presence and severity of inflammation may hinder the release of biological markers that are sequestered in the dentine matrix which are released by conditioning agents before eliciting apical bleeding (49). Therefore, relying on naturally occurring tissue-specific growth factors in the prepared scaffolds could be valuable. Therefore, growth factors that are present in the native pulp and essential for regenerative process, were quantified. TGF-β1 and bFGF are known to stimulate cell migration and contribute to cell proliferation and differentiation. Moreover, VEGF is crucial for neovascularization and enhancement of angiogenesis. While BMP-2 promotes odontoblastic differentiation and dentin formation. (9, 50, 51).
In this research, the release pattern of the forementioned growth factors was detected in the supernatants of both groups after gelation. TGF-β1, bFGF and VEGF were released from both hydrogels which was in accordance with the results of Alqahtani et al, where VEGF and TGF-β1 were detected after decellularization of pulp tissues. However, in their study, bFGF was not detected which may be due to different decellularization and sterilization protocols (9). VEGF showed almost sustained release up to 7 days which could be the crucial time during natural wound healing cascade (52, 53).
Although P-ECM + HA contained only half the concentration of ECM, it showed comparable results to P-ECM hydrogel regarding the growth factors release. This could be attributed to the physical nature of the combined P-ECM + HA scaffold having an increased density and decreased pore size which could explain the longer retention and sustained release of growth factors (21). The growth factor release at day 0 after gelation is comparable to that of native intact and inflamed pulp of other studies in which TGF-β1 had almost half amount that secreted by intact pulp and higher VEGF and bFGF release than intact pulp(54, 55).P-ECM hydrogel BMP-2 release at day 0 was in accordance with Jugoslav et al. in which it had less release than intact pulp but more release than injured pulp after indirect pulp capping (IPC) (56). While, P-ECM + HA BMP-2 release at day 0 had similar amount of release of injured pulp after IPC (56).
BMP-2 was detectable with low levels in P-ECM + HA in 0 and 1 days while it was undetectable in the rest of time points which may be due to affinity of hyaluronic acid to BMP-2 (57). Therefore it may need more tailoring to have a sustained BMP-2 release. In contrast, it was detectable in P-ECM in the 5 time points showing increasing release from 0 to 1 days then descending release till reaching 28 days.
Further research is still needed to assess other important growth factors related to dentin-pulp regeneration. Different concentrations of P-ECM and HA with different molecular weights should be compared. Moreover, the direct effects of the prepared hydrogels on stem cell functions should be evaluated paving the way for future in vivo applications. Despite the analysis of protein and different growth factors release, one of the limitations of the current study is that the functional activity of these scaffold hydrogels was not assessed. This would require performing extensive cellular assays as well as in vivo animal studies. Another additional limitation is that, due to the biological nature of these scaffolds, inherent variability in the results may be expected. However, this was overcome in the current study by pooling a large number of bovine pulps prior to initiating the processing.
Within the limitations of this study, it was concluded that a hydrogel form of decellularized P-ECM retained its architecture, collagen and protein content. P-ECM hydrogel had a larger pore area percentage, better rate of degradation, protein release, and BMP-2 release. The novel combination of P-ECM + HA had a larger fiber diameter, more dense structure and sustained growth factor release of TGF-β1, bFGF, and VEGF. These results highlight the promising potential of the prepared P-ECM-based scaffolds for further studies and comparison with other biomaterials in regenerative endodontic applications.