The alkaline pH (7.3–8) is crucial for the odontogenic differentiation of DPSCs . The alkalinity test results reveal a non-significant difference between hTDM and the control group (pH = 8.2). That ensures the ability of the hTDM to maintain the micro-environment pH. However, MTA raised the pH value to 9.8. After adding the MTA disc to the complete medium, its pink color changed into violet immediately. That indicates the high alkalinity of MTA. These results were consistent with studies of Tsai et al. and Luczaj-Cepowicz et al. that detected the immediate increase of MTA pH value from 10.2 to 12.5 and then decreased to 10.2 again after 3hrs .
The results of the antibacterial test reveal bacteriostatic activity of the hTDM. The PDGF & FGF released from the hTDM exhibit bacteriostatic activity against Gram-positive and Gram-negative bacteria  . Contrary to MTA, the results reveal a low antibacterial activity of the MTA against the cariogenic bacteria. These results were consistent with the study established by Huang et al. that investigated the antibacterial activity of MTA. It revealed the inferiority of the MTA when compared to Biodentine and Ca (OH) 2 .
The results of the Trans-well migration test revealed a higher ability of hTDM to induce DPSCs migration than MTA. That is due to the release of TGF-β1 from the hTDM . However, the high alkalinity of MTA may inhibit the DPSCs migration. These results were consistent with the study established by Avery et al. that detected a high bio-activity of hTDM via testing its ability to induce BMSCs proliferation and migration .
In the in vivo assessment, the teeth treated with MTA showed scores of 1 and 2 pulp inflammatory response after 3days. That reveals a prolonged inflammatory phase that delays the proliferative and regenerative phases. However, MTA inhibits the expression of Bcl-2 and Bcl-xL and upregulates Bax expression. Therefore, a superficial necrotic layer formed at the MTA-pulp tissue interface. During this time the pulp-self healing mechanism tries to tolerate the pulp injury and compensate for the necrotic layer that prolongs the inflammatory phase more than the physiologic level.
While in the second follow-up period (after three months), the teeth treated with MTA expressed a score (1) pulp inflammatory response even after calcific bridge formation. The calcific bridge thickness formed by MTA was thicker than the normal pre-dentin. Moreover, it was formed towards the pulp tissue obliterating the pulp chamber. These results are in agreement with the studies of Liu et al. and Oliveira et al. that detected uncontrolled mineralization and intra-pulpal calcification after using MTA as a DPC material ,.
However, the teeth treated with the hTDM scaffolds exhibited a non-inflammatory response score (0) besides the formation of new blood vessels at the exposure site after three days. The novel hTDM proves the extreme advantages of being a natural body product having high biocompatibility with the pulp tissue. Furthermore, it didn’t disturb the pH in the intra-pulpal environment.
Furthermore, the histologic sections’ examination of the teeth treated with hTDM after three months revealed the formation of an organized new pulp tissue with a distinct odontoblast layer along the hTDM surface. In addition, sealing the exposure site with newly formed dentin that adhered to the scaffold surface and the normal existing dentin makes the scaffold a part of the tooth structure. That explains the retention of the hTDM scaffold in histologic sections even after 18 months of the decalcification process.
The comparison between the new dentin and the pre-dentin thickness revealed new dentin formation at the physiologic rate. That is due to the presence of DGP in the hTDM scaffold (as a natural component) that inhibits bio-mineralization by preventing additional minerals’ deposition . In addition, the ability of the hTDM scaffold to release the endogenous cytokines and growth factors at the physiologic level . Optimizing the mineralization process is mandatory to avoid intra-pulpal calcification. Therefore using the hTDM scaffold as a direct pulp capping material didn’t alter the physiologic pulp-self healing mechanism.
The suggested healing mechanism induced by the hTDM scaffold involves four phases. The first one is hemostasis which starts a few seconds after the pulp exposure. The uncontrolled bleeding from the exposure site was intended before scaffold placement to allow wetting of the scaffold surface with blood. As a result of the high surface wettability of the scaffold, a resilient union formed between the blood clot and the scaffold surface . This union provides a sealing barrier at the exposure site . The growth factors released from the blood clot and hTDM co-ordinate to startup the inflammatory phase in the healing mechanism.
The inflammatory phase involves early and late inflammatory responses. The early inflammatory response started 24hr after the pulp exposure under the influence of PDGF and IL-6 released from the platelets in the blood clot and hTDM scaffold. IL-6 attracts neutrophils to the injury site, while PDGF activates the resident M2 macrophages and T-cells to release IL-1, IL-6, and IL-ß1 for attracting more neutrophils. The attracted neutrophils and activated M2-macrophages phagocytize the micro-organism and foreign bodies at the exposure site. At the end of the early inflammatory response, M2 macrophages release TNF-α inducing neutrophils apoptosis .
The late inflammatory response started after 48hr after the pulp exposure. The monocytes were attracted to the exposure site and activated into M1 macrophages under PDGF and IL-6 influence. The M1 macrophages completed the phagocytosis process and released collagenase for cleaning the injury site. After 3days, the inflammatory phase ended, and M1 macrophages underwent apoptosis . Therefore, after three days, the histologic sections of the teeth treated with hTDM scaffold showed a non-inflammatory reaction.
The third phase is the proliferative phase which starts immediately after the inflammatory phase. The released VEGF and FGF-2 from the hTDM scaffold and macrophages  orchestrate that phase. In response to VEGF, the pericytes detached from the outer wall of the blood vessels and then differentiated into new endothelial cells. After that, they migrated to the low oxygen tension area where they formed new blood vessels through angiogenesis. The formation of new blood vessels is a pre-requisite for the last phase of the healing mechanism “Regenerative phase” . After three days, the histologic sections of the teeth treated with the hTDM scaffold showed the formation of new blood vessels at the exposure site. This observation ensured the power of the hTDM in optimizing the healing process at the physiologic level without any deviation. However, FGF-2 induced proliferation and migration of the fibroblasts and DPSCs.
During the regenerative phase, after fibroblasts’ crawled from the cell rich-zone to the exposure site, they drilled tunnels in the provisional matrix of the fibrin clot, then produced collagen, fibronectin, and proteoglycan. These products of the fibroblasts were the components of the new extracellular matrix of the regenerated pulp tissue towards the hTDM surface. Under the influence of TGF-β1 released from the hTDM, the DPSCs differentiated into pre-odontoblasts . The hTDM scaffold released dentin sialophospho-protein (DSPP) that induced the pre-odontoblasts differentiation into mature odontoblasts . But, DSPP wasn’t the only factor that controlled the odontogenic differentiation of the pre-odontoblasts. Many previous studies ensured the nano-tubular pattern and high surface wettability of hTDM. These surface properties enhance DPSCs spreading, attachment, adhesion, and odontogenic differentiation. That alters their morphology by forming long cytoplasmic processes (odontoblastic processes) toward the opened dentinal tubules at the scaffold surface . Cellular migration to the hTDM scaffold explain its ability to direct the dentinogenesis towards the exposure site rather than the pulp tissue.