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The preservation of vital pulps is crucial for maintaining the physiological functions of teeth; however, vital pulp therapy (VPT) of pulpitis teeth remains a substantial challenge due to uncontrolled infection, excessive inflammation, and limited regenerative potential. Current pulp capping agents have restricted effects in the infectious and inflammatory microenvironment. To address this, a multifunctional hydrogel (TGH/DM) with antibacterial, immunomodulatory, and mineralization-promoting effects is designed. The antimicrobial peptide (AMP) and demineralized dentin matrix are incorporated into the hydrogel, achieving sustainable delivery of AMP and a cocktail of growth factors. In vitro results show that TGH/DM could kill endodontic microbiota, ameliorate inflammatory responses of human dental pulp stem cells (hDPSCs), and prompt odontogenic differentiation of inflammatory hDPSCs via activation of peroxisome proliferator-activated receptor gamma. In vivo results suggest that TGH/DM is capable of inducing M2 phenotype transformation of macrophages in mice and fostering the regeneration of the dentin-pulp complex in inflamed pulps of beagle dogs. Overall, this study first proposes the synergistic regulation of AMP and tissue-specific extracellular matrix for the treatment of pulpitis, and the advanced hydrogel provides a facile and effective way for VPT., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Regenerative endodontic therapy is a promising approach to restore the vitality of necrotic teeth, however, pulp regeneration in mature permanent teeth remains a substantial challenge due to insufficient developmental signals. The dentin is embryologically and histologically similar to the pulp, which contains a cocktail of pulp-specific structural proteins and growth factors, thus we proposed an optimizing strategy to obtain dentin matrix extracted proteins (DMEP) and engineered a DMEP functionalized double network hydrogel, whose physicochemical property was tunable by adjusting polymer concentrations to synchronize with regenerated tissues. In vitro models showed that the biomimetic hydrogel with sustained release of DMEP provided a beneficial microenvironment for the encapsulation, propagation and migration of human dental pulp stem cells (hDPSCs). The odontogenic and angiogenic differentiation of hDPSCs were enhanced as well. To elicit the mechanism hidden in the microenvironment to guide cell fate, RNA sequencing was performed and 109 differential expression of genes were identified, the majority of which enriched in cell metabolism, cell differentiation and intercellular communications. The involvement of ERK, p38 and JNK MAPK signaling pathways in the process was confirmed. Of note, in vivo models showed that the injectable and in situ photo-crosslinkable hydrogel was user-friendly for root canal systems and was capable of inducing the regeneration of highly organized and vascularized pulp-like tissues in root segments that subcutaneously implanted into nude mice. Taken together, this study reported a facile and efficient way to fabricate a cell delivery hydrogel with pulp-specific developmental cues, which exhibited promising application and translation potential in future regenerative endodontic fields., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background: To assess histologically the success of the pulp capping approach performed in traumatically exposed dogs' teeth using a novel injectable gelatin-treated dentin matrix light cured hydrogel (LCG-TDM) compared with LCG, MTA and TheraCal LC., Methods: Sixty-four dogs' teeth were divided into two groups (each including 32 teeth) based on the post-treatment evaluation period: group I: 2 weeks and group II: 8 weeks. Each group was further subdivided according to the pulp capping material into four subgroups (n = 8), with subgroup A (light-cured gelatin hydrogel) as the control subgroup, subgroup B (LCG-TDM), subgroup C (TheraCal LC), and subgroup D (MTA). Pulps were mechanically exposed in the middle of the cavity floor and capped with different materials. An assessment of periapical response was performed preoperatively and at 8 weeks. After 2 and 8-week intervals, the dogs were sacrificed, and the teeth were stained with hematoxylin-eosin and graded by using a histologic scoring system. Statistical analysis was performed using the chi-square and Kruskal-Wallis tests (p = 0.05)., Results: All subgroups showed mild inflammation with normal pulp tissue at 2 weeks with no significant differences between subgroups (p ≤ 0.05), except for the TheraCal LC subgroup, which exhibited moderate inflammation (62.5%). Absence of a complete calcified bridge was reported in all subgroups at 2 weeks, while at 8 weeks, the majority of samples in the LCG-TDM and MTA-Angelus subgroups showed complete dentin bridge formation and absence of inflammatory pulp response with no significant differences between them (p ≤ 0.05). However, the formed dentin in the LCG-TDM group was significantly thicker, with layers of ordered odontoblasts identified to create a homogeneous tubular structure and numerous dentinal tubule lines suggesting a favourable trend towards dentin regeneration. TheraCal LC samples revealed a reasonably thick dentin bridge with moderate inflammation (50%) and LCG showed heavily fibrous tissue infiltrates with areas of degenerated pulp with no signs of hard tissue formation., Conclusions: LCG-TDM, as an extracellular matrix-based material, has the potential to regenerate dentin and preserve pulp vitality, making it a viable natural alternative to silicate-based cements for healing in vivo dentin defects in direct pulp-capping procedures., (© 2024. The Author(s).)

Background: Treated dentin matrix hydrogel (TDMH) has been introduced as a novel injectable direct pulp capping material. In this regard, this study aimed to evaluate its marginal adaptation, physicochemical and rheological properties for the development of clinically feasible TDMH., Methods: TDMH was applied to the pulp floor of prepared Class I cavities (n = 5), marginal adaptation was assessed by SEM at 1000 X magnification to detect gap between dentin and filling material. Five syringes were filled with TDMH and placed between the compression plates of a universal testing machine to evaluate injectability and gelation time was also evaluated by test vial inverting method. The microstructures of lyophilized TDMH were observed by SEM. Moreover, TDMH discs (n = 5) were prepared and the water uptake (%) was determined based on the equilibrium swelling theory state of hydrogels. Its solubility was measured after one week by the ISO standard method. Rheological behaviours of TDMH (n = 5) were analysed with a rotational rheometer by computing their complex shear modulus G* and their associated storage modulus (G') and loss modulus (G''). Statistical analysis was performed using F test (ANOVA) with repeated measures and Post Hoc Test (p = 0.05)., Results: TDMH presented an overall 92.20 ± 2.95% of continuous margins. It exhibited gelation during the first minute, and injectability mean was 66 ± 0.36%. TDMH showed a highly porous structure, and the pores were interconnected with an average diameter about 5.09 ± 3.17 μm. Swelling equilibrium gradually reached at 6 days up to 377%. The prepared hydrogels and maintained their shape after absorbing over three times their original weight of water. TDMH fulfilled the requirements of ISO 6876, demonstrating a weight loss of 1.98 ± 0.09% and linear viscoelastic behaviour with G` 479.2 ± 12.7 and G`` 230.8 ± 13.8., Conclusions: TDMH provided good marginal adaptation, appropriate physicochemical and viscoelastic properties support its use as a novel direct pulp capping material in future clinical applications., (© 2023. The Author(s).)

Background: Transforming growth factor-beta 1 (TGF-β1) is a key morphogen in regenerative endodontics that plays a central role in regulating cellular functions. Various chelating agents have been shown to release this growth factor upon conditioning. The objective of the study was to evaluate TGF-β1 release from the dentin matrix upon conditioning with ethylene diamine tetra-acetate (EDTA), doxycycline hydrochloride, and propolis., Materials and Methods: Forty-two human 3 rd molar teeth were collected and coronal portion of the teeth was sectioned to obtain dentin blocks with dimensions 2 mm × 2 mm × 2 mm. The blocks were then randomly divided into three groups depending on the conditioning agent used; Group 1: 17% EDTA, Group 2: doxycycline hydrochloride (100 mg/mL), and Group 3: propolis (250 μg/mL). Conditioned blocks were placed in 0.5 mL of phosphate buffered saline and incubated for 1 week for quantification., Results: Highest TGF-β1 release was noted for propolis (0.21 ng/mL), followed by doxycycline hydrochloride (0.18 ng/mL) and 17% EDTA (0.14 ng/mL)., Conclusion: Doxycycline hydrochloride and propolis significantly enhanced the release of TGF-β1 from the dentin matrix compared to EDTA ( P < 0.05). No significant difference was observed between doxycycline hydrochloride and propolis ( P > 0.05). Doxycycline and propolis can be used as effective alternatives to EDTA during regenerative endodontic procedures., Competing Interests: There are no conflicts of interest., (Copyright: © 2023 Journal of Conservative Dentistry and Endodontics.)

Background: In recent years, treated dentin matrix (TDM) has been introduced as a bioactive hydrogel for dentin regeneration in DPC. However, no study has introduced TDM as a photocrosslinkable hydrogel with a natural photoinitiating system. Therefore, the present study aimed to explore the synthesis, characterizations and grafting optimization of injectable gelatin- glycidyl methacrylate (GMA)/TDM hydrogels as a novel photocrosslinkable pulp capping agent for dentin regeneration., Methods: G-GMA/TDM hydrogel was photocrosslinked using a new two-component photoinitiating system composed of riboflavin as a photoinitiator under visible light and glycine as a first time coinitiator with riboflavin. The grafting reaction conditions of G-GMA/TDM e.g. GMA concentration and reaction time were optimized. The kinetic parameters e.g. grafting efficiency (GE) and grafting percentage (GP%) were calculated to optimize the grafting reaction, while yield (%) was determined to monitor the formation of the hydrogel. Moreover, G-GMA/TDM hydrogels were characterized by swelling ratio, degradation degree, and cytotoxicity. The instrumental characterizations e.g. FTIR, 1 H-NMR, SEM and TGA, were investigated for verifying the grafting reaction. Statistical analysis was performed using F test (ANOVA) and Post Hoc Test (P = 0.05)., Results: The grafting reaction dramatically increased with an increase of both GMA concentration and reaction time. It was realized that the swelling degree and degradation rate of G-GMA/TDM hydrogels were significantly reduced by increasing the GMA concentration and prolonging the reaction time. When compared to the safe low and moderate GMA content hydrogels (0.048, 0.097 M) and shorter reaction times (6, 12, 24 h), G-GMA/TDM with high GMA contents (0.195, 0.391 M) and a prolonged reaction time (48 h) demonstrated cytotoxic effects against cells using the MTT assay. Also, the morphological surface of G-GMA/TDM freeze-dried gels was found more compacted, smooth and uniform due to the grafting process. Significant thermal stability was noticed due to the grafting reaction of G-GMA/TDM throughout the TGA results., Conclusions: G-GMA/TDM composite hydrogel formed by the riboflavin/glycine photoinitiating system is a potential bioactive and biocompatible system for in-situ crosslinking the activated-light pulp capping agent for dentin regeneration., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: The use of human dentin matrix could serve as an alternative to autologous, allogenic, and xenogeneic bone grafts due to its osteoinductive characteristics. The limitations of its use is tooth availability and that it is often necessary to mix it with a biomaterial., Aim: The aim of this study was to analyze a mix of two different graft materials with different reabsorption ranges when the dentin graft material was not sufficient for full socket preservation., Methods: Seven socket preservation surgeries were carried out employing a mixed graft material containing 50% dentin and 50% xenograft. After four months of recovery, the implants were positioned. At the time of the prosthesis placement and implant surgery, bone samples were collected., Results: The histologic analysis revealed no inflammatory or infective reaction against the seven biopsies. The histomorphometric graft analysis revealed an amount of New Bone of 29.03 ± 6.57% after 4 months and 34.11 ± 5.02% after 8 months., Conclusions: The two graft materials had a different volume reabsorption rate: 71% after 4 months and 90% after 8 months for dentin, and 6% after 4 months and 26% after 8 months for the xenograft. The space created by the dentin reabsorption increased the quantity of new bone.

Introduction: Human dentin is a natural acellular matrix with excellent reported biocompatibility. The aim was to fabricate a novel dentin matrix material from human dentin and investigate its applicative potential for vital pulp therapy., Methods: Digested dentin matrix extract (DDME) was fabricated using controlled enzymatic digestion under acidic conditions. The surfaces and biocompatibility of DDME were then investigated, with its effects on the odontogenic differentiation of human dental pulp cells (hDPCs) also studied. The ability of DDME to induce mineralization was assessed in a nude mouse model. The performance of DDME as a pulp capping agent was evaluated in an in situ rat model. The molecular mechanism was verified by mRNA sequencing., Results: A novel type of dentin matrix material with a uniform size of 8 μm was fabricated. DDME had a similar band compared with grinded dentin matrix, with a smaller size, and more uneven surface, as detected by Fourier-transform infrared spectrometer and X-ray photoelectron spectroscopy. DDME at low concentrations did not affect hDPC viability or proliferation, but enhanced runt-related transcription factor 2, dentin matrix acidic phosphoprotein 1, and COL1A1 (collagen type I alpha 1 chain) expression in hDPCs in vitro. DDME was superior to HA-TCP (hydroxyapatite-tricalcium phosphate) in dentin-like mineralized tissue formation after subcutaneous transplantation. In the rat model of pulpotomy, DDME showed visible curative effects. The underlying mechanism may be the inhibition of Hippo signaling following DDME treatment. DDME promoted Yes-associated protein (YAP) 1 nuclear influx, thereby enhancing the expression of DMP-1 (dentin matrix acidic phosphoprotein 1), which was reversed by YAP inhibitor treatment., Conclusions: Human DDME can be used as a biomaterial for dentin regeneration. The combined application of DDME and current pulp capping agents is a potential choice for vital pulp therapy., (Copyright © 2023 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background: A novel injectable mixture termed treated dentin matrix hydrogel (TDMH) has been introduced for restoring dentin defect in DPC. However, no study evaluated its physiological biodegradation. Therefore, the present study aimed to assess scaffold homogeneity, mechanical properties and biodegradability in vitro and in vivo and the regenerated dentin induced by TDMH as a novel pulp capping agent in human permanent teeth., Methods: Three TDMH discs were weighted, and dry/wet ratios were calculated in four slices from each disc to evaluate homogeneity. Hydrogel discs were also analyzed in triplicate to measure the compressive strength using a universal testing machine. The in vitro degradation behavior of hydrogel in PBS at 37 °C for 2 months was also investigated by monitoring the percent weight change. Moreover, 20 intact fully erupted premolars were included for assessment of TDMH in vivo biodegradation when used as a novel injectable pulp capping agent. The capped teeth were divided into four equal groups according to extraction interval after 2-, 8-, 12- and 16-weeks, stained with hematoxylin-eosin for histological and histomorphometric evaluation. Statistical analysis was performed using F test (ANOVA) and post hoc test (p = 0.05)., Results: No statistical differences among hydrogel slices were detected with (p = 0.192) according to homogeneity. TDMH compression modulus was (30.45 ± 1.11 kPa). Hydrogel retained its shape well up to 4 weeks and after 8 weeks completely degraded. Histological analysis after 16 weeks showed a significant reduction in TDMH area and a simultaneous significant increase in the new dentin area. The mean values of TDMH were 58.8% ± 5.9 and 9.8% ± 3.3 at 2 and 16 weeks, while the new dentin occupied 9.5% ± 2.8 at 2 weeks and 82.9% ± 3.8 at 16 weeks., Conclusions: TDMH was homogenous and exhibited significant stability and almost completely recovered after excessive compression. TDMH generally maintained their bulk geometry throughout 7 weeks. The in vivo response to TDMH was characterized by extensive degradation of the hydrogel and dentin matrix particles and abundant formation of new dentin. The degradation rate of TDMH matched the rate of new dentin formation., Trial Registration: PACTR201901866476410: 30/1/2019., (© 2023. The Author(s).)

Background/purpose: Demineralized dentin matrix (DDM) is used as a tissue regeneration scaffold. Effective preservation of DDM benefits clinical applications. Cryopreservation and freeze-drying may be effective methods to retain DDM mechanical properties and biological activity., Materials and Methods: Human periodontal ligament stem cells (hPDLSCs) isolated using enzymatic dissociation were identified by multidirectional differentiation and flow cytometry. DDM was prepared with EDTA and divided into four groups: fresh DDM (fDDM), room temperature-preserved DDM (rtDDM), cryopreserved DDM (cDDM) and freeze-dried DDM (fdDDM). The DDM surface morphology was observed, and microhardness was detected. Transforming growth factor-β1 (TGF-β1), fibroblast growth factor (FGF) and collagen-Ⅰ (COL-Ⅰ) concentrations in DDM liquid extracts were detected by enzyme-linked immunosorbent assay (ELISA). The hPDLSCs were cultured with DDM liquid extracts. The effect of DDM on cells proliferation was examined by CCK-8 assay. The effect of DDM on hPDLSC secreted phosphoprotein-1 (SPP1), periostin (POSTN) and COL-Ⅰ gene expression was examined by real-time qPCR., Results: cDDM dentinal tubules were larger than those of the other groups. The three storage conditions had no significant effect on DDM microhardness and COL-Ⅰ concentration. However, TGF-β1 and FGF concentrations decreased after storage, with the greatest change in rtDDM, followed by fdDDM and cDDM. The liquid extracts of fDDM, cDDM and fdDDM slightly inhibited hPDLSCs proliferation, but those of rtDDM had no significant effect. The hPDLSCs cultured with fDDM, cDDM and fdDDM liquid extracts showed increased SPP1, POSTN and COL-Ⅰ gene expression., Conclusion: Cryopreservation and freeze-drying better maintain the mechanical properties and biological activity of DDM., Competing Interests: The authors have no conflicts of interest relevant to this article., (© 2022 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V.)

Objectives: To assess clinically and radiographically the success of pulp capping procedure done in traumatically exposed permanent posterior teeth using a novel injectable treated dentin matrix hydrogel (TDMH), Biodentine, and MTA and to evaluate the formed dentin bridge under the capping materials using CBCT imaging., Materials and Methods: 45 patients subjected to accidental traumatic pulp exposures by undergraduate dental students are allocated for this study. For each patient, a pulp capping procedure was done. TDMH was formed of TDM powder and sodium alginate to be injected and then hardened in the defect area. Patients were assigned to 3 groups: TDMH, Biodentine, and MTA, respectively, and returned to the clinic after 3, 6, 12, 18, and 24 months for clinical and radiographic examinations. Tomographic data, including thickness and density of formed dentin bridges, were evaluated at the end of the study period compared to the base line. Pulp sensitivity was evaluated throughout the study period using thermal testing and electric pulp tester., Results: During the follow-up period, all patients were asymptomatic with no clinical signs and symptoms and revealed no radiographic signs of pathosis. However, tomographic evaluation showed the tested materials to have different levels of impact on formed dentin bridges with TDMH group resulted in significantly superior dentin bridges of a higher radiodensity and thickness than Biodentine and MTA., Conclusions: TDMH has a greater potential to induce dentin bridge formation than Biodentine and MTA under standardized conditions. Additionally, CBCT imaging was confirmed as a non-invasive and inclusive approach to evaluate the formed dentin bridges after pulp capping procedure., Clinical Relevance: Direct pulp capping can be done successfully with this novel injectable pulp capping material in future clinical applications., Trial Registration: PACTR201901866476410., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Commonly recognized mechanisms of the xenogeneic-extracellular matrix-based regenerative medicine include timely degradation, release of bioactive molecules, induced differentiation of stem cells, and well-controlled inflammation. This process is most feasible for stromal tissue reconstruction, yet unsuitable for non-degradable scaffold and prefabricated-shaped tissue regeneration, like odontogenesis. Treated dentin matrix (TDM) has been identified as a bioactive scaffold for dentin regeneration. This study explored xenogeneic porcine TDM (pTDM) for induced odontogenesis. The biological characteristics of pTDM were compared with human TDM (hTDM). To investigate its bioinductive capacities on allogeneic dental follicle cells (DFCs) in the inflammation microenvironment, pTDM populated with human DFCs were co-cultured with human peripheral blood mononuclear cells (hPBMCs), and pTDM populated with rat DFCs were transplanted into rat subcutaneous model. The results showed pTDM possessed similar mineral phases and bioactive molecules with hTDM. hDFCs, under the induction of pTDM and hTDM, expressed similar col-I, osteopontin and alkaline phosphatase (ALP) (all expressed by odontoblasts). Whereas, the expression of col-I, dentin matrix protein-1 (DMP-1) and bone sialoprotein (BSP) were down-regulated when cocultured with hPBMCs. The xenogeneic implants inevitably initiated Th1 inflammation (up-regulated CD8, TNF-α, IL-1 β , etc) in vivo . However, the biomineralization of pre-dentin and cementum were still processed, and collagen fibrils, odontoblast-like cells, fibroblasts contributed to odontogenesis. Although partially absorbed at 3 weeks, the implants were positively expressed odontogenesis-related-proteins like col-I and DMP-1. Taken together, xenogeneic TDM conserved ultrastructure and molecules for introducing allogeneic DFCs to odontogenic differentiation, and promoting odontogenesis and biomineralization in vivo . Yet effective immunomodulation methods warrant further explorations., (Creative Commons Attribution license.)

Objectives: To produce a novel injectable treated dentin matrix hydrogel (TDMH) to be used as a novel pulp-capping agent for dentin regeneration compared with Biodentine and MTA., Materials and Methods: Thirty intact fully erupted premolars scheduled to be extracted for orthodontic reasons were included. Pulps were mechanically exposed in the middle of the cavity floor. TDMH was composed of TDM powder (500-μm particle size) and sodium alginate as an injectable scaffold. The capped teeth were divided into three equal groups (n = 10): TDMH, Biodentine, and MTA respectively. Clinical examination and assessment of periapical response were performed. The teeth were extracted after 2-weeks and 2-month intervals, stained with hematoxylin-eosin, and categorized by using a histologic scoring system. Statistical analysis was performed using chi-square and Kruskal-Wallis test (p = 0.05)., Results: All teeth were vital during observation periods. Histological analysis after 2 months showed complete dentin bridge formation and absence of inflammatory pulp response with no significant differences between groups. However, the formed dentin was significantly thicker with the TDMH group with layers of well-arranged odontoblasts that were found to form a homogenous tubular structure with numerous dentinal tubule lines showing a positive trend to dentin regeneration., Conclusions: TDMH could achieve dentin regeneration and conservation of pulp vitality and might serve as a feasible natural substitute for silicate-based cements in restoring in vivo dentin defect in direct pulp-capping procedure., Trial Registration: PACTR201901866476410.

Objective: Periodontal tissue engineering is an attractive approach for restoring periodontal-supporting structures and functions. However, complete periodontal regeneration has not been accomplished. Previous studies demonstrated the feasibility of using cell sheets and treated dentin matrix (TDM) to regenerate bio-roots., Methods: In this study, we regenerated periodontal tissue using cell sheets combined with TDM particles (TDMPs). Human dental follicle cells (hDFCs) were isolated and characterized. Human dental follicle cells sheets (hDFCSs) and human TDMPs (hTDMP) were fabricated and characterized. The osteogenic effect of hTDMP was evaluated on human bone marrow stromal cells (hBMSCs) in vitro and a rat calvarial bone defect in vivo. Real-time PCR, western blotting, radiograph analysis, and histological analysis were performed to evaluate the periodontal induction capacity of hTDMP. One-wall periodontal intrabony defects were prepared to evaluate the periodontal regeneration capacity of TDMP/DFCSs on beagle dogs., Results: The results showed that hDFCs were mesenchymal stem cells. hTDMP promoted the proliferation and osteogenic differentiation of hBMSCs. New bone formation was observed in the rat calvarial bone defect zone in both the hTDMP and hydroxyapatite/β-tricalcium phosphate groups. Periodontal-like tissues showed better regeneration in the canine TDMP+DFCS group than in the other groups., Significance: These results demonstrate the potential of using TDMP/DFCSs in periodontal regeneration., (Copyright © 2019. Published by Elsevier Inc.)

Impact Statement: This research has been conducted with the aim to contribute to the development of treatment modalities for the reconstruction of lost/damaged mineralized tissues. Currently, determining the most appropriate stromal cell population and signaling cues stands at the core of developing effective treatments. We provide new insights into the effect of innate inductive cues found in human dentin matrix components, on the osteogenic differentiation of various human stromal cell types. The effects of dentin extracellular matrix components on umbilical cord mesenchymal stromal cells have not been investigated before. The findings of this study could underpin translational research based on the development of techniques for mineralized tissue engineering and will be of great interest for the readership of Tissue Engineering Part A.

Lesions on tissues and organs critically affect quality of life, due to severe tissue defects that are threatening. Tissue repair and functional reconstruction are concurrent challenges in modern medicine. Tissue engineering brings hope for tissue and organ regeneration. Scaffolds provide a microenvironment for cell growth, proliferation and differentiation. Moreover, scaffolds influence the size and morphology of regenerated tissues. Dentin matrix, which is a natural bioactive and biocompatible scaffold, has become a research hotspot in recent years and has been widely used in tissue engineering. Studies on the use of dentin matrix as scaffolds have made a series of important progress in tooth root, periodontal, dental pulp and bone regeneration. This review demonstrates the biological characteristics of dentin matrix as bioactive scaffolds, describes the application of dentin matrix in tissue regeneration and provides a theoretical basis for the use of a dentin matrix in clinical applications.

Objective: Matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) are two distinct enzymatic pathways responsible for the degradation of collagen fibrils in demineralized dentin. NaF and KF have been shown to inhibit salivary MMP-2, -9 and CCs. This study investigated the inhibitory effect of calcium fluoride (CaF 2 ) on the dentin matrix-bound MMPs and CCs., Design: Phosphoric acid (10%)-demineralized dentin beams (1 × 2×6 mm) were incubated at 37 °C in an 1 ml of artificial saliva (AS, control), or AS with 6, 12, 24, 48, 120. 179 and 238 mM F containing CaF 2 (n = 10/group) for 1, 7 and 21 days. All groups were further incubated in AS only for 6 months. Total MMP activity, dry mass loss, CTX and hydroxyproline (HYP) analyses were performed after each incubation. The beams were examined under scanning electron microscopy (SEM). MMP-2 and MMP-9 activities were screened with gelatin zymography. Data were analyzed by using ANOVA and Tukey HSD tests (p = .05)., Results: The total MMP activity was similar for all groups after 21 days and 6 months. After 21 days, the cumulative mass loss and CTX levels were lower compared to control for the CaF 2 ≥48 and CaF 2 ≥120 mM, respectively (p < .05). After 6 months, no significant difference was detected in the dry mass loss and CTX compared to the control (p > .05), whereas HYP level was higher with F 24 and 238 mM groups. CaF 2 -like minerals were observed on the beams under SEM. There was no gelatinase inhibition in zymography., Conclusion: CaF 2 does not prevent the degradation of demineralized dentin matrices due to the catalytic activity of MMPs and CCs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: The purpose of this study was to investigate the effects of aqueous solutions of different concentrations of titanium tetrafluoride (TiF 4 ) on dentin matrix stability up to six months., Design: Dentin specimens prepared from fifteen nonerupted molars were demineralized and randomly distributed into groups: 2.5% TiF 4 , 4% TiF 4, 1000 ppm NaF, and control (distilled water). The modulus of elasticity (ME) and dry masses of the dentin matrix were determined at baseline, and up to 6 months following treatment. Collagen solubilization was estimated by hydroxyproline (HYP) quantification in the simulated body fluid used to store the dentin specimens., Results: The 2.5% TiF 4 group presented higher ME immediately after treatment, and at 3 and 6 months storage, whereas 4% TiF 4 showed higher means at 3 and 6 months (p < 0.001). No significant differences were observed among the groups over time (p = 0.9325). However, the 2.5% TiF 4 group showed significantly higher ME than the control group, immediately after treatment. All the groups presented significantly higher mass change immediately, compared with 3 and 6 months (p < 0.0001). Except for the 4% TiF 4 group, HYP release was higher in the first quarter (p = 0.0152), when no significant differences were found among the groups. In the second quarter, the means were significantly higher in the 2.5% TiF 4 and 4% TiF 4 groups. The group treated with 2.5% TiF 4 had a statistically higher HYP release than the control group., Conclusion: An aqueous solution of 2.5% TiF 4 increases the immediate stiffness values, but does not stabilize the collagenous dentin matrix., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The purpose of this study is to investigate the interaction of neat ethanol with bound and non-bound water in completely demineralized dentin that is fully hydrated, using molecular dynamics (MD) simulation method. The key to creating ideal resin-dentin bonds is the removal of residual free water layers and its replacement by ethanol solvent in which resin monomers are soluble, using the ethanol wet-bonding technique. The test null hypotheses were that ethanol cannot remove any collagen-bound water, and that ethanol cannot infiltrate into the spacing between collagen triple helix due to narrow interlayer spacing. Collagen fibrillar structures of overlap and gap regions were constructed by aligning the collagen triple helix of infinite length in hexagonal packing. Three layers of the water molecules were specified as the layers of 0.15-0.22nm, 0.22-0.43nm and 0.43-0.63nm from collagen atoms by investigating the water distribution surrounding collagen molecules. Our simulation results show that ethanol molecules infiltrated into the intermolecular spacing in the gap region, which increased due to the lateral shrinkage of the collagen structures in contact with ethanol solution, while there was no ethanol infiltration observed in the overlap region. Infiltrated ethanol molecules in the gap region removed residual water molecules via modifying mostly the third water layer (50% decrease), which would be considered as a loosely-bound water layer. The first and second hydration layers, which would be considered as tightly bound water layers, were not removed by the ethanol molecules, thus maintaining the helical structures of the collagen molecules., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Objectives: To identify proteoglycans (PGs) and collagen fibrils (CF) within human dentin by means of a dual immunofluorescent labeling technique and to investigate the monomer infiltration of two etch-and-rinse adhesives to tosyl-phenylalanine chloromethyl-ketone (TPCK)-treated trypsin (TRY)-pretreated dentin., Methods: Thirty-micrometer sections of middle coronal dentin were obtained and etched with 37% phosphoric acid gel for 15 s. After preconditioning with or without TRY digestion, the sections were subjected to dual immunofluorescent labeling and observed with a confocal laser scanning microscope (CLSM). Demineralized dentin matrixes treated with or without TRY were observed with field emission scanning electron microscope (FE-SEM). Two etch-and-rinse adhesives, Adper Single Bond 2 (SB) and Prime & Bond NT (PBNT), were applied to the dentin surfaces that were pretreated with or without TRY. The thickness of the hybrid layers was evaluated under confocal micro-Raman spectroscopy and analyzed with a two-way ANOVA., Results: Green and red fluorescence was used to represent the PGs and the CF that were colocalized in the same section with different distributions. PGs were localized in the lumens of the dentin tubules and in peritubular dentin, while the type-I collagen fibrils were localized in intertubular dentin and peritubular dentin. After preconditioning with TRY digestion, the red fluorescence decreased or disappeared, the organic filaments in the lumens of the dentin tubules disappeared, the tubules were enlarged, and the hybrid layer thickness for adhesives bonded to the TRY-pretreated dentin surfaces were significantly increased (p<0.001 for both SB and PBNT)., Significance: The dual immunofluorescence labeling methodology can be used to study the human dentin matrix without decalcifying the entire dentin fragment. Proteoglycans were localized in the lumens of the dentin tubules and in peritubular dentin, which could depress the infiltration of the adhesive resin monomers. The use of TRY digestion increased the thickness of the hybrid layer created by the tested two-step etch-and-rinse adhesive., (Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Cryopreservation has been identified as an efficient approach to preserve tissue engineered products for a long term. Our prior studies have suggested that the treated dentin matrix (TDM) could be an ideal bioactive scaffold for dental tissue regeneration. In this study, we hypothesize that the cryopreservation could effectively maintain the survival and viability of dentinogenesis-related proteins of TDM and the cryopreserved dentin matrix (CDM) would provide the suitable biological scaffold and inductive microenvironment for the regeneration of dentin-pulp like tissue. CDM-3 and CDM-6 were prepared by cryopreserving TDM in liquid nitrogen (-196 °C) with cryoprotectant for 3 months and 6 months, respectively. Various biological characteristics of CDM, including mechanical properties, cell proliferation, and odontogenesis ability, were investigated. To further evaluate the inductive capacity of CDM, human dental follicle cells were encapsulated within CDM, and implanted the scaffold into a mouse model for 8 weeks, and the grafts were harvested and assessed histologically. The CDM showed superior mechanical properties than TDM. Compared to TDM, CDM can release more dentinogenesis-related proteins due to the larger pore diameter. Cell proliferation with the addition of CDM extract liquid was similar to that of TDM in the first five days. Human dental follicle cells, under the effect of CDM extract liquid, highly expressed bone sialoprotein, collagen-1, alkaline phosphatase, indicating that CDM, regarded as the inductive microenvironment, plays an important role in odontogenesis. Most importantly, in vivo, CDM could induce dental follicle cells to regenerate new dentin-pulp like tissues, such as dentinal tubules, predentin, collagen fibers, nerves, and blood vessels which were positive for dentin sialophosphoprotein, dental matrix protein-1, Tubulin, and collagen-1. In conclusion, CDM is an ideal biological scaffold material for human dentin-pulp like tissue regeneration. These findings indicated that TDM could be preserved as the tissue engineering scaffold that is readily available for patient treatments. Furthermore, the success of cryopreservation of TDM may also provide an insight into preserving other bioactive scaffold materials of tissue engineering., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The standard treatment modality for teeth with irreversibly damaged dental pulp is root canal therapy, which involves complete removal of the soft tissue and obturation with a synthetic material. So far, research studies show that the combination of stem cells with a suitable scaffold material and transplantation into the root canal may result in the generation of pulplike tissue and the formation of tubular dentin. Because of the technical challenges associated with such a procedure, cell-free alternatives that take advantage of the dental pulp's inherent regenerative capacity because of endogenous stem cell populations and bioactive dentin matrix components need to be considered and explored. Following the tissue engineering approach, this includes (1) a bioactive scaffold, (2) growth and differentiation factors from dentin, and (3) the recruitment of stem cells from resident populations within the pulp or from the periapical region. If this concept proved to be successful, cell-free therapies may be a safer, more practical, feasible, and affordable approach to dental pulp regeneration., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)