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Objective: This review evaluated the use of autogenous tooth as a bone graft material in guided bone regeneration (GBR). Moreover, it compared the results of GBR using autogenous demineralized dentin, partially demineralized dentin, and mineralized dentin with or without membrane to verify its clinical advantage, effectiveness, and safety. Methods: A search was conducted in PubMed/MEDLINE, Lilacs, Embase, Cochrane, and Scopus databases. Specific criteria were established for the inclusion and exclusion of studies, including types of studies considered, target population (clinical studies: humans), evaluated intervention (studies assessing and comparing autologous demineralized dentin, partially demineralized dentin, and mineralized dentin in GBR with or without resorbable membrane), and language and publication period of articles (English and published in the last 11 years). A detailed assessment of the methodological quality of the selected studies was conducted using the JBI critical appraisal tool. Results: Based on the analysis conducted, out of 174 potentially relevant articles obtained, only 19 publications met the inclusion criteria, with three papers showing medium quality/moderate risk of bias and the rest with high quality/low risk of bias. Comparison between groups revealed stability of the newly formed bone, low marginal bone loss, clinically acceptable primary and secondary implant stability quotient (ISQ) values, and high implant survival rates after using autogenous tooth biomaterial. Conclusions: The results of this review on the use of autogenous teeth as a bone graft material in guided bone regeneration indicated that the technique has the potential to be an effective and safe treatment option. Analysis of selected studies showed favorable evidence for the use of autogenous teeth in bone regeneration, suggesting clinical benefits, most for socket preservation. These results are relevant for guiding clinical practice and assisting dental professionals in having options for biomaterials for bone regeneration. [ABSTRACT FROM AUTHOR]

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.

Abstract 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.

Abstract 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, 1H-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.

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. [ABSTRACT FROM AUTHOR]

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 3rd 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. [ABSTRACT FROM AUTHOR]

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, 1H-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. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 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. 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. Cryopreservation and freeze-drying better maintain the mechanical properties and biological activity of DDM. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Objectives: To analyze the expression of tenascin, fibronectin, collagens I and III, osteonectin, and bone morphogenetic protein 4 (BMP4) in the extracellular matrix of pulp tissue in primary teeth during physiologic root resorption. Method and Materials: Eighteen teeth were decalcified and equally distributed into 3 groups (group I, teeth with two-thirds root length; group II, teeth with one-third root length; and group III, teeth lacking the root). Results: Immunohistochemical analysis showed that all the proteins were expressed. Tenascin, collagen I, and osteonectin showed strong and broad reactivity in group I, with weaker and rare reactivity in groups II and III. The expression of fibronectin, collagen III, and BMP4 did not vary with root resorption phase. Conclusion: The expression of tenascin, collagen I, and osteonectin was reduced in the extracellular matrix and odontoblasts during root resorption. This fact may be related to the decreasing pulp response to damage and treatment during the progression of root resorption. [ABSTRACT FROM AUTHOR]

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. 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. 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. These results demonstrate the potential of using TDMP/DFCSs in periodontal regeneration. [ABSTRACT FROM AUTHOR]

Demineralized dentin matrix (DDM), derived from human teeth, is an excellent scaffold material with exciting bioactive properties to enhance bone and dental tissue engineering efficacy. In this article, first the nano-structure and bioactive components of the dentin matrix were reviewed. Then the preparation methods of DDM and the resulting properties were discussed. Next, the efficacy of DDM as a bone substitute with in vitro and in vivo properties were analyzed. In addition, the applications of DDM in tooth regeneration with promising results were described, and the drawbacks and future research needs were also discussed. With the extraction of growth factors from DDM and the nano-structural properties of DDM, previous studies also broadened the use of DDM as a bioactive carrier for growth factor delivery. In addition, due to its excellent physical and biological properties, DDM was also investigated for incorporation into other biomaterials design and fabrication, yielding great enhancements in hard tissue regeneration efficacy. [ABSTRACT FROM AUTHOR]

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Highlights • Calcium fluoride in high doses reduced the activity of dentin matrix-bound CCs in the short-term. • Calcium fluoride did not prevent the proteolytic degradation of demineralized dentin matrices in the long-term. • CaF 2 -like mineral precipitations observed on dentin after subjected to CaF 2. Abstract 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Demineralized dentin matrix (DDM), derived from human teeth, is an excellent scaffold material with exciting bioactive properties to enhance bone and dental tissue engineering efficacy. In this article, first the nano-structure and bioactive components of the dentin matrix were reviewed. Then the preparation methods of DDM and the resulting properties were discussed. Next, the efficacy of DDM as a bone substitute with in vitro and in vivo properties were analyzed. In addition, the applications of DDM in tooth regeneration with promising results were described, and the drawbacks and future research needs were also discussed. With the extraction of growth factors from DDM and the nano-structural properties of DDM, previous studies also broadened the use of DDM as a bioactive carrier for growth factor delivery. In addition, due to its excellent physical and biological properties, DDM was also investigated for incorporation into other biomaterials design and fabrication, yielding great enhancements in hard tissue regeneration efficacy.

Matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) degrade the collagen fibrils of demineralized dentin. Sodium fluoride (NaF) has previously been shown to inhibit recombinant MMP-2 and MMP-9. This study aimed to evaluate the efficacy of NaF on the inhibition of dentin-bound MMPs and CCs. Dentin beams were completely demineralized in 10% phosphoric acid. The baseline total MMP activity and dry masses were measured. Beams were assigned to test groups based on similar MMP activity and dry mass (n = 10/group), and incubated in artificial saliva (control) or artificial saliva with NaF containing 6-238 mM fluoride for 1, 7 and 21 days. The dry mass loss and MMP activities were reassessed at each time point. The proteolytic activity was screened by gelatin zymography. ICTP and CTX released to the incubation medium were analyzed as indices of MMP and cathepsin K activity, respectively. The beams were examined under scanning electron microscopy. All NaF doses reduced the dry mass loss after 21 days (p < 0.05). NaF inhibition of the total MMP activity ranged between 5 and 80%. In gelatin zymography, the bands of MMP-2 and MMP-9 became less prominent with increasing NaF levels. NaF did not decrease the released ICTP (p > 0.05). Less CTX release was detected with F ≥179 mM (p < 0.05). CaF2-like minerals were observed on the beams. High levels of NaF may slow the degradation of the dentin matrix due to the inhibition of cathepsin K. Fluoride does not seem effective in the direct inhibition of proteolysis by dentin matrix-bound MMPs. [ABSTRACT FROM AUTHOR]

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 0.15–0.22 nm, 0.22–0.43 nm and 0.43–0.63 nm 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. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Small Heterodimer Partner (SHP) interacts with diverse transcription factors such as Runx2 and regulates many cellular events including differentiation, proliferation, and energy metabolism. SHP is reported to be a positive regulator of BMP2-induced bone formation. This study aimed to clarify the role of SHP in odontoblast differentiation and matrix mineralization. Rat tooth germs were isolated, and gene expression was determined by RT-PCR and real-time PCR. Localization of SHP protein expression was identified by immunofluorescent analysis. Primary human dental pulp cells (HDPCs) were cultured with BMP2 and/or Ad-siSHP. Matrix mineralization was evaluated by Alizarin red staining. Transient transfection experiment was performed with the SHP or Dlx5 expressional plasmids and the DSPP gene. In tooth germs from post-natal days 3 to 9, BMP-2 and SHP expression increased with DSPP and DMP1 mRNA expression. In an immunostaining study, SHP was expressed in odontoblasts and surrounding osteoblasts. When HDPCs were cultured with BMP2 in mineralization-inducing medium, SHP expression also increased with an increase in DSPP expression. Down-regulation of SHP by Ad-siSHP inhibited matrix mineralization. In transient transfection experiments, overexpression of SHP was shown to enhance DSPP promoter activity through interactions between SHP and Dlx5. These results suggest that SHP may mediate BMP2 signaling to promote mineralization of the dentin matrix. [ABSTRACT FROM PUBLISHER]

Recent studies have reported collagen cross-linking after exposure to riboflavin followed by ultraviolet-A (UVA) exposure. This study is the first to investigate the effect of a riboflavin-containing primer on adhesive interface stability and dentinal matrix metalloproteinase activity. Human dentin was etched with 35% phosphoric acid, treated with 0.1% riboflavin, exposed to UVA for 2 min, and bonded with a two-step etch-and-rinse adhesive. Adhesive was applied to control specimens without riboflavin/UVA. Specimens were subjected to microtensile bond strength tests and pulled to failure after storage for 24 hrs, 6 mos, or 1 yr. Interfacial nanoleakage was evaluated by light and transmission electron microscopy. To investigate dentinal matrix metalloproteinase activity, we performed correlative zymographic assays on protein extracts obtained from phosphoric-acid-etched dentin powder with or without riboflavin/UVA treatment and XP Bond. Ultraviolet-activated riboflavin treatment increased the immediate bond strength to dentin at all aging intervals (p < 0.05 vs. control) and decreased interfacial nanoleakage in aged specimens (1 yr; p < 0.05). Zymograms revealed that riboflavin/UVA pre-treatment inhibited dentinal matrix metalloproteinase activity (especially MMP-9). In conclusion, dentinal collagen cross-linking induced by riboflavin/UVA increased immediate bond strength, stabilized the adhesive interface, and inhibited dentin matrix metalloproteinases, thereby increasing the durability of resin-dentin bonds. [ABSTRACT FROM PUBLISHER]

Abstract: Objectives: While screening the activity of potential inhibitors of matrix metalloproteinases (MMPs), due to the limited water solubility of some of the compounds, they had to be solubilized in ethanol. When ethanol solvent controls were run, they were found to partially inhibit MMPs. Thus, the purpose of this study was to compare the MMP-inhibitory activity of a series of alcohols. Methods: The possible inhibitory activity of a series of alcohols was measured against soluble rhMMP-9 and insoluble matrix-bound endogenous MMPs of dentin in completely demineralized dentin. Increasing concentrations (0.17, 0.86, 1.71 and 4.28mol/L) of a homologous series of alcohols (i.e. methanol, ethanol, propanols, butanols, pentanols, hexanols, the ethanol ester of methacrylic acid, heptanols and octanol) were compared to ethanediol, and propanediol by regression analysis to calculate the molar concentration required to inhibit MMPs by 50% (i.e. the IC50). Results: Using two different MMP models, alcohols were shown to inhibit rhMMP-9 and the endogenous proteases of dentin matrix in a dose-dependent manner. The degree of MMP inhibition by alcohols increased with chain length up to 4 methylene groups. Based on the molar concentration required to inhibit rhMMP-9 fifty percent, 2-hydroxyethylmethacrylate (HEMA), 3-hexanol, 3-heptanol and 1-octanol gave the strongest inhibition. Significance: The results indicate that alcohols with 4 methylene groups inhibit MMPs more effectively than methanol or ethanol. MMP inhibition was inversely related to the Hoy''s solubility parameter for hydrogen bonding forces of the alcohols (i.e. to their hydrophilicity). [Copyright &y& Elsevier]

Abstract: An essential aspect of tooth tissue engineering is the identification of suitable scaffolding materials to support cell growth and tissue regeneration. Treated dentin matrix (TDM) from a rat has recently been shown to be a suitable scaffold for rat dentin regeneration. However, due to species-specific differences, it remains unclear whether a similar fabrication method can be extended to human TDM and human dentin regeneration. Therefore, this present study explored the biological response to a human TDM (hTDM) created using a modified dentin treatment method. Various biological characteristics, including cell proliferation, cell migration, cell viability, and cytotoxity were investigated. To assess the inductive capacity of hTDM, dental follicle cells (DFCs) were combined with hTDM and were implanted in vivo for 8 weeks in a mouse model. The resulting grafts were studied histologically. The results showed hTDM released dentinogenic factors, indicating that hTDM could play a sustained role in odontogenesis. DFC attachment, growth, viability, and cytotoxicity on the surface of hTDM showed a notable improvement over those on calcium phosphate controls. Most importantly, in vivo hTDM induced and supported regeneration of complete dentin tissues, which expressed dentin markers DSP and DMP-1. As cells in and around the regenerated dentin were positive for human mitochondria, implanted DFCs and hTDM were responsible for the regenerated dentin tissues. In conclusion, hTDM is indicated as an ideal biomaterial for human dentin regeneration. [Copyright &y& Elsevier]

Incompletely infiltrated collagen fibrils in acid-etched dentin are susceptible to degradation. We hypothesize that degradation can occur in the absence of bacteria. Partially demineralized collagen matrices (DCMs) prepared from human dentin were stored in artificial saliva. Control specimens were stored in artificial saliva containing proteolytic enzyme inhibitors, or pure mineral oil. We retrieved them at 24 hrs, 90 and 250 days to examine the extent of degradation of DCM. In the 24-hour experimental and 90- and 250-day control specimens, we observed 5- to 6- μm-thick layers of DCM containing banded collagen fibrils. DCMs were almost completely destroyed in the 250-day experimental specimens, but not when incubated with enzyme inhibitors or mineral oil. Functional enzyme analysis of dentin powder revealed low levels of collagenolytic activity that was inhibited by protease inhibitors or 0.2% chlorhexidine. We hypothesize that collagen degradation occurred over time, via host-derived matrix metalloproteinases that are released slowly over time. [ABSTRACT FROM AUTHOR]

Objectives. To evaluate the viscoelastic properties of demineralized dentin matrix. Stress–relaxation studies were done on matrices in tension and strain elongation or creep studies were done in both tension and compression.Methods. Mid-coronal dentin disks were prepared from extracted unerupted human third molars. Disks were 0.5 mm thick for stress–relaxation or tensile creep experiments and 0.2–0.3 mm thick for compressive creep studies. ‘I’ beam specimens were prepared from dentin disks and the middle region was demineralized in 0.5 M EDTA (pH 7) for 4 days. The specimens were held in miniature friction grips in water and pulled at 100 μm s−1 to strains of 5, 10, 15 or 20% and then held for 10 min to follow the decay of stress over time. Creep was determined on demineralized dentin immersed in water in tension and in compression. Compressive creep was measured using an LVDT contact probe with loads of 0.02–0.5 N. Strain data were converted to compliance–time curves (strain/stress) and expressed as total compliance (Jt), instantaneous elastic compliance (Jo), retarded elastic compliance (JR) and viscous response (t/η) or creep.Results. The dentin matrix exhibits both stress–relaxation and creep behavior. Stress–relaxation and tensile creep were independent of strain but compressive creep rates were inversely related to compressive strain. Creep values were about 10% at low compressive strains, but fell progressively to 1% at high strains. Compliance–time curves fell with stress and came closer together. However, tensile creep was about 3% regardless of the strain.Significance. The dentin matrix exhibits viscoelastic properties, but is not linearly viscoelastic. The relatively high creep rates of the matrix under low compressive loads may cause viscous deformations in poorly infiltrated hybrid layers in resin-bonded teeth under function. [Copyright &y& Elsevier]

Transforming growth factor beta 1 (TGFβ1) is generally considered to be a potent inducer of dentin formation. In order to further assess this role, we studied the influence of this factor in human dental pulp cells on the expression of osteoadherin (OSAD), a newly described proteoglycan found in bone and dentin and suspected to play a role in mineralization events. We performed TGFβ1 stimulation both in cultures of human tooth thick slices including mature odontoblasts and in pulp explant cultures giving rise to early secretory odontoblasts or pulpal fibroblasts. We first showed by immunohistochemistry that molecules involved in TGFβ1 signal transduction, that is, membrane receptors TβRI and TβRII and intracellular proteins SMAD-2, SMAD-3, and SMAD-4, were present in human dental cells in vivo and were all maintained after culture of thick-sliced teeth in cells undergoing TGFβ1 stimulation. In this culture system, OSAD synthesis was increased in mature odontoblasts close to the TGFβ1 delivery system. In explant cultures, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated that the growth factor stimulated OSAD gene expression in early secretory odontoblasts and in pulpal fibroblasts. Taken together, these results indicate that OSAD expression is stimulated by TGFβ1 in pulpal fibroblasts and in early secretory and mature odontoblasts. We suggest that TGFβ1 in this way could control the organization and the mineralization of the extracellular matrix deposited by these cells during dentin formation. [ABSTRACT FROM AUTHOR]

Studies of odontoblast differentiation and function have been limited due to difficulties in obtaining sufficient numbers of intact cells. We describe a novel approach of laser-capture microdissection to obtain homogenous populations of pre-odontoblasts and odontoblasts from tissue sections of mouse molar cusp tips. Fixation, processing, and staining conditions were assessed for the optimal retrieval of total RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of α1 (I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA from three-day-old captured odontoblasts was sufficient in quantity and quality. Odontoblast-specific gene expression was studied by RT-PCR analysis performed in a single streptavidin-coated tube. At E15.5, Days 0 and 3, gene expression in laser-captured odontoblasts resembled that seen in vivo by in situ hybridization. The use of LCM is thus a valuable means of retrieving quality RNA from discrete populations of odontoblasts at different stages of dentinogenesis. [ABSTRACT FROM AUTHOR]

In dentinogenesis, certain growth factors, matrix proteoglycans, and proteins are directly or indirectly dependent on growth hormone. The hypothesis that growth hormone up-regulates the expression of enzymes, sialoproteins, and other extracellular matrix proteins implicated in the formation and mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with growth hormone over 5 days. The molar teeth were processed for immunohistochemical demonstration of bone-alkaline phosphatase, bone morphogenetic proteins-2 and -4, osteocalcin, osteopontin, bone sialoprotein, and E11 protein. Odontoblasts responded to growth hormone by more cells expressing bone morphogenetic protein, alkaline phosphatase, osteocalcin, and osteopontin. No changes were found in bone sialoprotein or E11 protein expression. Thus, growth hormone may stimulate odontoblasts to express several growth factors and matrix proteins associated with dentin matrix biosynthesis in mature rat molars. [ABSTRACT FROM AUTHOR]

Recent atomic force microscopy studies have shown that acid-etching dentin causes the surface to recede. The purpose of this work was to test the hypothesis that acidic solutions can cause contraction of demineralized dentin matrix. Small beams of dentin were cut from extracted human third molars. The central region of each specimen was completely demineralized in EDTA. Specimens held in a tester were immersed in acidic solutions, and the amount of tension was recorded. Test variables included gauge length, cross-sectional area, pre-strain, and pH. The results showed that immersion of demineralized dentin in acidic solutions caused tension that was directly related both to H+ concentration at pH < 2 and to pre-strain. The contractile stress development (ca. 0.2-0.4 MPa) was sufficient to cause a collapse of demineralized dentin matrix. The mechanism for this effect probably involves H+-induced conformation changes in the collagen matrix. [ABSTRACT FROM AUTHOR]

The tissue compartmentalization of phosphophoryn, osteopontin (OPN) and bone sialoprotein (BSP) in dentin was biochemically analyzed. The radicular portion of rat incisor was excised and sequentially extracted; i.e., proteins non-covalently bound to matrix such as soft tissues were solubilized with 4 M guanidinium chloride (G1), mineral-binding proteins were then extracted with 0.5 M EDTA (E), proteins non-covalently bound to the collagen matrix were extracted with 4 M guanidinium chloride (G2) after E, and finally the proteins covalently bound to the collagen matrix were extracted by bacterial collagenase digestion (C). Stains-All and Rhodamine B staining, which selectively stain calcium-binding proteins, revealed a 50∼80 kDa broad band in the E extract on 10% polyacrylamide gels. This intense band disappeared after precipitation with CaCl, indicating that the protein is phosphophoryn. After removing phosphophoryn from the E extract, 67 kDa intense and 50 kDa weak bands appeared on the gels stained with Stains-All. The 67 kDa protein was observed similarly in the G2 and C extracts. Western blots using monoclonal antibodies revealed the presence of 67 kDa BSP and 50 kDa OPN. When the tissue compartmentalization was compared, these three proteins were not detected in the G1 extract, phosphophoryn and osteopontin were localized only in the E extract, and BSP was observed not only in the E extract but also in the G2 and C extracts. These findings indicate that phosphophoryn, OPN and BSP are rapidly incorporated into the mineralized fraction and that BSP also binds to collagen-related fraction, suggesting the association with intial mineralization in the dentin. [ABSTRACT FROM AUTHOR]

During resin-bonding procedures, dentin surfaces are treated with acidic conditioners to remove the smear layer and decalcify the surface to expose the collagen fibrils of the underlying matrix. These decalcified surfaces are then either air-dried or treated with dehydrating solvents, procedures which may modify the physical properties of the dentin matrix. The purpose of this study was to evaluate the effects of dehydration on the stiffness of the decalcified dentin matrix. Small (8 x 1.7 x 0.9 mm) beams of dentin were prepared from mid-coronal dentin of extracted human molars. The ends were covered with varnish for protection, and the specimens were placed in 0.5 M EDTA for 5 days to decalcify. The stiffness was measured by both the cantilever technique and by conventional stress-strain testing. Specimens tested by the cantilever technique were sequentially exposed to water, acetone, alcohol, HEMA, and glutaraldehyde. Specimens tested by conventional stress- strain testing were exposed either to water, acetone, or HEMA, or were allowed to air-dry. The results indicate that the stiffness of decalcified human dentin matrix is very low (ca. 7 MPa), if the specimens are wet with water. As they are dehydrated, either chemically in water-miscible organic solvents or physically in air, the stiffness increases 20- to 38- fold at low strains or three- to six-fold at high strains. These increases in modulus were rapidly reversed by rehydration in water. Exposure to glutaraldehyde also produced an increase in stiffness that was not reversible when the specimens were placed back in water. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]