Deletion or mutation of dentin matrix protein 1 （DMP1） leads to hypophosphatemic rickets and defects within the dentin. However, it is largely unknown if this pathological change is a direct role of DMP1 or an indir...Deletion or mutation of dentin matrix protein 1 （DMP1） leads to hypophosphatemic rickets and defects within the dentin. However, it is largely unknown if this pathological change is a direct role of DMP1 or an indirect role of phosphate （Pi） or both. It has also been previously shown that Klotho-deficient mice, which displayed a high Pi level due to a failure of Pi excretion, causes mild defects in the dentinal structure. This study was to address the distinct roles of DMP1 and Pi homeostasis in cell differentiation, apoptosis and mineralization of dentin and enamel. Our working hypothesis was that a stable Pi homeostasis is critical for postnatal tooth formation, and that DMP1 has an antiapoptotic role in both amelogenesis and dentinogenesis. To test this hypothesis, Dmpl-null （Dmpl-/-）, Klotho-deficient （kl/kl）, Dmpl/Klotho-double-deficient （Dmpl-/-/kl/kl） and wild-type （WT） mice were killed at the age of 6 weeks. Combinations of X-ray, microcomputed tomography （I^CT）, scanning electron microscopy （SEM）, histology, apoptosis and immunohistochemical methods were used for characterization of dentin, enamel and pulp structures in these mutant mice. Our results showed that Dmpl-/- （a low Pi level） or kl/kl（a high Pi level） mice displayed mild dentin defects such as thin dentin and a reduction of dentin tubules. Neither deficient mouse line exhibited any apparent changes in enamel or pulp structure. However, the double-deficient mice （a high Pi level） displayed severe defects in dentin and enamel structures, including loss of dentinal tubules and enamel prisms, as well as unexpected ectopic ossification within the pulp root canal. TUNEL assay showed a sharp increase in apoptotic cells in ameloblasts and odontoblasts. Based on the above findings, we conclude that DMP1 has a protective role for odontoblasts and ameloblasts in a pro-apoptotic environment （a high Pi level）.展开更多
The tooth root cementum is a thin, mineralized tissue covering the root dentin that is present primarily as acellular cementum on the cervical root and cellular cementum covering the apical root. While cementum shares...The tooth root cementum is a thin, mineralized tissue covering the root dentin that is present primarily as acellular cementum on the cervical root and cellular cementum covering the apical root. While cementum shares many properties in common with bone and dentin, it is a unique mineralized tissue and acellular cementum is critical for attachment of the tooth to the surrounding periodontal ligament （PDL）. Resources for methodologies for hard tissues often overlook cementum and approaches that may be of value for studying this tissue. To address this issue, this report offers detailed methodology, as well as comparisons of several histological and immunohistochemical stains available for imaging the cementum-PDL complex by light microscopy. Notably, the infrequently used Alcian blue stain with nuclear fast red counterstain provided utility in imaging cementum in mouse, porcine and human teeth. While no truly unique extracellular matrix markers have been identified to differentiate cementum from the other hard tissues, immunohistochemistry for detection of bone sialoprotein （BSP）, osteopontin （OPN）, and dentin matrix protein 1 （DMP1） is a reliable approach for studying both acellular and cellular cementum and providing insight into developmental biology of these tissues. Histoloeical and immunohistochemical aooroaches Drovide insight on developmental biology of cementum.展开更多
Dentin matrix protein 1（DMP1） is essential to odontogenesis. Its mutations in human subjects lead to dental problems such as dental deformities, hypomineralization and periodontal impairment. Primarily, DMP1 is cons...Dentin matrix protein 1（DMP1） is essential to odontogenesis. Its mutations in human subjects lead to dental problems such as dental deformities, hypomineralization and periodontal impairment. Primarily, DMP1 is considered as an extracellular matrix protein that promotes hydroxyapatite formation and activates intracellular signaling pathway via interacting with avb3 integrin. Recent in vitro studies suggested that DMP1 might also act as a transcription factor. In this study, we examined whether full-length DMP1 could function as a transcription factor in the nucleus and regulate odontogenesis in vivo. We first demonstrated that a patient with the DMP1M1 V mutation, which presumably causes a loss of the secretory DMP1 but does not affect the nuclear translocation of DMP1, shows a typical rachitic tooth defect. Furthermore, we generated transgenic mice expressingNLSDMP1, in which the endoplasmic reticulum（ER） entry signal sequence of DMP1 was replaced by a nuclear localization signal（NLS） sequence, under the control of a 3.6 kb rat type I collagen promoter plus a 1.6 kb intron 1. We then crossbred theNLSDMP1 transgenic mice with Dmp1 null mice to express the NLSDMP1 in Dmp1-deficient genetic background. Although immunohistochemistry demonstrated thatNLSDMP1 was localized in the nuclei of the preodontoblasts and odontoblasts, the histological, morphological and biochemical analyses showed that it failed to rescue the dental and periodontal defects as well as the delayed tooth eruption in Dmp1 null mice. These data suggest that the full-length DMP1 plays no apparent role in the nucleus during odontogenesis.展开更多
The blood-brain barrier （BBB） is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed mo...The blood-brain barrier （BBB） is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein I （DMP1）-proteoglycan （PG）, which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMPI-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 （S89） in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine （S89G）, which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that reg- ulated BBB formation, but also assigned a new function to DMP1-PG.展开更多
Treated dentin matrix(TDM)is an ideal scaffold material containing multiple extracellular matrix factors.The canonical Wnt signaling pathway is necessary for tooth regeneration.Thus,this study investigated whether the...Treated dentin matrix(TDM)is an ideal scaffold material containing multiple extracellular matrix factors.The canonical Wnt signaling pathway is necessary for tooth regeneration.Thus,this study investigated whether the TDM can promote the odontogenic differentiation of human dental pulp stem cells(hDPSCs)and determined the potential role of Wnt/β-catenin signaling in this process.Different concentrations of TDM promoted the dental differentiation of the hDPSCs and meanwhile,the expression of GSK3βwas decreased.Of note,the expression of the Wnt/β-catenin pathway-related genes changed significantly in the context of TDM induction,as per RNA sequencing(RNA seq)data.In addition,the experiment showed that new dentin was visible in rat mandible cultured with TDM,and the thickness was significantly thicker than that of the control group.In addition,immunohistochemical staining showed lower GSK3βexpression in new dentin.Consistently,the GSK3βknockdown hDPSCs performed enhanced odotogenesis compared with the control groups.However,GSK3βoverexpressing could decrease odotogenesis of TDM-induced hDPSCs.These results were confirmed in immunodeficient mice and Wistar rats.These suggest that TDM promotes odontogenic differentiation of hDPSCs by directly targeting GSK3βand activating the canonical Wnt/β-catenin signaling pathway and provide a theoretical basis for tooth regeneration engineering.展开更多
It is still a huge challenge for bone regenerative biomaterial to balance its mechanical,biological and biodegradable properties.In the present study,a new composite material including treated dentin matrix(TDM)andα-...It is still a huge challenge for bone regenerative biomaterial to balance its mechanical,biological and biodegradable properties.In the present study,a new composite material including treated dentin matrix(TDM)andα-calcium sulphate hemihydrate(α-CSH)was prepared.The optimal composition ratio between TDM andα-CSH was explored.The results indicate that both components were physically mixed and structurally stable.Its compressive strength reaches up to 5.027±0.035 MPa for 50%TDM/α-CSH group,similar to human cancellous bone tissues.Biological experiments results show that TDM/α-CSH composite exhibits excellent biocompatibility and the expression of osteogenic related genes and proteins(ALP,RUNX2,OPN)is significantly increased.In vivo experiments suggest that the addition of TDM for each group(10%,30%,50%)effectively promotes cell proliferation and osteomalacia.In addition,50%of the TDM/α-CSH combination displays optimal osteoconductivity.The novel TDM/α-CSH composite is a good candidate for certain applications in bone tissue engineering.展开更多
Background:Ankylosing spondylitis(AS)is the most common rheumatic condition that is slowly progressive and predominantly affects adolescents.Pathological bone formation associated with AS is an important cause of disa...Background:Ankylosing spondylitis(AS)is the most common rheumatic condition that is slowly progressive and predominantly affects adolescents.Pathological bone formation associated with AS is an important cause of disability.The aim of the study was to investigate the possible involvement of the genes related to endochondral ossification and ectopia ossification in genetic susceptibility to AS in a Chinese Han population.Methods:Sixty-eight single nucleotide polymorphisms(SNPs)from 13 genes were genotyped in discovery cohorts including 300 AS patients and 180 healthy controls.The rs10019009 in dentin matrix protein 1(DMP1)gene shown as association with AS after multiple testing corrections in discovery cohorts was replicated in a validation independent cohort of 620 AS patients and 683 healthy controls.The rs 10019009 was assessed with bioin fomlatics including phylogenetic context,F-SNP and FastSNP functional predictions,secondary structure prediction,and molecular modeling.We performed a functional analysis of rs10019009 via reverse transcription-polymerase chain reaction,alkaline phosphatase(ALP)activity in human osteosarcoma U2OS cells.Results:Interestingly,the SNP rs10019009 was associated with AS in both the discovery cohort(P=0.0012)and validation cohort(P=0.0349),as well as overall(P=0.0004)in genetic case-control association analysis.After a multivariate logistic regression analysis,the effect of this genetic variant was observed to be independent of linkage disequilibrium.Via bioinformatics analysis,it was found that the amino acid change of the rs 10019009 led to changes of SNP function,secondary structure,tertiary confomlation,and splice mode.Finally,functional analysis ofrsl0019009 in U2OS cells demonstrated that the risk T allele of the rsl0019009 increased enzymatic activity of ALP,compared to that of the nonrisk allele(P=0.0080).Conclusions:These results suggested that the DMP1 gene seems to be involved in genetic predisposition to AS,which may contribute to the ectopic mineralization or ossification in AS.In addition,DMP1 gene may be a promising intervention target for AS in the future.展开更多
基金supported by NIH grants Jian-Quan Feng (DE018486) and to Chun-Lin Qin (DE005092)State Key Laboratory of Oral Diseases Open Funding (SKLODOF2010-03) to Jian-Quan Feng
文摘Deletion or mutation of dentin matrix protein 1 （DMP1） leads to hypophosphatemic rickets and defects within the dentin. However, it is largely unknown if this pathological change is a direct role of DMP1 or an indirect role of phosphate （Pi） or both. It has also been previously shown that Klotho-deficient mice, which displayed a high Pi level due to a failure of Pi excretion, causes mild defects in the dentinal structure. This study was to address the distinct roles of DMP1 and Pi homeostasis in cell differentiation, apoptosis and mineralization of dentin and enamel. Our working hypothesis was that a stable Pi homeostasis is critical for postnatal tooth formation, and that DMP1 has an antiapoptotic role in both amelogenesis and dentinogenesis. To test this hypothesis, Dmpl-null （Dmpl-/-）, Klotho-deficient （kl/kl）, Dmpl/Klotho-double-deficient （Dmpl-/-/kl/kl） and wild-type （WT） mice were killed at the age of 6 weeks. Combinations of X-ray, microcomputed tomography （I^CT）, scanning electron microscopy （SEM）, histology, apoptosis and immunohistochemical methods were used for characterization of dentin, enamel and pulp structures in these mutant mice. Our results showed that Dmpl-/- （a low Pi level） or kl/kl（a high Pi level） mice displayed mild dentin defects such as thin dentin and a reduction of dentin tubules. Neither deficient mouse line exhibited any apparent changes in enamel or pulp structure. However, the double-deficient mice （a high Pi level） displayed severe defects in dentin and enamel structures, including loss of dentinal tubules and enamel prisms, as well as unexpected ectopic ossification within the pulp root canal. TUNEL assay showed a sharp increase in apoptotic cells in ameloblasts and odontoblasts. Based on the above findings, we conclude that DMP1 has a protective role for odontoblasts and ameloblasts in a pro-apoptotic environment （a high Pi level）.
基金supported (in part) by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health
文摘The tooth root cementum is a thin, mineralized tissue covering the root dentin that is present primarily as acellular cementum on the cervical root and cellular cementum covering the apical root. While cementum shares many properties in common with bone and dentin, it is a unique mineralized tissue and acellular cementum is critical for attachment of the tooth to the surrounding periodontal ligament （PDL）. Resources for methodologies for hard tissues often overlook cementum and approaches that may be of value for studying this tissue. To address this issue, this report offers detailed methodology, as well as comparisons of several histological and immunohistochemical stains available for imaging the cementum-PDL complex by light microscopy. Notably, the infrequently used Alcian blue stain with nuclear fast red counterstain provided utility in imaging cementum in mouse, porcine and human teeth. While no truly unique extracellular matrix markers have been identified to differentiate cementum from the other hard tissues, immunohistochemistry for detection of bone sialoprotein （BSP）, osteopontin （OPN）, and dentin matrix protein 1 （DMP1） is a reliable approach for studying both acellular and cellular cementum and providing insight into developmental biology of these tissues. Histoloeical and immunohistochemical aooroaches Drovide insight on developmental biology of cementum.
基金supported by NIH grants DE018486 and R56 DE022789 to Jian-Quan Feng, DE023365 to Yong-Bo Lu and a scholarship from the Chinese State Scholarship Fund to Shu-Xian Lin (2010627108)
文摘Dentin matrix protein 1（DMP1） is essential to odontogenesis. Its mutations in human subjects lead to dental problems such as dental deformities, hypomineralization and periodontal impairment. Primarily, DMP1 is considered as an extracellular matrix protein that promotes hydroxyapatite formation and activates intracellular signaling pathway via interacting with avb3 integrin. Recent in vitro studies suggested that DMP1 might also act as a transcription factor. In this study, we examined whether full-length DMP1 could function as a transcription factor in the nucleus and regulate odontogenesis in vivo. We first demonstrated that a patient with the DMP1M1 V mutation, which presumably causes a loss of the secretory DMP1 but does not affect the nuclear translocation of DMP1, shows a typical rachitic tooth defect. Furthermore, we generated transgenic mice expressingNLSDMP1, in which the endoplasmic reticulum（ER） entry signal sequence of DMP1 was replaced by a nuclear localization signal（NLS） sequence, under the control of a 3.6 kb rat type I collagen promoter plus a 1.6 kb intron 1. We then crossbred theNLSDMP1 transgenic mice with Dmp1 null mice to express the NLSDMP1 in Dmp1-deficient genetic background. Although immunohistochemistry demonstrated thatNLSDMP1 was localized in the nuclei of the preodontoblasts and odontoblasts, the histological, morphological and biochemical analyses showed that it failed to rescue the dental and periodontal defects as well as the delayed tooth eruption in Dmp1 null mice. These data suggest that the full-length DMP1 plays no apparent role in the nucleus during odontogenesis.
基金We thank Dr. Chunlin Qin （Bayler colleage of dentistry, Texas A＆M University） for providing us DMP1 antibody. This study was supported by China National Key Research and Development Program （2016YFA0100801 YS）, and the National Natural Science Foundation of China （Grant Nos. 8133030 YS and 31620103904 YS）, and grants： 2016YFC102705 YS 2014BAI04B07 WZL+1 种基金 81470715 YS TJ1504219036 WZL： 2017BR009 YS.
文摘The blood-brain barrier （BBB） is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein I （DMP1）-proteoglycan （PG）, which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMPI-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 （S89） in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine （S89G）, which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that reg- ulated BBB formation, but also assigned a new function to DMP1-PG.
基金This study was funded by the National Natural Science Foundation of China(grant numbers 31670994,U1904145,and 81901039)Nature Science Foundation of Henan province(grant number 182300410340)and Union project of Medical and Technology Research Program of Henan Province(grant number LHGJ20190191).
文摘Treated dentin matrix(TDM)is an ideal scaffold material containing multiple extracellular matrix factors.The canonical Wnt signaling pathway is necessary for tooth regeneration.Thus,this study investigated whether the TDM can promote the odontogenic differentiation of human dental pulp stem cells(hDPSCs)and determined the potential role of Wnt/β-catenin signaling in this process.Different concentrations of TDM promoted the dental differentiation of the hDPSCs and meanwhile,the expression of GSK3βwas decreased.Of note,the expression of the Wnt/β-catenin pathway-related genes changed significantly in the context of TDM induction,as per RNA sequencing(RNA seq)data.In addition,the experiment showed that new dentin was visible in rat mandible cultured with TDM,and the thickness was significantly thicker than that of the control group.In addition,immunohistochemical staining showed lower GSK3βexpression in new dentin.Consistently,the GSK3βknockdown hDPSCs performed enhanced odotogenesis compared with the control groups.However,GSK3βoverexpressing could decrease odotogenesis of TDM-induced hDPSCs.These results were confirmed in immunodeficient mice and Wistar rats.These suggest that TDM promotes odontogenic differentiation of hDPSCs by directly targeting GSK3βand activating the canonical Wnt/β-catenin signaling pathway and provide a theoretical basis for tooth regeneration engineering.
基金the National Natural Science Foundation of China(grant numbers 31670994,U1904145 and 81901039)Nature Science Foundation of Henan Province(grant numbers 182300410340,222300420569)+4 种基金Henan Medical Science and Technology Research Project(grant number SBGJ202002073,SBGJ202002075)Science and Technology Department of Science and Technology Research(grant number 182102310456)Henan Province Middle-Aged And Young Health Science and Technology Innovation Leader Training Project(grant number YXKC2021014)Scientific Research and Innovation Team of The First Affiliated Hospital of Zhengzhou University(grant number QNCXTD2023021)Union project of Medical and Technology Research Program of Henan Province(grant number LHGJ20190191).
文摘It is still a huge challenge for bone regenerative biomaterial to balance its mechanical,biological and biodegradable properties.In the present study,a new composite material including treated dentin matrix(TDM)andα-calcium sulphate hemihydrate(α-CSH)was prepared.The optimal composition ratio between TDM andα-CSH was explored.The results indicate that both components were physically mixed and structurally stable.Its compressive strength reaches up to 5.027±0.035 MPa for 50%TDM/α-CSH group,similar to human cancellous bone tissues.Biological experiments results show that TDM/α-CSH composite exhibits excellent biocompatibility and the expression of osteogenic related genes and proteins(ALP,RUNX2,OPN)is significantly increased.In vivo experiments suggest that the addition of TDM for each group(10%,30%,50%)effectively promotes cell proliferation and osteomalacia.In addition,50%of the TDM/α-CSH combination displays optimal osteoconductivity.The novel TDM/α-CSH composite is a good candidate for certain applications in bone tissue engineering.
基金Financial support and sponsorship This study was supported by grants from the National Natural Science Foundation of China(No.81371920)and the Natural Science Foundation of Liaoning Province(No.2013022060).
文摘Background:Ankylosing spondylitis(AS)is the most common rheumatic condition that is slowly progressive and predominantly affects adolescents.Pathological bone formation associated with AS is an important cause of disability.The aim of the study was to investigate the possible involvement of the genes related to endochondral ossification and ectopia ossification in genetic susceptibility to AS in a Chinese Han population.Methods:Sixty-eight single nucleotide polymorphisms(SNPs)from 13 genes were genotyped in discovery cohorts including 300 AS patients and 180 healthy controls.The rs10019009 in dentin matrix protein 1(DMP1)gene shown as association with AS after multiple testing corrections in discovery cohorts was replicated in a validation independent cohort of 620 AS patients and 683 healthy controls.The rs 10019009 was assessed with bioin fomlatics including phylogenetic context,F-SNP and FastSNP functional predictions,secondary structure prediction,and molecular modeling.We performed a functional analysis of rs10019009 via reverse transcription-polymerase chain reaction,alkaline phosphatase(ALP)activity in human osteosarcoma U2OS cells.Results:Interestingly,the SNP rs10019009 was associated with AS in both the discovery cohort(P=0.0012)and validation cohort(P=0.0349),as well as overall(P=0.0004)in genetic case-control association analysis.After a multivariate logistic regression analysis,the effect of this genetic variant was observed to be independent of linkage disequilibrium.Via bioinformatics analysis,it was found that the amino acid change of the rs 10019009 led to changes of SNP function,secondary structure,tertiary confomlation,and splice mode.Finally,functional analysis ofrsl0019009 in U2OS cells demonstrated that the risk T allele of the rsl0019009 increased enzymatic activity of ALP,compared to that of the nonrisk allele(P=0.0080).Conclusions:These results suggested that the DMP1 gene seems to be involved in genetic predisposition to AS,which may contribute to the ectopic mineralization or ossification in AS.In addition,DMP1 gene may be a promising intervention target for AS in the future.
