2种脱矿处理牙本质基质表面结构及促hPDLCs成骨性能研究
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摘要
目的比较不完全脱矿牙本质基质——经处理牙本质基质(treated dentin matrix,TDM)和完全脱矿牙本质基质——脱矿牙本质基质(demineralized dentin matrix,DDM)的材料制备、表面特性及对人牙周膜细胞(human periodontal ligament cells,hPDLCs)增殖与成骨分化的影响,为研究牙齿来源的骨替代材料治疗牙周骨缺损提供实验依据。方法以人离体牙分别采用乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)溶液梯度脱矿的方法制备TDM和通过盐酸浸泡完全脱矿的方法制备DDM,通过扫描电镜观察其表面结构;通过培养液浸泡法制备TDM和DDM的浸提液。将h PDLCs分为3组:TDM组(加入TDM浸提液)、DDM组(加入DDM浸提液)、对照组(加入含10%血清的培养液),分别诱导培养hPDLCs。通过CCK-8(Cell Counting Kit-8)法检测hPDLCs增殖情况,检测碱性磷酸酶(alkaline phosphatase,ALP)表达和矿化结节形成情况。结果 TDM表面疏松多孔、结构完整,三维结构性能优于DDM。TDM组和DDM组hPDLCs增殖能力均强于对照组,TDM组细胞增殖能力强于DDM组(F=36.480,P <0.05);TDM组细胞ALP活性高于DDM组;成骨诱导14 d后TDM组和DDM组均可见茜素红标记的矿化结节,TDM组多于DDM组。结论 TDM表面结构、促进hPDLCs增殖和成骨分化的能力均优于DDM,其有望作为牙周骨缺损的新型骨替代材料。
Objective To analyze the different fabrication methods and surface structure of treated dentin matrix(TDM) and demineralized dentin matrix(DDM) and their diverse function on promoting the proliferation and osteogenic differential capability of human periodontal ligament cells(hPDLCs). This study provides a preliminary basis for the treatment of periodontal bone defects with bone substitutes from teeth. Methods TDM was made from human dentinmatrices and demineralized incompletely by soaking in different concentrations of ethylene diamine tetra-acetic while DDM was made of human dentin matrices and demineralized completely by soaking in a hydrochloric acid solution followed by observation via SEM. The liquid extracts of TDM and DDM were collected according to the protocol of the International Standardization Organization(ISO 10993). Then, hPDLCs were divided into the following three groups: the TDM group(liquid extracts of TDM), the DDM group(liquid extracts of DDM), the control group(a-modified eagle medium with 10% fetal bovine serum), h PDLCs were cultured with liquid extracts of TDM or DDM, or a-modified eagle medium with 10% FBS). h PDLC proliferation was detected by a Cell Counting Kit-8(CCK-8). The alkaline phosphatase(ALP) expression and calcified nodules of hPDLCs were tested. Results TDM obtained a preferable surface structure compared to DDM due to more sufficiently exposed dentinal tubules and looser fiber bundles of the intertubular and peritubular dentin. Both TDM and DDM promoted the proliferation of hPDLCs compared with the control group, and the proliferation of h PDLCs was significantly greater in the TDM group compared to the DDM group(F = 36.480, P < 0.05).The ALP activity of hPDLCs in the TDM group was higher than the DDM group. After a 14-day osteogenic induction,Alizarin red staining mineral nodes were observed in both groups; however, the TDM group displayed more calcified nodules than the DDM group. Conclusion The advantages of TDM including the surface structure, proliferation and osteogenic differentiation of hPDLCs, are more prominent than those of DDM, suggesting that TDM is a potential promising bone graft substitute in periodontal regeneration.
Objective To analyze the different fabrication methods and surface structure of treated dentin matrix(TDM) and demineralized dentin matrix(DDM) and their diverse function on promoting the proliferation and osteogenic differential capability of human periodontal ligament cells(hPDLCs). This study provides a preliminary basis for the treatment of periodontal bone defects with bone substitutes from teeth. Methods TDM was made from human dentinmatrices and demineralized incompletely by soaking in different concentrations of ethylene diamine tetra-acetic while DDM was made of human dentin matrices and demineralized completely by soaking in a hydrochloric acid solution followed by observation via SEM. The liquid extracts of TDM and DDM were collected according to the protocol of the International Standardization Organization(ISO 10993). Then, hPDLCs were divided into the following three groups: the TDM group(liquid extracts of TDM), the DDM group(liquid extracts of DDM), the control group(a-modified eagle medium with 10% fetal bovine serum), h PDLCs were cultured with liquid extracts of TDM or DDM, or a-modified eagle medium with 10% FBS). h PDLC proliferation was detected by a Cell Counting Kit-8(CCK-8). The alkaline phosphatase(ALP) expression and calcified nodules of hPDLCs were tested. Results TDM obtained a preferable surface structure compared to DDM due to more sufficiently exposed dentinal tubules and looser fiber bundles of the intertubular and peritubular dentin. Both TDM and DDM promoted the proliferation of hPDLCs compared with the control group, and the proliferation of h PDLCs was significantly greater in the TDM group compared to the DDM group(F = 36.480, P < 0.05).The ALP activity of hPDLCs in the TDM group was higher than the DDM group. After a 14-day osteogenic induction,Alizarin red staining mineral nodes were observed in both groups; however, the TDM group displayed more calcified nodules than the DDM group. Conclusion The advantages of TDM including the surface structure, proliferation and osteogenic differentiation of hPDLCs, are more prominent than those of DDM, suggesting that TDM is a potential promising bone graft substitute in periodontal regeneration.
引文
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[15]Wu YC,Lin LK,Song CJ,et al.Comparisons of periodontal regenerative therapies:a meta-analysis on the long-term efficacy[J].JClin Periodontol,2017,44(5):511-519.
[16]Cortellini P,Tonetti MS.Clinical concepts for regenerative therapy in intrabony defects[J].J Clin Periodontol,2015,68(1):282-307.
[17]杨禾丰,胡瑜,孙晶晶,等.处理的牙本质基质对骨髓间充质干细胞成骨分化影响的研究[J].华西口腔医学杂志,2016,34(3):281-285.
[18]Reis-Filho CR,Silva ER,Martins AB,et al.Demineralised human dentine matrix stimulates the expression of VEGF and accelerates the bone repair in tooth sockets of rats[J].Arch Oral Biol,2012,57(5):469-476.
[19]Kim YK,Bang KM,Murata M,et al.Retrospective clinical study of allogenic demineralized dentin matrix for alveolar bone repair[J].Journal of Hard Tissue Biology,2017,26(1):95-102.
[20]Yang HS,Kang KJ,Lee MS,et al.In vitro and in vivo dentinogenic efficacy of human dental pulp-derived cells induced by demineralized dentin matrix and HA-TCP[J].Stem Cells Int,2017,6:1-15.
[21]Da Silva Beraldo?,Silva R,Da Gama Antunes A,et al.Scanning electron microscopic evaluation of smear layer removal using isolated or interweaving EDTA with sodium hypochlorite[J].Iran Endod J,2017,12(1):55-59.
[22]Yagihashi K,Miyazawa K,Togari K,et al.Demineralized dentin matrix acts as a scaffold for repair of articular cartilage defects[J].Calcif Tissue Int,2009,84(3):210-220.
[2]Pilipchuk SP,Plonka A B,Monje A,et al.Tissue engineering for bone regeneration and osseointegration in the oral cavity[J].Dent Mater,2015,31(4):317-338.
[3]Ning C,Zhou L,Tan G.Fourth-generation biomedical materials[J].Mater Today,2016,19(1):2-3.
[4]Ceccarelli G,Presta R,Benedetti L,et al.Emerging perspectives in scaffold for tissue engineering in oral surgery[J].Stem Cells Int,2017,10:4585401.
[5]Jordana F,LE Visage C,Weiss P.Bone substitutes[J].Med Sci(Paris),2017,33(1):60-65.
[6]Park M,Mah YJ,Kim DH,et al.Demineralized deciduous tooth as a source of bone graft material:its biological and physicochemical characteristics[J].Oral Surg Oral Med Oral Pathol Oral Radiol,2015,120(3):307-314.
[7]Kim SY,Kim YK,Park YH,et al.Evaluation of the healing potential of demineralized dentin matrix fixed with recombinant human bone morphogenetic protein-2 in bone grafts[J].Materials,2017,10(9):1049.
[8]Papadimitropoulos A,Scotti C,Bourgine P,et al.Engineered decellularized matrices to instruct bone regeneration processes[J].Bone,2015,70:66-72.
[9]Chen G,Chen J,Yang B,et al.Combination of aligned PLGA/Gelatin electrospun sheets,native dental pulp extracellular matrix and treated dentin matrix as substrates for tooth root regeneration[J].Biomaterials,2015,52(1):56-70.
[10]Li R,Guo W,Yang B,et al.Human treated dentin matrix as a natural scaffold for complete human dentin tissue regeneration[J].Biomaterials,2011,32(20):4525-4538.
[11]Guo W,Gong K,ShiI H,et al.Dental follicle cells and treated dentin matrix scaffold for tissue engineering the tooth root[J].Biomaterials,2012,33(5):1291-1302.
[12]Jiao L,Xie L,Yang B,et al.Cryopreserved dentin matrix as a scaffold material for dentin-pulp tissue regeneration[J].Biomaterials,2014,35(18):4929-4939.
[13]Eke PI,Dye BA,Wei L,et al.Update on prevalence of periodontitis in adults in the united states:NHANES 2009 to 2012[J].J Periodontol,2015,86(5):611-622.
[14]Preshaw PM,Henne K,Taylor JJ,et al.Age-related changes in immune function(immune senescence)in caries and periodontal diseases:a systematic review[J].J Clin Periodontol,2017,44(S18):S153-S177.
[15]Wu YC,Lin LK,Song CJ,et al.Comparisons of periodontal regenerative therapies:a meta-analysis on the long-term efficacy[J].JClin Periodontol,2017,44(5):511-519.
[16]Cortellini P,Tonetti MS.Clinical concepts for regenerative therapy in intrabony defects[J].J Clin Periodontol,2015,68(1):282-307.
[17]杨禾丰,胡瑜,孙晶晶,等.处理的牙本质基质对骨髓间充质干细胞成骨分化影响的研究[J].华西口腔医学杂志,2016,34(3):281-285.
[18]Reis-Filho CR,Silva ER,Martins AB,et al.Demineralised human dentine matrix stimulates the expression of VEGF and accelerates the bone repair in tooth sockets of rats[J].Arch Oral Biol,2012,57(5):469-476.
[19]Kim YK,Bang KM,Murata M,et al.Retrospective clinical study of allogenic demineralized dentin matrix for alveolar bone repair[J].Journal of Hard Tissue Biology,2017,26(1):95-102.
[20]Yang HS,Kang KJ,Lee MS,et al.In vitro and in vivo dentinogenic efficacy of human dental pulp-derived cells induced by demineralized dentin matrix and HA-TCP[J].Stem Cells Int,2017,6:1-15.
[21]Da Silva Beraldo?,Silva R,Da Gama Antunes A,et al.Scanning electron microscopic evaluation of smear layer removal using isolated or interweaving EDTA with sodium hypochlorite[J].Iran Endod J,2017,12(1):55-59.
[22]Yagihashi K,Miyazawa K,Togari K,et al.Demineralized dentin matrix acts as a scaffold for repair of articular cartilage defects[J].Calcif Tissue Int,2009,84(3):210-220.
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