牙本质基质—富血小板血浆凝胶构建牙髓组织工程支架的初步研究
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摘要
目的：探讨富血小板血浆(platelet-rich plasma, PRP)凝胶作为生长因子缓释系统的可行性；分别评价PRP凝胶和脱矿牙本质的细胞相容性,并探讨脱矿牙本质基质对乳牙牙髓细胞矿化能力的影响,为牙本质基质-PRP凝胶构建牙髓组织工程支架材料提供实验依据。
方法：
1.采用组织块酶消化法进行猪乳牙牙髓细胞体外分离培养,免疫组织化学染色法检测波形丝蛋白、角蛋白的表达。
2.抽取猪静脉血,以二次离心法制备PRP,对PRP和全血进行血小板计数；将激活剂分别与PRP和全血按比例混合,通过酶联免疫吸附法(enzyme-linked immunosorbent assay)分别测定7天内单位体积PRP或全血所形成凝胶释放的血小板衍化生长因子(platelet-derived growth factor,PDGF)、转化生长因子-β(transforming growth factor, TGF-β)和血管内皮生长因子(vascular endothelial growth factor, VEGF)的水平。
3.将第3代猪乳牙牙髓细胞分别与含50%和10%自体PRP溶液制备的凝胶支架复合,体外培养3周,进行组织学观察。
4.制备猪乳牙牙根段,依次浸泡于5.25%NaOC1溶液、17%EDTA溶液和PBS超声波处理,将第3代猪乳牙牙髓细胞接种于根管壁牙本质表面,扫描电镜观察细胞生长和增殖情况。通过茜素红染色,检测与脱矿牙本质基质共同培养的细胞矿化能力。
结果：
1.小型猪乳牙牙髓细胞形态呈多角形或成纤维细胞样,抗波形丝蛋白染色阳性,抗角蛋白染色阴性。
2.PRP和全血中血小板浓度分别为(1672.67±117.35)×109/L和(302.67±21.55)×109/L,PRP中血小板浓度是全血的(5.85±0.41)倍,差异有统计学意义,血小板的回收率为(66.45%±5.27%)。PRP和全血激活后第0.5、1d、3d、5d,PRP凝胶释放PDGF的水平均高于血凝块(P<0.05),第7d两者释放PDGF的水平无统计学差异(P>0.05)；第0.5、1d、3d,PRP凝胶释放TGF-p的水平均高于血凝块(P<0.05),第5d、7d两者释放TGF-p的水平无统计学差异(P>0.05)。PRP凝胶与血凝块释放VEGF的水平在各时间点无统计学差异(P>0.05)。
3.乳牙牙髓细胞在50%和10%PRP凝胶支架中呈三维分布,生长状态良好。细胞-50%PRP凝胶复合体7天收缩率为61.57%±3.82%,细胞-10%PRP凝胶复合体7天未发生明显收缩,高度塌陷程度较大。
4.猪乳牙牙髓细胞在脱矿牙本质基质表面生长和增殖情况良好。与脱矿牙本质基质共同培养的细胞茜素红染色阳性。
结论：PRP凝胶由于具有可塑性强、能持续释放多种生长因子的特征,可以作为生长因子缓释系统；10%PRP凝胶可作为牙髓组织工程支架材料。脱矿牙本质基质可以为乳牙牙髓细胞的增殖和矿化提供良好的微环境。
Objective:To explore the feasibility of platelet-rich plasma (PRP) gel as a delivery system for growth factors. To evaluate the cytocompatibility of PRP gel and demineralized dentin matrix, and investigate the effect of demineralized dentin matrix on the mineralization of dental pulp cells derived from miniature pig deciduous teeth, to provide a experimental basis for constructing dentin matrix-PRP gel as pulp tissue engineering scaffold.
To evaluate the performance of PRP gel as an injectable scaffold for constructing of tissue-engineered pulp by observing the growth of dental pulp cells derived from miniature pig deciduous teeth in autologous PRP gel. To investigate the effect of demineralized dentin matrix on the biological behavior of dental pulp cells derived from miniature pig deciduous teeth.
Methods:
1. Dental pulp cells derived from miniature pig deciduous teeth were isolated and cultured with tissue block enzyme digestion method. Vimentin and cytokeratin expression was detected with Immunohistochemical staining.
2. Venous blood samples were drawn from miniature pigs. PRP was prepared with two-step centrifugation procedure. Platelet concentration in PRP and whole blood was determined. The release of PDGF and TGF-β and VEGF from PRP gel and blood clot, which formed of activator mixed with PRP and whole blood respectively, was measured over7days by enzyme-linked immunosorbent assay.
3. Dental pulp cells at passage3were seeded in50%or10%PRP gel which containing50%or10%autologous PRP and cultivated in vitro for3weeks, then evaluated by histolody.
4. Root fragments of miniature pigs deciduous teeth were soaked in5.25%NaOCl following by17%EDTA then PBS, and meanwhile concussed by means of ultrasonic cleaner. Dental pulp cells derived from miniature pig deciduous teeth at passage3were seeded on the dentin surface of root canal wall. Cell growth and proliferation were observed by SEM. The demineralization ability of cells cultured with demineralized dentin matrix was determined with alizarin red staining.
Results:
1. Dental pulp cells derived from miniature pig deciduous teeth were polygonal or fibroblast-like. Positive immunohistochemical staining of vimentin and negative immunohistochemical staining of cytokeratin was observed.
2. Platelet concentration were (1672.67±117.35)×109/L and (302.67±21.55)×109/L in PRP and whole blood respectively. Platelet concentration of PRP is (5.85±0.41) times that of whole blood (P<0.05), and the recovery rate was (66.45%±5.27%). After PRP and whole blood were activated, the release of PDGF from PRP gel were higher than blood clots at day0.5,1,3and5(P <0.05), there was no significant difference in the release of PDGF between PRP gel and blood clot at day7(P>0.05); the release of TGF-β from PRP gel were higher than blood clots at day0.5,1and3(P<0.05); there was no significant difference in the release of TGF-β between PRP gel and blood clot at day5and7(P>0.05). And no significant difference was found in the release of VEGF between PRP gel and blood clot at each time point during7days (P>0.05).
3. Dental pulp cells derived from miniature pig deciduous teeth were well-distributed in the three-dimensional space of both50%and10%PRP gel. The retraction rate of cell-50%PRP gel composite at day7was61.57%±3.82%, while cell-10%PRP gel composite didn't retract significantly. Greater collapse was observed in cell-10%PRP gel composite.
4. Well-growth and proliferation of dental pulp cells derived from miniature pig deciduous teeth were observed on demineralized dentin matrix. Cells cultured with demineralized dentin matrix was positive for alizarin red staining.
Conclusion:The plasticity and the characteristics of continuously release of a variety of growth factors could probably promote the application of PRP gel as a delivery system for growth factors.10%PRP gel could serve as an injectable scaffold in pulp tissue engineering. Demineralized dentin matrix could possibly provide a good microenvironment for the proliferation and mineralization of dental pulp cells.
方法：
1.采用组织块酶消化法进行猪乳牙牙髓细胞体外分离培养,免疫组织化学染色法检测波形丝蛋白、角蛋白的表达。
2.抽取猪静脉血,以二次离心法制备PRP,对PRP和全血进行血小板计数；将激活剂分别与PRP和全血按比例混合,通过酶联免疫吸附法(enzyme-linked immunosorbent assay)分别测定7天内单位体积PRP或全血所形成凝胶释放的血小板衍化生长因子(platelet-derived growth factor,PDGF)、转化生长因子-β(transforming growth factor, TGF-β)和血管内皮生长因子(vascular endothelial growth factor, VEGF)的水平。
3.将第3代猪乳牙牙髓细胞分别与含50%和10%自体PRP溶液制备的凝胶支架复合,体外培养3周,进行组织学观察。
4.制备猪乳牙牙根段,依次浸泡于5.25%NaOC1溶液、17%EDTA溶液和PBS超声波处理,将第3代猪乳牙牙髓细胞接种于根管壁牙本质表面,扫描电镜观察细胞生长和增殖情况。通过茜素红染色,检测与脱矿牙本质基质共同培养的细胞矿化能力。
结果：
1.小型猪乳牙牙髓细胞形态呈多角形或成纤维细胞样,抗波形丝蛋白染色阳性,抗角蛋白染色阴性。
2.PRP和全血中血小板浓度分别为(1672.67±117.35)×109/L和(302.67±21.55)×109/L,PRP中血小板浓度是全血的(5.85±0.41)倍,差异有统计学意义,血小板的回收率为(66.45%±5.27%)。PRP和全血激活后第0.5、1d、3d、5d,PRP凝胶释放PDGF的水平均高于血凝块(P<0.05),第7d两者释放PDGF的水平无统计学差异(P>0.05)；第0.5、1d、3d,PRP凝胶释放TGF-p的水平均高于血凝块(P<0.05),第5d、7d两者释放TGF-p的水平无统计学差异(P>0.05)。PRP凝胶与血凝块释放VEGF的水平在各时间点无统计学差异(P>0.05)。
3.乳牙牙髓细胞在50%和10%PRP凝胶支架中呈三维分布,生长状态良好。细胞-50%PRP凝胶复合体7天收缩率为61.57%±3.82%,细胞-10%PRP凝胶复合体7天未发生明显收缩,高度塌陷程度较大。
4.猪乳牙牙髓细胞在脱矿牙本质基质表面生长和增殖情况良好。与脱矿牙本质基质共同培养的细胞茜素红染色阳性。
结论：PRP凝胶由于具有可塑性强、能持续释放多种生长因子的特征,可以作为生长因子缓释系统；10%PRP凝胶可作为牙髓组织工程支架材料。脱矿牙本质基质可以为乳牙牙髓细胞的增殖和矿化提供良好的微环境。
Objective:To explore the feasibility of platelet-rich plasma (PRP) gel as a delivery system for growth factors. To evaluate the cytocompatibility of PRP gel and demineralized dentin matrix, and investigate the effect of demineralized dentin matrix on the mineralization of dental pulp cells derived from miniature pig deciduous teeth, to provide a experimental basis for constructing dentin matrix-PRP gel as pulp tissue engineering scaffold.
To evaluate the performance of PRP gel as an injectable scaffold for constructing of tissue-engineered pulp by observing the growth of dental pulp cells derived from miniature pig deciduous teeth in autologous PRP gel. To investigate the effect of demineralized dentin matrix on the biological behavior of dental pulp cells derived from miniature pig deciduous teeth.
Methods:
1. Dental pulp cells derived from miniature pig deciduous teeth were isolated and cultured with tissue block enzyme digestion method. Vimentin and cytokeratin expression was detected with Immunohistochemical staining.
2. Venous blood samples were drawn from miniature pigs. PRP was prepared with two-step centrifugation procedure. Platelet concentration in PRP and whole blood was determined. The release of PDGF and TGF-β and VEGF from PRP gel and blood clot, which formed of activator mixed with PRP and whole blood respectively, was measured over7days by enzyme-linked immunosorbent assay.
3. Dental pulp cells at passage3were seeded in50%or10%PRP gel which containing50%or10%autologous PRP and cultivated in vitro for3weeks, then evaluated by histolody.
4. Root fragments of miniature pigs deciduous teeth were soaked in5.25%NaOCl following by17%EDTA then PBS, and meanwhile concussed by means of ultrasonic cleaner. Dental pulp cells derived from miniature pig deciduous teeth at passage3were seeded on the dentin surface of root canal wall. Cell growth and proliferation were observed by SEM. The demineralization ability of cells cultured with demineralized dentin matrix was determined with alizarin red staining.
Results:
1. Dental pulp cells derived from miniature pig deciduous teeth were polygonal or fibroblast-like. Positive immunohistochemical staining of vimentin and negative immunohistochemical staining of cytokeratin was observed.
2. Platelet concentration were (1672.67±117.35)×109/L and (302.67±21.55)×109/L in PRP and whole blood respectively. Platelet concentration of PRP is (5.85±0.41) times that of whole blood (P<0.05), and the recovery rate was (66.45%±5.27%). After PRP and whole blood were activated, the release of PDGF from PRP gel were higher than blood clots at day0.5,1,3and5(P <0.05), there was no significant difference in the release of PDGF between PRP gel and blood clot at day7(P>0.05); the release of TGF-β from PRP gel were higher than blood clots at day0.5,1and3(P<0.05); there was no significant difference in the release of TGF-β between PRP gel and blood clot at day5and7(P>0.05). And no significant difference was found in the release of VEGF between PRP gel and blood clot at each time point during7days (P>0.05).
3. Dental pulp cells derived from miniature pig deciduous teeth were well-distributed in the three-dimensional space of both50%and10%PRP gel. The retraction rate of cell-50%PRP gel composite at day7was61.57%±3.82%, while cell-10%PRP gel composite didn't retract significantly. Greater collapse was observed in cell-10%PRP gel composite.
4. Well-growth and proliferation of dental pulp cells derived from miniature pig deciduous teeth were observed on demineralized dentin matrix. Cells cultured with demineralized dentin matrix was positive for alizarin red staining.
Conclusion:The plasticity and the characteristics of continuously release of a variety of growth factors could probably promote the application of PRP gel as a delivery system for growth factors.10%PRP gel could serve as an injectable scaffold in pulp tissue engineering. Demineralized dentin matrix could possibly provide a good microenvironment for the proliferation and mineralization of dental pulp cells.
引文
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[18]Huang GT, Shagramanova K, Chan SW. Formation of odontoblast-like cells from cultured human dental pulp cells on dentin in vitro [J]. J Endod, 2006,32(11):1066-1073.
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[20]Guo W, He Y, Zhang X, et al. The use of dentin matrix scaffold and dental follicle cells for dentin regeneration [J]. Biomaterials,2009, 30(35):6708-6723.
[21]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.
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[23]Coimbra P, Alves P, Valente TA, et al. Sodium hyaluronate/chitosan poly electrolyte complex scaffolds for dental pulp regeneration:synthesis and characterization [J]. Int J Biol Macromol,2011,49(4):573-579.
[24]Galler KM, Cavender AC, Koeklue U, et al. Bioengineering of dental stem cells in a PEGylated fibrin gel [J]. Regen Med,2011,6(2):191-200.
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[1]Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis:new treatment protocol? [J] J Endod,2004,30(4): 196-200.
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[1]曹谊林,组织工程学理论与实践[M].上海：上海科学技术出版社2004：3.
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[4]Zhang W, Walboomers XF, van Kuppevelt TH, et al. The performance of human dental pulp stem cells on different three-dimensional scaffold materials [J]. Biomaterials,2006,27(33):5658-5668.
[5]Fujiwara S, Kumabe S, Iwai Y. Isolated rat dental pulp cell culture and transplantation with an alginate scaffold [J]. Okajimas Folia Anat Jpn,2006, 83(1):15-24.
[6]李珊,樊明文,梁素霞.成人牙髓干细胞,壳聚糖-磷酸三钙复合材料相容性试验研究[J].口腔医学研究,2006,22(3)：264-266.
[7]贺慧霞,金岩,史俊南等.两种钙磷材料表面壳聚糖修饰对人牙髓干细胞黏附和增殖的影响[J].牙体牙髓牙周病学杂志,2004,14(10)：544-548.
[8]Ohara T, Itaya T, Usami K, et al. Evaluation of scaffold materials for tooth tissue engineering [J]. J Biomed Mater Res A,2010,94(3):800-805.
[9]Ebrahimi M, Pripatnanont P, Monmaturapoj N, et al. Fabrication and characterization of novel nano hydroxyapatite/beta-tricalcium phosphate scaffolds in three different composition ratios [J]. J Biomed Mater Res A, 2012.
[10]Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo [J]. Proc Natl Acad Sci U S A,2000, 97(25):13625-13630.
[11]Wang J, Liu X, Jin X, et al. The odontogenic differentiation of human dental pulp stem cells on nanofibrous poly(l-lactic acid) scaffolds in vitro and in vivo [J]. Acta Biomater,2010.
[12]Mooney DJ, Powell C, Piana J, et al. Engineering dental pulp-like tissue in vitro [J]. Biotechnol Prog,1996,12(6):865-868.
[13]Bohl KS, Shon J, Rutherford B, et al. Role of synthetic extracellular matrix in development of engineered dental pulp [J]. J Biomater Sci Polym Ed, 1998,9(7):749-764.
[14]Huang GT, Yamaza T, Shea LD, et al. Stem/Progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model [J]. Tissue Eng Part A,2009,16(2):605-615.
[15]Cordeiro MM, Dong Z, Kaneko T, et al. Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth [J]. J Endod,2008, 34(8):962-969.
[16]Demarco FF, Casagrande L, Zhang Z, et al. Effects of morphogen and scaffold porogen on the differentiation of dental pulp stem cells [J]. J Endod, 2010,36(11):1805-1811.
[17]Casagrande L, Demarco FF, Zhang Z, et al. Dentin-derived BMP-2 and odontoblast differentiation [J]. J Dent Res,2010,89(6):603-608.
[18]Huang GT, Shagramanova K, Chan SW. Formation of odontoblast-like cells from cultured human dental pulp cells on dentin in vitro [J]. J Endod, 2006,32(11):1066-1073.
[19]Batouli S, Miura M, Brahim J, et al. Comparison of stem-cell-mediated osteogenesis and dentinogenesis [J]. J Dent Res,2003,82(12):976-981.
[20]Guo W, He Y, Zhang X, et al. The use of dentin matrix scaffold and dental follicle cells for dentin regeneration [J]. Biomaterials,2009, 30(35):6708-6723.
[21]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.
[22]Yang B, Chen G, Li J, et al. Tooth root regeneration using dental follicle cell sheets in combination with a dentin matrix-based scaffold [J]. Biomaterials,2012,33(8):2449-2461.
[23]Coimbra P, Alves P, Valente TA, et al. Sodium hyaluronate/chitosan poly electrolyte complex scaffolds for dental pulp regeneration:synthesis and characterization [J]. Int J Biol Macromol,2011,49(4):573-579.
[24]Galler KM, Cavender AC, Koeklue U, et al. Bioengineering of dental stem cells in a PEGylated fibrin gel [J]. Regen Med,2011,6(2):191-200.
[25]Yang X, Han G, Pang X, et al. Chitosan/collagen scaffold containing bone morphogenetic protein-7 DNA supports dental pulp stem cell differentiation in vitro and in vivo [J]. J Biomed Mater Res A,2012.
[26]Zheng L, Yang F, Shen H, et al. The effect of composition of calcium phosphate composite scaffolds on the formation of tooth tissue from human dental pulp stem cells [J]. Biomaterials,2011,32(29):7053-7059.
[27]Engler AJ, Sen S, Sweeney HL, et al. Matrix elasticity directs stem cell lineage specification [J]. Cell,2006,126(4):677-689.
[28]Tonomura A, Mizuno D, Hisada A, et al. Differential effect of scaffold shape on dentin regeneration [J]. Ann Biomed Eng,2010,38(4):1664-1671.
[29]Graziano A, d'Aquino R, Cusella-De Angelis MG, et al. Scaffold's surface geometry significantly affects human stem cell bone tissue engineering [J]. J Cell Physiol,2008,214(1):166-172.
[30]Wang J, Ma H, Jin X, et al. The effect of scaffold architecture on odontogenic differentiation of human dental pulp stem cells [J]. Biomaterials,2011,32(31):7822-7830.
[31]裴雪涛,再生医学——理论与技术[M].北京：科学出版社,2010：241.
[32]Yang X, Yang F, Walboomers XF, et al. The performance of dental pulp stem cells on nanofibrous PCL/gelatin/nHA scaffolds [J]. J Biomed Mater Res A,2010,93(1):247-257.
[1]Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis:new treatment protocol? [J] J Endod,2004,30(4): 196-200.
[2]Ding RY, Cheung GS, Chen J, et al. Pulp revascularization of immature teeth with apical periodontitis:a clinical study[J]. J Endod,2009,35(5) 745-749.
[3]Wang X, Thibodeau B, Trope M, et al. Histologic characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis [J]. J Endod,2010, 36(1):56-63.
[4]钟小奕,杨亦萍,陈文霞,等.年轻恒前牙感染根管内血管再生的动物 实验研究[J].华西口腔医学杂志,2010,28(6)：672-674.
[5]Cehreli ZC, Isbitiren B, Sara S, et al. Regenerative endodontic treatment (revascularization) of immature necrotic molars medicated with calcium hydroxide:a case series[J]. J Endod,2011,37(9):1327-1330.
[6]Guo W, He Y, Zhang X, et al. The use of dentin matrix scaffold and dental follicle cells for dentin regeneration [J]. Biomat,2009,30(35): 6708-6723.
[7]Huang GT, Sonoyama W, Chen J, et al. In vitro characterization of human dental pulp cells:various isolation methods and culturing environments [J]. Cell Tissue Res,2006,324(2):225-236.
[8]Batouli S, Miura M, Brahim J, et al. Comparison of stem-cell-mediated osteogenesis and dentinogenesis [J]. J Dent Res,2003,82(12):976-981.
[9]Gotlieb EL, Murray PE, Namerow KN, et al. An ultrastructural investigation of tissue-engineered pulp constructs implanted within endodontically treated teeth[J]. J Am Dent Assoc,2008,139(4):457-465.
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