牙根种植结合磷酸三钙/富含血小板血浆复合移植增高牙槽嵴的初步研究
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摘要
目的：① 由自体血液提取富含血小板的血浆(platelet-rich plasma，PRP)，诱导体外培养的骨髓基质细胞(bone marrow stromal cell，BMSCs)向成骨细胞分化，初步鉴定PRP对BMSCs的骨诱导作用。②探讨磷酸三钙／富含血小板血浆(TCP／PRP)复合材料的骨修复能力，希望获得一种更理想的骨移植材料。③评价牙根种植结合磷酸三钙／富含血小板血浆、引导骨再生术(guided bone regeneration，GBR)复合移植增高牙槽嵴的效果，探索牙槽嵴增高术(alveolar ridge augmentation，ARA)的新方法新理念，为临床应用提供理论基础和实验依据。
材料与方法：①抽取狗髂骨骨髓体外分离培养获得BMSCs，含有小牛血清的DMEM培养基进行原代培养。抽取上述狗自体静脉血，分离浓缩血小板，获得PRP。BMSCs传代培养中加入PRP 10ml／L进行诱导分化，用倒置相差显微镜进行细胞形态学和细胞生长情况观察，透射电镜观察细
胞的超微结构改变，应用流式细胞仪少C哟定期测量BMsCs内各周期
DNA含量，了解PRP对骨髓基质细胞增殖、分化及细胞合成方面的影响。
茜素红染色法检测体外矿化结节，细胞碱性磷酸酶染色(alkaline
Phosphate，A工p)和骨钙素(OC)含量测定，检测PRP对BMSCs的骨
诱导作用。②按设计牙位拔双侧上、下领前磨牙，去骨，建立局部牙槽
靖萎缩模型。③建模4周后，牙根种植结合磷酸三钙/富含血小板血浆复
合移植到实验性牙槽晴萎缩处，并覆盖聚四氟乙烯膜
(polytetrafluoroethylene，pTFE)。实验分组:实验组:牙根+TCp/P RP/
P刀飞;对照组:牙根+TC即叮FE。④分别于6周、12周处死动物取材，
组织切片染色，分析各组牙槽骨再生情况，观察牙根与骨组织接触情况，
测量牙槽晴增加的高度和宽度。
结果:①体外分离培养的骨髓基质细胞经PRP条件培养基诱导后表现出
明显的成骨活性，体外矿化结节的茜素红染色阳性，ALP染色阳性，OC
在各个时段的分泌量均高于未经PRI，诱导的BMSCs，PRP可促进BMSCs
代谢，刺激细胞由G夕G;期进入S期，经处理的细胞DNA含量明显增高。
②实验动物结果显示自体牙根种植结合TCp/P RP/GBR复合移植治疗可
以修复牙槽晴萎缩，组织学和X线检测显示，两组都有牙槽骨新生，种
植牙根与骨组织达到骨整合。实验组较对照组新生骨量更多更成熟、致
密。实验组和对照组平均新生牙槽骨高度和宽度分别为(3.17士0.53)
mm，(3 .23士0.45)nun和(2.61士0.35)mm，(2.67士0.31)mm。
结论:①RPR能够促进骨髓基质细胞DNA合成，增强其ALP活性表达，
刺激骨钙素分泌，诱导其向成骨细胞分化，具有骨诱导作用。②TCP田RP
复合材料可以修复实验性牙槽靖萎缩，能够促进牙槽骨再生，具有骨传
导和骨诱导能力，可作为一种良好的生物复合材料应用于临床骨移植及
组织工程学的研究。③自体牙根可作为固位装置，结合表面骨移植增高
牙槽晴，可达到良好的骨整合。④P即可以促进损伤愈合，加速骨组织
再生，可作为自身来源的生长因子应用于临床治疗牙周骨缺损。⑤牙根
种植结合TCP/PR卫/GBR复合移植治疗重症牙周病引起的牙槽蜡萎缩具
有可行性，有望成为增高术牙槽晴的新方法。
Objective: The purpose of this pilot study was as followed: (1)Bone marrow stromal cells isolated from dog treated with PRP which prepared from autologous blood and the effects of PRP on the induced osteoblast of the BMSCs were identified. (2) To explore the potential of using tricalciumphosphate and platelet-rich plasma for alveolar ridge bone healing. In search of an ideal bone grafting materials and substitutes. (3) To evaluate the effects of implantation of autogenous root and TCP/PRP in conjunction with guided bone regeneration(GBR) for alveolar ridge augmentation in the mongrel dogs. All what we did in this pilot study could provide enough theoretical evidence and experimental information for the new methods and technique of alveolar ridge augmentation.
Methods: (1) Bone marrow stromal cells isolated from the mongrel dogs were maintained in Dulbecco's Modified Eagle Medium (DMEM) basic medium supplemented with 10% fetal calf serum (FCS) for the primary culture. Cells
were treated with PRP (10ml/L) prepared from autologous venous blood for the subculture.The morphological characters of BMSCs were observed with phase contrast microscope and ultrastructure were observed under electron microscope, the cellular DNA contents were observed with flow cytometer, then the alkaline phosphatase activities were determined to record the amount of osteocalcin in the culture medium.The mineralized nodules in the culture dishes were stained with saturated solution of Alizarin Red. (2) In each jaw quadrant of both sides, a preclinical model of alveolar defects was surgically produced, following the extraction of the sixth premolar teeth according to the surgical protocols. (3) 4 weeks later, alveolar ridge defects were induced and implant autogenous root and TCP/PRP in conjunction with polytetrafluoroe-thylene membrane (PTFE)for alveolar ridge augmentation. Two groups were divided, Experimental group: dental roots+TCP/PRP/ PTFE; Control group: dental roots+TCP/ PTFE. (4) The animals were euthanized after a healing period of 6 weeks and 12 weeks ,and the experimental sites were analyzed histologically and histomorphometrically.
Results : (1) Bone marrow stromal cells were cultured well with addition of PRP (1%) in vitro. PRP altered the shape of cells and up-regulated cellular DNA contents at 72 hours. PRP's action on osteocalcin synthesis was detected, and the Alkaline phosphatase activities of the cells were observed, and the mineralized nodules in the culture dishes were stained by Alizarin Red histochemical staining methods. (2) Results from the animal studies have showned that the autogenous root in combination with B -tricalciumphosphate, platelet-rich plasma (PRP), and guided bone regeneration(GBR) could augment atrophic alveolar ridge. The outcomes were effective and the
osseointegration between root implants and alveolar bone were achieved.. The experimental groups showed excellent healing results including the quantity and quality of newly regenerated bone bone in contrast to the control groups by histologic and radiographic evaluation. The height and width of newly regenerated alveolar ridge were: (3.17+0.53) mm ,(3.23+0.48 )mm and (2.61 +0.35)mm, (2.67+0.31) mm.
Conclusions: (1) PRP could stimulate the synthesis of BMSCs DNA, and the expressions of ALP and the secretion amount of osteocalcin were increased greatly, PRP could promote BMSCs to differentiated into osteoblasts in vitro. (2) TCP/PRP could act as the bone graft materials in alveolar ridge augmentation procedures. (3) Autogenous root could be used for alveolar ridge augmentation, in combination with bone graft materials can be achieving osseointegration. (4) PRP had the ability to enhance bone regeneration and improve wound healing , which could be used to treat periodontal osseous defects as an adjuvant in clinical application. (5) A combination of autogenous root implants, platelet-rich plasma (PRP), tricalciumphosphate, and guided bone regeneration(GBR) had been shown to be effective in augmenting alveolar ridge bone. In our opinion, this t
材料与方法：①抽取狗髂骨骨髓体外分离培养获得BMSCs，含有小牛血清的DMEM培养基进行原代培养。抽取上述狗自体静脉血，分离浓缩血小板，获得PRP。BMSCs传代培养中加入PRP 10ml／L进行诱导分化，用倒置相差显微镜进行细胞形态学和细胞生长情况观察，透射电镜观察细
胞的超微结构改变，应用流式细胞仪少C哟定期测量BMsCs内各周期
DNA含量，了解PRP对骨髓基质细胞增殖、分化及细胞合成方面的影响。
茜素红染色法检测体外矿化结节，细胞碱性磷酸酶染色(alkaline
Phosphate，A工p)和骨钙素(OC)含量测定，检测PRP对BMSCs的骨
诱导作用。②按设计牙位拔双侧上、下领前磨牙，去骨，建立局部牙槽
靖萎缩模型。③建模4周后，牙根种植结合磷酸三钙/富含血小板血浆复
合移植到实验性牙槽晴萎缩处，并覆盖聚四氟乙烯膜
(polytetrafluoroethylene，pTFE)。实验分组:实验组:牙根+TCp/P RP/
P刀飞;对照组:牙根+TC即叮FE。④分别于6周、12周处死动物取材，
组织切片染色，分析各组牙槽骨再生情况，观察牙根与骨组织接触情况，
测量牙槽晴增加的高度和宽度。
结果:①体外分离培养的骨髓基质细胞经PRP条件培养基诱导后表现出
明显的成骨活性，体外矿化结节的茜素红染色阳性，ALP染色阳性，OC
在各个时段的分泌量均高于未经PRI，诱导的BMSCs，PRP可促进BMSCs
代谢，刺激细胞由G夕G;期进入S期，经处理的细胞DNA含量明显增高。
②实验动物结果显示自体牙根种植结合TCp/P RP/GBR复合移植治疗可
以修复牙槽晴萎缩，组织学和X线检测显示，两组都有牙槽骨新生，种
植牙根与骨组织达到骨整合。实验组较对照组新生骨量更多更成熟、致
密。实验组和对照组平均新生牙槽骨高度和宽度分别为(3.17士0.53)
mm，(3 .23士0.45)nun和(2.61士0.35)mm，(2.67士0.31)mm。
结论:①RPR能够促进骨髓基质细胞DNA合成，增强其ALP活性表达，
刺激骨钙素分泌，诱导其向成骨细胞分化，具有骨诱导作用。②TCP田RP
复合材料可以修复实验性牙槽靖萎缩，能够促进牙槽骨再生，具有骨传
导和骨诱导能力，可作为一种良好的生物复合材料应用于临床骨移植及
组织工程学的研究。③自体牙根可作为固位装置，结合表面骨移植增高
牙槽晴，可达到良好的骨整合。④P即可以促进损伤愈合，加速骨组织
再生，可作为自身来源的生长因子应用于临床治疗牙周骨缺损。⑤牙根
种植结合TCP/PR卫/GBR复合移植治疗重症牙周病引起的牙槽蜡萎缩具
有可行性，有望成为增高术牙槽晴的新方法。
Objective: The purpose of this pilot study was as followed: (1)Bone marrow stromal cells isolated from dog treated with PRP which prepared from autologous blood and the effects of PRP on the induced osteoblast of the BMSCs were identified. (2) To explore the potential of using tricalciumphosphate and platelet-rich plasma for alveolar ridge bone healing. In search of an ideal bone grafting materials and substitutes. (3) To evaluate the effects of implantation of autogenous root and TCP/PRP in conjunction with guided bone regeneration(GBR) for alveolar ridge augmentation in the mongrel dogs. All what we did in this pilot study could provide enough theoretical evidence and experimental information for the new methods and technique of alveolar ridge augmentation.
Methods: (1) Bone marrow stromal cells isolated from the mongrel dogs were maintained in Dulbecco's Modified Eagle Medium (DMEM) basic medium supplemented with 10% fetal calf serum (FCS) for the primary culture. Cells
were treated with PRP (10ml/L) prepared from autologous venous blood for the subculture.The morphological characters of BMSCs were observed with phase contrast microscope and ultrastructure were observed under electron microscope, the cellular DNA contents were observed with flow cytometer, then the alkaline phosphatase activities were determined to record the amount of osteocalcin in the culture medium.The mineralized nodules in the culture dishes were stained with saturated solution of Alizarin Red. (2) In each jaw quadrant of both sides, a preclinical model of alveolar defects was surgically produced, following the extraction of the sixth premolar teeth according to the surgical protocols. (3) 4 weeks later, alveolar ridge defects were induced and implant autogenous root and TCP/PRP in conjunction with polytetrafluoroe-thylene membrane (PTFE)for alveolar ridge augmentation. Two groups were divided, Experimental group: dental roots+TCP/PRP/ PTFE; Control group: dental roots+TCP/ PTFE. (4) The animals were euthanized after a healing period of 6 weeks and 12 weeks ,and the experimental sites were analyzed histologically and histomorphometrically.
Results : (1) Bone marrow stromal cells were cultured well with addition of PRP (1%) in vitro. PRP altered the shape of cells and up-regulated cellular DNA contents at 72 hours. PRP's action on osteocalcin synthesis was detected, and the Alkaline phosphatase activities of the cells were observed, and the mineralized nodules in the culture dishes were stained by Alizarin Red histochemical staining methods. (2) Results from the animal studies have showned that the autogenous root in combination with B -tricalciumphosphate, platelet-rich plasma (PRP), and guided bone regeneration(GBR) could augment atrophic alveolar ridge. The outcomes were effective and the
osseointegration between root implants and alveolar bone were achieved.. The experimental groups showed excellent healing results including the quantity and quality of newly regenerated bone bone in contrast to the control groups by histologic and radiographic evaluation. The height and width of newly regenerated alveolar ridge were: (3.17+0.53) mm ,(3.23+0.48 )mm and (2.61 +0.35)mm, (2.67+0.31) mm.
Conclusions: (1) PRP could stimulate the synthesis of BMSCs DNA, and the expressions of ALP and the secretion amount of osteocalcin were increased greatly, PRP could promote BMSCs to differentiated into osteoblasts in vitro. (2) TCP/PRP could act as the bone graft materials in alveolar ridge augmentation procedures. (3) Autogenous root could be used for alveolar ridge augmentation, in combination with bone graft materials can be achieving osseointegration. (4) PRP had the ability to enhance bone regeneration and improve wound healing , which could be used to treat periodontal osseous defects as an adjuvant in clinical application. (5) A combination of autogenous root implants, platelet-rich plasma (PRP), tricalciumphosphate, and guided bone regeneration(GBR) had been shown to be effective in augmenting alveolar ridge bone. In our opinion, this t
引文
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[48] Kim SG,Chung CH,Kim YK, et al.Use of particulate dentin-plaster of Paris combination with/without platelet-rich plasma in the treatment of bone defects around implants.Int J Oral Maxillofac Implants, 2002;17:86-94
[49] Wojtowicz A, Chaberek S, Kryst L, et al.Fourier and fractal analysis of maxillary alveolar ridge repair using platelet rich plasma (PRP) and inorganic bovine bone. Int J Oral Maxillofac Surg, February 1, 2003; 32(1): 84-6
[50] Weibrich G, Kleis Wilfried KG,Hafner G.Growth factor levels in the platelet-rich plasma produced by 2 different methods: Curasan-type PRP kit versus PCCS PRP system, lnt J Oral Maxillofac Implant, 2002;27:184-190
[51] Weibrich G,Kleis Wilfried KG,Buch R, et al.The Harvest Smart Prep system versus the Friadent-schütze PRP kit.Clin Oral lmpl Res,2003;14:233-239
[52] Nefussi JR,Brami G,Modrowski D. Sequential expression of bone matrix proteins during rat calvaria osteoblast differentiation and bone nodule formation in vitro.J Histochem cytochem,1997;45(4):493-503
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[56] Piché JE, Graves DT. Study of the growth factor requirements of human bone-derived cells: A comparison with human fibroblasts.Bone,1989;10:131-138.
[57] 李才良，刘宏伟，任肖华，等。rhBFGF、rhTGF-β、rhBMP-2对骨髓基质细胞体外培养增殖的影响。实用口腔医学杂志，2002；18(5)：456-459。
[58] 于世凤主编。口腔组织病理学。北京：人民卫生出版社，2000
[59] 曹采芳主编。牙周病学。北京：人民卫生出版社，2000
[60] 刘宏伟，欧龙，庞劲凡，等。自体骨髓基质细胞用于牙周骨缺损移植的动物实验研究。牙体牙髓牙周病学杂志，2000；10(3)：117-119
[61] Buser D,Dura K, Belser U,et al. Localized ridge augmentation using guided bone regeneration.Int J Periodontics Restorative Dent, 1993;13:29.
[62] Chen CC,Wang HL,Smith F, et al.Evaluation of a collagen membrane in treating periodontol intrabony defects.J Periodontol,1995;6:838
[63] Hutmacher DW, Kirsch A, Ackermann KL, et al. A tissue engineered cell-occlusive device for hard tissue regeneration--a preliminary report.Int J Periodontics Restorative Dent, Feb 2001; 21(1): 49-59.
[64] Donos N, Kostopoulos L, Karring T. Alveolar ridge augmentation using a resorbable copolymer membrane and autogenous bone grafts.An experimental study in the rat. Clin Oral Implants Res, 2002; 13(2): 203-13.
[65] Buser D,Dula K, Hess D, et al.Localized ridge augmentation with autografts. Periodontol 2000,1999;19:151-163
[66] Wikesjo UM, Lim WH,Razi SS, et al. Periodontal repair in dogs:a bioabsorbable calcium carbonate coral implant enhances space provision for alveolar bone regeneration in conjunction with guided tissue regeneration. J Periodontol, Jul 2003; 74(7): 957-64.
[67] Cochran DL,Jones A, Heijl L,et al.Periodontal regeneration with a combination of enamel matrix proteins and autogenous bone grafting. J Periodontol, Sep 2003; 74(9): 1269-81.
[68] Donos N, Lang NP, Karoussis IK, et al.Effect of GBR in combination with deproteinized bovine bone mineral and/or enamel matrix proteins on the healing of critical-size defects. Clin Oral Implants Res, Feb 2004; 15(1):101-11.
[69] Kazuhiro O, Tomoyuki K, Manabu M. Platelet-Rich Plasma Contains High Levels of Platelet-Derived Growth Factor and Transforming Growth Factor-β and Modulates the Proliferation of Periodontally Related Cells In
Vitro. J Periodontol 2003;74:849-857.
[70] Cheung LK, Leung AC. Dental implants in reconstructed jaws: implant longevity and peri-implant tissue outcomes.J Oral Maxillofac Surg, Nov 2003; 61(11): 1263-74.
[71] Verhoeven JW, Cune MS, Terlou M, et al. The combined use of endosteal implants and iliac crest onlay grafts-in the severely atrophic mandible: a longitudinal study. Int J Oral Maxillofac Surg, Oct 1997; 26(5): 351-7.
[72] Williamson RA. Rehabilitation of the resorbed maxilla and mandible using autogenous bone grafts and osseointegrated implants. Int J Oral Maxillofac Implants, Jul 1996; 11(4): 476-88.
[73] Chan MF,Howell RA,Cawood JI.Prosthetic rehabilitation of the atrophic maxilla using pre-implant surgery and endosseous implants. Br Dent J, Jul 1996; 181(2): 51-8.
[74] Froum SJ,Wallace SS,Tarnow DP, te al. Effect of platelet-rich plasma on bone growth and osscointegration in human maxillary sinus grafts: three bilateral case reports. Int J Periodontics Res Dent,2002; 22:45-53.
[75] Choi BH, Im CJ, Huh JY, et al.Effect of platelet-rich plasma on bone regeneration in autogenous bone graft.Int J Oral Maxillofac Surg, January 1,2004; 33(1): 56-9.