组织工程骨修复牙槽骨缺损与正畸牙齿移动
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摘要
组织工程是应用细胞生物学和工程学的原理，对病损组织结构和功能的修复与重建进行研究的一门新兴学科，骨组织工程是一个主要分支。骨髓基质细胞因为具备来源丰富、损伤小、取材方便以及易于体外扩增等优势，成为近年来组织工程骨研究中国内外学者首选的种子细胞。陶瓷化骨具有天然的骨小梁结构、易于传导成骨，避免了人工合成材料的孔隙率和孔径大小及交通等方面的制作难题，而且具有可降解、无免疫原性、来源丰富、制备简单、经济成本低等优点，是理想的骨组织工程支架材料。本实验采用陶瓷化骨作为支架材料，骨髓基质细胞为种子细胞，利用rhBMP-2和TGF-β骨形成促进作用，构建一种新型的骨组织工程修复方法。
第一部分：CBB／MSCs修复颌骨缺损的实验研究
首先进行大鼠骨髓基质细胞的分离、体外培养及使用矿化液(10~(-8)M地塞米松、0.01 mol／L β-甘油磷酸钠、50mg／L维生素C)诱导，然后应用细胞计数、MTT检测、ALP检测、免疫组织化学、Von Kossa等方法观察MSC在矿化液作用下生物学特性的改变。发现5天时已经有一部分骨髓基质细胞转化为成骨细胞，诱导20天后可见矿化结节。其次观察复合方式对骨髓基质细胞(MSC)的贴附、增殖、分化的影响。将诱导后的MSCs与CBB复合。体外培养，观察其对骨髓基质细胞成骨能力的影响。应用扫描电镜、ALP、免
第四军医人学博卜学位论文
疫组化等方法观察，证明MSCs在CBB上能够很好的贴附、伸展和
繁殖。将矿化诱导后的MSCs接种在CBB上，植于裸鼠及自体大鼠
皮下，观察thBMP一2和TGF一p等生长因子对MSCs成骨能力的影响，
通过HE、改良Mallory’s三色法、图像分析观察骨形成情况。结果
表明CBB复合MSCs在自体皮下能够形成编织骨、板层骨等组织，
thBMP一2和TGF一p具有协同作用，促进骨基质形成。其次进行大鼠下
领骨缺损的修复。通过HE、改良Mallory’s三色法、图像分析观察
新骨形成情况。2周后缺损处的支架材料周围有新骨形成，缺损处
与支架材料呈骨性连接。本实验证明:骨髓基质细胞在体外可以
经过诱导分化为成骨细胞作为种子细胞应用于骨组织工程，其与
陶瓷化骨复合后移植成骨明显，具有良好的骨组织工程应用前景。
第几部分:CBB/MSCs修复领骨缺损后牙齿移动研究
在组织工程骨修复领骨缺损ZW、4W和SW后，在大鼠下颇第
一磨牙和切牙间安装正畸矫治器，加力近中移动第一磨牙。然后
对移动牙齿牙周组织改建进行观察，并与正常牙周组织对比，以
了解组织工程骨修复领骨缺损后对正畸牙齿移动的影响。对牙齿
移动速度的研究表明，牙齿在缺损修复区的移动速度要快于正常
组织。对缺损修复区牙齿移动完成后牙根吸收和牙槽骨高度变化
的研究表明，牙根吸收值和牙槽骨高度丧失比对照组要小。证明
组织工程骨修复缺损不会对牙齿移动产生不良影响。进一步对牙
齿牙周组织改建进行研究发现，缺损修复区牙齿移动的牙周组织
改建规律与正常组织相同。但前者破骨细胞数量较多，骨陷窝数
量和面积也较大，BMP免疫组化染色阳性较强。提示骨缺损修复
区牙齿移动过程中牙周组织改建活跃，有利于正畸牙齿移动。
Tissue engineering is a rising research field which regenerate the
damaged tissue and organs by the principle of cell biology and mechenics, while bone tissue engineering is the main branch of it. Bone marrow stromal cells have got the priority in the seed cells choosing list because of their traits in rich resources , little trauma to the human body, easy to get and easy to proliferate. Ceramic bovine bone (CBB) has the natural trabecular structure, and it is easy to be conducted to generate ossification, at the same time, CBB do not produce such problems as the difficulty in making the proper hole rate and hole diameter and inner connection of artificial materials during the production process. On the contrary, CBB have many good traits such as decomposition available, not immunogenic, rich resources, easy to product, and low cost, so, it became the idea scaffold for bone tissue engineering in recent years. We use CBB as scaffold , BMSC as seed cells in our study, We also use rhBMP-2 and TGF-P for they can promote the bone formation. We try to regenerate a new kind of tis
sue engineering bone in our study.
Part I: experimental study on CBB/MBCs regenerate the defect of jaw First, we separated, cultivated, and induced(10nmol Dexamethasone 0.01mol/L P-Sodium Glycerophosphate, 50mg/L L-Vitamin C) SD rats MSCs out of their body and observed their biological characteristic alterations by inverted phase contrast microscope, the alkaline phosphatase level of cells, MTT chromometry, Von-Konssa staining and immunohistochemistry stainings.
Results: 5 days later,some BMSc differentiated to be osteoblast like cells; 20 days later, many minerized nodus formed.Second, we observed the influences of different combinations between BMSC and scaffolds on the absorption, proliferation and differentiation ability of BMSc.By the result of Scan Telescope and immuohistochemistry staining, we proved that MSCs can absorb, spread and proliferate well on the CBB scaffold. Then, we embedded the induced MSCs into the CBB scaffold, and imput them under the skin of SD rats. We make sure the MSCs were from the rats themselves. Next, we observed the influences of grow factor rhBMP-2 and TGF-B on the ossification of MSCs by HE staining, Mallary staining, and image analysis. Results: CBB combined with induced MSCs can form woven bone, lameller bone under the SD rats skin, rhBMP-2 and TGF-P can promote such effects. Third, we use the complex of induced MSCs and CBB to regenerate the bone defect of mandibular. Still, we observed by HE staining, Mallary staining, and image analysis. Results: 2 weeks later, there were new bone formation around the CBB scaffold, the ossification connection had been achieved between the bone defect area and CBB scaffold.
Conclusion: Our result proved that MSCs can be induced to be osteoblast like cells out of body and the induced MSCs combined with CBB can achieve prominent ossification, so, they have potential in bone tissue engineering applications.
Part II : Orthodontic tooth movement after CBB/MSCs regenerate the bone defection in jaw
After the jew defect was repaired 2weeks , 4 weeks, and 8 weeks later, we put the orthodontic appliance between the first molar and incisors of SD rats, adding force on the appliance to move the first molar moving forward. Then, we observed the rebuilding of periodontal ligament and compared the results to the normal periodontal ligament to understand the influence of the regeneration
new bone on the orthodontic tooth movement. The study on the tooth movement speed showed that: the tooth movement in the bone repair area was greater that in the normal bone tissue and the main movement style was bodily movement. Observing the apical absorption and the the alveolar height alterations in the repair area, we found that the apical absorption and the alveolar height loss was less than that in the normal condition. Thus, we proved that the regenerated bone by bone tissue engineering can not have negative influences on tooth movement. Further study done on periodontal ligament
第一部分：CBB／MSCs修复颌骨缺损的实验研究
首先进行大鼠骨髓基质细胞的分离、体外培养及使用矿化液(10~(-8)M地塞米松、0.01 mol／L β-甘油磷酸钠、50mg／L维生素C)诱导，然后应用细胞计数、MTT检测、ALP检测、免疫组织化学、Von Kossa等方法观察MSC在矿化液作用下生物学特性的改变。发现5天时已经有一部分骨髓基质细胞转化为成骨细胞，诱导20天后可见矿化结节。其次观察复合方式对骨髓基质细胞(MSC)的贴附、增殖、分化的影响。将诱导后的MSCs与CBB复合。体外培养，观察其对骨髓基质细胞成骨能力的影响。应用扫描电镜、ALP、免
第四军医人学博卜学位论文
疫组化等方法观察，证明MSCs在CBB上能够很好的贴附、伸展和
繁殖。将矿化诱导后的MSCs接种在CBB上，植于裸鼠及自体大鼠
皮下，观察thBMP一2和TGF一p等生长因子对MSCs成骨能力的影响，
通过HE、改良Mallory’s三色法、图像分析观察骨形成情况。结果
表明CBB复合MSCs在自体皮下能够形成编织骨、板层骨等组织，
thBMP一2和TGF一p具有协同作用，促进骨基质形成。其次进行大鼠下
领骨缺损的修复。通过HE、改良Mallory’s三色法、图像分析观察
新骨形成情况。2周后缺损处的支架材料周围有新骨形成，缺损处
与支架材料呈骨性连接。本实验证明:骨髓基质细胞在体外可以
经过诱导分化为成骨细胞作为种子细胞应用于骨组织工程，其与
陶瓷化骨复合后移植成骨明显，具有良好的骨组织工程应用前景。
第几部分:CBB/MSCs修复领骨缺损后牙齿移动研究
在组织工程骨修复领骨缺损ZW、4W和SW后，在大鼠下颇第
一磨牙和切牙间安装正畸矫治器，加力近中移动第一磨牙。然后
对移动牙齿牙周组织改建进行观察，并与正常牙周组织对比，以
了解组织工程骨修复领骨缺损后对正畸牙齿移动的影响。对牙齿
移动速度的研究表明，牙齿在缺损修复区的移动速度要快于正常
组织。对缺损修复区牙齿移动完成后牙根吸收和牙槽骨高度变化
的研究表明，牙根吸收值和牙槽骨高度丧失比对照组要小。证明
组织工程骨修复缺损不会对牙齿移动产生不良影响。进一步对牙
齿牙周组织改建进行研究发现，缺损修复区牙齿移动的牙周组织
改建规律与正常组织相同。但前者破骨细胞数量较多，骨陷窝数
量和面积也较大，BMP免疫组化染色阳性较强。提示骨缺损修复
区牙齿移动过程中牙周组织改建活跃，有利于正畸牙齿移动。
Tissue engineering is a rising research field which regenerate the
damaged tissue and organs by the principle of cell biology and mechenics, while bone tissue engineering is the main branch of it. Bone marrow stromal cells have got the priority in the seed cells choosing list because of their traits in rich resources , little trauma to the human body, easy to get and easy to proliferate. Ceramic bovine bone (CBB) has the natural trabecular structure, and it is easy to be conducted to generate ossification, at the same time, CBB do not produce such problems as the difficulty in making the proper hole rate and hole diameter and inner connection of artificial materials during the production process. On the contrary, CBB have many good traits such as decomposition available, not immunogenic, rich resources, easy to product, and low cost, so, it became the idea scaffold for bone tissue engineering in recent years. We use CBB as scaffold , BMSC as seed cells in our study, We also use rhBMP-2 and TGF-P for they can promote the bone formation. We try to regenerate a new kind of tis
sue engineering bone in our study.
Part I: experimental study on CBB/MBCs regenerate the defect of jaw First, we separated, cultivated, and induced(10nmol Dexamethasone 0.01mol/L P-Sodium Glycerophosphate, 50mg/L L-Vitamin C) SD rats MSCs out of their body and observed their biological characteristic alterations by inverted phase contrast microscope, the alkaline phosphatase level of cells, MTT chromometry, Von-Konssa staining and immunohistochemistry stainings.
Results: 5 days later,some BMSc differentiated to be osteoblast like cells; 20 days later, many minerized nodus formed.Second, we observed the influences of different combinations between BMSC and scaffolds on the absorption, proliferation and differentiation ability of BMSc.By the result of Scan Telescope and immuohistochemistry staining, we proved that MSCs can absorb, spread and proliferate well on the CBB scaffold. Then, we embedded the induced MSCs into the CBB scaffold, and imput them under the skin of SD rats. We make sure the MSCs were from the rats themselves. Next, we observed the influences of grow factor rhBMP-2 and TGF-B on the ossification of MSCs by HE staining, Mallary staining, and image analysis. Results: CBB combined with induced MSCs can form woven bone, lameller bone under the SD rats skin, rhBMP-2 and TGF-P can promote such effects. Third, we use the complex of induced MSCs and CBB to regenerate the bone defect of mandibular. Still, we observed by HE staining, Mallary staining, and image analysis. Results: 2 weeks later, there were new bone formation around the CBB scaffold, the ossification connection had been achieved between the bone defect area and CBB scaffold.
Conclusion: Our result proved that MSCs can be induced to be osteoblast like cells out of body and the induced MSCs combined with CBB can achieve prominent ossification, so, they have potential in bone tissue engineering applications.
Part II : Orthodontic tooth movement after CBB/MSCs regenerate the bone defection in jaw
After the jew defect was repaired 2weeks , 4 weeks, and 8 weeks later, we put the orthodontic appliance between the first molar and incisors of SD rats, adding force on the appliance to move the first molar moving forward. Then, we observed the rebuilding of periodontal ligament and compared the results to the normal periodontal ligament to understand the influence of the regeneration
new bone on the orthodontic tooth movement. The study on the tooth movement speed showed that: the tooth movement in the bone repair area was greater that in the normal bone tissue and the main movement style was bodily movement. Observing the apical absorption and the the alveolar height alterations in the repair area, we found that the apical absorption and the alveolar height loss was less than that in the normal condition. Thus, we proved that the regenerated bone by bone tissue engineering can not have negative influences on tooth movement. Further study done on periodontal ligament
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55. Minamide A, Tamaki T and Yoshida M. An experimental approach to spinal fusion using sintered bovine bone in a pig model. J Spinal Disord, 2000; 13(2): 156-170.
56．金丹，裴国献，王前等．骨髓基质细胞体外培养骨发生潜能及条件研究。中华矫形外科杂志，2000；7(6)：565-568．
57. Tenorio D, Foyle DM and Hughes FJ. The modulatory role of cementum matrix on osteoblastic cellsin vitro. J periodontal Res, 1997;32(4): 362-374.
58. Pockw inse SM, Stein JL, Lian JB, et al. Developmental stage spectific cellular responses to vitamin D and glucocoriticoids during differentiation
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60. Sous M, Bareille R, Rouais F, et al. Cellular biocompatibility and resistance to compression of macroporous beta-tricalcium phosphate ceramics. Biomaterials 1998 Dec; 19(23): 2147-2153.
61. Di Silvio L, Gurav N, Kayser MV, et al. Biodegradable microspheres: a new delivery system for growth hormone. Biomaterials 1994; 15(11): 931-936.
62. Hsu FY, Chueh, Wang YJ. Microspheres of hydroxyapatite/reconstituted collagen as supports for osteoblast cell growth. Biomaterials 1999; 20(20): 1931-1936.
63. Oldham JB, Lu L, Zhu X, et al. Biological activity of rhBMP-2 released from PLGA microspheres. J Biomech Eng, 2000; 22(3): 289-292.
64. Lu L, Yaszemski M J, Mikos AG. TGF-betal release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function. Bone Joint Surg Am, 2001; 83-A Suppl 1(Pt 2): S82-91.
65. Jin yan, Si xiaohui and Yang lianjia. Induction of new bone formation by ceramic bovine bone eomplexd with recombinant human bone morphogenetie protein-2 and transforming growth factor-β. Bone Morphonenetic Proteins Experimental And Clinical Aspects Today: International symposium. Finland, 1996
66．谭祖键，李起鸿，杨柳．转化生长因子-β1对骨形态发生蛋白诱导成骨的促进作用。第四军医大学学报2001，22(11)：998-1002．
67. Spinella-Jaegle S, Roman-Roman S, Faucheu C, et al. Opposite effects of
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69. Si X, Yang L, Jin Y. Effects of TGF beta on BMP-2 gene transfection cells in vitro. Hua Xi Kou Qiang Yi Xue Za Zhi, 1999,17(4): 321-324.
70. Minamide A, Kawakami M, Hashizume H, et al. Evaluation of carriers of bone morphogenetic protein for spinal fusion. Spine,2001; 26(8): 933-939.
71. Niedhart C, Maus U, Redmann E, et al. Stimulation of bone formation with an in situ setting tricalcium phosphate/rhBMP-2 composite in rats. J Biomed Mater Res 2003; 65A(1): 17-23.
72. Yumei Zhang, Tao Fu, Yong Han, et al. In vitro and in vivo tests of hydrothermally synthesized hydroxyapatite coating.Biomolecular Engineering, 2002, 19:57-61.
73. Carl G. Simon Jra, Chetan A. Preliminary report on the biocompatibility of a moldable, resorbable, composite bone graft consisting of calcium phosphate cement and poly(lactide-co-glycolide) microspheres. Journal of Orthopaedic Research, 2002, 20: 473-482.
74．张殿忠，范清宇，周勇等．牛煅烧骨颗粒复合骨水泥的生物力学性能评定．现代康复，2001，5(7)：48-49．
75. Minamide A, Tamaki T and Yoshida M.The use of sintered bone in spinal surgery. Eur Spine J, 2001, Oct; 10 Suppl 2: S185-188.
76. King Gj, Keeling SD,M ccoy EA et al. M easuring dental drift and o rthodontic too th movement in response to various initial fo rces in adult rats. A m J O rthod D entofacial O rthop, 1991; 99(4): 456-465
77. Zah row ski JJ, Patrick K. Force magnitude effects upon o steop regenitor cells during p remaxillary expansion in rats. A ng le O rthod ,1992; 62 (3):
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78. Wainwright WM Am JOrthod 1973; 64:278-286
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80．段银钟主编，口腔正畸生物学[M]．世界图书出版公司1994：109-145
81. Newmen WG. Possible Etialogio factors in externed root resorption[J]. AmJ Orthod, 1975,67:522-528
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84. Paitan K. Lnitial tissue beharior during apical root resorption[J]. Angle Orthod, 1974,44:68-71
85. Wainwright WM. Faciolingual tooth movement: its influence on the root and cartical Plate[J]. AmJ Orthod, 1973,64:278-284
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93．张丁 李小彤。成人与青少年牙槽骨中骨陷窝面积与数目的比较。口腔正畸学2000年第7卷第1期：18-19
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49. Hashizume H, Tamaki T, Oura H, et al. Changes in the extracellular matrix on the surface of sintered bovine bone implanted in the femur of a rabbit: an immunohistochemical study. J Orthop Sci, 1998; 3(1): 42-54.
50. Minamide A, Tamaki T, Kawakami M, et al. Experimental spinal fusion using sintered bovine bone coated with type Ⅰ collagen and recombinant human bone morphogenetic protein-2. Spine, 1999; 24(18): 1863-1872.
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54. Matsumoto T, Kawakami M, Kuribayashi K, et al. Effects of sintered bovine bone on cell proliferation, collagen synthesis, and osteoblastic expression in MC3T3-E1 osteoblast-like cells. J Orthop Res, 1999; 17(4): 586-598.
55. Minamide A, Tamaki T and Yoshida M. An experimental approach to spinal fusion using sintered bovine bone in a pig model. J Spinal Disord, 2000; 13(2): 156-170.
56．金丹，裴国献，王前等．骨髓基质细胞体外培养骨发生潜能及条件研究。中华矫形外科杂志，2000；7(6)：565-568．
57. Tenorio D, Foyle DM and Hughes FJ. The modulatory role of cementum matrix on osteoblastic cellsin vitro. J periodontal Res, 1997;32(4): 362-374.
58. Pockw inse SM, Stein JL, Lian JB, et al. Developmental stage spectific cellular responses to vitamin D and glucocoriticoids during differentiation
of osteoblast phenotype: interrelationship of morphology and gene expression by in situ hybridization. Exp Cell Res, 1995, 216(1): 244-260.
59. Matsumoto T, Kawakami M, Kuribayashi K, et al. Effects of sintered bovine bone on cell proliferation, collagen synthesis,and osteoblastic expression in MC3T3-E1 osteoblast-like cells.J Orthop Res 1999; 17(4): 586-592.
60. Sous M, Bareille R, Rouais F, et al. Cellular biocompatibility and resistance to compression of macroporous beta-tricalcium phosphate ceramics. Biomaterials 1998 Dec; 19(23): 2147-2153.
61. Di Silvio L, Gurav N, Kayser MV, et al. Biodegradable microspheres: a new delivery system for growth hormone. Biomaterials 1994; 15(11): 931-936.
62. Hsu FY, Chueh, Wang YJ. Microspheres of hydroxyapatite/reconstituted collagen as supports for osteoblast cell growth. Biomaterials 1999; 20(20): 1931-1936.
63. Oldham JB, Lu L, Zhu X, et al. Biological activity of rhBMP-2 released from PLGA microspheres. J Biomech Eng, 2000; 22(3): 289-292.
64. Lu L, Yaszemski M J, Mikos AG. TGF-betal release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function. Bone Joint Surg Am, 2001; 83-A Suppl 1(Pt 2): S82-91.
65. Jin yan, Si xiaohui and Yang lianjia. Induction of new bone formation by ceramic bovine bone eomplexd with recombinant human bone morphogenetie protein-2 and transforming growth factor-β. Bone Morphonenetic Proteins Experimental And Clinical Aspects Today: International symposium. Finland, 1996
66．谭祖键，李起鸿，杨柳．转化生长因子-β1对骨形态发生蛋白诱导成骨的促进作用。第四军医大学学报2001，22(11)：998-1002．
67. Spinella-Jaegle S, Roman-Roman S, Faucheu C, et al. Opposite effects of
bone morphogenetic protein-2 and transforming growth factor-betal on osteoblast differentiation. Bone, 2001; 29(4): 323-330.
68. Lu L, Yaszemski MJ, Mikos AG. TGF-betal release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function. Bone Joint Surg Am 2001; 83-A Suppl 1(Pt 2): S82-91.
69. Si X, Yang L, Jin Y. Effects of TGF beta on BMP-2 gene transfection cells in vitro. Hua Xi Kou Qiang Yi Xue Za Zhi, 1999,17(4): 321-324.
70. Minamide A, Kawakami M, Hashizume H, et al. Evaluation of carriers of bone morphogenetic protein for spinal fusion. Spine,2001; 26(8): 933-939.
71. Niedhart C, Maus U, Redmann E, et al. Stimulation of bone formation with an in situ setting tricalcium phosphate/rhBMP-2 composite in rats. J Biomed Mater Res 2003; 65A(1): 17-23.
72. Yumei Zhang, Tao Fu, Yong Han, et al. In vitro and in vivo tests of hydrothermally synthesized hydroxyapatite coating.Biomolecular Engineering, 2002, 19:57-61.
73. Carl G. Simon Jra, Chetan A. Preliminary report on the biocompatibility of a moldable, resorbable, composite bone graft consisting of calcium phosphate cement and poly(lactide-co-glycolide) microspheres. Journal of Orthopaedic Research, 2002, 20: 473-482.
74．张殿忠，范清宇，周勇等．牛煅烧骨颗粒复合骨水泥的生物力学性能评定．现代康复，2001，5(7)：48-49．
75. Minamide A, Tamaki T and Yoshida M.The use of sintered bone in spinal surgery. Eur Spine J, 2001, Oct; 10 Suppl 2: S185-188.
76. King Gj, Keeling SD,M ccoy EA et al. M easuring dental drift and o rthodontic too th movement in response to various initial fo rces in adult rats. A m J O rthod D entofacial O rthop, 1991; 99(4): 456-465
77. Zah row ski JJ, Patrick K. Force magnitude effects upon o steop regenitor cells during p remaxillary expansion in rats. A ng le O rthod ,1992; 62 (3):
197-202
78. Wainwright WM Am JOrthod 1973; 64:278-286
79. Harris E. F, Bulter ML: Patterns of incisor root resorption before and after orthodontic correction in cases with anterior openbites[J]. Am J Orthod, 1992,101-112
80．段银钟主编，口腔正畸生物学[M]．世界图书出版公司1994：109-145
81. Newmen WG. Possible Etialogio factors in externed root resorption[J]. AmJ Orthod, 1975,67:522-528
82. MC Fadden WM,Engstran C Engstran H, et al, A study of the retation ship betueen incisor intrution and root shortening[J].Am JOrthod, 1989,96:390
83. Kately J. Phillips C. factors retated to root resorption in edgeuise practice[J]. Angle Orthod, 1991; 61:125-130
84. Paitan K. Lnitial tissue beharior during apical root resorption[J]. Angle Orthod, 1974,44:68-71
85. Wainwright WM. Faciolingual tooth movement: its influence on the root and cartical Plate[J]. AmJ Orthod, 1973,64:278-284
86. Levander E. Malmgren O. Evaluation of the risk of root resorption during orthodontic treatment: a study of upper incirors[J]. Eur J Orthod, 1988,96:231-241
87. Parfitt AM. The cellular basis of bone turnover and bone loss. Clin Orthop Rel Res 1977; 127: 236-246.
88. Dillaman RM, Roser RD, Gay DM. Fluid movement in bone: theoretical an empirical. J Biomech 1991; 24: 163-171.
89. Haj AJ, Minter SL, Rawlinson SCF, et al. Cellular responses to me chanical loading in vitro. J Bone Miner Res 1990; 5: 923-931.
90. Cowin SC, Moss2Salentijn L, Moss ML. Candidates for mechanosensary system in bone. J Biomed Eng 1991; 113: 191-199.
91. Weinbaum S, Cowin SC, Zeng Y. A model for the excitation fo osteocytes
by mechanical loading2induced bone fluid shear stresses. J Biomech 1994; 27: 339-345.
92. Turner CH, Takano Y, Owan I. Aging changes mechanical loading thresh olds for bone formation in rats. J Bone Miner Res 1995; 10: 1544-1554.
93．张丁 李小彤。成人与青少年牙槽骨中骨陷窝面积与数目的比较。口腔正畸学2000年第7卷第1期：18-19
94．吴祖尧．诱导成骨与骨形态发生蛋白．中华骨科杂志，1988，8(3)：231-234．
95．金岩．牙齿与骨形成蛋白的关系．国外医学口腔医学分册，1992，19(6)：342-346．
96．金岩，杨连甲．骨形成蛋白在颌骨骨折愈合中的作用．实用口腔医学杂志，1989，5(4)：248-250．
97. Andrew JW,Hoyland J, Sharpe P. Demponstration of BMP mRNA expression in human fracture callus. J Bone J int Surg(Br),1993,75(3):281-288.
98. Kanatani M, Sugimoto T, Kaji H, et al. Stimulatory effect of bone morphogenetic protein-2 on osteoclast-like cell formation and bone-resorbing activity. J Bone Miner Res, 1995,10(11): 1681-1690