无机活性诱导元素支架材料对颌骨缺损骨创面骨祖细胞的生物诱导作用
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摘要
目的应用颌骨术后缺损骨创面周围骨松质分离培养和鉴定成骨细胞,并将其与无机活性诱导元素支架材料在体外复合生长,探讨无机活性诱导元素支架材料对骨祖细胞来源的成骨细胞生物学特性的生物诱导作用。
方法应用组织块贴壁法分离培养出成骨细胞,并用碱性磷酸酶(Burstone偶氮偶联法)染色法、茜素红染色法、透射电镜、流式细胞仪等对其碱性磷酸酶活性、钙结节形成情况、细胞的超微结构、细胞生长周期进行鉴定,然后将体外培养的成骨细胞与无机活性诱导元素支架材料体外复合生长,对复合体进行形态学、扫描电镜、细胞增殖能力、细胞周期变化的测定,评价细胞与材料的相容性。
结果颌骨术后缺损骨创面周围骨松质分离出的成骨细胞具有较强的碱性磷酸酶活性且有大量的钙结节形成,透射电镜观察其具有良好的分泌细胞特性,此成骨细胞与无机活性诱导元素支架材料体外复合培养14d,分布于支架材料的成骨细胞增殖良好,分泌细胞外基质并形成钙结节,细胞周期未见明显改变。
结论颌骨术后缺损骨创面周围骨松质中含有大量的骨祖细胞,其具有较强的向成骨细胞分化和繁殖能力。无机活性诱导元素支架材料均具有较好的细胞相容性,与成骨细胞共同培养有望获得具有三维立体结构的组织工程骨。
Objective To study the effect of bio-inductive effects of inorganic induced elements on osteogenitor cell near the postoperative jaw defects ,we isolate and culture osteoblasts from cancellous bone near the postoperative jaw defects and co-combine with cultured inorganic induced elements in vitro.
Methods By modified tissue explants-attached method,we can succeed to Isolate and culture osteoblasts. Alkaline pHospHatase (AKP) activity was determined by azo dye method,calcification nodule formed was observed by Alizarin Red S,the cell ultrastructure was identified by electron microscopy and the growth cycle was evaluated with flow cytometer.Then inorganic induced elements co-combined with cultured osteoblasts in vitro. The morpHological cytoskeleton characters,cell proliferation,mitotic cycle and DNA index were detected.
Results We found that Alkaline pHospHatase activity and Alizarin Red S was strongly positive. These cells possess good secreting characteristics. The cell cycle and pHysiological characteristics did not significantly change after co-cultured 14 days.
Conclusion Cancellous bone near the postoperative jaw defects contain a lot of osteogenitor cells which have osteogenic potential.The inorganic induced elements scaffold material has good cellular compatibility. So it is possible to achieve the tissue engineering bone which possesses three-dimensional stereoscopic structure by co-culturing them.
方法应用组织块贴壁法分离培养出成骨细胞,并用碱性磷酸酶(Burstone偶氮偶联法)染色法、茜素红染色法、透射电镜、流式细胞仪等对其碱性磷酸酶活性、钙结节形成情况、细胞的超微结构、细胞生长周期进行鉴定,然后将体外培养的成骨细胞与无机活性诱导元素支架材料体外复合生长,对复合体进行形态学、扫描电镜、细胞增殖能力、细胞周期变化的测定,评价细胞与材料的相容性。
结果颌骨术后缺损骨创面周围骨松质分离出的成骨细胞具有较强的碱性磷酸酶活性且有大量的钙结节形成,透射电镜观察其具有良好的分泌细胞特性,此成骨细胞与无机活性诱导元素支架材料体外复合培养14d,分布于支架材料的成骨细胞增殖良好,分泌细胞外基质并形成钙结节,细胞周期未见明显改变。
结论颌骨术后缺损骨创面周围骨松质中含有大量的骨祖细胞,其具有较强的向成骨细胞分化和繁殖能力。无机活性诱导元素支架材料均具有较好的细胞相容性,与成骨细胞共同培养有望获得具有三维立体结构的组织工程骨。
Objective To study the effect of bio-inductive effects of inorganic induced elements on osteogenitor cell near the postoperative jaw defects ,we isolate and culture osteoblasts from cancellous bone near the postoperative jaw defects and co-combine with cultured inorganic induced elements in vitro.
Methods By modified tissue explants-attached method,we can succeed to Isolate and culture osteoblasts. Alkaline pHospHatase (AKP) activity was determined by azo dye method,calcification nodule formed was observed by Alizarin Red S,the cell ultrastructure was identified by electron microscopy and the growth cycle was evaluated with flow cytometer.Then inorganic induced elements co-combined with cultured osteoblasts in vitro. The morpHological cytoskeleton characters,cell proliferation,mitotic cycle and DNA index were detected.
Results We found that Alkaline pHospHatase activity and Alizarin Red S was strongly positive. These cells possess good secreting characteristics. The cell cycle and pHysiological characteristics did not significantly change after co-cultured 14 days.
Conclusion Cancellous bone near the postoperative jaw defects contain a lot of osteogenitor cells which have osteogenic potential.The inorganic induced elements scaffold material has good cellular compatibility. So it is possible to achieve the tissue engineering bone which possesses three-dimensional stereoscopic structure by co-culturing them.
引文
1.王常勇,郭希民,费振祥.干细胞与组织工程,见裴雪涛主编.干细胞生物学[M].北京:科学出版社.2003:502-511.
2. Eugene Bell.(张姝江,杨志明译).组织工程的前景,见RP.Lanza,R.Langer,J.Vacanti主编(杨志明译)组织工程原理[M].北京:化学工业出版社,2006:1-7.
3. Y.Kinoshita,T.Amagasa.(林云峰,田卫东译).颌骨,见A.Atala,RP.Lanza主编(杨志明译)组织工程方法[M].北京:化学工业出版社,2006:1068-1073.
4.叶茂昌,周来生,陈柯,等.无机诱导因子复合性支架材料修复骨缺损的组织学观察[J].口腔颌面外科杂志.2005,15(3):247-250.
5.叶茂昌,周来生,康然刚,等.无机诱导因子支架材料行颌骨重建的放射学评价[J].中国口腔颌面外科杂志.2005,3(4):330-334.
6.陈梅梅,叶茂昌,周来生,等.无机诱导因子骨组织工程支架材料体外细胞相容性的实验研究[J].口腔医学研究.2005,21(5):513-516.
7.叶茂昌,周来生,李素梅,等.骨密度测量在评估骨支架材料促成骨作用中的意义[J].口腔医学研究.2005,21(6):615-617.
8.王来平,周来生,叶茂昌,等.无机诱导因子复合性骨支架材料的生物相容学研究兼论人体外复合细胞相容性观察[J].现代口腔医学杂志.2006,20(6):601-605.
9.陈志方,叶茂昌,丁晓红,等.无机诱导因子在牙槽骨缺损牙再植中的应用[J].中国口腔颌面外科杂志.2007,5:107-109.
10.邱蔚六.颌面外科理论与实践[M]第二版北京人民卫生出版社:1016-1020.
11. Tsao YP, Neiva R, Al-Shammari K,et al. Effects of a mineralized human cancellous bone allograft in regeneration of mandibular Class II furcation defects[J].J Periodontol. 2006,77(3):416-425.
12.袁捷,翁雨来,曹谊林.下颌骨缺损修复方法的研究进展[J].上海口腔医学2004,13(2):137-140.
13. Chou L,Al-Bazie S,Cottel D,et al. Atomie and molecular mechanisms underlying the osteogenic effects of bioglass materials[J]. Bioceramics.1998,11:265-268.
14.何川,邓廉夫,周来生,等.自体成骨细胞-nBGC复合物修复犬胫骨骨缺损[J].中华矫形外科杂志,2003,11(15):1047-1049.
15. Lyu K, Nathanson D, Cottrell D, Chou L. Induced osteonenesis in vitro by silicon, calcium, and pHospHorous ions[J]. Journal of Dental Research. 2001, 80:121.
16. Chou L, Cottrell D, Wu B. Clinical application of novel scaffolds for humans bone tissue engineering[J]. Journal of Dental Research. 2004, 304:2885.
17.叶茂昌,Chou L,王来平等.无机活性元素对山羊骨组织缺损重建与再造的生物诱导作用。中国组织工程研究与康复[J],2007,11(48):9627-9632.
18. Webster TI, Ergun C, Doremus RH, et al. Enhanced osteoclast-Like Cell function on nanopHase ceramics[J]. Biomaterials,2001,22(12):1327~1333.
19. Roostaeian J, Carlsen B, Simhaee D et al. Characterization of growth and osteogenic differentiation of rabbit bone marrow stromal cells[J]. J Surg Res. 2006,133(2):76-83.
20. M?dder UI, Khosla S. Skeletal stem/osteoprogenitor cells: Current concepts, alternate hypotheses, and relationship to the bone remodeling compartment[J]. J Cell Biochem. 2007 ,31.
21.王敏,崔磊,曹谊林.种子细胞培养技术.曹谊林.组织工程理论与实践[M]第一版上海上海科学技术出版社:114-117.
22. McDuffee LA, Anderson GI In vitro comparison of equine cancellous bone graft donor sites and tibial periosteum as sources of viable osteoprogenitors[J]. Vet Surg. 2003 ,32(5):455-63.
23. Trentz OA, Platz A, Helmy Net al. Response of osteoblast cultures to titanium, steel and hydroxyapatite implants[J]. Swiss Surg. 1998;(4):203-9.
24.王文勇,邓南生,张志培等.用细胞爬片和石蜡切片对HCV核心蛋白进行免疫组化染色的比较[J] .细胞与分子免疫学杂志,2005,21(3):392-393.
25. Mackie EJ.Osteoblasts: novel roles in orchestration of skeletal architecture[J]. Int J Biochem Cell Biol. 2003 ,35(9):1301-1305.
26. Sun JS, Wu SY, Lin FH. The role of muscle-derived stem cells in bone tissue engineering[J]. Biomaterials. 2005 ,26(18):3953-60.
27.周来生.生物医学工程学技术在创面修复领域的应用前景[J].中华烧伤杂志.2005,21:6-8.
28. Thomas V , J agani S , Johnson K, et al. Elect rospun bioactive nanocomposite scaffolds of polycaprolactone and nanohydroxyapaite for bone tissue engineering[J]. J Nanosci Nanotechnol , 2006 , 6 (2) :487.
29. Woo K M , Chen V J , Ma P X. Nano2fibrous scaffolding architecture selectively enhances protein adsorption cont ributing to cell attachment[J] . J Biomed Mater Res A , 2003 , 67(2) :531.
30. Mustafa K, Oden A , Wennerberg A , et al. The influence of surface topograpHy of ceramic abutment s on the attachment and proliferation of human oral fibroblast s[J]. Biomaterials , 2005 , 26 (4) : 373.
31. Chou L,Firth J,Uitto V,etal.Effect of surface topograpHy on fibronectin mRNA level.mRNA stability.secretion and assembly in human fibroblast[J].J Cell Science.1995,108:1563-1571.
1. Anderson JM. Multinucleated giant cells. Curr Opin Hematol, 2000,7(1): 40-47.
2.水野-乘,今村保忠,林利彦.蛋白質核酸酵素,2000,45(12):2161-2170.
3. Ingber DE. The architecture of life.Sci Am, 1998, 278(1):48-57.
4.筏义人.ティヲュ-ュァジニアンケ.金属.1998,36(3):23-32.
5. Rosales C, Juliano RL. Signal transduction by cell adhesion receptors in leuko- cytes.J Leukoc Biol, 1995,57(2):189-198.
6. Wei J,Shaw LM, Mercurio AM. Integrin signaling in leukocytes: lessons from the alpHa6beta1 integrin.J Leukoc Biol. 1997,61(4 ): 397-407.
7. Pakianathan DR. Extracellular matrix proteins and leukocyte function.J Leukoc Biol, 1995,57(5): 699-702.
8. Sevice RF. Tissue engineering build new bone .Science, 2000, 289 (5484):1498- 1500.
9. White SR, Dorscheid DR, Rabe KF, et al. Role of very late adhesion integrins in mediating repair of human airway epithelial cell monolayers after mechanical injury .Am J Respir Cell Mol Biol, 1999,20(4):787-796.
10. Li Y, Ma T, Kniss DA, et al. Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices . Biotechnol prog, 2001,17(5):935-944.
11. Hartgerink JD, Beniash E, Stupp SI. Self-assembly and mineralization of peptide- ampHipHile nanofibers .Science, 2001,294(5547):1684-1688.
12. Tian WM, Hou SP, Ma J, et al. Hyaluronic acid-poly-D-lysine-based three-dimen- sional hydrogel for traumatic brain injury. Tissue Eng, 2005,11(3-4):513-525.
13. Scholl I, Kopp T, Bohle B, et al. Biodegradable PLGA Particles for Improved Systemic and Mucosal Treatment of Type I Allergy .Immunol Allergy Clin North Am. 2006 ,26(2):349-64, ix. Review.
14. Li RH, Wozney JM. Delivering on the promise of bone morpHogenetic proteins .Trends Biotechnol, 2001,19(7):255-265.
15. Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chem Rev, 2001,101(7): 1869-1879.
16. Balkwill F. The molecular and cellular biology of the chemokines. J Viral Hepat, 1998,5(1):1-14.
17. Locati M, MurpHy PM. Chemokines and chemokine receptors:biology and clini- cal relevance in inflammation and AIDS. Annu Rev Med, 1999,50:425-440.
18. Vehof JW, Fisher JP, Dean D, et al. Nell-1-Induced Bone Regeneration in Calvarial Defects.J Biomed Mater Res, 2002,60(2):241-251.
19. Tanihara M, Suzuki Y, Yamamoto E, et al. Sustained release of basic fibroblast growth factor and angiogenesis in a novel covalently crosslinked gel ofheparin and alginate.J Biomed Mater Res, 2001,56(2):216-221.
20. Freier T, Kunze C, Nischan C, et al. In vitro and in vivo degradation studies for development of a biodegradable patch base on poly(3-hydroxybutyrate). Bioma- terials,2002,23(13):2649-2657.
21. Methe H, Edelman ER. Tissue engineering of endothelial cells and the immune response. Transplant Proc, 2006,38(10):3293-3299.
22. Temenoff JS, Mikos AG.Injectable biodegradable materials for orthopedic tissue engineering.Biomaterials, 2000,21(23):2405-2412.
2. Eugene Bell.(张姝江,杨志明译).组织工程的前景,见RP.Lanza,R.Langer,J.Vacanti主编(杨志明译)组织工程原理[M].北京:化学工业出版社,2006:1-7.
3. Y.Kinoshita,T.Amagasa.(林云峰,田卫东译).颌骨,见A.Atala,RP.Lanza主编(杨志明译)组织工程方法[M].北京:化学工业出版社,2006:1068-1073.
4.叶茂昌,周来生,陈柯,等.无机诱导因子复合性支架材料修复骨缺损的组织学观察[J].口腔颌面外科杂志.2005,15(3):247-250.
5.叶茂昌,周来生,康然刚,等.无机诱导因子支架材料行颌骨重建的放射学评价[J].中国口腔颌面外科杂志.2005,3(4):330-334.
6.陈梅梅,叶茂昌,周来生,等.无机诱导因子骨组织工程支架材料体外细胞相容性的实验研究[J].口腔医学研究.2005,21(5):513-516.
7.叶茂昌,周来生,李素梅,等.骨密度测量在评估骨支架材料促成骨作用中的意义[J].口腔医学研究.2005,21(6):615-617.
8.王来平,周来生,叶茂昌,等.无机诱导因子复合性骨支架材料的生物相容学研究兼论人体外复合细胞相容性观察[J].现代口腔医学杂志.2006,20(6):601-605.
9.陈志方,叶茂昌,丁晓红,等.无机诱导因子在牙槽骨缺损牙再植中的应用[J].中国口腔颌面外科杂志.2007,5:107-109.
10.邱蔚六.颌面外科理论与实践[M]第二版北京人民卫生出版社:1016-1020.
11. Tsao YP, Neiva R, Al-Shammari K,et al. Effects of a mineralized human cancellous bone allograft in regeneration of mandibular Class II furcation defects[J].J Periodontol. 2006,77(3):416-425.
12.袁捷,翁雨来,曹谊林.下颌骨缺损修复方法的研究进展[J].上海口腔医学2004,13(2):137-140.
13. Chou L,Al-Bazie S,Cottel D,et al. Atomie and molecular mechanisms underlying the osteogenic effects of bioglass materials[J]. Bioceramics.1998,11:265-268.
14.何川,邓廉夫,周来生,等.自体成骨细胞-nBGC复合物修复犬胫骨骨缺损[J].中华矫形外科杂志,2003,11(15):1047-1049.
15. Lyu K, Nathanson D, Cottrell D, Chou L. Induced osteonenesis in vitro by silicon, calcium, and pHospHorous ions[J]. Journal of Dental Research. 2001, 80:121.
16. Chou L, Cottrell D, Wu B. Clinical application of novel scaffolds for humans bone tissue engineering[J]. Journal of Dental Research. 2004, 304:2885.
17.叶茂昌,Chou L,王来平等.无机活性元素对山羊骨组织缺损重建与再造的生物诱导作用。中国组织工程研究与康复[J],2007,11(48):9627-9632.
18. Webster TI, Ergun C, Doremus RH, et al. Enhanced osteoclast-Like Cell function on nanopHase ceramics[J]. Biomaterials,2001,22(12):1327~1333.
19. Roostaeian J, Carlsen B, Simhaee D et al. Characterization of growth and osteogenic differentiation of rabbit bone marrow stromal cells[J]. J Surg Res. 2006,133(2):76-83.
20. M?dder UI, Khosla S. Skeletal stem/osteoprogenitor cells: Current concepts, alternate hypotheses, and relationship to the bone remodeling compartment[J]. J Cell Biochem. 2007 ,31.
21.王敏,崔磊,曹谊林.种子细胞培养技术.曹谊林.组织工程理论与实践[M]第一版上海上海科学技术出版社:114-117.
22. McDuffee LA, Anderson GI In vitro comparison of equine cancellous bone graft donor sites and tibial periosteum as sources of viable osteoprogenitors[J]. Vet Surg. 2003 ,32(5):455-63.
23. Trentz OA, Platz A, Helmy Net al. Response of osteoblast cultures to titanium, steel and hydroxyapatite implants[J]. Swiss Surg. 1998;(4):203-9.
24.王文勇,邓南生,张志培等.用细胞爬片和石蜡切片对HCV核心蛋白进行免疫组化染色的比较[J] .细胞与分子免疫学杂志,2005,21(3):392-393.
25. Mackie EJ.Osteoblasts: novel roles in orchestration of skeletal architecture[J]. Int J Biochem Cell Biol. 2003 ,35(9):1301-1305.
26. Sun JS, Wu SY, Lin FH. The role of muscle-derived stem cells in bone tissue engineering[J]. Biomaterials. 2005 ,26(18):3953-60.
27.周来生.生物医学工程学技术在创面修复领域的应用前景[J].中华烧伤杂志.2005,21:6-8.
28. Thomas V , J agani S , Johnson K, et al. Elect rospun bioactive nanocomposite scaffolds of polycaprolactone and nanohydroxyapaite for bone tissue engineering[J]. J Nanosci Nanotechnol , 2006 , 6 (2) :487.
29. Woo K M , Chen V J , Ma P X. Nano2fibrous scaffolding architecture selectively enhances protein adsorption cont ributing to cell attachment[J] . J Biomed Mater Res A , 2003 , 67(2) :531.
30. Mustafa K, Oden A , Wennerberg A , et al. The influence of surface topograpHy of ceramic abutment s on the attachment and proliferation of human oral fibroblast s[J]. Biomaterials , 2005 , 26 (4) : 373.
31. Chou L,Firth J,Uitto V,etal.Effect of surface topograpHy on fibronectin mRNA level.mRNA stability.secretion and assembly in human fibroblast[J].J Cell Science.1995,108:1563-1571.
1. Anderson JM. Multinucleated giant cells. Curr Opin Hematol, 2000,7(1): 40-47.
2.水野-乘,今村保忠,林利彦.蛋白質核酸酵素,2000,45(12):2161-2170.
3. Ingber DE. The architecture of life.Sci Am, 1998, 278(1):48-57.
4.筏义人.ティヲュ-ュァジニアンケ.金属.1998,36(3):23-32.
5. Rosales C, Juliano RL. Signal transduction by cell adhesion receptors in leuko- cytes.J Leukoc Biol, 1995,57(2):189-198.
6. Wei J,Shaw LM, Mercurio AM. Integrin signaling in leukocytes: lessons from the alpHa6beta1 integrin.J Leukoc Biol. 1997,61(4 ): 397-407.
7. Pakianathan DR. Extracellular matrix proteins and leukocyte function.J Leukoc Biol, 1995,57(5): 699-702.
8. Sevice RF. Tissue engineering build new bone .Science, 2000, 289 (5484):1498- 1500.
9. White SR, Dorscheid DR, Rabe KF, et al. Role of very late adhesion integrins in mediating repair of human airway epithelial cell monolayers after mechanical injury .Am J Respir Cell Mol Biol, 1999,20(4):787-796.
10. Li Y, Ma T, Kniss DA, et al. Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices . Biotechnol prog, 2001,17(5):935-944.
11. Hartgerink JD, Beniash E, Stupp SI. Self-assembly and mineralization of peptide- ampHipHile nanofibers .Science, 2001,294(5547):1684-1688.
12. Tian WM, Hou SP, Ma J, et al. Hyaluronic acid-poly-D-lysine-based three-dimen- sional hydrogel for traumatic brain injury. Tissue Eng, 2005,11(3-4):513-525.
13. Scholl I, Kopp T, Bohle B, et al. Biodegradable PLGA Particles for Improved Systemic and Mucosal Treatment of Type I Allergy .Immunol Allergy Clin North Am. 2006 ,26(2):349-64, ix. Review.
14. Li RH, Wozney JM. Delivering on the promise of bone morpHogenetic proteins .Trends Biotechnol, 2001,19(7):255-265.
15. Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chem Rev, 2001,101(7): 1869-1879.
16. Balkwill F. The molecular and cellular biology of the chemokines. J Viral Hepat, 1998,5(1):1-14.
17. Locati M, MurpHy PM. Chemokines and chemokine receptors:biology and clini- cal relevance in inflammation and AIDS. Annu Rev Med, 1999,50:425-440.
18. Vehof JW, Fisher JP, Dean D, et al. Nell-1-Induced Bone Regeneration in Calvarial Defects.J Biomed Mater Res, 2002,60(2):241-251.
19. Tanihara M, Suzuki Y, Yamamoto E, et al. Sustained release of basic fibroblast growth factor and angiogenesis in a novel covalently crosslinked gel ofheparin and alginate.J Biomed Mater Res, 2001,56(2):216-221.
20. Freier T, Kunze C, Nischan C, et al. In vitro and in vivo degradation studies for development of a biodegradable patch base on poly(3-hydroxybutyrate). Bioma- terials,2002,23(13):2649-2657.
21. Methe H, Edelman ER. Tissue engineering of endothelial cells and the immune response. Transplant Proc, 2006,38(10):3293-3299.
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