体外诱导BMSc在金属腔隙内成骨的实验研究
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摘要
目的：本课题利用中空多孔金属支架为骨组织再生工程构架，观察体外骨髓基质干细胞(bone marrow mesenchymal stem cells，BMSc)在支架内、外的分化生长情况以及成骨的可能性，并探讨体外应用骨诱导因子—骨形态发生蛋白—2(recombined human bone morphogenetic protein-2，rhBMP-2)提高成骨活性，促进金属假体与骨质相对一体化结合的可行性，为研制中空多孔人工关节假体或其他骨组织再生骨植入物提供基础实验依据。希望实验结果能够为生物固定的人工关节设计方法的改进提供一些理论及实验依据。
方法：自行设计中空金属试件，采用中空金属试件(A组)、中空金属试件假体+脱钙骨基质(decalcified bone matrix DBM)(B组)、中空金属试件+DBM+成骨诱导液(C组)、中空金属假体+DBM+rhBMP-2培养液(D组)，金属试件均为HA涂层，于BMSc混悬液中加10％胎牛血清DMEM培养液培养，在2、4周观察点取材，通过倒置相差显微镜、扫描电镜、成骨细胞表型检测等方法对中空金属试件表面、脱钙骨基质BMSc生长以及成骨分化进行观察、鉴定。
使用SPSS15.0软件包进行统计分析，计量资料以均数±标准差表示，组间同一时间各指标比较采用组间方差分析。
结果：实验发现：倒置相差显微镜、扫描电镜等方法显示中空金属试件表面及试件内三维细胞支架上BMSc细胞黏附正常，经成骨诱导后转为成骨细胞，并形成工程化组织。在实验设计的两种骨诱导方式中，成骨细胞的表达以成骨诱导液为明显，在统计学意义上其他对照组有明显差异。
结论：
一、体外DMSc经诱导向成骨细胞分化，并可在中空金属试件的空间结构中形成组织块。
二、中空金属假体的设计思路是可行的，在体外可以观察到BMSc在金属腔壁与腔内细胞支架之间紧密结合，并且在无细胞支架的情况下，1mm孔洞可以形成细胞间的直接连接。
因此中空结构的假体是一个值得探索的方向，希望对生物复合型假体的改进具有一定的指导意义。
Objective: Histological, histomorphological analysis were used to analyze the bony in-growth conditions in the interface of bone and prosthesis and inside hollow prosthesis of 4 groups. We hope that the results of our research can provide the clinical using new hollow prosthesis to improve biological fixation of artificial joint with some theoretical and experimental data.
Methods: We designed hollow prosthesis by self, and divided it into 5 group: hollow prosthesis (A group), hollow prosthesis with DBM (B group), hollow prosthesis with DBM in osteogenesis inducing nutrient liquid (D group), hollow prosthesis with DBM combined with rhBMP-2(D group). All hollow prostheses coated with hydroxyapatite. Then BMSc at the surface of the hollow prostheses and in the DBM was researched by inverted phase contrast microscope, scanning electron microscope, osteoblast phaenotype detection after 2and 4 weeks.
Measurement data were expressed as means+SD(x±s) and analyzed by t Test.
Results: Inverted phase contrast microscope and scanning electron microscope showed that BMSc at the surface and the hole of the hollow prosthese and in the DBM 3—D cytoskeleton can adhere normally and transform into osteoblast by inducing and form engineering tissue. Osteoblast phaenotype detection after 2 weeks shows the results: the present of osteoblast of C group is significant difference with other groups statistically
Conclusion : 1.BMSc at the surface and the hole of the hollow prostheses and in the DBM 3—D cytoskeleton can adhere normally and transform into osteoblast
by inducing and form engineering tissue in vitro.2.the desigen of the hollow prostheses is feasible. It shows in vitro that BMSc at the wall of the prosthesis can combine with the DBM 3—D cytoskeleton and BMSc in the 1 mm hole of the prosthesis without 3—D cytoskeleton can also connect directly.
So the hollow prosthesis is worthy with more researching, having some guidance in clinic.
方法：自行设计中空金属试件，采用中空金属试件(A组)、中空金属试件假体+脱钙骨基质(decalcified bone matrix DBM)(B组)、中空金属试件+DBM+成骨诱导液(C组)、中空金属假体+DBM+rhBMP-2培养液(D组)，金属试件均为HA涂层，于BMSc混悬液中加10％胎牛血清DMEM培养液培养，在2、4周观察点取材，通过倒置相差显微镜、扫描电镜、成骨细胞表型检测等方法对中空金属试件表面、脱钙骨基质BMSc生长以及成骨分化进行观察、鉴定。
使用SPSS15.0软件包进行统计分析，计量资料以均数±标准差表示，组间同一时间各指标比较采用组间方差分析。
结果：实验发现：倒置相差显微镜、扫描电镜等方法显示中空金属试件表面及试件内三维细胞支架上BMSc细胞黏附正常，经成骨诱导后转为成骨细胞，并形成工程化组织。在实验设计的两种骨诱导方式中，成骨细胞的表达以成骨诱导液为明显，在统计学意义上其他对照组有明显差异。
结论：
一、体外DMSc经诱导向成骨细胞分化，并可在中空金属试件的空间结构中形成组织块。
二、中空金属假体的设计思路是可行的，在体外可以观察到BMSc在金属腔壁与腔内细胞支架之间紧密结合，并且在无细胞支架的情况下，1mm孔洞可以形成细胞间的直接连接。
因此中空结构的假体是一个值得探索的方向，希望对生物复合型假体的改进具有一定的指导意义。
Objective: Histological, histomorphological analysis were used to analyze the bony in-growth conditions in the interface of bone and prosthesis and inside hollow prosthesis of 4 groups. We hope that the results of our research can provide the clinical using new hollow prosthesis to improve biological fixation of artificial joint with some theoretical and experimental data.
Methods: We designed hollow prosthesis by self, and divided it into 5 group: hollow prosthesis (A group), hollow prosthesis with DBM (B group), hollow prosthesis with DBM in osteogenesis inducing nutrient liquid (D group), hollow prosthesis with DBM combined with rhBMP-2(D group). All hollow prostheses coated with hydroxyapatite. Then BMSc at the surface of the hollow prostheses and in the DBM was researched by inverted phase contrast microscope, scanning electron microscope, osteoblast phaenotype detection after 2and 4 weeks.
Measurement data were expressed as means+SD(x±s) and analyzed by t Test.
Results: Inverted phase contrast microscope and scanning electron microscope showed that BMSc at the surface and the hole of the hollow prosthese and in the DBM 3—D cytoskeleton can adhere normally and transform into osteoblast by inducing and form engineering tissue. Osteoblast phaenotype detection after 2 weeks shows the results: the present of osteoblast of C group is significant difference with other groups statistically
Conclusion : 1.BMSc at the surface and the hole of the hollow prostheses and in the DBM 3—D cytoskeleton can adhere normally and transform into osteoblast
by inducing and form engineering tissue in vitro.2.the desigen of the hollow prostheses is feasible. It shows in vitro that BMSc at the wall of the prosthesis can combine with the DBM 3—D cytoskeleton and BMSc in the 1 mm hole of the prosthesis without 3—D cytoskeleton can also connect directly.
So the hollow prosthesis is worthy with more researching, having some guidance in clinic.
引文
[1] LaPorte DM, Mont MA, Hungerford DS. Proximally porous-coated ingrowth prostheses: limits of use [J]. Orthopedics, 1999, 22(12): 1154-1160.
[2] Song Y, Beaupre G, Goodman SB. Osseointegration of total hip arthroplasties: studies in humans and animals [J]. J Long Term Elf Med Implants, 1999, 9(1-2):77-112.
[3] Jeffrey O. Hollinger, Tomas A. Enhorn, Bruce A.Doll et.al.Bone Tissue Engineering[M]. Washington, D.C:CRC PRESS,2005:165-178.
[4] 司徒镇强，吴军正主编．细胞培养[M]．第1版．西安：世界图书出版公司，1996：111-115．
[5] Kalebo P, Buch F, Albrektsson. T Bone formation rate in osseointegrated titanium implants: Influence of locally applied haemostasis, peripheral blood, autologous bone marrow and fibrin adhesive system (FAS) [J]. Scand J Plast Reconstr Stag Hand Surg 1988,15(4):59-72.
[6] Albrektsson T, Jacobsson M, Kalebo P. The harvest chamber- a newly developed implant for analysis of bone (?)emodeling in situ[J]. Biomaterials and Biomechanics. Amsterdam: Elsevier Science Publishers B.V., 1984. 283-288.
[7] Goodman S, Song Y, Chun L, et al. Effects of local infusion of TGFbeta on bone ingrowth in rabbit chambers[J]. J Biomed Mater Res. 2000, 53(5):475-479.
[8] Sanchez RJ, Song S, Cardozo PF, et al. Adult bone marrow stromal cell differentiate into neural cels in vitro[J]. Exp Neurol, 2000, 164(2): 247～256.
[9] Benayahu D. Osteocalcin(BGP), gene expression, and protein production by marrow stromal adipocytes[J]. Biochem Biophys Res Commu, 1997, 233(1): 30～35.
[10] 杨林，陶天遵，刘枫晨，等．地塞米松对成人成骨细胞增殖和分化影响的实验研究[J]．中华骨科杂志，2001，21(8)：493-497．
[11] Cheng SL, Yang JW, Rifas L, et al. Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone[J]. Endocrinology, 1994, 134(1): 277—286.
[12] 李东，马鲁新，马玲，等．人骨髓基质干细胞体外三维立体培养的实验研究[J]．山东大学学报(医学版)，2006，44(9)：865-868．
[13] Watts NB. Clinical utility of biochemical markei's of bone remodeling[J]. Clin Chem, 1999, 45(8): 1359-1368.
[14] 李晓新，林琴，包振英．不同方法检测成骨细胞中碱性磷酸酶的比较[J]．中国骨质疏松杂志，2003，9(8)：211-213．
[15] 蔡谓．生物复合型中空人工关节假体[J]．世界医疗器械，2004，10(2)：12-15．
[16] Goodman SB. The effects of micromotion and particulate materials on tissue differentiation[J]. Bone chamber studies in rabbits. Acta Orthop Scand, 1994 ,Suppl:64-71.
[17] Goodman S, Song Y, Chun L, et al. Effects of local infusion of TGFbeta on bone ingrowth in rabbit chambers[J]. J Biomed Mater Res 2000,28 (7): 1428-1436.
[18] 刘侠，乐以伦．碱性磷酸酶与钙化[J]．中国生物医学工程学报，1997，16(1)：71-82
1. Urist MR. Bone: formation by autoinduction. Science. 1965;150:893-899
2. Cheng H, Jiang W, Phillips FM, et al. Osteogenic activity of the fourteen types of human bone morphogeneticproteins (BMPs). J Bone Joint Surg Am. 2003;85:1544-1552.
3. Barnes GL, Kostenuik PJ, Gerstenfeld LC, et al. Growth factor regulation of fracture repair. J Bone Miner Res. 1999;14:1805-1815.
4. Hay, E. Bone morphogenetic protein-2 promotes osteoblast apoptosis through a Smad-independent, protein kinase C-dependent signaling pathway. J. Biol. 2001; Chem. 276(31):29028-29036
5. Shi, W. Gremlin negatively modulates BMP-4 induction of embryonic mouse lung branching morphogenesis. Am. J. Physiol. Lung Cell Mol Physiol. 2001; 280(5): L1030- L1039.
6. Bahamonde, Matthew E, Lyons, et al, BMP-3: To Be or Not To Be a BMP. J Bone Joint Surg Am. 2001;83-A Supplement 1:56-62.
7. Mayer H, Scutt AM, Ankenbauer T. Subtle differences in the mitogenic effects of recombinant human bone morphogenetic proteins -2 to -7 on DNA synthesis on primary bone-forming cells and identification of BMP-2/4 receptor. Calcif Tissue Int. 1996:58:249-255.
8. Yasko, A.W. The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein. A radiographic, histological, and biomechanical study in rats. 1992; J. Bone Joint Surg. Am. 74(5): 659-670.
9. Sandhu, Harvinder S, Kanim, et al, Effective Doses of Recombinant Human Bone Morphogenetic Protein-2 in Experimental Spinal Fusion. Spine 1996:21:2115-2122.
10.Glaser DL, Economides AN, Wang L, et al. In vivo somatic cell gene transfer of an engineered Noggin mutein prevents BMP4-induced heterotopic ossification. J Bone Joint Surg Am. 2003:85:2332-2342.
11.Nohe, A. Cochran, D. L., Barboza, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signal ing pathways. J. biol. Chem. 2002;277(7): 5330 - 5338.
12.von Bubnoff A, Cho KW. Intracellular BMP signaling regulation in vertebrates: pathway or network? Dev Biol. 2001;239:1～14.
13.Seeherman H, Wozney J, Li R. Bone morphogenetic protein delivery systems. Spine. 2002;27(16 Suppl 1):S16-23.
14.Winn SR, Uludag H, Hollinger JO. Carrier systems for bone morphogenetic proteins. Clin Orthop Relat Res. 1999;367 Suppl:S95-106.
15.M. Borden, M. Attawia, Y. Khan, et al. Tissue-engineered bone formation in vivo using a novel sintered polymeric microsphere matrix. J Bone Joint Surg Br. 2004;86-B:1200-1208.
16.N. Saito, T. Okada, H. Horiuchi, et al. Local bone formation by injection of recombinant human bone morphigentic protein-2contained in polymer carriers. Bone 2003:32:381-386.
17.Dunn. A. S., Campbell. P. G., Marra. K. G., et al. The influence of polymer blend composition on the degradation of polymer/hydroxyapatite biomaterials. J. Mater Sci. Mater. Med. 2001; 12(8): 673-677.
18.Cooke. M. N., Fisher. J. P., Dean. D., et al. Use of stereolithography to manufacture critical-sized 3D biodegradable scaffolds for bone ingrowth. J. Biomed. Mater. Res. Part B: Appl Biomater. 2002;64B, 65 - 69.
[2] Song Y, Beaupre G, Goodman SB. Osseointegration of total hip arthroplasties: studies in humans and animals [J]. J Long Term Elf Med Implants, 1999, 9(1-2):77-112.
[3] Jeffrey O. Hollinger, Tomas A. Enhorn, Bruce A.Doll et.al.Bone Tissue Engineering[M]. Washington, D.C:CRC PRESS,2005:165-178.
[4] 司徒镇强，吴军正主编．细胞培养[M]．第1版．西安：世界图书出版公司，1996：111-115．
[5] Kalebo P, Buch F, Albrektsson. T Bone formation rate in osseointegrated titanium implants: Influence of locally applied haemostasis, peripheral blood, autologous bone marrow and fibrin adhesive system (FAS) [J]. Scand J Plast Reconstr Stag Hand Surg 1988,15(4):59-72.
[6] Albrektsson T, Jacobsson M, Kalebo P. The harvest chamber- a newly developed implant for analysis of bone (?)emodeling in situ[J]. Biomaterials and Biomechanics. Amsterdam: Elsevier Science Publishers B.V., 1984. 283-288.
[7] Goodman S, Song Y, Chun L, et al. Effects of local infusion of TGFbeta on bone ingrowth in rabbit chambers[J]. J Biomed Mater Res. 2000, 53(5):475-479.
[8] Sanchez RJ, Song S, Cardozo PF, et al. Adult bone marrow stromal cell differentiate into neural cels in vitro[J]. Exp Neurol, 2000, 164(2): 247～256.
[9] Benayahu D. Osteocalcin(BGP), gene expression, and protein production by marrow stromal adipocytes[J]. Biochem Biophys Res Commu, 1997, 233(1): 30～35.
[10] 杨林，陶天遵，刘枫晨，等．地塞米松对成人成骨细胞增殖和分化影响的实验研究[J]．中华骨科杂志，2001，21(8)：493-497．
[11] Cheng SL, Yang JW, Rifas L, et al. Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone[J]. Endocrinology, 1994, 134(1): 277—286.
[12] 李东，马鲁新，马玲，等．人骨髓基质干细胞体外三维立体培养的实验研究[J]．山东大学学报(医学版)，2006，44(9)：865-868．
[13] Watts NB. Clinical utility of biochemical markei's of bone remodeling[J]. Clin Chem, 1999, 45(8): 1359-1368.
[14] 李晓新，林琴，包振英．不同方法检测成骨细胞中碱性磷酸酶的比较[J]．中国骨质疏松杂志，2003，9(8)：211-213．
[15] 蔡谓．生物复合型中空人工关节假体[J]．世界医疗器械，2004，10(2)：12-15．
[16] Goodman SB. The effects of micromotion and particulate materials on tissue differentiation[J]. Bone chamber studies in rabbits. Acta Orthop Scand, 1994 ,Suppl:64-71.
[17] Goodman S, Song Y, Chun L, et al. Effects of local infusion of TGFbeta on bone ingrowth in rabbit chambers[J]. J Biomed Mater Res 2000,28 (7): 1428-1436.
[18] 刘侠，乐以伦．碱性磷酸酶与钙化[J]．中国生物医学工程学报，1997，16(1)：71-82
1. Urist MR. Bone: formation by autoinduction. Science. 1965;150:893-899
2. Cheng H, Jiang W, Phillips FM, et al. Osteogenic activity of the fourteen types of human bone morphogeneticproteins (BMPs). J Bone Joint Surg Am. 2003;85:1544-1552.
3. Barnes GL, Kostenuik PJ, Gerstenfeld LC, et al. Growth factor regulation of fracture repair. J Bone Miner Res. 1999;14:1805-1815.
4. Hay, E. Bone morphogenetic protein-2 promotes osteoblast apoptosis through a Smad-independent, protein kinase C-dependent signaling pathway. J. Biol. 2001; Chem. 276(31):29028-29036
5. Shi, W. Gremlin negatively modulates BMP-4 induction of embryonic mouse lung branching morphogenesis. Am. J. Physiol. Lung Cell Mol Physiol. 2001; 280(5): L1030- L1039.
6. Bahamonde, Matthew E, Lyons, et al, BMP-3: To Be or Not To Be a BMP. J Bone Joint Surg Am. 2001;83-A Supplement 1:56-62.
7. Mayer H, Scutt AM, Ankenbauer T. Subtle differences in the mitogenic effects of recombinant human bone morphogenetic proteins -2 to -7 on DNA synthesis on primary bone-forming cells and identification of BMP-2/4 receptor. Calcif Tissue Int. 1996:58:249-255.
8. Yasko, A.W. The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein. A radiographic, histological, and biomechanical study in rats. 1992; J. Bone Joint Surg. Am. 74(5): 659-670.
9. Sandhu, Harvinder S, Kanim, et al, Effective Doses of Recombinant Human Bone Morphogenetic Protein-2 in Experimental Spinal Fusion. Spine 1996:21:2115-2122.
10.Glaser DL, Economides AN, Wang L, et al. In vivo somatic cell gene transfer of an engineered Noggin mutein prevents BMP4-induced heterotopic ossification. J Bone Joint Surg Am. 2003:85:2332-2342.
11.Nohe, A. Cochran, D. L., Barboza, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signal ing pathways. J. biol. Chem. 2002;277(7): 5330 - 5338.
12.von Bubnoff A, Cho KW. Intracellular BMP signaling regulation in vertebrates: pathway or network? Dev Biol. 2001;239:1～14.
13.Seeherman H, Wozney J, Li R. Bone morphogenetic protein delivery systems. Spine. 2002;27(16 Suppl 1):S16-23.
14.Winn SR, Uludag H, Hollinger JO. Carrier systems for bone morphogenetic proteins. Clin Orthop Relat Res. 1999;367 Suppl:S95-106.
15.M. Borden, M. Attawia, Y. Khan, et al. Tissue-engineered bone formation in vivo using a novel sintered polymeric microsphere matrix. J Bone Joint Surg Br. 2004;86-B:1200-1208.
16.N. Saito, T. Okada, H. Horiuchi, et al. Local bone formation by injection of recombinant human bone morphigentic protein-2contained in polymer carriers. Bone 2003:32:381-386.
17.Dunn. A. S., Campbell. P. G., Marra. K. G., et al. The influence of polymer blend composition on the degradation of polymer/hydroxyapatite biomaterials. J. Mater Sci. Mater. Med. 2001; 12(8): 673-677.
18.Cooke. M. N., Fisher. J. P., Dean. D., et al. Use of stereolithography to manufacture critical-sized 3D biodegradable scaffolds for bone ingrowth. J. Biomed. Mater. Res. Part B: Appl Biomater. 2002;64B, 65 - 69.