成年恒河猴体内异位构建血管化组织工程骨的实验研究
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摘要
目的 (1) 对猴骨髓基质干细胞(rBMSc)进行体外培养及扩增,观察其原代及传代细胞的生长特点及生物学特点。(2) 观察恒河猴骨髓基质干细胞(rBMSc)与新型可吸收羟基磷灰石(HA)及AO人工骨β-磷酸三钙(β-TCP)的体外相容性。为在灵长类动物体内构建组织工程化骨作材料方面的准备。(3) 用血管束植入法异位构建血管化的组织工程骨,观察血管化的效果及对成骨作用的影响。(4) 用绿色荧光蛋白(GFP)标记rBMSc,观察其在组织工程骨体内构建中对种子细胞的示踪作用。(5) 对恒河猴体内构建组织工程骨进行安全性评价。
方法 (1) 从髂后上棘抽取成年恒河猴骨髓,用全骨髓培养法进行体外培养获得BMSc,胰酶消化传代,用条件培养基培养传代细胞。逐日倒置显微镜观察细胞生长情况,对传代细胞进行HE染色及碱性磷酸酶(ALP)染色。(2) 取第三代恒河猴骨髓基质细胞与材料复合培养。实验分三组:A组将rBMSc与HA复合培养,B组rBMSc与β-TCP复合培养,C组为对照,只有细胞不加材料。倒置相差显微镜、扫描电镜观察各组细胞形态及增殖情况,MTT法半定量检测细胞增殖情况。(3) 将可降解HA订制成中空的圆柱体,侧方开槽。实验分4组,① 可吸收羟基磷灰石(HA)组(M组):单将材料植于肌袋内;② HA+rBMSc组(MC):将材料接种诱导培养的rBMSc;③ HA+血管束植入组(MB):将材料内植入血管束,不加细胞;④ HA+血管束植入+rBMSc(MBC):材料接种细胞并植入血管束。需种细胞的组别,将经诱导培养的rBMSc以5×10~6/ml的密度接种于材料上。血管束植入组,将胸背动静脉分离,将其游离约4～5cm,将血管束从材料的侧槽内
Objectives To culcture bone marrow stromal cells (BMSc) of adult rhesus in ivtro, observe the biological characters of primary and passaged cells. study the biocompatibility of rhesus bone marrow stromal cells (rBMSc) and two biomaterials: novel hydroxyapatite(HA) and AO artificial bone β-tricalcium phosphate (6-TCP) in ivtro. Construct vascularized tissue engineering bone in latissinus dorsi muscle by implanting thoracodorsal vessels bundle. Observe the trace function of green fluorescent protein (GFP) transfected into rBMSc in tissue engineering bone formation in vivo. Study the biologic safety of tissue engineered bone constructed in rhesus.Methods Bone marrow of adult male rhesus were harvested from ilium, adherent cells were selected as BMScs when whole marrow were cultured. BMScs were digest by trypsin when confluent was seen and then passaged. Passaged cells were fed with conditioned medium which cotain dexamethasone 10~-8M, β-sodium glycerophosphate 10~-2M and vitamin C 50 μ g/ml. Cells were observed under inverted phase contrast microscope every day. Passaged cells were examined by HE stain and histochemisty stain for ALP. The third passage rhesus bone marrow stromal cells were cocultured with HA and β-TCP, rBMSc cultured without material as the control. The morphology and proliferation of rBMSc was observed by inverted phase contrast microscope and scanning electron microscope. MTT assay was used to semi-
quantitatively evaluate the proliferation of rBMSc. 24 absorbable HA were implanted in 12 rhesus under four conditions: (1) alone (reference group M); (2) coated with bone marrow stromal cells (group MC); (3) combined with a blood bundle (group MV); (4) coated with bone marrow stromal cells and combined with thoracodorsalis vascular bundle (group MBC). After operation, the physiological activity of all animals were observed. At 4, 8, 12 week postoperation, X rays were taken, Sample were harvested and decalcified tissue section were made. ECT were examined at 6 weeks to learn the vascularizing of the implants. Ad5.CMV-GFP was amplified by infecting QBI-293A cells, GFP was transfected into rBMSc by adenovirus vector. RBMScs with GFP were seeded on HA scaffold and implanted into latissinus dorsi muscles. Sample were harvested at 6 week and embedded in resin. Ground-bone sections were made and observed by laser scanning confocal microscope using PI staining. Long-term toxicity, muscle implanting test and genotoxicity were used to evaluate the safety of tissue engineering bone constructed in rhesus body.Results The BMSc of adult rhesus cultured grow and survived well in vitro, primarily rBMSc were confluent in 9-13 days, passaged cells took 4-7 days to cover the bottle bottom. Observed using HE staining, BMSc is monocyte, in spindle or multiangular shape. Passaged cells shew strong positive staining of ALP. The rBMScs cocultured with HA grew well when observed under inverted phase contrast microscope, and no significant difference was found between HA group and the control, there were some particals shed from 8-TCP, and a small part of cells die out. When observed by SEM, rBMScs adhensive well to HA and proliferate obviously. The adhensive ratio is not so high in 8-TCP group. Evaluated by MTT assay, the cells number have no significant difference between HA group and control, while the cell number of 6-TCP group is notable less than that of the control. All rhesus keeped in good health after operation, wound healed for 4-5 days. Macroscopic examination of
the implants showed no evidence of resorption or signs of exclusion. A fibrous capsule formed around the implants, as is observed with the implantation of any foreign material. All implants without bone marrow cells (M and MB) showed fibrovascular invasion into the pores and few bone formation. When the implants were combined with osteoprogenitor cells (MC) and blood vessel pedicle (MBC), bone ingrowth occurred. X rays show that the density of the implants of MBC and MC group get higher than that of M group at 8, 12 week. It is not so obvious in MB group. ECT indicated that the nuclide density of the group combined with blood vessel bundles is obviously higher, and combined with BMSc is also helpful. Histologic observasition show there were a little new bone formation on the border of the material's hole in MB, MC and MBC group at 4 week, no bone formation in M group but full of fibrous tissue. More bone formation in group MC and MBC, a few in MB, while still no in M group at 8 week. Mature bone tissue was seen in MBC group, more new bone tissue presented in MC group, increase of bone tissue in MB and M is not so significant at 12 week postoperation. The rBMScs grew well after GFP was transfected, green fluorescent could be seen after 24h, and became brighter after 48h, the positive rate of transfection was beyond 80%. 6 weeks after implanted, the rBMScs signed by GFP can be detected in ground-bone section by laser scanning confocal microscope. The rhesus' physiological status were well after the tissue engineering bone constructed in vivo, weight increased gradually. Blood routine, blood biochemical examination and myelogram analysis after the tissue engineering bone constructed 4, 8, 12 week were not significant different from those of preembed. A small quantity of inflammatory cell were observed in muscle around 2 weeks after implanting, but not found at 4 and 12 week. No allotype cell was observed in each section. Polychromatic erythrocyte was hardly observed in marrow smear, no micronuclei was not found in each group.
Conclusions The BMSc of adult rhesus cultured proliferated well and can be induced to differentiate into osteoblast in vitro, it can be used as the seed cell of bone tissue engineering. Novel absorbable HA have good biocompatibility with rBMSc of bone tissue engineering. AO artificial bone need to be improved asscaffold for rBMSc. Implanting blood vessel bundle can enhance vascularization and new bone formation of tissue engineering bone. GFP is an effective marker for living seed cells of tissue engineering bone constructed in vivo, the BMScs cultured in vitro were the main cell source of the bone formation of tissue engineering bone. Safety of constructing tissue engineering bone in rhesus is reliable.
方法 (1) 从髂后上棘抽取成年恒河猴骨髓,用全骨髓培养法进行体外培养获得BMSc,胰酶消化传代,用条件培养基培养传代细胞。逐日倒置显微镜观察细胞生长情况,对传代细胞进行HE染色及碱性磷酸酶(ALP)染色。(2) 取第三代恒河猴骨髓基质细胞与材料复合培养。实验分三组:A组将rBMSc与HA复合培养,B组rBMSc与β-TCP复合培养,C组为对照,只有细胞不加材料。倒置相差显微镜、扫描电镜观察各组细胞形态及增殖情况,MTT法半定量检测细胞增殖情况。(3) 将可降解HA订制成中空的圆柱体,侧方开槽。实验分4组,① 可吸收羟基磷灰石(HA)组(M组):单将材料植于肌袋内;② HA+rBMSc组(MC):将材料接种诱导培养的rBMSc;③ HA+血管束植入组(MB):将材料内植入血管束,不加细胞;④ HA+血管束植入+rBMSc(MBC):材料接种细胞并植入血管束。需种细胞的组别,将经诱导培养的rBMSc以5×10~6/ml的密度接种于材料上。血管束植入组,将胸背动静脉分离,将其游离约4～5cm,将血管束从材料的侧槽内
Objectives To culcture bone marrow stromal cells (BMSc) of adult rhesus in ivtro, observe the biological characters of primary and passaged cells. study the biocompatibility of rhesus bone marrow stromal cells (rBMSc) and two biomaterials: novel hydroxyapatite(HA) and AO artificial bone β-tricalcium phosphate (6-TCP) in ivtro. Construct vascularized tissue engineering bone in latissinus dorsi muscle by implanting thoracodorsal vessels bundle. Observe the trace function of green fluorescent protein (GFP) transfected into rBMSc in tissue engineering bone formation in vivo. Study the biologic safety of tissue engineered bone constructed in rhesus.Methods Bone marrow of adult male rhesus were harvested from ilium, adherent cells were selected as BMScs when whole marrow were cultured. BMScs were digest by trypsin when confluent was seen and then passaged. Passaged cells were fed with conditioned medium which cotain dexamethasone 10~-8M, β-sodium glycerophosphate 10~-2M and vitamin C 50 μ g/ml. Cells were observed under inverted phase contrast microscope every day. Passaged cells were examined by HE stain and histochemisty stain for ALP. The third passage rhesus bone marrow stromal cells were cocultured with HA and β-TCP, rBMSc cultured without material as the control. The morphology and proliferation of rBMSc was observed by inverted phase contrast microscope and scanning electron microscope. MTT assay was used to semi-
quantitatively evaluate the proliferation of rBMSc. 24 absorbable HA were implanted in 12 rhesus under four conditions: (1) alone (reference group M); (2) coated with bone marrow stromal cells (group MC); (3) combined with a blood bundle (group MV); (4) coated with bone marrow stromal cells and combined with thoracodorsalis vascular bundle (group MBC). After operation, the physiological activity of all animals were observed. At 4, 8, 12 week postoperation, X rays were taken, Sample were harvested and decalcified tissue section were made. ECT were examined at 6 weeks to learn the vascularizing of the implants. Ad5.CMV-GFP was amplified by infecting QBI-293A cells, GFP was transfected into rBMSc by adenovirus vector. RBMScs with GFP were seeded on HA scaffold and implanted into latissinus dorsi muscles. Sample were harvested at 6 week and embedded in resin. Ground-bone sections were made and observed by laser scanning confocal microscope using PI staining. Long-term toxicity, muscle implanting test and genotoxicity were used to evaluate the safety of tissue engineering bone constructed in rhesus body.Results The BMSc of adult rhesus cultured grow and survived well in vitro, primarily rBMSc were confluent in 9-13 days, passaged cells took 4-7 days to cover the bottle bottom. Observed using HE staining, BMSc is monocyte, in spindle or multiangular shape. Passaged cells shew strong positive staining of ALP. The rBMScs cocultured with HA grew well when observed under inverted phase contrast microscope, and no significant difference was found between HA group and the control, there were some particals shed from 8-TCP, and a small part of cells die out. When observed by SEM, rBMScs adhensive well to HA and proliferate obviously. The adhensive ratio is not so high in 8-TCP group. Evaluated by MTT assay, the cells number have no significant difference between HA group and control, while the cell number of 6-TCP group is notable less than that of the control. All rhesus keeped in good health after operation, wound healed for 4-5 days. Macroscopic examination of
the implants showed no evidence of resorption or signs of exclusion. A fibrous capsule formed around the implants, as is observed with the implantation of any foreign material. All implants without bone marrow cells (M and MB) showed fibrovascular invasion into the pores and few bone formation. When the implants were combined with osteoprogenitor cells (MC) and blood vessel pedicle (MBC), bone ingrowth occurred. X rays show that the density of the implants of MBC and MC group get higher than that of M group at 8, 12 week. It is not so obvious in MB group. ECT indicated that the nuclide density of the group combined with blood vessel bundles is obviously higher, and combined with BMSc is also helpful. Histologic observasition show there were a little new bone formation on the border of the material's hole in MB, MC and MBC group at 4 week, no bone formation in M group but full of fibrous tissue. More bone formation in group MC and MBC, a few in MB, while still no in M group at 8 week. Mature bone tissue was seen in MBC group, more new bone tissue presented in MC group, increase of bone tissue in MB and M is not so significant at 12 week postoperation. The rBMScs grew well after GFP was transfected, green fluorescent could be seen after 24h, and became brighter after 48h, the positive rate of transfection was beyond 80%. 6 weeks after implanted, the rBMScs signed by GFP can be detected in ground-bone section by laser scanning confocal microscope. The rhesus' physiological status were well after the tissue engineering bone constructed in vivo, weight increased gradually. Blood routine, blood biochemical examination and myelogram analysis after the tissue engineering bone constructed 4, 8, 12 week were not significant different from those of preembed. A small quantity of inflammatory cell were observed in muscle around 2 weeks after implanting, but not found at 4 and 12 week. No allotype cell was observed in each section. Polychromatic erythrocyte was hardly observed in marrow smear, no micronuclei was not found in each group.
Conclusions The BMSc of adult rhesus cultured proliferated well and can be induced to differentiate into osteoblast in vitro, it can be used as the seed cell of bone tissue engineering. Novel absorbable HA have good biocompatibility with rBMSc of bone tissue engineering. AO artificial bone need to be improved asscaffold for rBMSc. Implanting blood vessel bundle can enhance vascularization and new bone formation of tissue engineering bone. GFP is an effective marker for living seed cells of tissue engineering bone constructed in vivo, the BMScs cultured in vitro were the main cell source of the bone formation of tissue engineering bone. Safety of constructing tissue engineering bone in rhesus is reliable.
引文
1. Jadlowiec JA, Celil AB, Hollinger JO. Bone tissue engineering: recent advances and promising therapeutic agents. Expert Opin Biol Ther. 2003 Jun; 3(3): 409-23.
2.陈元霖,曾中兴,白寿昌.猕猴[M].第1版,北京:科学出版社,1985,1-1.
3. Sanchez-Ramos J, Song S, Cardozo-Pelaez F. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000, 164(2): 247-56.
4. Coelho MJ, Fernandes MH. Human bone cell cultures in biocompatibility testing. Part Ⅱ: effect ofascorbic acid, beta-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials. 2000, 21(11): 1095-102.
5. Aubin JE. Advance in the osteoblast lineage. Biochem Cell Biol, 1998, 76: 899-910.
6.任高宏,裴国献,王钢,等.成人成骨细胞体外培养.骨与关节损伤杂志,2003,18(2):118-121.
7.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.中国矫形外科杂志,2000,7(6):565-568.
8.陈滨,裴国献,王珂,等.筋膜瓣促组织工程骨体内再血管化的实验研究.解放军医学杂志,2002,27(6):482-484.
9. Oreffo RO, Driessens FC, Planell JA, Triffitt JT. Effects of novel calcium phosphate cements on human bone marrow fibroblastic cells. Tissue Eng. 1998, 4(3): 293-303.
10. Di Agostino S, Botti F, Di Carlo A. Meiotic progression of isolated mouse spermatocytes under simulated microgravity. Reproduction. 2004, 128(1): 25-32.
11.殷晓雪,陈仲强,郭昭庆,党耕町.转导人端粒酶逆转录酶基因致人成骨细胞永生化的研究.中华医学杂志,2003,83(14):1251-1254.
2.陈元霖,曾中兴,白寿昌.猕猴[M].第1版,北京:科学出版社,1985,1-1.
3. Sanchez-Ramos J, Song S, Cardozo-Pelaez F. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000, 164(2): 247-56.
4. Coelho MJ, Fernandes MH. Human bone cell cultures in biocompatibility testing. Part Ⅱ: effect ofascorbic acid, beta-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials. 2000, 21(11): 1095-102.
5. Aubin JE. Advance in the osteoblast lineage. Biochem Cell Biol, 1998, 76: 899-910.
6.任高宏,裴国献,王钢,等.成人成骨细胞体外培养.骨与关节损伤杂志,2003,18(2):118-121.
7.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.中国矫形外科杂志,2000,7(6):565-568.
8.陈滨,裴国献,王珂,等.筋膜瓣促组织工程骨体内再血管化的实验研究.解放军医学杂志,2002,27(6):482-484.
9. Oreffo RO, Driessens FC, Planell JA, Triffitt JT. Effects of novel calcium phosphate cements on human bone marrow fibroblastic cells. Tissue Eng. 1998, 4(3): 293-303.
10. Di Agostino S, Botti F, Di Carlo A. Meiotic progression of isolated mouse spermatocytes under simulated microgravity. Reproduction. 2004, 128(1): 25-32.
11.殷晓雪,陈仲强,郭昭庆,党耕町.转导人端粒酶逆转录酶基因致人成骨细胞永生化的研究.中华医学杂志,2003,83(14):1251-1254.