STUDY ON BONE MARROW MESENCHYMAL STEM CELLS DERIVED OSTEOBLASTS AND ENDOTHELIAL CELLS COMPOUND WITH CHITOSAN/HYDROXYAPATITE SCAFFOLD TO CONSTRUCT VASCULARIZED TISSUE ENGINEERED BONE_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HAO Zengtao ¹ , FENG Wei ² , HAO Ting ² , YU Bin. ¹

1Department of Trauma and Orthopaedics,Nanfang Hospital, Southern Medical University, Guangzhou Guangdong, 510515, P.R.China;;
2Department of Orthopaedics, Second Hospital Affiliated to Inner Mongolia Medical College.;

Corresponding author：

Keywords：

Bone marrow mesenchymal stem cells; Tissue engineered bone; Vascularization; Bone defect; Rat

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Objective To explore the osteogenesis and angiogenesis effect of bone marrow mesenchymal stem cells (BMSCs) derived osteoblasts and endothelial cells compound with chitosan/hydroxyapatite (CS/HA) scaffold in repairing radial
defect in rats. Methods The BMSCs were isolated from Sprague Dawley rats and the 3rd generation of BMSCs were induced into osteoblasts and endothelial cells. The endothelial cells, osteoblasts, and mixed osteoblasts and endothelial cells (1 ∶ 1) were compound with CS/HA scaffold in groups A, B, and C respectively to prepare the cell-scaffold composites. The cell proliferation was detected by MTT. The rat radial segmental defect model was made and the 3 cell-scaffolds were implanted, respectively. At 4, 8, and 12 weeks after transplantation, the graft was harvested to perform HE staining and CD34 immunohistochemistry staining. The mRNA expressions of osteopontin (OPN) and osteoprotegerin (OPG) were detected by RT-PCR. Results Alkal ine phosphatase staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days after osteogenic induction and the nuclei were stained red. CD34 immunocytochemical staining of the endothelial cells showed that there were brown grains in the cytoplasm at 14 days after angiogenesis induction. MTT test showed that the proliferation level of the cells in 3 groups increased with the time. HE staining showed that no obvious osteoid formation, denser microvessel, and more fibrous tissue were seen at 12 weeks in group A; homogeneous osteoid which distributed with cord or island, and many osteoblast-l ike cells were seen in groups B and C. The microvessel density was significantly higher in groups A and C than group B at 3 time points (P lt; 0.05), and in group A than in group C at 12 weeks (P lt; 0.05). The OPN and OPG mRNA expressions of group A were significantly lower than those of groups B and C at 3 time points (P lt; 0.05). In groups B and C, the OPN mRNA expressions reached peak t8 and 12 weeks, respectively, and OPG mRNA expressions reached peak at 4 weeks. Conclusion BMSCs derived steoblasts and endothelial cells (1 ∶ 1) compound with CS/HA porous scaffold can promote bone formation and vascularization in bone defect and accelerate the healing of bone defect.

Citation： HAO Zengtao,FENG Wei,HAO Ting,YU Bin.. STUDY ON BONE MARROW MESENCHYMAL STEM CELLS DERIVED OSTEOBLASTS AND ENDOTHELIAL CELLS COMPOUND WITH CHITOSAN/HYDROXYAPATITE SCAFFOLD TO CONSTRUCT VASCULARIZED TISSUE ENGINEERED BONE. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(4): 489-494. doi: Copy

1.	Samee M, Kasugai S, Kondo H, et al. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) transfection to human periosteal cells enhances osteoblast differentiation and bone formation. J Pharmacol Sci, 2008, 108(1): 18-31.
2.	Mott DA, Mailhot J, Cuenin MF, et al. Enhancement of osteoblast proliferation in vitro by selective enrichment of demineralized freeze-dried bone allograft with specific growth factors. J Oral Implantol, 2002, 28(2): 57-66.
3.	Ekenbäck SB, Linder LE, Lönnies H. Effect of four dental varnishes on the colonization of cariogenic bacteria on exposed sound root surfaces. Caries Res, 2000, 34(1): 70-74.
4.	Bodde EW, Spauwen PH, Mikos AG, et al. Closing capacity of segmental radius defects in rabbits. J Biomed Mater Res A, 2008, 85(1): 206-217.
5.	Zhao Q, Qian J, Zhou H, et al. In vitro osteoblast-like and endothelial cells’ response to calcium silicate/calcium phosphate cement. Biomed Mater, 2010, 5(3): 35004.
6.	Kagami H, Agata H, Tojo A. Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation. Int J Biochem Cell Biol, 2011, 43(3): 286-289.
7.	Lee WD, Hurtig MB, Kandel RA, et al. Membrane culture of bone marrow stromal cells yields better tissue than pellet culture for engineering cartilage-bone substitute biphasic constructs in a two-step process. Tissue Eng Part C Methods, 2011, 17(9): 939-948.
8.	Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 1968, 6(2): 230-247.
9.	Unger RE, Ghanaati S, Orth C, et al. The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature. Biomaterials, 2010, 31(27): 6959-6967.
10.	陈超, 李琪佳, 孙瑞军, 等. BMSCs来源的成骨细胞与血管内皮细胞复合于异体冻干颗粒骨的黏附性研究. 中国修复重建外科杂志, 2009, 23(9): 1129-1133.
11.	Zhang Y, Andrukhov O, Berner S, et al. Osteogenic properties of hydrophilic and hydrophobic titanium surfaces evaluated with osteoblast-like cells (MG63) in coculture with human umbilical vein endothelial cells (HUVEC). Dent Mater, 2010, 26(11): 1043-1051.
12.	Deckers MM, Karperien M, van der Bent C, et al. Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endocrinology, 2000, 141(5): 1667-1674.
13.	Kleinheinz J, Stratmann U, Joos U, et al. VEGF-activated angiogenesis during bone regeneration. Oral Maxillofac Surg, 2005, 63(9): 1310-1316.
14.	葛巍立, 谢志坚, 何剑锋. 兔下颌骨牵张成骨组织中c-fos和OPG及OPGL的表达. 浙江大学学报: 医学版, 2006, 35(5): 496-500.
15.	Shoji S, Tabuchi M, Miyazawa K, et al. Bisphosphonate inhibits bone turnover in OPG (-/-) mice via a depressive effect on both osteoclasts and osteoblasts. Calcif Tissue Int, 2010, 87(2): 181-192.
16.	Yan MZ, Xu Y, Gong YX, et al. Raloxifene inhibits bone loss and improves bone strength through an Opg-independent mechanism. Endocrine, 2010, 37(1): 55-61.
17.	Kon T, Cho TJ, Aiaawa T, et al. Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res, 2001, 16(6): 1004-1014.
18.	张建新, 徐展望, 常峰. 组织工程化人工骨修复骨缺损的实验研究. 中国矫形外科杂志, 2009, 17(16): 1258-1261.
19.	Cai L, Wang Q, Gu C, et al. Vascular and micro-environmental influences on MSC-coral hydroxyapatite construct-based bone tissue engineering. Biomaterials, 2011, 32(33): 8497-8505.
20.	Santos MI, Reis RL. Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges. Macromol Biosci, 2010, 10(1): 12-27.

1. Samee M, Kasugai S, Kondo H, et al. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) transfection to human periosteal cells enhances osteoblast differentiation and bone formation. J Pharmacol Sci, 2008, 108(1): 18-31.
2. Mott DA, Mailhot J, Cuenin MF, et al. Enhancement of osteoblast proliferation in vitro by selective enrichment of demineralized freeze-dried bone allograft with specific growth factors. J Oral Implantol, 2002, 28(2): 57-66.
3. Ekenbäck SB, Linder LE, Lönnies H. Effect of four dental varnishes on the colonization of cariogenic bacteria on exposed sound root surfaces. Caries Res, 2000, 34(1): 70-74.
4. Bodde EW, Spauwen PH, Mikos AG, et al. Closing capacity of segmental radius defects in rabbits. J Biomed Mater Res A, 2008, 85(1): 206-217.
5. Zhao Q, Qian J, Zhou H, et al. In vitro osteoblast-like and endothelial cells’ response to calcium silicate/calcium phosphate cement. Biomed Mater, 2010, 5(3): 35004.
6. Kagami H, Agata H, Tojo A. Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation. Int J Biochem Cell Biol, 2011, 43(3): 286-289.
7. Lee WD, Hurtig MB, Kandel RA, et al. Membrane culture of bone marrow stromal cells yields better tissue than pellet culture for engineering cartilage-bone substitute biphasic constructs in a two-step process. Tissue Eng Part C Methods, 2011, 17(9): 939-948.
8. Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 1968, 6(2): 230-247.
9. Unger RE, Ghanaati S, Orth C, et al. The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature. Biomaterials, 2010, 31(27): 6959-6967.
10. 陈超, 李琪佳, 孙瑞军, 等. BMSCs来源的成骨细胞与血管内皮细胞复合于异体冻干颗粒骨的黏附性研究. 中国修复重建外科杂志, 2009, 23(9): 1129-1133.
11. Zhang Y, Andrukhov O, Berner S, et al. Osteogenic properties of hydrophilic and hydrophobic titanium surfaces evaluated with osteoblast-like cells (MG63) in coculture with human umbilical vein endothelial cells (HUVEC). Dent Mater, 2010, 26(11): 1043-1051.
12. Deckers MM, Karperien M, van der Bent C, et al. Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endocrinology, 2000, 141(5): 1667-1674.
13. Kleinheinz J, Stratmann U, Joos U, et al. VEGF-activated angiogenesis during bone regeneration. Oral Maxillofac Surg, 2005, 63(9): 1310-1316.
14. 葛巍立, 谢志坚, 何剑锋. 兔下颌骨牵张成骨组织中c-fos和OPG及OPGL的表达. 浙江大学学报: 医学版, 2006, 35(5): 496-500.
15. Shoji S, Tabuchi M, Miyazawa K, et al. Bisphosphonate inhibits bone turnover in OPG (-/-) mice via a depressive effect on both osteoclasts and osteoblasts. Calcif Tissue Int, 2010, 87(2): 181-192.
16. Yan MZ, Xu Y, Gong YX, et al. Raloxifene inhibits bone loss and improves bone strength through an Opg-independent mechanism. Endocrine, 2010, 37(1): 55-61.
17. Kon T, Cho TJ, Aiaawa T, et al. Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res, 2001, 16(6): 1004-1014.
18. 张建新, 徐展望, 常峰. 组织工程化人工骨修复骨缺损的实验研究. 中国矫形外科杂志, 2009, 17(16): 1258-1261.
19. Cai L, Wang Q, Gu C, et al. Vascular and micro-environmental influences on MSC-coral hydroxyapatite construct-based bone tissue engineering. Biomaterials, 2011, 32(33): 8497-8505.
20. Santos MI, Reis RL. Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges. Macromol Biosci, 2010, 10(1): 12-27.

Chinese Journal of Reparative and Reconstructive Surgery

STUDY ON BONE MARROW MESENCHYMAL STEM CELLS DERIVED OSTEOBLASTS AND ENDOTHELIAL CELLS COMPOUND WITH CHITOSAN/HYDROXYAPATITE SCAFFOLD TO CONSTRUCT VASCULARIZED TISSUE ENGINEERED BONE

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