Effect of demineralized bone matrix modified by laminin α4 chain functional peptide on H-type angiogenesis and osteogenesis to promote bone defect repair_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

TANG Yong ^1,2 , LUO Keyu ¹ , CHEN Yueqi ¹ , GAO Xiaoliang ¹ , TAN Jiulin ¹ , DAI Qijie ¹ , XU Jianzhong ¹ ,  DONG Shiwu ^1,3 ,  LUO Fei ¹

1. Department of Orthopedics, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R.China;
2. Department of Orthopedics, the 72nd Group Army Hospital, Huzhou Zhejiang, 313000, P.R.China;
3. Department of Biomedical Engineering, Biomaterials, Army Medical University, Chongqing, 400038, P.R.China;

Corresponding author：

DONG Shiwu, Email: dongshiwu@163.com; LUO Fei, Email: luofeispine@126.com

Keywords：

Laminin α4 chains; endothelial progenitor cells; bone regeneration; H-type vessels; demineralized bone matrix; mouse

DOI：

10.7507/1002-1892.202006081

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Objective Based on the cell-extracellular matrix adhesion theory in selective cell retention (SCR) technology, demineralized bone matrix (DBM) modified by simplified polypeptide surface was designed to promote both bone regeneration and angiogenesis.Methods Functional peptide of α4 chains of laminin protein (LNα4), cyclic RGDfK (cRGD), and collagen-binding domain (CBD) peptides were selected. CBD-LNα4-cRGD peptide was synthesized in solid phase and modified on DBM to construct DBM/CBD-LNα4-cRGD scaffold (DBM/LN). Firstly, scanning electron microscope and laser scanning confocal microscope were used to examine the characteristics and stability of the modified scaffold. Then, the adhesion, proliferation, and tube formation properties of CBD-LNα4-cRGD peptide on endothelial progenitor cells (EPCs) were detected, respectively. Western blot method was used to verify the molecular mechanism affecting EPCs. Finally, 24 10-week-old male C57 mice were used to establish a 2-mm-length defect of femoral bone model. DBM/LN and DBM scaffolds after SCR treatment were used to repair bone defects in DBM/LN group (n=12) and DBM group (n=12), respectively. At 8 weeks after operation, the angiogenesis and bone regeneration ability of DBM/LN scaffolds were evaluated by X-ray film, Micro-CT, angiography, histology, and immunofluorescence staining [CD31, endomucin (Emcn), Ki67].Results Material related tests showed that the surface of DBM/LN scaffold was rougher than DBM scaffold, but the pore diameter did not change significantly (t=0.218, P=0.835). After SCR treatment, DBM/LN scaffold was still stable and effective. Compared with DBM scaffold, DBM/LN scaffold could adhere to more EPCs after the surface modification of CBD-LNα4-cRGD (P<0.05), and the proliferation rate and tube formation ability increased. Western blot analysis showed that the relative expressions of VEGF, phosphorylated FAK (p-FAK), and phosphorylated ERK1/2 (p-ERK1/2) proteins were higher in DBM/LN than in DBM (P<0.05). In the femoral bone defect model of mice, it was found that mice implanted with DBM/LN scaffold had stronger angiogenesis and bone regeneration capacity (P<0.05), and the number of CD31^hiEmcn^hi cells increased significantly (P<0.05).Conclusion DBM/LN scaffold can promote the adhesion of EPCs. Importantly, it can significantly promote the generation of H-type vessels and realize the effective coupling between angiogenesis and bone regeneration in bone defect repair.

Citation： TANG Yong, LUO Keyu, CHEN Yueqi, GAO Xiaoliang, TAN Jiulin, DAI Qijie, XU Jianzhong, DONG Shiwu, LUO Fei. Effect of demineralized bone matrix modified by laminin α4 chain functional peptide on H-type angiogenesis and osteogenesis to promote bone defect repair. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(12): 1594-1601. doi: 10.7507/1002-1892.202006081 Copy

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12.	Peng KY, Liu YH, Li YW, et al. Extracellular matrix protein laminin enhances mesenchymal stem cell (MSC) paracrine function through αvβ3/CD61 integrin to reduce cardiomyocyte apoptosis. J Cell Mol Med, 2017, 21(8): 1572-1583.
13.	Yeo IS, Min SK, Kang HK, et al. Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering. Biomaterials, 2015, 73: 96-109.
14.	Ljung K, Grönlund A, Felldin U, et al. Human fetal cardiac mesenchymal stromal cells differentiate in vivo into endothelial cells and contribute to vasculogenesis in immunocompetent mice. Stem Cells Dev, 2019, 28(5): 310-318.
15.	Shan N, Zhang X, Xiao X, et al. The role of laminin α4 in human umbilical vein endothelial cells and pathological mechanism of preeclampsia. Reprod Sci, 2015, 22(8): 969-979.
16.	Ishikawa T, Wondimu Z, Oikawa Y, et al. Monoclonal antibodies to human laminin α4 chain globular domain inhibit tumor cell adhesion and migration on laminins 411 and 421, and binding of α6β1 integrin and MCAM to α4-laminins. Matrix Biol, 2014, 36: 5-14.
17.	Schmohl KA, Mueller AM, Dohmann M, et al. Integrin αvβ3-mediated effects of thyroid hormones on mesenchymal stem cells in tumor angiogenesis. Thyroid, 2019, 29(12): 1843-1857.
18.	Bazzazi H, Zhang Y, Jafarnejad M, et al. Computational modeling of synergistic interaction between αVβ3 integrin and VEGFR2 in endothelial cells: Implications for the mechanism of action of angiogenesis-modulating integrin-binding peptides. J Theor Biol, 2018, 455: 212-221.
19.	Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature, 2014, 507(7492): 323-328.
20.	Verstappen J, Jin J, Kocer G, et al. RGD-functionalized supported lipid bilayers modulate pre-osteoblast adherence and promote osteogenic differentiation. J Biomed Mater Res A, 2020, 108(4): 923-937.

1. García-Gareta E, Coathup MJ, Blunn GW. Osteoinduction of bone grafting materials for bone repair and regeneration. Bone, 2015, 81: 112-121.
2. Sen MK, Miclau T. Autologous iliac crest bone graft: should it still be the gold standard for treating nonunions? Injury, 2007, 38 Suppl 1: S75-S80.
3. Hou T, Li Z, Luo F, et al. A composite demineralized bone matrix—self assembling peptide scaffold for enhancing cell and growth factor activity in bone marrow. Biomaterials, 2014, 35(22): 5689-5699.
4. Luo K, Gao X, Gao Y, et al. Multiple integrin ligands provide a highly adhesive and osteoinductive surface that improves selective cell retention technology. Acta Biomater, 2019, 85: 106-116.
5. Yang M, Li CJ, Xiao Y, et al. Ophiopogonin D promotes bone regeneration by stimulating CD31 hi EMCN hi vessel formation. Cell Prolif, 2020, 53(4): e12784.
6. Yousif LF, Di Russo J, Sorokin L. Laminin isoforms in endothelial and perivascular basement membranes. Cell Adh Migr, 2013, 7(1): 101-110.
7. Kikkawa Y, Sugawara Y, Harashima N, et al. Identification of laminin α5 short arm peptides active for endothelial cell attachment and tube formation. J Pept Sci, 2017, 23(7-8): 666-673.
8. Segarra M, Aburto MR, Cop F, et al. Endothelial Dab1 signaling orchestrates neuro-glia-vessel communication in the central nervous system. Science, 2018, 361(6404): eaao2861.
9. Dou C, Ding N, Luo F, et al. Graphene-based microRNA transfection blocks preosteoclast fusion to increase bone formation and vascularization. Adv Sci (Weinh), 2018, 5(2): 1700578.
10. Xu WL, Ong HS, Zhu Y, et al. In situ release of VEGF enhances osteogenesis in 3D porous scaffolds engineered with osterix-modified adipose-derived stem cells. Tissue Eng Part A, 2017, 23(9-10): 445-457.
11. Yu WL, Sun TW, Qi C, et al. Enhanced osteogenesis and angiogenesis by mesoporous hydroxyapatite microspheres-derived simvastatin sustained release system for superior bone regeneration. Sci Rep, 2017, 7: 44129.
12. Peng KY, Liu YH, Li YW, et al. Extracellular matrix protein laminin enhances mesenchymal stem cell (MSC) paracrine function through αvβ3/CD61 integrin to reduce cardiomyocyte apoptosis. J Cell Mol Med, 2017, 21(8): 1572-1583.
13. Yeo IS, Min SK, Kang HK, et al. Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering. Biomaterials, 2015, 73: 96-109.
14. Ljung K, Grönlund A, Felldin U, et al. Human fetal cardiac mesenchymal stromal cells differentiate in vivo into endothelial cells and contribute to vasculogenesis in immunocompetent mice. Stem Cells Dev, 2019, 28(5): 310-318.
15. Shan N, Zhang X, Xiao X, et al. The role of laminin α4 in human umbilical vein endothelial cells and pathological mechanism of preeclampsia. Reprod Sci, 2015, 22(8): 969-979.
16. Ishikawa T, Wondimu Z, Oikawa Y, et al. Monoclonal antibodies to human laminin α4 chain globular domain inhibit tumor cell adhesion and migration on laminins 411 and 421, and binding of α6β1 integrin and MCAM to α4-laminins. Matrix Biol, 2014, 36: 5-14.
17. Schmohl KA, Mueller AM, Dohmann M, et al. Integrin αvβ3-mediated effects of thyroid hormones on mesenchymal stem cells in tumor angiogenesis. Thyroid, 2019, 29(12): 1843-1857.
18. Bazzazi H, Zhang Y, Jafarnejad M, et al. Computational modeling of synergistic interaction between αVβ3 integrin and VEGFR2 in endothelial cells: Implications for the mechanism of action of angiogenesis-modulating integrin-binding peptides. J Theor Biol, 2018, 455: 212-221.
19. Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature, 2014, 507(7492): 323-328.
20. Verstappen J, Jin J, Kocer G, et al. RGD-functionalized supported lipid bilayers modulate pre-osteoblast adherence and promote osteogenic differentiation. J Biomed Mater Res A, 2020, 108(4): 923-937.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of demineralized bone matrix modified by laminin α4 chain functional peptide on H-type angiogenesis and osteogenesis to promote bone defect repair

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