Investigation of angiogenesis in bioactive 3-dimensional poly(d,l-lactide-co-glycolide)/nano-hydroxyapatite scaffolds by in vivo multiphoton microscopy in murine calvarial critical bone defect.
Reconstruction of critical size bone defects remains a major clinical challenge because of poor bone regeneration, which is usually due to poor angiogenesis during repair. Satisfactory vascularization is a prerequisite for the survival of grafts and the integration of new tissue with existing tissue. In this work, we investigated angiogenesis in 3D scaffolds by in vivo multiphoton microscopy during bone formation in a murine calvarial critical bone defect model and evaluated bone regeneration 8weeks post-implantation. The continuous release of bioactive lentiviral vectors (LV-pdgfb) from the scaffolds could be detected for 5days in vitro. In vivo, the released LV-pdgfb transfected adjacent cells and expressed PDGF-BB, facilitating angiogenesis and enhancing bone regeneration. The expression of both pdgfb and the angiogenesis-related genes vWF and VEGFR2 was significantly increased in the pdgfb gene-carrying scaffold (PHp) group. In addition, microCT scanning and histomorphology results proved that there was more new bone ingrowth in the PHp group than in the PLGA/nHA (PH) and control groups. MicroCT parameters, including BMD, BV/TV, Tb.Sp, and Tb.N indicated that there was significantly more new bone formation in the PHp group than in the other groups. With regard to neovascularization, 8weeks post-implantation, blood vessel areas (BVAs) were 9428±944μm(2), 4090±680.3μm(2), and none in the PHp, PH, and control groups, respectively. At each time point, BVAs in the PHp scaffolds were significantly higher than in the PH scaffolds. To our knowledge, this is the first use of multiphoton microscopy in bone tissue-engineering to investigate angiogenesis in scaffolds in vivo. This method represents a valuable tool for investigating neovascularization in bone scaffolds to determine if a certain scaffold is beneficial to neovascularization. We also proved that delivery of the pdgfb gene alone can improve both angiogenesis and bone regeneration Acronyms.



Reconstruction of critical size bone defects remains a major clinical challenge because of poor bone regeneration, which is usually due to poor angiogenesis during repair. Satisfactory vascularization is a prerequisite for the survival of grafts and the integration of new tissue with existing tissue. In this work, we investigated angiogenesis in 3D scaffolds by in vivo multiphoton microscopy during bone formation in a murine calvarial critical bone defect model and evaluated bone regeneration 8weeks post-implantation. To verify that pdgfb-expressing vectors carried by the scaffolds can promote angiogenesis in 3D-printed scaffolds in vivo, we monitored angiogenesis within the implants by multiphoton microscopy. To our knowledge, this is the first study to dynamically investigate angiogenesis in bone tissue engineering scaffolds in vivo.
Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China.
Jian
J
Li
Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China.
Qiang
Q
Xu
Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China.
Bin
B
Teng
Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China; Orthopedics Department, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215000, China.
Chen
C
Yu
Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China; Orthopedics Department, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215000, China.
Jian
J
Li
Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China.
Liang
L
Song
Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China.
Yu-Xiao
YX
Lai
Laboratory for Reproductive Health, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China.
Jian
J
Zhang
Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China. Electronic address: zhengwei@siat.ac.cn.
Wei
W
Zheng
Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China. Electronic address: pg.ren@siat.ac.cn.
Pei-Gen
PG
Ren
D000818:Animals; D001672:Biocompatible Materials / Q000494:pharmacology*; D001861:Bone Regeneration / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D004195:Disease Models, Animal; D017886:Durapatite / Q000737:chemistry*; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D019344:Lactic Acid / Q000737:chemistry*; D016086:Lentivirus / Q000378:metabolism; D008297:Male; D059630:Mesenchymal Stem Cells / Q000187:drug effects / Q000378:metabolism; D008807:Mice, Inbred BALB C; D008853:Microscopy / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000235:genetics; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D011100:Polyglycolic Acid / Q000737:chemistry*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D020574:Proto-Oncogene Proteins c-sis / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012886:Skull / Q000000981:diagnostic imaging / Q000473:pathology*; D054457:Tissue Scaffolds / Q000737:chemistry*; D055114:X-Ray Microtomography
D001672:Biocompatible Materials; D020574:Proto-Oncogene Proteins c-sis; D049452:Green Fluorescent Proteins; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D017886:Durapatite
3D biodegradable scaffold; Angiogenesis; Bone formation; Critical bone defects; Multiphoton microscopy