Name: visualizing angiogenesis by multiphoton microscopy in vivo in genetically modified 3d-plga/nhap scaffold for calvarial critical bone defect repair
Uploaded: 2017-09-07T12:30:00
Description: Watch this Scientific Journal Video about Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair at JoVE.com

This study was supported by the Shenzhen Peacock Program, China (No. 110811003586331), the Shenzhen Basic Research Program (No. JCYJ20150401150223631, No. JCYJ20150401145529020, and No. JCYJ20160331190714896), the Guangdong Public Research and Capacity Building Special Program (No. 2015A020212030), the National Natural Science Foundation of China (No. 81501893), the National Major Basic Research Program of China (2013CB945503), and the SIAT Innovation Program for Excellent Young Researchers (Y5G010).

The animal care was in compliance with the Guide for the Care and Use of Laboratory Animals of Guangdong Province. All procedures were performed under the supervision and approval of the Ethics Committee for Animal Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.
1. Lentiviral (LV) Production
<ol>
	<li>Clone the pdgf-b cDNA into a lentiviral expression vector (pLenti6/5-eGFP or LV-eGFP) at a custom multiple-cloning site downstream of the cytomegalov...

<ol>
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E., Mosheiff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+approaches+for+bone+repair:+concepts+and+evidence.">Tissue engineering approaches for bone repair: concepts and evidence.</a> Injury. 42 (6), 609-613 (2011).</li><li>Chiarello, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=allograft+and+bone+substitutes+in+reconstructive+orthopedic+surgery.">allograft and bone substitutes in reconstructive orthopedic surgery.</a> Aging Clin Exp Res. 25, S101-S103 (2013).</li><li>Elangovan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enhancement+of+bone+regeneration+by+gene+activated+matrix+encoding+for+platelet+derived+growth+factor.">The enhancement of bone regeneration by gene activated matrix encoding for platelet derived growth factor.</a> Biomaterials. 35 (2), 737-747 (2014).</li><li>Bouyer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+delivery+of+tunable+doses+of+BMP-2+from+an+adaptable+polymeric+scaffold+induces+volumetric+bone+regeneration.">Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration.</a> Biomaterials. 104, 168-181 (2016).</li><li>Rezwan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+and+bioactive+porous+polymer/inorganic+composite+scaffolds+for+bone+tissue+engineering.">Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering.</a> Biomaterials. 27 (18), 3413-3431 (2006).</li><li>Burg, K. J., Porter, S., Kellam, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+developments+for+bone+tissue+engineering.">Biomaterial developments for bone tissue engineering.</a> Biomaterials. 21 (23), 2347-2359 (2000).</li><li>Xiao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+of+collagen-based+biomaterials+with+immobilized+growth+factors+or+peptides.">Modifications of collagen-based biomaterials with immobilized growth factors or peptides.</a> Methods. 84, 44-52 (2015).</li><li>Chen, G., Lv, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+and+Application+of+Electrospun+Nanofiber+Scaffold-based+Growth+Factor+in+Bone+Tissue+Engineering.">Immobilization and Application of Electrospun Nanofiber Scaffold-based Growth Factor in Bone Tissue Engineering.</a> Curr Pharm Des. 21 (15), 1967-1978 (2015).</li><li>Kofron, M. D., Li, X., Laurencin, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-+and+gene-based+tissue+engineering+in+bone+repair.">Protein- and gene-based tissue engineering in bone repair.</a> Curr Opin Biotechnol. 15 (5), 399-405 (2004).</li><li>Chen, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+and+novel+applications+of+release+technology+for+periodontal+reconstructive+therapies.">New insights into and novel applications of release technology for periodontal reconstructive therapies.</a> J Control Release. 149 (2), 92-110 (2011).</li><li>Winn, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+approaches+for+modulating+bone+regeneration.">Gene therapy approaches for modulating bone regeneration.</a> Adv Drug Deliv Rev. 42 (1-2), 121-138 (2000).</li><li>Chang, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+Encoding+Human+Platelet-Derived+Growth+Factor-B+Delivered+to+Alveolar+Bone+Defects+Exhibits+Safety+and+Biodistribution+Profiles+Favorable+for+Clinical+Use.">Adenovirus Encoding Human Platelet-Derived Growth Factor-B Delivered to Alveolar Bone Defects Exhibits Safety and Biodistribution Profiles Favorable for Clinical Use.</a> Hum Gene Ther. 20 (5), 486-496 (2009).</li><li>Phipps, M. C., Xu, Y. Y., Bellis, S. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDGF-B+gene+therapy+accelerates+bone+engineering+and+oral+implant+osseointegration.">PDGF-B gene therapy accelerates bone engineering and oral implant osseointegration.</a> Gene Ther. 17 (1), 95-104 (2010).</li><li>Javed, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+the+platelet-derived+growth+factor+in+periodontal+tissue+regeneration.">Significance of the platelet-derived growth factor in periodontal tissue regeneration.</a> Arch Oral Biol. 56 (12), 1476-1484 (2011).</li><li>Murali, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+hybrid+porous+scaffolds+immobilized+with+platelet+derived+growth+factor-BB+promote+cellularization+and+vascularization+in+tissue+engineering.">Biomimetic hybrid porous scaffolds immobilized with platelet derived growth factor-BB promote cellularization and vascularization in tissue engineering.</a> J Biomed Mater Res A. 104 (2), 388-396 (2016).</li><li>Andrae, J., Gallini, R., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+platelet-derived+growth+factors+in+physiology+and+medicine.">Role of platelet-derived growth factors in physiology and medicine.</a> Genes Dev. 22 (10), 1276-1312 (2008).</li><li>Wosnitza, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+human+adipose+stem+cells+to+perform+adipogenic+and+endothelial+differentiation.">Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation.</a> Differentiation. 75 (1), 12-23 (2007).</li><li>Hankenson, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+bone+regeneration.">Angiogenesis in bone regeneration.</a> Injury. 42 (6), 556-561 (2011).</li><li>Schmidt, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+three-dimensional+quantification+of+VEGF-induced+scaffold+neovascularisation+by+microcomputed+tomography.">Rapid three-dimensional quantification of VEGF-induced scaffold neovascularisation by microcomputed tomography.</a> Biomaterials. 30 (30), 5959-5968 (2009).</li><li>Perng, C. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+critical+size+defect+as+an+experimental+model+to+test+bone+repair+materials.">The critical size defect as an experimental model to test bone repair materials.</a> J Craniofac Surg. 1 (1), 60-68 (1990).</li><li>Hollanders, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bevacizumab+Revisited:+Its+Use+in+Different+Mouse+Models+of+Ocular+Pathologies.">Bevacizumab Revisited: Its Use in Different Mouse Models of Ocular Pathologies.</a> Curr Eye Res. 40 (6), 611-621 (2015).</li><li>Gao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QSIM:+quantitative+structured+illumination+microscopy+image+processing+in+ImageJ.">QSIM: quantitative structured illumination microscopy image processing in ImageJ.</a> Biomed Eng Online. 14, 4 (2015).</li><li>Kobat, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+multiphoton+microscopy+using+longer+wavelength+excitation.">Deep tissue multiphoton microscopy using longer wavelength excitation.</a> Opt Express. 17 (16), 13354-13364 (2009).</li><li>Lohmann, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+regeneration+induced+by+a+3D+architectured+hydrogel+in+a+rat+critical-size+calvarial+defect.">Bone regeneration induced by a 3D architectured hydrogel in a rat critical-size calvarial defect.</a> Biomaterials. 113, 158-169 (2016).</li><li>Lv, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+angiogenesis+and+osteogenesis+in+critical+bone+defects+by+the+controlled+release+of+BMP-2+and+VEGF:+implantation+of+electron+beam+melting-fabricated+porous+Ti6Al4V+scaffolds+incorporating+growth+factor-doped+fibrin+glue.">Enhanced angiogenesis and osteogenesis in critical bone defects by the controlled release of BMP-2 and VEGF: implantation of electron beam melting-fabricated porous Ti6Al4V scaffolds incorporating growth factor-doped fibrin glue.</a> Biomed Mater. 10 (3), (2015).</li><li>Guldberg, R. 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Bone is a highly vascularized tissue with a unique capacity to continuously heal and remodel throughout the lifetime of an individual1. The level of vascularization is important for osteogenesis and defect repair. Low vascularization limits the wide clinical application of tissue-engineered bone. Constructing a highly vascularized tissue-engineered bone according to the theory of biomimetics has become a tool for repairing large segment bone defects. Various kinds of scaffolds have been successful...

Bone is a highly vascularized tissue that continues to remodel during the lifetime of an individual1. The rapid and effective bone regeneration of large bone defects resulting from trauma, nonunion, tumor resections, or craniofacial malformations is a complex physiological process. Traditional therapeutic approaches used for bone defect repair include autograft and allograft implantation, but their use involves several problems and limitations, such as limited availability, significant donor site morbidity, a high risk of infection, and host immune rejection2,3. However, artificial bone grafts offer an efficient alternative to alleviate these limitations. They can be made from biodegradable materials, are easy to be fabricate with a suitable pore size, and can be genetically modified4,5.
Currently, various tissue engineering scaffolds have been employed in the development of tissue-engineered bone6,7. To induce bone repair and regeneration more effectively, engineered biomaterials combined with growth factors have emerged and achieved good results8,9. Unfortunately, the short half-life, easy-to-lose activity, and supraphysiological dosage of growth factors for therapeutic efficacy limit their clinical application10. To overcome these problems, the delivery of growth factor genes instead of growth factors has been demonstrated as an effective approach to sustain bioactivity for the treatment of osseous defects and diseases11,12. Viral vectors are promising delivery tools for tissue regeneration due to their high expressing efficiency13.
Among growth factors, platelet-derived growth factor (PDGF-BB) was selected in this study because it is not only a mitogen and chemoattractant for mesenchymal and osteogenic cells, but also a stimulant for angiogenesis14,15. Previous preclinical and clinical studies showed that PDGF-BB could safely and effectively promote bone repair in periodontal osseous defects16,17. Recent studies revealed that PDGF-BB stimulates angiogenesis by motivating endothelial cell migration and proliferation in vivo18,19. Furthermore, PDGF-BB can also render mesenchymal stem cells (MSCs) capable of differentiating into endothelial cells20, and this further highlights the potential role of MSCs in neovascularization. Therefore, inducing the de novo formation of vasculature in scaffolds with PDGF-BB is an important step for the repair of tissue grown into scaffolds in bone tissue engineering.
Bone defect healing is a dynamic tissue morphogenetic process that requires coordinated osteogenesis and angiogenesis at the repairing positions21. Neoangiogenesis into implanted tissue-engineered scaffolds is an essential pre-requisite for supplying cells with nutrients and oxygen for growth and survival and for removing metabolic waste. Commonly used imaging methods, including X-ray micro-computed tomography (microCT), magnetic resonance imaging (MRI), scanning electron microscopy (SEM), optical coherence tomography (OCT), and confocal laser scanning microscopy, are applied instead of histological examination to obtain angiogenesis information22,23. However, these methods face various obstacles in visualizing and measuring neovasculature in 3D scaffolds in bone tissue engineering. Multiphoton microscopy (MPM) is a comparatively novel bio-imaging technique that has the distinct advantage of simultaneously visualizing cells, extracellular matrix, and surrounding vascular networks in vivo. It possesses an inherent three-dimensional imaging capability for deep tissue penetration and causes low photodamage. Hence, in the last decade, MPM has gained much attention in biomedical studies24, including in neuroscience, immunology, and stem cell dynamics. However, it is barely used in orthopedic research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly(D,L-lactide-co-glycolide) (PLGA)</td>
			<td>Sigma</td>
			<td>P1941</td>
			<td>L/G ratio 75:25, MW 66000-107000</td>
		</tr>
		<tr>
			<td>Hydroxyapatite nanoparticles</td>
			<td>Sigma</td>
			<td>702153</td>
			<td>Average diameter &#60; 200nm</td>
		</tr>
		<tr>
			<td>Chloroquine diphosphate salt</td>
			<td>Sigma</td>
			<td>C6628</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated 250-kD dextran</td>
			<td>Sigma</td>
			<td>FD250S</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-dioxane</td>
			<td>lingfeng,Shanghai</td>
			<td></td>
			<td>0.45 micron</td>
		</tr>
		<tr>
			<td>Stericup filters</td>
			<td>Merck Millipore Corporation</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-BB Cdna</td>
			<td>Sino Biological, Inc</td>
			<td>MZ50801-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PDGF-BB mouse polyclonal antibody</td>
			<td> BioVision, Inc</td>
			<td>5489-30T</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-BB recombinant protein</td>
			<td></td>
			<td>4489-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium-phosphate transfection solution</td>
			<td>Promega Corporation</td>
			<td>E1200</td>
			<td></td>
		</tr>
		<tr>
			<td>L-DMEM</td>
			<td>Hyclone</td>
			<td>SH30021.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Hyclone</td>
			<td>SH30028.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, Liquid</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10099-141</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell </td>
			<td>Corning</td>
			<td>3422</td>
			<td></td>
		</tr>
		<tr>
			<td>Male BALB/c mice</td>
			<td>Guangdong Medical Laboratory Animal Center </td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pentobarbital </td>
			<td>Merck</td>
			<td>1063180500</td>
			<td></td>
		</tr>
		<tr>
			<td>multiphoton microscopy</td>
			<td></td>
			<td></td>
			<td>A homemade in Shenzhen Institutes of Advanced Technology to detect two-photon excited fluorescence (TPEF) and second harmonic generation signal (SHG).</td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Keyuan, Shandong</td>
			<td>401750169</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript RT Master Mix (Perfect Real Time)</td>
			<td>Takara</td>
			<td>RR420B</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Premix Ex Taq (Tli RNaseH Plus)</td>
			<td>Takara</td>
			<td>RR036B</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin</td>
			<td>Beyotime</td>
			<td>C0105</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Leica </td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge OPtima L-100XP</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-temperature printer </td>
			<td>Tsinghua university</td>
			<td></td>
			<td>A homemade in Tsinghua university</td>
		</tr>
		<tr>
			<td>LightCycler 480 instrument </td>
			<td>Roche</td>
			<td>5815916001</td>
			<td></td>
		</tr>
		<tr>
			<td>microCT</td>
			<td>Bruker</td>
			<td>1176</td>
			<td></td>
		</tr>
		<tr>
			<td>commercial software</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing angiogenesis by multiphoton microscopy in vivo in genetically modified 3d-plga/nhap scaffold for calvarial critical bone defect repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Cylindrical Porous PLGA/nHAp scaffolds 0.6 mm in height and 4 mm in diameter were fabricated with a 3D printer. The morphologies of the scaffolds were analyzed via scanning electron microscopy and microCT. Figure 1A shows the photograph of the implanted scaffold. MicroCT scanning revealed that more than 85% of the pores had sizes ranging from 200 to 400 &#181;m (Figure 1B). SEM imaging demonstrated that the surface of the scaffold had a rough microtopogra...

Watch this Scientific Journal Video about Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair at JoVE.com

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

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