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

Summary

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.

Abstract

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.

Introduction

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 rejection^2,3. However, artificial bone....

Protocol

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

1. 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 cytomegalovirus promoter using Spe I and Sal I restriction sites to construct the pLenti6/5-PDGFB-eGFP plasmid (LV-pdgfb)²⁵.

Results

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 µm (Figure 1B). SEM imaging demonstrated that the surface of the scaffold had a rough microtopography, .......

Discussion

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.......

Materials

List of materials used in this article

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