Elsevier

Biomaterials

Volume 33, Issue 7, March 2012, Pages 2097-2108
Biomaterials

Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs

https://doi.org/10.1016/j.biomaterials.2011.11.053Get rights and content

Abstract

The successful clinical outcome of the implanted tissue-engineered bone is dependent on the establishment of a functional vascular network. A gene-enhanced tissue engineering represents a promising approach for vascularization. Our previous study indicated that hypoxia-inducible factor-1α (HIF-1α) can up-regulate the expression of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1) in bone mesenchymal stem cells (BMSCs). The angiogenesis is a co-ordinated process that requires the participation of multiple angiogenic factors. To further explore the angiogenic effect of HIF-1α mediated stem cells, in this study, we systematically evaluated the function of HIF-1α in enhancing BMSCs angiogenesis in vitro and in vivo. A constitutively active form of HIF-1α (CA5) was inserted into a lentivirus vector and transduced into BMSCs, and its effect on vascularization and vascular remodeling was further evaluated in a rat critical-sized calvarial defects model with a gelatin sponge (GS) scaffold. The expression of the key angiogenic factors including VEGF, SDF-1, basic fibroblast growth factor (bFGF), placental growth factor (PLGF), angiopoietin 1 (ANGPT1), and stem cell factor (SCF) at both mRNAs and proteins levels in BMSCs were significantly enhanced by HIF-1α overexpression compared to the in vitro control group. In addition, HIF-1α-over expressing BMSCs showed dramatically improved blood vessel formation in the tissue-engineered bone as analyzed by photography of specimen, micro-CT, and histology. These data confirm the important role of HIF-1α in angiogenesis in tissue-engineered bone. Improved understanding of the mechanisms of angiogenesis may offer exciting therapeutic opportunities for vascularization, vascular remodeling, and bone defect repair using tissue engineering strategies in the future.

Introduction

The possible risks of infection, rejection, the limited supply, and cost are serious limitations faced by autologous and allogeneic bone used to repair bone defects [1], [2]. The tissue-engineered bone offers new therapeutic strategies to repair tissue defects. When bone defects are repaired using tissue engineering technology, particularly larger bone defects, angiogenesis is a prerequisite step to achieve the successes of bone regeneration [3]. However, unlike organ transplants, where there is a pre-existing vascular supply, the tissue-engineered bone usually is devoid of pre-existing vasculature [4]. Therefore, how to promote the angiogenesis and vascularization of these bone constructs remains a big challenge in the clinic.

The importance of blood vessels in the formation of the skeleton and in bone repair was documented as early as the 1700s [5], [6]. The vasculature transports oxygen, nutrients, soluble factors, and numerous types of cells to the tissues of the body. Many of the factors that lead to the normal development of embryonic vasculature are recapitulated during neoangiogenesis in adults [7]. Factors involved in neoangiogenesis include VEGF, basic fibroblast growth factor (bFGF), various members of the transforming growth factor beta (TGF-β) family, Ang-1, placental growth factor (PLGF), angiopoietin 1 (ANGPT1), PDGF-BB, IGF-1, IGF-2, and SDF-1 [8]. Among these factors, VEGF, which is activated by hypoxia, plays a critical role in angiogenesis during the development of most tissues including bone [9]. It has been demonstrated that some growth factors can accelerate angiogenesis in the tissue-engineered bone, such as VEGF [10], [11]. However, the formation of blood vessels is a complex process that requires the coordination of multiple angiogenic factors, communication of cells with one another, and with their surrounding extracellular matrix (ECM) [12]. Besides VEGF, angiogenesis is also modulated by other growth factors, such as bFGF, PDGF, and SCF [13]. More recently, VEGF has been reported to be insufficient as a single agent to create the complex structures required for a functional vasculature. VEGF-induced vessels are often leaky and improperly connected to the existing vasculature [14]. To overcome these problems, several studies have used two angiogenic factors in combination to remodel and repair vasculature, such as Ang-1 and VEGF [15].

Hypoxia inducible factor-1 (HIF-1), which activates the transcription of hundreds of target genes in response to reduced O2 availability, is the master regulator of oxygen homeostasis in metazoans [16], [17], [18]. One of the main targets of HIF-1α-mediated gene induction is VEGF. As an upstream regulator, HIF-1α is known to activate the transcription of many other angiogenic genes, including TGF-β, Ang-1, PLGF, ANGPT1, PDGF-BB, stem cell factor (SCF), and SDF-1 [8], [19]. Therefore, HIF-1α should have more advantages in promoting angiogenesis compared with the above single agent gene therapies. Under normoxic conditions, HIF-1α is subject to degradation regulated by von Hippel-Lindau protein, which recruits an ubiquitin-protein ligase. A lack of oxygen, certain metabolic intermediates, or iron can inhibit prolyl-4-hydroxylase domain proteins (PHD) activity and stabilize HIF-1α [20], [21]. When stabilized, HIF-1α forms a heterodimer with HIF-1β and binds to the hypoxia response element to transactivate the expression of many downstream genes that are involved in angiogenesis.

We previously reported that a truncation mutant of HIF-1α (CA5), which contains a deletion (amino acids 392–520) and 2 substitutions (Pro567Thr and Pro658Gln), could effectively maintain the stability and activity of HIF-1α under normoxic conditions [22]. The expression of VEGF and SDF-1 in BMSCs was found up-regulated by HIF-1α overexpression [23]. However, whether HIF-1α could regulate angiogenesis in BMSCs through additional pathways and its in vivo role played in blood vessels formation in the tissue-engineered bone are largely unknown. In this study, we systematically explore the angiogenesis of HIF-1α mediated stem cells in vitro and evaluate the angiogenic function of CA5 gene inducing BMSCs in tissue-engineered bone in a rat model of critical-sized calvarial defects (CSD).

Section snippets

Culture of rat BMSCs

Male F344 rats were obtained from the Ninth People’s Hospital Animal Center (Shanghai, China), and all procedures were approved by the Animal Research Committee of the Shanghai Ninth People’s Hospital. Rat bone marrow was extracted from the femurs and tibias of 4-week-old male F344 rats, as described previously [22], and the BMSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, USA) containing 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO2 for

Cell culture and gene transduction

F344 rat BMSCs had characteristics of stem cells, including high expression of CD90 and CD105. Our previous data have showed that CD90 and CD105 were overexpression, whereas CD31 and CD34 are rarely detected in BMSCs of F344 rat [22]. After preliminary experiments to test for the correct dose of lentivirus, an MOI of 15 plaque forming units (pfu)/cell was determined to have the optimal effect on transfer efficiency in vitro. Four days after transduction with 15 pfu/cell Lenti-GFP, Lenti-WT, or

Discussion

The development of new therapeutic approaches that can enhance vascularized tissue growth has become one of the most active areas of tissue engineering. Due to the close association between angiogenesis and osteogenesis, neovascularization is considered as an important element in the repair of bone defects [26], [27]. This study investigated the angiogenesis of HIF-1α-transduced BMSCs in tissue-engineered bone that was used to repair a CSD. Our results indicate that overexpression of HIF-1α may

Conclusions

In summary, this study demonstrates that HIF-1α overexpression in BMSCs by gene therapy can increase angiogenesis in tissue-engineered bone, thereby promoting bone regeneration in a CSD in vivo. At the molecular level, the mRNA and protein expression of angiogenic factors in BMSCs were significantly up-regulated by CA5 in vitro. These results provide preclinical data for the potential applications of BMSCs engineered with CA5 to promote vascularization, vascular remodeling, and bone defect

Acknowledgments

The authors thank Xiuli Zhang, Xiaochen Zhang, Jun Zhao, and Shuhong Wang for their help with the animal studies and data collection. This work was supported by the National Natural Science Foundation of China (81070806, 81070864, 81100788, 31140007, 30973342), NCET-08-0353, the Science and Technology Commission of Shanghai Municipality (09411954800, 10JC1413300, 10430710900, 10dz2211600), and the Science and Technology Commission of Anhui Municipality (11040606M173, 11040606M204) and is a Key

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