Elsevier

Acta Biomaterialia

Volume 11, 1 January 2015, Pages 435-448
Acta Biomaterialia

Enhanced effect of β-tricalcium phosphate phase on neovascularization of porous calcium phosphate ceramics: In vitro and in vivo evidence

https://doi.org/10.1016/j.actbio.2014.09.028Get rights and content

Abstract

Neovascularization plays a key role in bone repair and regeneration. In the present study, four types of porous calcium phosphate (CaP) ceramics, namely hydroxyapatite (HA), biphasic calcium phosphates (BCP-1 and BCP-2) and β-tricalcium phosphate (β-TCP), with HA to β-TCP ratios of 100/0, 70/30, 30/70 and 2/98, respectively, were investigated in terms of their angiogenic induction. The in vitro cell culture revealed that the ceramics could promote proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs). This result could be achieved by stimulating CCD-18Co human fibroblasts to secrete angiogenic factors (vascular endothelial growth factor, basic fibroblast growth factor and transforming growth factor-β) as a paracrine effect, as well as by up-regulating HUVECs to express these angiogenic factors and their receptors (KDR, FGFR1 and ACVRL1) and the downstream eNOS as an autocrine effect. These effects were more significant in β-TCP and BCP-2, which had a higher content of β-TCP phase. In the in vivo implantation into the thigh muscles of mice, the process of neovascularization of the ceramics was initiated at 2 weeks and the mature vascular networks were formed at 4 weeks as visualized by hematoxylin and eosin staining and scanning electron microscopy. Microvessel density count confirmed that β-TCP and BCP-2 induced more microvessels to form than HA or BCP-1. This phenomenon was further confirmed by the significantly up-regulated expressions of angiogenesis-related genes in the ingrowth of cells into the inner pores of the two ceramics. All the results confirmed the angiogenic induction of porous CaP ceramics, and a higher content of β-TCP phase had an enhanced effect on the neovascularization of the ceramics.

Introduction

Bone defect is one of the major diseases threatening human health, and the treatment for large bone defects is still a clinical challenge. At present, autologous and allogenic bone grafts are the two main choices, but they have a number of drawbacks that are difficult to overcome, such as limited resources, donor site morbidity and immunological rejection [1], [2], [3]. In the past few decades, a large number of artificially synthesized biomaterials for the repair of bone defects have been developed. Among them, calcium phosphate (CaP)-based bioceramics are some of the most promising biomaterials for clinical application, not only because of their excellent biocompatibility and osteoconduction, but also due to their osteoinduction, which has been widely confirmed and accepted by researchers [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. A number of recent studies have confirmed that osteoinductive porous CaP ceramics without the addition of any growth factors or cells could be used to treat large bone defects of animals with an efficacy comparable to autologous bone grafts, which confirms their great clinical potential [15], [16], [17].

In normal bone, the vasculature transports oxygen, nutrients and soluble factors to various cells, and takes metabolic waste and carbon dioxide away [18]. Generally, angiogenesis is considered to be an essential process in fracture healing, and bone healing is highly dependent on adequate vascularization [18], [19], [20]. Hausman et al. [21] tested the effect of angiogenesis inhibitor on the healing of a closed femoral fracture in an established rat model and found that the treatment with angiogenesis inhibitor completely prevented the fracture from healing. Essentially, the repair of bone defects with bone grafts experiences similar molecular and cellular events as fracture healing, including hematoma, inflammation, new bone formation and remodeling. Therefore, the efficient vascularization of bone grafts after implantation could possibly be a crucial factor for the repair of large bone defects. To date, several methods have been developed to promote the process of neovascularization in various bone grafts. Various important angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-β, have been involved in the promotion of angiogenesis [20], [22], [23], [24]. Loading of these exogenous angiogenic factors onto a tissue engineering scaffold has been confirmed to be an effective method [25], [26], [27]. Wernike et al. [27] implanted VEGF-incorporated CaP ceramics into critical-sized cranial defects in BALB/c mice over 28 days, and the enhanced vascularization and bone formation in the ceramics were observed. Co-culture of endothelial cells and other types of cell on biocompatible tissue engineering scaffolds for construction of vascularized bone grafts could be another effective way [28], [29], [30], [31]. Santos et al. [28] co-cultured human dermal microvascular endothelial cells (HDMECs) and human osteoblasts on a 3-D starch-based scaffold and found that the HDMECs aligned and organized into microcapillary-like structures after 21 days of culture. Neovascularization of bone grafts could also be accelerated by optimizing pore size and interconnectivity of the porous scaffolds [32], [33], [34], [35]. Klenke et al. [32] investigated the impact of pore size on vascularization of biphasic calcium phosphate (BCP) ceramic particles by in vivo experiments. They found that the functional capillary density in BCP particles with a pore size greater than 140 μm was significantly higher compared with that in particles with a smaller pore size or dense particles.

In recent years, a simple strategy for promoting neovascularization of the scaffolds – stimulation by the biomaterial itself – has been proposed. Several studies have reported that silicate bioceramics have a strong ability to stimulate angiogenesis, in which the silicon ions played an important role [36], [37], [38]. Gorustovich et al. [39] made a detailed review on the effect of bioactive glasses on angiogenesis. As for porous CaP ceramics, new blood vessels and even Haversian canals in the inner pores were often observed after in vivo implantation [40], [41], [42], [43], indicating their potential to promote angiogenesis. However, to our knowledge, no systematic study on the process of neovascularization of porous CaP ceramics and the influencing factor involved has yet been reported.

It is known that angiogenesis requires a dynamic and temporally and spatially regulated interaction between endothelial cells, angiogenic factors and surrounding extracellular matrix proteins [44]. After implantation of a biomaterial, inflammatory cells, fibroblasts and stem cells would be recruited to the defective site and migrate into the inner pores of the implant. Among these cells, fibroblasts construct new extracellular matrix, which is necessary to support the growth of cells, and their importance to angiogenesis has been reported [44], [45]. The co-culture of fibroblasts and endothelial cells was confirmed to promote angiogenesis in porous tissue engineering scaffolds [31], [46]. Bioactive glass and calcium silicate ceramic were found to stimulate the secretion of VEGF and bFGF in fibroblasts and thus enhanced the proliferation and organization into the tube-like structure of endothelial cells [38], [47]. In the present study, we chose CCD-18Co human fibroblasts (HFs) and human umbilical vein endothelial cells (HUVECs) as cell models, and investigated the effect of various porous CaP ceramics with similar porous structure but different phase composition on proliferation and angiogenic factors secretion of the two types of cells by in vitro cell culture. Also, the role of the conditioned media collected from these cells in enhancing HUVECs proliferation and tubule formation was evaluated. In addition, we selected an intramuscular implantation mouse model, which was used to evaluate ectopic bone formation induced by porous CaP ceramics in our previous study [10], to investigate the neovascularization of the ceramics. Meanwhile, temporal gene expressions of the key angiogenic factors of the cells ingrowth into the inner pores of the ceramics were analyzed.

Section snippets

Materials

In this work, four types of porous CaP ceramics, namely hydroxyapatite (HA), biphasic calcium phosphates (BCP-1 and BCP-2) and β-tricalcium phosphate (β-TCP), with HA to β-TCP ratios of 100/0, 70/30, 30/70 and 2/98, respectively, were used for the subsequent in vitro and in vivo experiments. All the ceramics were fabricated by the same processing method as used in our previous work [10] and the same batch of ceramics was used. Briefly, the various CaP precursor powders synthesized by wet

Cell morphology

To evaluate effect of phase composition of porous CaP ceramics on cell morphology, HFs or HUVECs were seeded onto the four types of ceramic and cultured for 1, 4 and 7 days. Tissue culture plates served as controls. The CLSM observation of the cells’ growth on the ceramics is shown in Fig. 1. For both HFs and HUVECs, the amount of live cells increased significantly with time, while few dead cells could be found, indicating that all the ceramics support the growth of cells well.

Cell proliferation

Fig. 2 shows the

Discussion

Osteoinduction of porous CaP ceramics has been confirmed by many experimental studies, and is often dependent on the microstructure and phase composition of the ceramics [10], [11], [12], [43], [49], [50]. It is generally accepted that neovascularization is critical for bone repair and regeneration [18], [19], [20]; thus it might be correlated with the osteoinduction of biomaterials. Our previous study confirmed that porous CaP ceramics could induce ectopic bone formation in the thigh muscles

Conclusions

The current study investigated the neovascularization of porous CaP ceramics and assessed the effect of the phase composition of the ceramics and the action mechanism involved by using in vitro and in vivo evaluation. The results of the in vitro cell experiments confirmed that the ceramics could promote proliferation and angiogenesis of HUVECs by stimulating HFs to secrete angiogenic factors as a paracrine effect, and up-regulating HUVECs to express these angiogenic factors and their receptors

Acknowledgements

The authors wish to acknowledge the financial support from the National Natural Science Foundation of China (81190131, 31370973) and the National Basic Research Program of China (2011CB606201).

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