Elsevier

Biomaterials

Volume 29, Issue 31, November 2008, Pages 4217-4226
Biomaterials

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds

https://doi.org/10.1016/j.biomaterials.2008.07.024Get rights and content

Abstract

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.

Introduction

Current progress in tissue engineering research has revealed tremendous potential and new perspectives for the treatment of bone defects and fracture non-union. Reintroduction of in vitro expanded osteoblasts, bone marrow stromal cells, or mesenchymal stem cells in a state that guarantees their differentiation into functional bone matrix-producing cells is now widely considered a potential alternative to autologous bone grafting with a most promising future.

In a clinical study, Schimming et al. demonstrated that tissue engineered bone constructs for maxillary sinus augmentation, which is considered too small for most orthopedic and trauma surgery purposes, was not successful in many cases due to insufficient oxygen supply [1]. Other authors have suggested that the limited diffusion of oxygen and nutrients in a static cell culture environment as well as after in vivo implantation of tissue engineered bone may constrain cell growth and bone healing [2], [3]. This problem may be amplified in cases of long bone defects since extraosseous blood supply is often significantly impaired in fracture non-unions, and medullary vasculature is not sufficient to adequately supply the defect site [4], [5]. Therefore, the inducation of mechanisms to improve angiogenic processes in tissue engineered bone constructs is now a major focus of research in this field [6], [7], [8], [9], [10], [11], [12], [13].

The processes of angiogenesis and osteogenesis have been thought to be dependent on a closed interaction between endothelial cells and osteoblasts [6], [14], [15]. Meury et al. showed that osteoblastic differentiation of bone marrow stromal cells is controlled by endothelial cells by interfering with osterix (Osx) expression [16]. Furthermore, Villars et al. and Wang et al. demonstrated that endothelial cell differentiation and the crosstalk between endothelial cells and osteoblasts not only involve diffusible factors expressed by osteoblasts (e.g. VEGF) but are also influenced by cell membrane proteins and gap junctions in two-dimensional co-cultures [14], [17]. Although these studies provide new insights into the relationship between the two cell types, knowledge about their interaction in a more complex situation involving different biomaterials is limited.

In this study, we focused on the process of cell organization and neo-vessel formation in endothelial cell cultures on polyurethane scaffolds. In particular, we addressed the question of how the parameters of endothelial cell proliferation and differentiation would be influenced by differentiated human osteoblasts in a sophisticated three-dimensional long-term culture environment that is relevant for bone tissue engineering. This study should provide a new basis for the future development and biological improvement of complex bioengineered bone constructs.

Section snippets

Polyurethane synthesis and scaffold preparation

A biodegradable polyurethane was synthesized using hexamethylene-1,6-diisocyanate, poly(ɛ-caprolactone) diol (MW = 530 g mol−1) and 1,4,3,6-dianhydro-d-sorbitol, and the scaffold was prepared by a salt leaching-phase inverse process previously described [18]. The polyurethane sponge was analyzed by the buoyancy method, computer-assisted microtomography and scanning electron microscopy presented a porosity of 85%, an interconnectivity of 99 ± 39 μm, a macro- and a micro-average pore size equal to 216 ± 

Cell phenotype

In order to examine the interaction of HUVEC with human osteoblasts, both cell types were tested with regard to their specific cell phenotype. Cultures of HUVEC showed typical cobblestone-like morphology and grew in a typical monolayer fashion (Fig. 1A). Flow cytometry revealed that 80–90% of HUVEC used for the experiments were positive for CD31 (Fig. 1B and D). After hOB reached confluence, 80–90% of the cells were positive for ALP, whereas ALP-activity was negligible in HUVEC (Fig. 1C).

Discussion

Fracture healing is a complex process that requires activation, migration, and differentiation of various cells that are capable of extracellular matrix synthesis and formation of different morphological structures. Thereby, the process of angiogenesis is a key element in early wound and fracture healing and is considered one of the most important requirements for sufficient oxygen and nutrient supply as well as for directing inflammatory cells to the wound site. Therefore, the improvement of

Conclusion

In this study, we clearly demonstrate that human osteoblasts support cell proliferation, differentiation, and neo-vessel formation of human umbilical vein endothelial cells cultured on polyurethane scaffolds. In contrast, in a long-term monoculture, HUVEC do neither proliferate nor form any vessel-like structures. This three-dimensional, co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.

Acknowledgements

We gratefully acknowledge the excellent technical assistance of Angelika Ackermann. No benefits of any kind were received or will be received by the authors from a commercial party related directly or indirectly to the subject of this article. This work was supported by AO-Foundation grant 05-H63.

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    This work was performed at the Department of Trauma Surgery, Johannes Gutenberg University School of Medicine, Germany.

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