Engineering of axially vascularized bone tissue using natural coral scaffold and osteogenic bone marrow mesenchymal stem cell sheets

Abstract

Introduction

Blood supply remains one of the obstacles to large bone tissue engineering. This study aimed to generate vascularized bone tissue by inducing axial vascularization into a construct combining natural coral scaffold and a bone marrow mesenchymal stem cells (BMSCs) sheet.

Material and methods

Isolated BMSCs were cultured to form an osteogenic cell sheet using a continuous culture method. Natural coral scaffolds were prepared into customized shape with a cylinder of 20 mm length, 8 mm in outer diameter and 5 mm in inner diameter. Then, the freed superficial inferior epigastric vessel of rabbits was first wrapped with a cell sheet, and then inserted into the central passage of the scaffold, after being wrapped with another cell sheet, the complexes were implanted subcutaneously into a rabbit groin area. In contrast, the sheet-scaffold construct that implanted into groin subcutaneous area of the other side of the same rabbit with the distal end of the blood vessel was ligated, which was considered as control. New bone and vascularization formation were evaluated at 12 weeks postoperatively.

Results

The volume of new bone formation and amount of capillary infiltration in the vascular circulation group were significantly greater than that in the vascular ligation group, which suggested that insertion of axial vessels could significantly promote angiogenesis and osteogenesis of the tissue-engineered bone.

Conclusions

These findings indicate that inserting an arteriovenous bundle into the constructs of mesenchymal stem cell sheet and coral has great potential for clinical applications to repair large bone defects.

Introduction

Reconstruction of large bone defects caused by trauma, inflammation, tumor resectionremains a major clinical challenge in the field of orthopaedic and oral-maxillofacial surgery [1]. Autologous grafting has been the gold standard and the most frequently used approach for bone repair [2]. However, Autologous bone grafts still have drawbacks, such as the extra surgical procedure for harvesting the graft, the limited amount of bone, and the donor morbidity [3]. As an alternative, allogeneic grafting avoids the harvesting procedure and the amount of available tissue is no longer limited. Nevertheless, allografts also have several drawbacks, such as worse quality, higher cost, the possibility of disease transmission, and the immunogenic potential compared to autologous grafts [4].

With the development of biomaterials and life science technologies, bone tissue engineering (BTE) has proved an effective approach to generate bone tissue and reconstruct bone defects ectopically and orthotopically [5]. Conventional methods in BTE involve obtaining sufficient amount of cells by enzymatic digestion, expanding them in vitro and then seeding a single cell suspension of these cells onto a scaffold [6]. However, this conventional approach damages cell-cell contacts and the extracellular matrix (ECM) secreted by these cells, which are important for directing cell bioactivities and promoting tissue development. Moreover, there is often about 30%–40% loss of cells [7].

To avoid the limitations of the cell suspension seeding method described above, many researchers have investigated the strategy of cell sheet transplantation, which is a technique that enables collection of a sheet of interconnected cells, along with their natural extracellular environment [8,9]. In recent years, the cell sheet technology has been widely applied in the field of tissue engineering, such as the regeneration of cartilage, cornea and bone tissues [8,10,11]. Ma et al. injected cell sheets into nude mice and rabbits, and both showed significant bone development [7]. The effect of cell sheets for BTE has also been shown in a rat model [12]. In an earlier study, Gao et al. wrapped osteogenic cell sheets made from bone marrow mesenchymal stem cell around coral scaffolds and implanted subcutaneously [13]. The results showed some degree of neo bone formation, but mainly at the periphery of the scaffolds. Other studies have demonstrated this same pattern of neo bone tissue using medical-grade polycaprolactone–calcium phosphate scaffolds, and hydroxyapatite ceramic scaffolds [14,15]. Although many studies have previously demonstrated the promising potential of cell sheets for BTE applications, few researchers have constructed large tissue engineered bone grafts in an immune-competent animal model.

Insufficient blood supply after implantation proves to be the main limitation in large BTE using the cell sheet strategy [16]. For this reason, induction of vascularization is a core requirement for any successful BTE attempt [17]. Various strategies have been attempted to facilitate vascularization of engineered constructs, such as growth factor delivery, co-culture-based pre-vascularization, and utilization of suitable biomaterials [18]. However, these techniques usually result in random patterns of new vessel formation. Transplantation to the site of interest is impossible without destruction of the vascular network [19]. An alternative approach is intrinsic vascularization: angiogenesis is induced via a vessel located centrally in the scaffold [20]. The strategy of constructing tissue-engineered bone with an arteriovenous bundle has been investigated as an effective means of intrinsic vascularization [21].

In the present research, the aim of this study was to fabricate a vascularized large bone graft using natural coral scaffold by combining arteriovenous bundle implantation and cell sheet strategy in a rabbit model, and to investigate whether the use of this vessel bundle insertion enhanced bone formation and vascularization in a large cell sheet-coral construct.