Mineralization capacity of Runx2/Cbfa1-genetically engineered fibroblasts is scaffold dependent

Abstract

Development of tissue-engineered constructs for skeletal regeneration of large critical-sized defects requires the identification of a sustained mineralizing cell source and careful optimization of scaffold architecture and surface properties. We have recently reported that Runx2-genetically engineered primary dermal fibroblasts express a mineralizing phenotype in monolayer culture, highlighting their potential as an autologous osteoblastic cell source which can be easily obtained in large quantities. The objective of the present study was to evaluate the osteogenic potential of Runx2-expressing fibroblasts when cultured in vitro on three commercially available scaffolds with divergent properties: fused deposition-modeled polycaprolactone (PCL), gas-foamed polylactide-co-glycolide (PLGA), and fibrous collagen disks. We demonstrate that the mineralization capacity of Runx2-engineered fibroblasts is scaffold dependent, with collagen foams exhibiting ten-fold higher mineral volume compared to PCL and PLGA matrices. Constructs were differentially colonized by genetically modified fibroblasts, but scaffold-directed changes in DNA content did not correlate with trends in mineral deposition. Sustained expression of Runx2 upregulated osteoblastic gene expression relative to unmodified control cells, and the magnitude of this expression was modulated by scaffold properties. Histological analyses revealed that matrix mineralization co-localized with cellular distribution, which was confined to the periphery of fibrous collagen and PLGA sponges and around the circumference of PCL microfilaments. Finally, FTIR spectroscopy verified that mineral deposits within all Runx2-engineered scaffolds displayed the chemical signature characteristic of carbonate-containing, poorly crystalline hydroxyapatite. These results highlight the important effect of scaffold properties on the capacity of Runx2-expressing primary dermal fibroblasts to differentiate into a mineralizing osteoblastic phenotype for bone tissue engineering applications.

Introduction

Conventional orthopedic grafting templates based on autogenic bone, allogenic bone, or synthetic materials are widely utilized for the clinical treatment of non-healing skeletal defects. Although successful in many cases, these grafts remain limited by inadequate osseo-integration, donor site morbidity, poor mechanical properties, and/or the risk of disease transmission [1], [2], [3], [4], [5], [6]. Bone tissue engineering has emerged as a promising strategy to overcome complications associated with these traditional skeletal repair therapies [7], [8], [9], [10]. Tissue-engineered bone substitutes have been successfully developed through the integration of osteoinductive growth factors and/or osteogenic cells into an osteoconductive scaffolding matrix. Notably, several groups have demonstrated in vitro and in vivo mineralization and repair of bone defects by combining marrow-derived and mesenchymal stem cells with three-dimensional (3-D) scaffolds [11], [12], [13], [14], [15], [16]. Despite these advances, the development of mechanically robust skeletal grafts which are immunoaccepted by the host and are capable of healing large, critical-sized defects has not been realized.

One significant barrier toward the clinical application of tissue-engineered bone grafts is the inadequate availability of a sustained mineralizing cell source. In order to address this limitation, genetic engineering strategies have been developed for the induction of osteoblastic differentiation in nonosteogenic cells [17], [18], [19], [20]. In particular, gene delivery of soluble factors, such as BMP-2 and BMP-7, or osteogenic transcription factors, such as Runx2/Cbfa1, has been investigated for the conversion of primary fibroblasts and fibroblastic cell lines into an osteoblastic phenotype [21], [22], [23], [24]. We have recently demonstrated that retroviral Runx2 overexpression induces significant levels of mineral deposition in primary dermal fibroblast monolayer cultures [25]. These genetically modified fibroblasts have considerable potential as a cell source for bone tissue engineering applications because they are easily obtained from autologous donors through minimally invasive skin biopsy and have a high capacity for in vitro expansion.

In addition to the identification of an autologous mineralizing cell source, the successful development of bone grafting templates requires careful optimization of scaffold architecture and surface properties. Biomaterial scaffolds typically function as a three-dimensional structural support, which facilitates tissue integration into the skeletal defect site and promotes cell attachment, proliferation, and differentiation into functional osteoblasts. Various classes of materials have been considered for bone grafting applications, including ceramics, natural and synthetic polymers, and their composites [26]. Among these, scaffolds based on naturally derived collagen and synthetic polycaprolactone (PCL) and polylactide-co-glycolide (PLGA) polymers were selected for investigation in this study because of their widespread use in tissue engineering applications, well-documented biodegradation profile, FDA-approval, and commercial availability [26], [27], [28], [29]. These scaffolds present a broad range of architectural and surface properties (e.g. topography, surface chemistry, roughness) that may potentially influence the biological response of seeded cells [30]. The objective of the present work was to evaluate the ability of three commonly utilized, commercially available scaffolds to support in vitro matrix mineralization when seeded with Runx2-expressing fibroblasts.