Composite scaffolds of nano-hydroxyapatite and silk fibroin enhance mesenchymal stem cell-based bone regeneration via the interleukin 1 alpha autocrine/paracrine signaling loop

Abstract

Composite scaffolds of nano-hydroxyapatite (nHAp) and silk fibroin (SF) have been reported to promote bone regeneration mainly through signaling pathways associated with cell–biomaterial interaction. However, it is unclear whether soluble factors also play a role in osteoinduction with nHAp-SF. In this study, we confirmed the biocompatibility and superior osteoinductivity of nHAp-SF scaffolds versus SF scaffolds both in vitro and on a calvarial defect model in vivo. This was followed by further analysis with microarray assay. The cDNA microarray results identified 247 differentially expressed genes in bone marrow mesenchymal stem cells (BMSCs) cultured on SF-nHAp scaffolds versus SF scaffolds. The greatest disparity in gene expression levels were observed with Il1α and Ilr2. Real-time PCR assay validated the results. The addition of IL-1α into cultures of BMSCs with SF significantly increased both Bmp2 and Ilr2 expression. However, with BMSCs alone, the Il1r2 expression increased substantially, whereas Bmp2 expression exhibited a decrease rather than increase. These data suggested that nHAp may exert osteoinductive effects on BMSCs via the secretion of IL-1α in an autocrine/paracrine fashion, and IL-1α activity could be regulated through the synthesis of IL1R2 by BMSCs upon interaction with nHAp. These results complemented our understanding of the underlying mechanisms of biomaterial osteoinductivity.

Introduction

As one of the three major elements of regenerative medicine, biomaterial scaffolds play an important role in orchestrating the interaction of cells with soluble bioactive factors during reconstruction of bone tissues. Among various biomaterials, hydroxyapatite (HAp) has attracted much attention for large bone defects regeneration [1], due to its excellent osteoconductivity and similarity to the mineral phase and crystalline structure of bone. However, its low compressive strength limits its application to non/low-load bearing bone repairs [2]. With the advancement of nano-technology, much progress has been made in fabricating functional scaffolds from natural polymers, which can be incorporated together with nano-HAp (nHAp) to achieve both controllable porosity and mechanical strength [3], [4]. Silk fibroin (SF) is such a natural biopolymer with remarkable mechanical strength, controllable biodegradability, excellent biocompatibility, and ease of processing [5]. Various different techniques have been utilized for SF-nHAp fabrication [6], [7], [8], [9]. Available data from the scientific literature suggest that SF-nHAp scaffolds can be an excellent source for bone regeneration [6], [10], [11]. However, the underlying mechanisms of osteoinduction by SF-nHAp are not completely understood.

It was believed that the major osteoinductive component of SF-nHAp scaffolds is nHAp [11]. To date, most theories about the osteoinductivity of HAp (including nHAp) focused on interaction of biomaterials with the surface molecules of osteo-progenitor cells i.e. integrin superfamily [12] and focal adhesion components [13]. These interactions subsequently trigger cytoskeletal rearrangement [13] and multiple intracellular signaling cascades, such as ERK/Sox9 [12], BMP/Smad [14], Wnt [15], TGF-β, MAPK, and Notch signaling pathways [16]. Lin and colleagues found that soluble factors produced by mesenchymal stem cells (MSCs) play an important role in osteoinduction with HAp scaffolds [17]. However, the authors did not elucidate the identity of these soluble factors. In this study, we attempted to rigorously investigate differential expression of genes involved in osteoinduction by rat bone marrow MSCs (BMSCs) cultured on SF-nHAp in comparison to SF, utilizing cDNA microarray technology. It is expected that this approach could lead to the identification of novel key regulators and mechanistic pathways of the osteoinduction process.