Elsevier

Biomaterials

Volume 32, Issue 11, April 2011, Pages 2812-2820
Biomaterials

Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds

https://doi.org/10.1016/j.biomaterials.2010.12.058Get rights and content

Abstract

We describe a composite hydroxyapatite (HA)–silk fibroin scaffold designed to induce and support the formation of mineralized bone matrix by human mesenchymal stem cells (hMSCs) in the absence of osteogenic growth factors. Porous three-dimensional silk scaffolds were extensively used in our previous work for bone tissue engineering and showed excellent biodegradability and biocompatibility. However, silk is not an osteogenic material and has a compressive stiffness significantly lower than that of native bone. In the present study, we explored the incorporation of silk sponge matrices with HA (bone mineral) micro-particles to generate highly osteogenic composite scaffolds capable of inducing the in vitro formation of tissue-engineered bone. Different amounts of HA were embedded in silk sponges at volume fractions of 0%, 1.6%, 3.1% and 4.6% to enhance the osteoconductive activity and mechanical properties of the scaffolds. The cultivation of hMSCs in the silk/HA composite scaffolds under perfusion conditions resulted in the formation of bone-like structures and an increase in the equilibrium Young’s modulus (up to 4-fold or 8-fold over 5 or 10 weeks of cultivation, respectively) in a manner that correlated with the initial HA content. The enhancement in mechanical properties was associated with the development of the structural connectivity of engineered bone matrix. Collectively, the data suggest two mechanisms by which the incorporated HA enhanced the formation of tissue engineered bone: through osteoconductivity of the material leading to increased bone matrix production, and by providing nucleation sites for new mineral resulting in the connectivity of trabecular-like architecture.

Introduction

Bone repair procedures often require a replacement graft to restore the function of damaged or diseased tissue. These grafts are in most cases derived from tissues harvested from a second anatomic location of the same patient (autografts) or from other patients (allografts). Autografts have been considered the gold standard for bone repair. However, limited supplies of suitable bone grafts, donor site morbidity and difficulties in shaping explanted bone have posed significant problems. On the other hand, allografts have a risk of disease transmission [1]. These limitations provide incentives for finding alternative methods. Tissue engineered bone offers a promising alternative treatment for clinical use, as well as a controllable model system for studies of cell function, developmental biology and pathogenesis [2], [3].

Successfully engineered bone grafts must be biocompatible and meet certain minimal mechanical requirements to be functional. The scaffold material provides many of the mechanical properties of the engineered graft. Organic- and polymer-based scaffolds are easily fabricated into different structures but often do not have the desired compressive modulus [4], [5], [6], [7]. Alternatively, ceramic scaffolds are stiffer but are often fragile and have low porosity, resulting in loosening or fracture of implants in clinical applications [8]. Combining both types of materials to form composite scaffolds can enhance the mechanical and biochemical properties of scaffolds used for bone tissue engineering. In this study, silk protein and hydroxyapatite (HA) ceramic were chosen because of their biocompatibility and osteoconductivity, and ease and reproducibility of fabrication. Silk sponges have been used extensively in bone tissue engineering approaches with human mesenchymal stem cells (hMSCs) and shown to facilitate bone formation in vitro and in vivo [7], [9], [10], [11]. Silk prepared with organic solvent (hexafluoroisopropanol: HFIP) and salt leeching allows the fabrication of biocompatible scaffolds with high silk content, high porosity, and good inter-pore connectivity [7], [9], [12]. HA also has excellent biocompatibility and bioactivity, is osteoinductive and is slowly replaced by host bone after implantation [13], [14], [15], [16]. We hypothesized that embedding HA micro-particles within the walls of silk sponges would improve scaffold stiffness and enhance hMSC differentiation resulting in the development of tissue engineered bone grafts with higher mineral content and improved compressive stiffness. We therefore examined the effects of scaffold properties on the structural and mechanical outcomes of engineered bone grafts by incorporating various amounts of HA mineral in porous silk scaffolds.

Bone-like constructs have been prepared in vitro by culturing hMSCs seeded into biomaterial scaffolds. HMSCs offer several advantages: they can be obtained autologously, expanded in vitro to provide sufficient cell numbers, differentiated into osteoblasts [17], [18], [19], [20] and have shown promising results in clinical models [21]. In this study, silk-HA scaffolds were seeded with hMSCs and cultured in perfusion bioreactors, which improve cell distribution and bone formation inside the scaffolds [22], [23], [24]. Perfusion provides adequate nutrient and oxygen supplies as well as cell stimulation through fluid shear stress, which enhances hMSCs osteogenic differentiation [24], [25], [26], [27]. Constructs were cultured for up to 10 weeks before being harvested and analyzed for bone tissue formation.

Section snippets

Scaffold fabrication

All reagents were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise specified. Silk fibroin was extracted from Bombyx mori cocoons utilizing our previously developed methods [12]. Briefly, the sericin was removed by boiling the cocoons in a 0.02 m Na2CO3 solution for 30 min. The resulting fibers were then dissolved in 9.3 m LiBr for 4 h at 60 °C and then subsequently dialyzed against ultrapure water for 48 h to remove residual LiBr. The aqueous silk solutions were lyophilized and

Scaffold fabrication

The inter-pore connectivity of the scaffolds was maintained with the incorporation of HA into the silk sponges, while minimally reducing the porosity as seen in low magnification SEM images (Fig. 2). The pore size of the scaffolds in all groups ranged between 400 and 600 μm, which is equivalent to the size of the salt particles that were used in the process. High magnification SEM images showed an increase in scaffold surface roughness qualitatively as more HA was added, but with less distinct

Discussion

Silk has shown significant promise as a biomaterial for bone tissue engineering scaffolds [4], [31], [32]. However, silk by itself is not osteogenic, and the mechanical properties of silk scaffolds are considerably lower than those of native bone (Young’s moduli ∼ 100 kPa vs. ∼ 10 MPa for bone). In the present study, we investigated the potential of HA micro-particles to improve the osteogenic and mechanical properties of silk scaffolds, and enhance the in vitro formation of bone-like tissues

Conclusions

The effect of incorporating the HA mineral into porous silk scaffolds was investigated for tissue engineered bone formation with hMSCs. The HA mineral enhanced hMSCs osteogenic differentiation and provided a platform for bone-like structure formation when adequate HA content was incorporated. The HA mineral provided a platform for the formation of engineered bone by hMSCs, both through the osteoconductivity of the material and by providing nucleation sites for the newly produced mineral.

Conflict of interest

Authors declare no conflict of interest.

Acknowledgment

The NIH support of this work (DE016525, P41EB002520, EB003210) is gratefully acknowledged.

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