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

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.

Figure 1

(A) Silk-HA composite scaffold fabrication process. (B) Experimental design to study the effects of embedded HA content in silk sponge in formation of tissue engineered bone constructs. Four types of scaffolds (0% HA, 1.6% HA, 3.1% HA, and 4.6% HA) were seeded with hMSCs and cultured in osteogenic media under perfusion bioreactor for 5 and 10 weeks. Unseeded 0% HA and 4.6% HA were also cultured for 5 weeks.

Figure 2

Calculated volume fraction of silk and HA mineral and SEM images of 0% HA, 1.6% HA, 3.1% HA, and 4.6% HA scaffolds. 200× SEM image showed porous scaffold with inter-pore connectivity (bar: 200 μm). 1000× SEM image illustrated the different in surface topography of the scaffolds (bar: 20 μm).

Figure 3

(A) DNA content before and after cultivation in perfusion bioreactor for 5 and 10 weeks. (Line represents a statistically significant difference between time point of the same scaffold group; ^{a, b} represent statistically significant differences from 0% HA and 1.6% HA, respectively, at the same time point). (B) Live/Dead image of cells inside the scaffolds before and after cultivation for 10 weeks. (scale bar: 200 μm)

Figure 4

Reconstructed 3D μCT images of the tissue engineered bone construct before and after cultivation for 5 and 10 weeks of all groups (scale bar: 2mm)

Figure 5

Development of tissue engineered bone constructs over 5 and 10 weeks of cultivation: (A) Equilibrium Young's Modulus, (B) calcium content, and bone structural parameters determined by μCT analysis; (C) BV, (D) BVF, (E) Conn.D, (F) Tb.N, (G) Tb.Th, and (H) Tb.Sp. (Dash line indicates average value of decellularized native trabecular bovine bone. Solid tree line represents a statistically significant difference between time point of the same scaffold group; ^{a, b, c} represent statistically significant differences from 0% HA, 1.6% HA, and 3.1% HA, respectively, at the same time point.

Figure 6

Histology and immunohistochemistry of the constructs before and after cultivation: (A) H&E, (B) Von Kossa, (C) Collagen Type I, and (D) Bone sialoprotein (bar: 200 μm)

Figure 7

Schematic of mineralization process. Yellow, blue and red regions represent silk, premineralized HA, and new mineral, respectively. Arrows indicate the connection of the new mineral structure. In 0% HA, newly produced mineral localized within the pore space and grew larger in size over time resulting in spherical-like mineral structure. In 1.6% HA, new mineral nucleated from premineralized HA as well as was deposited into the pore space. In 3.1-4.6% HA, the newly produced mineral nucleated from the premineralized HA. As the structure grew, structural connections occurred.