Nanofiber-microsphere (nano-micro) matrices for bone regenerative engineering: a convergence approach toward matrix design

Abstract

Bone is an essential organ for health and quality of life. Due to current shortfalls in therapy for bone tissue engineering, scientists have sought the application of synthetic materials as bone graft substitutes. As a composite organic/inorganic material with significant extra cellular matrix (ECM), one way to improve bone graft substitutes may be to engineer a synthetic matrix that is influenced by the physical appearance of natural ECM networks. In this work, the authors evaluate composite, hybrid scaffolds for bone tissue engineering based on composite ceramic/polymer microsphere scaffolds with synthetic ECM-mimetic networks in their pore spaces. Using thermally induced phase separation, nanoscale fibers were deposited in the pore spaces of structurally sound microsphere-based scaffold with a density proportionate to the initial polymer concentration. Porosimetry and mechanical testing indicated no significant changes in overall pore characteristics or mechanical integrity as a result of the fiber deposition process. These scaffolds displayed adequate mechanical integrity on the scale of human trabecular bone and supported the adhesion and proliferation of cultured mouse calvarial osteoblasts. Drawing from natural cues, these scaffolds may represent a new avenue forward for advanced bone tissue engineering scaffolds.

Keywords: bioceramics; bone; medical device; nanobiomaterials.

Figure 1.

(A and B) 50× and 500× SEM view of sintered composite microsphere matrix. The pore spaces between microspheres form an interconnected pore network throughout the scaffold. Note in Fig. 1B a detail of the empty pore space.

Figure 2.

(A–C) 50× (left scale bar 100 µm), 500× (middle, scale bar 50 µm) and 15 000× (right, scale bar 1 µm) SEM of 0.25% nanofiber solution in a sintered composite microsphere matrix. On 500× magnification, the fibrous network is visible in the pore spaces between microspheres that was previously unoccupied by control scaffolds. A detailed view in Fig. 2C shows numerous fibers less than 500 nm in diameter.

Figure 3.

(A–C) 50× (left), 500× (middle) and 15 000× (right) views of 1% nanofiber solution. Notice the difference in lower scales to the 0.25% solution shown in Fig. 2B, but similarity to the lower concentration when viewed at high magnification (2C and 3C).

Figure 4.

(A–C) 50× (left), 500× (middle) and 15 000× (right) views of 2% nanofiber solution. As with the comparison between 0.25% and 1%, there appear to be differences in pore size on larger scales (10–100 µm), but not on smaller scales (10–100 µm).

Figure 5.

(A and B) Representative images of control scaffold with no nanofibers (A-left) and 0.25% hybrid scaffold (B-right). Although differences are notable between the control and hybrid nanofiber preparations, most importantly both scaffolds maintain a pore distribution that includes abundant pores around 100 µm in diameter. It is believed that this is the lower limit of pores that are conducive to bone tissue formation.

Figure 6.

(A–C) Live/dead assay at 7 days. 2 × 10⁵ cells were seeded onto control and 1% scaffolds (425–600 µm in diameter) and cultured for 7 days in growth medium to determine whether cells could adhere only to nanofibers alone. (A-left) The controls scaffolds, viable cells shown in green adhere solely to the surface of the microspheres (scale bar 100 µm). (B-middle) The pore space of a nanofiber-permeated pore space in a sintered microsphere scaffold. Cells appear to adhere not only to the surface of the microspheres but also to nanofibers. The detail of the pore space shown in C is indicated with a yellow box (scale bar 100 µm). (C-right) The detail of the pore space of a hybrid scaffolds showing a viable cell on nanofibers indicated with a yellow arrow (scale bar 10 µm).

Figure 7.

DNA content assay; 2 × 10⁵ MC3T3 cells were seeded onto scaffolds and cultured for 21 days in growth medium. At 1, 3, 7, 14 and 21 day time points, the scaffolds were evaluated for DNA content. Although these data showed that the indirect marker for cell proliferation, DNA, increased from day 1 to day 21, there were no statistical differences in proliferation between groups. These results indicate that the presence of nanofibers in the pore spaces of the scaffold do not inhibit the underlying proliferative capacity of cells seeded on the sintered microsphere matrix.