Effects of Scaffold Shape on Bone Regeneration: Tiny Shape Differences Affect the Entire System

Abstract

Although studies on scaffolds for tissue generation have mainly focused on the chemical composition and pore structure, the effects of scaffold shape have been overlooked. Scaffold shape determines the scaffold surface area (SA) at the single-scaffold level (i.e., microscopic effects), although it also affects the amount of interscaffold space in the tissue defect at the whole-system level (i.e., macroscopic effects). To clarify these microscopic and macroscopic effects, this study reports the osteogenesis abilities of three types of carbonate apatite granular scaffolds with different shapes, namely, irregularly shaped dense granules (DGs) and two types of honeycomb granules (HCGs) with seven hexagonal channels (∼255 μm in length between opposite sides). The HCGs possessed either 12 protuberances (∼75 μm in length) or no protuberances. Protuberances increased the SA of each granule by 3.24 mm² while also widening interscaffold spaces and increasing the space percentage in the defect by ∼7.6%. Interscaffold spaces were lower in DGs than HCGs. On DGs, new bone formed only on the surface, whereas on HCGs, bone simultaneously formed on the surface and in intrascaffold channels. Interestingly, HCGs without protuberances formed approximately 30% more new bone than those with protuberances. Thus, even tiny protuberances on the scaffold surface can affect the percentage of interscaffold space, thereby exerting dominant effects on osteogenesis. Our findings demonstrate that bone regeneration can be improved by considering macroscopic shape effects beyond the microscopic effects of the scaffold.

Keywords: bone; granule; honeycomb; regenerative medicine; scaffold; tissue engineering.

Figure 1

Schematic illustration of the manufacture of CAp HCGs and animal experiments: (A) extrusion process; (B) cutting of green honeycomb samples; (C) prepared green honeycomb granules (HCGs); (D) heat treatment of HCGs to obtain CaCO₃ HCGs via debinding and sintering; (E) phosphatization of CaCO₃ HCGs by immersion in Na₂HPO₄ solution; (F) preparation of CAp HCGs; (G) implantation of CAp HCGs (with or without protuberances) or DGs into a critical size bone defect in the femur condyle of rabbits.

Figure 2

(A) XRD patterns and (B) FTIR spectra of HCGs with and without protuberances and DGs. Commercial CAp and HAp powder were used as references, respectively.

Figure 3

SEM images of (A) DGs and (B) HCGs with and (C) without protuberances. (D–F) High-magnification SEM images of panels A–C. (G–I) High-magnification SEM images of panels D–F. The characters “∗” and “#,” white arrows, and yellow arrowheads indicate intrascaffold channels, struts, protuberances, and micropores, respectively. Scale bars: 500 μm (A–C), 50 μm (D–F), and 5 μm (G–I).

Figure 4

Three-dimensional reconstructed CT images of (A) DGs, (B) HCG with protuberances, and (C) HCGs without protuberances filled in a mold. The yellow arrow indicates an interscaffold space on the surface of a granular aggregate. CT images in the center regions of aggregates of (D) DGs and HCG (E) with and (F) without protuberances filled in a mold. (G) Volume percentages of interscaffold spaces acquired using CT image analysis. (H) The number of granules filled in a mold per square centimeter. Porous structure characteristics measured via mercury intrusion porosimetry: (I) pore size distribution and (J) cumulative pore volume versus pore size. Schematic illustration of the correlation between scaffold shape and (K) interscaffold space volume, (L) region functioning as cell scaffold in the system, and (M) surface area of each granular scaffold. *p < 0.05 and **p < 0.01.

Figure 5

(A) Calcium and (B) phosphate ions released from HCGs and DGs in buffer solutions at pH 7.4 and 5.5. *p < 0.01.

Figure 6

(A) Cell proliferation assays during a culture period of 7 d. (B) Fluorescent images after 7 d of culture (scale bar = 100 μm). Cellular F-actin and nuclei were stained with phalloidin and Hoechst 33258, respectively. (C) ALP activities after 7 and 14 d of culture. (D) Optical images of cultures stained with Alizarin Red S after 21 d of culture (scale bar = 1 mm). (E) Absorbance of alizarin extracts for quantification of mineral nodule formation. *p < 0.05 and **p < 0.01

Figure 7

μ-CT images at (A–D) 4 and (E–H) 12 weeks after surgery. (A,E) DGs and HCGs (B,F) with and (C,G) without protuberances-groups. (D, H) Blank group (negative control). Yellow arrowheads indicate granular scaffolds. Quantitative analyses of μ-CT images: (G) MV/TV, (H) BV/TV, and (I) TbTh. *p < 0.05 and **p < 0.01. DG- and HCG-implanted groups: n = 6. Blank group: n = 4.

Figure 8

Histological sections of the DG- and HCG implanted groups with and without protuberances and blank group at 4 weeks after surgery. (A–H) HE, (G–L) MT, (M–R) TRAP, (M–R) OCN, and (U–X) CD31-stained sections. Panels E–X showed magnified images of the regions corresponding to the regions enclosed by squares in panels A–D. NB, BV, OB, OC, VEC, FCT, and “#” indicate new bone, blood vessel, osteoblast, osteoclast, vascular endothelial cell, fibroconnective tissue, and remaining scaffold, respectively. Scale bars: 500 μm (A–D) and 20 μm (E–X).

Figure 9

Histological sections of the DG- and HCG implanted groups with and without protuberances and blank group at 12 weeks after surgery. (A–H) HE, (G–L) MT, (M–R) TRAP, (M–R) OCN, and (U–X) CD31-stained sections. Panels E–X are magnified images of the regions corresponding to the regions enclosed by squares in the panels A–D. NB, BV, OB, OC, VEC, AT, and “#” indicate new bone, blood vessel, osteoblast, osteoclast, vascular endothelial cell, adipose tissue, and remaining scaffold, respectively. Scale bars: 500 μm (A–D) and 20 μm (E–X).

Figure 10

Percentages of (a) new bone and (b) remaining materials and (c) number of osteoclasts on granular scaffolds. *p < 0.05, **p < 0.01. ^†Significant difference compared with the results at 4 weeks after surgery. DG- and HCG-implanted groups: n = 6. Blank group: n = 4.