Evaluation of dense polylactic acid/beta-tricalcium phosphate scaffolds for bone tissue engineering

Abstract

Advances in biomaterial fabrication have introduced numerous innovations in designing scaffolds for bone tissue engineering. Often, the focus has been on fabricating scaffolds with high and interconnected porosity that would allow for cellular seeding and tissue ingrowth. However, such scaffolds typically lack the mechanical strength to sustain in vivo ambulatory stresses in models of load bearing cortical bone reconstruction. In this study, we investigated the microstructural and mechanical properties of dense PLA and PLA/beta-TCP (85:15) scaffolds fabricated using a rapid volume expansion phase separation technique, which embeds uncoated beta-TCP particles within the porous polymer. PLA scaffolds had a volumetric porosity in the range of 30 to 40%. With the embedding of beta-TCP mineral particles, the porosity of the scaffolds was reduced in half, whereas the ultimate compressive and torsional strength were significantly increased. We also investigated the properties of the scaffolds as delivery vehicles for growth factors in vitro and in vivo. The low-surface porosity resulted in sub optimal retention efficiency of the growth factors, and burst release kinetics reflecting surface coating rather than volumetric entrapment, regardless of the scaffold used. When loaded with BMP2 and VEGF and implanted in the quadriceps muscle, PLA/beta-TCP scaffolds did not induce ectopic mineralization but induced a significant 1.8-fold increase in neo vessel formation. In conclusion, dense PLA/beta-TCP scaffolds can be engineered with enhanced mechanical properties and potentially be exploited for localized therapeutic factor delivery.

Figure 1

Micro computed tomography (micro CT) and Scanning electron microscopy (SEM) assessment of the microstructure and porosity of the scaffolds. Micro CT rendering of the PLA (A) and PLA/βTCP (D) scaffolds, respectively (Red in D represents the embedded βTCP mineral particles). SEM micrographs demonstrate the low porosity of the external surface of the PLA (B) and PLA/βTCP (E) scaffolds, compared to the internal (cross-section) porosity (C and F, respectively). Inset in E shows a magnified micrograph of the exposed mineral surface of a βTCP particle partially embedded (yellow arrow) within the PLA polymer scaffold.

Figure 2

Multivariable linear regression analysis of the compressive mechanical properties with the scaffold porosity and mineral content. Adjusted R2 and the regression equations are indicated on each graph. The dotted diagonal line represents the identity line (predicted=measured). The solid line represents the regression line.

Figure 3

The cumulative release kinetics of rhBMP-2 from PLA and PLA/βTCP into PBS in vitro. The scaffolds were loaded using the soak and coat method in PBS or collagen type I solutions containing 200 ng of rhBMP-2. (A) The release kinetics from each scaffold was determined by measuring the concentration of rhBMP-2 in the PBS supernatant using ELISA. (B) The release time constant (τ) is estimated by modeling the release data over time using the equation Mt / M∞ = 1 – exp(t/τ). Data presented as mean ± SEM (n=3). Asterisks indicate significant differences (p<0.05) between the collagen coated and non-coated scaffolds, for both scaffold types.

Figure 4

Ectopic assessment of scaffold-mediated delivery and in vivo biologic activity of rhBMP-2 and rmVEGF120. PLA/βTCP scaffolds loaded with rhBMP-2 and rmVEGF120, or control scaffolds without the growth factors were implanted in muscular pockets in the thighs of mice, as illustrated by x-ray (arrow in A). The constructs were harvested at 8 weeks post-implantation and analyzed using micro CT to quantify the vascularization in a standard ROI containing the construct and immediate surrounding soft tissue. Representative 3D renderings of the mineralized non-loaded (B) and loaded (C) scaffolds, and the lead-chromate perfused contrast in the surrounding vessels are shown. Rendering of the vessels in the ROI after subtracting the scaffolds demonstrates the differences in vascularity between the non-loaded (D) and loaded scaffolds (E). (F) The mean vessel volume in the ROI of the control vs. VEGF treated constructs is presented as mean ± SEM (n=6). Asterisks indicate significant differences (p<0.01) between the growth factor loaded and non-loaded scaffolds.