In vitro and in vivo bioactivity assessment of a polylactic acid/hydroxyapatite composite for bone regeneration

Abstract

Synthetic bone graft substitutes based on composites consisting of a polymer and a calcium-phosphate (CaP) ceramic are developed with the aim to satisfy both mechanical and bioactivity requirements for successful bone regeneration. In the present study, we have employed extrusion to produce a composite consisting of 50 wt.% poly(D,L-lactic acid) (PLA) and 50 wt.% nano-sized hydroxyapatite (HA) powder, achieving homogeneous distribution of the ceramic within the polymeric phase. In vitro, in both a simulated physiological saline (SPS) and a simulated body fluid (SBF), a greater weight loss was observed for PLA/HA than for PLA particles upon 12-week immersion. Furthermore, in SPS, a continuous release of calcium and phosphate from the composite was measured, whereas in SBF, decrease of the amount of the two ions in the solution was observed both for PLA and PLA/HA accompanied with the formation of a CaP layer on the surface. In vitro characterization of the composite bioactivity was performed by culturing human mesenchymal stromal cells (hMSCs) and assessing proliferation and osteogenic differentiation, with PLA as a control. Both PLA/HA composite and PLA control were shown to support hMSCs proliferation over a period of two weeks. In addition, the composite significantly enhanced alkaline phosphatase (ALP) activity of hMSCs in osteogenic medium as compared with the polymer control. A novel implant design was employed to develop implants from dense, extruded materials, suitable for testing osteoinductivity in vivo. In a preliminary study in dogs, PLA/HA composite implants induced heterotopic bone formation upon 12-week intramuscular implantation in all animals, in contrast to PLA control, which was not osteoinductive. Unlike in vitro, a more pronounced degradation of PLA was observed in vivo as compared with PLA/HA composite.

Keywords: bone regeneration; composite; hydroxyapatite; osteoinduction; polylactic acid.

Figure 1. Implant design. Digital photograph (A) and schematic representation with dimensions (B) of implants used for intramuscular implantation in a canine model to assess osteoinductivity.

Figure 2. Characterization of PLA, PLA/HA and HA. XRD spectrum of nano-HA powder (A), SEM micrograph of nano-HA powder (B), and FTIR spectra of nano-HA powder, PLA and PLA/HA composite (C).

Figure 3. Composite homogeneity. SEM micrograph (A), EDAX spectrum (B) and elemental maps of calcium (C) and phosphorous (D) atoms of PLA/HA pellet after polishing and sterilization. (scale bars: 20 μm).

Figure 4. In vitro degradation and calcium phosphate release. Wet and dry weights of the PLA and PLA/HA particles upon immersion in SPS and SBF over a period of 12 wk. (A) pH of the solutions. (B) Calcium and phosphate release from PLA/HA particles in SPS (C and Drespectively) and of PLA and PLA/HA in SBF (E and F respectively). Statistical analysis was performed using one way ANOVA with Tukey’s multiple comparison post-hoc test (P < 0.05 and n = 3).

Figure 5. HMSCs proliferation and osteogenic differentiation. DNA content (A) and ALP activity corrected for DNA content (B) of hMSCs cultured on PLA and PLA/HA pellets in basic and osteogenic medium for 7 and 14 d. Statistical analysis was performed using one way ANOVA with Tukey’s multiple comparison post-hoc test (P < 0.05 and n = 3).

Figure 6. In vivo degradation of implants and formation of fibrous capsule. Images of methylene blue and basic fuchsin stained histological sections of implants after 12-wk intramuscular implantation in dogs. Digital photographs of a PLA (A) and a PLA/HA (B) implant section (scale bars: 5 mm), showing a more pronounced degradation of the polymer as compared with the composite implant. High magnification micrographs of a PLA (C) and a PLA/HA (D) implant section, showing fibrous capsule (FC) that had formed around both implant types and the surrounding muscle tissue (M) (scale bars: 100 µm).

Figure 7. In vivo bone formation in composite implants. Low magnification micrographs (AandB) and enlargements of black square areas in (A and B), respectively (CandD) of representative methylene blue and basic fuchsin stained sections of PLA/HA composite implants from two dogs. Images (A and B) show the entrance of the protective gap between the two composite plates surrounded with fibrous tissue (FT). Bone formation can be observed inside the gap (Bone). The newly formed bone was normal in appearance, with osteoid containing osteocytes and aligned with a layer of osteoblasts (CandD). Bone formation was also observed surrounding composite particles formed upon degradation (indicated by arrows, scale bars: A and B, 500 µm; C and D, 50 µm; E, 200 µm).