Biomimetic Mineralization on 3D Printed PLA Scaffolds: On the Response of Human Primary Osteoblasts Spheroids and In Vivo Implantation

Abstract

This study aimed to assess the response of 3D printed polylactic acid (PLA) scaffolds biomimetically coated with apatite on human primary osteoblast (HOb) spheroids and evaluate the biological response to its association with Bone Morphogenetic Protein 2 (rhBMP-2) in rat calvaria. PLA scaffolds were produced via 3D printing, soaked in simulated body fluid (SBF) solution to promote apatite deposition, and characterized by physical-chemical, morphological, and mechanical properties. PLA-CaP scaffolds with interconnected porous and mechanical properties suitable for bone repairing were produced with reproducibility. The in vitro biological response was assessed with human primary osteoblast spheroids. Increased cell adhesion and the rise of in vitro release of growth factors (Platelet-Derived Growth Factor (PDGF), Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF) was observed for PLA-CaP scaffolds, when pre-treated with fetal bovine serum (FBS). This pre-treatment with FBS was done in a way to enhance the adsorption of serum proteins, increasing the number of bioactive sites on the surface of scaffolds, and to partially mimic in vivo interactions. The in vivo analysis was conducted through the implantation of 3D printed PLA scaffolds either alone, coated with apatite (PLA-CaP) or PLA-CaP loaded with rhBMP-2 on critical-sized defects (8 mm) of rat calvaria. PLA-CaP+rhBMP2 presented higher values of newly formed bone (NFB) than other groups at all in vivo experimental periods (p < 0.05), attaining 44.85% of NFB after six months. These findings indicated two new potential candidates as alternatives to autogenous bone grafts for long-term treatment: (i) 3D-printed PLA-CaP scaffold associated with spheroids, since it can reduce the time of repair in situ by expression of biomolecules and growth factors; and (ii) 3D-printed PLA-CaP functionalized rhBMP2 scaffold, a biocompatible, bioactive biomaterial, with osteoconductivity and osteoinductivity.

Keywords: 3D printed scaffold; 3D printing; biomimetic; biomimetic apatite; bone morphogenetic protein 2; bone repair; poly (lactic acid); spheroids.

Figure 1

PLA scaffold design. The computer-aided design (CAD) model of scaffolds (A); Representative macrographs of printed PLA scaffolds (B) (Scale bar = 1 mm).

Figure 2

Surgical procedures for scaffold implantation in 8 mm calvaria defect. Surgical bone defect (A), scaffold implantation in situ (B) and defect sutured (C).

Figure 3

SEM micrographs of scaffolds before (A–C) and after CaP coating (E–G).

Figure 4

EDS spectrum of the top surface uncoated PLA (A) and PLA-CaP (B) scaffolds. Element mapping image for calcium, imaged in pink, (C) and phosphorous, imaged in blue, (D) of the fracture surface of PLA-CaP scaffold.

Figure 5

XRD (A) and FTIR (B) spectra of PLA and PLA-CaP scaffolds. TGA (C) and DTG (D) curves of uncoated PLA and PLA-CaP scaffolds exhibited single-step decomposition.

Figure 6

Cytocompatibility evaluation of the scaffolds, as evaluated by XTT assay. Bars indicate mean ± SD results normalized as a percentage of the unexposed control group (C). Extracts of polystyrene beads were used as negative control (C−) and fragments of latex as a positive control (C+). An asterisk indicates a significant difference with all other groups (p < 0.05).

Figure 7

Fluorescence microscopy images of HOb osteospheres cultivated for 7 days over surfaces of PLA, PLA-CaP scaffolds, pre-treated or not with FBS. Cell nuclei appear in blue, and actin microfilaments marked in green. Images obtained either with ×10 objectives. Bars indicate 200 µm.

Figure 8

Estimative of the adhered cells to each scaffold, as measured by the release of cytoplasmic lactate dehydrogenase (A) or total protein content (B), after solubilization with the detergent Triton X-100. Bars indicate the mean and SD of NADH reduction in culture media 25 min after the addition of substrates and solubilizer. The asterisk indicates significant difference from all groups (p < 0.05, Kruskal-Wallis Test with Dunn post-test).

Figure 9

Evaluation of the release of biological markers into the culture media by human osteoblast spheroids after 7 days incubation inside the tested scaffolds. (A). Concentration of released Basic Fibroblastic Growth factor (FGF-2). (B). Concentration of released Vascular Endothelial Growth Factor (VEGF). (C) Concentration of released Platelet-Derived Growth Factor (PDGF). (D) Alkaline Phosphatase (ALP) activity. Bars indicate mean and standard deviation of three biological replicates with five technical replicated each. Bars with different letters are significantly different (p < 0.05). UE: units of enzyme activity.

Figure 10

Representative photomicrographs of calvaria defect after 1 month. Histological section stained with hematoxylin/eosin from the region of control without biomaterial (A,B), biomaterial implantation in the PLA (C,D), PLA-CaP (E,F) and PLA-CaP-rhBMP-2 (G,H) groups—1 month after implantation. The region occupied by pre-existing bone is indicated with (PEB), newly formed bone (NFB); connective tissue (CT) and biomaterial/PLA scaffold (BM). Results are representative of 5 rat/group.

Figure 11

Representative photomicrographs of calvaria defect after 3 month. Histological section stained with hematoxylin/eosin from the region of control without biomaterial (A,B), biomaterial implantation in the PLA (C,D), PLA-CaP (E,F) and PLA-CaP-rhBMP-2 (G,H) groups—3 month after implantation. The region occupied by pre-existing bone is indicated with (PEB), newly formed bone (NFB); connective tissue (CT) and biomaterial/PLA scaffold (BM). Results are representative of 5 rat/group.

Figure 12

Representative photomicrographs of calvaria defect after 6 months. Histological section stained with hematoxylin/eosin from the region of control without biomaterial (A,B), biomaterial implantation in the PLA (C,D), PLA-CaP (E,F) and PLA-CaP-rhBMP-2 (G,H) groups—6 months after implantation. The region occupied by pre-existing bone is indicated with (PEB), newly formed bone (NFB); connective tissue (CT) and biomaterial/PLA scaffold (BM). Results are representative of 5 rat/group.

Figure 13

Box plot comparing the volume of new formed bone (NFB), biomaterial (BM) and connective tissue (CT) (%) of PLA, PLA-CaP and PLA-CaP-BMP2 groups (n = 5) after experimental periods. Horizontal bars represent statistical differences between same group at different experimental periods and its respective p values. (a) represents significant statistical differences compared to PLA group. (b) represents significant statistical differences compared to PLA-CaP group (ANOVA and Tukey post-test, p < 0.05).