The Healing of Bone Defects by Cell-Free and Stem Cell-Seeded 3D Printed PLA Tissue Engineered Scaffolds

One of the main issues in bone tissue engineering is to realize the response of the host to the engineered scaffolds. In this paper, the in-vivo healing of critical-sized bony defects by cell-free and stem cell-seeded 3D printed PLA scaffolds was studied in rat calvaria bone. First, the scaffolds were 3D printed based on a designed computer model and half of them were seeded by with bone marrow-derived mesenchymal stem cells (BMSCs). The SEM images of the surfaces of PLA and PLA+Cell scaffolds were taken for morphological analysis. All the scaffolds were implanted in the defect sites of rat calvaria bones and histological analysis was conducted after 8 and 12 weeks. The results showed that both cell-free and stem cell-seeded scaffolds exhibited superb healing compared with the empty defect controls. The histological observation revealed the formation of both new bone and connective tissues in the healing site after 8 and 12 weeks, postoperatively. The bone cells including osteoblasts and osteocytes with lacuna were also observed. The higher filled area and the higher bone formation and bone maturation were observed after 12 weeks and in particular for PLA+Cell scaffolds. Furthermore, the systemic toxicity evaluation of the scaffolds using ALT and AST tests reject any toxicity for both cell-free and stem cell-seeded scaffolds. It can be concluded that the 3D printed PLA scaffold with BMSCs seeding has well osteogenic potential to be used for bone defect healing.

Osteoconductive Properties of a Volume-Stable Collagen Matrix in Rat Calvaria Defects: A Pilot Study

Volume-stable collagen matrices (VSCM) are conductive for the connective tissue upon soft tissue augmentation. Considering that collagen has osteoconductive properties, we have investigated the possibility that the VSCM also consolidates with the newly formed bone. To this end, we covered nine rat calvaria circular defects with a VSCM. After four weeks, histology, histomorphometry, quantitative backscattered electron imaging, and microcomputed tomography were performed. We report that the overall pattern of mineralization inside the VSCM was heterogeneous. Histology revealed, apart from the characteristic woven bone formation, areas of round-shaped hypertrophic chondrocyte-like cells surrounded by a mineralized extracellular matrix. Quantitative backscattered electron imaging confirmed the heterogenous mineralization occurring within the VSCM. Histomorphometry found new bone to be 0.7 mm2 (0.01 min; 2.4 max), similar to the chondrogenic mineralized extracellular matrix with 0.7 mm2 (0.0 min; 4.2 max). Microcomputed tomography showed the overall mineralized tissue in the defect to be 1.6 mm3 (min 0.0; max 13.3). These findings suggest that in a rat cranial defect, VSCM has a limited and heterogeneous capacity to support intramembranous bone formation but may allow the formation of bone via the endochondral route.

Repair of Critical Size Bone Defects Using Synthetic Hydroxyapatite or Xenograft with or without the Bone Marrow Mononuclear Fraction: A Histomorphometric and Immunohistochemical Study in Rat Calvaria

Bone defects are a challenging clinical situation, and the development of hydroxyapatite-based biomaterials is a prolific research field that, in addition, can be joined by stem cells and growth factors in order to deal with the problem. This study compares the use of synthetic hydroxyapatite and xenograft, used pure or enriched with bone marrow mononuclear fraction for the regeneration of critical size bone defects in rat calvaria through histomorphometric (Masson’s staining) and immunohistochemical (anti-VEGF, anti-osteopontin) analysis. Forty young adult male rats were divided into five groups (n = 8). Animals were submitted to critical size bone defects (Ø = 8 mm) in the temporoparietal region. In the control group, there was no biomaterial placement in the critical bone defects; in group 1, it was filled with synthetic hydroxyapatite; in group 2, it was filled with xenograft; in group 3, it was filled with synthetic hydroxyapatite, enriched with bone marrow mononuclear fraction (BMMF), and in group 4 it was filled with xenograft, enriched with BMMF. After eight weeks, all groups were euthanized, and histological section images were captured and analyzed. Data analysis showed that in groups 1, 2, 3 and 4 (received biomaterials and biomaterials plus BMMF), a significant enhancement in new bone matrix formation was observed in relation to the control group. However, BMMF-enriched groups did not differ from hydroxyapatite-based biomaterials-only groups. Therefore, in this experimental model, BMMF did not enhance hydroxyapatite-based biomaterials’ potential to induce bone matrix and related mediators.

Bone Regeneration Assessment of Polycaprolactone Membrane on Critical-Size Defects in Rat Calvaria

Biomaterials for use in guided bone regeneration (GBR) are constantly being investigated and developed to improve clinical outcomes. The present study aimed to comparatively evaluate the biological performance of different membranes during the bone healing process of 8 mm critical defects in rat calvaria in order to assess their influence on the quality of the newly formed bone. Seventy-two adult male rats were divided into three experimental groups (n = 24) based on the membranes used: the CG—membrane-free control group (only blood clot, negative control), BG—porcine collagen membrane group (Bio-Guide®, positive control), and the PCL—polycaprolactone (enriched with 5% hydroxyapatite) membrane group (experimental group). Histological and histometric analyses were performed at 7, 15, 30, and 60 days postoperatively. The quantitative data were analyzed by two-way ANOVA and Tukey’s test (p < 0.05). At 7 and 15 days, the inflammatory responses in the BG and PCL groups were significantly different (p < 0.05). The PCL group, at 15 days, showed a large area of newly formed bone. At 30 and 60 days postoperatively, the PCL and BG groups exhibited similar bone healing, including some specimens showing complete closure of the critical defect (p = 0.799). Thus, the PCL membrane was biocompatible, and has the potential to help with GBR procedures.

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

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.