Correction of large jawbone defect in the mouse using immature osteoblast-like cells and a 3D polylactic acid scaffold.

Bone tissue engineering has been developed using a combination of mesenchymal stem cells (MSCs) and calcium phosphate-based scaffolds. However, these complexes cannot regenerate large jawbone defects. To overcome this limitation of MSCs and ceramic scaffolds, a novel bone regeneration technology must be developed using cells possessing high bone forming ability and a scaffold that provides space for vertical bone augmentation. To approach this problem in our study, we developed alveolar bone-derived immature osteoblast-like cells (HAOBs), which have the bone regenerative capacity to correct a large bone defect when used as a grafting material in combination with polylactic acid fibers that organize the 3D structure and increase the strength of the scaffold material (3DPL). HAOB-3DPL constructs could not regenerate bone via xenogeneic transplantation in a micromini pig alveolar bone defect model. However, the autogenic transplantation of mouse calvaria-derived immature osteoblast-like cells (MCOBs) isolated using the identical protocol for HAOBs and mixed with 3DPL scaffolds successfully regenerated the bone in a large jawbone defect mouse model, compared to the 3DPL scaffold alone. Nanoindentation analysis indicated that the regenerated bone had a similar micromechanical strength to native bone. In addition, this MCOB-3DPL regenerated bone possesses osseointegration ability wherein a direct structural connection is established with the titanium implant surface. Hence, a complex formed between a 3DPL scaffold and immature osteoblast-like cells such as MCOBs represents a novel bone tissue engineering approach that enables the formation of vertical bone with the micromechanical properties required to treat large bone defects.

Alkali treatment facilitates functional nano-hydroxyapatite coating of 3D printed polylactic acid scaffolds.

Autografting is currently the gold standard for treatment of bone defects, but has shown disadvantages in the limited volume of and donor site morbidity associated with harvested bone. Customized bone scaffolds that mimic the mechanical and biological properties of native bone are needed to augment the currently limited bone regeneration strategies. To achieve this goal, a repeated cross-hatch structure with uniform cubic pores was designed and 3D printed using polylactic acid (PLA) via fused deposition modeling (FDM). PLA surfaces were modified by wet chemical (alkali) treatment for either 1 h (1hAT) or 6 h (6hAT), followed by coating with nano-hydroxyapatite (nHA). Our hypotheses were that: (i) 6-hour (but not 1-hour) alkali treatment would enhance nHA coating, (ii) the nHA coating on the 6-hour alkali-treated surface would increase hydrophilicity and cell attachment/proliferation, and (iii) stiffness, but not effective Young's modulus, would be reduced by 6-hour alkali treatment. The effects of AT and nHA coating on scaffold morphology was observed by scanning electron microscopy and quantified using a custom MATLAB script. Chemical composition and hydrophilicity were evaluated via energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy, and water contact angle analyses, respectively. Mechanical testing and in vitro cell culture were further employed to analyze compressive properties, and cell attachment and proliferation, respectively. As expected, 6hAT led to reduced strut width and stiffness, while improving the nHA coating and hydrophilicity. Interestingly, PLA/6hAT but not PLA/6hAT/nHA demonstrated a reduction in effective modulus compared to PLA and PLA/nHA scaffolds. From in vitro experiments, the combined PLA/6hAT/nHA modification resulted in the greatest extent of cell attachment but not proliferation. These results collectively demonstrate that the PLA/6hAT/nHA scaffold exhibits properties that may prove beneficial for cancellous bone regeneration.

Mineral-Doped Poly(L-lactide) Acid Scaffolds Enriched with Exosomes Improve Osteogenic Commitment of Human Adipose-Derived Mesenchymal Stem Cells.

Exosomes derived from mesenchymal stem cells are extracellular vesicles released to facilitate cell communication and function. Recently, polylactic acid (PLA), calcium silicates (CaSi), and dicalcium phosphate dihydrate (DCPD) have been used to produce bioresorbable functional mineral-doped porous scaffolds-through thermally induced phase separation technique, as materials for bone regeneration. The aim of this study was to investigate the effect of mineral-doped PLA-based porous scaffolds enriched with exosome vesicles (EVs) on osteogenic commitment of human adipose mesenchymal stem cells (hAD-MSCs). Two different mineral-doped scaffolds were produced: PLA-10CaSi-10DCPD and PLA-5CaSi-5DCPD. Scaffolds surface micromorphology was investigated by ESEM-EDX before and after 28 days immersion in simulated body fluid (HBSS). Exosomes were deposited on the surface of the scaffolds and the effect of exosome-enriched scaffolds on osteogenic commitment of hAD-MSCs cultured in proximity of the scaffolds has been evaluated by real time PCR. In addition, the biocompatibility was evaluated by direct-contact seeding hAD-MSCs on scaffolds surface-using MTT viability test. In both formulations, ESEM showed pores similar in shape (circular and elliptic) and size (from 10-30 µm diameter). The porosity of the scaffolds decreased after 28 days immersion in simulated body fluid. Mineral-doped scaffolds showed a dynamic surface and created a suitable bone-forming microenvironment. The presence of the mineral fillers increased the osteogenic commitment of hAD-MSCs. Exosomes were easily entrapped on the surface of the scaffolds and their presence improved gene expression of major markers of osteogenesis such as collagen type I, osteopontin, osteonectin, osteocalcin. The experimental scaffolds enriched with exosomes, in particular PLA-10CaSi-10DCPD, increased the osteogenic commitment of MSCs. In conclusion, the enrichment of bioresorbable functional scaffolds with exosomes is confirmed as a potential strategy to improve bone regeneration procedures.