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(1) Objectives: The effect of cell-processing protocols on the clinical efficacy of bone tissue engineering is not well-known. To maximize efficacy, we optimized the cell-processing protocol for bone-marrow-derived mesenchymal stromal cells for bone tissue engineering. In this study, the efficacy of bone tissue engineering using this modified protocol was compared to that of the original protocol. (2) Materials and Methods: This single-arm clinical study included 15 patients. Cells were obtained from bone marrow aspirates and expanded in culture flasks containing basic fibroblast growth factor. The cells were seeded onto β-tricalcium phosphate granules and induced into osteogenic cells for two weeks. Then, the cell–scaffold composites were transplanted into patients with severe atrophic alveolar bone. Radiographic evaluations and bone biopsies were performed. The results were compared with those of a previous clinical study that used the original protocol. (3) Results: Panoramic X-ray and computed tomography showed bone regeneration at the transplantation site in all cases. The average bone area in the biopsy samples at 4 months was 44.0%, which was comparable to that in a previous clinical study at 6 months (41.9%) but with much less deviation. No side effects related to cell transplantation were observed. In regenerated bone, 100% of the implants were integrated. (4) Conclusions: Compared to the original protocol, the non-inferiority of this protocol was proven. The introduction of an optimized cell-processing protocol resulted in a comparable quality of regenerated bone, with less fluctuation. Optimized cell-processing protocols may contribute to stable bone regeneration.

Delivering and retaining cells in areas of interest is an ongoing challenge in tissue engineering. Here we introduce a novel approach to fabricate osteoblast-loaded titanium suitable for cell delivery for bone integration, regeneration, and engineering. We hypothesized that titanium age influences the efficiency of protein adsorption and cell loading onto titanium surfaces. Fresh (newly machined) and 1-month-old (aged) commercial grade 4 titanium disks were prepared. Fresh titanium surfaces were hydrophilic, whereas aged surfaces were hydrophobic. Twice the amount of type 1 collagen and fibronectin adsorbed to fresh titanium surfaces than aged titanium surfaces after a short incubation period of three hours, and 2.5-times more fibronectin than collagen adsorbed regardless of titanium age. Rat bone marrow-derived osteoblasts were incubated on protein-adsorbed titanium surfaces for three hours, and osteoblast loading was most efficient on fresh titanium adsorbed with fibronectin. The number of osteoblasts loaded using this synergy between fresh titanium and fibronectin was nine times greater than that on aged titanium with no protein adsorption. The loaded cells were confirmed to be firmly attached and functional. The number of loaded cells was strongly correlated with the amount of protein adsorbed regardless of the protein type, with fibronectin simply more efficiently adsorbed on titanium surfaces than collagen. The role of surface hydrophilicity of fresh titanium surfaces in increasing protein adsorption or cell loading was unclear. The hydrophilicity of protein-adsorbed titanium increased with the amount of protein but was not the primary determinant of cell loading. In conclusion, the osteoblast loading efficiency was dependent on the age of the titanium and the amount of protein adsorption. In addition, the efficiency of protein adsorption was specific to the protein, with fibronectin being much more efficient than collagen. This is a novel strategy to effectively deliver osteoblasts ex vivo and in vivo using titanium as a vehicle.

This study aimed to induce bone-like tissue from immature muscular tissue (IMT) in vitro using commercially available recombinant human bone morphogenetic protein (rhBMP)-2, rhBMP-4, and rhBMP-7, and then implanting this tissue into a calvarial defect in rats to assess healing. IMTs were extracted from 20-day-old Sprague-Dawley (SD) fetal rats, placed on expanded polytetrafluoroethylene (ePTFE) with 10 ng/μL each of rhBMP-2, BMP-4, and BMP-7, and cultured for two weeks. The specimens were implanted into calvarial defects in 3-week-old SD rats for up to three weeks. Relatively strong radiopacity was observed on micro-CT two weeks after culture, and bone-like tissue, comprising osteoblastic cells and osteoids, was partially observed by H&E staining. Calcium, phosphorus, and oxygen were detected in the extracellular matrix using an electron probe micro analyzer, and X-ray diffraction patterns and Fourier transform infrared spectroscopy spectra of the specimen were found to have typical apatite crystal peaks and spectra, respectively. Furthermore, partial strong radiopacity and ossification were confirmed one week after implantation, and a dominant novel bone was observed after two weeks in the defect site. Thus, rhBMP-2, BMP-4, and BMP-7 differentiated IMT into bone-like tissue in vitro, and this induced bone-like tissue has ossification potential and promotes the healing of calvarial defects. Our results suggest that IMT is an effective tissue source for bone tissue engineering.

Large bone defects with limited intrinsic regenerative potential represent a major surgical challenge and are associated with a high socio-economic burden and severe reduction in the quality of life. Tissue engineering approaches offer the possibility to induce new functional bone regeneration, with the biomimetic scaffold serving as a bridge to create a microenvironment that enables a regenerative niche at the site of damage. Magnetic nanoparticles have emerged as a potential tool in bone tissue engineering that leverages the inherent magnetism of magnetic nano particles in cellular microenvironments providing direction in enhancing the osteoinductive, osteoconductive and angiogenic properties in the design of scaffolds. There are conflicting opinions and reports on the role of MNPs on these scaffolds, such as the true role of magnetism, the application of external magnetic fields in combination with MNPs, remote delivery of biomechanical stimuli in-vivo and magnetically controlled cell retention or bioactive agent delivery in promoting osteogenesis and angiogenesis. In this review, we focus on the role of magnetic nanoparticles for bone-tissue-engineering applications in both disease modelling and treatment of injuries and disease. We highlight the materials-design pathway from implementation strategy through the selection of materials and fabrication methods to evaluation. We discuss the advances in this field and unmet needs, current challenges in the development of ideal materials for bone-tissue regeneration and emerging strategies in the field.

Chitosan is a deacetylated polysaccharide from chitin, the natural biopolymer primarily found in shells of marine crustaceans and fungi cell walls. Upon deacetylation, the protonation of free amino groups of the d-glucosamine residues of chitosan turns it into a polycation, which can easily interact with DNA, proteins, lipids, or negatively charged synthetic polymers. This positive-charged characteristic of chitosan not only increases its solubility, biodegradability, and biocompatibility, but also directly contributes to the muco-adhesion, hemostasis, and antimicrobial properties of chitosan. Combined with its low-cost and economic nature, chitosan has been extensively studied and widely used in biopharmaceutical and biomedical applications for several decades. In this review, we summarize the current chitosan-based applications for bone and dental engineering. Combining chitosan-based scaffolds with other nature or synthetic polymers and biomaterials induces their mechanical properties and bioactivities, as well as promoting osteogenesis. Incorporating the bioactive molecules into these biocomposite scaffolds accelerates new bone regeneration and enhances neovascularization in vivo.

Bone regeneration after injury or after surgical bone removal due to disease is a serious medical challenge. A variety of materials are being tested to replace a missing bone or tooth. Regeneration requires cells capable of proliferation and differentiation in bone tissue. Although there are many possible human cell types available for use as a model for each phase of this process, no cell type is ideal for each phase. Osteosarcoma cells are preferred for initial adhesion assays due to their easy cultivation and fast proliferation, but they are not suitable for subsequent differentiation testing due to their cancer origin and genetic differences from normal bone tissue. Mesenchymal stem cells are more suitable for biocompatibility testing, because they mimic natural conditions in healthy bone, but they proliferate more slowly, soon undergo senescence, and some subpopulations may exhibit weak osteodifferentiation. Primary human osteoblasts provide relevant results in evaluating the effect of biomaterials on cellular activity; however, their resources are limited for the same reasons, like for mesenchymal stem cells. This review article provides an overview of cell models for biocompatibility testing of materials used in bone tissue research.

Pure titanium is used in dental implants owing to its excellent biocompatibility and physical properties. However, the aging of the material during storage is detrimental to the long-term stability of the implant after implantation. Therefore, in this study, we attempted to improve the surface properties and circumvent the negative effects of material aging on titanium implants by using a portable handheld nonthermal plasma device capable of piezoelectric direct discharge to treat pure titanium discs with nitrogen gas. We evaluated the osteogenic properties of the treated samples by surface morphology and elemental analyses, as well as in vitro and in vivo experiments. The results showed that nonthermal atmospheric-pressure nitrogen plasma can improve the hydrophilicity of pure titanium without damaging its surface morphology while introducing nitrogen-containing functional groups, thereby promoting cell attachment, proliferation, and osseointegration to some extent. Therefore, nitrogen plasma treatment may be a promising method for the rapid surface treatment of titanium implants.

The craniofacial area is prone to trauma or pathologies often resulting in large bone damages. One potential treatment option is the grafting of a tissue‐engineered construct seeded with adult mesenchymal stem cells (MSCs). The dental pulp appears as a relevant source of MSCs, as dental pulp stem cells display strong osteogenic properties and are efficient at bone formation and repair. Fibroblast growth factor‐2 (FGF‐2) and/or hypoxia primings were shown to boost the angiogenesis potential of dental pulp stem cells from human exfoliated deciduous teeth (SHED). Based on these findings, we hypothesized here that these primings would also improve bone formation in the context of craniofacial bone repair. We found that both hypoxic and FGF‐2 primings enhanced SHED proliferation and osteogenic differentiation into plastically compressed collagen hydrogels, with a much stronger effect observed with the FGF‐2 priming. After implantation in immunodeficient mice, the tissue‐engineered constructs seeded with FGF‐2 primed SHED mediated faster intramembranous bone formation into critical size calvarial defects than the other groups (no priming and hypoxia priming). The results of this study highlight the interest of FGF‐2 priming in tissue engineering for craniofacial bone repair. Stem Cells Translational Medicine 2019;8:844&857 Priming stem cells from human exfoliated deciduous teeth with fibroblast growth factor‐2 within plastically compressed collagen hydrogels improves osteoformation in vivo in the context of craniofacial bone repair with a faster intramembranous bone formation into calvarial bone critical size defects when compared with either of the 2 other groups (no priming and hypoxia priming) that produced smaller amounts of new bone.

Bone tissue engineering is an alternative therapeutic intervention to repair or regenerate lost bone. This technique requires three essential components: stem cells that can differentiate into bone cells, growth factors that stimulate cell behavior for bone formation, and scaffolds that mimic the extracellular matrix. Among the various kinds of scaffolds, highly porous nanofibrous scaffolds are a potential candidate for supporting cell functions, such as adhesion, delivering growth factors, and forming new tissue. Various fabricating techniques for nanofibrous scaffolds have been investigated, including electrospinning, multi-axial electrospinning, and melt writing electrospinning. Although electrospun fiber fabrication has been possible for a decade, these fibers have gained attention in tissue regeneration owing to the possibility of further modifications of their chemical, biological, and mechanical properties. Recent reports suggest that post-modification after spinning make it possible to modify a nanofiber’s chemical and physical characteristics for regenerating specific target tissues. The objectives of this review are to describe the details of recently developed fabrication and post-modification techniques and discuss the advanced applications and impact of the integrated system of nanofiber-based scaffolds in the field of bone tissue engineering. This review highlights the importance of nanofibrous scaffolds for bone tissue engineering.

Bone regeneration repairs bone tissue lost due to trauma, fractures, and tumors, or absent due to congenital disorders. The extracellular matrix (ECM) is an intricate dynamic bio-environment with precisely regulated mechanical and biochemical properties. In bone, ECMs are involved in regulating cell adhesion, proliferation, and responses to growth factors, differentiation, and ultimately, the functional characteristics of the mature bone. Bone ECM can induce the production of new bone by osteoblast-lineage cells, such as MSCs, osteoblasts, and osteocytes and the absorption of bone by osteoclasts. With the rapid development of bone regenerative medicine, the osteoinductive, osteoconductive, and osteogenic potential of ECM-based scaffolds has attracted increasing attention. ECM-based scaffolds for bone tissue engineering can be divided into two types, that is, ECM-modified biomaterial scaffold and decellularized ECM scaffold. Tissue engineering strategies that utilize the functional ECM are superior at guiding the formation of specific tissues at the implantation site. In this review, we provide an overview of the function of various types of bone ECMs in bone tissue and their regulation roles in the behaviors of osteoblast-lineage cells and osteoclasts. We also summarize the application of bone ECM in bone repair and regeneration. A better understanding of the role of bone ECM in guiding cellular behavior and tissue function is essential for its future applications in bone repair and regenerative medicine.