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As an attractive biomaterial for bone reconstruction, injectable biomaterials have many prominent characteristics such as good biocompatibility and bone-filling ability. However, there are weak as load-bearing scaffolds. In this study, polyvinyl alcohol (PVA) and bioactive glass (BAG) were interpenetrated into sodium alginate (SA) network to obtain self-enhanced injectable hydrogel. The optimum ratio of PVA/SA/BAG hydrogel was determined based on injectability, gelation time and chemical characterization. Results showed that the selected ratio had the shortest gelation time of 3.5min, and the hydrogel had a rough surface and good coagulation property. The hydrogel was capable of carrying 1kg of weight by mineralization for 14 d The compressive strength, compressive modulus, and fracture energy of the hydrogel reached 0.12MPa, 0.376MPa and 17.750kJ m-2, respectively. Meanwhile, the hydrogel had high moisture content and dissolution rate, and it was sensitive to temperature and ionic strength. Hydroxyapatite was generated on the hydrogel surface, and the hydrogel pores increased, and the pore size enlarged. The biocompatibility of PVA/SA/BAG hydrogel was analyzed using hemolysis and cytotoxicity assays. Results revealed its good biocompatibility with low hemolysis rate and no cytotoxicity to MC3T3-E1 cells. The hydrogel was also found to promote the differentiation of MC3T3-E1 cells with significantly increased in ALP activity and expression of relevant differentiation factors. In vitro mineralization assay showed an increase in calcium nodules and calcification area, indicating the ability of hydrogel to promote mineralization MC3T3-E1 cells. These findings indicated that PVA/SA/BAG hydrogel had potential uses in the field of irregular bone-defect repair due to its injectability, cytocompatibility, and tailorable functionality.

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The utilization of guided tissue regeneration membranes is a significant approach for enhancing bone tissue growth in areas with bone defects. Biodegradable magnesium alloys are increasingly being used as guided tissue regeneration membranes due to their outstanding osteogenic properties. However, the degradation rates of magnesium alloy bone implants documented in the literature tend to be rapid. Moreover, many studies focus only on the initial 3-month period post-implantation, limiting their applicability and impeding clinical adoption. Furthermore, scant attention has been given to the interplay between the degradation of magnesium alloy implants and the adjacent tissues. To address these gaps, this study employs a well-studied magnesium-aluminum (Mg-Al) alloy membrane with a slow degradation rate. This membrane is implanted into rat skull bone defects and monitored over an extended period of up to 48 weeks. Observations are conducted at various intervals (2, 4, 8, 12, 24, and 48 weeks) following the implantation. Assessment of degradation behavior and tissue regeneration response is carried out using histological sections, micro-CT scans, and scanning electron microscopy (SEM). The findings reveal that the magnesium alloy membranes demonstrate remarkable biocompatibility and osteogenic capability over the entire observation duration. Specifically, the Mg-Al alloy membranes sustain their structural integrity for 8 weeks. Notably, their osteogenic ability is further enhanced as a corrosion product layer forms during the later stages of implantation. Additionally, our in vitro experiments employing extracts from the magnesium alloy display a significant osteogenic effect, accompanied by a notable increase in the expression of osteogenic-related genes. Collectively, these results strongly indicate the substantial potential of Mg-Al alloy membranes in the context of guided tissue regeneration.

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Tomographic volumetric bioprinting (VBP) has recently emerged as a powerful tool for rapid solidification of cell-laden hydrogel constructs within seconds. However, its practical applications in tissue engineering requires a detailed understanding of how different printing parameters (concentration of resins, laser dose) affect cell activity and tissue formation. Herein, we explore a new application of VBP in bone tissue engineering by merging a soft gelatin methacryloyl (GelMA) bioresin (<5 kPa) with 3D endothelial co-culture to generate heterocellular bone-like constructs with enhanced functionality. To this, a series of bioresins with varying concentrations of GelMA and lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP) photoinitiator were formulated and characterized in terms of photo-reactivity, printability and cell-compatibility. A bioresin with 5% GelMA and 0.05% LAP was identified as the optimal formulation for VBP of complex perfusable constructs within 30 s at high cell viability (>90%). The fidelity was validated by micro-computed tomography and confocal microscopy. Compared to 10% GelMA, this bioresin provided a softer and more permissive environment for osteogenic differentiation of human mesenchymal stem cells (hMSCs). The expression of osteoblastic markers (collagen-I, ALP, osteocalcin) and osteocytic markers (podoplanin, Dmp1) was monitored for 42 days. After 21 days, early osteocytic markers were significantly increased in 3D co-cultures of hMSCs with human umbilical vein endothelial cells (HUVECs). Additionally, we demonstrate VBP of a perfusable, pre-vascularized model where HUVECs self-organized into an endothelium-lined channel. Altogether, this work leverages the benefits of VBP and 3D co-culture, offering a promising platform for fast scaled biofabrication of 3D bone-like tissues with unprecedented functionality. STATEMENT OF SIGNIFICANCE: This study explores new strategies for ultrafast bio-manufacturing of bone tissue models by leveraging the advantages of tomographic volumetric bioprinting (VBP) and endothelial co-culture. After screening the properties of a series of photocurable gelatin methacryloyl (GelMA) bioresins, a formulation with 5% GelMA was identified with optimal printability and permissiveness for osteogenic differentiation of human mesenchymal stem cells (hMSC). We then established 3D endothelial co-cultures to test if the heterocellular interactions may enhance the osteogenic differentiation in the printed environments. This hypothesis was evidenced by increased gene expression of early osteocytic markers in 3D co-cultures after 21 days. Finally, VBP of a perfusable cell-laden tissue construct is demonstrated for future applications in vascularized tissue engineering.

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Cells with osteogenic potential are believed to be an ideal source for bone tissue bioengineering. Large bone defects require temporary substitution of the damaged parts. In this respect, the transplantation of bone cells cultured on osteogenic substrates has been investigated. To use the natural bone matrix, one approach is the so-called demineralized bone matrix (DBM). In this study, we evaluated the interaction of human fetal osteoblasts (hFOB 1.19 cells, a human fetal osteoblastic cell line) with DBM fragments. No additional bone differentiation inducer was used other than the DBM itself. The samples were processed, had adhesion pattern evaluated and analyzed by light microscopy (cytochemical and immunocytochemical analysis) and electron microscopy (scanning and transmission). The adhesion pattern of hFOB cells on DBM was similar to what was observed on the cell culture plate. Morphological analysis showed that the hFOB cells had emitted filopodia and cellular projections on both controls and DBM. On DBM, the adhered cells emitted prolongations and migrated into the matrix. The monolayer growth pattern was observed as well as the accumulation of filamentous and reticulate extracellular materials when hFOB cells were cultured on the DBM surface. EDS analysis revealed the deposition of calcium on DBM. Immunocytochemical data showed that the hFOB cells were able to secrete extracellular matrix molecules such as fibronectin and laminin on DBM. Our data indicate that DBM successfully stimulates the osteoblastic phenotype of osteoblast-like cells and corroborate with the fact that DBM is a considerable natural matrix that promotes fractured-bone healing.

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BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

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The field of bone tissue engineering has seen the advancement of a variety of biomaterials with a diverse range of material properties. Biomaterial properties such as particle shape and size, stiffness, and pore size all influence the osteogenic capacity of biomaterials, typically evaluated in vitro by analyzing their potential to promote osteogenesis in mesenchymal stem cells (MSCs). There is now accumulating evidence highlighting the role of macrophages in driving bone regeneration responses. In this study, we evaluated the osteogenic capacity of collagen scaffolds functionalized with hydroxyapatite particles of varying shapes (needle vs spherical) and sizes (5 µm vs 100 µm) using an in vitro culture system of MSCs alone and in coculture with macrophages. We show that macrophage response to HA particles was elevated in the presence of a scaffold with 5 µm needle-shaped particles (Coll N5), with an increase in the expression and secretion of both pro-inflammatory (TNFα, IL6, and MIP1α) and anti-inflammatory (IL10 and IL1Ra) factors. When MSCs alone were cultured on the scaffolds, we show that scaffolds with HA particles were highly osteogenic, with superior osteogenesis observed in scaffolds with large 30 µm spherical particles (Coll S30) compared to small 5 µm needle-shaped particles (Coll N5). A coculture of MSCs with macrophages increased osteogenesis in all groups, with the most dramatic increase on Coll N5 scaffolds, leading to an elimination of the differences observed during monoculture. Through gene expression analysis, we showed that this correlated with an enhanced pro-osteogenic macrophage phenotype on Coll N5 scaffolds. These results highlight the potential of modulating material properties such as particle shape and size to develop osteoimmunomodulatory materials that direct osteogenic responses by influencing macrophage response.

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There is currently an interest in "active" implantable biomedical devices that include mechanical stimulation as an integral part of their design. This paper reports the experimental use of a porous scaffold made of interconnected networks of slender ferromagnetic fibers that can be actuated in vivo by an external magnetic field applying strains to in-growing cells. Such scaffolds have been previously characterized in terms of their mechanical and cellular responses. In this study, it is shown that the shape changes induced in the scaffolds can be used to promote osteogenesis in vitro. In particular, immunofluorescence, gene and protein analyses reveal that the actuated networks exhibit higher mineralization and extracellular matrix production, and express higher levels of osteocalcin, alkaline phosphatase, collagen type 1α1, runt-related transcription factor 2 and bone morphogenetic protein 2 than the static controls at the 3-week time point. The results suggest that the cells filling the inter-fiber spaces are able to sense and react to the magneto-mechanically induced strains facilitating osteogenic differentiation and maturation. This work provides evidence in support of using this approach to stimulate bone ingrowth around a device implanted in bone and can pave the way for further applications in bone tissue engineering.

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The rapid development and application of nanotechnology to biological interfaces has impacted the bone implant field, allowing researchers to finely modulate the interface between biomaterials and recipient tissues. In the present study, oxidative anodization was exploited to generate two alumina surfaces with different pore diameters. The former displayed surface pores in the mean range of 16-30 nm, while in the latter pores varied from to 65 to 89 nm. The samples were characterized by Field Emission Scanning Electron Microscopy (FESEM) and Energy Dispersive X-ray spectroscopy (EDX) analysis prior to being tested with pre-osteoblastic MC3T3-E1 cells. In vitro cell response was studied in terms of early cell adhesion, viability, and morphology, including focal adhesion quantification. Both the alumina samples promoted higher cell adhesion and viability than the control condition represented by the standard culture dish plastic. Osteogenic differentiation was assessed through alkaline phosphatase activity and extracellular calcium deposition, and it was found that of the two nano-surfaces, one was more efficient than the other. By comparing for the first time two nano-porous alumina surfaces with different pore diameters, our data supported the role of nano-topography in inducing cell response. Modulating a simple aspect of surface texture may become an attractive route for guiding bone healing and regeneration around implantable metals.

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Limitations associated to the use of growth factors represent a major hurdle to musculoskeletal regeneration. On the one hand, they are needed to induce neo-tissue formation for the substitution of a necrotic or missing tissue. On the other hand, these factors are used in supraphysiological concentrations, are short lived and expensive and result in many side effects. Here we develop a gene transfer strategy based on the use of chemically modified mRNA (cmRNA) coding for human bone morphogenetic protein 2 (hBMP-2) that is non-immunogenic and highly stable when compared to unmodified mRNA. Transfected stem cells secrete hBMP-2, show elevated alkaline phosphatase levels and upregulated expression of RunX2, ALP, Osterix, Osteocalcin, Osteopontin and Collagen Type I genes. Mineralization was induced as seen by positive Alizarin red staining. hBMP-2 cmRNA transfected human fat tissue also yielded an osteogenic response in vitro as indicated by expression of hBMP-2, RunX2, ALP and Collagen Type I. Delivering hBMP-2 cmRNA to a femur defect in a rat model results in new bone tissue formation as early as 2 weeks after application of very low doses. Overall, our studies demonstrate the feasibility and therapeutic potential of a new cmRNA-based gene therapy strategy that is safe and efficient. When applied clinically, this approach could overcome BMP-2 growth factor associated limitations in bone regeneration.

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Poly(epsilon-caprolactone) (PCL) is a hydrophobic bioplastic under development for bone tissue engineering applications. Limited information is available on the role of internal geometry and cell-surface attachment on osseous integration potential. We tested the hypothesis that human bone marrow mesenchymal stem cells (MSCs) deposit more mineral inside porous 3D PCL scaffolds with fully interconnected 84 or 141 µm pores, when the surfaces are coated with chitosan via Layer-by-Layer (LbL)-deposited polyelectrolytes. Freshly trypsinized MSCs were seeded on PCL 3D cylinders using a novel static cold seeding method in 2% serum to optimally populate all depths of the scaffold discs, followed by 10 days of culture in proliferation medium and 21 additional days in osteogenic medium. MSCs were observed by SEM and histology to spread faster and to proliferate more on chitosan-coated pore surfaces. Most pores, with or without chitosan, became filled by collagen networks sparsely populated with fibroblast-like cells. After 21 days of culture in osteogenic medium, sporadic matrix mineralization was detected histologically and by micro-CT in highly cellular surface layers that enveloped all scaffolds and in cell aggregates in 141 µm pores near the edges. LbL-chitosan promoted punctate mineral deposition on the surfaces of 84 µm pores (p < 0.05 vs. PCL-only) but not the 141 µm pores. This study revealed that LbL-chitosan coatings are sufficient to promote MSC attachment to PCL but only enhance mineral formation in 84 µm pores, suggesting a potential inhibitory role for MSC-derived fibroblasts in osteoblast terminal differentiation.

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