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Current challenges in the biofabrication of stable, cell-seeded fibrin gels are faced with a lack of microstructural and mechanical properties, limiting their use as bone extracellular matrix-mimicking constructs. While current approaches focus on generating fibrin gels of various fibrinogen and thrombin concentrations, i.e. 5–50 mg mL−1 and 1–250 units mL−1, respectively, processes that modulate gel fibrillar alignment and mechanical properties have yet to be explored. Herein, it was demonstrated that the automated gel aspiration-ejection (GAE) of precursor fibrin hydrogels of low concentration (i.e. 1 mg mL−1 fibrinogen and 0.5 units mL−1 thrombin) resulted in their immediate compaction while tailoring their fibrillar alignment and mechanical properties for potential target applications. Furthermore, this gel microenvironment accelerated the osteoblastic differentiation of seeded mesenchymal stem cells and their matrix mineralization, in vitro. Therefore, the ability to modulate the properties of dense fibrin constructs through automated GAE may be a novel biofabrication approach for cell delivery in bone regeneration.

There is an increasing need to generate novel materials for the treatment and augmentation of bone defects, affecting millions of people worldwide. Fibrillar type I collagen is the most abundant ti...

Collagen based hydrogels are frequently used as templates to mimic the native biomineralization process. However, a lack of structural control and their inherently poor mineralization capability represent challenges when used as bone-extracellular-matrix mimicking constructs. The aspiration-ejection of highly-hydrated collagen gels allows for their densification and fibrillar remodelling, leading to the production of injectable dense collagen (I-DC) gel scaffolds characterized by an osteoid-like structure. In this study, silk-extracted sericin (SS), a negatively-charged protein that is rich in anionic amino-acids such as Asp and Glu, was hybridized into I-DC gels to induce hydroxyapatite deposition and stimulate the osteoblastic differentiation of seeded mesenchymal stem cells (MSCs). The effect of SS content on the acellular mineralization of I-DC gels in simulated body fluid (SBF) and on modulating the proliferation and osteogenesis of seeded MSCs, in vitro, were investigated. Methylene blue staining indicated increasingly negatively charged gels through SS incorporation. Attributable to the carboxyl groups provided by the acidic SS amino-acids, serving as calcium-phosphate nucleation sites, there was a time dependent increase in hydroxyapatite deposition, approaching 90 wt% by day 14 in SBF. Three dimensionally seeded MSCs attached and proliferated in all gel types and SS-incorporation led to an increase in their metabolic activity. Relative to neat I-DC gels, alkaline phosphatase (at day 7), runt related transcription factor 2 (at day 21) and osteocalcin (at days 14 and 21) expression was higher in MSCs when seeded in SS-incorporated I-DC gels. Cell-induced mineralization was accelerated in SS-incorporated I-DC gels suggesting its osteostimulative potential. In sum, SS incorporation into clinically relevant I-DC gels can provide a strategy to design scaffolds with potential applications in bone tissue engineering.

The mineralized extracellular matrix of bone is an organic–inorganic nanocomposite consisting primarily of carbonated hydroxyapatite, fibrous type I collagen, noncollagenous proteins, proteoglycans, and diverse biomolecules such as pyrophosphate and citrate. While much is now known about the mineralization-regulating role of pyrophosphate, less is known about the function of citrate. In order to assess the effect of negatively charged citrate on collagen mineralization, citrate-functionalized, bone osteoid-mimicking dense collagen gels were exposed to simulated body fluid for up to 7 days to examine the multiscale evolution of intra- and interfibrillar collagen mineralization. Here, we show by increases in methylene blue staining that the net negative charge of collagen can be substantially augmented through citrate functionalization. Structural and compositional analyses by transmission and scanning electron microscopy (including X-ray microanalysis and electron diffraction), and atomic force microscopy, all demonstrated that citrate-functionalized collagen fibrils underwent extensive intrafibrillar mineralization within 12 h in simulated body fluid. Time-resolved, high-resolution transmission electron microscopy confirmed the temporal evolution of intrafibrillar mineralization of single collagen fibrils. Longer exposure to simulated body fluid resulted in additional interfibrillar mineralization, all through an amorphous-to-crystalline transformation towards apatite (assessed by X-ray diffraction and attenuated total reflection-Fourier-transform infrared spectroscopy). Calcium deposition assays indicated a citrate concentration-dependent temporal increase in mineralization, and micro-computed tomography confirmed that >80 vol% of the collagen in the gels was mineralized by day 7. In conclusion, citrate effectively induces mesoscale intra- and interfibrillar collagen mineralization, a finding that advances our understanding of the role of citrate in mineralized tissues.

Tissue engineering endeavours to repair and regenerate living tissue with compositions, structures, and functions comparable to native tissues. It involves the assembly of a tissue equivalent usually by combining cells and a porous three-dimensional (3D) scaffold. Therefore, developing 3D constructs with both the appropriate microarchitecture and microenvironment to surround and influence cells, and mimic natural tissues, is one approach to increasing the functionality of these scaffolds. In an effort to imitate features of the molecular and structural properties of extracellular matrices in native tissues, collagen has been widely used as a biomaterial in a range of different tissue engineering applications, including those related to bone and cartilage. The extracellular matrix (ECM) is typically a complex, cell-influencing environment that is composed not only of collagen fibrils but also contains other macromolecules such as adhesive and mineral-binding proteins, and proteoglycans (including glycosaminoglycans; GAGs). Chitosan, a natural polysaccharide that is comprised of N-acetylglucosamine and glucosamine units, structurally and compositionally resembles GAGs, and can be used as an ECM. This chapter explores the use of collagen and chitosan, and composites of the two, as tissue-equivalent scaffolds for the engineering of bone and cartilage.

While silk fibroin (SF)-based fibrous matrices are often considered as templates to mimic the native biomineralization process, their limited ability to induce apatite deposition hinders their potential applications in bone tissue engineering. In this study, it was hypothesized that the incorporation of anionic fibroin derived polypeptides (Cs), generated through the α-chymotrypsin digestion of SF, into SF would induce apatite deposition. The effect of Cs incorporation and content on the mineralization of fibrous, electrospun (ES) SF matrices, was assessed in simulated body fluid (SBF). Moreover, the potential role of Cs in mediating the proliferation and osteoblastic differentiation of seeded mesenchymal stem cells (MSCs), in vitro, was also investigated. Methylene blue staining indicated that the ES SF matrices became increasingly negatively charged with an increase in Cs content. Furthermore, the mechanical properties of the ES SF matrices were modulated through variations in Cs content. Their subsequent immersion in SBF demonstrated rapid mineralization, attributable to the carboxyl groups provided by the negatively charged Cs polypeptides, which served as nucleation sites for apatite deposition. Seeded MSCs attached on all scaffold types with differences observed in metabolic activities when cultured in osteogenic medium. Relative to basal medium, there was an up-regulation of alkaline phosphatase, runt related transcription factor 2 and osteocalcin in osteogenic medium (at days 14 and 21). Cell-induced mineralized matrix deposition appeared to be accelerated on Cs incorporated ES SF suggesting an osteoinductive potential of these polypeptides. In sum, the ability to incorporate Cs into SF scaffolds offers promise in bone tissue engineering applications.