Mineralization of Bone Extracellular Matrix-like Scaffolds Fabricated as Silk Sericin-Functionalized Dense Collagen-Fibrin Hybrid Hydrogels.

The design of hydrogels that combine both the biochemical cues needed to direct seeded cellular functions and mineralization to provide the structural and mechanical properties approaching those of mineralized native bone extracellular matrix (ECM) represents a significant challenge in bone tissue engineering. While fibrous hydrogels constituting of collagen or fibrin (and their hybrids) can be considered as scaffolds that mimic to some degree native bone ECM, their insufficient mechanical properties limit their application. In the present study, an automated gel aspiration-ejection (automated GAE) method was used to generate collagen-fibrin hybrid gel scaffolds with micro-architectures and mechanical properties approaching those of native bone ECM. Moreover, the functionalization of these hybrid scaffolds with negatively charged silk sericin accelerated their mineralization under acellular conditions in simulated body fluid and modulated the proliferation and osteoblastic differentiation of seeded MC3T3-E1 pre-osteoblastic cells. In the latter case, alkaline phosphatase activity measurements indicated that the hybrid gel scaffolds with seeded cells showed accelerated osteoblastic differentiation, which in turn led to increased matrix mineralization. In summary, the design of dense collagen-fibrin hybrid gels through an automated GAE process can provide a route to tailoring specific biochemical and mechanical properties to different types of bone ECM-like scaffolds, and can provide a model to better understand cell-matrix interactions in vitro for bioengineering purposes.

Effect of Fibrillization pH on Gelation Viscoelasticity and Properties of Biofabricated Dense Collagen Matrices via Gel Aspiration-Ejection.

Reconstituted hydrogels based on the self-assembly of acid-solubilized collagen molecules have been extensively used as in vitro models and precursors in biofabrication processes. This study investigated the effect of fibrillization pH-ranging from 4 to 11-on real-time rheological property changes during the gelation of collagen hydrogels and its interplay with the properties of subsequently biofabricated dense collagen matrices generated via automated gel aspiration-ejection (GAE). A contactless, nondestructive technique was used to characterize the temporal progression in shear storage modulus (G', or stiffness) during collagen gelation. There was a relative increase in G' of the hydrogels from 36 to 900 Pa with an increase in gelation pH. Automated GAE, which simultaneously imparts collagen fibrillar compaction and alignment, was then applied to these precursor collagen hydrogels to biofabricate native extracellular matrix-like densified gels. In line with viscoelastic properties, only hydrogels fibrillized in the 6.5 < pH ≤ 10 range could be densified via GAE. There was an increase in both fibrillar density and alignment in the GAE-derived matrices with an increase in gelation pH. These factors, combined with a higher G' in the alkaline precursor hydrogels, led to a significant increase in the micro-compressive modulus of GAE-densified gels of pH 9 and 10. Furthermore, NIH/3T3 fibroblast-seeded GAE-derived matrices densified from gels fibrillized in the pH range of 7 to 10 exhibited low cell mortality with >80% viability. It is anticipated that the results of this study can be potentially applicable to other hydrogel systems, as well as biofabrication techniques involving needles or nozzles, such as injection and bioprinting.

Effect of Menin Deletion in Early Osteoblast Lineage on the Mineralization of an In Vitro 3D Osteoid-like Dense Collagen Gel Matrix.

Bone has a complex microenvironment formed by an extracellular matrix (ECM) composed mainly of mineralized type I collagen fibres. Bone ECM regulates signaling pathways important in the differentiation of osteoblast-lineage cells, necessary for bone mineralization and in preserving tissue architecture. Compared to conventional 2D cell cultures, 3D in vitro models may better mimic bone ECM and provide an environment to support osteoblastic differentiation. In this study, a biomimetic 3D osteoid-like dense collagen gel model was used to investigate the role of the nuclear protein menin plays in osteoblastic differentiation and matrix mineralization. Previous in vitro and in vivo studies have shown that when expressed at later stages of osteoblastic differentiation, menin modulates osteoblastogenesis and regulates bone mass in adult mice. To investigate the role of menin when expressed at earlier stages of the osteoblastic lineage, conditional knockout mice in which the Men1 gene is specifically deleted early (i.e., at the level of the pluripotent mesenchymal stem cell lineage), where generated and primary calvarial osteoblasts were cultured in plastically compressed dense collagen gels for 21 days. The proliferation, morphology and differentiation of isolated seeded primary calvarial osteoblasts from knockout (Prx1-Cre; Men1f/f) mice were compared to those isolated from wild-type (Men1f/f) mice. Primary calvarial osteoblasts from knockout and wild-type mice did not show differences in terms of proliferation. However, in comparison to wild-type cells, primary osteoblast cells derived from knockout mice demonstrated deficient mineralization capabilities and an altered gene expression profile when cultured in 3D dense collagen gels. In summary, these findings indicate that when expressed at earlier stages of osteoblast differentiation, menin is important in maintaining matrix mineralization in 3D dense collagen gel matrices, in vitro.

Multiscale structural evolution of citrate-triggered intrafibrillar and interfibrillar mineralization in dense collagen gels.

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.

Bioinspired mineralization of a functionalized injectable dense collagen hydrogel through silk sericin incorporation.

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.

Functionalization of silk fibroin through anionic fibroin derived polypeptides.

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.