Recapitulation of the form and function of complex tissue organization using appropriate biomaterials impacts success in tissue engineering endeavors. The annulus fibrosus (AF) represents a complex, multilamellar, hierarchical structure consisting of collagen, proteoglycans, and elastic fibers. To mimic the intricacy of AF anatomy, a silk protein-based multilayered, disc-like angle-ply construct was fabricated, consisting of concentric layers of lamellar sheets. Scanning electron microscopy and fluorescence image analysis revealed cross-aligned and lamellar characteristics of the construct, mimicking the native hierarchical architecture of the AF. Induction of secondary structure in the silk constructs was confirmed by infrared spectroscopy and X-ray diffraction. The constructs showed a compressive modulus of 499.18 ± 86.45 kPa. Constructs seeded with porcine AF cells and human mesenchymal stem cells (hMSCs) showed ∼2.2-fold and ∼1.7-fold increases in proliferation on day 14, respectively, compared with initial seeding. Biochemical analysis, histology, and immunohistochemistry results showed the deposition of AF-specific extracellular matrix (sulfated glycosaminoglycan and collagen type I), indicating a favorable environment for both cell types, which was further validated by the expression of AF tissue-specific genes. The constructs seeded with porcine AF cells showed ∼11-, ∼5.1-, and ∼6.7-fold increases in col I, sox 9, and aggrecan genes, respectively. The differentiation of hMSCs to AF-like tissue was evident from the enhanced expression of the AF-specific genes. Overall, the constructs supported cell proliferation, differentiation, and ECM deposition resulting in AF-like tissue features based on ECM deposition and morphology, indicating potential for future studies related to intervertebral disc replacement therapy.

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Hydrogels have received considerable attention in the field of tissue engineering because of their unique structural and compositional resemblance to the highly hydrated human tissues. In addition, controlled fabrication processes benefit them with desirable physicochemical features for injectability in minimally invasive manner and cell survival within hydrogels. Formulation of biologically active hydrogels with desirable characteristics is one of the prerequisites for successful applications like nucleus pulposus (NP) tissue engineering to address disc degeneration. To achieve such a benchmark, in this study, two naturally derived silk fibroin proteins (Bombyx mori, BM SF; and Antheraea assamensis, AA SF) were blended together to allow self-assembly and transformation to hydrogels in absence of any cross-linker or external stimuli. A comprehensive study on sol–gel transition of fabricated hydrogels in physiological fluid microenvironment (pH, temperature, and ionic strength) was conducted using optical and fluorescence analysis. Tunable gelation time (∼8–40 min) was achieved depending on combinations. The developed hydrogels were validated by extensive physicochemical characterizations which include confirmation of secondary structure, surface morphology, swelling and degradation. Mechanical behavior of the hydrogels was further analyzed in various in vitro-physiological-like conditions with varying pH, ionic strength, diameter, storage time, and strain values to determine their suitability in native physiological environments. Rheological study, cytocompatibility using primary porcine NP cells and ex vivo biomechanics of hydrogels were explored to validate their in situ applicability in minimally invasive manner toward potential disc regeneration therapy.

Development of a hierarchically analogous biphasic scaffold fabricated in a facile and minimalistic method for repair of osteochondral defect.

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An alkali metal ion assisted Michael addition reaction between acrylate and amine groups is strategically exploited in the synthesis of a chemically reactive and tailored hierarchical topography for addressing important fundamental aspects of biomimicked interfaces.

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The limited self-regenerative capacity of adult cartilage has steered the upsurge in tissue engineered replacements to combat the problem of osteoarthritis. In the present study, the potential of fiber-reinforced silk composites from mulberry (Bombyx mori) and non-mulberry (Antheraea assamensis) silk has been investigated for cartilage tissue engineering. The fabricated composites were physico-chemically characterized and analyzed for cellular viability, proliferation, extracellular matrix formation and immunocompatibility. Both mulberry and non-mulberry silk composites showed effective swelling (25%-30%) and degradation (10%-30%) behavior, owing to their interconnected porous nature. The non-mulberry fiber-reinforced composite scaffolds showed slower degradation (∼90% mass remaining) than mulberry silk over a period of 28 days. The reinforcement of silk fibers within silk solution resulted in an increased compressive modulus and stiffness (nearly eight-fold). The biochemical analysis revealed significant increase in DNA content, sulphated glycosaminoglycan (sGAG) (∼1.5 fold) and collagen (∼1.4 fold) in reinforced composites as compared to pure solution scaffolds (p ≤ 0.01). Histological and immunohistochemical (IHC) staining corroborated enhanced deposition of sGAG and localization of collagen type II in fiber-reinforced composites. This was further substantiated by real time polymerase chain reaction studies, which indicated an up-regulation (∼1.5 fold) of cartilage-specific gene markers namely collagen type II, sox-9 and aggrecan. The minimal secretion of tumor necrosis factor-α (TNF-α) by murine macrophages further demonstrated in vitro immunocompatibility of the scaffolds. Taken together, the results signified the potential of silk fiber-reinforced composite (particularly non-mulberry, A. assamensis) scaffolds as viable alternative biomaterial for cartilage tissue engineering.