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.
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.
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