PhD Theses (Biosciences and Bioengineering)

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Browsing PhD Theses (Biosciences and Bioengineering) by Subject "3D Bioprinting"

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    Biomimetic Silk-Bioceramic Based Composites for Bone Tissue Engineering Applications
    (2022) Moses, Joseph Christakiran
    Bone, a structurally and functionally important organ is hierarchically built through bottom-up approach using organic - collagen biopolymer and inorganic - apatite bioceramic. Composited into a biomineralized tissue, bone is considered to be nature’s most robust, nanoassembled biological structures contributing to its strength and fracture toughness. Though the bone possess s innate healing abilities, clinical interventions are necessitated for bone defect repair during pathological or degenerative conditions. In this thesis, few of the major long bone defects which arise due to degeneration, infection or trauma were identified. Biomaterials-based regenerative strategies were explored in addressing these issues through five objectives which are progressively presented in this thesis. Inspired by bone’s nano-architecture, different fabrication strategies were investigated towards recreating biomimetic, cell instructive composites using resorbable and bioactive biomaterials. Silk fibroin was chosen as the bioactive biopolymer, sourced from mulberry (Bombyx mori) or non-mulberry (Antheraea assama) silk types, for development of composites along with sol-gel derived bioactive glass or wet chemical synthesized apatites (as the ceramic constituents). In the first objective, electrospun silk-bioactive glass mats were investigated as prospective bilayered grafts for osteochondral lesion management. The hierarchically structured composite electrospun mats helped in preserving the chondrogenic and osteogenic phenotype of seeded chondrocytes and osteoblasts preferentially due to the innate physicochemical cues presented by the biomimetic mats. In the second objective, functionalised silk microfiberreinforced freeze-dried composite silk sponges were investigated as resorbable bone grafts for volumetric bone defect management. The copper doped bioactive glass used for functionalising the microfibers, helped attribute proangiogenic traits to the scaffold, which aided in restoration of volumetric defects in rabbit femur with total resorption of implants noticed after 3 months. In the third objective, advanced additive manufacturing technique was investigated to develop 3D bioprinted cellularized osteochondral grafts, thereby circumventing drawbacks associated with conventional acellular grafts. Similarly, in the fourth objective, diaphyseal cross-sectional unit was biofabricated with an outer mechanically robust bioprinted cortical bone shell, encompassing an engineered bone marrow towards serving as orthobiologic substitute for atrophic non-union repairs. In the fifth objective, to improve implant patency of metal implants used as fixtures or prosthesis in orthopaedic reconstruction, silk-bioactive glass nanocomposites were used to create multifunctional interface on these metal surfaces. Conformal coatings of these mesoporous nanocomposites enabled in releasing antibiotics or glucocorticoids towards preventing implant associated infection and improving osseointegration of these metal implants. Thus, the conventional and additive manufacturing strategies demonstrated in this thesis helped develop pro-regenerative, cell instructive matrices in different length scales, to suit the clinical need of the investigated bone pathology. These matrices were functionally validated under both in vitro and preclinical in vivo conditions. Thus, the positive findings from this work hold promise for the viable clinical translation of these interventions for bone tissue engineering applications.
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    Development of Silk Based Matrices for Cartilage and Osteochondral Tissue Engineering
    (2021) Singh, Yogendra Pratap
    The osteochondral tissue of the knee joint is a multi-tissue organ composed of articular cartilage, subchondral bone, and the synovial membrane, with effective functioning dependent on sustained joint homeostasis. The articular cartilage is incapable of self-healing and affects entire osteochondral tissue when damaged. In the absence of intervention, it ultimately leads to Osteoarthritis (OA) and limits the functioning of the entire joint. The extant non-availability of a cure for OA mandates the use of external regenerative strategies to repair the damaged osteochondral tissue. The current thesis explored silk fibroin as a biomaterial-based regenerative strategy, sourced from mulberry (Bombyx mori) and non-mulberry/wild (Antheraea assamensis, Antheraea mylitta, and Philosamia ricini) silk types. The subsequent fabrication of matrices for cartilage and osteochondral tissue engineering was achieved through conventional or manual fabrication methods such as freeze-drying. The developed agarose and silk fibroin blended hydrogel nurtured the positive facets of the agarose gold standard and offset the negative aspects. It resulted in hydrogels that were biodegradable and immunocompatible, with the ability to support extracellular matrix (ECM) synthesis. The subsequent requirement of mechanical compliance, crucial in load-bearing articular joints, was achieved through fiber-reinforced composite scaffolds. The fiber-reinforced scaffolds supported improved growth of chondrocytes with an increased compressive modulus and stiffness (nearly 8-fold), in comparison to the fiber-free control groups. Since the entire osteochondral unit is implicated in OA, a biphasic silk scaffold was developed that mimicked the native osteochondral joint. It contained a spongy fiber-free phase for cartilage, a fiber-reinforced phase for bone revival, and a connecting interface, fabricated using a facile reproducible process. The developed hierarchically structured biphasic silk scaffold displayed phase-specific porous structures, with suitable mechanical strength, and positive characteristics of in vitro ECM deposition, and in vivo regeneration (in rabbit) of osteochondral tissue. Efforts were subsequently directed towards advanced additive manufacturing techniques such as 3D bioprinting. The self-gelling ability of silk fibroin blends (B. mori and P. ricini) was used along with gelatin as a bulking agent to develop the bioink, to encapsulate chondrocytes for cartilage bioprinting. The bioprinted constructs demonstrated in vitro cartilage-specific ECM formation and in vivo biocompatibility. The developed bioink showed good print fidelity for bioprinting cartilage grids along with state-of-art anatomical structures like the human ear. Disease model development was central to the final objective of understanding the molecular mechanisms of OA initiation and progression. An inflamed in vitro model was generated by 3D bioprinting an osteochondral unit using primed stem cells encapsulated silk-based bioink and cytokine-induced pathological conditions. The model was validated using antiinflammatory drugs viz. Rhein and Celecoxib. It successfully mimicked the inflamed OA unit observed in the early stages of OA, and displayed the mitigative effects of the applied drugs, consequently, catering to the demand for a robust, high-throughput platform for screening novel anti-inflammatory drugs towards OA therapeutics. Therefore, the current thesis explored and developed novel silk biomaterial-based matrices and bioinks for cartilage and osteochondral repair and regeneration; and validated their pre-clinical functionality both in vitro and in vivo.