To accurately mimic the native tissue environment, tissue engineered scaffolds often need to have a highly controlled and varied display of three-dimensional (3D) architecture and geometrical cues. Additive manufacturing in tissue engineering has made possible the development of complex scaffolds that mimic the native tissue architectures. As such, architectural details that were previously unattainable or irreproducible can now be incorporated in an ordered and organized approach, further advancing the structural and chemical cues delivered to cells interacting with the scaffold. This control over the environment has given engineers the ability to unlock cellular machinery that is highly dependent upon the intricate heterogeneous environment of native tissue. Recent research into the incorporation of physical and chemical gradients within scaffolds indicates that integrating these features improves the function of a tissue engineered construct. This review covers recent advances on techniques to incorporate gradients into polymer scaffolds through additive manufacturing and evaluate the success of these techniques. As covered here, to best replicate different tissue types, one must be cognizant of the vastly different types of manufacturing techniques available to create these gradient scaffolds. We review the various types of additive manufacturing techniques that can be leveraged to fabricate scaffolds with heterogeneous properties and discuss methods to successfully characterize them.

Synthetic hydrogels are investigated extensively in tissue engineering for their tunable physicochemical properties but are bioinert and lack the tissue-specific cues to produce appropriate biological responses. To introduce tissue-specific biochemical cues to these hydrogels, we have developed a modular hydrogel cross-linker, poly(glycolic acid)–poly(ethylene glycol)–poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT), that can be functionalized with small peptide-based cues and large macromolecular cues simply by mixing PdBT in water with the appropriate biomolecules at room temperature. Cartilage- and bone-specific PdBT macromers were generated by functionalization with a cartilage-associated hydrophobic N-cadherin peptide, a hydrophilic bone morphogenetic protein peptide, and a cartilage-derived glycosaminoglycan, chondroitin sulfate. These biofunctionalized PdBT macromers can spontaneously cross-link polymers such as poly(N-isopropylacrylamide) to produce rapidly cross-linking, highly swollen, cytocompatible, and hydrolytically degradable hydrogels suitable for mesenchymal stem cell encapsulation. These favorable properties, combined with PdBT’s modular design and ease of functionalization, establish strong potential for its usage in tissue engineering applications.

Although the etiology of ALS remains poorly understood, impaired proteostasis is a common feature of different forms of ALS. Mutations in Ubiquilins, UBQLN2 and UBQLN4, cause familial ALS. The role of UBQLNs in proteasomal degradation is well established but their role in autophagy-lysosomal clearance is poorly defined. Here, we describe a crosstalk between ER stress, mTOR signaling, and autophagic flux in Drosophila and mammalian cells lacking Ubiquilins. We found that loss of Ubiquilins leads to ER stress, impairs mTORC1 activity, promotes autophagy, and causes the demise of neurons. We show that ubiquilin mutants display defective autophagic flux due to reduced lysosome acidification. Ubiquilins are required to maintain proper levels of V0a/V100 subunit of the v-ATPase and lysosomal pH. Feeding flies acidic nanoparticles alleviates defective autophagic flux in ubiquilin mutants. Hence, our studies reveal a conserved role for Ubiquilins as regulators of autophagy by controlling v-ATPase activity and mTOR signaling.

Although skeletal muscle has a high potential for self-repair, volumetric muscle loss can result in impairment beyond the endogenous regenerative capacity. There is a clinical need to improve on current clinical treatments that fail to fully restore the structure and function of lost muscle. Decellularized extracellular matrix (dECM) scaffolds have been an attractive platform for regenerating skeletal muscle, as dECM contains many biochemical cues that aid in cell adhesion, proliferation, and differentiation. However, there is limited capacity to tune physicochemical properties in current dECM technologies to improve outcome. In this study, we aim to create a novel, high-throughput technique to fabricate dECM scaffolds with tunable physicochemical properties while retaining proregenerative matrix components. We demonstrate a successful decellularization protocol that effectively removes DNA. We also identified key steps for the successful production of electrospun muscle dECM without the use of a carrier polymer; electrospinning allows for rapid scaffold fabrication with high control over material properties, which can be optimized to mimic native muscle. To this end, fiber orientation and degree of crosslinking of these dECM scaffolds were modulated and the corollary effects on fiber swelling, mechanical properties, and degradation kinetics were investigated. Beyond application in skeletal muscle, the versatility of this technology has the potential to serve as a foundation for dECM scaffold fabrication in a variety of tissue engineering applications.

Large mandibular defects are clinically challenging to reconstruct due to the complex anatomy of the jaw and the limited availability of appropriate tissue for repair. We envision leveraging current advances in fabrication and biomaterials to create implantable devices that generate bone within the patients themselves suitable for their own specific anatomical pathology. The in vivo bioreactor strategy facilitates the generation of large autologous vascularized bony tissue of customized geometry without the addition of exogenous growth factors or cells. To translate this technology, we investigated its success in reconstructing a mandibular defect of physiologically relevant size in sheep. We fabricated and implanted 3D-printed in vivo bioreactors against rib periosteum and utilized biomaterial-based space maintenance to preserve the native anatomical mandibular structure in the defect site before reconstruction. Nine weeks after bioreactor implantation, the ovine mandibles were repaired with the autologous bony tissue generated from the in vivo bioreactors. We evaluated tissues generated in bioreactors by radiographic, histological, mechanical, and biomolecular assays and repaired mandibles by radiographic and histological assays. Biomaterial-aided mandibular reconstruction was successful in a large superior marginal defect in five of six (83%) sheep. Given that these studies utilized clinically available biomaterials, such as bone cement and ceramic particles, this strategy is designed for rapid human translation to improve outcomes in patients with large mandibular defects.