Bilayered extracellular matrix derived scaffolds with anisotropic pore architecture guide tissue organization during osteochondral defect repair.

While some clinical advances in cartilage repair have occurred, osteochondral (OC) defect repair remains a significant challenge, with current scaffold-based approaches failing to recapitulate the complex, hierarchical structure of native articular cartilage (AC). To address this need, we fabricated bilayered extracellular matrix (ECM)-derived scaffolds with aligned pore architectures. By modifying the freeze-drying kinetics and controlling the direction of heat transfer during freezing, it was possible to produce anisotropic scaffolds with larger pores which supported homogenous cellular infiltration and improved sulfated glycosaminoglycan deposition. Neo-tissue organization in vitro could also be controlled by altering scaffold pore architecture, with collagen fibres aligning parallel to the long-axis of the pores within scaffolds containing aligned pore networks. Furthermore, we used in vitro and in vivo assays to demonstrate that AC and bone ECM derived scaffolds could preferentially direct the differentiation of mesenchymal stromal cells (MSCs) towards either a chondrogenic or osteogenic lineage respectively, enabling the development of bilayered ECM scaffolds capable of spatially supporting unique tissue phenotypes. Finally, we implanted these scaffolds into a large animal model of OC defect repair. After 6 months in vivo, scaffold implantation was found to improve cartilage matrix deposition, with collagen fibres preferentially aligning parallel to the long axis of the scaffold pores, resulting in a repair tissue that structurally and compositionally was more hyaline-like in nature. These results demonstrate how scaffold architecture and composition can be spatially modulated to direct the regeneration of complex interfaces such as the osteochondral unit, enabling their use as cell-free, off-the-shelf implants for joint regeneration. STATEMENT OF SIGNIFICANCE: The architecture of the extracellular matrix, while integral to tissue function, is often neglected in the design and evaluation of regenerative biomaterials. In this study we developed a bilayered scaffold for osteochondral defect repair consisting of tissue-specific extracellular matrix (ECM)-derived biomaterials to spatially direct stem/progenitor cell differentiation, with a tailored pore microarchitecture to promote the development of a repair tissue that recapitulates the hierarchical structure of native AC. The use of this bilayered scaffold resulted in improved tissue repair outcomes in a large animal model, specifically the ability to guide neo-tissue organization and therefore recapitulate key aspects of the zonal structure of native articular cartilage. These bilayer scaffolds have the potential to become a new therapeutic option for osteochondral defect repair.

Drug delivery to the pediatric upper airway.

Pediatric upper airway disorders are frequently life-threatening and require precise assessment and intervention. Targeting these pathologies remains a challenge for clinicians due to the high complexity of pediatric upper airway anatomy and numerous potential etiologies; the most common treatments include systemic delivery of high dose steroids and antibiotics or complex and invasive surgeries. Furthermore, the majority of innovative airway management technologies are only designed and tested for adults, limiting their widespread implementation in the pediatric population. Here, we provide a comprehensive review of the most recent challenges of managing common pediatric upper airway disorders, describe the limitations of current clinical treatments, and elaborate on how to circumvent those limitations via local controlled drug delivery. Furthermore, we propose future advancements in the field of drug-eluting technologies to improve pediatric upper airway management outcomes.

Reinforcing interpenetrating network hydrogels with 3D printed polymer networks to engineer cartilage mimetic composites.

Engineering constructs that mimic the complex structure, composition and biomechanics of the articular cartilage represents a promising route to joint regeneration. Such tissue engineering strategies require the development of biomaterials that mimic the mechanical properties of articular cartilage whilst simultaneously providing an environment supportive of chondrogenesis. Here three-dimensional (3D) bioprinting is used to develop polycaprolactone (PCL) fibre networks to mechanically reinforce interpenetrating network (IPN) hydrogels consisting of alginate and gelatin methacryloyl (GelMA). Inspired by the significant tension-compression nonlinearity of the collagen network in articular cartilage, we printed reinforcing PCL networks with different ratios of tensile to compressive modulus. Synergistic increases in compressive modulus were observed when IPN hydrogels were reinforced with PCL networks that were relatively soft in compression and stiff in tension. The resulting composites possessed equilibrium and dynamic mechanical properties that matched or approached that of native articular cartilage. Finite Element (FE) modelling revealed that the reinforcement of IPN hydrogels with specific PCL networks limited radial expansion and increased the hydrostatic pressure generated within the IPN upon the application of compressive loading. Next, multiple-tool biofabrication techniques were used to 3D bioprint PCL reinforced IPN hydrogels laden with a co-culture of bone marrow-derived stromal cells (BMSCs) and chondrocytes (CCs). The bioprinted biomimetic composites were found to support robust chondrogenesis, with encapsulated cells producing hyaline-like cartilage that stained strongly for sGAG and type II collagen deposition, and negatively for type X collagen and calcium deposition. Taken together, these results demonstrate how 3D bioprinting can be used to engineer constructs that are both pro-chondrogenic and biomimetic of the mechanical properties of articular cartilage.

Integrating finite element modelling and 3D printing to engineer biomimetic polymeric scaffolds for tissue engineering.

The suitability of a scaffold for tissue engineering is determined by a number of interrelated factors. The biomaterial should be biocompatible and cell instructive, with a porosity and pore interconnectivity that facilitates cellular migration and the transport of nutrients and waste products into and out of the scaffolds. For the engineering of load bearing tissues, the scaffold may also be required to possess specific mechanical properties and/or ensure the transfer of mechanical stimuli to cells to direct their differentiation. Achieving these design goals is challenging, but could potentially be realised by integrating computational tools such as finite element (FE) modelling with three-dimensional (3D) printing techniques to assess how scaffold architecture and material properties influence the performance of the implant. In this study we first use Fused Deposition Modelling (FDM) to modulate the architecture of polycaprolactone (PCL) scaffolds, exploring the influence of varying fibre diameter, spacing and laydown pattern on the structural and mechanical properties of such scaffolds. We next demonstrate that a simple FE modelling strategy, which captures key aspects of the printed scaffold's actual geometry and material behaviour, can be used to accurately model the mechanical characteristics of such scaffolds. We then show the utility of this strategy by using FE modelling to help design 3D printed scaffolds with mechanical properties mimicking that of articular cartilage. In conclusion, this study demonstrates that a relatively simple FE modelling approach can be used to inform the design of 3D printed scaffolds to ensure their bulk mechanical properties mimic specific target tissues.

Electrospinning of highly porous yet mechanically functional microfibrillar scaffolds at the human scale for ligament and tendon tissue engineering.

Electrospun fibers offer tremendous potential for tendon and ligament tissue engineering, yet developing porous scaffolds mimicking the size, stiffness and strength of human tissues remains a challenge. Previous studies have rolled, braided, or stacked electrospun sheets to produce three-dimensional (3D) scaffolds with tailored sizes and mechanical properties. A common limitation with such approaches is the development of low porosity scaffolds that impede cellular infiltration into the body of the implant, thereby limiting their regenerative potential. Here, we demonstrate how varying the rotational speed of the collecting mandrel during the electrospinning of poly(ε-caprolactone) (PCL) can be used to limit inter-fiber fusion (or fiber welding). Increasing the fraction of unfused fibers reduced the flexural rigidity of the electrospun sheets, which in turn allowed us to bundle the fibers into 3D scaffolds with similar dimensions to the human anterior cruciate ligament (ACL). These unfused fibers allowed for higher levels of porosity (up to 95%) that facilitated the rapid migration of mesenchymal stem cells (MSCs) into the body of the scaffolds. Mechanical testing demonstrated that the fiber-bundles possessed a Young's modulus approaching that of the native human ACL. The scaffolds were also capable of supporting the differentiation of MSCs towards either the fibrocartilage or ligament/tendon lineage. This novel electrospinning strategy could be used to produce mechanically functional, yet porous, scaffolds for a wide range of biomedical applications.

Fiber Reinforced Cartilage ECM Functionalized Bioinks for Functional Cartilage Tissue Engineering.

Focal articular cartilage (AC) defects, if left untreated, can lead to debilitating diseases such as osteoarthritis. While several tissue engineering strategies have been developed to promote cartilage regeneration, it is still challenging to generate functional AC capable of sustaining high load-bearing environments. Here, a new class of cartilage extracellular matrix (cECM)-functionalized alginate bioink is developed for the bioprinting of cartilaginous tissues. The bioinks are 3D-printable, support mesenchymal stem cell (MSC) viability postprinting and robust chondrogenesis in vitro, with the highest levels of COLLII and ACAN expression observed in bioinks containing the highest concentration of cECM. Enhanced chondrogenesis in cECM-functionalized bioinks is also associated with progression along an endochondral-like pathway, as evident by increases in RUNX2 expression and calcium deposition in vitro. The bioinks loaded with MSCs and TGF-ß3 are also found capable of supporting robust chondrogenesis, opening the possibility of using such bioinks for direct "print-and-implant" cartilage repair strategies. Finally, it is demonstrated that networks of 3D-printed polycaprolactone fibers with compressive modulus comparable to native AC can be used to mechanically reinforce these bioinks, with no loss in cell viability. It is envisioned that combinations of such biomaterials can be used in multiple-tool biofabrication strategies for the bioprinting of biomimetic cartilaginous implants.

Regeneration of Osteochondral Defects Using Developmentally Inspired Cartilaginous Templates.

IMPACT STATEMENT: Successfully treating osteochondral defects involves regenerating both the damaged articular cartilage and the underlying subchondral bone, in addition to the complex interface that separates these tissues. In this study, we demonstrate that a cartilage template, engineered using bone marrow-derived mesenchymal stem cells, can enhance the regeneration of such defects and promote the development of a more mechanically functional repair tissue. We also use a computational mechanobiological model to understand how joint-specific environmental factors, specifically oxygen levels and tissue strains, regulate the conversion of the engineered template into cartilage and bone in vivo.