Microfluidic systems for modeling human development.

The proper development and patterning of organs rely on concerted signaling events emanating from intracellular and extracellular molecular and biophysical cues. The ability to model and understand how these microenvironmental factors contribute to cell fate decisions and physiological processes is crucial for uncovering the biology and mechanisms of life. Recent advances in microfluidic systems have provided novel tools and strategies for studying aspects of human tissue and organ development in ways that have previously been challenging to explore ex vivo. Here, we discuss how microfluidic systems and organs-on-chips provide new ways to understand how extracellular signals affect cell differentiation, how cells interact with each other, and how different tissues and organs are formed for specialized functions. We also highlight key advancements in the field that are contributing to a broad understanding of human embryogenesis, organogenesis and physiology. We conclude by summarizing the key advantages of using dynamic microfluidic or microphysiological platforms to study intricate developmental processes that cannot be accurately modeled by using traditional tissue culture vessels. We also suggest some exciting prospects and potential future applications of these emerging technologies.

Intra-Operative Bioprinting of Hard, Soft, and Hard/Soft Composite Tissues for Craniomaxillofacial Reconstruction.

Reconstruction of complex craniomaxillofacial (CMF) defects is challenging due to the highly organized layering of multiple tissue types. Such compartmentalization necessitates the precise and effective use of cells and other biologics to recapitulate the native tissue anatomy. In this study, intra-operative bioprinting (IOB) of different CMF tissues, including bone, skin, and composite (hard/soft) tissues, is demonstrated directly on rats in a surgical setting. A novel extrudable osteogenic hard tissue ink is introduced, which induced substantial bone regeneration, with ≈80% bone coverage area of calvarial defects in 6 weeks. Using droplet-based bioprinting, the soft tissue ink accelerated the reconstruction of full-thickness skin defects and facilitated up to 60% wound closure in 6 days. Most importantly, the use of a hybrid IOB approach is unveiled to reconstitute hard/soft composite tissues in a stratified arrangement with controlled spatial bioink deposition conforming the shape of a new composite defect model, which resulted in ≈80% skin wound closure in 10 days and 50% bone coverage area at Week 6. The presented approach will be absolutely unique in the clinical realm of CMF defects and will have a significant impact on translating bioprinting technologies into the clinic in the future.

The Role of Concentration on Drop Formation and Breakup of Collagen, Fibrinogen, and Thrombin Solutions during Inkjet Bioprinting.

The influence of protein concentration on drop formation and breakup of aqueous solutions of fibrous proteins collagen and fibrinogen and globular protein thrombin in different concentration regimes has been investigated during drop-on-demand (DOD) inkjet printing. The capillary-driven thinning and breakup of dilute collagen, fibrinogen, and thrombin solutions, the solutions in which protein molecules are far away from each other, are predominantly resisted by inertial force. Although the capillary-driven thinning and breakup of semidilute unentangled collagen and fibrinogen solutions, the solutions in which protein molecules begin to interpenetrate each other, are predominantly resisted by inertial force on the initial onset of necking, the breakup of droplets is delayed because of the resistance of elastic force. The resistance of viscous force to the necking and breakup of both the dilute and semidilute unentangled protein solutions is negligible. Aggregates or subvisible particles (between 1 and 100 µm) constantly disrupt the formation of droplets for the semidilute unentangled protein solutions, even when their inverse Ohnesorge number (Z) is within the printability range of 4 ≤ Z ≤ 14. Although aggregates are present in the dilute protein solutions, they do not disrupt the formation of droplets.

Rheological investigation of collagen, fibrinogen, and thrombin solutions for drop-on-demand 3D bioprinting.

Collagen, fibrinogen, and thrombin proteins in aqueous buffer solutions are widely used as precursors of natural biopolymers in three-dimensional (3D) bioprinting applications. The proteins are sourced from animals and their quality may vary from batch to batch, inducing differences in the rheological properties of such solutions. In this work, we investigate the rheological response of collagen, fibrinogen, and thrombin protein solutions in bulk and at the solution/air interface. Interfacial rheological measurements show that fibrous collagen, fibrinogen and globular thrombin proteins adsorb and aggregate at the solution/air interface, forming a viscoelastic solid film at the interface. The viscoelastic film corrupts the bulk rheological measurements in rotational rheometers by contributing to an apparent yield stress, which increases the apparent bulk viscosity up to shear rates as high as 1000 s-1. The addition of a non-ionic surfactant, such as polysorbate 80 (PS80) in small amounts between 0.001 and 0.1 v/v%, prevents the formation of the interfacial layer, allowing the estimation of true bulk viscosity of the solutions. The estimation of viscosity not only helps in identifying those protein solutions that are potentially printable with drop-on-demand (DOD) inkjet printing but also detects inconsistencies in flow behavior among the batches.

3D bioprinting-a model for skin aging.

Human lifespan continues to extend as an unprecedented number of people reach their seventh and eighth decades of life, unveiling chronic conditions that affect the older adult. Age-related skin conditions include senile purpura, seborrheic keratoses, pemphigus vulgaris, bullous pemphigoid, diabetic foot wounds and skin cancer. Current methods of drug testing prior to clinical trials require the use of pre-clinical animal models, which are often unable to adequately replicate human skin response. Therefore, a reliable model for aged human skin is needed. The current challenges in developing an aged human skin model include the intrinsic variability in skin architecture from person to person. An ideal skin model would incorporate innate functionality such as sensation, vascularization and regeneration. The advent of 3D bioprinting allows us to create human skin equivalent for use as clinical-grade surgical graft, for drug testing and other needs. In this review, we describe the process of human skin aging and outline the steps to create an aged skin model with 3D bioprinting using skin cells (i.e. keratinocytes, fibroblasts and melanocytes). We also provide an overview of current bioprinted skin models, associated limitations and direction for future research.

Porous tissue strands: avascular building blocks for scalable tissue fabrication.

The scalability of cell aggregates such as spheroids, strands, and rings has been restricted by diffusion of nutrient and oxygen into their core. In this study, we introduce a novel concept in generating tissue building blocks with micropores, which represents an alternative solution for vascularization. Sodium alginate porogens were mixed with human adipose-derived stem cells, and loaded into tubular alginate capsules, followed by de-crosslinking of the capsules. The resultant cellular structure exhibited a porous morphology and formed cell aggregates in the form of strands, called 'porous tissue strands (pTSs).' Three-dimensional reconstructions show that pTSs were able to maintain ∼25% porosity with a high pore interconnectivity (∼85%) for 3 weeks. Owing to the porous structure, pTSs showed up-regulated cell viability and proliferation rate as compared to solid counterparts throughout the culture period. pTSs also demonstrated self-assembly capability through tissue fusion yielding larger-scale patches. In this paper, chondrogenesis and osteogenesis of pTSs were also demonstrated, where the porous microstructure up-regulated both chondrogenic and osteogenic functionalities indicated by cartilage- and bone-specific immunostaining, quantitative biochemical assessment and gene expression. These findings indicated the functionality of pTSs, which possessed controllable porosity and self-assembly capability, and had great potential to be utilized as tissue building blocks in distinct applications such as cartilage and bone regeneration.

Bioprinting of osteochondral tissues: A perspective on current gaps and future trends.

Osteochondral tissue regeneration has remained a critical challenge in orthopaedic surgery, especially due to complications of arthritic degeneration arising out of mechanical dislocations of joints. The common gold standard of autografting has several limitations in presenting tissue engineering strategies to solve the unmet clinical need. However, due to the complexity of joint anatomy, and tissue heterogeneity at the interface, the conventional tissue engineering strategies have certain limitations. The advent of bioprinting has now provided new opportunities for osteochondral tissue engineering. Bioprinting can uniquely mimic the heterogeneous cellular composition and anisotropic extra-cellular matrix (ECM) organization, while allowing for targeted gene delivery to achieve heterotypic differentiation. In this perspective, we discuss the current advances made towards bioprinting of composite osteochondral tissues and present an account of challenges-in terms of tissue integration, long-term survival, and mechanical strength at the time of implantation-required to be addressed for effective clinical translation of bioprinted tissues. Finally, we highlight some of the future trends related to osteochondral bioprinting with the hope of in-clinical translation.

A comprehensive review on droplet-based bioprinting: Past, present and future.

Droplet-based bioprinting (DBB) offers greater advantages due to its simplicity and agility with precise control on deposition of biologics including cells, growth factors, genes, drugs and biomaterials, and has been a prominent technology in the bioprinting community. Due to its immense versatility, DBB technology has been adopted by various application areas, including but not limited to, tissue engineering and regenerative medicine, transplantation and clinics, pharmaceutics and high-throughput screening, and cancer research. Despite the great benefits, the technology currently faces several challenges such as a narrow range of available bioink materials, bioprinting-induced cell damage at substantial levels, limited mechanical and structural integrity of bioprinted constructs, and restrictions on the size of constructs due to lack of vascularization and porosity. This paper presents a first-time review of DBB and comprehensively covers the existing DBB modalities including inkjet, electrohydrodynamic, acoustic, and micro-valve bioprinting. The recent notable studies are highlighted, the relevant bioink biomaterials and bioprinters are expounded, the application areas are presented, and the future prospects are provided to the reader.

Alginate gelation-induced cell death during laser-assisted cell printing.

Modified laser-induced forward transfer has emerged as a promising bioprinting technique. Depending on the operating conditions and cell properties, laser cell printing may cause cell injury and even death, which should be carefully elucidated for it to be a viable technology. This study has investigated the effects of alginate gelation, gelation time, alginate concentration, and laser fluence on the post-transfer cell viability of NIH 3T3 fibroblasts. Sodium alginate and calcium chloride are used as the gel precursor and gel reactant solution to form cell-laden alginate microspheres. It is found that the effects of gelation depend on the duration of gelation. Two-minute gelation is observed to increase the cell viability after 24 h incubation, mainly due to the protective cushion effect of the forming gel membrane during droplet landing. Despite the cushion effect from 10 min gelation, it is observed that the cell viability decreases after 24 h incubation because of the forming thick gel membrane that reduces nutrient and oxygen diffusion from the culture medium. In addition, the cell viability after 24 h incubation decreases as the laser fluence or alginate concentration increases.