[n]Cycloparaphenylenes as Compatible Fluorophores for Melt Electrowriting.

Fluorescent probes are an indispensable tool in the realm of bioimaging technologies, providing valuable insights into the assessment of biomaterial integrity and structural properties. However, incorporating fluorophores into scaffolds made from melt electrowriting (MEW) poses a challenge due to the sustained, elevated temperatures that this processing technique requires. In this context, [n]cycloparaphenylenes ([n]CPPs) serve as excellent fluorophores for MEW processing with the additional benefit of customizable emissions profiles with the same excitation wavelength. Three fluorescent blends are used with distinct [n]CPPs with emission wavelengths of either 466, 494, or 533 nm, identifying 0.01 wt% as the preferred concentration. It is discovered that [n]CPPs disperse well within poly(ε-caprolactone) (PCL) and maintain their fluorescence even after a week of continuous heating at 80 °C. The [n]CPP-PCL blends show no cytotoxicity and support counterstaining with commonly used DAPI (Ex/Em: 359 nm/457 nm), rhodamine- (Ex/Em: 542/565 nm), and fluorescein-tagged (Ex/Em: 490/515 nm) phalloidin stains. Using different color [n]CPP-PCL blends, different MEW fibers are sequentially deposited into a semi-woven scaffold and onto a solution electrospun membrane composed of [8]CPP-PCL as a contrasting substrate for the [10]CPP-PCL MEW fibers. In general, [n]CPPs are potent fluorophores for MEW, providing new imaging options for this technology.

Fundamentals and Applications of Photo-Cross-Linking in Bioprinting.

This review provides a detailed overview of the rapidly advancing field of biofabrication, particularly with regards to the use of photo-cross-linking (i.e., light-based) techniques. The major emphasis of this review is on the fundamentals of photo-cross-linking and key criteria identified for the successful design and implementation of photo-cross-linked bioinks and bioresins in extrusion-based and lithography-based bioprinting. The general mechanisms associated with photo-cross-linking (e.g., free-radical chain polymerization, thiol-ene, photomediated redox) of natural and synthetic materials are described to inform bioink and bioresin design, which includes the selection of polymers, functional group modifications, photoinitiators, and light sources that enable facile and cytocompatible photo-cross-linking. Depending on material selection and the bioprinting technique of interest, we describe the specific bioink or bioresin properties and criteria that must be achieved to ensure optimal printability and utility. Finally, examples of current state-of-the-art applications of light-based bioprinting for in vitro tissue models, tissue engineering, and regenerative medicine are provided to further motivate future opportunities within the bioprinting landscape that are facilitated with light.

Bioassembly of hemoglobin-loaded photopolymerizable spheroids alleviates hypoxia-induced cell death.

The delivery of oxygen within tissue engineered constructs is essential for cell survivability; however, achieving this within larger biofabricated constructs poses a significant challenge. Efforts to overcome this limitation often involve the delivery of synthetic oxygen generating compounds. The application of some of these compounds is problematic for the biofabrication of living tissues due to inherent issues such as cytotoxicity, hyperoxia and limited structural stability due to oxygen inhibition of radical-based crosslinking processes. This study aims to develop an oxygen delivering system relying on natural-derived components which are cytocompatible, allow for photopolymerization and advanced biofabrication processes, and improve cell survivability under hypoxia (1% O2). We explore the binding of human hemoglobin (Hb) as a natural oxygen deposit within photopolymerizable allylated gelatin (GelAGE) hydrogels through the spontaneous complex formation of Hb with negatively charged biomolecules (heparin, hyaluronic acid, and bovine serum albumin). We systematically study the effect of biomolecule inclusion on cytotoxicity, hydrogel network properties, Hb incorporation efficiency, oxygen carrying capacity, cell viability, and compatibility with 3D-bioassembly processes within melt electrowritten (MEW) scaffolds. All biomolecules were successfully incorporated within GelAGE hydrogels, displaying controllable mechanical properties and cytocompatibility. Results demonstrated efficient and tailorable Hb incorporation within GelAGE-Heparin hydrogels. The developed system was compatible with microfluidics and photopolymerization processes, allowing for the production of GelAGE-Heparin-Hb spheres. Hb-loaded spheres were assembled into MEW polycaprolactone scaffolds, significantly increasing the local oxygen levels. Ultimately, cells within Hb-loaded constructs demonstrated good cell survivability under hypoxia. Taken together, we successfully developed a hydrogel system that retains Hb as a natural oxygen deposit post-photopolymerization, protecting Hb from free-radical oxidation while remaining compatible with biofabrication of large constructs. The developed GelAGE-Heparin-Hb system allows for physoxic oxygen delivery and thus possesses a vast potential for use across broad tissue engineering and biofabrication strategies to help eliminate cell death due to hypoxia.

Droplet-based microfluidics for engineering shape-controlled hydrogels with stiffness gradient.

Current biofabrication strategies are limited in their ability to replicate native shape-to-function relationships, that are dependent on adequate biomimicry of shape, size and spatial heterogeneity, within cell-laden hydrogels. In this study, a diffusion-based microfluidics platform is presented that meets these needs in a two-step process. In the first step, a hydrogel-precursor solution is dispersed into a continuous oil phase within the microfluidics tubing. By adjusting the dispersed and oil phase flow rates, the physical architecture of hydrogel-precursor phases can be adjusted to generate spherical and plug-like structures, as well as continuous meter-long hydrogel-precursor phases (up to 1.75 m). The second step involves the controlled introduction a small molecule-containing aqueous phase through a T-shaped tube connector to enable controlled small molecule diffusion across the interface of the aqueous phase and hydrogel-precursor. Application of this system is demonstrated by diffusing co-initiator sodium persulfate (SPS) into hydrogel-precursor solutions, where the controlled SPS diffusion into the hydrogel-precursor and subsequent photo-polymerization allows for the formation of unique radial stiffness patterns across the shape- and size-controlled hydrogels, as well as allowing the formation of hollow hydrogels with controllable internal architectures. Mesenchymal stromal cells are successfully encapsulated within hollow hydrogels and hydrogels containing radial stiffness gradient. The cells are observed to respond to the microscale spatial heterogeneity as evidenced by increased cell elongation in softer core regions of the hydrogel as compared to the peripheral stiffer hydrogel regions, as well as stiffness-dependent nuclear accumulation of the yes-associated protein mechano-regulator. Finally, breast cancer cells are found to phenotypically switch in response to stiffness gradients, causing a shift in their ability to aggregate, which may have implications for metastasis. The diffusion-based microfluidics will mimic native shape-to-function relationship and provides a platform to further study the roles of micro- and macroscale architectural features that exist within native tissues.

Harnessing Macromolecular Chemistry to Design Hydrogel Micro- and Macro-Environments.

Cell encapsulation within three-dimensional hydrogels is a promising approach to mimic tissues. However, true biomimicry of the intricate microenvironment, biophysical and biochemical gradients, and the macroscale hierarchical spatial organizations of native tissues is an unmet challenge within tissue engineering. This review provides an overview of the macromolecular chemistries that have been applied toward the design of cell-friendly hydrogels, as well as their application toward controlling biophysical and biochemical bulk and gradient properties of the microenvironment. Furthermore, biofabrication technologies provide the opportunity to simultaneously replicate macroscale features of native tissues. Biofabrication strategies are reviewed in detail with a particular focus on the compatibility of these strategies with the current macromolecular toolkit described for hydrogel design and the challenges associated with their clinical translation. This review identifies that the convergence of the ever-expanding macromolecular toolkit and technological advancements within the field of biofabrication, along with an improved biological understanding, represents a promising strategy toward the successful tissue regeneration.

Hybrid fabrication of photo-clickable vascular hydrogels with additive manufactured titanium implants for enhanced osseointegration and vascularized bone formation.

Bone regeneration of critical-sized bone defects, bone fractures or joint replacements remains a significant clinical challenge. Although there has been rapid advancement in both the fields of bone tissue engineering and additive manufacturing, functional bone implants with rapid vascularization capacity to ensure osseointegration and long-term biological fixation in large bone defects remains limited in clinics. In this study, we developed anin vitrovascularized bone implant by combining cell-laden hydrogels with direct metal printed (DMP) porous titanium alloys (Ti-6Al-4V). A 5 wt% allylated gelatin (GelAGE), was utilized to co-encapsulate human mesenchymal stromal cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) to investigate concurrent osteogenic and vasculogenic performance. DMP macro-porous Ti-6Al-4V scaffolds were subsequently infused/enriched with cell-laden GelAGE to examine the feasibility to deliver cells and engineer vascular-like networks in the hybrid implant. Furthermore, as a proof of concept, a full-scale porous Ti-6Al-4V acetabular cup was impregnated with cell-laden hydrogel to validate the clinical potential of this strategy. The vasculogenic potential was evaluated by examining micro-capillary formation coupled with capillary network maturation and stabilization. Osteogenic differentiation was assessed via alkaline phosphatase activity as well as osteocalcin and osteopontin expression. Our results suggested that GelAGE supported HUVECs spreading and vascular-like network formation, along with osteogenesis of hMSCs. Titanium hybrid constructs with cell-laden hydrogel demonstrated enhanced osteogenesis with similar vasculogenic capability compared to the cell-laden hydrogel alone constructs. The full-scale implant with cell-laden hydrogel coating similarly showed cell distribution and spreading, implying the potential for further clinical application. Our study presents the feasibility of integrating bio-functional hydrogels with porous titanium implants to fabricate a vascularized hybrid construct with both mechanical support and preferable biological functionality (osteogenesis/vasculogenesis), which paves the way for improved strategies to enhance bone regeneration in complex large bone defects achieving long-term bone-implant fixation.

Strategies for inclusion of growth factors into 3D printed bone grafts.

There remains a critical need to develop new technologies and materials that can meet the demands of treating large bone defects. The advancement of 3-dimensional (3D) printing technologies has allowed the creation of personalized and customized bone grafts, with specific control in both macro- and micro-architecture, and desired mechanical properties. Nevertheless, the biomaterials used for the production of these bone grafts often possess poor biological properties. The incorporation of growth factors (GFs), which are the natural orchestrators of the physiological healing process, into 3D printed bone grafts, represents a promising strategy to achieve the bioactivity required to enhance bone regeneration. In this review, the possible strategies used to incorporate GFs to 3D printed constructs are presented with a specific focus on bone regeneration. In particular, the strengths and limitations of different methods, such as physical and chemical cross-linking, which are currently used to incorporate GFs to the engineered constructs are critically reviewed. Different strategies used to present one or more GFs to achieve simultaneous angiogenesis and vasculogenesis for enhanced bone regeneration are also covered in this review. In addition, the possibility of combining several manufacturing approaches to fabricate hybrid constructs, which better mimic the complexity of biological niches, is presented. Finally, the clinical relevance of these approaches and the future steps that should be taken are discussed.

Probing Multicellular Tissue Fusion of Cocultured Spheroids-A 3D-Bioassembly Model.

While decades of research have enriched the knowledge of how to grow cells into mature tissues, little is yet known about the next phase: fusing of these engineered tissues into larger functional structures. The specific effect of multicellular interfaces on tissue fusion remains largely unexplored. Here, a facile 3D-bioassembly platform is introduced to primarily study fusion of cartilage-cartilage interfaces using spheroids formed from human mesenchymal stromal cells (hMSCs) and articular chondrocytes (hACs). 3D-bioassembly of two adjacent hMSCs spheroids displays coordinated migration and noteworthy matrix deposition while the interface between two hAC tissues lacks both cells and type-II collagen. Cocultures contribute to increased phenotypic stability in the fusion region while close initial contact between hMSCs and hACs (mixed) yields superior hyaline differentiation over more distant, indirect cocultures. This reduced ability of potent hMSCs to fuse with mature hAC tissue further underlines the major clinical challenge that is integration. Together, this data offer the first proof of an in vitro 3D-model to reliably study lateral fusion mechanisms between multicellular spheroids and mature cartilage tissues. Ultimately, this high-throughput 3D-bioassembly model provides a bridge between understanding cellular differentiation and tissue fusion and offers the potential to probe fundamental biological mechanisms that underpin organogenesis.

Stepwise Control of Crosslinking in a One-Pot System for Bioprinting of Low-Density Bioinks.

Extrusion-based 3D bioprinting is hampered by the inability to print materials of low-viscosity. In this study, a single initiating system based on ruthenium (Ru) and sodium persulfate (SPS) is utilized for a sequential dual-step crosslinking approach: 1) primary (partial) crosslinking in absence of light to alter the bioink's rheological profile for print fidelity, and 2) subsequent secondary post-printing crosslinking for shape maintenance. Allyl-functionalized gelatin (Gel-AGE) is used as a bioink, allowing thiol-ene click reaction between allyl moieties and thiolated crosslinkers. A systematic investigation of primary crosslinking reveals that a thiol-persulfate redox reaction facilitates thiol-ene crosslinking, mediating an increase in bioink viscosity that is controllable by tailoring the Ru/SPS, crosslinker, and/or Gel-AGE concentrations. Thereafter, subsequent photoinitiated secondary crosslinking then facilitates maximum conversion of thiol-ene bonds between AGE and thiol groups. The dual-step crosslinking method is applicable to a wide biofabrication window (3-10 wt% Gel-AGE) and is demonstrated to allow printing of low-density (3 wt%) Gel-AGE, normally exhibiting low viscosity (4 mPa s), with high shape fidelity and high cell viability (>80%) over 7 days of culture. The presented approach can therefore be used as a one-pot system for printing low-viscous bioinks without the need for multiple initiating systems, viscosity enhancers, or complex chemical modifications.

Visible Light Cross-Linking of Gelatin Hydrogels Offers an Enhanced Cell Microenvironment with Improved Light Penetration Depth.

In this study, the cyto-compatibility and cellular functionality of cell-laden gelatin-methacryloyl (Gel-MA) hydrogels fabricated using a set of photo-initiators which absorb in 400-450 nm of the visible light range are investigated. Gel-MA hydrogels cross-linked using ruthenium (Ru) and sodium persulfate (SPS), are characterized to have comparable physico-mechanical properties as Gel-MA gels photo-polymerized using more conventionally adopted photo-initiators, such as 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one (Irgacure 2959) and lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP). It is demonstrated that the Ru/SPS system has a less adverse effect on the viability and metabolic activity of human articular chondrocytes encapsulated in Gel-MA hydrogels for up to 35 days. Furthermore, cell-laden constructs cross-linked using the Ru/SPS system have significantly higher glycosaminoglycan content and re-differentiation capacity as compared to cells encapsulated using I2959 and LAP. Moreover, the Ru/SPS system offers significantly greater light penetration depth as compared to the I2959 system, allowing thick (10 mm) Gel-MA hydrogels to be fabricated with homogenous cross-linking density throughout the construct. These results demonstrate the considerable advantages of the Ru/SPS system over traditional UV polymerizing systems in terms of clinical relevance and practicability for applications such as cell encapsulation, biofabrication, and in situ cross-linking of injectable hydrogels.