Evaluation of surgical fixation methods for the implantation of melt electrowriting-reinforced hyaluronic acid hydrogel composites in porcine cartilage defects.

The surgical repair of articular cartilage remains an ongoing challenge in orthopedics. Tissue engineering is a promising approach to treat cartilage defects; however, scaffolds must (i) possess the requisite material properties to support neocartilage formation, (ii) exhibit sufficient mechanical integrity for handling during implantation, and (iii) be reliably fixed within cartilage defects during surgery. In this study, we demonstrate the reinforcement of soft norbornene-modified hyaluronic acid (NorHA) hydrogels via the melt electrowriting (MEW) of polycaprolactone to fabricate composite scaffolds that support encapsulated porcine mesenchymal stromal cell (pMSC, three donors) chondrogenesis and cartilage formation and exhibit mechanical properties suitable for handling during implantation. Thereafter, acellular MEW-NorHA composites or MEW-NorHA composites with encapsulated pMSCs and precultured for 28 days were implanted in full-thickness cartilage defects in porcine knees using either bioresorbable pins or fibrin glue to assess surgical fixation methods. Fixation of composites with either biodegradable pins or fibrin glue ensured implant retention in most cases (80%); however, defects treated with pinned composites exhibited more subchondral bone remodeling and inferior cartilage repair, as evidenced by micro-computed tomography (micro-CT) and safranin O/fast green staining, respectively, when compared to defects treated with glued composites. Interestingly, no differences in repair tissue were observed between acellular and cellularized implants. Additional work is required to assess the full potential of these scaffolds for cartilage repair. However, these results suggest that future approaches for cartilage repair with MEW-reinforced hydrogels should be carefully evaluated with regard to their fixation approach for construct retention and surrounding cartilage tissue damage.

Linguistic Analysis Identifies Emergent Biomaterial Fabrication Trends for Orthopaedic Applications.

The expanse of publications in tissue engineering (TE) and orthopedic TE (OTE) over the past 20 years presents an opportunity to probe emergent trends in the field to better guide future technologies that can make an impact on musculoskeletal therapies. Leveraging this trove of knowledge, a hierarchical systematic search method and trend analysis using connected network mapping of key terms is developed. Within discrete time intervals, an accelerated publication rate for anatomic orthopedic tissue engineering (AOTE) of osteochondral defects, tendons, menisci, and entheses is identified. Within these growing fields, the top-listed key terms are extracted and stratified into evident categories, such as biomaterials, delivery method, or 3D printing and biofabrication. It is then identified which categories decreased, remained constant, increased, or emerged over time, identifying the specific emergent categories currently driving innovation in orthopedic repair technologies. Together, these data demonstrate a significant convergence of material types and descriptors used across tissue types. From this convergence, design criteria to support future research of anatomic constructs that mimic both the form and function of native tissues are formulated. In summary, this review identifies large-scale trends and predicts new directions in orthopedics that will define future materials and technologies.

Synthesis, Characterization, and Digital Light Processing of a Hydrolytically Degradable Hyaluronic Acid Hydrogel.

Numerous chemical modifications of hyaluronic acid (HA) have been explored for the formation of degradable hydrogels that are suitable for a variety of biomedical applications, including biofabrication and drug delivery. Thiol-ene step-growth chemistry is of particular interest due to its lower oxygen sensitivity and ability to precisely tune mechanical properties. Here, we utilize an aqueous esterification route via reaction with carbic anhydride to synthesize norbornene-modified HA (NorHACA) that is amenable to thiol-ene crosslinking to form hydrolytically unstable networks. NorHACA is first synthesized with varying degrees of modification (∼15-100%) by adjusting the ratio of reactive carbic anhydride to HA. Thereafter, NorHACA is reacted with dithiol crosslinker in the presence of visible light and photoinitiator to form hydrogels within tens of seconds. Unlike conventional NorHA, NorHACA hydrogels are highly susceptible to hydrolytic degradation through enhanced ester hydrolysis. Both the mechanical properties and the degradation timescales of NorHACA hydrogels are tuned via macromer concentration and/or the degree of modification. Moreover, the degradation behavior of NorHACA hydrogels is validated through a statistical-co-kinetic model of ester hydrolysis. The rapid degradation of NorHACA hydrogels can be adjusted by incorporating small amounts of slowly degrading NorHA macromer into the network. Further, NorHACA hydrogels are implemented as digital light processing (DLP) resins to fabricate hydrolytically degradable scaffolds with complex, macroporous structures that can incorporate cell-adhesive sites for cell attachment and proliferation after fabrication. Additionally, DLP bioprinting of NorHACA hydrogels to form cell-laden constructs with high viability is demonstrated, making them useful for applications in tissue engineering and regenerative medicine.

Simultaneous One-Pot Interpenetrating Network Formation to Expand 3D Processing Capabilities.

The incorporation of a secondary network into traditional single-network hydrogels can enhance mechanical properties, such as toughness and loading to failure. These features are important for many applications, including as biomedical materials; however, the processing of interpenetrating polymer network (IPN) hydrogels is often limited by their multistep fabrication procedures. Here, a one-pot scheme for the synthesis of biopolymer IPN hydrogels mediated by the simultaneous crosslinking of two independent networks with light, namely: i) free-radical crosslinking of methacrylate-modified hyaluronic acid (HA) to form the primary network and ii) thiol-ene crosslinking of norbornene-modified HA with thiolated guest-host assemblies of adamantane and ß-cyclodextrin to form the secondary network, is reported. The mechanical properties of the IPN hydrogels are tuned by changing the network composition, with high water content (≈94%) hydrogels exhibiting excellent work of fracture, tensile strength, and low hysteresis. As proof-of-concept, the IPN hydrogels are implemented as low-viscosity Digital Light Processing resins to fabricate complex structures that recover shape upon loading, as well as in microfluidic devices to form deformable microparticles. Further, the IPNs are cytocompatible with cell adhesion dependent on the inclusion of adhesive peptides. Overall, the enhanced processing of these IPN hydrogels will expand their utility across applications.

Fabrication of MSC-laden composites of hyaluronic acid hydrogels reinforced with MEW scaffolds for cartilage repair.

Hydrogels are of interest in cartilage tissue engineering due to their ability to support the encapsulation and chondrogenesis of mesenchymal stromal cells (MSCs). However, features such as hydrogel crosslink density, which can influence nutrient transport, nascent matrix distribution, and the stability of constructs during and after implantation must be considered in hydrogel design. Here, we first demonstrate that more loosely crosslinked (i.e. softer, ∼2 kPa) norbornene-modified hyaluronic acid (NorHA) hydrogels support enhanced cartilage formation and maturation when compared to more densely crosslinked (i.e. stiffer, ∼6-60 kPa) hydrogels, with a >100-fold increase in compressive modulus after 56 d of culture. While soft NorHA hydrogels mature into neocartilage suitable for the repair of articular cartilage, their initial moduli are too low for handling and they do not exhibit the requisite stability needed to withstand the loading environments of articulating joints. To address this, we reinforced NorHA hydrogels with polycaprolactone (PCL) microfibers produced via melt-electrowriting (MEW). Importantly, composites fabricated with MEW meshes of 400µm spacing increased the moduli of soft NorHA hydrogels by ∼50-fold while preserving the chondrogenic potential of the hydrogels. There were minimal differences in chondrogenic gene expression and biochemical content (e.g. DNA, GAG, collagen) between hydrogels alone and composites, whereas the composites increased in compressive modulus to ∼350 kPa after 56 d of culture. Lastly, integration of composites with native tissue was assessedex vivo; MSC-laden composites implanted after 28 d of pre-culture exhibited increased integration strengths and contact areas compared to acellular composites. This approach has great potential towards the design of cell-laden implants that possess both initial mechanical integrity and the ability to support neocartilage formation and integration for cartilage repair.

Enhancing Biopolymer Hydrogel Functionality through Interpenetrating Networks.

Traditional hydrogels are strong candidates for biomedical applications; however, they may suffer from drawbacks such as weak mechanics, static properties, and an inability to fully replicate aspects of the cellular microenvironment. These challenges can be addressed through the incorporation of second networks to form interpenetrating polymer network (IPN) hydrogels. The objective of this review is to establish clear trends on the enhanced functionality achieved by incorporating secondary networks into traditional, biopolymer-based hydrogels. These include mechanical reinforcement, 'smart' systems that respond to external stimuli, and the ability to tune cell-material interactions. Through attention to network structure and chemistry, IPN hydrogels may advance to meet challenging criteria for a wide range of biomedical fields.

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.

3D bioprinting via an in situ crosslinking technique towards engineering cartilage tissue.

3D bioprinting is a promising approach for the repair of cartilage tissue after damage due to injury or disease; however, the design of 3D printed scaffolds has been limited by the availability of bioinks with requisite printability, cytocompatibility, and bioactivity. To address this, we developed an approach termed in situ crosslinking that permits the printing of non-viscous, photocrosslinkable bioinks via the direct-curing of the bioink with light through a photopermeable capillary prior to deposition. Using a norbornene-modified hyaluronic acid (NorHA) macromer as a representative bioink and our understanding of thiol-ene curing kinetics with visible light, we varied the printing parameters (e.g., capillary length, flow rate, light intensity) to identify printing conditions that were optimal for the ink. The printing process was cytocompatible, with high cell viability and homogenous distribution of mesenchymal stromal cells (MSCs) observed throughout printed constructs. Over 56 days of culture in chondrogenic media, printed constructs increased in compressive moduli, biochemical content (i.e., sulfated glycosaminoglycans, collagen), and histological staining of matrix associated with cartilage tissue. This generalizable printing approach may be used towards the repair of focal defects in articular cartilage or broadly towards widespread biomedical applications across a range of photocrosslinkable bioinks that can now be printed.

Influence of hyaluronic acid modification on CD44 binding towards the design of hydrogel biomaterials.

Hyaluronic acid (HA) is a linear polysaccharide of d-glucuronic acid and N-acetyl-d-glucosamine that is native to many tissues and interacts with cells via cell-surface receptors (e.g., CD44). HA has been extensively explored as a chemically-modified macromer for crosslinking into biomaterials, such as hydrogels and macroporous scaffolds. However, the influence of the extent and type of HA modification on its binding to CD44 is not well understood or quantified. To address this, we modified HA at either the carboxylic acid or the primary alcohol with various chemical groups (e.g., norbornenes, methacrylates) and magnitudes (~10, 20, or 40% of disaccharides) and then characterized binding in both soluble and hydrogel forms. HA binding to CD44 immobilized on plates or presented by cells was influenced by the extent and type of its modification, where increased modification (i.e., ~40%) generally decreased binding. The adhesion of CD44-modified beads to hydrogels as measured by atomic force microscopy revealed a similar trend, particularly with decreased adhesion with hydrophobic modifications to the carboxylic acid. Further, the chondrogenesis of mesenchymal stromal cells when encapsulated in hydrogels fabricated from modified HA macromers was reduced at high modification, behaving similarly to inert hydrogel controls. This work suggests that the types and extents of modification of polysaccharides are important factors that should be considered in preserving their biological function when processed as hydrogels.

Tailoring supramolecular guest-host hydrogel viscoelasticity with covalent fibrinogen double networks.

Supramolecular chemistry has enabled the design of tunable biomaterials that mimic the dynamic and viscoelastic characteristics of the extracellular matrix. However, the noncovalent nature of supramolecular bonds renders them inherently weak, limiting their applicability to many biomedical applications. To address this, we formulated double network (DN) hydrogels through a combination of supramolecular and covalent networks to tailor hydrogel viscoelastic properties. Specifically, DN hydrogels were formed through the combination of supramolecular guest-host (GH) hyaluronic acid (HA) networks with covalent networks from the photocrosslinking of acrylated poly(ethylene glycol) modified fibrinogen (PEG-fibrinogen) and PEG diacrylate. DN hydrogels exhibited higher compressive moduli, increased failure stresses, and increased toughness when compared to purely covalent networks. While GH concentration had little influence on the compressive moduli across DN hydrogels, an increase in the GH concentration resulted in more viscous behavior of DN hydrogels. High viability of encapsulated bovine mesenchymal stromal cells (MSCs) was observed across groups with enhanced spreading and proliferation in DN hydrogels with increased GH concentration. This combination of supramolecular and covalent chemistries enables the formation of dynamic hydrogels with tunable properties that can be customized towards repair of viscoelastic tissues.

Gallol-derived ECM-mimetic adhesive bioinks exhibiting temporal shear-thinning and stabilization behavior.

3D bioprinting is an attractive technique to fabricate well-organized, cell-laden constructs for tissue repair and disease modeling. Although numerous hydrogel bioinks have been developed, materials are still needed that mimic the cellular microenvironment, have the appropriate viscosity and stabilization for printing, and are cytocompatible. Here, we present a unique gallol-modified extracellular matrix (ECM) hydrogel ink that is inspired by rapid fruit browning phenomena. The gallol-modification of ECM components (e.g., hyaluronic acid, gelatin) allowed (i) immediate gelation and shear-thinning properties by dynamic hydrogen bonds on short time-scales and (ii) further auto-oxidation and covalent crosslinking for stabilization on longer time-scales. The gallol ECM hydrogel ink was printable using an extrusion-based 3D printer by exploiting temporal shear-thinning properties, and further showed cytocompatibility (∼95% viability) and on-tissue printability due to adhesiveness of gallol moieties. Printed cell-laden filaments degraded and swelled with culture over 6 days, corresponding to increases in cell density and spreading. Ultimately, this strategy is useful for designing hydrogel inks with tunable properties for 3D bioprinting. STATEMENT OF SIGNIFICANCE: 3D bioprinting is a promising technique for the fabrication of cell-laden constructs for applications as in vitro models or for therapeutic applications. Despite the previous development of numerous hydrogel bioinks, there still remain challenging considerations in the design of bioinks. In this study, we present a unique cytocompatible hydrogel ink with gallol modification that is inspired by rapid fruit browning phenomena. The gallol hydrogel ink has three important properties: i) it shows immediate gelation by dynamic, reversible bonds for shear-thinning extrusion, ii) it allows spontaneous stabilization by subsequent covalent crosslinking after printing, and iii) it is printable on tissues by adhesive properties of gallol moieties. As such, this work presents a new approach in the design of hydrogel inks.

Three-dimensional extrusion bioprinting of single- and double-network hydrogels containing dynamic covalent crosslinks.

The fabrication of three-dimensional (3D) scaffolds is indispensable to tissue engineering and 3D printing is emerging as an important approach towards this. Hydrogels are often used as inks in extrusion-based 3D printing, including with encapsulated cells; however, numerous challenging requirements exist, including appropriate viscosity, the ability to stabilize after extrusion, and cytocompatibility. Here, we present a shear-thinning and self-healing hydrogel crosslinked through dynamic covalent chemistry for 3D bioprinting. Specifically, hyaluronic acid was modified with either hydrazide or aldehyde groups and mixed to form hydrogels containing a dynamic hydrazone bond. Due to their shear-thinning and self-healing properties, the hydrogels could be extruded for 3D printing of structures with high shape fidelity, stability to relaxation, and cytocompatibility with encapsulated fibroblasts (>80% viability). Forces for extrusion and filament sizes were dependent on parameters such as material concentration and needle gauge. To increase scaffold functionality, a second photocrosslinkable interpenetrating network was included that was used for orthogonal photostiffening and photopatterning through a thiol-ene reaction. Photostiffening increased the scaffold's modulus (∼300%) while significantly decreasing erosion (∼70%), whereas photopatterning allowed for spatial modification of scaffolds with dyes. Overall, this work introduces a simple approach to both fabricate and modify 3D printed scaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 865-875, 2018.