Mechanics of 3D Cell-Hydrogel Interactions: Experiments, Models, and Mechanisms.

Hydrogels are highly water-swollen molecular networks that are ideal platforms to create tissue mimetics owing to their vast and tunable properties. As such, hydrogels are promising cell-delivery vehicles for applications in tissue engineering and have also emerged as an important base for ex vivo models to study healthy and pathophysiological events in a carefully controlled three-dimensional environment. Cells are readily encapsulated in hydrogels resulting in a plethora of biochemical and mechanical communication mechanisms, which recapitulates the natural cell and extracellular matrix interaction in tissues. These interactions are complex, with multiple events that are invariably coupled and spanning multiple length and time scales. To study and identify the underlying mechanisms involved, an integrated experimental and computational approach is ideally needed. This review discusses the state of our knowledge on cell-hydrogel interactions, with a focus on mechanics and transport, and in this context, highlights recent advancements in experiments, mathematical and computational modeling. The review begins with a background on the thermodynamics and physics fundamentals that govern hydrogel mechanics and transport. The review focuses on two main classes of hydrogels, described as semiflexible polymer networks that represent physically cross-linked fibrous hydrogels and flexible polymer networks representing the chemically cross-linked synthetic and natural hydrogels. In this review, we highlight five main cell-hydrogel interactions that involve key cellular functions related to communication, mechanosensing, migration, growth, and tissue deposition and elaboration. For each of these cellular functions, recent experiments and the most up to date modeling strategies are discussed and then followed by a summary of how to tune hydrogel properties to achieve a desired functional cellular outcome. We conclude with a summary linking these advancements and make the case for the need to integrate experiments and modeling to advance our fundamental understanding of cell-matrix interactions that will ultimately help identify new therapeutic approaches and enable successful tissue engineering.

Hydrolytically degradable Poly (ß-amino ester) resins with tunable degradation for 3D printing by projection micro-stereolithography.

Applications of 3D printing that range from temporary medical devices to environmentally responsible manufacturing would benefit from printable resins that yield polymers with controllable material properties and degradation behavior. Towards this goal, poly(ß-amino ester) (PBAE)-diacrylate resins were investigated due to the wide range of available chemistries and tunable material properties. PBAE-diacrylate resins were synthesized from hydrophilic and hydrophobic chemistries and with varying electron densities on the ester bond to provide control over degradation. Hydrophilic PBAE-diacrylates led to degradation behaviors characteristic of bulk degradation while hydrophobic PBAE-diacrylates led to degradation behaviors dominated initially by surface degradation and then transitioned to bulk degradation. Depending on chemistry, the crosslinked PBAE-polymers exhibited a range of degradation times under accelerated conditions, from complete mass loss in 90 min to minimal mass loss at 45 days. Patterned features with 55 µm resolution were achieved across all resins, but their fidelity was dependent on PBAE-diacrylate molecular weight, reactivity, and printing parameters. In summary, simple chemical modifications in the PBAE-diacrylate resins coupled with projection microstereolithography enables high resolution 3D printed parts with similar architectures and initial properties, but widely different degradation rates and behaviors.

Effects of Kinetic Chain Length on the Degradation of Poly(ß-amino ester)-Based Networks and Use in 3D Printing by Projection Microstereolithography.

Poly(ß-amino ester)-diacrylates (PBAE-dAs) are promising resins for three-dimensional (3D) printing. This study investigated the degradation of two PBAEs with different chemistries and kinetic chain lengths. PBAE-dA monomers were synthesized from benzhydrazide and poly(ethylene glycol) (A6) or butanediol (B6) diacrylate and then photopolymerized with pentaerythritol tetrakis(3-mercaptopropionate), which formed thiol-polyacrylate kinetic chains. This tetrathiol acts as a cross-linker and chain-transfer agent that controls the polyacrylate kinetic chain length. A6 networks exhibited bulk degradation, while B6 networks exhibited surface degradation, which transitioned to a combined surface and bulk degradation. Increasing the tetrathiol concentration shortened the polyacrylate kinetic chain and time-to-reverse gelation but degradation mode was unaffected. Hydrolysis occurred primarily through the ß-amino ester. As network hydrophilicity increased, the slower degrading ester in the thiol-polyacrylate chains contributed to degradation. Overall, this work demonstrates control over network degradation rate, mode of degradation, and time-to-reverse gelation in PBAE networks and their application in 3D printing.

A 3D printed mimetic composite for the treatment of growth plate injuries in a rabbit model.

Growth plate injuries affecting the pediatric population may cause unwanted bony repair tissue that leads to abnormal bone elongation. Clinical treatment involves bony bar resection and implantation of an interpositional material, but success is limited and the bony bar often reforms. No treatment attempts to regenerate the growth plate cartilage. Herein we develop a 3D printed growth plate mimetic composite as a potential regenerative medicine approach with the goal of preventing limb length discrepancies and inducing cartilage regeneration. A poly(ethylene glycol)-based resin was used with digital light processing to 3D print a mechanical support structure infilled with a soft cartilage-mimetic hydrogel containing chondrogenic cues. Our biomimetic composite has similar mechanical properties to native rabbit growth plate and induced chondrogenic differentiation of rabbit mesenchymal stromal cells in vitro. We evaluated its efficacy as a regenerative interpositional material applied after bony bar resection in a rabbit model of growth plate injury. Radiographic imaging was used to monitor limb length and tibial plateau angle, microcomputed tomography assessed bone morphology, and histology characterized the repair tissue that formed. Our 3D printed growth plate mimetic composite resulted in improved tibial lengthening compared to an untreated control, cartilage-mimetic hydrogel only condition, and a fat graft. However, in vivo the 3D printed growth plate mimetic composite did not show cartilage regeneration within the construct histologically. Nevertheless, this study demonstrates the feasibility of a 3D printed biomimetic composite to improve limb lengthening, a key functional outcome, supporting its further investigation as a treatment for growth plate injuries.

Biomimetic and mechanically supportive 3D printed scaffolds for cartilage and osteochondral tissue engineering using photopolymers and digital light processing.

Successful 3D scaffold designs for musculoskeletal tissue engineering necessitate full consideration of the form and function of the tissues of interest. When designing structures for engineering cartilage and osteochondral tissues, one must reconcile the need to develop a mechanically robust system that maintains the health of cells embedded in the scaffold. In this work, we present an approach that decouples the mechanical and biochemical needs and allows for the independent development of the structural and cellular niches in a scaffold. Using the highly tuned capabilities of digital light processing-based stereolithography, structures with complex architectures are achieved over a range of effective porosities and moduli. The 3D printed structure is infilled with mesenchymal stem cells and soft biomimetic hydrogels, which are specifically formulated with extracellular matrix analogs and tethered growth factors to provide selected biochemical cues for the guided differentiation towards chondrogenesis and osteogenesis. We demonstrate the ability to utilize these structures to (a) infill a focal chondral defect and mitigate macroscopic and cellular level changes in the cartilage surrounding the defect, and (b) support the development of a stratified multi-tissue scaffold for osteochondral tissue engineering.

Highly Tunable Thiol-Ene Photoresins for Volumetric Additive Manufacturing.

Volumetric additive manufacturing (VAM) forms complete 3D objects in a single photocuring operation without layering defects, enabling 3D printed polymer parts with mechanical properties similar to their bulk material counterparts. This study presents the first report of VAM-printed thiol-ene resins. With well-ordered molecular networks, thiol-ene chemistry accesses polymer materials with a wide range of mechanical properties, moving VAM beyond the limitations of commonly used acrylate formulations. Since free-radical thiol-ene polymerization is not inhibited by oxygen, the nonlinear threshold response required in VAM is introduced by incorporating 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) as a radical scavenger. Tuning of the reaction kinetics is accomplished by balancing inhibitor and initiator content. Coupling this with quantitative measurements of the absorbed volumetric optical dose allows control of polymer conversion and gelation during printing. Importantly, this work thereby establishes the first comprehensive framework for spatial-temporal control over volumetric energy distribution, demonstrating structures 3D printed in thiol-ene resin by means of tomographic volumetric VAM. Mechanical characterization of this thiol-ene system, with varied ratios of isocyanurate and triethylene glycol monomers, reveals highly tunable mechanical response far more versatile than identical acrylate-based resins. This broadens the range of materials and properties available for VAM, taking another step toward high-performance printed polymers.

Stereolithographic 3D Printing for Deterministic Control over Integration in Dual-Material Composites.

This work introduces a rapid and facile approach to predictably control integration between two materials with divergent properties. Programmed integration between photopolymerizable soft and stiff hydrogels was investigated for their promise in applications such as tissue engineering where heterogeneous properties are often desired. Spatial control afforded by grayscale 3D printing was leveraged to define regions at the interface that permit diffusive transport of a second material in-filled into the 3D printed part. The printing parameters (i.e., effective exposure dose) for the resin were correlated directly to mesh size to achieve controlled diffusion. Applying this information to grayscale exposures led to a range of distances over which integration was achieved with high fidelity. A prescribed finite distance of integration between soft and stiff hydrogels led to a 33% increase in strain to failure under tensile testing and eliminated failure at the interface. The feasibility of this approach was demonstrated in a layer-by-layer 3D printed part fabricated by stereolithography, which was subsequently infilled with a soft hydrogel containing osteoblastic cells. In summary, this approach holds promise for applications where integration of multiple materials and living cells is needed by allowing precise control over integration and reducing mechanical failure at contrasting material interfaces.

Rabbit Model of Physeal Injury for the Evaluation of Regenerative Medicine Approaches.

Physeal injuries can lead to bony repair tissue formation, known as a bony bar. This can result in growth arrest or angular deformity, which is devastating for children who have not yet reached their full height. Current clinical treatment involves resecting the bony bar and replacing it with a fat graft to prevent further bone formation and growth disturbance, but these treatments frequently fail to do so and require additional interventions. Novel treatments that could prevent bone formation but also regenerate the injured physeal cartilage and restore normal bone elongation are warranted. To test the efficacy of these treatments, animal models that emulate human physeal injury are necessary. The rabbit model of physeal injury quickly establishes a bony bar, which can then be resected to test new treatments. Although numerous rabbit models have been reported, they vary in terms of size and location of the injury, tools used to create the injury, and methods to assess the repair tissue, making comparisons between studies difficult. The study presented here provides a detailed method to create a rabbit model of proximal tibia physeal injury using a two-stage procedure. The first procedure involves unilateral removal of 25% of the physis in a 6-week-old New Zealand white rabbit. This consistently leads to a bony bar, significant limb length discrepancy, and angular deformity within 3 weeks. The second surgical procedure involves bony bar resection and treatment. In this study, we tested the implantation of a fat graft and a photopolymerizable hydrogel as a proof of concept that injectable materials could be delivered into this type of injury. At 8 weeks post-treatment, we measured limb length, tibial angle, and performed imaging and histology of the repair tissue. By providing a detailed, easy to reproduce methodology to perform the physeal injury and test novel treatments after bony bar resection, comparisons between studies can be made and facilitate translation of promising therapies toward clinical use. Impact Statement This study provides details to create a rabbit model of physeal injury that can facilitate comparisons between studies and test novel regenerative medicine approaches. Furthermore, this model mimics the human, clinical situation that requires a bony bar resection followed by treatment. In addition, identification of a suitable treatment can be seen in the correction of the growth deformity, allowing this model to facilitate the development of novel physeal cartilage regenerative medicine approaches.

Understanding and Improving Mechanical Properties in 3D printed Parts Using a Dual-Cure Acrylate-Based Resin for Stereolithography.

Application of 3D printed structures via stereolithography (SLA) is limited by imprecise dimensional control and inferior mechanical properties. These challenges is attributed to poor understanding ofpolymerization behavior during the printing process and inadequate post-processing methods. The former via a modified version of Jacob's working curve equation that incorporates the resin's sub-linear response to irradiation intensity is addressed by the authors. This new model provides a more accurate approach to select 3D printing parameters given a desired z-resolution and conversion profile along the depth of the printed part. The authors use this improved model to motivate a novel material design that can be post-processed to be indistinguishable from the polymer at 100% conversion. This approach employs a dual initiating system in which photo-initiated printing is followed by a thermal post-cure to achieve uniform conversion. The authors show that this approach enables fast printing times (10 s per layer), exceptional horizontal resolution (1-10 microns), precise control over vertical resolution, and decreased surface corrugations on a 10's of microns scale. The techniques described herein use an acrylate-based SLA resin, but the approach can be extended to other monomer systems to simultaneously achieve predictable properties and dimensions that are critical for application of additive manufacturing in load-bearing applications.

A Stereolithography-Based 3D Printed Hybrid Scaffold for In Situ Cartilage Defect Repair.

Damage to articular cartilage can over time cause degeneration to the tissue surrounding the injury. To address this problem, scaffolds that prevent degeneration and promote neotissue growth are needed. A new hybrid scaffold that combines a stereolithography-based 3D printed support structure with an injectable and photopolymerizable hydrogel for delivering cells to treat focal chondral defects is introduced. In this proof of concept study, the ability to a) infill the support structure with an injectable hydrogel precursor solution, b) incorporate cartilage cells during infilling using a degradable hydrogel that promotes neotissue deposition, and c) minimize damage to the surrounding cartilage when the hybrid scaffold is placed in situ in a focal chondral defect in an osteochondral plug that is cultured under mechanical loading is demonstrated. With the ability to independently control the properties of the structure and the injectable hydrogel, this hybrid scaffold approach holds promise for treating chondral defects.