Controlled Mechanical Property Gradients Within a Digital Light Processing Printed Hydrogel-Composite Osteochondral Scaffold.

Tissue engineered scaffolds are needed to support physiological loads and emulate the micrometer-scale strain gradients within tissues that guide cell mechanobiological responses. We designed and fabricated micro-truss structures to possess spatially varying geometry and controlled stiffness gradients. Using a custom projection microstereolithography (µSLA) system, using digital light projection (DLP), and photopolymerizable poly(ethylene glycol) diacrylate (PEGDA) hydrogel monomers, three designs with feature sizes < 200 µm were formed: (1) uniform structure with 1 MPa structural modulus ( E ) designed to match equilibrium modulus of healthy articular cartilage, (2) E = 1 MPa gradient structure designed to vary strain with depth, and (3) osteochondral bilayer with distinct cartilage ( E = 1 MPa) and bone ( E = 7 MPa) layers. Finite element models (FEM) guided design and predicted the local mechanical environment. Empty trusses and poly(ethylene glycol) norbornene hydrogel-infilled composite trusses were compressed during X-ray microscopy (XRM) imaging to evaluate regional stiffnesses. Our designs achieved target moduli for cartilage and bone while maintaining 68-81% porosity. Combined XRM imaging and compression of empty and hydrogel-infilled micro-truss structures revealed regional stiffnesses that were accurately predicted by FEM. In the infilling hydrogel, FEM demonstrated the stress-shielding effect of reinforcing structures while predicting strain distributions. Composite scaffolds made from stiff µSLA-printed polymers support physiological load levels and enable controlled mechanical property gradients which may improve in vivo outcomes for osteochondral defect tissue regeneration. Advanced 3D imaging and FE analysis provide insights into the local mechanical environment surrounding cells in composite scaffolds.

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.

Microscale Photopatterning of Through-thickness Modulus in a Monolithic and Functionally Graded 3D Printed Part.

3D printing is transforming traditional processing methods for applications ranging from tissue engineering to optics. To fulfill its maximum potential, 3D printing requires a robust technique for producing structures with precise three-dimensional (x, y and z) control of mechanical properties. Previous efforts to realize such spatial control of modulus within 3D printed parts have largely focused on low-resolution (mm to cm scale) multi-material processes and grayscale approaches that spatially vary the modulus in the x-y plane and energy dose-based (E = I 0 t exp) models that do not account for the resin's sub-linear response to irradiation intensity. Here, we demonstrate a novel approach for through-thickness (z) voxelated control of mechanical properties within a single-material, monolithic part. Control over the local modulus is enabled by a predictive model that incorporates the observed non-reciprocal dose response of the material. The model is validated by an application of atomic force microscopy to map the through-thickness modulus on multi-layered 3D parts. Overall, both smooth gradations (30 MPa change over ≈75 µm) and sharp step-changes (30 MPa change over ≈5 µm) in modulus are realized in poly(ethylene glycol) diacrylate based 3D constructs, paving the way for advancements in tissue engineering, stimuli-responsive 4D printing and graded metamaterials.

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.