Post-mortem changes of anisotropic mechanical properties in the porcine brain assessed by MR elastography.

Knowledge of the mechanical properties of brain tissue in vivo is essential to understanding the mechanisms underlying traumatic brain injury (TBI) and to creating accurate computational models of TBI and neurosurgical simulation. Brain white matter, which is composed of aligned, myelinated, axonal fibers, is structurally anisotropic. White matter in vivo also exhibits mechanical anisotropy, as measured by magnetic resonance elastography (MRE), but measurements of anisotropy obtained by mechanical testing of white matter ex vivo have been inconsistent. The minipig has a gyrencephalic brain with similar white matter and gray matter proportions to humans and therefore provides a relevant model for human brain mechanics. In this study, we compare estimates of anisotropic mechanical properties of the minipig brain obtained by identical, non-invasive methods in the live (in vivo) and dead animals (in situ). To do so, we combine wave displacement fields from MRE and fiber directions derived from diffusion tensor imaging (DTI) with a finite element-based, transversely-isotropic nonlinear inversion (TI-NLI) algorithm. Maps of anisotropic mechanical properties in the minipig brain were generated for each animal alive and at specific times post-mortem. These maps show that white matter is stiffer, more dissipative, and more anisotropic than gray matter when the minipig is alive, but that these differences largely disappear post-mortem, with the exception of tensile anisotropy. Overall, brain tissue becomes stiffer, less dissipative, and less mechanically anisotropic post-mortem. These findings emphasize the importance of testing brain tissue properties in vivo. Statement of Significance: In this study, MRE and DTI in the minipig were combined to estimate, for the first time, anisotropic mechanical properties in the living brain and in the same brain after death. Significant differences were observed in the anisotropic behavior of brain tissue post-mortem. These results demonstrate the importance of measuring brain tissue properties in vivo as well as ex vivo, and provide new quantitative data for the development of computational models of brain biomechanics.

Structural tuning of anisotropic mechanical properties in 3D-Printed hydrogel lattices.

We investigated the ability to tune the anisotropic mechanical properties of 3D-printed hydrogel lattices by modifying their geometry (lattice strut diameter, unit cell size, and unit cell scaling factor). Many soft tissues are anisotropic and the ability to mimic natural anisotropy would be valuable for developing tissue-surrogate "phantoms" for elasticity imaging (shear wave elastography or magnetic resonance elastography). Vintile lattices were 3D-printed in polyethylene glycol di-acrylate (PEGDA) using digital light projection printing. Two mechanical benchtop tests, dynamic shear testing and unconfined compression, were used to measure the apparent shear storage moduli (G') and apparent Young's moduli (E) of lattice samples. Increasing the unit cell size from 1.25 mm to 2.00 mm reduced the Young's and shear moduli of the lattices by 91% and 85%, respectively. Decreasing the strut diameter from 300 µm to 200 µm reduced the apparent shear moduli of the lattices by 95%. Increasing the geometric scaling ratio of the lattice unit cells from 1.00 × to 2.00 × increased mechanical anisotropy in shear (by a factor of 3.1) and in compression (by a factor of 2.9). Both simulations and experiments show that the effects of unit cell size and strut diameter are consistent with power law relationships between volume fraction and apparent elastic moduli. In particular, experimental measurements of apparent Young's moduli agree well with predictions of the theoretical Gibson-Ashby model. Thus, the anisotropic mechanical properties of a lattice can be tuned by the unit cell size, the strut diameter, and scaling factors. This approach will be valuable in designing tissue-mimicking hydrogel lattice-based composite materials for elastography phantoms and tissue engineered scaffolds.

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.

Isolation and Characterization of the Murine Uterosacral Ligaments and Pelvic Floor Organs.

Pelvic organ prolapse (POP) is a condition that affects the integrity, structure, and mechanical support of the pelvic floor. The organs in the pelvic floor are supported by different anatomical structures, including muscles, ligaments, and pelvic fascia. The uterosacral ligament (USL) is a critical load-bearing structure, and injury to the USL results in a higher risk of developing POP. The present protocol describes the dissection of murine USLs and the pelvic floor organs alongside the acquisition of unique data on the USL biochemical composition and function using Raman spectroscopy and the evaluation of mechanical behavior. Mice are an invaluable model for preclinical research, but dissecting the murine USL is a difficult and intricate process. This procedure presents an approach to guide the dissection of murine pelvic floor tissues, including the USL, to enable multiple assessments and characterization. This work aims to aid the dissection of pelvic floor tissues by basic scientists and engineers, thus expanding the accessibility of research on the USL and pelvic floor conditions and the preclinical study of women's health using mouse models.

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.

The heterogeneous mechanical properties of adolescent growth plate cartilage: A study in rabbit.

The growth plate is a cartilaginous tissue that functions to lengthen bones in children. When fractured, however, the growth plate can lose this critical function. Our understanding of growth plate fracture and mechanobiology is currently hindered by sparse information on the growth plate's microscale spatial gradients in mechanical properties. In this study, we performed microindentation across the proximal tibia growth plate of 9-week-old New Zealand White rabbits (n = 15) to characterize spatial variations in mechanical properties using linear elastic and nonlinear poroelastic material models. Mean indentation results for Hertz reduced modulus ranged from 380 to 690 kPa, with a peak in the upper hypertrophic zone and significant differences (p < 0.05) between neighboring zones. Using a subset of these animals (n = 7), we characterized zonal structure and extracellular matrix content of the growth plate through confocal fluorescent microscopy and Raman spectroscopy mapping. Comparison between mechanical properties and matrix content across the growth plate showed that proteoglycan content correlated with compressive modulus. This study is the first to measure poroelastic mechanical properties from microindentation across growth plate cartilage and to discern differing mechanical properties between the upper and lower hypertrophic zones. This latter finding may explain the location of typical growth plate fractures. The spatial variation in our reported mechanical properties emphasize the heterogeneous structure of the growth plate which is important to inform future regenerative implant design and mechanobiological models.

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.

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.

The effects of dynamic compressive loading on human mesenchymal stem cell osteogenesis in the stiff layer of a bilayer hydrogel.

Bilayer hydrogels with a soft cartilage-like layer and a stiff bone-like layer embedded with human mesenchymal stem cells (hMSCs) are promising for osteochondral tissue engineering. The goals of this work were to evaluate the effects of dynamic compressive loading (2.5% applied strain, 1 Hz) on osteogenesis in the stiff layer and spatially map local mechanical responses (strain, stress, hydrostatic pressure, and fluid velocity). A bilayer hydrogel was fabricated from soft (24 kPa) and stiff (124 kPa) poly (ethylene glycol) hydrogels. With hMSCs embedded in the stiff layer, osteogenesis was delayed under loading evident by lower OSX and OPN expressions, alkaline phosphatase activity, and collagen content. At Day 28, mineral deposits were present throughout the stiff layer without loading but localized centrally and near the interface under loading. Local strains mapped by particle tracking showed substantial equivalent strain (~1.5%) transferring to the stiff layer. When hMSCs were cultured in stiff single-layer hydrogels subjected to similar strains, mineralization was inhibited. Finite element analysis revealed that hydrostatic pressures ≥~600 Pa correlated to regions lacking mineralization in both hydrogels. Fluid velocities were low (~1-10 nm/s) in the hydrogels with no apparent correlation to mineralization. Mineralization was recovered by inhibiting ERK1/2, indicating cell-mediated inhibition. These findings suggest that high strains (~1.5%) combined with higher hydrostatic pressures negatively impact osteogenesis, but in a manner that depends on the magnitude of each mechanical response. This work highlights the importance of local mechanical responses in mediating osteogenesis of hMSCs in bilayer hydrogels being studied for osteochondral tissue engineering.