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This manuscript explores the nonlinear viscoelasticity of agarose hydrogels by conceptualizing them as biopolymer networks with transient bond dynamics.

Prostaglandin E

Poly(β-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 β-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.

In the present work, the photocatalytic activity was investigated toward a thiol-Michael reaction of different combinations of 9-fluorenylmethyl carbamate (Fmoc) derivatives and photocaged amines (PCA) as base amplifiers and the catalytic base, respectively. This phototriggering approach was systematically studied for evaluating its effect on kinetics between thiol and Michael acceptors like acrylate or sulfone wherein, butyl 3-mercaptopropionate (BMP), 1-hexyl acrylate (HA), and ethyl vinyl sulfone (EVS) were used as model reactants. Interestingly, PCAs exhibited low quantum yields by themselves; NPPOC-Hex (2.5 mol %) which when used with BMP and HA, resulted in only 25% thiol conversion; however, when used along with Fmoc-Hex or Fmoc-TMG (2.5 or 5 mol %), it resulted in a higher thiol conversion of 50–60%. Furthermore, use of NPPOC-DEA (5 mol %) with 1 mol % Fmoc-TMG resulted in >70% thiol conversion for the same system. Upon using BMP and EVS nearly complete conversion of functional groups with 5 mol % NPPOC-DEA and 5 mol % Fmoc-DEA was obtained. This enhancement in reaction kinetics and conversion upon addition of an Fmoc derivative to a monofunctional thiol-Michael system was extended to multifunctional derivatives for polymerizing cross-linked polymer networks. Moreover, the kinetic study on model reactants also demonstrated efficient dark curing, resulting in 50–75% thiol conversion with only 30 s irradiation time, leading to validation of the efficacy of Fmoc derivatives and PCAs as photocatalysts for dark cure. Upon precise characterization in cross-linked systems using Raman spectroscopy for TMPTA/PETMP in the presence of 20 mol % NPPOC-DEA and 1 mol % Fmoc-TMG, the extent of dark cure was evaluated for a distance of 16.5 mm, which was observed to undergo maximum conversion and high dark cure propagation upon heating to 70 °C. Therefore, Fmoc-PCA catalysis is a practically useful approach for improving the photoinitiated efficiency of the thiol-Michael reaction and enabling photopolymerization in the dark with a marked improvement in photosensitivity.

Metalattices are artificial 3D solids, periodic on sub-100 nm length scales, that enable the functional properties of materials to be tuned. However, because of their complex structure, predicting and characterizing their properties is challenging. Here we demonstrate the first nondestructive measurements of the mechanical and structural properties of metalattices with feature sizes down to 14 nm. By monitoring the time-dependent diffraction of short wavelength light from laser-excited acoustic waves in the metalattices, we extract their acoustic dispersion, Young's modulus, filling fraction, and thicknesses. Our measurements are in excellent agreement with macroscopic predictions and potentially destructive techniques such as nanoindentation and scanning electron microscopy, with increased accuracy over larger areas. This is interesting because the transport properties of these metalattices do not obey bulk predictions. Finally, this approach is the only way to validate the filling fraction of metalattices over macroscopic areas. These combined capabilities can enable accurate synthesis of nanoenhanced materials.

Osteocytes reside within a heavily mineralized matrix making them difficult to study in vivo and to extract for studies in vitro. IDG-SW3 cells are capable of producing mineralized collagen matrix and transitioning from osteoblasts to mature osteocytes, thus offering an alternative to study osteoblast to late osteocyte differentiation in vitro. The goal for this work was to develop a 3D degradable hydrogel to support IDG-SW3 differentiation and deposition of bone ECM. In 2D, the genes Mmp2 and Mmp13 increased during IDG-SW3 differentiation and were used as targets to create a MMP-sensitive poly(ethylene glycol) hydrogel containing the peptide crosslink GCGPLG-LWARCG and RGD to promote cell attachment. IDG-SW3 differentiation in the MMP-sensitive hydrogels improved over non-degradable hydrogels and standard 2D culture. Alkaline phosphatase activity at day 14 was higher, Dmp1 and Phex were 8.1-fold and 3.8-fold higher, respectively, and DMP1 protein expression was more pronounced in the MMP-sensitive hydrogels compared to non-degradable hydrogels. Cell-encapsulation density (cells/ml precursor) influenced formation of dendrite-like cellular process and mineral and collagen deposition with 80×10(6) performing better than 2×10(6) or 20×10(6), while connexin 43 was not affected by cell density. The cell density effects were more pronounced in the MMP-sensitive hydrogels over non-degradable hydrogels. This study identified that high cell encapsulation density and a hydrogel susceptible to cell-mediated degradation enhanced mineralized collagen matrix and osteocyte differentiation. Overall, a promising hydrogel is presented that supports IDG-SW3 cell maturation from osteoblasts to osteocytes in 3D.

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.

Mechanical unloading decreases bone volume and strength. In humans and mice, bone mineral density is highly heritable, and in mice the response to changes in loading varies with genetic background. Thus, genetic variability may affect the response of bone to unloading. As a first step to identify genes involved in bones' response to unloading, we evaluated the effects of unloading in eight inbred mouse strains: C57BL/6J, PWK/PhJ, WSB/EiJ, A/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, and CAST/EiJ. C57BL/6J and NOD/ShiLtJ mice had the greatest unloading-induced loss of diaphyseal cortical bone volume and strength. NZO/HlLtJ mice had the greatest metaphyseal trabecular bone loss, and C57BL/6J, WSB/EiJ, NOD/ShiLtJ, and CAST/EiJ mice had the greatest epiphyseal trabecular bone loss. Bone loss in the epiphyses displayed the highest heritability. With immobilization, mineral:matrix was reduced, and carbonate:phosphate and crystallinity were increased. A/J mice displayed the greatest unloading-induced loss of mineral:matrix. Changes in gene expression in response to unloading were greatest in NOD/ShiLtJ and CAST/EiJ mice. The most upregulated genes in response to unloading were associated with increased collagen synthesis and extracellular matrix formation. Our results demonstrate a strong differential response to unloading as a function of strain. Diversity outbred (DO) mice are a high-resolution mapping population derived from these eight inbred founder strains. These results suggest DO mice will be highly suited for examining the genetic basis of the skeletal response to unloading., Highlights • Mouse strain affects bone's response to immobilization. • Magnitude of bone loss from immobilization is heritable. • Bone transcriptomic response to immobilization is influenced by genetic variation.

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.

Genome-wide association studies (GWASs) for bone mineral density (BMD) have identified over 1,100 associations to date. However, identifying causal genes implicated by such studies has been challenging. Recent advances in the development of transcriptome reference datasets and computational approaches such as transcriptome-wide association studies (TWASs) and expression quantitative trait loci (eQTL) colocalization have proven to be informative in identifying putatively causal genes underlying GWAS associations. Here, we used TWAS/eQTL colocalization in conjunction with transcriptomic data from the Genotype-Tissue Expression (GTEx) project to identify potentially causal genes for the largest BMD GWAS performed to date. Using this approach, we identified 512 genes as significant (Bonferroni PPP6R3, the gene with the strongest support from our analysis which was not previously implicated in the regulation of BMD, for further investigation. We observed that Ppp6r3 deletion in mice decreased BMD. In this work, we provide an updated resource of putatively causal BMD genes and demonstrate that PPP6R3 is a putatively causal BMD GWAS gene. These data increase our understanding of the genetics of BMD and provide further evidence for the utility of combined TWAS/colocalization approaches in untangling the genetics of complex traits.

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.

Individuals with type 2 diabetes (T2D) are at increased risk of fracture, often despite normal bone density. This observation suggests deficits in bone quality in the setting of abnormal glucose homeostasis. The goal of this article is to review recent developments in our understanding of how advanced glycation end products (AGEs) are incorporated into the skeleton with resultant deleterious effects on bone health and structural integrity in patients with T2D.The adverse effects of skeletal AGE accumulation on bone remodeling and the ability of the bone to deform and absorb energy prior to fracture have been demonstrated both at the bench as well as in small human studies; however, questions remain as to how these findings might be better explored in large, population-based investigations.Hyperglycemia drives systemic, circulating AGE formation with subsequent accumulation in the bone tissue. In those with T2D, studies suggest that AGEs diminish fracture resistance, though larger clinical studies are needed to better define the direct role of longstanding AGE accumulation on bone strength in humans as well as to motivate potential interventions to reverse or disrupt skeletal AGE deposition with the goal of fracture prevention.

Purpose of review Individuals with type 2 diabetes (T2D) are at increased risk of fracture, often despite normal bone density. This observation suggests deficits in bone quality in the setting of abnormal glucose homeostasis. The goal of this article is to review recent developments in our understanding of how advanced glycation end products (AGEs) are incorporated into the skeleton with resultant deleterious effects on bone health and structural integrity in patients with T2D. Recent findings The adverse effects of skeletal AGE accumulation on bone remodeling and the ability of the bone to deform and absorb energy prior to fracture have been demonstrated both at the bench as well as in small human studies; however, questions remain as to how these findings might be better explored in large, population-based investigations. Summary Hyperglycemia drives systemic, circulating AGE formation with subsequent accumulation in the bone tissue. In those with T2D, studies suggest that AGEs diminish fracture resistance, though larger clinical studies are needed to better define the direct role of longstanding AGE accumulation on bone strength in humans as well as to motivate potential interventions to reverse or disrupt skeletal AGE deposition with the goal of fracture prevention.

Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.

Focal defects in articular cartilage are unable to self-repair and, if left untreated, are a leading risk factor for osteoarthritis. This study examined cartilage degeneration surrounding a defect and then assessed whether infilling the defect prevents degeneration. We created a focal chondral defect in porcine osteochondral explants and cultured them ex vivo with and without dynamic compressive loading to decouple the role of loading. When compared to a defect in a porcine knee four weeks post-injury, this model captured loss in sulfated glycosaminoglycans (sGAGs) along the defect's edge that was observed in vivo, but this loss was not load dependent. Loading, however, reduced the indentation modulus of the surrounding cartilage. After infilling with in situ polymerized hydrogels that were soft (100 kPa) or stiff (1 MPa) and which produced swelling pressures of 13 and 310 kPa, respectively, sGAG loss was reduced. This reduction correlated with increased hydrogel stiffness and swelling pressure, but was not affected by loading. This ex vivo model recapitulates sGAG loss surrounding a defect and, when infilled with a mechanically supportive hydrogel, degeneration is minimized.

Highlights • Machine learning approaches allow for the simultaneous analysis to an entire microCT dataset to minimize bias and demonstrated that collective microarchitectural changes. • K-Means clusters and Support Vector Machine classification visualization provide intuitive interpretations of the differences in bone structure and microarchitecture between groups. • These techniques are complimentary to common statistical testing and provide additional ways of showing differences between microCT outcomes., The current standard approach for analyzing cortical bone structure and trabecular bone microarchitecture from micro-computed tomography (microCT) is through classic parametric (e.g., ANOVA, Student's T-test) and nonparametric (e.g., Mann-Whitney U test) statistical tests and the reporting of p-values to indicate significance. However, on their own, these univariate assessments of significance fall prey to a number of weaknesses, including an increased chance of Type 1 error from multiple comparisons. Machine learning classification methods (e.g., unsupervised, k-means cluster analysis and supervised Support Vector Machine classification, SVM) simultaneously utilize an entire dataset comprised of many cortical structure or trabecular microarchitecture measures, thus minimizing bias and Type 1 error that are generated through multiple testing. Through simultaneous evaluation of an entire dataset, k-means and SVM thus provide a complementary approach to classic statistical analysis and enable a more robust assessment of microCT measures., Graphical abstract Image, graphical abstract

Cells embedded in the extracellular matrix of tissues play a critical role in maintaining homeostasis while promoting integration and regeneration following damage or disease. Emerging engineered biomaterials utilize decellularized extracellular matrix as a tissue-specific support structure; however, many dense, structured biomaterials unfortunately demonstrate limited formability, fail to promote cell migration, and result in limited tissue repair. Here, we developed a reinforced composite material of densely packed acellular extracellular matrix microparticles in a hydrogel, termed tissue clay, that can be molded and crosslinked to mimic native tissue architecture. We utilized hyaluronic acid-based hydrogels, amorphously packed with acellular articular cartilage tissue particulated to ~125-250 microns in diameter and defined a percolation threshold of 0.57 (v/v) beyond which the compressive modulus exceeded 300kPa. Remarkably, primary chondrocytes recellularized particles within 48 hours, a process driven by chemotaxis, exhibited distributed cellularity in large engineered composites, and expressed genes consistent with native cartilage repair. We additionally demonstrated broad utility of tissue clays through recellularization and persistence of muscle, skin, and cartilage composites in a subcutaneous in vivo mouse model. Our findings suggest optimal strategies and material architectures to balance concurrent demands for large-scale mechanical properties while also supporting integration of dense musculoskeletal and connective tissues.

Mechanical unloading decreases bone volume and strength. In humans and mice, bone mineral density is highly heritable, and in mice the response to changes in loading varies with genetic background. Thus, genetic variability may affect the response of bone to unloading. As a first step to identify genes involved in bone response to unloading, we evaluated the effects of unloading in eight inbred mouse strains: C57BL/6J, PWK/PhJ, WSB/EiJ, A/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, and CAST/EiJ. C57BL/6J and NOD/ShiLtJ mice had the greatest unloading induced loss of diaphyseal cortical bone volume and strength. NZO/HlLtJ mice had the greatest metaphyseal trabecular bone loss, and C57BL/6J, WSB/EiJ, NOD/ShiLtJ, and CAST/EiJ mice had the greatest epiphyseal trabecular loss. Bone loss in the epiphyses displayed the highest heritability. With immobilization, mineral:matrix was reduced, and carbonate:phosphate and crystallinity were increased. A/J mice displayed the greatest unloading induced loss of mineral:matrix. Changes in gene expression in response to unloading were greatest in NOD/ShiLtJ and CAST/EiJ mice. The most upregulated genes in response to unloading were associated with increased collagen synthesis and extracellular matrix formation. Our results demonstrate a strong differential response to unloading as a function of strain. Diversity outbred (DO) mice are a high-resolution mapping population derived from these eight inbred founder strains. These results suggest DO mice will be highly suited for examining the genetic basis of the skeletal response to unloading.

Summary Objective Quantitative, micrometer length scale assessment of human articular cartilage is essential to enable progress toward new functional tissue engineering approaches, including utilization of emerging 3D bioprinting technologies, and for improved computational modeling of the osteochondral unit. Thus the objective of this study was to characterize the structural organization, material properties, and chemical composition of human skeletally mature articular cartilage with respect to depth and defined morphological features: normal to the articulating surface, parallel to the split-line, and transverse to the split-line. Method Three samples from the lateral femoral condyles of 4 healthy adult donors (55–61 years old) were evaluated via histology, second harmonic generation, microindentation, and Raman spectroscopy. All metrics were evaluated as a function of depth and direction relative to the split-line. Results All donors presented with intact and healthy tissue. Collagen fiber orientation varied significantly between testing directions and with increasing depth from the articular surface. Both compressive and tensile modulus increased significantly with depth and differed across the middle and deep zones and depended on orthogonal direction relative to the split-line. Similarly, matrix components varied with both depth and direction, where chondroitin sulfate steadily increased with depth while collagen prevalence was highest in the surface layer. Conclusions Microscale measurements of human articular cartilage demonstrate that properties are both depth-dependent and orthotropic and depend on the underlying tissue structure and composition. These findings improve upon existing knowledge establishing more accurate measurements, with greater degree of depth and spatial specificity, as inputs for tissue engineering and computational modeling.

Bone mineral density and systemic factors are used to assess skeletal health in astronauts. Yet, even in a general population, these measures fail to accurately predict when any individual will fracture. This review considers how long-duration human spaceflight requires evaluation of additional bone structural and material quality measures that contribute to microgravity-induced skeletal fragility. In both humans and small animal models following spaceflight, bone mass is compromised via reduced bone formation and elevated resorption levels. Concurrently, bone structural quality (e.g., trabecular microarchitecture) is diminished and the quality of bone material is reduced via impaired tissue mineralization, maturation, and maintenance (e.g., mediated by osteocytes). Bone structural and material quality are both affected by microgravity and may, together, jeopardize astronaut operational readiness and lead to increased fracture risk upon return to gravitational loading. Future studies need to directly evaluate how bone quality combines with diminished bone mass to influence bone strength and toughness (e.g., resistance to fracture). Bone quality assessment promises to identify novel biomarkers and therapeutic targets.

PURPOSE OF REVIEW: This article reviews recent publications on the effect of type 1 diabetes (T1D) on fracture risk, bone mineral density (BMD), bone structure, and bone tissue quality. Possible fracture prevention strategies for patients with T1D have also been reviewed. RECENT FINDINGS: T1D is associated with substantially elevated fracture risk and modestly low BMD at the femoral neck. However, BMD alone does not explain higher observed fracture risk in T1D. T1D also affects bone macro- and microstructure, characterized by thinner cortices and trabecular bone changes such as thinner and more widely spaced trabeculae. Structural bone deficit is pronounced in the presence of microvascular complications. Tissue-level changes, such as accumulation of advanced glycation endproducts, detrimental alterations of the mineral phase because of low bone turnover, and occlusion of vascular channels in bone by mineralized tissue, are implicated in pathophysiology of bone fragility in T1D. There are no guidelines on screening and prevention of osteoporotic fractures in T1D. SUMMARY: More studies are needed to understand the influence of T1D on structural bone quality and tissue material properties. There is a need for a prospective study to evaluate better screening strategies for diagnosis and treatment of osteoporosis in T1D.

Osteocytes can participate in systemic mineral homeostasis through perilacunar maintenance and remodeling, where changes to osteocyte lacunar morphology may affect bone structural integrity, tissue strains, and osteocyte mechanosensitivity. Though aging is associated with both decreased bone quality and altered mineral metabolism, it is not known if osteocyte lacunae undergo age-related changes in geometry. In order to survey lacunar changes with age, we developed an open-source program whereby 3D osteocyte lacunae are automatically segmented and then subsequently reconstructed from confocal laser scanning microscopy (CLSM) depth stacks for quantitative analysis of geometry and orientation. This approach takes advantage of the availability and speed of CLSM while avoiding time-consuming and bias-prone manual segmentation. Unlike conventional approaches used to quantify osteocyte lacunar morphology, CLSM enables facile analysis in three-dimensions with clear identification of osteocyte lacunae. We report that 3D osteocyte lacunae measured by CLSM become smaller, more spherical, more oblate, more spatially disorganized, and more sparsely populated with increased age in C57Bl/6 mouse cortical bone in groups spanning 6–24 months old. Critically, these age-related changes are in large part not observed in 2D analyses from the same samples. These results (1) demonstrate proof-of-concept of an efficient method to quantitatively assess osteocyte lacunae in 3D for application to a wide range of studies and (2) motivate further inquiry into how changes to osteocyte lacunar geometries and perilacunar material contribute to diminished bone quality in aging.

The physis, or growth plate, is a cartilaginous region at the end of children's long bones that serves as the primary center for longitudinal growth and characterizes the immature skeleton. Musculoskeletal injury, including fracture, infection, malignancy, or iatrogenic damage, has risk of physeal damage. Physeal injuries account for 30% of pediatric fractures and may result in impaired bone growth. Once damaged, cartilage tissue within the physis is often replaced by unwanted bony tissue, forming a “bony bar” that can lead to complications such as complete growth arrest, angular or rotational deformities, and altered joint mechanics. Children with a bony bar occupying 50% of their physis undergo corrective osteotomy or bone lengthening procedures. These approaches are complex and have variable success rates. As such, there is a critical need for regenerative approaches to not only prevent initial bony bar formation but also regenerate healthy physeal cartilage following injury. This review describes physeal anatomy, mechanisms of physeal injury, and current treatment options with associated limitations. Furthermore, we provide an overview of the current research using cell-based therapies, growth factors, and biomaterials in the different animal models of injury along with strategic directions for modulating intrinsic injury pathways to inhibit bony bar formation and/or promote physeal tissue formation. Pediatric physeal injuries constitute a unique niche within regenerative medicine for which there is a critical need for research to decrease child morbidity related to this injurious process.

The age at which astronauts experience microgravity is a critical consideration for skeletal health and similarly has clinical relevance for musculoskeletal disuse on Earth. While astronauts are extensively studied for bone and other physiological changes, rodent studies enable direct evaluation of skeletal changes with microgravity. Yet, mouse spaceflight studies have predominately evaluated tissues from young, growing mice. We evaluated bone microarchitecture in tibiae and femurs from Young (9-week-old) and Mature (32-weeks-old) female, C57BL/6N mice flown in microgravity for ~2 and ~3 weeks, respectively. Microgravity-induced changes were both compartment- and site-specific. Changes were greater in trabecular versus cortical bone in Mature mice exposed to microgravity (−40.0% Tb. BV/TV vs −4.4% Ct. BV/TV), and bone loss was greater in the proximal tibia as compared to the distal femur. Trabecular thickness in Young mice increased by +25.0% on Earth and no significant difference following microgravity. In Mature mice exposed to microgravity, trabecular thickness rapidly decreased (−24.5%) while no change was detected in age-matched mice that were maintained on Earth. Mature mice exposed to microgravity experienced greater bone loss than Young mice with net skeletal growth. Moreover, machine learning classification models confirmed that microgravity exposure-driven decrements in trabecular microarchitecture and cortical structure occurred disproportionately in Mature than in Young mice. Our results suggest that age of disuse onset may have clinical implications in osteoporotic or other at-risk populations on Earth and may contribute to understanding bone loss patterns in astronauts.

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.

Cells embedded in the extracellular matrix of tissues play a critical role in maintaining homeostasis while promoting integration and regeneration following damage or disease. Emerging engineered biomaterials utilize decellularized extracellular matrix as a tissue-specific support structure; however, many dense, structured biomaterials unfortunately demonstrate limited formability, fail to promote cell migration, and result in limited tissue repair. Here, we developed a reinforced composite material of densely packed acellular extracellular matrix microparticles in a hydrogel, termed tissue clay, that can be molded and crosslinked to mimic native tissue architecture. We utilized hyaluronic acid-based hydrogels, amorphously packed with acellular articular cartilage tissue particulated to ~125–250 microns in diameter and defined a percolation threshold of 0.57 (v/v) beyond which the compressive modulus exceeded 300kPa. Remarkably, primary chondrocytes recellularized particles within 48 hours, a process driven by chemotaxis, exhibited distributed cellularity in large engineered composites, and expressed genes consistent with native cartilage repair. We additionally demonstrated broad utility of tissue clays through recellularization and persistence of muscle, skin, and cartilage composites in a subcutaneous in vivo mouse model. Our findings suggest optimal strategies and material architectures to balance concurrent demands for large-scale mechanical properties while also supporting recellularization and integration of dense musculoskeletal and connective tissues. TABLE OF CONTENTS ENTRY: We present a new design framework for regenerative articular cartilage scaffolds using acellular extracellular matrix particles, packed beyond a percolation threshold, and crosslinked within chondroinductive hydrogels. Our results suggest that the architecture and the packing, rather than altering the individual components, creates a composite material that can balance mechanics, porosity to enable migration, and tissue specific biochemical interactions with cells. Moreover, we provide a technique that we show is applicable to other tissue types.

Multi-layer hydrogels are promising for tissue engineering due to the ability to control the local properties within each layer. However, the interface that forms between each layer has the potential to affect the performance of the hydrogel. The goals of this study were to characterize how the interface forms via its thickness and mechanical properties, identify its impact on the overall hydrogel properties, and provide new insights into how to control the interface. A photo-clickable poly(ethylene glycol) hydrogel was used to form bilayer hydrogels that were sequentially polymerized in a step-and-repeat process. Different processing conditions were studied: the time (0–20 min) before initiating polymerization of the second layer (soak time, ts) and the hydrogel crosslink density (the same, less crosslinked, or more crosslinked) of the first layer as compared to the second layer. Interface thickness was characterized by confocal microscopy, monomer transport by Fickian diffusion, single and bilayer hydrogel mechanics by bulk moduli measurements, and interface moduli measurements using AFM, nanoindentation, and strain mapping. The interface thickness ranged from ~70 to 600 μm (1–10% of total height) depending on processing conditions, but did not affect the bulk hydrogel modulus. Analysis of monomer transport revealed that convection, due to changes in hydrogel swelling, and diffusion contribute to interface thickness. Nanomechanical analysis of bilayer hydrogels formed from soft (75 kPa) and stiff (250 kPa) layers showed a gradient in elastic modulus across the interface, which corresponded to strain maps. In summary, this work identifies that diffusive and convective transport of monomers across the interface controls its thickness and that a mechanically robust interface forms, which does not affect the hydrogel modulus. By controlling the processing conditions, the thickness of the interface can be tuned without affecting the mechanical properties of the bulk 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.

Bone fragility fractures are caused by low bone mass or impaired bone quality. Osteoblast/osteoclast coordination determines bone mass, but the factors that control bone quality are poorly understood. Osteocytes regulate osteoblast and osteoclast activity on bone surfaces but can also directly reorganize the bone matrix to improve bone quality through perilacunar/canalicular remodeling; however, the molecular mechanisms remain unclear. We previously found that deleting the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-motif (TAZ) from osteoblast-lineage cells caused lethality in mice due to skeletal fragility. Here, we tested the hypothesis that YAP and TAZ regulate osteocyte-mediated bone remodeling by conditional ablation of both YAP and TAZ from mouse osteocytes using 8 kb-DMP1-Cre. Osteocyte-conditional YAP/TAZ deletion reduced bone mass and dysregulated matrix collagen content and organization, which together decreased bone mechanical properties. Further, YAP/TAZ deletion impaired osteocyte perilacunar/canalicular remodeling by reducing canalicular network density, length, and branching, as well as perilacunar flourochrome-labeled mineral deposition. Consistent with recent studies identifying TGF-β as a key inducer of osteocyte expression of matrix-remodeling enzymes, YAP/TAZ deletion in vivo decreased osteocyte expression of matrix proteases MMP13, MMP14, and CTSK. In vitro, pharmacologic inhibition of YAP/TAZ transcriptional activity in osteocyte-like cells abrogated TGF-β-induced matrix protease gene expression. Together, these data show that YAP and TAZ control bone matrix accrual, organization, and mechanical properties by regulating osteocyte-mediated bone remodeling. Elucidating the signaling pathways that control perilacunar/canalicular remodeling may enable future therapeutic targeting of bone quality to reverse skeletal fragility. © 2019 American Society for Bone and Mineral Research.

Osteocytes are bone matrix-entombed cells that form an interconnected network of processes called the lacunar/canalicular system, which enables osteocytes to coordinate bone formation and resorption. Osteocytes indirectly regulate osteoblast and osteoclast activity on bone surfaces but also directly resorb and deposit their surrounding bone matrix through perilacunar/canalicular remodeling. However, the molecular mechanisms by which osteocytes control bone remodeling remain unclear. We previously reported that the transcriptional regulators Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-motif (TAZ) promote bone acquisition in osteoblast-lineage cells. Here, we tested the hypothesis that YAP and TAZ regulate osteocyte-mediated bone remodeling by conditional ablation of both YAP and TAZ from mouse osteocytes using 8kb-DMP1-Cre. Osteocyte conditional YAP/TAZ deletion reduced bone mass and dysregulated matrix collagen content and organization, which together impaired bone mechanical properties. YAP/TAZ deletion reduced osteoblast number and activity and increased osteoclast activity. In addition, YAP/TAZ deletion directly impaired osteocyte lacunar/canalicular network remodeling, reducing canalicular density, length, and branching, but did not alter lacunar size or shape. Further, consistent with recent studies identifying TGF-β signaling as a key inducer of perilacunar/canalicular remodeling through expression of matrix-remodeling enzymes, YAP/TAZ deletion in vivo decreased osteocyte expression of matrix proteases Mmp13, Mmp14, and Cathepsin K. In vitro, pharmacologic inhibition of YAP/TAZ transcriptional activity in osteocyte-like cells abrogated TGF-β-induced protease gene expression. Together, these data show that YAP and TAZ act downstream of TGF-β in osteocytes to control bone matrix accrual, organization, and mechanical properties indirectly by coordinating osteoblast/osteoclast activity and directly by regulating perilacunar/canalicular remodeling.

A predominant challenge in tissue engineering is the need of a robust technique for producing structures with precise three-dimensional control of mechanical properties. In an effort to understand how to control the mechanical and chemical properties of materials patterned using stereolithography, this study focuses on the development of a model that allows for orthogonally programmable elastic modulus and geometry in a 3D hydrogel structure. In this study, we use pediatric physeal tissue engineering as a model application. Pediatric physeal injuries can be detrimental to children because damaged cartilage within the physis can lead to assymetric growth arrest. A challenge related to physeal tissue regeneration is the presence of three distinct zones within the physis or growth plate, where cells evolve differently and are known to be sensitive to the mechanical environment. We use a poly(ethylene glycol) diacrylate based photopolymerization chain-growth reaction with a modulus that can range from 600KPa to 39 MPa to spatially control the mechanical properties in our 3D parts and match the growth plate tissue environment. We apply Fourier transform infrared spectroscopy (FTIR) to quantify the degree of monomer conversion over time and intensity, while comparing it with a mechanical model that relates conversion to modulus. To evaluate the spatially controlled mechanical properties in printed structures, nanoindentation is performed to extract the modulus in each biomimetic zone. In this work we present the implementation of both a gradual change in mechanical properties, as well as a step function in 3D scaffolds on the order of 100 microns. This work enables predictive models of structural properties that can be translated into tissue engineering microstructures that match the native biomechanical environment of any tissue.

There is evidence that spaceflight poses acute and late risks to the central nervous system. To explore possible mechanisms, the proteomic changes following spaceflight in mouse brain were characterized. Space Shuttle Atlantis (STS-135) was launched from the Kennedy Space Center (KSC) on a 13-day mission. Within 3&ndash, 5 h after landing, brain tissue was collected to evaluate protein expression profiles using quantitative proteomic analysis. Our results showed that there were 26 proteins that were significantly altered after spaceflight in the gray and/or white matter. While there was no overlap between the white and gray matter in terms of individual proteins, there was overlap in terms of function, synaptic plasticity, vesical activity, protein/organelle transport, and metabolism. Our data demonstrate that exposure to the spaceflight environment induces significant changes in protein expression related to neuronal structure and metabolic function. This might lead to a significant impact on brain structural and functional integrity that could affect the outcome of space missions.

There is evidence that spaceflight poses acute and late risks on the central nervous system. To explore possible mechanisms, the proteomic changes following spaceflight in mouse brain were characterized. Space Shuttle Atlantis (STS-135) was launched at the Kennedy Space Center on a 13-day mission. Within 3–5 hours after landing, brain tissue was collected to evaluate protein expression profiles using quantitative proteomic analysis. Our results showed that there were 26 proteins that were significantly altered after spaceflight in the grey and/or white matter. While there was no overlap between the white and grey matter in terms of individual proteins, there was overlap in terms of function, synaptic plasticity, vesical activity, protein/organelle transport, and metabolism. Our data demonstrate that exposure to the spaceflight environment induces significant changes in protein expression related to neuronal structure and metabolic function. This might lead to a significant impact on brain structural and functional integrity that could affect the outcome of space missions.

The need for smaller, lighter, and longer lasting rechargeable batteries is projected to increase rapidly in the coming years because of high demand for portable electronics and electric vehicles. While traditional Li-ion batteries use liquid-phase electrolytes, these suffer from safety risks and low energy density. Solid-phase electrolytes can avoid these issues by enabling a Li metal anode, but tend to fail during cycling due to Li metal dendrite growth between the electrodes. Because Li dendrite nucleation and growth can be viewed in terms of the mechanical behavior of the battery components, it is critical to understand the mechanical response of candidate electrolyte materials. In this work, we use nanoindentation and bulk acoustic techniques to characterize the mechanical properties of β-Li3PS4, a promising Li-ion conducting ceramic. We find that the bulk and shear moduli of an 80% dense bulk LPS sample are 10–12 GPa and 5–6 GPa, respectively. Although this value of shear modulus may be too low to p...

Vessel ligation using energy-based surgical devices is steadily replacing conventional closure methods during minimally invasive and open procedures. In exploring the molecular nature of thermally-induced tissue bonds, novel applications for surgical resection and repair may be revealed. This work presents an analysis of the influence of unbound water and hydrophilic glycosaminoglycans on the formation and resilience of vascular seals via: (a) changes in pre-fusion tissue hydration, (b) the enzymatic digestion of glycosaminoglycans (GAGs) prior to fusion and (c) the rehydration of vascular seals following fusion. An 11% increase in pre-fusion unbound water led to an 84% rise in vascular seal strength. The digestion of GAGs prior to fusion led to increases of up to 82% in seal strength, while the rehydration of native and GAG-digested vascular seals decreased strengths by 41 and 44%, respectively. The effects of increased unbound water content prior to fusion combined with the effects of seal rehydration after fusion suggest that the heat-induced displacement of tissue water is a major contributor to tissue adhesion during energy-based vessel sealing. The effects of pre-fusion GAG-digestion on seal integrity indicate that GAGs are inhibitory to the bond formation process during thermal ligation. GAG digestion may allow for increased water transport and protein interaction during the fusion process, leading to the formation of stronger bonds. These findings provide insight into the physiochemical nature of the fusion bond, its potential for optimization in vascular closure and its application to novel strategies for vascular resection and repair.

Chronic kidney disease (CKD) increases bone fracture risk. While the causes of bone fragility in CKD are not clear, the disrupted mineral homeostasis inherent to CKD may cause material quality changes to bone tissue. In this study, 11-week old male C57Bl/6J mice underwent either 5/6th nephrectomy (5/6 Nx) or sham procedures. Mice were fed a normal chow diet and euthanized 11 weeks post-surgery. Moderate CKD with high bone turnover was established in the 5/6 Nx group as determined through serum chemistry and bone gene expression assays. We compared nanoindentation modulus and mineral volume fraction (assessed through quantitative backscattered scanning electron microscopy) at matched sites in arrays placed on the cortical bone of the tibia mid-diaphysis. Trabecular and cortical bone microarchitecture (μCT) and whole bone strength were also evaluated. We found that moderate CKD minimally affected bone microarchitecture and did not influence whole bone strength. Meanwhile, bone material quality decreased with CKD; a pattern of altered tissue maturation was observed with 5/6 Nx whereby the newest 60 micrometers of bone tissue adjacent to the periosteal surface had lower indentation modulus and mineral volume fraction than more interior, older bone. The variance of modulus and mineral volume fraction were also altered following 5/6 Nx, implying that tissue-scale heterogeneity may be negatively affected by CKD. The observed lower bone material quality may play a role in the decreased fracture resistance that is clinically associated with human CKD.