Objective Women have a higher prevalence and burden of joint injuries and pathologies involving articular cartilage than men. Although knee injuries affecting young women are on the rise, most studies related to sexual dimorphism target postmenopausal women. We hypothesize that sexual dimorphism in cartilage structure and mechanics is present before menopause, which can contribute to sex disparities in cartilage pathologies. Design Bovine knee was used as a model to study healthy adult cartilage. We compared elastic moduli under compression, abundances of extracellular and pericellular matrix (PCM) proteins using proteomics, and PCM constituency with tissue immunofluorescence. The gene expression of matrix-related genes under basal, anabolic, and catabolic conditions was assessed by quantitative polymerase chain reaction (qPCR). Results The equilibrium modulus was higher in male cartilage compared with female cartilage. Proteoglycans were not associated with this biomechanical dimorphism. Proteomic and pathway analyses of tissue showed dimorphic enriched pathways in extracellular matrix (ECM)-related proteins in which male cartilage was enriched in matrix interconnectors and crosslinkers that strengthen the ECM network. Moreover, male and female tissue differed in enriched PCM components. Females had more abundance of collagen type VI and decorin, suggesting different PCM mechanics. Furthermore, the activation of regenerative and catabolic function in chondrocytes triggered sex-dependent signatures in gene expression, indicating dimorphic genetic regulation that is dependent on stimulation. Conclusions We provide evidence for sexual dimorphism in cartilage before menopause. Some differences are intrinsic to chondrocytes’ gene expression defined by their XX versus XY chromosomal constituency.

Intravascular stents for pediatric patients that degrade without inhibiting vessel growth remain a clinical challenge. Here, poly(L-lactide) fibers (DH-BDS) at two thicknesses, 250 μm and 300 μm, were assembled into large, pediatric-sized stents (Ø10 - Ø20 mm). Fibers were characterized mechanically and thermally, then stent mechanical properties were compared to metal controls, while mass loss and degradation kinetics modeling estimated total stent degradation time. Thicker fibers displayed lower stiffness (1969 ± 44 vs 2126 ± 37 MPa) and yield stress (117 ± 12 vs 137 ± 5 MPa) than thinner counterparts, but exhibited similar fail strength (478 ± 28 vs 476 ± 16 MPa) at higher strains (47 ± 2 vs 44 ± 2%). Stents all exhibited crystallinity between 51.3 - 54.4% and fiber glass transition temperatures of 88.6 ± 0.5 °C and 84.6 ± 0.5 °C were well above physiological ranges. Radial strength (0.31 ± 0.01 - 0.34 ± 0.02 N/mm) in thinner stents was similar to metal stents (0.24 - 0.41 N/mm) up to Ø14 mm with no foreshortening and thicker coils granted comparable radial strength (0.32 ± 0.02 - 0.34 ± 0.02 N/mm) in stents larger than Ø14 mm. Both 10 mm (1.17 ± 0.02 % and 0.86 ± 0.1 %) and 12 mm (1.1 ± 0.03% and 0.89 ± 0.1%) stents exhibited minimal weight loss over one year. Degradation kinetics models predicted full stent degradation within 2.8 - 4.5 years depending on thickness. DH-BDS exhibiting hoop strength similar to metal stents and demonstrating minimal degradation and strength loss over the first year before completely disappearing within 3 to 4.5 years show promise as a pediatric interventional alternative to current strategies.

Bioresorbable materials have been frequently used to three-dimensional (3D) print biomedical structures. In this study, we developed a technique to 3D print poly(glycerol sebacate fumarate) gadodiamide (Rylar)-poly(ethylene glycol) diacrylate (PEGDA) samples and investigated their mechanical and thermal properties as a function of (PS) and ultraviolet intensity (UVI). The Young's modulus (E), ultimate tensile strength (UTS), failure strain (ɛF ), and glass transition temperature (Tg ) showed strong correlation with PS and UVI. Results showed E to be between 1.31 and 3.12 MPa, UTS between 0.07 and 0.43 MPa, and ɛF between 7 and 20% with brittle failure. The Tg was observed to lie between -54.48 and -49.10 °C without secondary/tertiary transitions. Dominant elastic behavior was observed from the dynamic mechanical testing viscoelastic data. Testing results were used to develop a regression predictive model for E as a function of PS and UVI. The model performance was evaluated experimentally with an average absolute error of 3.62%. The E and stress-strain response of our 3D printed samples show agreement with published data for human tracheal cartilage, and the mechanical properties were comparable to other published soft polymeric scaffolds/patches. The E' moduli were also similar to bovine articular cartilage. We have successfully demonstrated that Rylar, a novel bioresorbable radiopaque polymer, when blended with PEGDA can be 3D printed controllably for soft tissue applications such as airway obstructions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 664-671, 2019.

Severe burn causes systemic inflammation and hypercatabolism, resulting in damage to multiple organs distant to the burn site, including the musculoskeletal system. Bone mass and muscle loss have been reported. However, tendon that connects bone and muscle has not been studied in comparable detail. Here we aimed to characterize the molecular and functional changes in Achilles tendon triggered by severe burn. Forty male Sprague-Dawley rats received 40% total body surface area scald burn. Achilles tendons were collected up to 14 days postburn. Sham-treated animals served as a control group. We analyzed tendons for changes in expression of IL-6, IL-1β, TNF, MMP9, MMP13, TGFβ1, Collagens I and III, and for morphological and biomechanical changes. Gene expression of IL-6 and IL-1β as well as MMP9 and MMP13 increased in rat tendon 3 days after burn. Col3a1 increased at day 3 and col1a1 at day 7. At day 14, TGFβ1 increased, whereas the protein ratio for collagens I/III decreased, indicating tendon remodeling. Histological analysis with H&E and Picrosirius red staining further revealed a decrease in organized collagen fibers 14 days after burn. Biomechanical analysis showed a decrease in stiffness and ultimate force of tendons in burn rats.We conclude that tendinopathy was observed in Achilles tendon 14 days after severe burn, via the induction of inflammation and remodeling. The present study provides a model of tendinopathy that may be used for the development of therapeutic approaches after burn.

Human embryonic stem cell-derived cardiomyocytes (hESC-CM) are being developed for tissue repair and as a model system for cardiac physiology and pathophysiology. However, the signaling requirements of their growth have not yet been fully characterized. We showed that hESC-CM retain their capacity for increase in size in long-term culture. Exposing hESC-CM to hypertrophic stimuli such as equiaxial cyclic stretch, angiotensin II, and phenylephrine (PE) increased cell size and volume, percentage of hESC-CM with organized sarcomeres, levels of ANF, and cytoskeletal assembly. PE effects on cell size were separable from those on cell cycle. Changes in cell size by PE were completely inhibited by p38-MAPK, calcineurin/FKBP, and mTOR blockers. p38-MAPK and calcineurin were also implicated in basal cell growth. Inhibitors of ERK, JNK, and CaMK II partially reduced PE effects; PKG or GSK3β inhibitors had no effect. The role of p38-MAPK was confirmed by an additional pharmacological inhibitor and adenoviral infection of hESC-CM with a dominant-inhibitory form of p38-MAPK. Infection of hESC-CM with constitutively active upstream MAP2K3b resulted in an increased cell size, sarcomere and cytoskeletal assembly, elongation of the cells, and induction of ANF mRNA levels. siRNA knockdown of p38-MAPK inhibited PE-induced effects on cell size. These results reveal an important role for active protein kinase signaling in hESC-CM growth and hypertrophy, with potential implications for hESC-CM as a novel in vitro test system. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

Regeneration or repair of the damaged myocardium requires different strategies including engineered constructs for more efficient cell delivery. This study was undertaken to examine the potential of a new nanostructured elastomer to deliver embryonic stem cell-derived cardiomyocytes (ESC-CM) to an infarcted area of the myocardium. Engineered materials were biocompatible, mechanically stable, and elastomeric nanocomposites serving as substrates for delivery of ESC-CM and as a left ventricular support device in myocardial regeneration strategies. Materials investigated were soft and strong poly(aliphatic/aromatic-ester) multiblock thermoplastic elastomers with poly(ethylene terephthalate) (PET) hard segments and dimerized fatty acid, i.e., dilinoleic acid (DLA) soft segments, respectively, with and without addition of 0.2 wt% TiO2 nanoparticles to form nanocomposites. The PET/DLA-TiO2 nanocomposite exhibited over 8 MPa tensile strength and 900% elongation at break. Addition of TiO2 nanoparticles significantly altered surface roughness and enhanced adhesion and spreading of ESC-CM derived from mouse and human embryonic stem cells. The newly developed materials did not affect the functional activity of spontaneously beating hESC-CM, as demonstrated by unaltered rate of their beating, and the cells continued to demonstrate contractile activity on the materials for more than two months in culture (the longest time tested). Quantitative proliferation and survival assays using fibroblasts confirmed the ability of the new materials to support cells as well as or better than the present commercial-type thermoplastic elastomer analog. The results indicate that PET/DLA and PET/DLA-TiO2 are promising candidates for the manufacture of engineered patches to deliver ESC-CMs.

We hypothesize that a combinatorial approach of ventricle constraint and stem cell therapy would offer a greater benefit for the treatment of heart failure than either strategy alone. A heart patch would serve two therapeutic purposes: biomechanical support and cell delivery. In this study, we describe a hybrid heart patch engineered from a synthetic elastomer, poly(glycerol sebacate) (PGS), supplemented with cardiomyocytes differentiated from human embryonic stem cells (hESCs). In line with two therapeutically relevant considerations, i.e. biocompatibility and cell delivery efficiency, the PGS was (a) pre-conditioned in culture medium for 6 days, and (b) prepared without gelatin coatings to facilitate detachment and delivery of cardiomyocytes following patch implantation. Following pre-conditioning under physiological conditions, the PGS patch material without gelatin coating was found to satisfactorily support cardiomyocyte viability and attachment, with active cell beating for periods of longer than 3 months until interrupted. Dynamic culture studies revealed that cells detached more efficiently from the uncoated surface of PGS than from gelatin-coated PGS. No significant differences were detected between the beating rates of human embryonic stem cell-derived cardiomyocytes on tissue culture plate and the pre-conditioned and gelatin-uncoated PGS. PGS patches sutured over the left ventricle of rats in vivo remained intact over a 2 week period without any deleterious effects on ventricular function. We conclude that PGS is a suitable biomaterial for stem cell-based regeneration strategies to restore cardiomyocyte function, and the hybrid heart patch engineered under optimal conditions would be a promising support device for the cardiac repair.

Treatment of human disease with human embryonic stem cell (hESC)-derived cells is now close to reality, but little is known of their responses to physiological and pathological insult. The ability of cells to respond via activation of Toll like receptors (TLR) is critical in innate immune sensing in most tissues, but also extends to more general danger sensing, e.g. of oxidative stress, in cardiomyocytes. We used biomarker release and gene-array analysis to compare responses in hESC before and after differentiation, and to those in primary human endothelial cells. The presence of cardiomyocytes and endothelial cells was confirmed in differentiated cultures by immunostaining, FACS-sorting and, for cardiomyocytes, beating activity. Undifferentiated hESC did not respond with CXCL8 release to Gram positive or Gram negative bacteria, or a range of PAMPs (pathogen associated molecular patterns) for TLRs 1-9 (apart from flagellin, an activator of TLR5). Surprisingly, lack of TLR-dependent responses was maintained over 4 months of differentiation of hESC, in cultures which included cardiomyocytes and endothelial cells. In contrast, primary cultures of human aortic endothelial cells (HAEC) demonstrated responses to a broad range of PAMPs. Expression of downstream TLR signalling pathways was demonstrated in hESC, and IL-1beta, TNFalpha and INFgamma, which bypass the TLRs, stimulated CXCL8 release. NFkappaB pathway expression was also present in hESC and NFkappaB was able to translocate to the nucleus. Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals. TLR5 levels were similar between differentiated hESC and HAEC, and siRNA knockdown of TLR5 abolished the response to flagellin. These findings have potential implications for survival and function of grafted hESC-derived cells.

Human embryonic stem cells can be differentiated into the cardiomyocyte phenotype (hESCM), and these cells are a subject of study for cardiac repair and as a model system for investigation of cardiac physiology. Endothelin is a key hormone in cardiac physiology and pathophysiology: we have therefore characterised the responses of hESCM to this agent. HESCM were derived from the H7 hESC line via embryoid body (EB) formation in 20% FCS. Individual EBs were transferred to a bath on a Nikon TE2000 microscope and superfused at 32 °C in 2 mM Ca. Spontaneous beating was tracked using the IonOptix system. After the first 30 min, beating was stable in rate and amplitude for 3–4 h. EBs on glass coverslips could be returned to culture and studied on different days: rate of beating increased significantly with time in culture. Endothelin-1 (ET1, 10 nM) increased beating rate of hESCM from 15.3±2.8 beats per min (bpm, mean±sem) to 27.7±5.3 bpm (P<0.05, n=6) in early cultures at day 17 of differentiation or less. Increased rate was not blocked by 1 μM of ETA antagonist ABT627 (10.0±2.0 vs. 17.2±2.0 bpm, n=6, P<0.05). After day 43 of differentiation, ET-1 no longer increased rate significantly (70.7±8.6 vs. 78.5±12.2, n=6, NS) and at days 69–70 there could be a slight decrease. The presence of ETB antagonist A-192621 (100 nM) revealed a significant decrease in rate after addition of 10 nM ET1, in hESCM between days 43–79 (71.3±19.1 vs. 62.1±18.1, n=7, P<0.05). This suggests that ET receptors control rate in hESCM, and that an increase in ETA receptor signalling with day of development may progressively oppose an ETB-mediated positive chronotropic response.