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MK-8722, a systemic pan-AMPK activator improves glucose homeostasis in animal models of diabetes, but causes cardiac hypertrophy # . Two- and 3-dimensional tissue (2D,3D) constructs of human induced pluripotent stem cell derived cardiomyocytes (CM) (iCells, Fujifilm/CDI) were used to probe the translatability of cardiac hypertrophy and the role of AMPK in cardiomyocyte function. Chronic 10-day exposure (2 μM and 10 μM MK-8722) of iCell cardiomyocyte 2D monolayers, probed via monitoring of cellular impedance (CardioECR, Agilent), revealed a decrease in beat amplitude (-13%,2μM;-49%,10μM) as well as an increase in excitability, based on the ability to follow higher pacing frequencies (+81%,2uM;+34%,10uM). Direct force measurements in 3D iCell CM tissues (Biowire II, TARA Biosystems) reported loss of contractility when stimulated at 1Hz (-74%,2μM;-90%,10 μM), as well as changes in passive resting tension (+18%,2μM;-75%,10 μM) over chronic six-week exposure. MK-8722 caused a marked increase in Biowire excitability as revealed by a large drop in the voltage threshold of entrainment of paced tissues (-36%,2μM;-43%,10 μM). The increased excitability of both 2D and 3D CM models suggests a cellular correlate of the increased electrocardiographic QRS amplitude observed in rhesus monkeys upon chronic dosing with MK-8722 # . Hypertrophy of the Biowires could be clearly observed at the end of the 6-week incubation and presented as increased microtissue widths (+5%,2μM; +10%,10μM) and thickened regions of attachment to the chamber force sensors at each end of the Biowire. Volume estimation revealed increases (+22.3%, 2μM; +31.4%, 10 μM) in Biowire tissue volumes that were comparable to heart hypertrophic changes in preclinical test species # (mouse, rat, and rhesus monkey). The effect of 10μM MK-8722 was more pronounced when treatment began before tissues had completed a multi-week electrical stimulation maturation protocol, rather than after. The functional effects in both 2D and 3D CM models, and the contractile and structural readouts of the 3D Biowire model were highly informative in the interpretation of the clinical applicability of the pleiotropic cardiac effects of MK-8722 observed in preclinical testing. # Myers, et al. Science 357, p 507-511 (2017)

In primary osteoarthritis (OA), normal ‘wear and tear’ associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

Joint disorders can be detrimental to quality of life. There is an unmet need for precise functional reconstruction of native-like cartilage and bone tissues in the craniofacial space and particularly for the temporomandibular joint (TMJ). Current surgical methods suffer from lack of precision and comorbidities and frequently involve multiple operations. Studies have sought to improve craniofacial bone grafts without addressing the cartilage, which is essential to TMJ function. For the human-sized TMJ in the Yucatan minipig model, we engineered autologous, biologically, and anatomically matched cartilage-bone grafts for repairing the ramus-condyle unit (RCU), a geometrically intricate structure subjected to complex loading forces. Using image-guided micromilling, anatomically precise scaffolds were created from decellularized bone matrix and infused with autologous adipose-derived chondrogenic and osteogenic progenitor cells. The resulting constructs were cultured in a dual perfusion bioreactor for 5 weeks before implantation. Six months after implantation, the bioengineered RCUs maintained their predefined anatomical structure and regenerated full-thickness, stratified, and mechanically robust cartilage over the underlying bone, to a greater extent than either autologous bone-only engineered grafts or acellular scaffolds. Tracking of implanted cells and parallel bioreactor studies enabled additional insights into the progression of cartilage and bone regeneration. This study demonstrates the feasibility of TMJ regeneration using anatomically precise, autologous, living cartilage-bone grafts for functional, personalized total joint replacement. Inclusion of the adjacent tissues such as soft connective tissues and the TMJ disc could further extend the functional integration of engineered RCUs with the host.

When cultured with sufficient nutrient supply, engineered cartilage synthesizes proteoglycans rapidly, producing an osmotic swelling pressure that destabilizes immature collagen and prevents the development of a robust collagen framework, a hallmark of native cartilage. We hypothesized that mechanically constraining the proteoglycan-induced tissue swelling would enhance construct functional properties through the development of a more stable collagen framework. To test this hypothesis, we developed a novel “cage” growth system to mechanically prevent tissue constructs from swelling while ensuring adequate nutrient supply to the growing construct. The effectiveness of constrained culture was examined by testing constructs embedded within two different scaffolds: agarose and cartilage-derived matrix hydrogel (CDMH). Constructs were seeded with immature bovine chondrocytes and cultured under free swelling (FS) conditions for 14 days with transforming growth factor-β before being placed into a constraining cage for the remainder of culture. Controls were cultured under FS conditions throughout. Agarose constructs cultured in cages did not expand after the day 14 caging while FS constructs expanded to 8 × their day 0 weight after 112 days of culture. In addition to the physical differences in growth, by day 56, caged constructs had higher equilibrium (agarose: 639 ± 179 kPa and CDMH: 608 ± 257 kPa) and dynamic compressive moduli (agarose: 3.4 ± 1.0 MPa and CDMH 2.8 ± 1.0 MPa) than FS constructs (agarose: 193 ± 74 kPa and 1.1 ± 0.5 MPa and CDMH: 317 ± 93 kPa and 1.8 ± 1.0 MPa for equilibrium and dynamic properties, respectively). Interestingly, when normalized to final day wet weight, cage and FS constructs did not exhibit differences in proteoglycan or collagen content. However, caged culture enhanced collagen maturation through the increased formation of pyridinoline crosslinks and improved collagen matrix stability as measured by α-chymotrypsin solubility. These findings demonstrate that physically constrained culture of engineered cartilage constructs improves functional properties through improved collagen network maturity and stability. We anticipate that constrained culture may benefit other reported engineered cartilage systems that exhibit a mismatch in proteoglycan and collagen synthesis.

The ability to maintain living articular cartilage tissue in long-term culture can serve as a valuable analytical research tool, allowing for direct examination of mechanical or chemical perturbations on tissue behavior. A fundamental challenge for this technique is the recreation of the salient environmental conditions of the synovial joint in culture that are required to maintain native cartilage homeostasis. Interestingly, conventional media formulations used in explanted cartilage tissue culture investigations often consist of levels of metabolic mediators that deviate greatly from their concentrations in synovial fluid (SF). Here, we hypothesize that the utilization of a culture medium consisting of near-physiologic levels of several highly influential metabolic mediators (glucose, amino acids, cortisol, insulin, and ascorbic acid) will maintain the homeostasis of cartilage explants as assessed by their mechanical properties and extracellular matrix (ECM) contents. Results demonstrate that the aforementioned mediators have a strong effect on the mechanical and biochemical stability of skeletally immature bovine cartilage explants. Most notably, (1) in the absence of cortisol, explants exhibit extensive swelling and tissue softening and (2) in the presence of supraphysiologic levels of anabolic mediators (glucose, amino acids, insulin), explants exhibit increased matrix accumulation and tissue stiffening. In contrast, the administration of physiologic levels of these mediators (as present in native SF) greatly improves the stability of live cartilage explants over one month of culture. These results may have broad applicability for articular cartilage and other musculoskeletal tissue research, setting the foundation for important culture formulations required for examinations into tissue behavior.

This study investigated wear damage of immature bovine articular cartilage using reciprocal sliding of tibial cartilage strips against glass or cartilage. Experiments were conducted in physiological buffered saline (PBS) or mature bovine synovial fluid (SF). A total of 63 samples were tested, of which 47 exhibited wear damage due to delamination of the cartilage surface initiated in the middle zone, with no evidence of abrasive wear. There was no difference between the friction coefficient of damaged and undamaged samples, showing that delamination wear occurs even when friction remains low under a migrating contact area configuration. No difference was observed in the onset of damage or in the friction coefficient between samples tested in PBS or SF. The onset of damage occurred earlier when testing cartilage against glass versus cartilage against cartilage, supporting the hypothesis that delamination occurs due to fatigue failure of the collagen in the middle zone, since stiffer glass produces higher strains and tensile stresses under comparable loads. The findings of this study are novel because they establish that delamination of the articular surface, starting in the middle zone, may represent a primary mechanism of failure. Based on preliminary data, it is reasonable to hypothesize that delamination wear via subsurface fatigue failure is similarly the primary mechanism of human cartilage wear under normal loading conditions, albeit requiring far more cycles of loading than in immature bovine cartilage.

In this study, we explored the application of lipid-shelled, gas-filled microbubbles as a method for creating on-demand microporous hydrogels for cartilage tissue engineering. The technique allowed for homogenous distribution of cells and micropores within the scaffold, increasing the absorption coefficient of large solutes (70kDa dextran) over controls in a concentration-dependent manner. The stability of the gas phase of the microbubbles depended on several factors, including the initial size distribution of the microbubble suspension, as well as the temperature and pressure during culture. Application of pressure cycles provided controlled release of the gas phase to generate fluid-filled micropores with remnant lipid. The resulting microporous agarose scaffolds were biocompatible, leading to a twofold increase in engineered cartilage properties (E(Y)=492±42kPa for the bubble group vs. 249±49kPa for the bubble-free control group) over a 42-day culture period. Our results suggest that microbubbles offer a simple and robust method of modulating mass transfer in cell-seeded hydrogels through mild pressurization, and the methodology may be expanded in the future to include focused ultrasound for improved spatio-temporal control.

This study presents a damage mechanics framework that employs observable state variables to describe damage in isotropic or anisotropic fibrous tissues. In this mixture theory framework, damage is tracked by the mass fraction of bonds that have broken. Anisotropic damage is subsumed in the assumption that multiple bond species may coexist in a material, each having its own damage behaviour. This approach recovers the classical damage mechanics formulation for isotropic materials, but does not appeal to a tensorial damage measure for anisotropic materials. In contrast with the classical approach, the use of observable state variables for damage allows direct comparison of model predictions to experimental damage measures, such as biochemical assays or Raman spectroscopy. Investigations of damage in discrete fibre distributions demonstrate that the resilience to damage increases with the number of fibre bundles; idealizing fibrous tissues using continuous fibre distribution models precludes the modelling of damage. This damage framework was used to test and validate the hypothesis that growth of cartilage constructs can lead to damage of the synthesized collagen matrix due to excessive swelling caused by synthesized glycosaminoglycans. Therefore, alternative strategies must be implemented in tissue engineering studies to prevent collagen damage during the growth process.

Transforming growth factor beta (TGF-β) has become one of the most widely utilized mediators of engineered cartilage growth. It is typically exogenously supplemented in the culture medium in its active form, with the expectation that it will readily transport into tissue constructs through passive diffusion and influence cellular biosynthesis uniformly. The results of this investigation advance three novel concepts regarding the role of TGF-β in cartilage tissue engineering that have important implications for tissue development. First, through the experimental and computational analysis of TGF-β concentration distributions, we demonstrate that, contrary to conventional expectations, media-supplemented exogenous active TGF-β exhibits a pronounced concentration gradient in tissue constructs, resulting from a combination of high-affinity binding interactions and a high cellular internalization rate. These gradients are sustained throughout the entire culture duration, leading to highly heterogeneous tissue growth; biochemical and histological measurements support that while biochemical content is enhanced up to 4-fold at the construct periphery, enhancements are entirely absent beyond 1 mm from the construct surface. Second, construct-encapsulated chondrocytes continuously secrete large amounts of endogenous TGF-β in its latent form, a portion of which undergoes cell-mediated activation and enhances biosynthesis uniformly throughout the tissue. Finally, motivated by these prior insights, we demonstrate that the alternative supplementation of additional exogenous latent TGF-β enhances biosynthesis uniformly throughout tissue constructs, leading to enhanced but homogeneous tissue growth. This novel demonstration suggests that latent TGF-β supplementation may be utilized as an important tool for the translational engineering of large cartilage constructs that will be required to repair the large osteoarthritic defects observed clinically.

The frictional response of whole human joints over durations spanning activities of daily living has not been reported previously. This study measured the friction of human glenohumeral joints during 24 h of reciprocal loading in a pendulum testing device, at moderate (0.2 mm/s, 4320 cycles) and low (0.02 mm/s, 432 cycles) sliding speeds, under a 200 N load. The effect of joint congruence was also investigated by testing human humeral heads against significantly larger mature bovine glenoids. Eight human joints and six bovine joints were tested in four combinations: human joints tested at moderate (hHCMS, n=6) and low speed (hHCLS, n=3), human humeral heads tested against bovine glenoids at moderate speed (LCMS, n=3), and bovine joints tested at moderate speed (bHCMS, n=3). In the first half hour the mean±standard deviation of the friction coefficient was hHCMS: 0.0016±0.0011, hHCLS: 0.0012±0.0002, LCMS: 0.0008±0.0002 and bHCMS: 0.0024±0.0008; in the last four hours it was hHCMS: 0.0057±0.0025, hHCLS: 0.0047±0.0017, LCMS: 0.0012±0.0003 and bHCMS: 0.0056±0.0016. The initial value was lower than the final value (p

The objective of this study was to measure the wear response of immature bovine articular cartilage tested against glass or alloys used in hemiarthroplasties. Two cobalt chromium alloys and a stainless steel alloy were selected for these investigations. The surface roughness of one of the cobalt chromium alloys was also varied within the range considered acceptable by regulatory agencies. Cartilage disks were tested in a configuration that promoted loss of interstitial fluid pressurization to accelerate conditions believed to occur in hemiarthroplasties. Results showed that considerably more damage occurred in cartilage samples tested against stainless steel (10 nm roughness) and low carbon cobalt chromium alloy (27 nm roughness) compared to glass (10 nm) and smoother low or high carbon cobalt chromium (10 nm). The two materials producing the greatest damage also exhibited higher equilibrium friction coefficients. Cartilage damage occurred primarily in the form of delamination at the interface between the superficial tangential zone and the transitional middle zone, with much less evidence of abrasive wear at the articular surface. These results suggest that cartilage damage from frictional loading occurs as a result of subsurface fatigue failure leading to the delamination. Surface chemistry and surface roughness of implant materials can have a significant influence on tissue damage, even when using materials and roughness values that satisfy regulatory requirements.

Previously, microbubbles have been studied for a number of different medical applications including ultrasound imaging contrast and drug delivery [1]. Microbubbles are comprised of a gas enclosed in a lipid shell. Recent research has shown that the inclusion of microbubbles in tissue engineered cartilage constructs has been shown to enhance mechanical and biochemical growth [2,3]. This modification of the tissue engineering scaffold by incorporation of gas-filled microbubbles has been shown to homogenize depth-dependent mechanical properties (Fig. 1) [3], which, in standard constructs, resembles a “U-shaped” strain profile with the stiffest regions on the edges surrounding a soft center [4]. In addition, these microbubble containing constructs are described by a higher partition coefficient than standard constructs, indicating increased solute transport [3]. These results led us to propose the hypothesis that the incorporation of microbubbles: a) increases nutrient transport upon microbubble dissolution, b) creates fluid-filled pores upon gas efflux and subsequent influx of culture media [3]. In this study, the aforementioned hypothesis is interrogated through analysis of local solute diffusivity.Copyright © 2012 by ASME

Residual stress plays an important role in determining arterial wall stress and wall remodeling. We studied the effect of collagenase treatment on the opening angle of arteries. Our results demonstrated that collagenase treatment significantly reduced the opening angle of arteries and diminished the effect of smooth muscle contraction on the opening angles. Changes in opening angle need to be considered in studying arterial wall stress and buckling associated with wall remodeling and diseases.