Activation of Wnt/ß-catenin signalling and HIF1α stabilisation alters pluripotency and differentiation/proliferation properties of human-induced pluripotent stem cells.

BACKGROUND INFORMATION: Wnt/ß-catenin signalling, in the microenvironment of pluripotent stem cells (PSCs), plays a critical role in their differentiation and proliferation. Contradictory reports on the role of Wnt/ß-catenin signalling in PSCs self-renewal and differentiation, however, render these mechanisms largely unclear. RESULTS: Wnt/ß-catenin signalling pathway in human-induced pluripotent stem cells (hiPSCs) was activated by inhibiting glycogen synthase kinase 3 (GSK3), driving the cells into a mesodermal/mesenchymal state, exhibiting proliferative, invasive and anchorage-independent growth properties, where over 70% of cell population became CD 44 (+)/CD133 (+). Wnt/ß-catenin signalling activation also altered the metabolic state of hiPSCs from aerobic glycolysis to oxidative metabolism and changed their drug and oxidative stress sensitivities. These effects of GSK3 inhibition were suppressed in HIF1α-stabilised cells. CONCLUSIONS: Persistent activation of Wnt/ß-catenin signalling endows hiPSCs with proliferative/invasive 'teratoma-like' states, shifting their metabolic dependence and allowing HIF1α-stabilisation to inhibit their proliferative/invasive properties. SIGNIFICANCE: The hiPSC potential to differentiate into 'teratoma-like' cells suggest that stem cells may exist in two states with differential metabolic and drug dependency.

Viable Vitreous Grafts of Whole Porcine Menisci for Transplant in the Knee and Temporomandibular Joints.

The shortage of suitable donor meniscus grafts from the knee and temporomandibular joint (TMJ) impedes treatments for millions of patients. Vitrification offers a promising solution by transitioning these tissues into a vitreous state at cryogenic temperatures, protecting them from ice crystal damage using high concentrations of cryoprotectant agents (CPAs). However, vitrification's success is hindered for larger tissues (>3 mL) due to challenges in CPA penetration. Dense avascular meniscus tissues require extended CPA exposure for adequate penetration; however, prolonged exposure becomes cytotoxic. Balancing penetration and reducing cell toxicity is required. To overcome this hurdle, a simulation-based optimization approach is developed by combining computational modeling with microcomputed tomography (µCT) imaging to predict 3D CPA distributions within tissues over time accurately. This approach minimizes CPA exposure time, resulting in 85% viability in 4-mL meniscal specimens, 70% in 10-mL whole knee menisci, and 85% in 15-mL whole TMJ menisci (i.e., TMJ disc) post-vitrification, outperforming slow-freezing methods (20%-40%), in a pig model. The extracellular matrix (ECM) structure and biomechanical strength of vitreous tissues remain largely intact. Vitreous meniscus grafts demonstrate clinical-level viability (≥70%), closely resembling the material properties of native tissues, with long-term availability for transplantation. The enhanced vitrification technology opens new possibilities for other avascular grafts.

Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor.

Cardiac ryanodine receptor (RyR2) is a homotetramer of 560 kDa polypeptides regulated by calmodulin (CaM), which decreases its open probability at diastolic and systolic Ca(2+) concentrations. Point mutations in the CaM-binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2(ADA)) in mice result in severe cardiac hypertrophy, poor left ventricle contraction and death by postnatal day 16, suggesting that CaM inhibition of RyR2 is required for normal cardiac function. Here, we report on Ca(2+) signalling properties of enzymatically isolated, Fluo-4 dialysed whole cell clamped cardiac myocytes from 10-15-day-old wild-type (WT) and homozygous Ryr2(ADA/ADA) mice. Spontaneously occurring Ca(2+) spark frequency, measured at -80 mV, was 14-fold lower in mutant compared to WT myocytes. ICa, though significantly smaller in mutant myocytes, triggered Ca(2+) transients that were of comparable size to those of WT myocytes, but with slower activation and decay kinetics. Caffeine-triggered Ca(2+) transients were about three times larger in mutant myocytes, generating three- to four-fold bigger Na(+)-Ca(2+) exchanger NCX currents (INCX). Mutant myocytes often exhibited Ca(2+) transients of variable size and duration that were accompanied by similarly alternating and slowly activating INCX. The data suggest that RyR2(ADA) mutation produces significant reduction in ICa density and ICa-triggered Ca(2+) release gain, longer but infrequently occurring Ca(2+) sparks, larger sarcoplasmic reticulum Ca(2+) loads, and spontaneous Ca(2+) releases accompanied by activation of large and potentially arrhythmogenic inward INCX.

Sexual dimorphisms in three-dimensional masticatory muscle attachment morphometry regulates temporomandibular joint mechanics.

Temporomandibular joint (TMJ) disorders disproportionally affect females, with female to male prevalence varying from 3:1 to 8:1. Sexual dimorphisms in masticatory muscle attachment morphometry and association with craniofacial size, critical for understanding sex-differences in TMJ function, have not been reported. The objective of this study was to determine sex-specific differences in three-dimensional (3D) TMJ muscle attachment morphometry and craniofacial sizes and their impact on TMJ mechanics. Human cadaveric TMJ muscle attachment morphometry and craniofacial anthropometry (10Males; 11Females) were determined by previously developed 3D digitization and imaging-based methods. Sex-differences in muscle attachment morphometry and craniofacial anthropometry, and their correlation were determined, respectively using multivariate general linear and linear regression statistical models. Subject-specific musculoskeletal models of the mandible were developed to determine effects of sexual dimorphisms in mandibular size and TMJ muscle attachment morphometry on joint loading during static biting. There were significant sex-differences in craniofacial size (p = 0.024) and TMJ muscle attachment morphometry (p < 0.001). TMJ muscle attachment morphometry was significantly correlated with craniofacial size. TMJ contact forces estimated from biomechanical models were significantly, 23% on average (p < 0.001), greater for females compared to those for males when generating the same bite forces. There were significant linear correlations between TMJ contact force and both 3D mandibular length (R2 = 0.48, p < 0.001) and muscle force moment arm ratio (R2 = 0.68, p < 0.001). Sexual dimorphisms in masticatory muscle morphology and craniofacial sizes play critical roles in subject-specific TMJ biomechanics. Sex-specific differences in the TMJ mechanical environment should be further investigated concerning mechanical fatigue of TMJ discs associated with TMJ disorders.

A noninvasive fluorescence imaging-based platform measures 3D anisotropic extracellular diffusion.

Diffusion is a major molecular transport mechanism in biological systems. Quantifying direction-dependent (i.e., anisotropic) diffusion is vitally important to depicting how the three-dimensional (3D) tissue structure and composition affect the biochemical environment, and thus define tissue functions. However, a tool for noninvasively measuring the 3D anisotropic extracellular diffusion of biorelevant molecules is not yet available. Here, we present light-sheet imaging-based Fourier transform fluorescence recovery after photobleaching (LiFT-FRAP), which noninvasively determines 3D diffusion tensors of various biomolecules with diffusivities up to 51 µm2 s-1, reaching the physiological diffusivity range in most biological systems. Using cornea as an example, LiFT-FRAP reveals fundamental limitations of current invasive two-dimensional diffusion measurements, which have drawn controversial conclusions on extracellular diffusion in healthy and clinically treated tissues. Moreover, LiFT-FRAP demonstrates that tissue structural or compositional changes caused by diseases or scaffold fabrication yield direction-dependent diffusion changes. These results demonstrate LiFT-FRAP as a powerful platform technology for studying disease mechanisms, advancing clinical outcomes, and improving tissue engineering.

Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity.

Environmental factors are the largest contributors to cardiovascular disease. Here we show that cardiac organoids that incorporate an oxygen-diffusion gradient and that are stimulated with the neurotransmitter noradrenaline model the structure of the human heart after myocardial infarction (by mimicking the infarcted, border and remote zones), and recapitulate hallmarks of myocardial infarction (in particular, pathological metabolic shifts, fibrosis and calcium handling) at the transcriptomic, structural and functional levels. We also show that the organoids can model hypoxia-enhanced doxorubicin cardiotoxicity. Human organoids that model diseases with non-genetic pathological factors could help with drug screening and development.

Limb bud and flank mesoderm have distinct "physical phenotypes" that may contribute to limb budding.

Limb bud outgrowth in chicken embryos is initiated during the third day of development by Fibroblast Growth Factor 8 (FGF8) produced by the newly formed apical ectodermal ridge (AER). One of the earliest effects of this induction is a change in the properties of the limb field mesoderm leading to bulging of the limb buds from the body wall. Heintzelman et al. [Heintzelman, K.F., Phillips, H.M., Davis, G.S., 1978. Liquid-tissue behavior and differential cohesiveness during chick limb budding. J. Embryol. Exp. Morphol. 47, 1-15.] suggested that budding of the limbs is caused by a higher liquid-like cohesivity of limb bud tissue compared with flank. We sought additional evidence relevant to this hypothesis by performing direct measurements of the effective surface tension, a measure of relative tissue cohesivity, of 4-day embryonic chicken wing and leg bud mesenchymal tissue, and adjacent flank mesoderm. As predicted, the two types of limb tissues were 1.5- to 2-fold more cohesive than the flank tissue. These differences paralleled cell number and volume density differences: 4-day limb buds had 2- to 2.5-fold as many cells per unit area of tissue as surrounding flank, a difference also seen at 3 days, when limb budding begins. Exposure of flank tissue to exogenous FGF8 for 24 h increased its cell number and raised its cohesivity to limb-like values. Four-day flank tissue exhibited a novel and unique active rebound response to compression, which was suppressed by the drug latrunculin and therefore dependent on an intact actin cytoskeleton. Correspondingly, flank at this stage expressed high levels of alpha-smooth muscle actin (SMA) mRNA and protein and a dense network of microfilaments. Treatment of flank with FGF8 eliminated the rebound response. We term material properties of tissues, such as cohesivity and mechanical excitability, the "physical phenotype", and propose that changes thereof are driving forces of morphogenesis. Our results indicate that two independent aspects of the physical phenotype of flank mesoderm can be converted to a limb-like state in response to treatment with FGF8. The higher tissue cohesivity induced by this effect will cause the incipient limb bud to phase separate from the surrounding flank, while the active mechanical response of the flank could help ensure that the limb bud bulges out from, rather than becoming engulfed by, this less cohesive tissue.

Comparison of Oxygen Consumption Rates of Nondegenerate and Degenerate Human Intervertebral Disc Cells.

STUDY DESIGN: In vitro measurements of the oxygen consumption rates (OCR) of human intervertebral disc (IVD) cells. OBJECTIVE: The aim of this study was to determine the differences in the OCR of nondegenerate and degenerate human annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP) cells at different glucose concentrations. SUMMARY OF BACKGROUND DATA: The avascular nature of the IVD creates a delicate balance between rate of nutrient transport through the matrix and rate of disc cell consumption necessary to maintain tissue health. Previous studies have shown a dependence of OCR for animal (e.g., bovine and porcine) IVD cells on oxygen level and glucose concentration. However, the OCR of nondegenerate human IVD cells compared to degenerate human IVD cells at different glucose concentrations has not been investigated. METHODS: IVD cells were isolated from the AF, NP, and CEP regions of human cadaver spines and surgical samples. The changes in oxygen concentration were recorded when cells were sealed in a metabolic chamber. The OCR of cells was determined by curve fitting using the Michaelis-Menton equation. RESULTS: Under identical cell culture conditions, the OCR of degenerate human IVD cells was three to five times greater than that of nondegenerate human IVD cells. The degenerate IVD cells cultured in low-glucose medium (1 mmol/L) exhibited the highest OCR compared to degenerate cells cultured at higher glucose levels (i.e., 5 mmol/L, 25 mmol/L), whereas no significant differences in OCR were found among the nondegenerate IVD cells for all glucose levels. CONCLUSION: Considering the significantly higher OCR and unique response to glucose of degenerate human IVD cells, the degeneration of the IVD is associated with a cell phenotypic change related to OCR. The OCR of IVD cells reported in this study will be valuable for understanding human IVD cellular behavior and tissue nutrition in response to disc degeneration. LEVEL OF EVIDENCE: N/A.

Three-dimensional temporomandibular joint muscle attachment morphometry and its impacts on musculoskeletal modeling.

In musculoskeletal models of the human temporomandibular joint (TMJ), muscles are typically represented by force vectors that connect approximate muscle origin and insertion centroids (centroid-to-centroid force vectors). This simplification assumes equivalent moment arms and muscle lengths for all fibers within a muscle even with complex geometry and may result in inaccurate estimations of muscle force and joint loading. The objectives of this study were to quantify the three-dimensional (3D) human TMJ muscle attachment morphometry and examine its impact on TMJ mechanics. 3D muscle attachment surfaces of temporalis, masseter, lateral pterygoid, and medial pterygoid muscles of human cadaveric heads were generated by co-registering measured attachment boundaries with underlying skull models created from cone-beam computerized tomography (CBCT) images. A bounding box technique was used to quantify 3D muscle attachment size, shape, location, and orientation. Musculoskeletal models of the mandible were then developed and validated to assess the impact of 3D muscle attachment morphometry on joint loading during jaw maximal open-close. The 3D morphometry revealed that muscle lengths and moment arms of temporalis and masseter muscles varied substantially among muscle fibers. The values calculated from the centroid-to-centroid model were significantly different from those calculated using the 'Distributed model', which considered crucial 3D muscle attachment morphometry. Consequently, joint loading was underestimated by more than 50% in the centroid-to-centroid model. Therefore, it is necessary to consider 3D muscle attachment morphometry, especially for muscles with broad attachments, in TMJ musculoskeletal models to precisely quantify the joint mechanical environment critical for understanding TMJ function and mechanobiology.

Electrical Conductivity Method to Determine Sexual Dimorphisms in Human Temporomandibular Disc Fixed Charge Density.

To investigate potential mechanisms associated with the increased prevalence of temporomandibular joint (TMJ) disorders among women, the study objective was to determine sex-dependent and region-dependent differences in fixed charge density (FCD) using an electrical conductivity method. Seventeen TMJ discs were harvested from nine males (77 ± 4 years) and eight females (86 ± 4 years). Specimens were prepared from the anterior band, posterior band, intermediate zone, medial disc and lateral disc. FCD was determined using an electrical conductivity method, assessing differences among disc regions and between sexes. Statistical modeling showed significant effects for donor sex (p = 0.002), with cross-region FCD for male discs 0.051 ± 0.018 milliequivalent moles per gram (mEq/g) wet tissue and 0.043 ± 0.020 mEq/g wet tissue for female discs. FCD was significantly higher for male discs compared to female discs in the posterior band, with FCD 0.063 ± 0.015 mEq/g wet tissue for male discs and 0.032 ± 0.020 mEq/g wet tissue for female discs (p = 0.050). These results indicate FCD contributes approximately 20% towards TMJ disc compressive modulus, through osmotic swelling pressure regulation. Additionally, FCD regulates critical extracellular ionic/osmotic and nutrient environments. Sexual dimorphisms in TMJ disc FCD, and resulting differences in extracellular ionic/osmotic and nutrient environments, could result in altered mechano-electro-chemical environments between males and females and requires further study.

Quantifying Baseline Fixed Charge Density in Healthy Human Cartilage Endplate: A Two-point Electrical Conductivity Method.

STUDY DESIGN: Regional measurements of fixed charge densities (FCDs) of healthy human cartilage endplate (CEP) using a two-point electrical conductivity approach. OBJECTIVE: The aim of this study was to determine the FCDs at four different regions (central, lateral, anterior, and posterior) of human CEP, and correlate the FCDs with tissue biochemical composition. SUMMARY OF BACKGROUND DATA: The CEP, a thin layer of hyaline cartilage on the cranial and caudal surfaces of the intervertebral disc, plays an irreplaceable role in maintaining the unique physiological mechano-electrochemical environment inside the disc. FCD, arising from the carboxyl and sulfate groups of the glycosaminoglycans (GAG) in the extracellular matrix of the disc, is a key regulator of the disc ionic and osmotic environment through physicochemical and electrokinetic effects. Although FCDs in the annulus fibrosus (AF) and nucleus pulposus (NP) have been reported, quantitative baseline FCD in healthy human CEP has not been reported. METHODS: CEP specimens were regionally isolated from human lumbar spines. FCD and ion diffusivity were concurrently investigated using a two-point electrical conductivity method. Biochemical assays were used to quantify regional GAG and water content. RESULTS: FCD in healthy human CEP was region-dependent, with FCD lowest in the lateral region (P = 0.044). Cross-region FCD was 30% to 60% smaller than FCD in NP, but similar to the AF and articular cartilage (AC). CEP FCD (average: 0.12 ±â€Š0.03 mEq/g wet tissue) was correlated with GAG content (average: 31.24 ±â€Š5.06 µg/mg wet tissue) (P = 0.005). In addition, the cross-region ion diffusivity in healthy CEP (2.97 ±â€Š1.00 × 10 cm/s) was much smaller than the AF and NP. CONCLUSION: Healthy human CEP acts as a biomechanical interface, distributing loads between the bony vertebral body and soft disc tissues and as a gateway impeding rapid solute diffusion through the disc. LEVEL OF EVIDENCE: N/A.

Tensile biomechanical properties of human temporomandibular joint disc: Effects of direction, region and sex.

Approximately 30% of temporomandibular joint (TMJ) disorders include degenerative changes to the articular disc, with sex-specific differences in prevalence and severity. Limited tensile biomechanical properties of human TMJ discs have been reported. Stress relaxation tests were conducted on TMJ disc specimens harvested bilaterally from six males and six females (68.9±7.9 years), with step-strain increments of 5%, 10%, 15%, 20% and 30%, at 1% strain-per-second. Stress versus strain plots were constructed, and Young׳s Modulus, Instantaneous Modulus and Relaxed Modulus were determined. The effects of direction, region, and sex were examined. Regional effects were significant (p<0.01) for Young׳s Modulus and Instantaneous Modulus. Anteroposteriorly, the central region was significantly stiffer than medial and lateral regions. Mediolaterally, the posterior region was significantly stiffer than central and anterior regions. In the central region, anteroposteriorly directed specimens were significantly stiffer compared to mediolateral specimens (p<0.04). TMJ disc stiffness, indicated by Young׳s Modulus and Instantaneous Modulus, was higher in directions corresponding to high fiber alignment. Additionally, human TMJ discs were stiffer for females compared to males, with higher Young׳s Modulus and Instantaneous Modulus, and female TMJ discs relaxed less. However, sex effects were not statistically significant. Using second-harmonic generation microscopy, regional collagen fiber organization was identified as a potentially significant factor in determining the biomechanical properties for any combination of direction and region. These findings establish structure-function relationships between collagen fiber direction and organization with biomechanical response to tensile loading, and may provide insights into the prevalence of TMJ disorders among women.

Fabrication of a biomimetic elastic intervertebral disk scaffold using additive manufacturing.

A custom-designed three-dimensional additive manufacturing device was developed to fabricate scaffolds for intervertebral disk (IVD) regeneration. This technique integrated a computer with a device capable of 3D movement allowing for precise motion and control over the polymer scaffold resolution. IVD scaffold structures were designed using computer-aided design to resemble the natural IVD structure. Degradable polyurethane (PU) was used as an elastic scaffold construct to mimic the elastic nature of the native IVD tissue and was deposited at a controlled rate using ultra-fine micropipettes connected to a syringe pump. The elastic PU was extruded directly onto a collecting substrate placed on a freezing stage. The three-dimensional movement of the computer-controlled device combined with the freezing stage enabled precise control of polymer deposition using extrusion. The addition of the freezing stage increased the polymer solution viscosity and hardened the polymer solution as it was extruded out of the micropipette tip. This technique created scaffolds with excellent control over macro- and micro-structure to influence cell behavior, specifically for cell adhesion, proliferation, and alignment. Concentric lamellae were printed at a high resolution to mimic the native shape and structure of the IVD. Seeded cells aligned along the concentric lamellae and acquired cell morphology similar to native tissue in the outer portion of the IVD. The fabricated scaffolds exhibited elastic behavior during compressive and shear testing, proving that the scaffolds could support loads with proper fatigue resistance without permanent deformation. Additionally, the mechanical properties of the scaffolds were comparable to those of native IVD tissue.

Patterns of muscular strain in the embryonic heart wall.

The hypothesis that inner layers of contracting muscular tubes undergo greater strain than concentric outer layers was tested by numerical modeling and by confocal microscopy of strain within the wall of the early chick heart. We modeled the looped heart as a thin muscular shell surrounding an inner layer of sponge-like trabeculae by two methods: calculation within a two-dimensional three-variable lumped model and simulated expansion of a three-dimensional, four-layer mesh of finite elements. Analysis of both models, and correlative microscopy of chamber dimensions, sarcomere spacing, and membrane leaks, indicate a gradient of strain decreasing across the wall from highest strain along inner layers. Prediction of wall thickening during expansion was confirmed by ultrasonography of beating hearts. Degree of stretch determined by radial position may thus contribute to observed patterns of regional myocardial conditioning and slowed proliferation, as well as to the morphogenesis of ventricular trabeculae and conduction fascicles. Developmental Dynamics 238:1535-1546, 2009. (c) 2009 Wiley-Liss, Inc.

Periostin promotes a fibroblastic lineage pathway in atrioventricular valve progenitor cells.

Differentiation of prevalvular mesenchyme into valve fibroblasts is an integral step towards the development of functionally mature cardiac valves. Although clinically relevant, little is known regarding the molecular and cellular mechanisms by which this process proceeds. Genes that are regulated in a spatio-temporal pattern during valve remodeling are candidates for affecting this differentiation process. Based on its expression pattern, we have focused our studies on the role of the matricellular gene, periostin, in regulating the differentiation of cushion mesenchymal cells into valve fibroblasts. Herein, we demonstrate that periostin expression is coincident with and regulates type I collagen protein production, a major component of mature valve tissue. Adenoviral-mediated knock-down of periostin in atrioventricular mesenchyme resulted in a decrease in collagen I protein expression and aberrant induction of myocyte markers indicating an alteration in AV mesenchyme differentiation. In vitro analyses using a novel "cardiotube" assay further demonstrated that expression of periostin regulates lineage commitment of valve precursor cells. In these cells, expression of periostin and collagen I are regulated, in part, by TGFbeta-3. We further demonstrate that TGFbeta-3, through a periostin/collagen pathway, enhances the viscoelastic properties of AV cushion tissue surface tension and plays a crucial role in regulating valve remodeling. Thus, data presented here demonstrate that periostin, a TGFbeta-3 responsive gene, functions as a crucial mediator of chick AV valve maturation via promoting mesenchymal-to-fibroblast differentiation while blocking differentiation of alternative cell types (myocytes).