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An attractive alternative to bone autografts is the use of autologous mesenchymal progenitor cells (MSCs) in combination with biomaterials. We compared the therapeutic potential of different sources of mesenchymal stem cells in combination with biomaterials in a bone nonunion model. A critical‐size defect was created in Sprague–Dawley rats. Animals were divided into six groups, depending on the treatment to be applied: bone defect was left empty (CTL); treated with live bone allograft (LBA); hrBMP‐2 in collagen scaffold (CSBMP2); acellular polycaprolactone scaffold (PCL group); PCL scaffold containing periosteum‐derived MSCs (PCLPMSCs) and PCL containing bone marrow‐derived MSCs (PCLBMSCs). To facilitate cell tracking, both MSCs and bone graft were isolated from green fluorescent protein (GFP)‐transgenic rats. CTL group did not show any signs of healing during the radiological follow‐up (n = 6). In the LBA group, all the animals showed bone bridging (n = 6) whereas in the CSBMP2 group, four out of six animals demonstrated healing. In PCL and PCLPMSCs groups, a reduced number of animals showed radiological healing, whereas no healing was detected in the PCLBMSCs group. Using microcomputed tomography, the bone volume filling the defect was quantified, showing significant new bone formation in the LBA, CSBMP2, and PCLPMSCs groups when compared with the CTL group. At 10 weeks, GFP positive cells were detected only in the LBA group and restricted to the outer cortical bone in close contact with the periosteum. Tracking of cellular implants demonstrated significant survival of the PMSCs when compared with BMSCs. In conclusion, PMSCs improve bone regeneration being suitable for mimetic autograft design.

Background & Aims Liver stiffness is increased in advanced chronic liver disease (ACLD) and accurately predicts prognosis in this population. Recent data suggest that extracellular matrix stiffness per se may modulate the phenotype of liver cells. We aimed at investigating the effect of matrix stiffness on the phenotype of liver cells of rats with cirrhosis, assessing its influence on their response to antifibrotic strategies and evaluating associated molecular mechanisms. Methods Hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells were isolated from healthy rats or rats with cirrhosis (carbon tetrachloride or thioacetamide), and cultured on polyacrylamide gels with different physiologically relevant stiffness for 72 h. Results All cell types of rats with cirrhosis cultured at low stiffness showed a significant phenotype amelioration vs. rigid matrix (assessed by quantitative morphology, mRNA expression, protein synthesis, and electron microscopy imaging). Additionally, stiffness modified the antifibrotic effects of liraglutide in stellate cells of rats with cirrhosis. Finally, evaluation of nuclear morphology revealed that high stiffness induced nuclei deformation in all cell types, an observation confirmed in cells from human livers. Disconnecting the nucleus from the cytoskeleton by cytoskeleton disruption or a defective form of nesprin 1 significantly recovered spherical nuclear shape and quiescent phenotype of cells. Conclusions The environment's stiffness per se modulates the phenotype of healthy rats and liver cells of rats with cirrhosis by altering the nuclear morphology through cytoskeleton-derived mechanical forces. The reversibility of this mechanism suggests that targeting the stiffness-mediated intracellular mechanical tensions may represent a novel therapeutic strategy for ACLD. Lay summary During cirrhosis, the liver becomes scarred, stiff, and unable to perform its normal functions efficiently. In this study, we demonstrated that cells from diseased (stiff) livers recovered their functionality when placed in a soft environment (as that of a healthy liver). Furthermore, treatments aimed at tricking liver cells into believing they are in a healthy, soft liver improved their function and could potentially contribute to treat cirrhosis., Graphical abstract, Highlights • Low stiffness directly improves the phenotype of hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells. • Stiffness modulates sinusoidal crosstalk in advanced chronic liver disease (ACLD). • Stiffness may determine the efficacy of antifibrotic strategies. • Mechanical forces applied to the nucleus through the LINC complex mediate the effects of stiffness on liver cell phenotype.

We investigated the anatomical localization, biomechanical properties, and molecular phenotype of myocardial collagen tissue in 40 patients with severe aortic stenosis with preserved ejection fraction and symptoms of heart failure.Two transmural biopsies were taken from the left ventricular free wall. Mysial and nonmysial regions of the collagen network were analyzed. Myocardial collagen volume fraction (CVF) was measured by picrosirius red staining. Young's elastic modulus (YEM) was measured by atomic force microscopy in decellularized slices to assess stiffness. Collagen types I and III were measured as CCompared with controls, patients exhibited increased mysial and nonmysial CVF and nonmysial:mysial CVF ratio (P.05). In patients, nonmysial CVF (r = 0.330; P = .046) and the nonmysial:mysial CVF ratio (r = 0.419; P = .012) were directly correlated with the ratio of maximal early transmitral flow velocity in diastole to early mitral annulus velocity in diastole. Both the CThese findings suggest that, in patients with severe aortic stenosis with preserved ejection fraction and symptoms of heart failure, diastolic dysfunction is associated with increased nonmysial deposition of collagen, predominantly type I, resulting in increased extracellular matrix stiffness. Therefore, the characteristics of collagen tissue may contribute to diastolic dysfunction in these patients.

Resumen Introduccion y objetivos Se ha estudiado la localizacion anatomica, las propiedades biomecanicas y el fenotipo molecular del colageno miocardico tisular en 40 pacientes con estenosis aortica grave, fraccion de eyeccion conservada y sintomas de insuficiencia cardiaca. Metodos Se obtuvieron 2 biopsias transmurales de la pared libre del ventriculo izquierdo. La fraccion del volumen de colageno (FVC) se cuantifico mediante rojo picrosirio y la rigidez, mediante el modulo elastico de Young (YEM) evaluado con microscopia de fuerza atomica en regiones misiales y no misiales. Las FVC de tipos I y III se cuantificaron mediante microscopia confocal en areas con determinacion del YEM. Resultados Comparados con sujetos de control, la FVC misial y no misial y el cociente FVC no misial:misial (p I :FVC III y el YEM aumentaban (p ≤ 0,001) en regiones no misiales respecto de las misiales, con correlacion entre ellos (r = 0,895; p Conclusiones En la estenosis aortica grave con fraccion de eyeccion conservada y sintomas de insuficiencia cardiaca, la disfuncion diastolica se asocia con un deposito no misial de colageno aumentado, predominantemente de tipo I y con mayor rigidez. Las caracteristicas del colageno tisular pueden contribuir a la disfuncion diastolica en estos pacientes.

In the treatment of bone non-unions, an alternative to bone autografts is the use of bone morphogenetic proteins (BMPs), e.g., BMP&ndash, 2, BMP&ndash, 7, with powerful osteoinductive and osteogenic properties. In clinical settings, these osteogenic factors are applied using absorbable collagen sponges for local controlled delivery. Major side effects of this strategy are derived from the supraphysiological doses of BMPs needed, which may induce ectopic bone formation, chronic inflammation, and excessive bone resorption. In order to increase the efficiency of the delivered BMPs, we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic) or trabecular bone (random distributed porosity, isotropic). We hypothesize that an anisotropic structure would enhance the osteoconductive properties of the scaffolds by increasing the regenerative performance of the provided rhBMP&ndash, 2. In vitro, both scaffolds presented similar mechanical properties, rhBMP&ndash, 2 retention and delivery capacity, as well as scaffold degradation time. In vivo, anisotropic scaffolds demonstrated better bone regeneration capabilities in a rat femoral critical-size defect model by increasing the defect bridging. In conclusion, anisotropic cryostructured collagen scaffolds improve bone regeneration by increasing the efficiency of rhBMP&ndash, 2 mediated bone healing.

YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation.

Tissue rigidity regulates processes in development, cancer and wound healing. However, how cells detect rigidity, and thereby modulate their behaviour, remains unknown. Here, we show that sensing and adaptation to matrix rigidity in breast myoepithelial cells is determined by the bond dynamics of different integrin types. Cell binding to fibronectin through either α5β1 integrins (constitutively expressed) or αvβ6 integrins (selectively expressed in cancer and development) adapts force generation, actin flow and integrin recruitment to rigidities associated with healthy or malignant tissue, respectively. In vitro experiments and theoretical modelling further demonstrate that this behaviour is explained by the different binding and unbinding rates of both integrin types to fibronectin. Moreover, rigidity sensing through differences in integrin bond dynamics applies both when integrins bind separately and when they compete for binding to fibronectin.

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved., Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.