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It has been suggested that curvature progression in adolescent idiopathic scoliosis occurs through irreversible changes in the intervertebral discs. Strains of mice have been identified who differ in their disc wedging response upon extended asymmetrical compression. Annulus fibrosus tissue remodeling could contribute to the faster disc wedging progression previously observed in these mice. Differences in collagen remodeling capacity of annulus fibrosus cells between these in-bred mice strains was compared using an in vitro micro-tissue system. Annulus fibrosus cells of 8-10 week-old LG/J ("fast-healing") and C57BL/6J ("normal healing") mice were embedded in a micro-tissue platform and cultured for 48 hours. Hereafter, tissues were partially released and cultured for another 96 hours. Micro-tissue surface area and waistcoat contraction, collagen orientation and collagen content were measured. After 96 hours post-release, micro-tissues with annulus fibrosus cells of LG/J mice showed more surface area contraction (p < 0.001) and waistcoat contraction (p = 0.002) than C57BL/6J micro-tissues. Collagen orientation did not differ at 24 hours after partial release. However, at 96 hours, collagen in the micro-tissues from LG/J annulus fibrosus cells was aligned more than in those from C57BL/6J mice (p < 0.001). Collagen content did not differ between micro-tissues at 96 hours. Annulus fibrosus cells of inbred LG/J mice were better able to remodel and realign their collagen fibers than those from C57BL/6J mice. The remodeling of annulus fibrosus tissue could be contributing to the faster disc wedging progression observed in LG/J mice. This article is protected by copyright. All rights reserved.

Mechanobiology explores how forces regulate cell behaviors and what molecular machinery are responsible for the sensing, transduction, and modulation of mechanical cues. To this end, probing of cells cultured on planar substrates has served as a primary experimental setting for many decades. However, native extracellular matrices (ECMs) consist of fibrous protein assemblies where the physical properties spanning from the individual fiber to the network architecture can influence the transmission of forces to and from the cells. Here, a robotic manipulation platform that allows wireless, localized, and programmable deformation of an engineered fibrous ECM is introduced. A finite-element-based digital twin of the fiber network calibrated against measured local and global parameters enables the calculation of deformations and stresses generated by different magnetic actuation schemes across a range of network properties. Physiologically relevant mechanical forces are applied to cells cultured on the fiber network, statically or dynamically, revealing insights into the effects of matrix-borne forces and deformations as well as force-mediated matrix remodeling on cell migration and intracellular signaling. These capabilities are not matched by any existing approach, and this versatile platform has the potential to uncover fundamental mechanisms of mechanobiology in settings with greater relevance to living tissues.

Here we present an experimental model for human luminal progenitor cells that enables single, primary cells isolated from normal tissue to generate complex branched structures resembling the ductal morphology of low-grade carcinoma of no special type. Thereby, we find that ductal structures are generated through invasive branching morphogenesis via matrix remodeling and identify reduced actomyosin contractility as a prerequisite for invasion. In addition, we show that knockout of E-cadherin causes a dissolution of duct formation as observed in invasive lobular carcinoma, a subtype of invasive carcinomas where E-cadherin function is frequently lost. Thus, our model shows that invasive capacity can be elicited from normal luminal cells in specific environments, which results in low-grade no special type morphology. This assay offers a platform to investigate the dynamics of luminal cell invasion and unravel the impact of genetic and non-genetic aberrations on invasive morphology. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Stem cell‐based cartilage regeneration strategies take benefits from functional tissue engineering concepts that effectively exploit cells native mechanotransduction phenomenon for the development of in vitro cartilage constructs. In the present study, stem cell‐seeded constructs were subjected to a dynamic compression of 10% strain, 1 Hz, 4 hours/1 hour each for 7 days. Integrin and Integrin‐associated protein expression was evaluated using real‐time PCR to elucidate the possible mechanotransduction pathway. Matrix remodeling was analyzed by evaluating chondrocyte‐specific extracellular matrix genes as well as by expression of matrix metalloproteinases. The results suggest that dynamic compression significantly influences chondrogenesis by enhancing the expression of chondrocyte‐specific extracellular matrix genes. The results also propose the involvement of Integrin‐mediated signaling pathways in the mechanotransduction events on biomechanical stimuli‐assisted chondrogenesis of stem cells.

The anterior cruciate ligament (ACL)-to-bone interface constitutes a complex, multi-tissue structure comprised of contiguous ligament, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone regions. This composite structure enables load transfer between structurally and functionally dissimilar tissues and is critical for ligament homeostasis and joint stability. Presently, there is a lack of quantitative understanding of the matrix composition and organization across this junction, especially after the onset of skeletal maturity. The objective of this study is to characterize the adult bovine ACL-to-bone interface using Fourier transform infrared spectroscopic imaging (FTIRI), testing the hypothesis that regional changes in collagen, proteoglycan, and mineral distribution, as well as matrix organization, persist at the mature insertion. It was observed that while collagen content increases continuously across the adult interface, collagen alignment decreases between ligament and bone. Proteoglycans were primarily localized to the fibrocartilage region and an exponential increase in mineral content was observed between the non-mineralized and mineralized regions. These observations reveal significant changes in collagen distribution and alignment with maturity, and these trends underscore the role of physiologic loading in postnatal matrix remodeling. Findings from this study provide new insights into interface organization and serve as benchmark design criteria for interface regeneration and integrative soft tissue repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2513-2523, 2017.

SUMMARY Tissue equivalents represent excellent model systems for elucidating principles of mechanobiology and for exploring methods to improve the functionality of tissue-engineered constructs. The simplest tissue equivalent is the free-floating fibroblast-populated collagen lattice. Although introduced over 30 years ago, the associated mechanics of the cell-mediated compaction of this lattice was only recently analyzed in detail. The goal of this paper was to build on this recent stress analysis by developing a computational model of the evolving geometry, regionally varying material properties and cell stresses, and overall residual stress fields during the first two days of compaction. Baseline results were found to agree well with most experimental observations, namely evolving changes in radius, thickness, and material symmetry, yet hypothesis testing revealed aspects of the mechanobiology that require more experimental attention. Given the generality of the proposed framework, we submit that modifications and refinements can be used to study many similar systems and thereby help guide future experiments. Copyright © 2014 John Wiley & Sons, Ltd.

Neural progenitor cells (NPCs) are a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Material‐based strategies, including culture within 3D hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. It has recently been demonstrated that cell‐mediated remodeling of 3D hydrogels is necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remains unknown. By culturing NPCs within engineered protein hydrogels susceptible to degradation by NPC‐secreted proteases, it is identified that a critical amount of remodeling is necessary to enable NPC differentiation, even in highly degradable gels. Chemical induction of differentiation after sufficient remodeling time results in differentiation into astrocytes and neurotransmitter‐responsive neurons. Matrix remodeling modulates expression of the transcriptional co‐activator Yes‐associated protein, which drives expression of NPC stemness factors and maintains NPC differentiation capacity, in a cadherin‐dependent manner. Thus, cell‐remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use.

Podosomes are recently rediscovered highly dynamic actin-rich structural and functional modules that form close contact with the surrounding substrate. They play a role in the control of migration, tissue invasion, and matrix remodeling of highly motile cells, including lymphocytes, macrophages, dendritic cells, and osteoclasts. In osteoclasts, the compaction of podosomes induces the formation of a tight adhesive contact, the sealing zone, which defines a subosteoclastic environment specialized for bone resorption. Integrins and the Rho family small GTPases are key regulators of podosome rearrangements. However, it remains to be determined how the activation of integrins and Rho family GTPases is regulated during osteoclast podosome rearrangements. Here, we demonstrate a crucial role for the FERM domain-containing guanine nucleotide exchange factor (GEF), FARP2, in osteoclast podosome rearrangements and resorbing activity. We determine by live cell imaging and biochemical assays that FARP2 is required for localized activation of GTP-bound Rac1 into podosome-ring like structures. In addition, FARP2 is relevant to integrin β3 activity during osteoclastogenesis. Furthermore, FARP2 deficiency results in reduced formation of multinucleated osteoclasts and resorption pits compared to wild-type osteoclasts (controls). Collectively, our findings reveal an integral role of FARP2 for regulation of Rac1 and integrin β3 throughout podosome rearrangement in osteoclastogenesis.

Lysyl hydroxylase 3 (LH3), the multifunctional enzyme associated with collagen biosynthesis that possesses lysyl hydroxylase and collagen glycosyltransferase activities, has been characterized in the extracellular space in this study. Lysine modifications are known to occur in the endoplasmic reticulum (ER) prior to collagen triple-helix formation, but in this study we show that LH3 is also present and active in the extracellular space. Studies with in vitro cultured cells indicate that LH3, in addition to being an ER resident, is secreted from the cells and is found both in the medium and on the cell surface associated with collagens or other proteins with collagenous sequences. Furthermore, in vivo, LH3 is present in serum. LH3 protein levels correlate with the galactosylhydroxylysine glucosyltransferase (GGT) activity of mouse tissues. This, together with other data, indicates that LH3 is responsible for GGT activity in the tissues and that GGT activity assays can be used to quantify LH3 in tissues. LH3 in vivo is located in two compartments, in the ER and in the extracellular space, and the partitioning varies with tissue type. In mouse kidney the enzyme is located mainly intracellularly, whereas in mouse liver it is located solely in the extracellular space. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, nondenaturated conformation reveals a new dynamic in extracellular matrix remodeling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine-linked carbohydrates in collagenous proteins.

Biochemical, biomechanical and ultrastructural properties of the connective tissue matrix were investigated during the early remodeling phase of tissue repair in experimentally tenotomized and repaired rabbit Achilles tendons. Sterile surgical tenotomy was performed on the right Achilles tendons of 14 rabbits and allowed to heal for 15 days. The animals were euthanized and the Achilles tendons excised from both limbs. The left contralateral Achilles tendon of each rabbit was used as a control in the experiments. Prior to biochemical analysis, both intact and healing tendons were tested for their biomechanical integrity. The results revealed that the healing tendons had regained some of their physicochemical characteristics, but differed significantly from the intact left tendons. The healing tendons regained 48% tensile strength, 30% energy absorption, 20% tensile stress, and 14% Young's modulus of elasticity of intact tendons. In contrast, biochemical analysis showed that the healing tendons had 80% of the collagen and 60% of the collagen crosslinks (hydroxypyridinium) of normal tendons. Sequential extraction of collagen from the tissues yielded more soluble collagen in the healing tendons than intact tendons, suggesting either an increase in collagen synthesis and/or enhanced resorption of mature collagen in healing tendons compared to intact tendons. Electron microscopic studies revealed remarkable differences in the ultrastructure between intact and healing tendons. These observations could explain, in part, the connective tissue response to healing during the early phases of tissue remodeling.

Parstatin, a novel protease activated receptor-1 (PAR-1) derived peptide is a potent inhibitor of angiogenesis. We and others have reported that imbalance between angiogenic growth factors and anti-angiogenic factors results in transition from compensatory cardiac hypertrophy to heart failure in a pressure overload condition. Though cardio protective role of parstatin was shown previously in ischemic cardiac injury, its role in pressure overload cardiac injury is yet to unveil. We hypothesize that supplementing anti-parstatin antibody during pressure overload condition augments angiogenesis and ameliorate left ventricular dysfunction and heart failure. To verify this, we created ascending aortic banding in mice to mimic pressure overload condition and then treated mice with anti-parstatin antibody. Left ventricular function was assessed by echocardiography and pressure-volume loop study. Angiogenic growth factors and anti-angiogenic factors along with MMP-2,-9 were evaluated by western blot and immunohistochemistry. Results: our results showed an improved left ventricular function in anti-parstatin treated aortic banding hearts compared to their corresponding wild type controls. Expression of angiogenic growth factor, VEGF, MMP-2 and CD31 expression was increased in treated aortic banding hearts compared to their corresponding wild type controls. Our results suggest that treating pressure overload mice with anti-parstatin antibody augments angiogenesis and ameliorates left ventricular dysfunction.

We have correlated early material and biochemical changes in articular cartilage in a surgical model for cartilage degeneration. Medial meniscectomy was performed on the left knee of 17 adult, female New Zealand white rabbits. The equilibrium Young's modulus of cartilage was assessed by an indentation test in situ at defined sites on the medial and lateral tibial plateaus of the operated and control knees; the cartilage was then excised and analyzed biochemically. Focal changes were consistently observed in the medial surface of the operated knee. The equilibrium modulus and the glycosaminoglycan content fell rapidly, reaching a minimum by 2 weeks after surgery; the lateral tibial surface was essentially unaffected. Six months after surgery, the glycosaminoglycan content had returned to normal and the modulus to near normal. Independent measurements on cored plugs from the medial surface 2 weeks after surgery revealed a significant decrease in both the dynamic stiffness and the streaming potential in the operated knee compared with the control. The findings suggest that normal ambulatory loads in vivo will deform the affected medial cartilage much more than normal. It remains to be seen if altered mechanical stresses are solely responsible for initiating and sustaining matrix remodeling by the chondrocytes.