Changes in stiffness induced by hindlimb suspension in rat Achilles tendon

The aim of this study was to measure the effects of hindlimb suspension on mechanical properties of the rat Achilles tendon. Adult male Wistar rats were randomly assigned to groups to be either suspended, or a control. After 21 days, Achilles tendons were removed for mechanical analysis. Classical tests of tensile performance were made, and mechanical parameters were derived from a stress-strain relationship. The tendons of animals that had been suspended presented values for maximal stress and tangent modulus which were 37.5% (P < 0.01) and 41% (P < 0.01), respectively, lower than the tendons of the control rats. In a similar way, the energy absorption capacity had largely decreased in animals that had been suspended. However, the maximal strain was similar in the two groups. These results showed that hindlimb suspension in rats has an important detrimental effect on mechanical properties of the Achilles tendon. Differences in tendon stiffness obtained here, along with those found by other investigators, encourage the hypothesis that homeostatic responses of soft tissues are due to changes in limb loadings. This study may be useful in providing a better understanding of the adaptation of human skeletal muscle when exposed to microgravity. Accepted: 3 September 1999     

Partial regeneration and reconstruction of the rat uterus through recellularization of a decellularized uterine matrix

Despite dramatic progress in infertility treatments and assisted reproduction, no effective therapies exist for complete loss of uterine structure and/or function. For such patients, genetic motherhood is possible only through gestational surrogacy or uterine transplantation. However, many ethical, social, technical and safety challenges accompany such approaches. A theoretical alternative is to generate a bioartificial uterus, which requires engineering of uterine architecture and appropriate cellular constituents. Here, rat uteri decellularization by aortic perfusion with detergents produced an underlying extracellular matrix together with an acellular, perfusable vascular architecture. Uterine-like tissues were then regenerated and maintained in vitro for up to 10 d through decellularized uterine matrix (DUM) reseeding with adult and neonatal rat uterine cells and rat mesenchymal stem cells followed by aortic perfusion in a bioreactor. Furthermore, DUM placement onto a partially excised uterus yielded recellularization and regeneration of uterine tissues and achievement of pregnancy nearly comparable to the intact uterus. These results suggest that DUM could be used for uterine regeneration, and provides insights into treatments for uterine factor infertility.     

Age-related changes in biomechanical properties of the Achilles tendon in rabbits

We investigated age-related changes in the mechanical properties of rabbit Achilles tendon. The animals used were immature (age 3 weeks, body mass 380 g), young adult (age 8–10 months, body mass 4.1 kg) and old (age 4–5 years, body mass 5.1 kg) rabbits. The cross-sectional area of the tendon increased with growth and the tensile strength of the young adult [67.3 (SEM 4.2) MPa] and old [66.7 (SEM 3.8) MPa] tendon was significantly higher than that of the immature tendon [23.9 (SEM 3.8) MPa]. However, there was no statistically significant difference in tensile strength between mature and old tendons. These differences may be attributable to the change in body mass. The gradient of the stress-strain curves, that is, the tangent modulus of the mature tendon [618.0 (SEM 87.0) MPa], was higher than that of the immature [281.0 (SEM 104.6) MPa] and old [530.5 (SEM 91.0) MPa] tendon, although the difference was not significant. The elongation at failure was approximately 16% for all age groups. These results would suggest that rabbit Achilles tendon is highly compliant during growth.     

Development and ageing of phenotypically distinct fibrocartilages associated with the rat Achilles tendon

Summary We describe by routine histology and by immunohistochemistry three phenotypically and developmentally distinct fibrocartilages associated with the Achilles tendon of the rat. All the fibrocartilages develop after birth and show significant age-related changes in the composition of their extracellular matrix. Attachment-zone fibrocartilage occurs at the insertion of the tendon on the calcaneus. It derives from the cartilage rudiment of the calcaneus and from the region where the tendon merges with the perichondrium. The extracellular matrix contain type II collagen and chondroitin sulphate. Compressive tendon fibrocartilage occurs in the deep part of the tendon where it presses against the calcaneus, and is derived by metaplasia of tendon cells. The cells label strongly for the intermediate filament vimentin, and the extracellular matrix contains chondroitin and keratan sulphates, but type II collagen only in very old animals (>2 years). Calcaneal fibrocartilage covered the posterior surface of the calcaneus where it was in contact with the Achilles tendon. It labelled intensely for type II collagen and contained chondroitin and keratan sulphates. The cells were rich in vimentin. This fibrocartilage was derived from the calcaneal perichondrium.     

The phenotype of cancer cell invasion controlled by fibril diameter and pore size of 3D collagen networks

The behavior of cancer cells is strongly influenced by the properties of extracellular microenvironments, including topology, mechanics and composition. As topological and mechanical properties of the extracellular matrix are hard to access and control for in-depth studies of underlying mechanisms in vivo, defined biomimetic in vitro models are needed. Herein we show, how pore size and fibril diameter of collagen I networks distinctively regulate cancer cell morphology and invasion. Three-dimensional collagen I matrices with a tight control of pore size, fibril diameter and stiffness were reconstituted by adjustment of concentration and pH value during matrix reconstitution. At first, a detailed analysis of topology and mechanics of matrices using confocal laser scanning microscopy, image analysis tools and force spectroscopy indicate pore size and not fibril diameter as the major determinant of matrix elasticity. Secondly, by using two different breast cancer cell lines (MDA-MB-231 and MCF-7), we demonstrate collagen fibril diameter – and not pore size – to primarily regulate cell morphology, cluster formation and invasion. Invasiveness increased and clustering decreased with increasing fibril diameter for both, the highly invasive MDA-MB-231 cells with mesenchymal migratory phenotype and the MCF-7 cells with amoeboid migratory phenotype. As this behavior was independent of overall pore size, matrix elasticity is shown to be not the major determinant of the cell characteristics. Our work emphasizes the complex relationship between structural-mechanical properties of the extracellular matrix and invasive behavior of cancer cells. It suggests a correlation of migratory and invasive phenotype of cancer cells in dependence on topological and mechanical features of the length scale of single fibrils and not on coarse-grained network properties.     

The synergetic effect of hydrogel stiffness and growth factor on osteogenic differentiation

Cells respond to various chemical signals as well as environmental aspects of the extracellular matrix (ECM) that may alter cellular structures and functions. Hence, better understanding of the mechanical stimuli of the matrix is essential for creating an adjuvant material that mimics the physiological environment to support cell growth and differentiation, and control the release of the growth factor. In this study, we utilized the property of transglutaminase cross-linked gelatin (TG-Gel), where modification of the mechanical properties of TG-Gel can be easily achieved by tuning the concentration of gelatin. Modifying one or more of the material parameters will result in changes of the cellular responses, including different phenotype-specific gene expressions and functional differentiations. In this study, stiffer TG-Gels itself facilitated focal contact formation and osteogenic differentiation while soft TG-Gel promoted cell proliferation. We also evaluated the interactions between a stimulating factor (i.e. BMP-2) and matrix rigidity on osteogenesis both in vitro and in vivo. The results presented in this study suggest that the interactions of chemical and physical factors in ECM scaffolds may work synergistically to enhance bone regeneration.     

Engineering of a bio-functionalized hybrid off-the-shelf heart valve

Currently available heart valve replacements are limited in long-term performance or fail due to leaflet thickening, lack of growth or remodeling potential. In order to address these issues, it is necessary to mimic multiple factors of the native valvular extracellular matrix (ECM) such as architecture, mechanical behavior and biochemical signals. Here, we successfully generated an electrospun PEGdma–PLA scaffold adapted to the structure and mechanical properties of native valve leaflets. Valvular interstitial cells (VICs) and valvular endothelial cells (VECs) were seeded on the scaffold and when cultured under physiological conditions in a bioreactor, the construct performed like a native leaflet. Atomic force microscopy (AFM) was employed to obtain detailed mechanical information from the leaflets, which enabled the first layer-specific measurement of the Young's modulus. Interestingly, spongiosa stiffness was much lower compared to the fibrosa and ventricularis. Moreover, investigations into human fetal heart valve development identified collagen type I and versican as important structural proteins. As a proof of principle, these proteins were introduced to the scaffold, demonstrating the ability to bio-functionalize the hybrid valve based on natures' blueprint.     

Influence of the stiffness of three-dimensional alginate/collagen-I interpenetrating networks on fibroblast biology

Wound dressing biomaterials are increasingly being designed to incorporate bioactive molecules to promote healing, but the impact of matrix mechanical properties on the biology of resident cells orchestrating skin repair and regeneration remains to be fully understood. This study investigated whether tuning the stiffness of a model wound dressing biomaterial could control the behavior of dermal fibroblasts. Fully interpenetrating networks (IPNs) of collagen-I and alginate were fabricated to enable gel stiffness to be tuned independently of gel architecture, polymer concentration or adhesion ligand density. Three-dimensional cultures of dermal fibroblasts encapsulated within matrices of different stiffness were shown to promote dramatically different cell morphologies, and enhanced stiffness resulted in upregulation of key-mediators of inflammation such as IL-10 and COX-2. These findings suggest that simply modulating the matrix mechanical properties of a given wound dressing biomaterial deposited at the wound site could regulate the progression of wound healing.     

Endogenous overexpression of hyaluronan synthases within dynamically cultured collagen gels: Implications for vascular and valvular disease

Hyaluronan is a ubiquitous component of the extracellular matrix with important roles in cell and tissue functions. Hyaluronan content is often elevated in cardiovascular diseases, such as mitral valve disease and atherosclerosis. The objective of this study was to determine the impact of endogenously produced hyaluronan dynamically cultured three-dimensional model of collagenous tissues. Collagen gels containing excess HA and hyaluronan synthase (has) overexpressing cells were grown in a cyclic strain environment to simulate cell-mediated matrix organization. Cyclic strain caused a significant elevation in the collagen fibril density, cell number, and hyaluronan content of the resulting collagen gels compared to those grown under a static strain regimen. The material behavior of collagen gels containing has overexpressing cells was also notably weakened compared to controls. Transmission electron microscopy and immunohistochemistry showed that proteoglycan distribution was influenced by both strain and has overexpression. The results were also dependent on the specific has isozyme overexpressed. This investigation helps to identify the mechanism by which hyaluronan acts in vivo to alter tissue material behavior in cardiovascular diseases such as myxomatous mitral valve disease and atherosclerosis.     

Role of moderate exercising on Achilles tendon collagen crimping patterns and proteoglycans

Abstract

In this study, the morphological and morphometric changes in the collagen crimping pattern of Achilles tendon and metabolism/expression of tenocytes explanted from tendons of running (RUN) and sedentary (SED) rats were investigated to assess the effects of 12 weeks moderate running exercise. The number, the top angle width and the base length of each crimp in three different regions (proximal, central and distal) of RUN and SED tendons were measured with a polarized light microscope. The most significant morphometric differences in the crimps were detectable in the central region of the RUN tendons. In this region, crimps were fewer, larger and more flattened than those of other regions as a consequence of a functional adaptation of extracellular matrix to running, in order to increase tendon stiffness and force transmission efficiency. Conversely, the top angle width of the crimps reduced in proximal and distal regions of the RUN tendons, suggesting that these crimps might act as more reactive mechanical springs, able to store and improve the release of the stored strain energy in most loaded regions. Tenocytes explanted from Achilles tendons of both RUN and SED groups were cultured. Running influenced tenocytes which showed a significant increase in collagen type-I synthesis and proteoglycans production, suggesting enhancement of the loading transmission efficiency and facilitate inter-fibril and inter-fiber sliding.     

Effects of freezing/thawing on the biomechanical properties of human tendons

Abstract Numerous biomechanical studies using osteoarticular complex need frozen cadaveric specimens. Some of these studies deal with the resistance of the tendinous structures, for example the resistance of some autografts, such as the patellar ligament and the semitendinosus and gracilis tendons for reconstruction of the anterior cruciate ligament. The aim of this study was the in-vitro evaluation of the mechanical modifications induced by freezing/thawing on human tendons. The long head of the biceps brachii tendon was used as the reference. Eight pairs of tendons of the long head of the biceps brachii were taken from eight fresh cadavers. After drawing lots, one was tested immediately, the other was deep-frozen and then thawed. With an Instron material-testing machine, we performed a relaxation test and a uniaxial tensile test, to estimate the ultimate tensile failure and the elastic modulus of each pair of tendons. Freezing had no influence on the tendinous relaxation, but altered significantly the ultimate tensile failure and Young’s modulus of the tendons.     

Towards the development of a bioengineered uterus: Comparison of different protocols for rat uterus decellularization

Uterus transplantation (UTx) may be the only possible curative treatment for absolute uterine factor infertility, which affects 1 in every 500 females of fertile age. We recently presented the 6-month results from the first clinical UTx trial, describing nine live-donor procedures. This routine involves complicated surgery and requires potentially harmful immune suppression to prevent rejection. However, tissue engineering applications using biomaterials and stem cells may replace the need for a live donor, and could prevent the required immunosuppressive treatment. To investigate the basic aspects of this, we developed a novel whole-uterus scaffold design for uterus tissue engineering experiments in the rat. Decellularization was achieved by perfusion of detergents and ionic solutions. The remaining matrix and its biochemical and mechanical properties were quantitatively compared from using three different protocols. The constructs were further compared with native uterus tissue composition. Perfusion with Triton X-100/dimethyl sulfoxide/H₂O led to a compact, weaker scaffold that showed evidence of a compromised matrix organization. Sodium deoxycholate/dH₂O perfusion gave rise to a porous scaffold that structurally and mechanically resembled native uterus better. An innovative combination of two proteomic analyses revealed higher fibronectin and versican content in these porous scaffolds, which may explain the improved scaffold organization. Together with other important protocol-dependent differences, our results can contribute to the development of improved decellularization protocols for assorted organs. Furthermore, our study shows the first available data on decellularized whole uterus, and creates new opportunities for numerous in vitro and in vivo whole-uterus tissue engineering applications.     

Age-dependent regulation of tendon crimp structure,cell length and gap width with strain

The black-and-white patterning of tendon fascicles when visualized by light microscopy, also known as crimp, is a well-known feature of fiber-forming collagens. However, not much is known about its development, function and response to strain. The objective of this study is to investigate the interaction of tenocyte and crimp morphology as well as their changes with increasing age and acute strain. In contrast to previous studies, which used indirect measures, such as polarized light, to investigate the crimp structure, this study visualizes internal crimp structure in three dimensions without freezing, sectioning, staining or fixing the tissue, via two-photon imaging of green fluorescent protein expressing cells within mouse tail tendon fascicles. This technique further allows straining of the live tissue while visualizing changes in crimp morphology and cell shape with increasing specimen length. Combining this novel microscopy technique with computational image and data analysis revealed a complex relationship between tenocytes and the extracellular matrix that evolves with increasing age. While the reduction of crimping with strain was observed as expected, most of the crimps were gone at 0–1% strain already. Even relatively low strains of 3% led to pronounced changes in the crimp structure after relaxation, particularly in the young animals, which could not be seen with bright-field imaging. Cell length and gap width increased with strain. However, while the cells were able to return to their original length even after high strains of 6%, the gaps between the cells widened, which may imply modified cell–cell communication after overstretching.     

A tunable synthetic hydrogel system for culture of retinal ganglion cells and amacrine cells

The central nervous system consists of complex groups of individual cells that receive electrical, chemical and physical signals from their local environment. Standard in vitro cell culture methods rely on two-dimensional (2-D) substrates that poorly simulate in vivo neural architecture. Neural cells grown in three-dimensional (3-D) culture systems may provide an opportunity to study more accurate representations of the in vivo environment than 2-D cultures. Furthermore, each specific type of neuron depends on discrete compositions and physical properties of their local environment. Previously, we developed a library of hydrogels composed of poly(ethylene glycol) and poly(l-lysine) which exhibit a wide range of mechanical properties. Here, we identified specific scaffolds from this library that readily support the survival, migration and neurite outgrowth of purified retinal ganglion cells and amacrine cells. These data address important biological questions about the interaction of neurons with the physical and chemical properties of their local environment and provide further insight for engineering neural tissue for cell-replacement therapies following injury.     

基于RP的组织工程细胞外基质--载体制造问题研究

针对组织工程中急需具有适当尺寸的孔隙及孔隙度结构的细胞外基质-载体和组织载体的骨架材料的问题,本文引入了目前正在飞速发展的快速成形(RP)技术.应用这种技术可以在短时间内制出既具有精确解剖学形态、又具有适当尺寸的孔隙及孔隙度的三维立体结构的支架.它可以控制载体骨架材料内部微孔的数量、大小、分布及形状,从而提供给细胞的生存以最适宜的空间和营养条件.     

材料生物力学2021年研究进展

机体组织具有复杂的三维动态结构,且受到多种形式的作用力。细胞从细胞外基质（extracellular matrix, ECM）中感受力学刺激,ECM构建的力学微环境调控细胞不同生物学功能。制备可模拟机体组织ECM力学微环境的生物材料是生物力学领域研究的热点和难点之一。生物材料的不同理化性质赋予材料特定的力学性能,进而影响细胞行为和功能。本文结合2021年材料生物力学领域的最新文献,特别关注新型材料生物力学对细胞生物学行为的调控和在组织工程中的应用,并探讨材料生物力学研究领域的未来发展方向。     

Green tea and glycine aid in the recovery of tendinitis of the Achilles tendon of rats

Abstract

Purpose: Green tea (GT) is widely used due to its anti-inflammatory properties. Previous studies have shown beneficial effects of a glycine diet on the remodeling process in inflamed tendons. Tendinitis is commonly observed in athletes and is of concern to surgeons due to the slowness of the recovery process. Our hypothesis is that GT?+?a glycine diet may improve tendinitis. Aim of the study: To analyze the effect of GT and/or glycine in the diet on tendinitis. Materials and methods: Wistar rats were divided into seven groups (G): control group (C); G1 and G4, tendinitis; G2 and G5, tendinitis supplied with GT; and G3 and G6, tendinitis supplied with GT and a glycine diet for 7 or 21 days, respectively. We performed zymography for metalloproteinase, biochemical, morphological and biomechanics tests. Results: G2, G3 and G5 showed high levels of hydroxyproline in relation to G1, while G4 showed high levels of glycosaminoglycans. High activity of metalloproteinase-2 was detected in G3. The organization of collagen bundles was better in G2 and G3. G5 showed similar birefringence measurements compared with C. G5 withstood a larger load compared with G4. Conclusions: The presence of metalloproteinase-2 indicates that a tissue is undergoing a remodeling process. High birefringence suggests a better organization of collagen bundles. After 21 days, G5 sustained a high load before rupture, unlike G4. The results suggest that GT?+?a glycine diet has beneficial effects that aid in the recovery process of the tendon after tendinitis.     

Effects of nitric oxide on the adhesion of human melanocytes to extracellular matrix components

The aim of the present study was to explore whether nitric oxide (NO) interferes with the attachment of human melanocytes to the extracellular matrix (ECM) components. Consequently, the effects have been investigated of the NO-releasing compounds 3-morpholino-sydnonimine (SIN-1) and S-nitroso-glutathione (GSNO) on the in vitro adhesion of human melanocytic cells to fibronectin. The NO donors induced a concentration-dependent reduction in the adhesion of both ⁵¹CrO₄²⁻-labelled melanocytes and melanoma cells to fibronectin. Pigmented M14 melanoma cells were more susceptible to the effect of SIN-1 (half-maximal inhibiting effect at about 0·5 mm) than normal human melanocytes and also than the non-pigmented melanoma cells Mel57 (half-maximal inhibiting effects between 0·9 and 2 mm). This effect of SIN-1 also appeared to be related to the melanin content of normal melanocytes, whereas GSNO was significantly less active. Both flow cytometric analysis and immunocytochemical staining showed expression of neuronal NO synthase in all cell lines. The results of this study suggest that aberrant in vivo production of NO during infection and inflammation may contribute to loss of melanocytes in, for example, vitiligo, by reducing de novo attachment of melanocytes to the ECM. These findings could also be important for understanding the process of metastasis. © 1997 John Wiley & Sons, Ltd.