Mechanical homeostasis in tissue equivalents: a review.
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ABSTRACT:
SUBMITTER: Eichinger JF
PROVIDER: S-EPMC8154823 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Soft biological tissues consist of cells and extracellular matrix (ECM), a network of diverse proteins, glycoproteins, and glycosaminoglycans that surround the cells. The cells actively sense the surrounding ECM and regulate its mechanical state. Cell-seeded collagen or fibrin gels, so-called tissue equivalents, are simple but powerful model systems to study this phenomenon. Nevertheless, few quantitative studies document the stresses that cells establish and maintain in such gels; moreover, most prior data were collected via uniaxial experiments whereas soft tissues are mainly subject to multiaxial loading in vivo. To begin to close this gap between existing experimental data and in vivo conditions, we describe here a computer-controlled bioreactor that enables accurate measurements of the evolution of mechanical tension and deformation of tissue equivalents under well-controlled biaxial loads. This device allows diverse studies, including how cells establish a homeostatic state of biaxial stress and if they maintain it in response to mechanical perturbations. It similarly allows, for example, studies of the impact of cell and matrix density, exogenous growth factors and cytokines, and different types of loading conditions (uniaxial, strip-biaxial, and biaxial) on these processes. As illustrative results, we show that NIH/3T3 fibroblasts establish a homeostatic mechanical state that depends on cell density and collagen concentration. Following perturbations from this homeostatic state, the cells were able to recover biaxial loading similar to homeostatic. Depending on the precise loads, however, they were not always able to fully maintain that state.
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Project description:Tissue mechanical properties are determined mainly by the extracellular matrix (ECM) and actively maintained by resident cells. Despite its broad importance to biology and medicine, tissue mechanical homeostasis remains poorly understood. To explore cell-mediated control of tissue stiffness, we developed mutations in the mechanosensitive protein talin 1 to alter cellular sensing of ECM. Mutation of a mechanosensitive site between talin 1 rod-domain helix bundles R1 and R2 increased cell spreading and tension exertion on compliant substrates. These mutations promote binding of the ARP2/3 complex subunit ARPC5L, which mediates the change in substrate stiffness sensing. Ascending aortas from mice bearing these mutations showed less fibrillar collagen, reduced axial stiffness, and lower rupture pressure. Together, these results demonstrate that cellular stiffness sensing contributes to ECM mechanics, directly supporting the mechanical homeostasis hypothesis and identifying a mechanosensitive interaction within talin that contributes to this mechanism.
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Project description:Macrophages play a key role in orchestrating the host immune response toward invading organisms or non-self molecules in the oral mucosa. Three-dimensional (3D) oral mucosal equivalents (OME) containing oral fibroblasts and keratinocytes are used extensively to mimic the human oral mucosa where they have been employed to examine innate immune responses to both bacterial and fungal pathogens as well as to biomaterials. Although the presence of immune cells is critical in generating an immune response, very few studies have incorporated leukocytes into OME, and to date, none have contained primary human macrophages. In this study, we report the generation of an immunocompetent OME to investigate immune responses toward bacterial challenge. Primary human monocyte-derived macrophages (MDM) were as responsive to bacterial lipopolysaccharide (LPS) challenge when cultured within a 3D hydrogel in terms of proinflammatory cytokine (IL-6, CXCL8, and TNF-α) gene expression and protein secretion compared with culture as two-dimensional monolayers. MDM were incorporated into a type 1 collagen hydrogel along with oral fibroblasts and the apical surface seeded with oral keratinocytes to generate an MDM-containing OME. Full-thickness MDM-OME displayed a stratified squamous epithelium and a fibroblast-populated connective tissue containing CD68-positive MDM that could be readily isolated to a single-cell population for further analysis by collagenase treatment followed by flow cytometry. When stimulated with LPS, MDM-OME responded with increased proinflammatory cytokine secretion, most notably for TNF-α that increased 12-fold when compared with OME alone. Moreover, this proinflammatory response was inhibited by pretreatment with dexamethasone, showing that MDM-OME are also amenable to drug treatment. Dual-labeled immunofluorescence confocal microscopy revealed that MDM were the sole source of TNF-α production within MDM-OME. These data show functional activity of MDM-OME and illustrate their usefulness for investigations aimed at monitoring the immune response of the oral mucosa to pathogens, biomaterials, and for tissue toxicity and anti-inflammatory drug delivery studies. Impact statement Three-dimensional in vitro models of the oral mucosa have been used extensively to investigate the host response to pathogens, but, to date, few have contained primary leukocytes. In this report, we describe the successful incorporation of primary human macrophages into oral mucosal equivalents (OME). These macrophage-containing models were histologically similar to the oral mucosa and immunoresponsive to bacterial lipopolysaccharides by upregulation of key proinflammatory markers. These advanced OME will significantly aid research into host-pathogen interaction, biomaterial toxicity, and drug delivery studies where the presence of an immune cell component is critical to better represent host oral tissue.
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Project description:Tissue mechanical homeostasis, the concept that cells sense mechanical properties and alter rates of ECM synthesis, assembly and degradation, is of broad interest in biology and medicine, but there is little direct support or insight into mechanisms. Tissue mechanical properties such as elasticity and stiffness are determined primarily by the extracellular matrix (ECM). We therefore set out to test the mechanical homeostasis hypothesis by developing mutations in the mechanosensitive protein talin1 that alter cells’ sensing of ECM stiffness. We identified the side-to-side interaction between talin1 rod domain helix bundles 1 and 2 (R1 and R2) as a novel mechanosensitive site. Mutations that decrease the affinity of the interaction result in a leftward shift in cellular stiffness sensing curves, enabling cells to spread and exert tension on softer substrates. Opening of the R1-R2 interface promotes binding of the Arp2/3 subunit ArpC5L, which is required for the altered stiffness sensing. Introduction of these talin mutations into mice resulted in softer tissue in the ascending aorta with less fibrillar collagen and rupture under lower pressure ex vivo. Together, these results demonstrate that altering cellular stiffness sensing results in altered ECM deposition, tissue stiffness and strength, thus providing direct support for the mechanical homeostasis hypothesis. These results also identify a novel mechanosensitive interaction in the talin rod domain that contributes to this mechanism.
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Project description:Articular cartilage is integral to the mechanical function of many joints in the body. When injured, cartilage lacks the capacity to self-heal, and thus, therapies and replacements have been developed in recent decades to treat damaged cartilage. Given that the primary function of articular cartilage is mechanical in nature, rigorous physical evaluation of cartilage tissues undergoing treatment and cartilage constructs intended for replacement is an absolute necessity. With the large number of groups developing cartilage tissue engineering strategies, however, a variety of mechanical testing protocols have been reported in the literature. This lack of consensus in testing methods makes comparison between studies difficult at times, and can lead to misinterpretation of data relative to native tissue. Therefore, the purpose of this study was to systematically review mechanical testing of articular cartilage and cartilage repair constructs over the past 10 years (January 2009-December 2018), to highlight the most common testing configurations, and to identify key testing parameters. For the most common tests, key parameters identified in this systematic review were validated by characterizing both cartilage tissue and hydrogels commonly used in cartilage tissue engineering. Our findings show that compression testing was the most common test performed (80.2%; 158/197), followed by evaluation of frictional properties (18.8%; 37/197). Upon further review of those studies performing compression testing, the various modes (ramp, stress relaxation, creep, dynamic) and testing configurations (unconfined, confined, in situ) are described and systematically reviewed for parameters, including strain rate, equilibrium time, and maximum strain. This systematic analysis revealed considerable variability in testing methods. Our validation testing studies showed that such variations in testing criteria could have large implications on reported outcome parameters (e.g., modulus) and the interpretation of findings from these studies. This analysis is carried out for all common testing methods, followed by a discussion of less common trends and directions in the mechanical evaluation of cartilage tissues and constructs. Overall, this work may serve as a guide for cartilage tissue engineers seeking to rigorously evaluate the physical properties of their novel treatment strategies. Impact Statement Articular cartilage tissue engineering has made significant strides with regard to treatments and replacements for injured tissue. The evaluation of these approaches typically involves mechanical testing, yet the plethora of testing techniques makes comparisons between studies difficult, and often leads to misinterpretation of data compared with native tissue. This study serves as a guide for the mechanical testing of cartilage tissues and constructs, highlighting recent trends in test conditions and validating these common procedures. Cartilage tissue engineers, especially those unfamiliar with mechanical testing protocols, will benefit from this study in their quest to physically evaluate novel treatment and regeneration approaches.
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