Changes in Physiological Tendon Substrate Stiffness Have Moderate Effects on Tendon-Derived Cell Growth and Immune Cell Activation

Abstract

Tendinopathy is characterised by pathological changes in tendon matrix composition, architecture, and stiffness, alterations in tendon resident cell characteristics, and fibrosis, with inflammation also emerging as an important factor in tendinopathy progression. The sequence of pathological changes in tendinopathy and the cellular effects of the deteriorating matrix are largely unknown. This study investigated the effects of substrate stiffness on tendon-derived cells (TDCs) and THP-1 macrophages using PDMS substrates representing physiological tendon stiffness (1.88 MPa), a stiff gel (3.17 MPa) and a soft gel (0.61 MPa). Human TDCs were cultured on the different gel substrates and on tissue culture plastic. Cell growth was determined by alamarBlue™ assay, cell morphology was analysed in f-actin labelled cells, and phenotypic markers were analysed by real-time PCR. We found that in comparison to TDCs growing on gels with physiological stiffness, cell growth increased on soft gels at 48 h (23%, p = 0.003). Cell morphology was similar on all three gels. SCX expression was slightly reduced on the soft gels (1.4-fold lower, p = 0.026) and COL1A1 expression increased on the stiff gels (2.2-fold, p = 0.041). Culturing THP-1 macrophages on soft gels induced increased expression of IL1B (2-fold, p = 0.018), and IL8 expression was inhibited on the stiffer gels (1.9-fold, p = 0.012). We also found that culturing TDCs on plastic increased cell growth, altered cell morphology, and inhibited the expression of SCX, SOX9, MMP3, and COL3. We conclude that TDCs and macrophages respond to changes in matrix stiffness. The magnitude of responses measured in TDCs were minor on the range of substrate stiffness tested by the gels. Changes in THP-1 macrophages suggested a more inflammatory phenotype on substrates with non-physiological stiffness. Although cell response to subtle variations in matrix stiffness was moderate, it is possible that these alterations may contribute to the onset and progression of tendinopathy.

Diagnostic utility of shear wave elastography in musculoskeletal injuries: A narrative review

“…Degenerative tendinopathy, on the other hand, represents a noninflammatory proliferative cell and matrix response with increases in type III collagen production, extracellular matrix disorganization, and cell death, which can manifest as increased tissue stiffness. 43 In studies of the axial spine, it was observed that in certain cases there was no notable variance in tissue stiffness within the trapezius muscle or sciatic nerve when comparing patients with pathology to healthy control participants. 22,23 However, several studies indicated that comparing side-to-side evaluations of Young's modulus or SWV in the same individual, between the symptomatic and asymptomatic side, will provide helpful diagnostic information, as noted in comparison of sciatic nerve stiffness between the symptomatic and asymptomatic limbs.…”

Novel In Vitro Platform for Studying the Cell Response to Healthy and Diseased Tendon Matrices

“…Along with the matrix architecture, matrix stiffness is also crucial in maintaining the tenogenic phenotype. Compared to the native tendon, culturing tendon cells in the plastic substrate led to a significant decrease in the SCX expression, whereas tendons mimicking substrate stiffness maintained a tenogenic phenotype. , Human mesenchymal stem cells (hMSCs) cultured on substrates with stiffness ranging from 0.1 to 10 MPa observed the increased tenogenic potential of hMSCs on the stiffness of 1 and 10 MPa with increased expression of SCX and type-I collagen . However, the previous in vitro models fail to account for the appropriate substrate stiffness and architecture.…”

“…Numerous studies have highlighted the importance of appropriate substrate stiffness and architecture in regulating cell phenotype. − SCX is one of the primary markers for the tenogenic phenotype, − and previous studies have demonstrated the importance of substrate stiffness and architecture in maintaining cell morphology and SCX expression. − , Studies have found that tenocytes in anisotropic matrices exhibit an elongated cell morphology and have increased levels of SCX expression compared to the cells on isotropic matrix, while the loss of matrix anisotropy leads to decreased SCX expression. ,, Not only in tenocytes but tendon progenitor stem cells also cultured on an anisotropic matrix express increased levels of SCX compared to that of cells on an isotropic matrix . Along with the matrix architecture, matrix stiffness is also crucial in maintaining the tenogenic phenotype.…”

“…Numerous in vitro models have aimed to capture some biophysical aspects of the tendon microenvironment. However, these models fail to fully capture the physiological architecture or stiffness of the native tendon tissue simultaneously. ,− Tissue culture plates, which are commonly used for in vitro studies, expose the cells to a highly stiff, flat substrate that does not resemble the tendon microenvironment and causes a loss of tenogenic phenotype. , In contrast, aligned tendon matrix models prepared by electrospinning polycaprolactone or anisotropic collagen gels fail to incorporate the native tendon stiffness and do not mimic the tendon fibrillar architecture. ,,− , Therefore, in vitro models that manage to capture the complexity of the native tissue at scale for stiffness and architecture would be a valuable tool for studying cell behavior in healthy and diseased tendon matrices. Decellularized matrices closely mimic the in vivo tissue characteristics and thereby offer an ideal platform as an in vitro tissue mimetic model. − Although there have been previous studies that have decellularized tendons for tissue engineering purposes, − to our knowledge, this is the first study to utilize decellularized tendons as an in vitro model, and further characterizing cell phenotype is yet to be explored.…”

Applications of functionally-adapted hydrogels in tendon repair

“…Substrate stiffness is one of the most common factors that stem cells sense in the ECM microenvironment. Previous researches suggested that substrate stiffness plays a pivotal role in the proliferation, migration and differentiation of stem cells ( Engler et al, 2006 ; Konar et al, 2022 ). In terms of tendon tissues, substrate stiffness has been suggested to affect the tenogenic differentiation of stem cells ( Islam et al, 2017 ).…”