ABSTRACT
Although tendon predominantly experiences longitudinal tensile forces, transverse forces due to impingement from bone are implicated in both physiological and pathophysiological processes. However, prior studies have not characterized the micromechanical strain environment in the context of tendon impingement. To address this knowledge gap, mouse hindlimb explants are imaged on a multiphoton microscope, and image stacks of the same population of tendon cells are obtained in the Achilles tendon before and after dorsiflexion-induced impingement by the heel bone. Based on the acquired images, multiaxial strains are measured at the extracellular matrix (ECM), pericellular matrix (PCM), and cell scales. Impingement generated substantial transverse compression at the matrix-scale, which led to longitudinal stretching of cells, increased cell aspect ratio, and enormous volumetric compression of the PCM. These experimental results are corroborated by a finite element model, which further demonstrated that impingement produces high cell surface stresses and strains that greatly exceed those brought about by longitudinal tension. Moreover, in both experiments and simulations, impingement-generated microscale stresses and strains are highly dependent on initial cell-cell gap spacing. Identifying factors that influence the microscale strain environment generated by impingement could contribute to a more mechanistic understanding of impingement-induced tendinopathies.
ABSTRACT
Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone, and cartilage development and homeostasis, knowledge about Ca2+ signaling and the source of Ca2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca2+ signaling through CaV 1.2 voltage-gated Ca2+ channel in tendon formation. Using a reporter mouse, we found that CaV 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;CaV 1.2TS mice that express a gain-of-function mutant CaV 1.2 in tendon. We found that mutant tendons were hypertrophic, with more tendon fibroblasts but decreased cell density. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendons. Biomechanical testing revealed that the hypertrophic tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomic analysis showed no significant difference in the abundance of type I and III collagens, but mutant tendons had about two-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2, and cathepsin K. Taken together, these data highlight roles for increased Ca2+ signaling through CaV 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.
Subject(s)
Mechanotransduction, Cellular , Myostatin , Animals , Mice , Biomechanical Phenomena , Collagen/metabolism , Myostatin/metabolism , Proteomics , Tendons/metabolismABSTRACT
Articular cartilage (AC) is a load-bearing tissue that covers long bones in synovial joints. The biphasic/poroelastic mechanical properties of AC help it to protect joints by distributing loads, absorbing impact forces, and reducing friction. Unfortunately, alterations in these mechanical properties adversely impact cartilage function and precede joint degeneration in the form of osteoarthritis (OA). Thus, understanding what factors regulate the poroelastic mechanical properties of cartilage is of great scientific and clinical interest. Transgenic mouse models provide a valuable platform to delineate how specific genes contribute to cartilage mechanical properties. However, the poroelastic mechanical properties of murine articular cartilage are challenging to measure due to its small size (thickness â¼ 50 microns). In the current study, our objective was to test whether the poroelastic mechanical properties of murine articular cartilage can be determined based solely on time-dependent cell death measurements under constant loading conditions. We hypothesized that in murine articular cartilage subjected to constant, sub-impact loading from an incongruent surface, cell death area and tissue strain are closely correlated. We further hypothesized that the relationship between cell death area and tissue strain can be used-in combination with inverse finite element modeling-to compute poroelastic mechanical properties. To test these hypotheses, murine cartilage-on-bone explants from different anatomical locations were subjected to constant loading conditions by an incongruent surface in a custom device. Cell death area increased over time and scaled linearly with strain, which rose in magnitude over time due to poroelastic creep. Thus, we were able to infer tissue strain from cell death area measurements. Moreover, using tissue strain values inferred from cell death area measurements, we applied an inverse finite element modeling procedure to compute poroelastic material properties and acquired data consistent with previous studies. Collectively, our findings demonstrate in the key role poroelastic creep plays in mediating cell survival in mechanically loaded cartilage and verify that cell death area can be used as a surrogate measure of tissue strain that enables determination of murine cartilage mechanical properties.
Subject(s)
Cartilage, Articular , Osteoarthritis , Animals , Mice , Chondrocytes/physiology , Stress, Mechanical , Cartilage, Articular/physiology , Cell DeathABSTRACT
Chondrocytes, the resident cells in articular cartilage, carry the burden of producing and maintaining the extracellular matrix (ECM). However, as these cells have a low proliferative capacity and are not readily replaced, chondrocyte death due to extreme forces may contribute to the pathogenesis of osteoarthritis (OA) after injury or may inhibit healing after osteochondral transplantation, a restorative procedure for damaged cartilage that requires a series of mechanical impacts to insert the graft. Consequently, there is a need to understand what factors influence the vulnerability of in situ chondrocytes to mechanical trauma. To this end, the objective of this study was to investigate how altering cell volume by different means (hydrostatic pressure, uniaxial load, and osmotic challenge with and without inhibition of regulatory volume decrease) affects the vulnerability of in situ chondrocytes to extreme mechanical forces. Using a custom experimental platform enabling testing of viable and intact murine cartilage-on-bone explants, we established a strong correlation between chondrocyte volume and vulnerability to impact injury wherein reduced volume was protective. Moreover, we found that the volume-perturbing interventions did not affect cartilage ECM mechanical properties, suggesting that their effects on chondrocyte vulnerability occurred at the cellular level. The findings of this study offer new avenues for novel strategies aimed at preventing chondrocyte loss during osteochondral grafting or to halting the progression of cell death after a joint destabilizing injury.
Subject(s)
Cell Size , Chondrocytes , Extracellular Matrix , Menisci, Tibial , Tibial Meniscus Injuries , Animals , Chondrocytes/metabolism , Chondrocytes/pathology , Disease Models, Animal , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Female , Menisci, Tibial/metabolism , Menisci, Tibial/pathology , Mice , Mice, Inbred BALB C , Tibial Meniscus Injuries/metabolism , Tibial Meniscus Injuries/pathologyABSTRACT
Corneal endothelial cell (CEC) loss occurs from tissue manipulation during anterior segment surgery and corneal transplantation as well as from contact with synthetic materials like intraocular lenses and tube shunts. While several studies have quantified CEC loss for specific surgical steps, the vulnerability of CECs to isolated, controllable and measurable mechanical forces has not been assessed previously. The purpose of this study was to develop an experimental testing platform where the susceptibility of CECs to controlled mechanical trauma could be measured. The corneal endothelial surfaces of freshly dissected porcine corneas were subjected to a range of indentation forces via a spherical stainless steel bead. A cell viability assay in combination with high-resolution fluorescence microscopy was used to visualize and quantify injured/dead CEC densities before and after mechanical loading. In specimens subjected to an indentation force of 9â¯mN, the mean⯱â¯SD peak contact pressure P0 was 18.64⯱â¯3.59â¯kPa (139.81⯱â¯26.93â¯mmHg) in the center of indentation and decreased radially outward. Injured/dead CEC densities were significantly greater (pâ¯≤â¯0.001) after mechanical indentation of 9â¯mN (167⯱â¯97â¯cells/mm2) compared to before indentation (39⯱â¯52â¯cells/mm2) and compared to the sham group (34⯱â¯31â¯cells/mm2). In specimens subjected to "contact only" - defined as an applied indentation force of 0.65â¯mN - the peak contact pressure P0 was 7.31⯱â¯1.5â¯kPa (54.83⯱â¯11.25â¯mmHg). In regions where the contact pressures was below 78% of P0 (<5.7â¯kPa or 42.75â¯mmHg), injured/dead CEC densities were within the range of CEC loss observed in the sham group, suggesting negligible cell death. These findings indicate that CECs are highly susceptible to mechanical trauma via indentation, supporting the established "no-touch" policy for ophthalmological procedures. While CECs can potentially remain viable below contact pressures of 5.7â¯kPa (42.75â¯mmHg), this low threshold suggests that prevention of indentation-associated CEC loss may be challenging.
Subject(s)
Corneal Endothelial Cell Loss/etiology , Elasticity Imaging Techniques , Endothelium, Corneal/injuries , Endothelium, Corneal/pathology , Eye Injuries/etiology , Microscopy, Fluorescence , Stress, Mechanical , Wounds, Nonpenetrating/etiology , Animals , Cell Count , Cell Survival , Corneal Endothelial Cell Loss/diagnostic imaging , Corneal Endothelial Cell Loss/physiopathology , Endothelium, Corneal/diagnostic imaging , Eye Injuries/diagnostic imaging , Eye Injuries/physiopathology , Microspheres , Swine , Wounds, Nonpenetrating/diagnostic imaging , Wounds, Nonpenetrating/physiopathologyABSTRACT
With the onset and progression of osteoarthritis (OA), articular cartilage (AC) mechanical properties are altered. These alterations can serve as an objective measure of tissue degradation. Although the mouse is a common and useful animal model for studying OA, it is extremely challenging to measure the mechanical properties of murine AC due to its small size (thickness < 50 µm). In this study, we developed novel and direct approach to independently quantify two quasi-static mechanical properties of mouse AC: the load-dependent (nonlinear) solid matrix Young's modulus (E) and drained Poisson's ratio (ν). The technique involves confocal microscope-based multiaxial strain mapping of compressed, intact murine AC followed by inverse finite element analysis (iFEA) to determine E and ν. Importantly, this approach yields estimates of E and ν that are independent of the initial guesses used for iterative optimization. As a proof of concept, mechanical properties of AC on the medial femoral condyles of wild-type mice were obtained for both trypsin-treated and control specimens. After proteolytic tissue degradation induced through trypsin treatment, a dramatic decrease in E was observed (compared to controls) at each of the three tested loading conditions. A significant decrease in ν due to trypsin digestion was also detected. These data indicate that the method developed in this study may serve as a valuable tool for comparative studies evaluating factors involved in OA pathogenesis using experimentally induced mouse OA models.
Subject(s)
Cartilage, Articular , Elastic Modulus , Nonlinear Dynamics , Animals , Biomechanical Phenomena , Cartilage, Articular/metabolism , Female , Glycosaminoglycans/metabolism , Materials Testing , Mice , Mice, Inbred BALB C , Trypsin/metabolismABSTRACT
OBJECTIVES: To examine (1) the validity of ultrasound imaging to measure osteophytes and (2) the association between osteophytes and insertional Achilles tendinopathy (IAT). DESIGN: Case-control study. SETTING: Academic medical center. PARTICIPANTS: Persons with chronic unilateral IAT (n=20; mean age, 58.7±8.3y; 10 [50%] women) and age- and sex-matched controls (n=20; mean age, 57.4±9.8y; 10 [50%] women) participated in this case-control study (N=40). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Symptom severity was assessed using the Foot and Ankle Ability Measure, the Victorian Institute of Sport Assessment-Achilles questionnaire, and the numerical rating scale. Length of osteophytes was measured bilaterally in both groups using ultrasound imaging, as well as on the symptomatic side of the IAT group using radiography. The intraclass correlation coefficient was used to examine the agreement between ultrasound and radiograph measures. McNemar, Wilcoxon signed-rank, and Fisher exact tests were used to compare the frequency and length of osteophytes between sides and groups. Pearson correlation was used to examine the association between osteophyte length and symptom severity. RESULTS: There was good agreement (intraclass correlation coefficient, ≥.75) between ultrasound and radiograph osteophyte measures. There were no statistically significant differences (P>.05) in the frequency of osteophytes between sides or groups. Osteophytes were larger on the symptomatic side of the IAT group than on the asymptomatic side (P=.01) and on the left side of controls (P=.03). There was no association between osteophyte length and symptom severity. CONCLUSIONS: Ultrasound imaging is a valid measure of osteophyte length, which is associated with IAT. Although a larger osteophyte indicates tendinopathy, it does not indicate more severe IAT symptoms.
Subject(s)
Achilles Tendon , Osteophyte/diagnostic imaging , Osteophyte/epidemiology , Tendinopathy/diagnostic imaging , Tendinopathy/epidemiology , Academic Medical Centers , Aged , Case-Control Studies , Female , Humans , Male , Middle Aged , Reproducibility of Results , Severity of Illness Index , UltrasonographyABSTRACT
Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer.
Subject(s)
Adipose Tissue/cytology , Breast Neoplasms/therapy , Stem Cell Transplantation , Animals , Breast Neoplasms/pathology , Cell Differentiation , Cell Line, Tumor , Disease Progression , Extracellular Matrix , Female , Humans , Mice , Neoplasm TransplantationABSTRACT
Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing.
Subject(s)
Cicatrix , Myofibroblasts , Humans , Myofibroblasts/metabolism , Cicatrix/metabolism , Periostin , Fibrosis , Cell Differentiation , TendonsABSTRACT
Injury and degeneration of tendon, the soft tissue that mechanically links muscle and bone, can cause substantial pain and loss of function. This review discusses the composition and function of healthy tendon and describes the structural, biological, and mechanical changes initiated during the process of tendon healing. Biochemical pathways activated during repair, experimental injury models, and parallels between tendon healing and tendon development are emphasized, and cutting-edge strategies for the enhancement of tendon healing are discussed.
Subject(s)
Tendon Injuries/physiopathology , Tendon Injuries/therapy , Tendons/physiology , Wound Healing/physiology , Animals , Biomechanical Phenomena , Bone and Bones/physiopathology , Cell Culture Techniques , Chickens , Dogs , Elasticity , Horses , Humans , Mice , Rabbits , Rats , Regeneration , Stress, Mechanical , Tendons/pathologyABSTRACT
Tendons like the flexor carpi ulnaris (FCU) that contain region-specific distributions of proteoglycans (PGs) as a result of the heterogeneous, multi-axial loads they are subjected to in vivo provide valuable models for understanding structure-function relationships in connective tissues. However, the contributions of specific PGs to FCU tendon mechanical properties are unknown. Therefore, the objective of this study was to determine how the location-dependent, viscoelastic mechanical properties of the FCU tendon are impacted individually by PG-associated glycosaminoglycans (GAGs) and by two small leucine-rich proteoglycans (SLRPs), biglycan and decorin. Full length FCU tendons from biglycan- and decorin-null mice were compared to wild-type (WT) mice to evaluate the effects of specific SLRPs, while chondroitinase ABC digestion of isolated specimens removed from the tendon midsubstance was used to determine how chondroitin/dermatan sulfate (CS/DS) GAGs impact mechanics in mature FCU tendons. A novel combined genetic knockout/digestion technique also was employed to compare SLRP-null and WT tendons in the absence of CS/DS GAGs that may impact properties in the mature state. In all genotypes, mechanical properties in the FCU tendon midsubstance were not affected by GAG digestion. Full-length tendons exhibited complex, multi-axial deformation under tension that may be associated with their in vivo loading environment. Mechanical properties were adversely affected by the absence of biglycan, and a decreased modulus localized in the center of the tendon was measured. These results help elucidate the role that local alterations in PG levels may play in processes that adversely impact tendon functionality including injury and pathology.
Subject(s)
Proteoglycans/metabolism , Tendons/metabolism , Animals , Biglycan/deficiency , Biglycan/metabolism , Biomechanical Phenomena , Chondroitin Sulfates/metabolism , Dermatan Sulfate/metabolism , Elastic Modulus , Female , Mice , Mice, Inbred C57BL , Stress, MechanicalABSTRACT
The mechanical properties of the human supraspinatus tendon (SST) are highly heterogeneous and may reflect an important adaptive response to its complex, multiaxial loading environment. However, these functional properties are associated with a location-dependent structure and composition that have not been fully elucidated. Therefore, the objective of this study was to determine the concentrations of types I, II and III collagen in six distinct regions of the SST and compare changes in collagen concentration across regions with local changes in mechanical properties. We hypothesized that type I collagen content would be high throughout the tendon, type II collagen would be restricted to regions of compressive loading and type III collagen content would be high in regions associated with damage. We further hypothesized that regions of high type III collagen content would correspond to regions with low tensile modulus and a low degree of collagen alignment. Although type III collagen content was not significantly higher in regions that are frequently damaged, all other hypotheses were supported by our results. In particular, type II collagen content was highest near the insertion while type III collagen was inversely correlated with tendon modulus and collagen alignment. The measured increase in type II collagen under the coracoacromial arch provides evidence of adaptation to compressive loading in the SST. Moreover, the structure-function relationship between type III collagen content and tendon mechanics established in this study demonstrates a mechanism for altered mechanical properties in pathological tendons and provides a guideline for identifying therapeutic targets and pathology-specific biomarkers.
Subject(s)
Collagen Type III/metabolism , Collagen Type II/metabolism , Collagen Type I/metabolism , Tendons/anatomy & histology , Tendons/metabolism , Elastic Modulus , Enzyme-Linked Immunosorbent Assay , Humans , Middle Aged , Reference StandardsABSTRACT
Though remarkably robust, articular cartilage becomes susceptible to damage at high loading rates, particularly under shear. While several studies have measured the local static and steady-state shear properties of cartilage, it is the local viscoelastic properties that determine the tissue's ability to withstand physiological loading regimens. However, measuring local viscoelastic properties requires overcoming technical challenges that include resolving strain fields in both space and time and accurately calculating their phase offsets. This study combined recently developed high-speed confocal imaging techniques with three approaches for analyzing time- and location-dependent mechanical data to measure the depth-dependent dynamic modulus and phase angles of articular cartilage. For sinusoidal shear at frequencies f = 0.01 to 1 Hz with no strain offset, the dynamic shear modulus |G*| and phase angle δ reached their minimum and maximum values (respectively) approximately 100 µm below the articular surface, resulting in a profound focusing of energy dissipation in this narrow band of tissue that increased with frequency. This region, known as the transitional zone, was previously thought to simply connect surface and deeper tissue regions. Within 250 µm of the articular surface, |G*| increased from 0.32 ± 0.08 to 0.42 ± 0.08 MPa across the five frequencies tested, while δ decreased from 12 deg ± 1 deg to 9.1 deg ± 0.5 deg. Deeper into the tissue, |G*| increased from 1.5 ± 0.4 MPa to 2.1 ± 0.6 MPa and δ decreased from 13 deg ± 1 deg to 5.5 deg ± 0.2 deg. Viscoelastic properties were also strain-dependent, with localized energy dissipation suppressed at higher shear strain offsets. These results suggest a critical role for the transitional zone in dissipating energy, representing a possible shift in our understanding of cartilage mechanical function. Further, they give insight into how focal degeneration and mechanical trauma could lead to sustained damage in this tissue.
Subject(s)
Cartilage, Articular/physiology , Shear Strength , Weight-Bearing , Animals , Cattle , Materials Testing , Stress, Mechanical , ViscosityABSTRACT
While useful models have been proposed to predict the mechanical impact of damage in tendon and other soft tissues, the applicability of these models for describing in vivo injury and age-related degeneration has not been investigated. Therefore, the objective of this study was to develop and validate a simple damage model to predict mechanical alterations in mouse patellar tendons after aging, injury, or healing. To characterize baseline properties, uninjured controls at age 150 days were cyclically loaded across three strain levels and five frequencies. For comparison, damage was induced in mature (120 day-old) mice through either injury or aging. Injured mice were sacrificed at three or six weeks after surgery, while aged mice were sacrificed at either 300 or 570 days old. Changes in mechanical properties (relative to baseline) in the three week post-injury group were assessed and used to develop an empirical damage model based on a simple damage parameter related to the equilibrium stress at a prescribed strain (6%). From the derived model, the viscoelastic properties of the 300 day-old, 570 day-old, and six week post-injury groups were accurately predicted. Across testing conditions, nearly all correlations between predicted and measured parameters were statistically significant and coefficients of determination ranged from R² = 0.25 to 0.97. Results suggest that the proposed damage model could exploit simple in vivo mechanical measurements to predict how an injured or aged tendon will respond to complex physiological loading regimens.
Subject(s)
Aging , Computer Simulation , Patellar Ligament/injuries , Animals , Female , Mice , Mice, Inbred C57BL , Models, Biological , Patellar Ligament/physiology , Stress, MechanicalABSTRACT
Aged tendons have disrupted homeostasis, increased injury risk, and impaired healing capacity. Understanding mechanisms of homeostatic disruption is crucial for developing therapeutics to retain tendon health through the lifespan. Here, we developed a novel model of accelerated tendon extracellular matrix (ECM) aging via depletion of Scleraxis-lineage cells in young mice (Scx-DTR). Scx-DTR recapitulates many aspects of tendon aging including comparable declines in cellularity, alterations in ECM structure, organization, and composition. Single-cell RNA sequencing demonstrated a conserved decline in tenocytes associated with ECM biosynthesis in aged and Scx-DTR tendons, identifying the requirement for Scleraxis-lineage cells during homeostasis. However, the remaining cells in aged and Scx-DTR tendons demonstrate functional divergence. Aged tenocytes become pro-inflammatory and lose proteostasis. In contrast, tenocytes from Scx-DTR tendons demonstrate enhanced remodeling capacity. Collectively, this study defines Scx-DTR as a novel model of accelerated tendon ECM aging and identifies novel biological intervention points to maintain tendon function through the lifespan.
Subject(s)
Extracellular Matrix , Tendons , Mice , Animals , Extracellular Matrix/genetics , Aging , Homeostasis , Phenotype , Basic Helix-Loop-Helix Transcription Factors/geneticsABSTRACT
Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.
Subject(s)
Achilles Tendon , Tendinopathy , Mice , Animals , Achilles Tendon/surgery , Achilles Tendon/pathology , Lower Extremity , Pressure , Collagen/metabolismABSTRACT
Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing.
ABSTRACT
Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca 2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone and cartilage development and homeostasis, knowledge about Ca 2+ signaling and the source of Ca 2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca 2+ signaling through Ca V 1.2 voltage-gated Ca 2+ channel in tendon formation. Using a reporter mouse, we found that Ca V 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;Ca V 1.2 TS mice that express a gain-of-function mutant Ca V 1.2 channel (Ca V 1.2 TS ) in tendon. We found that tendons in the mutant mice were approximately 2/3 larger and had more tendon fibroblasts, but the cell density of the mutant mice decreased by around 22%. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendon. Biomechanical testing revealed that the hypertrophic Achilles tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomics analysis reveals no significant difference in the abundance of major extracellular matrix (ECM) type I and III collagens, but mutant mice had about 2-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor of TGF-ß family myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2 and cathepsin K. Taken together, these data highlight roles for increased Ca 2+ signaling through Ca V 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.
ABSTRACT
Tendon injuries are very common and result in significant impairments in mobility and quality of life. During healing, tendons produce a scar at the injury site, characterized by abundant and disorganized extracellular matrix and by permanent deficits in mechanical integrity compared to healthy tendon. Although a significant amount of work has been done to understand the healing process of tendons and to develop potential therapeutics for tendon regeneration, there is still a significant gap in terms of assessing the direct effects of therapeutics on the functional and material quality specifically of the scar tissue, and thus, on the overall tendon healing process. In this study, we focused on characterizing the mechanical properties of only the scar tissue in flexor digitorum longus (FDL) tendons during the proliferative and early remodeling healing phases and comparing these properties with the mechanical properties of the composite healing tissue. Our method was sensitive enough to identify significant differences in structural and material properties between the scar and tendon-scar composite tissues. To account for possible inaccuracies due to the small aspect ratio of scar tissue, we also applied inverse finite element analysis (iFEA) to compute mechanical properties based on simulated tests with accurate specimen geometries and boundary conditions. We found that the scar tissue linear tangent moduli calculated from iFEA were not significantly different from those calculated experimentally at all healing timepoints, validating our experimental findings, and suggesting the assumptions in our experimental calculations were accurate. Taken together, this study first demonstrates that due to the presence of uninjured stubs, testing composite healing tendons without isolating the scar tissue overestimates the material properties of the scar itself. Second, our scar isolation method promises to enable more direct assessment of how different treatment regimens (e.g., cellular ablation, biomechanical and/or biochemical stimuli, tissue engineered scaffolds) affect scar tissue function and material quality in multiple different types of tendons.
Subject(s)
Cicatrix , Quality of Life , Animals , Biomechanical Phenomena , Cicatrix/pathology , Mice , Tendons/pathology , Wound HealingABSTRACT
Tendons are composed of a heterogeneous cell environment, with Scleraxis-lineage (ScxLin) cells being the predominant population. Although ScxLin cells are required for maintenance of tendon homeostasis, their functions during tendon healing are unknown. To this end, we first characterized the spatiotemporal dynamics of ScxLin cells during tendon healing, and identified that the overall ScxLin pool continuously expands up to early remodeling healing phase. To better define the function of ScxLin cells during the late proliferative phase of healing, we inducibly depleted ScxLin cells from day 14-18 post-surgery using the Scx-Cre; Rosa-DTR mouse model, with local administration of diphtheria toxin inducing apoptosis of ScxLin cells in the healing tendon. At D28 post-surgery, ScxLin cell depleted tendons (DTRScxLin) had substantial impairments in structure and function, relative to WT, demonstrating the importance of ScxLin cells during tendon healing. Next, bulk RNAseq was utilized to identify the underlying mechanisms that were impaired with depletion and revealed that ScxLin depletion induced molecular and morphological stagnation of the healing process at D28. However, this stagnation was transient, such that by D56 tendon mechanics in DTRScxLin were not significantly different than wildtype repairs. Collectively, these data offer fundamental knowledge on the dynamics and roles of ScxLin cells during tendon healing.