Type V collagen regulates the structure and biomechanics of TMJ condylar cartilage: A fibrous-hyaline hybrid

doi:10.1016/j.matbio.2021.07.002

Matrix Biology

Volume 102, August 2021, Pages 1-19

https://doi.org/10.1016/j.matbio.2021.07.002 Get rights and content

Highlights

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Type V collagen regulates the collagen fibril nanostructure and micromechanics of both the fibrocartilage and hyaline cartilage layers in temporomandibular joint condyle.
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Reduction of type V collagen leads to decreased cell density and aberrant cell clustering in both fibrous and hyaline layers.
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Loss of type V collagen leads to reduced cell proliferation and β-catenin expression in the fibrous layer, indicating its role in maintaining the progenitor cell niche in condylar cartilage.
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Ablation of type V collagen at the post-weaning age results in pronounced thinning of the hyaline layer, highlighting the interplay between type V collagen and mechanoregulation of condylar cartilage growth.
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The role of type V collagen in regulating cell fate is specific to the progenitor cells in condylar cartilage, and is absent in knee cartilage.

Abstract

This study queried the role of type V collagen in the post-natal growth of temporomandibular joint (TMJ) condylar cartilage, a hybrid tissue with a fibrocartilage layer covering a secondary hyaline cartilage layer. Integrating outcomes from histology, immunofluorescence imaging, electron microscopy and atomic force microscopy-based nanomechanical tests, we elucidated the impact of type V collagen reduction on TMJ condylar cartilage growth in the type V collagen haploinsufficiency and inducible knockout mice. Reduction of type V collagen led to significantly thickened collagen fibrils, decreased tissue modulus, reduced cell density and aberrant cell clustering in both the fibrous and hyaline layers. Post-natal growth of condylar cartilage involves the chondrogenesis of progenitor cells residing in the fibrous layer, which gives rise to the secondary hyaline layer. Loss of type V collagen resulted in reduced proliferation of these cells, suggesting a possible role of type V collagen in mediating the progenitor cell niche. When the knockout of type V collagen was induced in post-weaning mice after the start of physiologic TMJ loading, the hyaline layer exhibited pronounced thinning, supporting an interplay between type V collagen and occlusal loading in condylar cartilage growth. The phenotype in hyaline layer can thus be attributed to the impact of type V collagen on the mechanically regulated progenitor cell activities. In contrast, knee cartilage does not contain the progenitor cell population at post-natal stages, and develops normal structure and biomechanical properties with the loss of type V collagen. Therefore, in the TMJ, in addition to its established role in regulating the assembly of collagen I fibrils, type V collagen also impacts the mechanoregulation of progenitor cell activities in the fibrous layer. We expect such knowledge to establish a foundation for understanding condylar cartilage matrix development and regeneration, and to yield new insights into the TMJ symptoms in patients with classic Ehlers-Danlos syndrome, a genetic disease due to autosomal mutation of type V collagen.

Introduction

Temporomandibular joint (TMJ) disorder, known as TMD, afflicts 5–12% of the US population, leading to limited jaw motion and chronic pain [1]. TMD, especially TMJ osteoarthritis (OA), is associated with degeneration of the mandibular condylar cartilage, a pivotal unit to the load bearing and energy dissipation functions for everyday jaw activities such as speaking and chewing [2]. Successful regeneration of condylar cartilage holds the potential for restoring joint function for TMD patients without causing common complications, including bone resorption and revision surgery, from standard treatments such as prosthetics and autografts [3]. However, one major roadblock for developing effective regeneration strategy [4] is the lack of understanding of the formation and degeneration of condylar cartilage extracellular matrix (ECM) [5]. The ECM has a hybrid structure integrating a fibrocartilage layer covering a secondary hyaline cartilage layer [6,7]. This is distinct from the articular surfaces of other diarthrodial joints that are dominated by hyaline cartilage [8]. To this day, there is little knowledge on the molecular activities that govern the formation of this unique hybrid ECM. Establishing the structure-mechanics principles of condylar cartilage ECM will provide the necessary benchmark for designing regenerative strategies [9]. Furthermore, the integrity of ECM is pivotal to cell mechanotransduction, and aberrant remodeling of the ECM is a major driving force of tissue dysfunction and perturbed cell signaling in disease [10], [11], [12]. Understanding the activities of ECM molecules will enable a more targeted design of disease intervention and regeneration strategies for this unique tissue [13,14].

Type V collagen (collagen V) could be a central player in the establishment of condylar cartilage ECM. In vivo, collagen V serves as the co-nucleator for initiating collagen I fibrillogenesis [15], and its partially processed N-propeptide projects outward from the fibril surface to limit aberrant fibril lateral growth [16]. In tension-bearing fibrous tissues such as skin [15], tendon [17] and cornea [18], reduction of collagen V leads to abnormally thickened collagen fibrils, reduced fibril numbers and impaired mechanical properties. Complete deletion of Col5a1 gene in mice results in embryonic lethality due to the incapability of collagen I fibrillogenesis [19]. Since the top layer of TMJ condylar cartilage consists of collagen I-dominated fibrocartilage [6], collagen V could play an essential role in regulating the formation and maintenance of this layer. In fact, the importance of collagen V to TMJ health is highlighted by the higher propensity towards TMD in patients with classic Ehlers-Danlos syndrome (cEDS) [20], a human autosomal dominant disorder (prevalence of ∼ 1:20,000) due to the mutation of COL5A1 or COL5A2 gene [21].

This study aimed to elucidate the role of collagen V in the structure and biomechanics of TMJ condylar cartilage in vivo. To assess the impact of collagen V deficiency on condylar cartilage ECM, we studied the murine model of cEDS (Col5a1^+/− [15]) at 3 months of age. To determine the age-dependent impact of collagen V loss, we tested the recently established inducible collagen V knockout mice (Col5a1^iKO [18]). In these mice, we maintained the normal level of collagen V in embryonic and neo-natal development, induced the knockout of Col5a1 gene expression at 1 week (pre-weaning) and 1 month (post-weaning) of ages, and analyzed the resulting phenotype at 1 and 2 months of ages, respectively. We assessed condylar cartilage morphology and cellularity, major ECM molecule distribution, collagen fibril nanostructure, as well as tissue modulus. In these tests, the fibrous and hyaline layers were analyzed separately to delineate the role of collagen V in these two distinct units. In addition, to determine if the observed defects of condylar cartilage are associated with tissue-specific activities of collagen V, we also tested the structure and biomechanics of knee cartilage in these mice, and compared cell proliferation and other phenotypic changes between condylar cartilage and knee cartilage. Our findings pointed to a crucial role of collagen V in regulating both matrix assembly and mechanosensitive cell activities during the post-natal growth of condylar cartilage.

Section snippets

Distribution of ECM molecules in TMJ condylar cartilage

To assess the distribution of collagen V in condylar cartilage, we applied immunofluorescence (IF) imaging to the TMJ of 3-month-old wild-type (WT) and Col5a1^+/− mice. In WT condylar cartilage, collagen V was found to be highly expressed in the fibrous layer with a preferential localization in the pericellular domain (Fig. 1a), similar to its distribution pattern in tendon [22]. In the hyaline layer, collagen V was present at much lower concentration, with trace amount detected in the

Role of collagen V in the post-natal growth of condylar cartilage hyaline layer

This study highlights a distinctive role of collagen V in the establishment of TMJ condylar cartilage ECM, a fibrous-hyaline hybrid (Fig. 10). In condylar cartilage, we found that collagen V impacts the structure and biomechanics of not only the collagen I-dominated fibrous layer, but also the collagen II-rich secondary hyaline layer. In the fibrous layer, the relative high concentration of collagen V (Fig. 1a), as well as the phenotype observed in Col5a1^+/− and Col5a1^iKO mice, support the

Conclusions

In summary, this study is the first to query the matrix molecular activities that give rise to the fibrous-hyaline hybrid ECM of TMJ condylar cartilage. Results highlight a crucial role of collagen V in this process. In addition to its expected role in regulating the fibrillar structure of fibrous layer, collagen V also regulates the formation of hyaline layer. The impact of collagen V on the hyaline layer can be attributed to its role in regulating the proliferation of progenitor cells in the

Animal model

Col5a1^+/− [15] and Col5a1^iKO (Col5a1^flox/flox/Rosa26Cre^ER) [18] mice in the C57BL/6 strain were generated as previously described and were housed in the Calhoun animal facility at Drexel University. To induce the knockout of Col5a1 gene, tamoxifen (T5648, Sigma) was intraperitoneally (i.p.) injected into Col5a1^iKO mice at 4.5 mg per 40 g of body weight per day for 3 consecutive days in the form of 20 mg/mL suspension in sesame oil (S3547, Sigma) with 1% benzyl alcohol (305197, Sigma). By day 5,

Author contributions

Conceptualization: L.H.; Supervision: L.H.; Data Collection and Analysis: P.C., B.K., B.H., S.M.A., C.W., D.R.C.; Data Interpretation: P.C., B.K., B.H., R.L.M., N.A.D., X.L.L., D.B.F., E.K., D.E.B., L.H.; Writing: P.C., B.K., B.H., X.L.L., E.K., L.H.; Funding Acquisition: L.H. All authors intellectually contributed and provided approval for publication.

Acknowledgements

This work was financially supported by the National Science Foundation (NSF) Grant CMMI-1751898 (to LH), National Institutes of Health (NIH) Grant R21DE029567 (to LH), as well as NIH Grant P30AR069619 to the Penn Center for Musculoskeletal Disorders. We thank the Singh Center for Nanotechnology at the University of Pennsylvania for the use of TIRF MFP-3D, which is part of the National Nanotechnology Coordinated Infrastructure Program supported by the NSF Grant NNCI-1542153. We thank Dr. L. J.

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Citation Excerpt :
Reduction of collagen V was found to impair the micromodulus of PCM in the hyaline layer in the TMJ condyle of Col5a1+/− mice. This was attributed to the altered proliferation and chondrogenesis of progenitor cells with the loss of collagen V in the fibrous layer, and thus, impaired matrix development (Chandrasekaran et al., 2021). Besides the aforementioned matrix molecules, there are many other constituents that are either exclusively or preferentially distributed in the PCM, such as collagens IX, XI, matrilines, chondroadherin and cartilage oligometric matrix protein (COMP) (Batista et al., 2014; Han et al., 2011b; Heinegård, 2009; Heinegård and Saxne, 2011).
Mechanosensing at the interface of a cell and its surrounding microenvironment is an essential driving force of physiological processes. Understanding molecular activities at the cell-matrix interface has the potential to provide novel targets for improving tissue regeneration and early disease intervention. In the past few decades, the advancement of atomic force microscopy (AFM) has offered a unique platform for probing mechanobiology at this crucial microdomain. In this review, we describe key advances under this topic through the use of an integrated system of AFM (as a biomechanical testing tool) with complementary immunofluorescence (IF) imaging (as an in situ navigation system). We first describe the body of work investigating the micromechanics of the pericellular matrix (PCM), the immediate cell micro-niche, in healthy, diseased, and genetically modified tissues, with a focus on articular cartilage. We then summarize the key findings in understanding cellular biomechanics and mechanotransduction, in which, molecular mechanisms governing transmembrane ion channel-mediated mechanosensing, cytoskeleton remodeling, and nucleus remodeling have been studied in various cell and tissue types. Lastly, we provide an overview of major technical advances that have enabled more in-depth studies of mechanobiology, including the integration of AFM with a side-view microscope, multiple optomicroscopy, a fluorescence recovery after photobleaching (FRAP) module, and a tensile stretching device. The innovations described here have contributed greatly to advancing the fundamental knowledge of extracellular matrix biomechanics and cell mechanobiology for improved understanding, detection, and intervention of various diseases.
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Type V collagen regulates the structure and biomechanics of TMJ condylar cartilage: A fibrous-hyaline hybrid

Highlights

Abstract

Introduction

Section snippets

Distribution of ECM molecules in TMJ condylar cartilage

Role of collagen V in the post-natal growth of condylar cartilage hyaline layer

Conclusions

Animal model

Author contributions

Acknowledgements

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