Tenascin-C Expression Controls the Maturation of Articular Cartilage In

Gruber et al. BMC Res Notes (2020) 13:78 https://doi.org/10.1186/s13104-020-4906-8 BMC Research Notes RESEARCH NOTE Open Access Tenascin-C expression controls the maturation of articular cartilage in mice Bastian L. Gruber1, Michael J. Mienaltowski2,4, James N. MacLeod2, Johannes Schittny3, Stephanie Kasper1 and Martin Flück1,3* Abstract Objective: Expression of the de-adhesive extracellular matrix protein tenascin-C (TNC) is associated with the early postnatal development of articular cartilage which is both load-dependent and associated with chondrocyte diferentiation. We assessed morphological changes in the articular cartilage of TNC defcient mice at postnatal ages of 1, 4 and 8 weeks compared to age-matched wildtype mice. Results: Cartilage integrity was assessed based on hematoxylin and eosin stained-sections from the tibial bone using a modifed Mankin score. Chondrocyte density and cartilage thickness were assessed morphometrically. TNC expression was localized based on immunostaining. At 8 weeks of age, the formed tangential/transitional zone of the articular cartilage was 27% thicker and the density of chondrocytes in the articular cartilage was 55% lower in wildtype than the TNC-defcient mice. TNC protein expression was associated with chondrocytes. No relevant changes were found in mice at 1 and 4 weeks of age. The fndings indicate a role of tenascin-C in the post-natal maturation of the extracellular matrix in articular cartilage. This might be a compensatory mechanism to strengthen resilience against mechanical stress. Keywords: Tenascin C, Knock-out mouse, Articular cartilage, Cell density, Cartilage defect, Load, Adhesion Introduction adhesions [3–5]. Tis de-adhesive action of TNC allows Tenascin-C (TNC) is a hexameric glycoprotein of the quiescent cells to enter an intermediate adhesive state extracellular matrix (ECM) that shapes mechanical and that is compatible with tissue remodelling during mor- biochemical cues within the cellular microenvironment phogenesis, wound healing and oncogenic transforma- of various tissues by the modulation of cell adhesion tion [1, 3, 6, 7]. [1]. TNC has a modular composition containing a hep- Expression of TNC is regulated by growth factor- and tad repeat region, epidermal growth factor (EGF)-like cytokine-activated signaling pathways [1, 8–10] and is domains, fbronectin-type III repeats, and a fbrinogen- subject to direct and indirect, damage-related regulation like globe enabling alternatively spliced TNC isoforms by mechanical stress in connective tissue cells [1, 11–13]. to bind diferent ECM proteins, including syndecan, Enhanced TNC expression is especially implicated in the fbronectin and diferent integrins [1, 2] and subsequently adaptive response of musculoskeletal tissues (i.e. skeletal modify the organization of the cytoskeleton and down- muscle, tendon and bone) to mechanical stress [11–15], stream signalling pathways via the dissolution of focal which governs the post-natal diferentiation, and regenerative response subsequent to the impact of a mechanical challenge or insult, of this tissue family [14, 16–19]. *Correspondence: [email protected] Based on its particularly high abundance in the con- 1 Laboratory for Muscle Plasticity, Department of Orthopedics, University of Zurich, Balgrist Campus, Lengghalde 5, 8008 Zurich, Switzerland densed mesenchyme, TNC has also been implicated Full list of author information is available at the end of the article in the diferentiation of chondrocytes during cartilage © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gruber et al. BMC Res Notes (2020) 13:78 Page 2 of 6 maturation in the embryo before TNC expression in University of Berne, Switzerland. Animal health sta- these cartilage anlagen is lost and chondrocytes pro- tus was daily inspected and the microbiological status duce cartilage-specifc extracellular matrix proteins inspected in sentinels. Genotype was determined by PCR [20–22]. Later, up to 4 weeks postpartum TNC expres- on tail DNA [14]. Tail cutting was done after euthanasia. sion reappears in the peripheral perichondrium [21, 23] and remains expressed in articular cartilage, but not in Sample preparation and histology the growth plate, and decreases thereafter [16, 20, 24]. Te mice were anaesthetized with 5% isofurane (Provet Recently, TNC has been found to be re-expressed subse- AG, Burgdorf, Switzerland) and euthanized by decapita- quent to traumatic joint loading of the developed articu- tion. Skeletal elements of the explanted hindlimbs were lar cartilage and to promote cartilage repair via a switch fxed in 4% paraformaldehyde and shipped to the Uni- in extracellular matter synthesis [25]. versity of Kentucky. Te tissue was processed by decal- Although proposed [26], and suggested by TNC’s con- cifcation as described [30, 31], embedded in parafn, tribution to musculoskeletal remodelling [11–15] and sectioned at 5 μm thickness in parallel direction to the load-dependent regenerative functional adaptations of tibial axes, and subjected to standard hematoxylin and joints after birth [27, 28]; it had never been tested experi- eosin (H&E) staining. Slides representing the distal femur mentally whether TNC participates in articular chon- from the diaphysis (proximal) to the articular surface of drocyte development and diferentiation in long bone the knee (distal) and the proximal tibia from the articular models, and remains functional throughout postnatal surface to the diaphysis were shipped to the University of life. We thus hypothesized that TNC-defcient mice Zurich for morphological analysis. would demonstrate structural aberrations of articular cartilage in the frst 2 months after birth when knee joints are frst subjected to gravitational loading and chondro- Assessment of structural cartilage defcits cyte volume and extracellular matrix production undergo H&E stained sections of the coronary tibia were recorded marked alterations [24]. at a four- and tenfold magnifcation on an IX50 micro- scope via a DP72 digital camera (Olympus, Volketswil, Main text Switzerland). A modifed Mankin score was used to grade Methods cartilage integrity from 0 to 7 points (i.e. normal struc- Study design ture to complete cartilaginous destruction) based on the TNC-defcient mice (TNC / ) and homozygous staining of the cartilage structure and tidemark (Addi- − − tional fle 1: Figure S1, Additional fle 2: Table S1; [32]). wildtype mice (TNC +/+) were generated by the breeding of homozygous TNC-defcient mice, and homozy- Te employed scoring rubric has been shown to strongly gous wildtype mice, respectively. Mice were ear marked, correlate with the OARSI scoring [33] and has been used genotyped within the frst 2 weeks after birth and subse- consistently in rodent [34, 35] and human specimens to quently housed in groups of 2–6 animals per cage. Te grade mild to moderate cartilage defects [33]. Cell den- parental homozygous TNC-defcient mice and homozy- sity within the articular cartilage was detected by point gous wildtype mice were derived from the breeding of counting using a 25 by 25 µm grid that was placed on a randomly selected tenfold magnifed microscopic feld of heterozygous TNC-defcient mice (TNC +/+/). Skeletal tissue was collected from euthanized mice irrespective of each sample under application of the forbidden line rule. sex, at 4 or 8 weeks of age and subjected to histological Tickness of the tangential/transitional zone of articular processing. Te assessment of structural defcits (modi- cartilage was determined from the average of three meas- fed Mankin score, cell density in the articular cartilage, urements for the tangential distance in the center of the TNC expression in articular cartilage) was carried out in joint with the cellSens software (version 1.6, Olympus, a blinded fashion. Volketswil, Switzerland). Animals Immunohistochemical detection of tenascin‑C TNC-defcient mice were derived from the original strain Parafn sections were processed essentially as described with a targeted insertion of a β-lactamase cassette in the [36] but without preincubation with proteolytic enzymes. NcoI site of exon 2 of the TNC gene [29] and back crossed Deparafnized sections were incubated with afn- with WT 129/SV mice. Mice were housed with 12:12-h ity purifed TNC specifc antibody from rabbit (#473, light/dark cycle at a constant temperature of 22 °C in 1:100; [37]) or a negative control (rabbit antibody against Macrolon type III cages (Indulab, Italy) under specifc- serum response factor, [38]) and subsequently with horse pathogen-free conditions with standard chow and water radish peroxidase-coupled goat anti-rabbit antibody ad libitum at the Department of Clinical Research, [#55676 (1:200; MP Biomedicals, Ohio, USA)]. Signal was Gruber et al. BMC Res Notes (2020) 13:78 Page 3 of 6 detected using AEC high sensitivity substrate (DAKO, Baar, Switzerland) and microscopically recorded. Statistical analysis We used SPSS by IBM (Armonk, NY, USA) for the statistical analysis and graphic representation of the data. A two-way ANOVA for the factors genotype and age fol- lowed by Bonferroni post hoc analysis was performed, when equality of variance could be assumed based on a Levene’s test.

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