Properties and Mechanobiological Behavior of Bovine Nasal Septum Cartilage

Abstract

Bovine nasal septum (BNS) is a source of non-load bearing hyaline cartilage. Little information is available on its mechanical and biological properties. The aim of this work was to assess the characteristics of BNS cartilage and investigate its behavior in in vitro mechanobiological experiments. Mechanical tests, biochemical assays, and microscopic assessment were performed for tissue characterization. Compressions tests showed that the tissue is viscoelastic, although values of elastic moduli differ from the ones of other cartilaginous tissues. Water content was 78 ± 1.4%; glycosaminoglycans and collagen contents—measured by spectrophotometric assay and hydroxyproline assay—were 39 ± 5% and 25 ± 2.5% of dry weight, respectively. Goldner’s Trichrome staining and transmission electron microscopy proved isotropic cells distribution and results of earlier cell division. Furthermore, gene expression was measured after uniaxial compression, showing variations depending on compression time as well as trends depending on equilibration time. In conclusion, BNS has been characterized at several levels, revealing that bovine nasal tissue is regionally homogeneous. Results suggest that, under certain conditions, BNS could be used to perform in vitro cartilage loading experiments.

Appendix

Mechanical Model of the Tissue

A linear solid model (Kelvin model) was chosen to characterize the tissue. This model is a combination of a linear spring in parallel with a series of a linear spring and a dashpot with a step response such as:

$$\sigma (t) = E_{R} \left[ {1 - \left( {1 - \frac{{\tau_{\sigma } }}{{\tau_{\varepsilon } }}} \right)e^{{{\raise0.7ex\hbox{${ - t}$} \!\mathord{\left/ {\vphantom {{ - t} {\tau_{\varepsilon } }}}\right.\kern-0pt} \!\lower0.7ex\hbox{${\tau_{\varepsilon } }$}}}} } \right]\varepsilon$$

(1)

where the stress σ(t) and the strain ε are related through the relaxed elastic modulus E _R and the time constants τ _σ and τ _ε . The instantaneous elastic modulus E ₀ can be written as:

$$E_{0} = E_{R} \frac{{\tau_{\sigma } }}{{\tau_{\varepsilon } }}$$

(2)

Quantification of GAG

The method used was modified from the literature.6 We incubated 100 μL of diluted sample aliquots (papain digested cartilage) with 1 mL of DMMB solution (16 mg/L DMMB in 0.2 M GuHCl, 1 g/L sodium formate and 1 mL/L formic acid) and mixed continuously for 30 min in order to promote the formation of the GAG-DMMB complex. After centrifugation at 12,000 g for 10 min, the precipitated complex was resuspended in 1 mL water and again centrifuged as before, in order to remove any dye excess. The complex was finally dissociated by adding 800 μL of decomplexation solution (50 mM sodium acetate buffer, pH 6.8, containing 10% n-propanol and 4 M GuHCl). The absorbance of 200 μL solution was read at 656 nm using a spectrophotometer plate reader (Synergy HT multi-Mode Microplate Reader, BioTek Instruments Inc., Winooski VT, USA). GAG concentrations were calculated on the basis of the Chondroitin-sulphate-A standard curve.

Quantification of Collagen

Collagen content was quantified by means of a hydroxyproline assay with some modifications.55 100 μL of diluted papain digested cartilage as well as 100 μL of standard hydroxyproline (Hyp) solution (2–40 μg/mL) were mixed with 100 μL of 4 N sodium hydroxide (1:1 v/v), gently mixed and hydrolyzed by autoclaving at 120 °C for 20 min. Autoclaved samples (100 μL) as well as standard solution (100 μL) were mixed with 425 μL of chloramine-T solution (0.056 M chloramine-T in 50% n-propanol) and incubated at room temperature for 25 min. Then, 475 µL of Ehrlich’s solution (1 M p-dimethylaminobenzaldehyde in n-propanol/perchloric acid (2:1 v/v)) were added to the oxidized samples or to the standard solution and incubated at 64 °C for 20 min. Absorbance was read at 550 nm in a plate reader and the amount of Hyp in the samples was determined by comparison with a Hyp standard curve. Thereafter, since Hyp represents approximately 14.4% of the aminoacidic composition of collagen, a factor of 6.94 was applied to calculate the total collagen content from the measured Hyp quantity.

RNA Extraction

The methods used were described in the literature.19 Finely sliced cartilage sub-explants (≈50 mg) were placed in Eppendorf tubes and homogenized twice for 1 min in 800 μL TRIzol reagent (Invitrogen Inc., Carlsbad CA, USA). After 5 min equilibration at room temperature, 200 μL of chloroform were added and the tubes were vigorously shaken, mixed and incubated for 2 min at room temperature. Following centrifugation at 9500×g for 30 min at 4 °C, the obtained aqueous phases were recovered, extracted with 200 μL of chloroform and treated as previously described. The recovered supernatants were transferred into 2 mL tubes, gently mixed with 500 μL of isopropanol, incubated for 10 min at room temperature and consecutively centrifuged at 9500×g for 40 min at 4 °C. The supernatants were discarded and the pellets resuspended in 900 μL of lysis buffer (RNeasy Mini Kit^®; Qiagen GmbH, Hilden, Germany) supplemented with 90 μL β-mercaptoethanol (Sigma-Aldrich Inc., St. Louis MO, USA). After adding 900 μL ethanol (75%), the RNA was purified using Qiagen RNeasy mini kit (Qiagen GmbH, Hilden, Germany) whereas genomic DNA was digested with DNase kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions.