Introduction: Intracellular calcium concentration ([Ca 2+] i) is a critical parameter in cellular homeostasis, including articular chondrocytes. Perturbed [Ca 2+] i of chondrocytes may be associated with joint disease. The objective of the study was to compare large animal models for investigating Ca 2+ homeostasis in chondrocytes. Materials and Methods: The gross anatomy of the metacarpophalangeal joint (MCP) of cattle and sheep was compared, along with the effect of various manoeuvres used to study the mechanisms of Ca 2+ homeostasis in chondrocytes from load-bearing areas. The gross anatomy was observed before and after dissection, and internal architecture was examined after sectioning. Cartilage thickness was measured with a digital micrometer. Chondrocyte yield was determined after isolation. Chondrocytes were incubated with Fura-2 and Ca 2+ i followed in different extracellular conditions. A hypotonic shock (HTS) was used to mimic removal of a load. Results: The results showed that ovids and bovids were skeletally immature and aspects of Ca 2+ homeostasis were similar. Ovine chondrocytes had higher resting fluorescence, consistent with elevated resting Ca 2+ levels. Results from ion substitution experiments were consistent with a role for Na +/Ca 2+ exchange, and swelling-induced Ca 2+ enters into the cytoplasm via the plasma membrane and intracellular stores. Conclusions: Ca 2+ homeostasis in chondrocytes from both species behaved in a similar manner to HTS and ion substitutions. Differences in resting [Ca 2+] i could be associated with species, stage of maturation, or Fura-2 itself and require further investigation. These findings contribute to our understanding of the physiology of articular cartilage in different species, and their potential use as models for studying joint disease in humans.
Due to a lack of human tissue availability, when investigating the physiology or pathophysiology of articular chondrocytes, it is necessary that appropriate animal models are used for comparative studies. The supply of cattle and sheep material from abattoirs is often more reliable than that from horses due to restricted age range. As a result there is considerable literature relating to these two large animal species and horses [
The gross anatomy of the right (off-fore) MCP joint, internal architecture of the metacarpus (cannon bone) and thickness of the cartilage at load-bearing positions were studied. In addition, chondrocyte yield and intracellular Ca2+ levels (from both or one forelimbs) before and after a 50% hypotonic shock (HTS) were investigated. HTS was used to mimic cellular swelling, which will occur after removal of mechanical load from joints, and was chosen as it has often been used as a paradigm in this context [
It was expected that the thickness of the cartilage would be greater in cattle compared to sheep due to allometric scaling [
Standard saline comprised (in mM): NaCl (145), KCl (5), CaCl2 (2), MgSO4 (1), D+ glucose (10) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10), pH 7.40 at 37˚C (290 ± 5 mOsmol∙kg−1). To investigate Ca2+-free conditions, CaCl2 was omitted and the Ca2+ chelator EGTA (1 mM) added; for Na+-free saline, N-methyl-D-glucamine (NMDG+) replaced Na+. Cells were always prepared in standard saline and only exposed to these Ca2+- or Na+-free solutions for a few minutes. Stock solutions of digitonin and Fura-2-AM were dissolved in DMSO. Fura-2 was obtained from Calbiochem (Merck, Darmstadt, Germany) and other chemicals such as Dulbecco’s modified Eagle’s medium (DMEM) from Sigma-Aldrich (Poole, UK). Ultrapure (Milli-Q, Merck Millipore, Mass, USA) water was used to dilute salines for a 50% HTS. Cell viability was not affected by any of these manoeuvres and remained at >95% (as assessed by Trypan blue which is excluded from viable cells).
Ethical approval for the research was obtained from the Department of Veterinary Medicine (University of Cambridge). Bovine feet (from animals aged 18 - 36 months) and ovine feet (<12 months) from humanely slaughtered animals were obtained from local, UK abattoirs. All tissue samples were refrigerated (5˚C) or frozen (−20˚C), and processed within 72 h of death.
The lower forelimb (below the carpus) of bovids, equids and ovids were skinned, cleaned and frozen (−20˚C, 48 h) before cutting through the MCP joint and cannon bone, longitudinally with a band saw.
After cleaning and thawing, a digital micrometer was used to measure the thickness of the cartilage overlying the load-bearing area. The cartilage was clearly visible and was measured from the articular surface to the delineation between cartilage and bone. Blinded measurements were taken from central positions at the lateral and medial trochlears, and at the upper most part of the condyles. The effects of freezing and thawing were not determined. Longitudinal slices through the frozen joint and cannon bone also allowed comparison of the internal architecture of the metacarpal bone of all species.
Immediately after collection, the MCP joints of bovids (n = 18) and ovids (n = 5) were skinned, cleaned and stored at 5˚C. The joints were opened aseptically in a flow hood within 72 hours of death. Full depth hyaline cartilage shavings from the MCP joint were taken at ambient O2 tension and placed in DMEM containing penicillin (100 IU∙ml−1), streptomycin (0.1 μg.ml−1) and fungizone (2.5 μg∙ml−1). They were incubated at 37˚C, 5% CO2 for 16 - 18 h at 20% O2 whilst matrix was digested with 0.1% (w/v) collagenase type I (16 h) in cattle and sheep. Isolated chondrocytes were re-suspended in DMEM lacking phenol red (which is known to affect pH measurements and subsequently Ca2+ levels) and a cell count performed using a haemocytometer. Cell viability was determined by the Trypan Blue exclusion test, at >95% before and after experimental regimes (within 3 h post isolation).
Intracellular Ca2+ levels ([Ca2+]i) from multiple individuals (bovids n = 9 or n = 4, ovids n = 3 or n = 4) were measured using Fura-2 [
[ Ca 2 + ] i = K d × [ ( R − R min ) / ( R max − R ) ] × ( S f 2 / S b 2 )
The dissociation (Kd) of Fura-2 was taken as 224 mM although this may alter with different intracellular environments. R is the 340:380 nm fluorescence ratio of the indicator at an unknown [Ca2+]. Rmin is the ratio in the absence of Ca2+ and Rmax is the maximum fluorescence ratio at saturating [Ca2+]. The Sf2/Sb2 is the ratio of the long wavelength (380 nm) in the absence of Ca2+ (Sf2) and presence of Ca2+ (Sb2). The autofluorescence in the absence of dye at the 340 and 380 nm wavelength was subtracted in order to calculate [Ca2+]i [
Results are presented as the means ± standard error of the mean (SEM) for n individual animals unless otherwise stated, and statistical analysis performed by the GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, California, USA). Gross anatomy: Multiple animal joints were used for all measurements (bovids n = 6, ovids n = 5). Cartilage thickness: The average cartilage thickness overlying the medial and lateral trochlears and condyles from the same animals was calculated, and the standard deviation (SD) and SEM determined. An Independent t-test was employed to ascertain statistical significance (P < 0.05) between cartilage thickness at these points in different species. Ca2+i levels: Each experiment was repeated in triplicate using tissue from individual animals (bovids n = 9, n = 4, n = 4 and ovids n = 3, n = 4 and n = 4 respectively for chondrocytes suspended in normocalcaemic, Ca2+-free or Na+-free salines). However, chondrocytes in suspension tend to descend to the bottom of eppenddorfs over a period of time, and despite attempts to resuspend them this can cause currents that result in a reduction in the number of chondrocytes placed in the cuvette and subsequently, a decrease in the fluorescence signal emitted. This has been reported previously in similar studies and resulted in the data being pooled for analysis. To calculate Ca2+i levels, numerical analysis of ratiometric measurements and [Ca2+]i was performed. The autofluorescence was measured and deducted from 340 and 380 nm wavelength recordings and controls were taken. The results were compared before and after HTS in each case for chondrocytes suspended in normocalcaemic saline (control group), or Ca2+-free and Na+-free salines (test groups). The mean, percentage increase, SD and SEM were determined. The Student’s paired t-test was utilised to determine statistical significance (P < 0.05) on changes of [Ca2+]i following HTS within control and test groups of the same species. The Independent t-test was employed to ascertain statistical significance (P < 0.05) between resting Ca2+ levels and those following HTS for both control and test groups of the same species (dependant on saline) and different species. For example, the resting [Ca2+]i of chondrocytes suspended in standard saline and the resting [Ca2+]i of chondrocytes suspended in Ca2+-free saline of one species, or both. The mean ± SEM was expressed in all histograms and statistical significance denoted with an asterisk (*) or hash sign (#).
The distal metacarpal bones of cattle (18 - 36 months) and sheep were very similar in structure (
Chondrocytes from cattle and sheep articular cartilage were isolated overnight by collagenase digestion (0.1% w/v) Following this procedure, the number of chondrocytes from 1 g of shavings obtained was similar in bovine (1 ± 0.2 × 107, n = 18) and ovine (1.2 ± 0.3 × 107, n = 5) tissue. Cell viability, ascertained by the Trypan Blue exclusion test was found to be >95% for both species. There were no noticeable differences in the staining or size of chondrocytes.
The intracellular Ca2+ levels of bovine and ovine chondrocytes were followed fluorimetrically before and after HTS over a 300 s time course. This is shown in representative traces in normocalcaemic (2 mM Ca2+) saline and also in the absence of Ca2+ and Na+ (
Ca2+ influx from the extracellular fluid was investigated in bovine (n = 4) and ovine (n = 4) chondrocytes by suspending them in Ca2+-free saline (Ca2+ replaced by 1 mM EGTA). The absence of Ca2+ caused an immediate reduction in the mean steady state Ca2+ levels. For bovine chondrocytes, a significant reduction of 50% was observed (P < 0.001, Independent t-test;
chondrocytes, a reduction of 30% was observed in response to suspension in Ca2+-free saline, although the change was not significant. Notwithstanding the absence of extracellular Ca2+, HTS evoked a rise in intracellular Ca2+ levels, by approximately60% in both species (P < 0.04, Student’s paired t-test;
Experiments were performed to investigate the function and role of extracellular Na+ in maintaining [Ca2+]i in bovine and ovine chondrocytes. In bovids and ovids, the suspension of chondrocytes in Na+-free saline (Na+ replaced by 145 mM NMDG+) caused a rise in the steady state resting Ca2+ levels in comparison to controls (
The present study provides a comparison of the architecture of the MCP joints of two herbivores (cattle and sheep) together with Ca2+ homeostasis in articular chondrocytes. These large animals represent potential model species for investigating the pathophysiology of joint disease in humans, from which tissue is much less readily available. Ca2+ homeostasis was qualitatively similar in both species when ratiometric recordings are taken into consideration too. The results are consistent with a role for Ca2+/Na+ exchange in reduction of intracellular Ca2+, with the elevation in [Ca2+]i following hypotonic shock being mediated by both Ca2+ entry across the plasma membrane and release from intracellular stores.
The main difference in joint architecture between the two species was the stage of skeletal maturation. Epiphyseal growth plates were still present in both
cattle and sheep, indicative of their skeletal immaturity. Longitudinal growth of the metacarpal bone continues until 6 months of age in sheep and 16 months of in age in cattle [
Ca2+ homeostasis of articular chondrocytes appeared similar across bovids and ovids. Thus [Ca2+]i fell on removal of extracellular Ca2+, indicative of entry across the plasma membrane. Removal of extracellular Na+ led to elevation of [Ca2+]i implying active Na+/Ca2+ exchange (NCE) operating to remove intracellular Ca2+. HTS which mimics chondrocyte unloading caused [Ca2+]i to rise, over a 60 s timescale. The rise in Ca2+ was greater in the presence of extracellular Ca2+ consistent with a role for Ca2+ entry from extracellular fluid, likely through stretch-activated Ca2+ channels [
Quantitative differences were observed between the two species. The steady state, resting [Ca2+]i of bovine articular chondrocytes taken from the MCP joint of the fore limbs was 60 ± 5 nM. This is considerably less than 100 nM recorded in previous experiments [
[Ca2+]i of ovine chondrocytes has not been reported previously. In this study, ovine chondrocytes appeared to have a much higher [Ca2+]i than bovine chondrocytes. Three possibilities may result in this and require further investigation. First, it could be due to differences in the structure, function and metabolism of chondrocytes from immature and mature articular cartilage [
In conclusion, chondrocytes from all species behave in similar manner to HTS and Ca2+ homeostasis appears to be controlled by similar transport mechanisms. It appears that there are species differences in [Ca2+]i of chondrocytes that may be affected by the age and stage of maturation of an animal. Further investigations are required to ascertain the stage of musculoskeletal maturation in different species and compare the [Ca2+]i of chondrocytes in mature and immature animals, and also [Ca2+]i from diseased or damaged tissue to those of normal, healthy tissue. The behaviour of Fura-2 could also account for these differences and further studies are required to elucidate this before ovids are used as a large animal model for investigating the physiology or pathophysiology of chondrocytes when access to human tissue is limited.
RW helped plan the experiments, carried them out, analysed the data and prepared the manuscript. JSG planned the experiments and helped write the manuscript. All authors have read and approved the final manuscript. This work was supported by a BBSRC Studentship held by RW. RW received financial support from University Centre Myerscough for the publication of this article.
The Author(s) declare that there are no conflicts of interests.
White, R. and Gibson, J.S. (2018) Calcium Homeostasis in Articular Chondrocytes of Two Different Animal Species. Open Journal of Veterinary Medicine, 8, 119-133. https://doi.org/10.4236/ojvm.2018.88012