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Description
Introduction
Mammalian bone growth in length occurred through the process of enochondral bone formation through sequential chains of chondrocytes proliferation and differentiation at the growth plate (GP) 1. The endochondral ossification (EO) process is highly regulated to ensure normal post natal bone development at the epiphyseal growth plate (EGP) 2. The cascade of events during ED is regulated by paracrine and other signalling pathways which bring about linear bone growth 3.
The mechanism through which proliferative chondrocytes differentiate and mature to hypertrophic chondrocytes is poorly understood. This study aimed to determined the possible roles of the NHE1 (antiporter) and AE2 (anion exchanger) plasma membrane proteins in longitudinal bone growth.
Methodology
This study was approved by the ICUC of the Universiti Putra Malaysia, (ref. R028/2015). Twenty (20), P10, female Sprague Dawley rat pups were used. The pups were divided into four treatment groups with (n=5) each; EIPA and DIDS plasma membrane inhibitors treatments at respective concentrations of 444µM and 250µM and their respective controls (DMSO for EIPA) and KHCO3 for DIDS). The tibial bones were carefully dissected along with intact proximal articular cartilages with the help of dissecting stereo-microscope after euthanizing the pups. The dissected limbs were temporarily placed in dissecting medium prior to4.
Digital images of the bones were captured and measured at baseline (0 hours), and 48 hours after ex vivo incubation with the dissecting stereo-microscope fitted to PC with imaging software. The lengths of the bones were measured before and after incubation at 48 hours in culture media.
Standard histology of the bones stained with toluidine blue O using was performed to measure the GPs, various zones of GP, GP length and chondrocytes density at GP using fluorescence microscope as described by Bush et al.5 using NIS-Element BR4.20.00 64-bit analytical software.
Results
The mean tibial growth rates in DMSO and KHCO3 control medium were higher compare to their respective inhibitors with significant differences (Table 1).
EIPA treatment had the highest growth velocity inhibition rate (9.90 ± 1.62 µm day-1) compared to DIDS group (16.88 19.86 ± 2.70 µm day-1) (Figure 1).
There were no significant differences in total GP length among the groups. The length of hypertrophic GP and GP chondrocytes density were significantly differed in all the groups.
Conclusions
The Na+/H+ and HCO3- exchange across the plasma membrane of growth plate chondrocytes could have important role in longitudinal bone growth and GP lengthening regulation, majorly driven by increase in the overall GP chondrocytes and hypertrophic chondrocytes density.
References
Abubakar, A. A. et al., Bone Joint Res 5, 610-618 (2016).
Staines, K. A. et al., J. Endocrinol 219(1), R1-R12 (2013).
Rahman, S. et al., Bone Res 3, 15005 (2015).
Abubakar, A. A. et al., Animal Models Exp Med. 2(1), 1-10 (2019).
Bush, P. G. et al., J Bone Mine Res 25(7), 1594-1603 (2010).
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