Distinct developmental changes in the distribution of calcium, phosphorus and sulphur during fetal growth-plate development

Abstract

Gradients in the concentrations of free phosphate (Pi) and calcium (Ca) exist in fully developed growth zones of long bones and ribs, with the highest concentrations closest to the site of mineralization. As high concentrations of Pi and Ca induce chondrocyte maturation and apoptosis, it has been hypothesized that Ca and Pi drive chondrocyte differentiation in growth plates. This study aimed to determine whether gradients in the important spectral elements phosphorus (P), Ca and sulphur (S) are already present in early stages of development, or whether they gradually develop with maturation of the growth zone. We quantified the concentration profiles of Ca, P, S, chloride and potassium at four different stages of early development of the distal growth plates of the porcine femurs, using particle-induced X-ray emission and forward- and backward-scattering spectrometry with a nuclear microprobe. A Ca concentration gradient towards the mineralized area and a stepwise increase in S was found to develop slowly with tissue maturation. The increase in S co-localizes with the onset of proliferation. A P gradient was not detected in the earliest developmental stages. High Ca levels, which may induce chondrocyte maturation, are present near the mineralization front. As total P concentrations do not correspond with former free Pi measurements, we hypothesize that the increase of free Pi towards the bone-forming site results from enzymatic cleavage of bound phosphate.

Fig 1

Schematic principle of sample analysis with a nuclear microprobe: a high-energy proton beam is scanned across the sample in a two-dimensional grid. Upon interaction with the protons, two processes occur. First, X-rays are emitted from target atoms when hit by a proton. These X-rays are detected during the PIXE measurement. Second, transmitted protons are scattered from their straight path in either a forward (FS) or a backward (RBS) direction.

Fig 2

Example of a raw data set, which contains some characteristics that can be used for orientation. These markers can clearly be traced in all raw images, illustrating the accuracy of the method. Note that such biasing aspects are not present in the actual measurements. Images are marked with the name of the element that is plotted. The image indicated with CNO contains the sum of all detected elements C, N and O. From blue to yellow, the amounts of detected elements increase. The bottom right image shows the unstained sample on Formvar foil.

Fig 3

Concentration profiles of Ca, K, Cl, S and P (mg g⁻¹ dry weight) of the samples (A to D correspond to femurs A to D in Fig. 1). These concentrations are obtained after correction for the local tissue density using H, C, N and O. The Ca and P concentrations are scaled such that the gradients in the growth plate become visible. As a result, the peak values in the bone are not visible. Distinct gradients in Ca, K, Cl and S slowly develop with maturation of the growth plate from sample A to D. They are most clearly visible in sample C. Owing to the presence of the secondary ossification centre at the left side in sample D, gradients develop in both directions.

Fig 4

Average element concentrations in the four samples (A–D) in the proliferative (left) and hypertrophic (right) zones over areas 350–550 µm (left) and 650–950 µm (right) as indicated in Fig. 3. These areas are selected because they are parts of, respectively, the proliferative and hypertrophic zones in all animals. The only exception is for the concentrations of Cl and K in the hypertrophic zones in animals C and D. Because these elements develop steep gradients towards the mineralized area (Fig. 3C,D), they are averaged over 105–107 µm (animal C) and 98–100 µm (animal D).

Fig 5

Alkaline phosphatase (blue) in the hypertrophic zones of a growth plate of a late fetal porcine bone (sample 4). Left: primary ossification centre; right: secondary ossification centre. The distributions of most elements under consideration show a similar asymmetric distribution over a growth plate at this stage of development (Fig. 3D).