Abstract

Numerous studies of experimental hypo- and hypervitaminosis A have long suggested that retinoic acid (RA) is involved in chondrocyte maturation during endochondral ossification and skeletogenesis. However, the specific and direct roles of RA in these complex processes remain unclear. Based on recent studies from our laboratories, we tested the hypothesis that RA induces the expression of genes associated with the terminal mineralization phase of chondrocyte maturation and promotes apatite deposition in the extracellular matrix. Cell populations containing chondrocytes at advanced stages of maturation were isolated from the upper portion of Day 18 chick embryo sterna and grown for 2 weeks in monolayer until confluent. The cells were then treated with low doses (10-100 nM) of RA for up to 6 days in the presence of a phosphate donor (beta-glycerophosphate) but in the absence of ascorbic acid. Within 4 days of treatment, RA dramatically induced expression of the alkaline phosphatase (APase), osteonectin, and osteopontin genes, caused a several-fold increase in APase activity, and provoked massive mineral formation while it left type X collagen gene expression largely unchanged. The mineral had a mean Ca/Pi molar ratio of 1.5; Fourier transform infrared spectra confirmed that it represented hydroxyapatite. Mineralization was completely abolished by treatment with parathyroid hormone; this profound effect confirmed that RA induced cell-mediated mineralization and not nonspecific precipitation. When cultures were treated with both RA and ascorbic acid, there was a slight further increase in APase activity and increased calcium accumulation. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum; however, RA only had minimal effects on mineralization and gene expression in these cells. Thus, RA appears to be a rapid, potent, maturation-dependent, ascorbate-independent promoter of terminal maturation and matrix calcification in chondrocytes.

Analysis & Interpretation:

From the 1st study, the first thing that really stands out is that the researchers note that both too much and too little vitamin A results in mammalian bone longitudinal growth abnormalities. It seems that Vitamin A is the precursor for retinoic acids, in all of its many variations. The researchers hypothesized that retinoic acid would inhibit long bone longitudinal growth. It seems that just 1 oral dose of the RA by a lab rate resulted in thinner growth plates. It seems that the RA decreased chondrocyte proliferation, hypertrophy, and cartilage extracellular matrix formation. What is the big discovery to us as height increase researchers is what the abstract says next…

“The growth-inhibiting effects of RA were completely reversed by a retinoic acid receptor (RAR) antagonist. In the absence of exogenous RA, this antagonist accelerated bone growth, as did an RA-specific neutralizing antibody, suggesting that endogenous RA negatively regulates growth plate chondrogenesis”

This is a major discovery. It seems that if we can get exogenous RAR close to the growth plates, the longitudinal growth of the long bones can be accelerated. It seems that the RA actually acts through the RAR though. So any endogenous RA will have this effect. This means that since the balance of RA and RAR in the human body is important to keep the bones from being completely inhibited or growth uncontrollably, the concentrations of RA and RAR is kept at a sort of metastatic equilibirum. I would then suggest that we get an exogenous source of RAR which we apply close to the growth plates through injection, or even taken orally can possibly lead to the growth plates having accelerated lengthening and growth.

As for study #2, the researchers state the same initial hypothesis as made by the researchers of study #1. The Retinoic acid causes endochondral ossification to mature aka stop. For their specific study, …

“…we tested the hypothesis that RA induces the expression of genes associated with the terminal mineralization phase of chondrocyte maturation and promotes apatite deposition in the extracellular matrix…”

Chondrocytes were extracted from the chest cartilage of days old chicken. They were grown in a monolayer until they became confluent. The cells were then treated with low doses of RA for 6 days. After 4 days it seems that the chondrocytes showed a very high increase in the expression of alkaline phosphatase,osteonectin, and osteopontin genes. There was a huge accumulation of mineral formation. When the mineral that was formed was testing, it was concluded that the mineral was hydroxyapatite. What is even more interesting is how the researchers made the mineralization go away….

“Mineralization was completely abolished by treatment with parathyroid hormone;…”

It seems that by using the PTH, the researchers made the mineralized hard material of calcium deposits aka hydroxyapatite dissolve.

So the researchers concluded that…

“…this profound effect confirmed that RA induced cell-mediated mineralization and not nonspecific precipitation…”

It seems that if you take the RA with ascorbic acid, there is an even higher level of alkaline phosphatase release or expression and the mineralization and calcification increased too.

What is really strange twist is that if we then take non-mature chondrocytes, the RA injections seem to not have as dramatic of an effect on them as the mature chondrocytes. It would seem that “fresh” chondrocytes don’t get mineralized as easily as mature chondrocytes.

The researchers conclude with this statement which puts the nail on the coffin on the debate on how retinoic acid would effect chondrocytes and growth plates.

“…Thus, RA appears to be a rapid, potent, maturation-dependent, ascorbate-independent promoter of terminal maturation and matrix calcification in chondrocytes.”