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Oxandrolone, administered to severely burned children over the first year postburn, produces increased lean body mass by 6 months; however, an increase in total body bone mineral requires 12 months. Consequently, this bone mineral response may be due to increased muscle mass. Alternatively, oxandrolone may act directly on bone. The current study seeks to determine whether oxandrolone can transactivate the androgen receptor in osteoblasts.
Collagen, alkaline phosphatase, osteocalcin, osteoprotegerin, and androgen receptor abundance were determined by qRT-PCR, confocal laser scanning microscopy, or immunoquantitative assay. Short-term treatment produced no significant effects, but at 5 days androgen receptor levels were increased while collagen levels were significantly decreased, with little effect on alkaline phosphatase, osteocalcin, or osteoprotegerin.
These data suggest oxandrolone can stimulate production of osteoblast differentiation markers in proliferating osteoblastic cells, most likely through the androgen receptor; however, with longer treatment in mature cells, oxandrolone decreases collagen expression. Thus it is possible that oxandrolone given to burned children acts directly on immature osteoblasts to stimulate collagen production, but also may have positive effects to increase bone mineral through other mechanisms.
Long-term use of the orally administered anabolic agent oxandrolone has been shown to increase both lean body mass and bone mineral content in severely burned children when given over the first year postburn.
Increased endogenous glucocorticoid production is likely responsible for the acute bone loss observed in severely burned patients.
Oxandrolone is an anabolic steroid with the ability to transactivate the androgen receptor AR , which may be one mechanism underlying the anabolic response to therapy. The aim of our study is to determine whether oxandrolone can increase osteoblastic production of type I collagen and whether this action is mediated by AR signaling. Freshly discarded human cancellous bones were obtained from healthy young patients undergoing osteotomy.
The supernatant containing the released cells was recovered. Washing and recovering were repeated 3 times. The cell pellet was resuspended in 5 mL of fresh medium. After confluence, adherent cells were collected by trypsinizing with 0.
Only passages 4 to 8 were used in this study. Medium was changed every other day. Osteoblasts in vitro progress through several developmental stages that correlate with osteoblast development in vivo: Total RNA was isolated for gene expression analysis as described below. Immunohistochemical staining was carried out for both the AR 8 , 9 and type I collagen. Stock solution containing 2. The polyclonal antibody used for collagen I was applied overnight at the dilution of 1: Optical sections were obtained from the cells by capturing single images of central cell focal planes.
Three microscopic fields were captured for the control group of each cell line and the same was done for cells treated with oxandrolone. After 24 hours of treatment, the levels of alkaline phosphatase activity were determined using a commercial kit Pierce Biotechnology, Inc, Rockford, IL. The cells were washed with cold PBS and subjected to 3 freeze-thaw cycles. These samples were assayed for enzymatic activity with p -nitrophenyl phosphate as a substrate.
Sample absorbance was measured at nm with microplate reader. After washing with PBS, cells were exposed to p -nitrophenyl phosphate containing levamisole to inhibit the generation of p -nitrophenol by endogenous alkaline phosphatase for 8 minutes. The values OD were normalized with protein concentration OD.
Total RNA was isolated from osteocytic cultures and gene expression characterized by qRT-PCR analysis using human primers from Qiagen Valencia, CA specific for AR and the osteoblast marker proteins alkaline phosphatase, type I collagen, osteocalcin, and osteoprotegerin. The statistical significance of intergroup differences was tested using the Student t test when the variances were equal.
Although the affinity for oxandrolone for AR is approximately fold lower than testosterone, oxandrolone treatment still results in AR transactivation. Confocal scanning laser microscopic analysis of human osteoblastic cells after oxandrolone treatment. Compare to Figures 1c and 1d. We next assessed the effect of oxandrolone treatment on type I collagen, the major constituent of the bone matrix.
Immunohistochemistry for collagen type I on cultured osteoblast cells demonstrated an increase in collagen that was dose dependent. To determine the effect of oxandrolone on additional markers of osteoblast activity, osteoblastic cultures were treated with or without oxandrolone.
We also performed immunoquantitative assays for type I collagen and osteocalcin on osteoblastic cells. Thus, after multiple repetitions of the experiment, the data confirmed the results observed using immunofluorescence.
Stimulation of alkaline phosphatase activity by oxandrolone in proliferating human osteoblastic cultures. Increased osteocalcin levels after oxandrolone treatment. We next assessed the levels of osteocalcin, an important biochemical indicator of bone turnover. Thus, the production of osteocalcin mirrored the pattern of alkaline phosphatase activity. Enhanced type I collagen secretion after oxandrolone exposure. AR concentrations increase as osteoblasts differentiate, reaching their highest levels in osteocytic cultures.
We therefore determined the effect of oxandrolone treatment on gene expression in mature osteocytic cultures. Although there was little effect of oxandrolone after 24 hours of treatment in osteocytic cells on type I collagen, alkaline phosphatase, osteocalcin, or osteoprotegerin mRNA abundance, a nonsignificant increase in AR mRNA was noted.
This is consistent with other reports documenting an increase in AR after androgen treatment in osteoblasts. Thus, longer treatments with oxandrolone in osteocytic cultures reduced collagen expression. Consequence of oxandrolone treatment on gene expression in normal human osteocytes: In this study we have shown that stimulation of cultured human osteoblasts by oxandrolone results in immunofluorescent detection of AR in the nucleus and increased osteomarkers in these osteoblasts.
These data suggest that oxandrolone directly targets human osteoblasts by means of the AR, resulting in increased expression of osteoblast differentiation markers after short-term treatment. Therefore, oxandrolone may act directly on the osteoblast in addition to effects that result in increasing skeletal loading. Immunohistochemistry and confocal laser scanning microscopy CLSM were used to evaluate the expression of AR and type I collagen in osteoblasts, with several advantages over regular fluorescence microscopy.
The increased expression of type I collagen by osteoblasts was also determined by immunoassay confirming the results observed with immunohistochemistry. The AR is a steroid receptor that generally mediates biologic responses to androgens. The increased expression of AR in the present study is consistent with that documented in the literature. Alkaline phosphatase is well recognized as a marker that reflects osteoblastic activity.
In a clinical study, Murphy et al 1 have shown that oxandrolone administration increases levels of serum alkaline phosphatase in treated patients by 6 months versus controls. In the present study, the cells treated with oxandrolone produced a greater level of alkaline phosphatase. The elevated activity of this cellular enzyme suggests an early increase in the osteoblast differentiation process.
While the in vivo function of osteocalcin remains unclear, its affinity for bone mineral constituents implies a role in bone formation. Hence it has been shown that osteocalcin is a biochemical indicator of bone turnover. To test the effect of oxandrolone on osteocyte-like cells differentiated from osteoblasts at the molecular level, the human osteoblastic cells were cultured in a differentiation medium and then exposed to oxandrolone. The results showed that short-term treatment produced little effect on type I collagen, alkaline phosphatase, osteocalcin, osteoprotegerin, or AR mRNA.
Long-term treatment decreased type I collagen expression, consistent with decreased collagen expression observed by Wiren et al 16 in AR-transgenic mice with targeted AR overexpression in the osteoblast lineage. The dramatic inhibition at the endosteal envelope may be responsible for a modest decrease in cortical bone area and reductions in biomechanical properties observed. Murphy et al 1 have recently shown that oxandrolone administration increases lean body mass 3 to 6 months before an increase in bone mineral content is observed.
On the other hand, we have found an increase in collagen production when high doses of oxandrolone were used to stimulate osteoblasts. The applicability of our findings in this study to the in vivo effects of oxandrolone in burned children 1 is not clear.
While an in vitro environment may not adequately reproduce the in vivo situation, many potential variables are eliminated during in vitro studies, and it is possible to investigate a specific effect of a drug on cells. The time frame of in vivo and in vitro settings is plainly different, and the concentration of oxandrolone used to stimulate cells in the in vitro setting is probably higher than the one used in the clinical trial. Although this study demonstrates that oxandrolone is capable of affecting the osteoblast AR and stimulating type I collagen synthesis, regulatory mechanisms that are present only in a complex in vivo situation may account for increased collagen turnover or degradation after synthesis.
Moreover, the delay in the increase of total body bone mineral content reported in the clinical study by Murphy et al 1 may be explained in part by the acute inhibition of bone formation and osteoblast differentiation after a severe burn as previously reported. In conclusion, the use of high-dose oxandrolone results in increased nuclear fluorescence of the osteoblast AR, increased osteoblast differentiation markers in cultured osteoblasts and decreased bone marker in cultured osteocyte-like cells in vitro.
Oxandrolone may have the ability to directly stimulate bone collagen synthesis, over and above its effect on skeletal loading effected through an increase in lean body mass following long-term treatment. However, longer exposure may no longer be directly anabolic in mature bone cells. This observation may help to explain the variability and confusion regarding androgen actions on the skeleton.
National Center for Biotechnology Information , U. Journal List J Burns Wounds v. Oliveira , MD, c, e, f Gordon L. Klein , MD, b, f Elgene G. Herndon , MD c, f.
This article has been cited by other articles in PMC. Osteocytic cell culture Osteoblasts in vitro progress through several developmental stages that correlate with osteoblast development in vivo: Immunohistochemistry for collagen type I and AR Immunohistochemical staining was carried out for both the AR 8 , 9 and type I collagen.
Real-time quantitative reverse transcription—polymerase chain reaction qRT-PCR on cultured human osteocytic cells Total RNA was isolated from osteocytic cultures and gene expression characterized by qRT-PCR analysis using human primers from Qiagen Valencia, CA specific for AR and the osteoblast marker proteins alkaline phosphatase, type I collagen, osteocalcin, and osteoprotegerin.
Open in a separate window. Effects of long-term oxandrolone administration on severely burned children. Attenuation of post-traumatic muscle catabolism and osteopenia by long-term growth hormone therapy. Histomorphometric and biochemical characterization of bone following acute severe burns in children.
Evidence supporting a role of glucocorticoids in short-term bone loss in burned children. Progressive development of the rat osteoblast phenotype in vitro: Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: