Fracture repair involves all the processes in bone development including proliferation, differentiation, and mineralization of osteoblasts. Critical-size defect fractures do not heal completely without the aid of mechanical supports and growth factors. The use of BMP-2 loaded scaffold to stabilize the fracture and to initiate bone regeneration has been shown to be an effective treatment for segmental defects 
. Although BMP-2 has high osteoinductive potency and can improve bone healing, its high cost and potential adverse effects make this approach unfavorable. Since testosterone has anabolic effects on bone development, we tested its effects on the repair of critical-size segmental fractures and compared its efficacy to that of BMP-2. We found that treating the fracture with a scaffold containing BMP-2 resulted in callus formation 14 days after initiation of the treatment, whereas no callus was formed if the fracture was treated with a scaffold containing no BMP-2 or testosterone during the entire period (35 days) of the study (, ). Interestingly, similar results were observed at the same time (day 14) if the fracture was treated with a scaffold containing testosterone (). Results of micro-CT examinations showed that the degrees of callus formation and bone regeneration were comparable between testosterone and BMP-2 treated fractures (). Analyses of structure model index (SMI) revealed that the fractures treated with testosterone plus BMP-2 (1.665) had a significant lower SMI (SMI
1.665) than that treated with BMP-2 alone (2.065) (), suggesting that testosterone plus BMP2 is more efficacious than BMP-2 alone in promoting bone regeneration. Histological examinations on both trabecular and cortical bone showed no significant difference between BMP-2 and testosterone treated fractures (). Together, these results suggest that testosterone is as effective as BMP-2 in promoting the healing of critical-size segmental defects of femoral bone in mice.
BMP-2 is known to activate bone regeneration by two different mechanisms, depending on the type of receptors bound. Two different BMP-2 receptor proteins, types I and II, with serine/threonine kinase activity exist. A functional BMP-2 receptor is a heterodimer of these two receptor proteins. Type II receptor phosphorylates type I receptor which then activates Smad 1, 5, and 8 
. These activated Smad proteins interact with Runx-2/Cbfa-1 to promote maturation of chondrocytes 
. Binding of BMP-2 to its receptor also induces formation of more heterodimeric receptors. When BMP-2 binds to these newly formed receptors, it activates the MAPK pathway 
, leading to activation of many transcription factors such as members of the Fos/Jun family and the activating transcription factor-2 and subsequently increased production of fibronectin and osteopontin 
. Androgens have been shown to modulate the expression of genes in the BMP signaling pathway including genes for chordin, SMAD specific E3 ubiquitin protein ligase 1 (Smurf1), Six3, and sclerostin 
. In this study, we found that BMP-2 promoted bone healing in ARKO mice, suggesting that BMP-2 can be used to aid fracture repairs in individuals deficient in the androgen/AR pathway. It is likely that BMP-2 and testosterone promote bone regeneration by different mechanisms. Since we found that combination therapy with testosterone and BMP-2 was superior to single therapy (–), these mechanisms may be complementary or synergistic leading to a more speedy fracture healing.
Testosterone may affect bone 
development directly or indirectly by being converted to estrogen through aromatization. In direct action, testosterone binds to and activates AR, enabling it to be transported into the nucleus to bind to the androgen response element. As a result, the transcription of many target genes such as the osteoblast genes AKP2
, and Bglap
are activated to promote bone mineralization 
. Testosterone also has nongenomic effects by activating PI3K/Akt signaling pathways in osteoblasts 
and altering the activity of Elk-1, CCAAT enhancer binding protein-β, and cAMP-response element binding protein resulting in activation of the Src/Shc/ERK pathway. Testosterone can also interact with c-Jun/c-Fos leading to down-regulation of the c-Jun N-terminal kinase. Since these actions are anti-apoptotic, they are beneficial to the differentiation of osteoblasts. Testosterone may also affect bone regeneration by increasing the production of TGF-β and IGFs and decreasing the production of IL-6 receptors 
. IGFs and IGF-binding proteins can enhance osteoblast proliferation and differentiation 
. In addition to the anabolic effect, testosterone is also anti-resorptive as a decline in bone resorption is seen in hypogonadal men after testosterone replacement 
. It is possible that these pathways all play important roles at different stages of bone healing. However, it remains unclear as to how androgens and AR coordinate the expression of genes in these pathways. We have also detected the presence of osteoclasts in regenerating calluses by TRAP staining (). Since bone remodeling is the balance in action between osteoblasts and osteoclasts 
, it is conceivable that the osteoclasts were present in the regenerating calluses. We also found that the number of osteoclasts in the calluses of fractures treated with both testosterone and BMP-2 were significantly higher (~120 vs. 80 per section) than those treated with either alone (). This result suggests that osteoclastic activities were promoted by both testosterone and BMP-2 during bone fracture healing.
Testosterone has different effects on different types of skeletal cells. It has been shown to decrease osteoblast and osteocyte apoptosis 
, stimulate proliferation of osteoblast progenitors and differentiation of mature osteoblasts, and promote the apoptosis of osteoclasts 
and epiphyseal growth and maturation 
. Using AR transgenic mice under the control of the 2.3-kb alpha (I)-collagen promoter, Wiren et al. showed that anabolic effects of testosterone exhibited exclusively on periosteal surfaces, suggesting that the consequences of androgen action could be compartment specific 
. While AR over expression has been postulated to limit the effect of androgen on osteoblast differentiation and mineralization 
, our results suggest that local short-term testosterone treatment can initiate bone regeneration.
Since no callus formation on the fractured femur treated with a scaffold containing testosterone in ARKO mice was observed during the entire 35-day period of the study (), it is likely that testosterone promotes bone regeneration through the genomic instead of the nongenomic pathway by activating AR. This is the first finding that AR is required for testosterone to promote bone fracture repair. This result also suggests that estrogen derived from aromatization of testosterone plays little role in fracture repair. However, this possibility requires to be verified. Since this study was done in male mice, the effects of testosterone and the roles of AR in promoting fracture healing in females remain to be investigated.
The results of this study strongly suggest that testosterone can be used to promote fracture healing. This finding is consistent with that described by Zaifirau et al. 
who found that traumatized bone of Sprague-Dawley rats implanted with twelve-hour calcined hydroxyapatide ceramics containing testosterone healed faster than the controls. Similarly, Gordon et al. 
reported that a self-setting zinc sulfate calcium phosphate containing testosterone was integrated into traumatized femurs of albino Holtzman rats. Benghuzzi et al. 
found that delivery of dihydrotestosterone using the tricalcium phosphate lysine delivery system to fractured femurs of Sprague-Dawley rats resulted in stimulation of osteoblastic activities and increased cortical bone density. The tricalcium phosphate lysine system was also used to deliver simvastatin (inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A reductase) to promote the healing of segmental bone fractures 
. Although all of these studies found testosterone to be effective in promoting fracture repair, its efficacy was not clear because it was not compared to that of any well-established treatment method. In this study, we compared the efficacy of testosterone for fracture repair with that of BMP-2 which is FDA approved for treatment of acute, open tibial shaft fractures as described above and found that testosterone is as effective as BMP-2 in promoting fracture healing. We also used scaffolds that have been shown to effectively deliver BMP-2 and antibiotics to fractured bone 
. Although the scaffolds used to bridge the fractures in this study were not resorbed during the 35-day period of the investigation, their biodegradable nature had been firmly established 
and had been shown to be resorbed in 6 months in a canine model 
. This slow resorption rate of the scaffold is beneficial as it provides sufficient time for the fractured bone to heal to the point where it can stand weights.
Although testosterone was found to be effective in promoting fracture repair, it has not been used clinically to treat fractures, perhaps due to the concern over its virilizing side effects as well as the possibility of altering lipoprotein profiles and increasing the levels of endothelin-1, C-reactive protein, and total homocysteine 
. However, we found that the dose of testosterone required to promote fracture repair is very low. In this study, we used 100 µg and loaded it onto a scaffold used to bridge the fracture on femur in mice. Since mice are 12 times more resistant to drugs than humans 
, only 8.3 µg of testosterone would be required to treat a segmental defect of the same size in humans and only approximately 1.8 ng/ml increase in plasma testosterone levels would result if all of it is absorbed at once. This would not significantly alter the physiological levels of testosterone. Therefore, adverse effects would be minimal as evidenced by our demonstration of the lack of effects on BMD, seminal vesicles, testis, and prostate (). We also found that the combination of BMP-2 and testosterone works better than either BMP-2 or testosterone alone. Since testosterone is much cheaper than BMP-2, its short-term local application in fracture treatment would be more feasible. As increasing number of adverse effects are found to be associated with BMP-2 
, alternative treatments are needed. Our findings may provide a more economic method using testosterone or a more efficient means using a combination of BMP-2 and testosterone to treat fractures with critical-size segmental defects of a long bone.