This article presents two important main findings; one is the effects of ALN on the liver and kidneys after using alendronate in bone repair. These two organs had been chosen due to their direct relation with the drug. Glucocorticoids are metabolized by the hepatic route, and they are involved in the suspension of bone formation and the promotion of bone resorption, inducing osteoporosis. The kidney evaluation was important because this organ is responsible for ALN excretion (17).
As far as systemic involvement is concerned, many studies ( 2,5,13,12,18–24,) have used this type of drug at different dosages and concentrations (aiming to accelerate bone repair) without evaluating the general health implications since the ALN had a nonlinear uptake by bone, accompanied by simultaneous accumulation in noncalcified tissues at high doses (25). This study has shown that the treatment with 3 mg/kg of ALN significantly increases liver and kidney damage compared to a placebo. A dose of 1 mg/kg could be used therapeutically in humans, as its effects are not as serious as a dose of 3 mg/kg. A study by Erlebacher et al. (26) examining collapsed focal segmental glomerulosclerosis in a patient receiving liver transplantation due to alendronate use for osteopenia—concluded that the drug was not related to renal toxicity at a dose of 0.3 mg/kg. However, it should be emphasized that bisphosphonates must be used with caution in patients with low glomerular filtration rate (27).
A previous study observed that a dose of 3 mg/kg caused systemic alterations in animals, with hepatic impairment (28). In Deliberador et al. (15) work, local applications of 1 mg/kg alendronate in rat calvaria defects were delivered, the authors concluded that the kidneys showed normal glomeruli, with capillaries surrounding these structures. No impact on the kidneys was observed in any group of their work. This study did not analyze blood biochemistry, which is an important indicator of possible future systemic complications. Li, F et al. and Srisubut et al. had also studied the effects of local delivery of ALN and suggest that the drug have increased bone formation (29, 30). Wang, YH et al. linked PLGA (lactic-co-glycolic acid) to ALN for slow and local application and found an excellent graft potential (31).
The second main finding is the effect of different doses of alendronate on bone repair. We chose to use the human-based dose, which corresponds to 1 mg/kg, as well as a dose triple that. Previous research has already shown that reduced doses of alendronate may improve osteoblast differentiation and bone matrix formation (25, 32), although the literature has not expounded on whether the ideal dosage of the drug can benefit bone repair. A greater dosage of ALN has been proven to be more effective in qualitative microtomographic analysis of bone repair. This could be seen through the greater number of trabeculae and the decreased spacing between the trabeculae in the 3 mg/kg group (6, 16, 33). More bone density suggests that a higher dosage influences bone quality, which encouraged the researchers in this experiment to use both 1 mg/kg and 3 mg/kg.
Types I and III collagen are the main collagens found in the connective tissue matrix. Type I can be found in tendons, fibrous cartilage, and bone (among others); type III can be found in a wide range of tissues, varying from spleen to granulation tissue, and it plays a special role in tissue regeneration, given the early increase in its expression following tissue injury (34). Reduced collagen III has been demonstrated to decrease bone formation and remodeling alterations during fracture healing. In 2015, Miedel et al. (35) showed that collagen III levels begin to increase in the first two days following a long bone fracture and remain elevated for the first three weeks; the collagen can be found throughout immature woven bone of murine fracture callus, although it is less prevalent (by comparison) in the cartilaginous matrix. ALN act increasing bone volume and the level of mature collagen I reserve (36). That data suggests that collagen III is less likely to play a significant role in endochondral ossification, which might explain why the researchers of this study could not find significant differences among groups in terms of the time of euthanasia and callus remodeling.
In terms of quantitative effect on bone repair, in this study no correlations were found between doses and area of trabecular bone in the femur. One hypothesis is the duration of drug delivery. The researchers believe that a longer time period (more than 45 days) is critical. A study of dose-response relationship for ALN treatment in elderly osteoporotic women showed that treatment with ALN (1, 2.5, or 5 mg/day) decreased bone absorption markers and reduced markers of bone formation in a dose-related but delayed manner, with all doses evaluated (27). Study of Ramchand et al. shown that a chronic use of BF, decrease the bone remodeling as the bone volume (37).
Another hypothesis for no association is that alendronate was only used after the surgical period. Other studies show pre-surgery use, both with and without success. Wan Rong et al. (38) had a total of 82 osteoporotic patients whose humerus fractures were stabilized with plaques. They divided the sample into two groups: group A (initiation of bisphosphonate treatment within two weeks after surgery) and group B (control group, initiation of treatment three months after surgery). They concluded that all patients had fracture union, and the mean time to radiographic union was similar in groups A and B.
Another important point of this study was the choice of the TGF-β1 marker to accomplish the immunohistochemical evaluation. This marker is responsible for maintaining a constant bone mass, acting directly on the regulation of osteoblast differentiation and promoting polymorphonuclear cell chemotaxis, fibroblast proliferation, and collagen I synthesis (5). TGF-β1 is a physiological regulator of osteoblast differentiation and a key mediator of the coupling of osteoblast differentiation and osteoclastic bone resorption, which is required for skeletal homeostasis (26). Study of Vieira JS et al. demonstrated that ALN influences marker TGF- β1 changing the production and remodelation of the chondroid extracellular matrix (39). Other studies had also proved the relation between them, suggesting that ALN acts directly on this marker, increasing its level (5, 6) A smaller dosage of ALN (1 mg/kg) contributed with less negative impact on the TGF-β1 marker compared to the higher dosage (3 mg/kg), as noted in this study. A study by Jei et al. (5) concluded that prolonged reduction of bone turnover after alendronate treatment in female rats increased TGF-β1 production by bone marrow cells and periosteal osteoprogenitors.