Recent studies in fracture healing showed that successful fracture healing is a complex process involving the growth and differentiation of mesenchymal stem cells, regulation of cytokines (inflammatory and osteogenic) and the resorption of extracellular matrix [
25]. The golden standard in non-union therapy is the transplantation of autologous bone graft [
26‐
29], more recently this therapy was combined with the application of BMP-7 and studies show a better outcome after non-union treatment if BMP-7 was applied [
18,
30,
31]. The underlying mechanism of the clinical success of BMP-7 application remains uncertain. In the current study we sought to determine the influence of local BMP-7 application on successful bone healing utilizing quantitative serum measurement of osteogenic cytokine expression pattern. Transforming growth factor beta is initially secreted from thrombocytes in areas of hematoma after injury, but later from chondrocytes and osteoblasts during fracture healing. It stimulates the proliferation of different cells including osteoblasts, chrondroblasts, and chondrocytes [
22,
23]. Regarding the serum concentration of TGF-ß our results showed that patients in the BMP-7 group had lower levels after the first week compared to the group with physiological bone healing suggesting that osseous biology was still impaired at this time. However local application of BMP-7 lead to an increase in TGF-β after the second week that lasted longer than in every other group. It has been shown that the concentrations of bone morphogenic proteins were decreased in patients suffering from non-union, thereby indicating that a down-regulation in expression of osteogenic BMPs might be responsible for failed fracture healing [
32,
33]. In our study patients with failed fracture healing were treated with adjunct application of BMP-7 serving as a substitution of known lower BMP concentrations in patients with failed fracture healing, thereby modulating the microenviroment in the fracture gap and increasing local concentration of BMP-7. BMPs and TGF-ß are both members of TGF-ß protein superfamily and promote bone formation. The most important effect of TGF-ß is the stimulation of the synthesis of components of the extracellular matrix (e.g. collagen I and III). This has been shown to be pivotal in the early stages of bone healing as scaffolding for later bone mineralization [
34]. BMPs facilitate osteogenic differentiation due to various pathways in order to achieve successful bone healing [
34]. Peak concentrations in the BMP-7 group were measured after week 4, suggesting that BMP-7 was able to stimulate osteoblast biology and lead to higher expression of TGF-β. BMP-7 appears to have its most significant impact between week 2 and 4 [
35]. A steeper decrease in the non-union group between week 2 and 4 compared to other groups could be attributed to a decrease in hard callus formation, which plays a crucial role in non-union formation. In our previous publications, we attributed this decrease to hard callus formation between week 2 and 4 and proposed this phase to be crucial in nonunion formation. It is known that TGF-β plays an important role in the formation of cartilage-like tissue, whereas a decrease in the expression of TGF-β accounts for an impaired potential in the formation of cartilage-like tissue. Hereby indicating that impaired expression of TGF-β, as seen in decreased plasma levels, may provide a predisposing factor of an dysfunctional cartilage and osseous regeneration [
24].
In conclusion it is known that local and systemic concentrations of different osteogenic cytokines increase during physiological fracture healing, in particular sufficient serum concentrations of TGF-ß have been shown to be pivotal to fracture healing [
36]. It has been reported that TGF-ß and BMPs are capable of inducing each others expressions [
33,
34], however patients with failed bone healing are known to have impaired local BMP concentration, furthermore the current study showed that the concentration of TGF-ß in concerned patients is decreased during the initial 12 months; indicating that BMP-7 and TGF-ß have to be present in fracture healing in a sufficient concentration [
37]. Our results show that local application of BMP-7 serves as a substitution of impaired BMP-7 levels in non-union patients and thereby modulating the microenviroment in the fracture gap and promoting bone regeneration. The results of our current study accentuate the findings of previous studies indicating that lower circulating levels of TGF-ß could be utilized as a predictor of delayed bone healing and non-union [
11].