**7. New perspectives**

Despite the several clinical studies that support the benefits of HBOT in patients afflicted with osteonecrosis [1, 5], therapy is still not worldwide approved. This could be due to several factors including the apparent high number of HBO treatments necessary plus limited amount of clinical evidence for FHN, the majority of the evidence relies on ex vivo and in vitro studies, and the molecular mechanisms responsible for the regenerative responses of HBOT are still debatable. Thus, the underlying mechanism of action is still unclear.

A recent study investigated HBO upregulation on serum osteoprotegerin (OPG) and/or inhibition of osteoclast activation [50]. Twenty-three patients suffering from unilateral FHN at stage I, II, and III consented to the study: patients received standard HBOT; nineteen patients completed the study. Serum OPG levels were obtained at the beginning of HBOT (T0 ), after 15 sessions (T1 ), after 30 sessions (T2 ), after a 30-day break (T<sup>3</sup> ), and at the end of our treatment protocol, after 60 sessions (T4 ). Magnetic resonance imaging (MRI) was obtained at T0 and about 1 year from the end of HBO treatments to compare pre-HBOT with post-HBOT lesion size.

The findings were:


These evidences proposed an influence of HBO on the immune system and inflammatory processes. Indeed, one of the initial studies by Lukich et al. suggested an immunosuppressive effect of HBO therapy in patients suffering from rheumatoid arthritis [51]. Later, other studies reinforced these findings. Specifically, authors showed TNF-alpha and interleukin-1β (IL-1β) inhibition after HBO therapy in indomethacin-induced enteropathy and in chronic constriction injury-induced neuropathy [52, 53]. The following are some examples of tissue cytokine changes proposed after HBO.

IL-1β is a pro-inflammatory cytokine that responds to injury or infection by binding to the type-1 IL-1 receptor (IL-1R) and IL-1R accessory protein [54]. Although belonging to a structurally different cytokine class, IL-1 resembles many of the biological activities of TNF-α; IL-1 activation results in downstream activation of NF-kB and JNK [55, 56]. Fukushima et al. found that IL-1β has a stimulatory effect on osteoclast formation via increasing expression of RANKL [57]. IL-1β, like TNF-α and IL-6, is produced by stromal cells and monocytes. These cytokines can synergistically stimulate osteoclast differentiation leading to a net increase in RANKL activity and control their own expression [56].

IL-6 has been demonstrated to exhibit a dual effect on osteoclastic differentiation. During the inflammatory process, monocytes/macrophages produce IL-6, which can directly stimulate pre-osteoclast cells to be differentiated and activated [56]. In addition, IL-6 can stimulate stromal/osteoblastic cells to produce certain effectors, namely, IL-6, which will then promote osteoclastic differentiation [56]. Thus, there is a dual contribution, direct and indirect interaction, in which IL-6 can upregulate bone turnover. In vivo studies of IL-6 found that in transgenic mice with overexpressed IL-6 there is a greater bone turnover, reduced osteoblasts, and increased osteoclasts leading to osteopenia [58]. Correspondingly, IL-6-deficient mice displayed reduced osteoclasts and lower levels of bone erosion [59]. Kurokouchi and colleagues found TNF-α to increase the expression of IL-6 and ICAM-1 genes [60]. Hence, lower levels of TNF-α resulting from HBOT exposure could explain the reduction in IL-6. This effect, in summation with the synergistic effects of TNF-α, IL-6, and RANKL, could ultimately lead to decreased levels of osteoclastogenesis and, hence, greater resolution for the patient [56]. In the case of FHN, HBOT results in a decreased amount of circulating TNF-α. We propose the following mechanism: HBOT leads to reduced levels of TNF-α leading to decreased binding of TNF-α to the p55r type 1 receptor and thus decreased levels of NF-kB activation [56]. This reduction in RANKL would tip the balance of OPG/RANKL in the direction of osteoblast activation [50].

Understanding the HBOT's molecular mechanism of action remains the best approach in order to gain greater recognition for this treatment and to achieve earlier resolution for patients.
