Life expectancy has increased rapidly in the last century due to economic growth. This has led to reduced mortality, improved quality of life, as well as greater availability of health care. In fact, there are more elderly people than at any other time in our history, and it is anticipated that within the next few years there will be more older adults than children. This forecast makes it essential for people to reach this age in good health, to avoid increased healthcare costs due to longer hospital stays, readmissions and demand for healthcare resources. One of the most common disorders associated with aging is osteoporosis, the most fatal consequence of which is fracture. Approximately half of the clinical fractures that occur in postmenopausal women do not present criteria for osteoporosis according to their bone mineral density [1]. In fact, the highest percentage of fractures occur in osteopenic women. Thus, other variables or tools are needed that allow the identification of people at high risk of fractures, a determining factor of morbidity and mortality in the elderly population.

Aromatase inhibitors (AIs) are the recommended adjuvant therapy to treat estrogen receptor-positive breast cancer [1,2]. Its effectiveness in reducing the risk of recurrence and mortality is acknowledged [3]. However, AIs are also associated with various side effects that affect patients’ quality of life and therefore compromise adherence to treatment and associated mortality [4].
Reportedly, 30% of patients prescribed with AI discontinue their treatment due to adverse events [5], mainly musculoskeletal [6,7]. Among them, the most frequent are arthralgias [8] and accelerated loss of bone mass [9] associated with an increase in osteoporotic fracture [10,11]. To prevent the loss of bone mass, treating patients with antiresorptives is recommended, with bisphosphonates (BP) being the most used [12-14].
BP use has been associated with improved mortality associated with reduced bone metastases [13]. Similarly, a study published in a Korean population showed the use of BP was associated with improved adherence [15].

The generation of functional tissue through tissue engineering has a high impact in various areas of regenerative medicine, among which is skeletal tissue. The first implants were used in the field of medicine, in 1909, when Kirschner wires and Steinman nails were developed for the fixation of bone fractures, where stainless steel was used. Over the years, steel has been improved, making it more resistant to corrosion and not causing harmful effects on the human body. In 1940, the study of titanium (Ti) began as a biomaterial for bone implantation [1].
The phase change determines the change in the crystalline structure of the material when subjected to temperature changes. Titanium’s allotropic transformation occurs at 882ºC and goes from an α phase, which has a compact and hexagonal structure (HCP), little deformable and resistant at room temperature, to a β phase characterized by a cubic structure centered on the body (BCC), which is easily deformable and allows for carrying out heat treatments to optimize the material’s properties [2].

Pleitropin (PTN) is a cytokine secreted by multiple tissues during embryonic development, and which in adulthood is abundantly expressed in the brain and bone [1,2]. PTN is composed of 136 amino acids and its sequence is very rich in lysine and cysteine. Together with midkine (Mdk), with which it shares 50% homology, this cytokine constitutes the heparin-binding family of growth and differentiation factors, both having affinity for bone extracellular matrix [3-5]. PTN is also known as osteoblast stimulating factor 1 (OSF-1) or heparin-linked growth factor (HB-GAM) [6]. This cytokine was initially isolated from the bone and neuronal tissues of newborn rats [2,7,8] and subsequently its homologues have been found in many species including humans, with 90% homology between the different species [9,10].
PTN reportedly exerts its effects through its binding to glucosamin-glucans of several receptors such as N-syndecan, also called syndecan 3 [11], syndecans 1 and 4 [12], integrin αvβ3 [13] and the receptor protein tyrosine phosphatase beta/zeta (PTRPβ/ζ) [14]. It has also been suggested that nucleolin may be a low-affinity receptor for PTN [15] and that anaplastic lymphoma kinase (ALK) may play a role in PTN signaling [16].

Osteoporosis is the most frequent bone disease, characterized by low bone mass and alteration of the microstructure. This is due to an imbalance between bone formation and bone resorption that causes loss of connections among the different bone trabeculae, a greater thinning and cortical bone porosity. Consequently, there is greater bone fragility and an increased risk of fractures (Fx) [1,2].
Osteoblasts, cells specialized in bone formation, originate from the differentiation of mesenchymal stem cells (MSCs) [3]. These cells are multipotent and can differentiate into a wide variety of mesoderm cell types, such as osteoblasts, adipocytes, or chondrocytes. MSCs are highly interesting candidates for regenerative medicine, because they migrate to skeletal lesions where they have the capacity to form new bone [4]. The many relevant published studies show the importance of MSCs in tissue engineering and regenerative medicine [5,6]. In addition, there are currently more than 250 clinical trials with MSCs, as reflected in the clinical trial database (clinicaltrials.gov).

Every year 8.9 million osteoporosis-related fractures occur worldwide, representing one fracture every 3 seconds [1], with vertebral being the most common osteoporotic fractures [2].
Dual-energy X-ray absorptiometry (DXA) is the standard test for diagnosing osteoporosis and evaluating fracture risk [3,4], as it is a low-radiation, inexpensive technique. The DXA provides two-dimensional (2D) images that measure the bone mineral density of the area (aBMD) projected along the anteroposterior (AP) direction. Various studies show that a low aBMD value, measured in AP DXA explorations, is among the highest fracture risks [3-5]. The decrease of the aBMD standard deviation leads to an increase from 1.5 up to 3.0 times the risk of fracture, depending on its location and its measurement’s location [5]. Nevertheless, a low BMD value is not enough to explain every fracture. Recent studies suggest that the risk of fracture is high when the BMD value is low, but this does not mean that fracture risk is negligible when the BMD value is normal [3-8].
Most osteoporosis-related vertebral fractures are located in the vertebral body [9]. In AP DXA images of the spinal column, the vertebral body overlaps the posterior vertebral elements, so the BMD in the vertebral body cannot be estimated separately. On the other hand, the risk of fracture depends on the architecture of the trabecular bone and the thickness of the cortical bone [10]. However, the trabecular and cortical bone compartments are difficult to assess separately on AP DXA scans.

Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD), was defined in 2009 as a set of systemic disorders of the bone and mineral metabolism due to chronic kidney disease, resulting in a combination of the following manifestations [1,2]:
I) Abnormalities of the metabolism of calcium, phosphorus, paratohormone or vitamin D.
II) Anomalies of bone remodeling, mineralization, volume, linear growth or resistance.
III) Vascular and other soft tissue calcifications.
This recently updated definition [3], and the consensus documents of various scientific societies [4], have highlighted the importance of the role of vascular calcification in the morbidity and mortality of patients with chronic kidney disease.

The increase in the elderly population and the growing concern about the consequences of fractures, together with insufficient rates of detection of situations of bone fragility [1,2], has increased the indication of the assessment of fracture risk in people of both sexes older than 64 years [3]. The dual-energy X-ray absorptiometry (DXA) technique is currently the clinical standard for this type of bone measurement.
Nowadays, when evaluating the risk of fracture, different methods are applied, although the most widely used include the presence of clinical risk factors and the measurement of areal bone mineral density (BMD). Bone measurements are made in the proximal femur and lumbar spine using DXA. However, BMD only allows a limited assessment of the mechanical determinants of bone fracture [4,5].
Finite element analysis (FE) has been applied to assess bone resistance in volumetric bone models, based on computed tomography (CT) scans, precisely identifying the subject-specific mechanical determinants of fracture. This type of analysis includes the three-dimensional geometry of the bone, the quantity and distribution of bone tissue, and the loads to which the bone is subjected [6]. With this process, the limitations of the BMDa are overcome. CT-based models of FE have been extensively validated ex vivo [7-12], and have shown better performance compared to a BMD in predicting proximal femur resistance in vitro [6,13]. A significant association between bone fractures and estimated resistance with FE has also been reported in an in vivo study [14].

Currently, aromatase inhibitors (AI) are used as first-line adjuvant therapy for women diagnosed with breast cancer with positive hormonal receptors. Although its effectiveness in reducing the risk of recurrence and mortality is well known [1], AIs have also been associated with side effects that can negatively affect the patient’s quality of life, adherence to treatment and associated mortality [2].
In AI treatment, there is a marked reduction in circulating estrogens in postmenopausal women by blocking the conversion by the enzyme aromatase from androgens to estrogens. This action leaves the woman without residual estrogens, such as estradiol and estrone, after menopause. One of the most common side effects is accelerated bone loss, which is associated with an increased risk of osteoporotic fractures [3,4]. Along these lines, there are different meta-analyzes that include randomized controlled clinical trials that have shown an association between prolonged treatment with AI and an increased risk of bone fractures, with an increase between 34% and 59% [5,6].

Primary hyperparathyroidism (HPT) is a very common bone mineral metabolic disease consisting of autonomous overproduction of parathyroid hormone (PTH), which leads to an increase in serum calcium [1]. It is the most frequent cause of hypercalcemia.
A lesser known clinical variant of HPT is the so-called “normocalcemic primary hyperparathyroidism” (NHPT), which has normal blood calcium levels and elevated parathyroid hormone (PTH) values, not knowing the mechanism by which this differential fact occurs [2-4]. These patients do not have clear causes that justify secondary elevations of PTH such as chronic renal damage [5], vitamin D deficiency (less than 30 ng/ml) [6], renal hypercalciuria or drugs [7]. Although NHPT was first formally recognized in the Third International Workshop on the Management of Asymptomatic Primary Hyperparathyroidism in 2008 [8], all clinical features are not yet known, particularly with regard to its epidemiology, natural history, management and prognosis [9,10]. Therefore, this clinical variety of the disease is less studied [11] and there is less bibliography. All of which has motivated us to carry out this study.

Selective estrogen-receptor modulators (SERMs) are synthetic, nonsteroidal agents with estrogenic agonist-antagonist activity in different target tissues [1]. Their estrogenic responses are mediated by estrogen receptors (α and β). SERMs may present agonistic or antagonistic behavior depending on the tissue type [2,3]. In general, SERMs exhibit agonist activity in the liver, the digestive tube, the skeleton and the heart, but antagonist activity in the breast. In the uterus some SERMs manifest agonist activity while others show an antagonist behavior [1]. Several co-regulatory proteins modify the behavior of the SERMs on gene expression and contribute to their tissue-selective pharmacology.
Tamoxifen is a SERM used as a mammary antiestrogen for preventing and treating breast cancer with estrogen agonistic activity in the uterus. Raloxifene has been used for the prevention and treatment of osteoporosis and prevents breast cancer but presents some estrogenic activity [4]. Bazedoxifene is a 3rd generation SERM with agonistic effects on the bone and additional positive effects on lipids, the uterus and the breast tissue [5,6].

Oxygen is required to produce cellular energy and is involved in numerous processes, such as enzymatic activation, molecular signaling and regulation of gene expression [1]. Also in angiogenesis, the maintenance of hematopoietic stem cells and bone formation [2]. In fact, changes in the partial pressure of oxygen can influence the function of osteoblasts and osteoclasts [3]. In hypoxia, bone formation and mineralization decreases, while resorption increases [4-6]. In the opposite direction, hyperoxia could have a beneficial effect on the bone. Treatment with high concentration of oxygen in the hyperbaric chamber has proven useful in osteomyelitis and osteonecrosis of the jaw caused by radiotherapy or by the use of bisphosphonates [7-9]. HC accelerates osteogenic differentiation of mesenchymal cells and decreases the activation of osteoclasts [10-12].
In this work we wanted to analyze the actions of oxygen at high concentration in HBO on the expression of genes related to bone metabolism in osteoblastic cell lines and human bone [5,6,13,14].