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To date, the dogma in the field has been that RANKL, an essential cytokine in osteoclast maturation, is released by osteoblasts as a way to coordinate bone growth and bone loss during adult bone remodeling. Now, Hiroshi Takayanagi and colleagues, as well as Charles O'Brien and colleagues, have independently found that osteocytes are the predominant source of RANKL in the adult mouse. As RANKL signaling is a key target in treating osteoporosis, these results have potentially important implications for disease management. Osteoclasts resorb the mineralized matrices formed by chondrocytes or osteoblasts. The cytokine receptor activator of nuclear factor-κB ligand (RANKL) is essential for osteoclast formation and thought to be supplied by osteoblasts or their precursors, thereby linking bone formation to resorption. However, RANKL is expressed by a variety of cell types, and it is unclear which of them are essential sources for osteoclast formation. Here we have used a mouse strain in which RANKL can be conditionally deleted and a series of Cre-deleter strains to demonstrate that hypertrophic chondrocytes and osteocytes, both of which are embedded in matrix, are essential sources of the RANKL that controls mineralized cartilage resorption and bone remodeling, respectively. Moreover, osteocyte RANKL is responsible for the bone loss associated with unloading. Contrary to the current paradigm, RANKL produced by osteoblasts or their progenitors does not contribute to adult bone remodeling. These results suggest that the rate-limiting step of matrix resorption is controlled by cells embedded within the matrix itself.

Bone fragility is a major health concern, as the increased risk of bone fractures has devastating outcomes in terms of mortality, decreased autonomy, and healthcare costs. Efforts made to address this problem have considerably increased our knowledge about the mechanisms that regulate bone formation and resorption. In particular, we now have a much better understanding of the cellular events that are triggered when bones are mechanically stimulated and how these events can lead to improvements in bone mass. Despite these findings at the molecular level, most exercise intervention studies reveal either no effects or only minor benefits of exercise programs in improving bone mineral density (BMD) in osteoporotic patients. Nevertheless, and despite that BMD is the gold standard for diagnosing osteoporosis, this measure is only able to provide insights regarding the quantity of bone tissue. In this article, we review the complex structure of bone tissue and highlight the concept that its mechanical strength stems from the interaction of several different features. We revisited the available data showing that bone mineralization degree, hydroxyapatite crystal size and heterogeneity, collagen properties, osteocyte density, trabecular and cortical microarchitecture, as well as whole bone geometry, are determinants of bone strength and that each one of these properties may independently contribute to the increased or decreased risk of fracture, even without meaningful changes in aBMD. Based on these findings, we emphasize that while osteoporosis (almost) always causes bone fragility, bone fragility is not always caused just by osteoporosis, as other important variables also play a major role in this etiology. Furthermore, the results of several studies showing compelling data that physical exercise has the potential to improve bone quality and to decrease fracture risk by influencing each one of these determinants are also reviewed. These findings have meaningful clinical repercussions as they emphasize the fact that, even without leading to improvements in BMD, exercise interventions in patients with osteoporosis may be beneficial by improving other determinants of bone strength.

Vascularization is crucial for bone regeneration after the transplantation of tissue-engineered bone grafts in the clinical setting. Growing evidence suggests that mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are potently pro-angiogenic both in vitro and in vivo . In the current study, we fabricated a novel EV-functionalized scaffold with enhanced pro-angiogenic and pro-bone regeneration activities by coating decalcified bone matrix (DBM) with MSC-derived EVs. EVs were harvested from rat bone marrow-derived MSCs and the pro-angiogenic potential of EVs was investigated in vitro . DBM scaffolds were then coated with EVs, and the modification was verified by scanning electron microscopy and confocal microscopy. Next, the pro-angiogenic and pro-bone regeneration activities of EV-modified scaffolds were evaluated in a subcutaneous bone formation model in nude mice. Micro-computed tomography scanning analysis showed that EV-modified scaffolds with seeded cells enhanced bone formation. Enhanced bone formation was confirmed by histological analysis. Immunohistochemical staining for CD31 proved that EV-modified scaffolds promoted vascularization in the grafts, thereby enhancing bone regeneration. This novel scaffold modification method provides a promising way to promote vascularization, which is essential for bone tissue engineering.

Osteocytes are key in bone remodeling, responding to mechanical stimuli. The piezoelectric bone matrix converts these stimuli into electrical signals, influencing remodeling. To delve deeper into this, we created an osteocyte model within a piezoelectric bone matrix, incorporating the lacuna-canalicular system and mechanosensors such as integrins, cytoskeleton, and primary cilia. Upon subjecting the bone matrix to triaxial dynamic displacement loads, we examined the electric potential and flow velocity distributions and analyzed the mechanical signals of six mechanosensors. The results show that the strain is greater when the bone matrix is piezoelectric than non-piezoelectric. The maximum average potential of the surface structure of the cell membrane is about 69.3 mV. Piezoelectricity significantly increases the fluid velocity and changes the trend. The cytoskeleton and integrins in cell process experience greater stress than in cell body. Microtubules experience greater stress than actin filaments. Among all integrins, those in contact with collagen hillocks experience the greatest stress. In individual integrin, the β subunit has higher stress than α subunit, and the stress of legs connected to cytoskeleton is higher than head contacted with fluid. Within the cytoplasm, the stress of integrin increases with a decrease of the surrounding cytoskeleton density. Moreover, collagen hillocks have the greatest fluid shear stress and stress. Integrins, primary cilia, and cytoskeleton all exhibit significant displacement signal amplification, especially integrins. In conclusion, this study illuminates the complex process of mechanosensing in osteocytes within a piezoelectric environment. The established model offers valuable insights into the mechanism of osteomechanical signal transduction.

Bone is a complex tissue of which the principal function is to resist mechanical forces and fractures. Bone strength depends not only on the quantity of bone tissue but also on the quality, which is characterized by the geometry and the shape of bones, the microarchitecture of the trabecular bones, the turnover, the mineral, and the collagen. Different determinants of bone quality are interrelated, especially the mineral and collagen, and analysis of their specific roles in bone strength is difficult. This review describes the interactions of type I collagen with the mineral and the contribution of the orientations of the collagen fibers when the bone is submitted to mechanical forces. Different processes of maturation of collagen occur in bone, which can result either from enzymatic or nonenzymatic processes. The enzymatic process involves activation of lysyl oxidase, which leads to the formation of immature and mature crosslinks that stabilize the collagen fibrils. Two type of nonenzymatic process are described in type I collagen: the formation of advanced glycation end products due to the accumulation of reducible sugars in bone tissue, and the process of racemization and isomerization in the telopeptide of the collagen. These modifications of collagen are age-related and may impair the mechanical properties of bone. To illustrate the role of the crosslinking process of collagen in bone strength, clinical disorders associated with bone collagen abnormalities and bone fragility, such as osteogenesis imperfecta and osteoporosis, are described.

Peak bone mass (PBM) is regarded as the most important determinant of osteoporosis. Growing evidence suggests a role of intrauterine programming in skeletal development. We examined PBM and trabecular bone score (TBS) in adults born preterm with very low birth weight (VLBW) or small for gestational age (SGA) at term compared with term-born controls. This follow-up cohort study included 186 men and women (25 to 28 years); 52 preterm VLBW (≤1500 g), 59 term-born SGA (<10th percentile), and 75 controls (>10th percentile). Main outcome was bone mineral density (BMD) by dual x-ray absorptiometry. Secondary outcomes were bone mineral content (BMC), TBS, and serum bone markers. VLBW adults had lower BMC and BMD vs controls, also when adjusted for height, weight, and potential confounders, with the following BMD Z-score differences: femoral neck, 0.6 standard deviation (SD) (P = 0.003); total hip, 0.4 SD (P = 0.01); whole body, 0.5 SD (P = 0.007); and lumbar spine, 0.3 SD (P = 0.213). The SGA group displayed lower spine BMC and whole-body BMD Z-scores, but not after adjustment. Adjusted odds ratios for osteopenia/osteoporosis were 2.4 and 2.0 in VLBW and SGA adults, respectively. TBS did not differ between groups, but it was lower in men than in women. Serum Dickkopf-1 was higher in VLBW subjects vs controls; however, it was not significant after adjustment for multiple comparisons. Both low-birth-weight groups displayed lower PBM and higher frequency of osteopenia/osteoporosis, implying increased future fracture risk. The most pronounced bone deficit was seen in VLBW adults.

Demineralized bone matrix (DBM) is widely used for bone regeneration. Since DBM is prepared in powder form its handling properties are not optimal and limit the clinical use of this material. Various synthetic and biological carriers have been used to enhance the DBM handling. In this study we evaluated the effect of gamma irradiation on the physical-chemical properties of Pluronic and on bone morphogenetic proteins (BMPs) amount in DBM samples. In vivo studies were carried out to investigate the effect on bone regeneration of a gamma irradiated DBM-Pluronic F127 (DBM-PF127) composite implanted in the femur of rats. Gamma irradiation effects (25 kGy) on physical-chemical properties of Pluronic F127 were investigated by rheological and infrared analysis. The BMP-2/BMP-7 amount after DBM irradiation was evaluated by ELISA. Bone regeneration capacity of DBM-PF127 containing 40% (w/w) of DBM was investigated in transcortical holes created in the femoral diaphysis of Wistar rat. Bone porosity, repaired bone volume and tissue organization were evaluated at 15, 30 and 90 days by Micro-CT and histological analysis. The results showed that gamma irradiation did not induce significant modification on physical-chemical properties of Pluronic, while a decrease in BMP-2/BMP-7 amount was evidenced in sterilized DBM. Micro-CT and histological evaluation at day 15 post-implantation revealed an interconnected trabeculae network in medullar cavity and cellular infiltration and vascularization of DBM-PF127 residue. In contrast a large rate of not connected trabeculae was observed in Pluronic filled and unfilled defects. At 30 and 90 days the DBM-PF127 samples shown comparable results in term of density and thickness of the new formed tissue respect to unfilled defect. In conclusion a gamma irradiated DBM-PF127 composite, although it may have undergone a significant decrease in the concentration of BMPs, was able to maintains bone regeneration capability.

Autologous bone grafts, used mainly in extensive bone loss, are considered the gold standard treatment in regenerative medicine, but still have limitations mainly in relation to the amount of bone available, donor area, morbidity and creation of additional surgical area. This fact encourages tissue engineering in relation to the need to develop new biomaterials, from sources other than the individual himself. Therefore, the present study aimed to investigate the effects of an elastin and collagen matrix on the bone repair process in critical size defects in rat calvaria. The animals (Wistar rats, n = 30) were submitted to a surgical procedure to create the bone defect and were divided into three groups: Control Group (CG, n = 10), defects filled with blood clot; E24/37 Group (E24/37, n = 10), defects filled with bovine elastin matrix hydrolyzed for 24 h at 37 °C and C24/25 Group (C24/25, n = 10), defects filled with porcine collagen matrix hydrolyzed for 24 h at 25 °C. Macroscopic and radiographic analyses demonstrated the absence of inflammatory signs and infection. Microtomographical 2D and 3D images showed centripetal bone growth and restricted margins of the bone defect. Histologically, the images confirmed the pattern of bone deposition at the margins of the remaining bone and without complete closure by bone tissue. In the morphometric analysis, the groups E24/37 and C24/25 (13.68 ± 1.44; 53.20 ± 4.47, respectively) showed statistically significant differences in relation to the CG (5.86 ± 2.87). It was concluded that the matrices used as scaffolds are biocompatible and increase the formation of new bone in a critical size defect, with greater formation in the polymer derived from the intestinal serous layer of porcine origin (C24/25).

Advanced glycation end products (AGEs) accumulate in bone extracellular matrix as people age. Although previous evidence shows that the accumulation of AGEs in bone matrix may impose significant effects on bone cells, the effect of matrix AGEs on bone formation in vivo is still poorly understood. To address this issue, this study used a unique rat model with autograft implant to investigate the in vivo response of bone formation to matrix AGEs. Fluorochrome biomarkers were sequentially injected into rats to label the dynamic bone formation in the presence of elevated levels of matrix AGEs. After sacrificing animals, dynamic histomorphometry was performed to determine mineral apposition rate (MAR), mineralized surface per bone surface (MS/BS), and bone formation rate (BFR). Finally, nanoindentation tests were performed to assess mechanical properties of newly formed bone tissues. The results showed that MAR, MS/BS, and BFR were significantly reduced in the vicinity of implant cores with high concentration of matrix AGEs, suggesting that bone formation activities by osteoblasts were suppressed in the presence of elevated matrix AGEs. In addition, MAR and BFR were found to be dependent on the surrounding environment of implant cores (i.e., cortical or trabecular tissues). Moreover, MS/BS and BFR were also dependent on how far the implant cores were away from the growth plate. These observations suggest that the effect of matrix AGEs on bone formation is dependent on the biological milieu around the implants. Finally, nanoindentation test results indicated that the indentation modulus and hardness of newly formed bone tissues were not affected by the presence of elevated matrix AGEs. In summary, high concentration of matrix AGEs may slow down the bone formation process in vivo, while imposing little effects on bone mineralization.

Background and Objectives: Demineralized bone matrix (DBM) is a widely used bone graft substitute due to its osteoconductive and osteoinductive properties. However, its efficacy varies due to differences in donor, processing, and storage conditions. Synthetic alternatives, such as iFactor®, combine non-organic bone mineral and a small peptide (P-15) to enhance the cellular attachment and osteogenesis. To compare the osteogenic potential and bone matrix maturity of iFactor® and a commercial DBM scaffold through calcium nodule formation and Fourier transform infrared spectroscopy (FTIR) analysis. Materials and Methods: Human mesenchymal stem cells (hMSCs) were cultured and exposed to iFactor® or DBM in paracrine culture conditions for 21 days. Calcium nodule formation was assessed using alizarin red staining and quantified spectrophotometrically. The FTIR analysis of hMSCs exposed to the scaffolds for three months evaluated the biomolecular composition and bone matrix maturity. Results: Calcium nodules formed in both groups but in smaller quantities than in the positive control (p < 0.05). The biomolecular components of the DBM were similar to healthy bone (p > 0.05) than those of the iFactor® group (p < 0.005). A different rate of bone regeneration was observed through the formation of a greater number of calcium nodule aggregates identified in the extracellular matrix of mesenchymal stem cell cultures exposed to iFactor® compared to those cultures enriched with DBM. Conclusions: Both experimental matrices demonstrated similar osteogenic potential at the 3-month follow-up. Although DBM has a closer biomolecular composition and carbonate substitution compared to healthy bone, iFactor® showed faster matrix maturity expressed through the formation of a greater number of calcium nodule aggregates and higher hMSCs proliferation.