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Purpose of Review: Bone regeneration plays an important role in contemporary clinical treatment. Bone tissue engineering should result in successful bone regeneration to restore congenital or acquired bone defects in the human skeleton. Osteocytes are thought to have a governing role in bone remodeling by regulating osteoclast and osteoblast activity, and thus bone loss and formation. In this review, we address the so far largely unknown role osteocytes may play in bone tissue regeneration., Recent Findings: Osteocytes release biochemical signaling molecules involved in bone remodeling such as prostaglandins, nitric oxide, Wnts, and insulin-like growth factor-1 (IGF-1). Treatment of mesenchymal stem cells in bone tissue engineering with prostaglandins (e.g., PGE 2 , PGI 2 , PGF 2α ), nitric oxide, IGF-1, or Wnts (e.g., Wnt3a) improves osteogenesis. This review provides an overview of the functions of osteocytes in bone tissue, their interaction with other bone cells, and their role in bone remodeling. We postulate that osteocytes may have a pivotal role in bone regeneration as well, and consequently that the bone regeneration process may be improved effectively and rapidly if osteocytes are optimally used and stimulated.

Osteocytes are the most abundant (~95%) cells in bone with the longest half-life (~25 years) in humans. In the past osteocytes have been regarded as vestigial cells in bone, since they are buried inside the tough bone matrix. However, during the last 30 years it has become clear that osteocytes are as important as bone forming osteoblasts and bone resorbing osteoclasts in maintaining bone homeostasis. The osteocyte cell body and dendritic processes reside in bone in a complex lacuno-canalicular system, which allows the direct networking of osteocytes to their neighboring osteocytes, osteoblasts, osteoclasts, bone marrow, blood vessels, and nerves. Mechanosensing of osteocytes translates the applied mechanical force on bone to cellular signaling and regulation of bone adaptation. The osteocyte lacuno-canalicular system is highly efficient in transferring external mechanical force on bone to the osteocyte cell body and dendritic processes via displacement of fluid in the lacuno-canalicular space. Osteocyte mechanotransduction regulates the formation and function of the osteoblasts and osteoclasts to maintain bone homeostasis. Osteocytes produce a variety of proteins and signaling molecules such as sclerostin, cathepsin K, Wnts, DKK1, DMP1, IGF1, and RANKL/OPG to regulate osteoblast and osteoclast activity. Various genetic abnormality-associated rare bone diseases are related to disrupted osteocyte functions, including sclerosteosis, van Buchem disease, hypophosphatemic rickets, and WNT1 and plastin3 mutation-related disorders. Meticulous studies during the last 15 years on disrupted osteocyte function in rare bone diseases guided for the development of various novel therapeutic agents to treat bone diseases. Studies on genetic, molecular, and cellular mechanisms of sclerosteosis and van Buchem disease revealed a role for sclerostin in bone homeostasis, which led to the development of the sclerostin antibody to treat osteoporosis and other bone degenerative diseases. The mechanism of many other rare bone diseases and the role of the osteocyte in the development of such conditions still needs to be investigated. In this review, we mainly discuss the knowledge obtained during the last 30 years on the role of the osteocyte in rare bone diseases. We speculate about future research directions to develop novel therapeutic drugs targeting osteocyte functions to treat both common and rare bone diseases., (Copyright © 2020 Pathak, Bravenboer and Klein-Nulend.)

In patients with postmenopausal osteoporosis low bone volume is associated with high bone marrow adipose tissue (MAT). Moreover, high MAT is associated with increased fracture risk. This suggests an interaction between MAT and bone turnover, however literature remains equivocal. Estrogen treatment decreases MAT, but the effect of raloxifene, a selective estrogen receptor modulator (SERM) registered for treatment of postmenopausal osteoporosis, on MAT is not known. The aim of this study is 1] to determine the effect of raloxifene on MAT and 2] to determine the relationship between MAT and bone turnover in patients with osteoporosis. Bone biopsies from the MORE trial were analyzed. The MORE trial investigated the effects of raloxifene 60 or 120mg per day versus placebo on bone metabolism and fracture incidence in patients with postmenopausal osteoporosis. We quantified MAT in iliac crest biopsies obtained at baseline and after 2years of treatment (n=53; age 68.2±6.2years). Raloxifene did not affect the change in MAT volume after 2years compared to baseline (placebo: 1.89±10.84%, raloxifene 60mg: 6.31±7.22%, raloxifene 120mg: -0.77±10.72%), nor affected change in mean adipocyte size (placebo: 1.45 (4.45) μm, raloxifene 60mg: 1.45 (4.35) μm, raloxifene 120mg: 0.81 (5.21) μm). Adipocyte number tended to decrease after placebo treatment (-9.92 (42.88) cells/mm 2 ) and tended to increase during raloxifene 60mg treatment (13.27 (66.14) cells/mm 2 ) while adipocyte number remained unchanged in the raloxifene 120mg group, compared to placebo (3.06 (39.80) cells/mm 2 , Kruskal-Wallis p=0.055, post hoc: placebo vs raloxifene 60mg p=0.017). MAT volume and adipocyte size were negatively associated with osteoclast number at baseline (R 2 =0.123, p=0.006 and R 2 =0.098, p=0.016 respectively). Furthermore adipocyte size was negatively associated with osteoid surface (R 2 =0.067, p=0.049). Finally, patients with vertebral fractures had higher MAT volume (50.82 (8.80)%) and larger adipocytes (55.75 (3.14) μm) compared to patients without fractures (45.58 (12.72)% p=0.032, 52.77 (3.73) μm p=0.004 respectively). In conclusion, raloxifene did not affect marrow adipose tissue, but tended to increase adipocyte number compared to placebo. At baseline MAT volume and adipocyte size were associated with bone resorption, and adipocyte size was associated with osteoid surface, suggesting an interaction between bone marrow adipocytes and bone turnover. In addition, we found that high MAT volume and larger adipocyte size are associated with prevalent vertebral fractures in postmenopausal women with osteoporosis, indicating that adipocyte size affects bone quality independent of bone volume., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Patients with Crohn's disease (CD) are at increased risk of developing osteoporosis. The mechanism underlying bone loss in CD patients is only partly understood. Inflammation is thought to contribute by causing a disturbed bone remodeling. In this study, we aimed to compare functional characteristics of osteoblasts from CD patients and controls, as osteoblasts are one of the effector cells in bone remodeling. The study included 18 patients with quiescent CD and 18 healthy controls. Bone cells obtained from iliac crest biopsies were cultured in the absence and presence of the inflammatory cytokines IL-1α, IL-1β, IL-6, TNF-α, IL-10, and TGF-β. At various time points, cell proliferation and differentiation were analyzed. Bone cells from CD patients showed a prolonged culture period to reach confluence and a decreased cell number at confluence. CD patient-derived bone cell cultures produced higher alkaline phosphatase levels, whereas osteocalcin levels were considerably reduced compared to control cultures. At the proliferation level, the responsiveness to inflammatory cytokines was similar in bone cells from CD patients and controls. At the differentiation level, CD cultures showed an increased responsiveness to IL-6 and a decreased responsiveness to TGF-β. Responsiveness to the other cytokines tested was unaffected. In summary, we show a reduced growth potential and impeded maturation of bone cells from quiescent CD patients in vitro. These disease-related alterations combined with an unchanged sensitivity of CD patient-derived bone cells to inflammatory cytokines, provide a new insight in the understanding of CD-associated bone loss., (Copyright © 2012 Wiley Periodicals, Inc.)

Mechanical loading and estrogen play important roles in bone homeostasis. The aim of this study was to evaluate the effects of mechanical loading on trabecular bone in the proximal femur of ovariectomized rats. We hypothesized that mechanical loading suppresses bone resorption and increases bone formation, which differs from the suppressive effects of estrogen on both resorption and formation. Furthermore, we expected to find changes in trabecular architecture elicited by the effects of mechanical loading and estrogen deficiency. Sixty female Wistar rats, 12 weeks old, were assigned to either the sedentary groups sham surgery (SED), ovariectomy (SED+OVX), and ovariectomy with estrogen replacement (SED+OVX+E2) or to the exercise groups EX, EX+OVX, EX+OVX+E2. Following ovariectomy, 5 microg 17beta-estradiol was given once weekly to the estrogen replacement groups. Exercise consisted of running with a backpack (load +/-20% of body weight) for 15 minutes/day, 5 days/week, for 19 weeks. Dual-energy X-ray absorptiometry (DXA) scans were performed before (T0), during (T6), and after (T19) the exercise period to obtain bone mineral content (BMC) and bone mineral density (BMD) data. After the exercise program, all rats were killed and right and left femora were dissected and prepared for micro-CT scanning and histomorphometric analysis of the proximal femoral metaphysis. After 19 weeks, increases in BMC (P = 0.010) and BMD (P = 0.031) were significant. At T19, mechanical loading had a significant effect on BMC (P = 0.025) and BMD (P = 0.010), and an interaction between mechanical loading and estrogen (P = 0.023) was observed. Bone volume and trabecular number decreased significantly after ovariectomy, while trabecular separation, mineralizing surface, bone formation rate, osteoclast surface, degree of anisotropy, and structure model index increased significantly after ovariectomy (P < 0.05). Trabecular bone turnover and structural parameters in the proximal femur were not affected by exercise. Estrogen deficiency resulted in a less dense and more oriented trabecular bone structure with increased marrow cavity and a decreased number of trabeculae. In conclusion, mechanical loading has beneficial effects on BMC and BMD of the ovariectomized rat. This indicates that the load in the backpack was high enough to elicit an osteogenic response sufficient to compensate for the ovariectomy-induced bone loss. The results confirm that estrogen suppresses both bone resorption and bone formation in the proximal metaphysis in the femoral head of our rat-with-backpack model. The effects of mechanical loading on the trabecular bone of the femoral head were not significant. This study suggests that the effect of mechanical loading in the rat-with-backpack model mainly occurs at cortical bone sites.

In fibrous dysplasia/McCune-Albright syndrome (FD/MAS), mosaic mutations in the GNAS gene lead to locally abnormal bone turnover. Additionally, patients with FD/MAS, particularly with thoracic lesions, have an increased risk for breast cancer. Development and progression of breast cancer has been associated with expression of Receptor Activator of NF-κB ligand (RANKL) in mammary tissue, and due to the GNAS mutation, RANKL is systemically increased in patients with FD/MAS. Yet it is unknown whether breast cancer in FD/MAS is also dependent on RANKL. We hypothesized that the GNAS mutation might induce RANKL overproduction and an oncogenic niche in mammary tissue, and examined RANKL expression in breast cancer tissue of patients with FD/MAS compared to controls. Nine patients with FD/MAS and breast cancer were included and clinical data were retrieved. Patients were matched to controls with breast cancer without FD/MAS based on age and tumor type. Three pregnant breast cancer patients were included as positive controls. Immunohistochemical detection of RANKL was performed on formalin-fixed paraffin-embedded breast cancer specimens. Staining intensity was classified as weak, moderate or intense. The area of positive RANKL staining divided by the total ductal-lobular area was assessed (positive area percentage, PAP). Number of patients with RANKL expression was compared between FD/MAS and control group by chi-square (χ2) test, the PAP by Mann-Whitney U test (MWU). RANKL expression was observed in 3 patients with FD/MAS (38 %), mainly in healthy tissue, and none of the control patients (χ2 p = 0.055). The FD/MAS group demonstrated considerably more intense staining than the control group, comparable to positive controls. The median PAP was 0.64 % (range 0.14–2.04 %) in the 3 FD/MAS patients with RANKL expression, 0.01 % (Q1-Q3: 0.0003–0.514 %) in the entire FD/MAS group, 0.006 % (Q1-Q3: 0.001–0.012 %) in the control group (MWU = 0.574), and 0.19 % (0.08–0.32 %) in the pregnant patients. All patients with FD/MAS and RANKL expression had thoracic bone lesions, but no correlation was observed between RANKL expression and presence of the GNAS mutation or FD disease burden. The triad of a higher number of patients, higher positive area percentage and stronger intensity in the FD/MAS compared to the control group indicates that RANKL may be upregulated in mammary tissue in a subset of patients with FD/MAS, which may explain the increased risk for breast cancer, although the clinical significance remains unclear. Further research is needed to establish risk profiles for the development of RANKL-positive breast cancer and to improve early screening and treatment. • Patients with fibrous dysplasia/McCune-Albright syndrome may develop breast cancer. • A subset of patients expressed RANKL in breast tissue, mainly in healthy tissue. • Control subjects with breast cancer without the disease expressed no RANKL. • RANKL expression is also known to be elevated in diseased bone in fibrous dysplasia. • RANKL possibly links the bone abnormalities with the increased breast cancer risk. [ABSTRACT FROM AUTHOR]

Animal models show that vitamin D deficiency may have severe consequences for skeletal health. However, most studies have been performed in young rodents for a relatively short period, while in older adult rodents the effects of long-term vitamin D deficiency on skeletal health have not been extensively studied. Therefore, the first aim of this study was to determine the effects of long-term vitamin D deficiency on bone structure, remodeling and mineralization in bones from older adult mice. The second aim was to determine the effects of long-term vitamin D deficiency on mRNA levels of genes involved in vitamin D metabolism in bones from older adult mice. Ten months old male C57BL/6 mice were fed a diet containing 0.5% calcium, 0.2% phosphate and 0 ( n = 8) or 1 ( n = 9) IU vitamin D 3 /gram for 14 months. At an age of 24 months, mice were sacrificed for histomorphometric and micro-computed tomography (micro-CT) analysis of humeri as well as analysis of CYP27B1, CYP24 and VDR mRNA levels in tibiae and kidneys using RT-qPCR. Plasma samples, obtained at 17 and 24 months of age, were used for measurements of 25-hydroxyvitamin D (25(OH)D) (all samples), phosphate and parathyroid hormone (PTH) (terminal samples) concentrations. At the age of 17 and 24 months, mean plasma 25(OH)D concentrations were below the detection limit (<4 nmol/L) in mice receiving vitamin D deficient diets. Plasma phosphate and PTH concentrations did not differ between both groups. Micro-CT and histomorphometric analysis of bone mineral density, structure and remodeling did not reveal differences between control and vitamin D deficient mice. Long-term vitamin D deficiency did also not affect CYP27B1 mRNA levels in tibiae, while CYP24 mRNA levels in tibiae were below the detection threshold in both groups. VDR mRNA levels in tibiae from vitamin D deficient mice were 0.7 fold lower than those in control mice. In conclusion, long-term vitamin D deficiency in older adult C57BL/6 mice, accompanied by normal plasma PTH and phosphate concentrations, does not affect bone structure, remodeling and mineralization. In bone, expression levels of CYP27B1 are also not affected by long-term vitamin D deficiency in older adult C57BL/6 mice. Our results suggest that mice at old age have a low or absent response to vitamin D deficiency probably due to factors such as a decreased bone formation rate or a reduced response of bone cells to 25(OH)D and 1,25(OH) 2 D. Older adult mice may therefore be less useful for the study of the effects of vitamin D deficiency on bone health in older people. [ABSTRACT FROM AUTHOR]

Gaucher disease (GD) is a lysosomal storage disorder characterized by accumulation of glucosylceramide in macrophages, so-called Gaucher cells, as a result of a deficiency of the lysosomal enzyme glucocerebrosidase. Bone complications are an important cause of morbidity of GD and are thought to result from imbalance in bone remodeling. Bone manifestations among GD patients demonstrate a large variation including increased osteoclastic bone resorption, low bone formation and osteonecrosis. The purpose of the current case series is to describe the histological features observed in undecalcified bone samples, obtained from three GD patients, and evaluate the relationship with clinical features in these patients. Bone fragments were obtained from three adult type 1 GD patients with variable degrees of bone disease during orthopedic surgery. Specimens were embedded without prior decalcification in methylmethacrylate and prepared for histology according to standardized laboratory procedures. Histology revealed a heterogeneous pattern of bone involvement. High cellularity of bone marrow, abundant presence of Gaucher cells (GCs) and high turnover were observed in a patient with a history of multiple bone complications, while minimal bone turnover and few GCs were detected in the mildest affected patient in this series. An intermediate picture with relatively low bone turnover and a substantial amount of Gaucher cells was demonstrated in the third, moderately affected patient. No gross abnormalities in three biochemical markers of bone turnover (osteocalcin, N-terminal propeptide of type 1 procollagen and type 1 collagen C-terminal telopeptide) were noted. Plastic embedding and subsequent Goldner and TRAP staining offered a unique possibility to study bone histological findings in GD. Our data show that bone manifestations in GD may vary both clinically as well as histologically and bone disease in GD will likely require a personalized approach. [ABSTRACT FROM AUTHOR]