The Effects of Acute and Chronic Alcohol Administration and Withdrawal on Bone Microstructure, Mechanical Strength, and Remodeling Protein Expression and Their Relation to an Antioxidant and FGF23 In Vivo.

Alcohol's detrimental effects on bone health are well established, yet some literature suggests moderate consumption may offer benefits. With alcohol use on the rise, we investigate the impact of acute and chronic alcohol administration, along with withdrawal, on male Wistar rat femurs. We observed a transient cortical thickness increase with acute alcohol (AA) compared to chronic exposure (CA) but no significant changes in trabecular parameters or mechanical properties. High osteocalcin and osteopontin expression levels were noted in AA, alongside elevated RANKL expression. Conversely, CA showed low TRAP levels. FGF23 expression significantly increased during alcohol withdrawal (AW), while GPX decreased after chronic exposure but rose during withdrawal. Although mechanical strength changes were insignificant, biochemical shifts suggest alcohol exposure promotes bone resorption, reduces antioxidant protection, and potentially hampers active vitamin D and phosphate reabsorption via FGF23 upregulation.

Changes of the subchondral bone microchannel network in early osteoarthritis.

OBJECTIVE: We have identified a 3D network of subchondral microchannels that connects the deep zone of cartilage to the bone marrow (i.e., cartilage-bone marrow microchannel connectors; CMMC). However, the pathological significance of CMMC is largely unknown. Here, we quantitatively evaluated how the CMMC microarchitecture is related to cartilage condition, as well as regional differences in early idiopathic osteoarthritis (OA). METHODS: Two groups of cadaveric female human femoral heads (intact cartilage vs early cartilage lesions) were identified, and a biopsy-based high-resolution micro-CT imaging was employed. Subchondral bone (SB) thickness, CMMC number, maximum and minimum CMMC size, and the CMMC morphology were quantified and compared between the two groups. The effect of joint's region and cartilage condition was examined on each dependent variable. RESULTS: The CMMC number and morphology were affected by region of the joint, but not by cartilage condition. On the other hand, the minimum and maximum CMMC size was changed by both the location on the joint, as well as the cartilage condition. The smallest CMMC were consistently detected at the load-bearing region (LBR) of the joint. Compared to non-pathological subjects, the size of the microchannels was enlarged in early OA, most noticeably at the non-load-bearing region (NLBR) and the peripheral rim (PR) of the femoral head. Furthermore, subchondral bone thinning was observed in early OA as a localized occurrence linked with areas of partial chondral defect. CONCLUSION: Our data point to an enlargement of the SB microchannel network, and a collective structural deterioration of SB in early idiopathic OA.

Lifelong Excess in GH Elicits Sexually Dimorphic Effects on Skeletal Morphology and Bone Mechanical Properties.

Excess in growth hormone (GH) levels, seen in patients with acromegaly, is associated with increases in fractures. This happens despite wider bones and independent of bone mineral density. We used the bovine GH (bGH) transgenic mice, which show constitutive excess in GH and insulin-like growth factor 1 (IGF-1) in serum and tissues, to study how lifelong increases in GH and IGF-1 affect skeletal integrity. Additionally, we crossed the acid labile subunit (ALS) null (ALSKO) to the bGH mice to reduce serum IGF-1 levels. Our findings indicate sexually dimorphic effects of GH on cortical and trabecular bone. Male bGH mice showed enlarged cortical diameters, but with marrow cavity expansion and thin cortices as well as increased vascular porosity that were associated with reductions in diaphyseal strength and stiffness. In contrast, female bGH mice presented with significantly smaller-diameter diaphysis, with greater cortical bone thickness and with a slightly reduced tissue elastic modulus (by microindentation), ultimately resulting in overall stronger, stiffer bones. We found increases in C-terminal telopeptide of type 1 collagen and procollagen type 1 N propeptide in serum, independent of circulating IGF-1 levels, indicating increased bone remodeling with excess GH. Sexual dimorphism in response to excess GH was also observed in the trabecular bone compartment, particularly at the femur distal metaphysis. Female bGH mice preserved their trabecular architecture during aging, whereas trabecular bone volume in male bGH mice significantly reduced and was associated with thinning of the trabeculae. We conclude that pathological excess in GH results in sexually dimorphic changes in bone architecture and gains in bone mass that affect whole-bone mechanical properties, as well as sex-specific differences in bone material properties. © 2022 American Society for Bone and Mineral Research (ASBMR).

Direct Assessment of Rabbit Cortical Bone Basic Multicellular Unit Longitudinal Erosion Rate: A 4D Synchrotron-Based Approach.

Cortical bone remodeling is carried out by basic multicellular units (BMUs), which couple resorption to formation. Although fluorochrome labeling has facilitated study of BMU formative parameters since the 1960s, some resorptive parameters, including the longitudinal erosion rate (LER), have remained beyond reach of direct measurement. Indeed, our only insights into this spatiotemporal parameter of BMU behavior come from classical studies that indirectly inferred LER. Here, we demonstrate a 4D in vivo method to directly measure LER through in-line phase contrast synchrotron imaging. The tibias of rabbits (n = 15) dosed daily with parathyroid hormone were first imaged in vivo (synchrotron micro-CT; day 15) and then ex vivo 14 days later (conventional micro-CT; day 29). Mean LER assessed by landmarking the co-registered scans was 23.69 ± 1.73 µm/d. This novel approach holds great promise for the direct study of the spatiotemporal coordination of bone remodeling, its role in diseases such as osteoporosis, as well as related treatments. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Investigating the Microchannel Architectures Inside the Subchondral Bone in Relation to Estimated Hip Reaction Forces on the Human Femoral Head.

The interplay between articular cartilage (AC) and subchondral bone (SB) plays a pivotal role in cartilage homeostasis and functionality. As direct connective pathways between the two are poorly understood, we examined the location-dependent characteristics of the 3D microchannel network within the SB that connects the basal cartilage layer to the bone marrow (i.e. cartilage-bone marrow microchannel connectors; CMMC). 43 measuring points were defined on five human cadaveric femoral heads with no signs of osteoarthritis (OA) (age ≤ 60), and cartilage-bone cylinders with diameters of 2.00 mm were extracted for high-resolution scanning (n = 215). The micro-CT data were categorized into three groups (load-bearing region: LBR, n = 60; non-load-bearing region: NLBR, n = 60; and the peripheral rim: PR, n = 95) based on a gait analysis estimation of the joint reaction force (young, healthy cohort with no signs of OA). At the AC-SB interface, the number of CMMC in the LBR was 1.8 times and 2.2 times higher compared to the NLBR, and the PR, respectively. On the other hand, the median Feret size of the CMMC were smallest in the LBR (55.2 µm) and increased in the NLBR (73.5 µm; p = 0.043) and the PR (89.1 µm; p = 0.043). AC thickness was positively associated with SB thickness (Pearson's r = 0.48; p < 1e-13), CMMC number. (r = 0.46; p < 1e-11), and circularity index (r = 0.61; p < 1e-38). In conclusion, our data suggest that regional differences in the microchannel architecture of SB might reflect regional differences in loading.

Multiscale characterization of ovariectomized rat femur.

Estrogen deficiency activates bone resorbing cells (osteoclasts) and to a lesser extent bone forming cells (osteoblasts), resulting in a gap between resorption and formation that leads to a net loss of bone. These cell activities alter bone architecture and tissue composition. Thus, the objective of this study is to examine whether multiscale (10-2 to 10-7 m) characterization can provide more integrated information to understand the effects of estrogen deficiency on the fracture risk of bone. This is the first study to examine the effects of estrogen deficiency on multiscale characteristics of the same bone specimen. Sprague-Dawley female rats (6 months old) were obtained for a bilateral ovariectomy (OVX) or a sham operation (sham). Micro-computed tomography of rat femurs provided bone volumetric, mineral density, and morphological parameters. Dynamic mechanical analysis, static elastic and fracture mechanical testing, and nanoindentation were also performed using the same femur. As expected, the current findings indicate that OVX reduces bone quantity (mass and bone mineral density) and quality (morphology, and fracture displacement). Additionally, they demonstrated reductions in amount and heterogeneity of tissue mineral density (TMD) and viscoelastic properties. The current results validate that multiscale characterization for the same bone specimen can provide more comprehensive insights to understand how the bone components contributed to mechanical behavior at different scales.

CD11b promotes the differentiation of osteoclasts induced by RANKL through the spleen tyrosine kinase signalling pathway.

Macrophage surface antigen-1 (Mac-1, CD11b/CD18) has been implicated in the regulation of osteoclastogenesis. In the synovial tissues of patients with aseptic loosening after total hip replacement, CD11b was up-regulated, which indicated that CD11b is closely involved in osteolysis around the prosthesis. We found that CD11b, but not CD18, promoted osteoclast (OC) maturation. Here, we show CD11b up-regulated the levels of spleen tyrosine kinase (Syk), c-Fos and nuclear factor of activated T cells, cytoplasmic-1 (NFATc1), as well as the activity of extracellular-regulated kinase (Erk), and as a result, osteoclast precursors (OCPs) differentiated and became tartrate-resistant acid phosphatase (TRAP)-positive. In addition, increased tumour necrosis factor-α (TNF-α) induced by ultra-high molecular weight polyethylene (UHMWPE) particles up-regulated the level of CD11b. Taken together, these findings suggest that CD11b is a positive regulator of osteoclastogenesis and that it functions by activating the Syk signalling pathway, while CD18 does not have the same effect.

Physiological exercise loading suppresses post-traumatic osteoarthritis progression via an increase in bone morphogenetic proteins expression in an experimental rat knee model.

OBJECTIVE: To evaluate the dose-response relationship of exercise loading in the cartilage-subchondral bone (SB) unit in surgically-induced post-traumatic osteoarthritis (PTOA) of the knee. DESIGN: Destabilized medial meniscus (DMM) surgery was performed on the right knee of 12-week-old male Wistar rats, and sham surgery was performed on the contralateral knee. Four weeks after the surgery, the animals were subjected to moderate (12 m/min) or intense (21 m/min) treadmill exercises for 30 min/day, 5 days/week for 4 weeks. PTOA development in articular cartilage and SB was examined using histological and immunohistochemical analyses, micro-computed tomography (micro-CT) analysis, and biomechanical testing at 8 weeks after surgery. Gremlin-1 was injected to determine the role of bone morphogenetic protein (BMP) signaling on PTOA development following moderate exercise. RESULTS: Moderate exercise increased BMP-2, BMP-4, BMP-6, BMP receptor 2, pSmad-5, and inhibitor of DNA binding protein-1 expression in the superficial zone chondrocytes and suppressed cartilage degeneration, osteophyte growth, SB damage, and osteoclast-mediated SB resorption. However, intense exercise had little effect on BMP expression and even caused progression of these osteoarthritis (OA) changes. Gremlin-1 injection following moderate exercise caused progression of the PTOA development down to the level of the non-exercise DMM-operated knee. CONCLUSIONS: Exercise regulated cartilage-SB PTOA development in DMM-operated knees in a dose-dependent manner. Our findings shed light on the important role of BMP expression in superficial zone chondrocytes in attenuation of PTOA development following physiological exercise loading. Further studies to support a mechanism by which BMPs would be beneficial in preventing PTOA progression are warranted.

Exercise intervention increases expression of bone morphogenetic proteins and prevents the progression of cartilage-subchondral bone lesions in a post-traumatic rat knee model.

OBJECTIVE: This study aimed to determine whether treadmill walking (TW) prevents the progression of post-traumatic osteoarthritic changes in cartilage-subchondral bone unit, and whether the exercise timing changes the exercise efficacy in destabilized medial meniscus (DMM) rat knees. DESIGN: Twelve-week-old male Wistar rats underwent DMM surgery on their right knees and sham surgery on their left knees and were assigned to either the sedentary (n = 10) or walking (n = 24) groups. The rats in the walking group were subjected to TW from day 2 through 4 weeks, from 4 through 8 weeks, or from day 2 through 8 weeks (n = 8 per group). Osteoarthritic changes of cartilage and subchondral bone were assessed with micro-computed tomography, histology, and immunohistochemistry 8 weeks after surgery. RESULTS: TW prevented the progression of cartilage and subchondral bone lesions induced by the DMM, and increased bone morphogenetic protein (BMP)-2 and -6 expressions in superficial zone chondrocytes and bone-lining cells including osteoblasts. Furthermore, the TW-induced increase in BMPs varied with the exercise timing. Beginning TW 4 weeks after DMM surgery was the best option for increasing BMPs, coinciding with the most robust prevention of osteoarthritic changes. CONCLUSIONS: TW increased the expression of BMPs and prevented the progression of cartilage-subchondral bone lesions in rat knees with a DMM. Selective exercise timing may be a key factor in the development of an exercise regimen for preventing the progression of post-traumatic osteoarthritis (PTOA). Furthermore, exercise may have favorable effects even after the PTOA has been developed.

Biological reaction to polyethylene particles in a murine calvarial model is highly influenced by age.

Particle-induced osteolysis is driven by multiple factors including bone metabolism, inflammation, and age. The objective of this study was to determine the influence of age on polyethylene (PE) particle-induced osteolysis in a murine calvarial model comparing 2-month-old (young) versus 24-month-old (old) mice. After PE particle implantation, calvaria were assessed at days (D) 3, D7, D14, and D21 via chemoluminescent imaging for inflammation (L-012 probe). In addition micro-computed tomography (micro-CT) and histomorphometry end points addressed the bone reaction. Inflammation peaked at D7 in young mice and D14 in old mice. Using micro-CT, a nadir of mature bone was recorded at D7 for young mice, versus D21 for old mice. Besides, regenerating bone peaked at distinct timepoints: D7 for young mice versus D21 for old mice. In the young mice group, the histomorphometric findings correlated with micro-CT regenerating bone findings at D7, associated with ample osteoïd deposition. No osteoïd could be histologically quantified in the old mice group at D7. This study demonstrated that the biological reaction to polyethylene particles is highly influenced by age.