Svetlana V. Komarova

Mutations in the gene coding G6b-B, Mpig6b in mice, and MPIG6B in humans lead to myelofibrosis, progressive splenomegaly and osteosclerosis. Aberrant megakaryocyte function in bone marrow and spleen may affect bone cells through cell-cell contact or soluble factors. We aimed to assess the role of circulating factors produced by splenic cells in mediating osteosclerosis in Mpig6b ^−/− mice using splenectomy, a procedure commonly used in clinics. Splenectomy or sham surgery was performed on 32-week-old female Mpig6b ^−/− and littermate wildtype (WT) mice, when splenomegaly and long bone osteosclerosis were established. Twenty weeks later, the hematopoietic parameters and bone structure of the femur and lumbar vertebra were analyzed. Splenectomy significantly improved platelet count, but the larger platelet size persisted in Mpig6b ^−/− mice. The Mpig6b ^−/− femurs with established osteosclerosis were not affected by splenectomy. In contrast, splenectomy prevented the development of osteosclerosis in vertebrae of Mpig6b ^−/− mice. Significant vertebral bone loss and increased circulating TRACP 5b were demonstrated in splenectomized Mpig6b ^−/− mice compared to sham-operated and WT mice. Using quantitative plasma proteomics, we identified intercellular adhesion molecule 2 (ICAM2) as significantly increased in Mpig6b ^−/− mice compared to WT and reversed to normal levels after splenectomy. The interaction of membrane-bound ICAM2 and MAC1 was reported to be critical for osteoclast fusion. Our in vitro study revealed that soluble recombinant ICAM2 dose-dependently inhibited osteoclast fusion, suggesting that competitive binding of MAC1 with soluble ICAM2 prevented its interaction with membrane-bound ICAM2. Thus, we identified the soluble ICAM2 as a novel inhibitor of osteoclast fusion and mediator of osteosclerosis in Mpig6b ^−/− mice.

Background/Objectives: Osteoarthritis (OA) is partly driven by an imbalance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs). Collagen hydrolysates (CHs) are widely used for OA symptom management, with potential benefits attributed to bioactive peptides (BAPs) released during digestion. However, their direct effects on bone cells remain unclear. Methods: Two bovine-sourced CHs, CH-GL and CH-OPT, were digested in vitro and tested on primary murine OC and OB cultures. Osteoclastogenesis was evaluated mainly under standard RANKL conditions (50 ng/mL), with exploratory analysis under high RANKL conditions (100 ng/mL). OC cultures were exposed to CH digesta at 0.01, 0.05, 0.1, or 0.5 mg/mL, while OB cultures were exposed to 0.01 or 0.1 mg/mL. Results: Under standard RANKL conditions, CH-GL significantly reduced OC area by over 50% across all tested concentrations, while OC number was not significantly altered. This coincided with decreased expression of selected osteoclastogenic markers, including Nfatc1, Ctsk, Dc-stamp, and Oscar. In OB cultures, CH-GL increased Runx2, Osterix, and Alp expression at 0.1 mg/mL and reduced Mmp9 expression at both tested doses. It also modestly increased mineralization-related signal intensity and collagen-associated matrix area. For CH-OPT, selected reductions in Dc-stamp and Oscar expression were observed, while OC number, OC area, and most OB-related outcomes were not significantly altered. Conclusions: CH digesta modulated selected OC- and OB-related outcomes in primary murine cells. These preliminary in vitro findings support further investigation of CH-derived bioactives in models that more closely reproduce OA-associated joint pathology.

Osteogenesis imperfecta (OI) is a rare bone fragility disorder. Previously, in a severe OI mouse model ( Col1a1 ^Jrt/+ ), a sex- and age-dependent metabolic phenotype was observed, correlating with elevated levels of the bone-derived hormone osteocalcin (OCN). This hormone is known to play a crucial role in managing energy metabolism, including glucose regulation and fat mass. In fact, upon high-fat diet (HFD) exposure, OI mice developed a metabolic syndrome linked to sex and OCN. To assess OCN’s role in OI, Col1a1 ^Jrt/+ mice were crossed with OCN-deficient mice ( Bglap ). Under regular chow and HFD conditions, both OCN-dependent and OCN-independent metabolic alterations were identified. OCN-dependent processes were adipose tissue, liver, and insulin metabolism in a sex-, age-, and diet-dependent manner. OCN-independent traits included the pancreas in juvenile mice, HFD-induced pancreatic insulin levels and glucose intolerance, besides overall growth, fertility, and bone phenotype. Notably, increased juvenile energy expenditure was OCN-independent, while HFD-induced changes were OCN-driven. These findings demonstrate OCN’s role in shaping the metabolic phenotype while revealing distinct OCN-independent effects, emphasizing the complex genetic regulation of metabolism in OI.

Disuse-induced bone loss is a common consequence of spaceflight and prolonged bed rest. Intraosseous blood vessel volume and number are decreased in rodents after sciatic nerve resection, and femoral and tibial perfusion and blood flow to the femoral shaft and marrow are reduced after hindlimb unloading. However, it is unclear if alterations in the flow of blood contribute to botulinum toxin (BTX)-induced bone loss. The objective of this study was to assess patterns of tibial bone loss and alterations in blood flow in murine hindlimbs following BTX injection. We hypothesize that flow of blood to the affected hindlimb will diminish along with bone mass and structure. Skeletally mature C57Bl/6J female were injected with BTX (n = 15) or vehicle (n = 14). Paralysis was confirmed using digit abduction, wire hang tests, and activity analysis. In vivo microCT and ex vivo synchrotron tomography were used to assess bone mass, microstructure, (re)modeling, as well as vascular and lacunar porosity. Blood flow in the hindlimbs and cardiac structure/function was monitored by echocardiography. After 3 wk, BTX-injected tibiae had 16% lower cortical thickness and 66% lower trabecular bone volume fraction compared to baseline. MicroCT-based timelapse morphometry showed bone loss was predominantly at endocortical surfaces. Bone loss in the contralateral limb was coincident with reduced rearing capability of BTX-injected mice compared to vehicle controls. Bony vascular canal thickness and surface area were reduced, but there was no change in lacunar properties due to BTX. In vivo ultrasound demonstrated increased velocity time integral for blood flow due to BTX injection in femoral and popliteal but not in saphenous arteries. Thus, BTX led to significant bone loss in hindlimbs, while increasing blood velocity in the femoral popliteal arteries and decreasing vascular porosity. The vascular response to BTX differs from what has been observed in other hindlimb unloading models.

Bone mineralization is a complex process tightly regulated by both biological factors such as collagen maturation as well as physicochemical factors such as pH. A previous model of biological mineralization captured the biological regulation of bone mineralization dynamics, but not the impact of bone microenvironment such as ion availabilities which may be altered in hypo or hyperphosphatemia. To build an integrated model of bone mineralization, we utilized two previously developed models which addressed a distinct aspect of bone mineralization. The first model described the processes of the extracellular matrix formation and maturation, inhibitor and nucleator formation and removal and their combined action in regulating bone mineralization. The second model simulated the bone interstitial fluid (BIF) permissive to precipitation of hydroxyapatite and described the physicochemical process of hydroxyapatite precipitation. The resulting bone mineralization model accounts for biological and physicochemical aspects of the process. The integrated model was analyzed for the impact of physicochemical factors (pH, levels of calcium and phosphate) on the mineralization dynamics. Model predictions were compared to experimental findings using two outcomes characterizing mineralization dynamics: mineralization delay that corresponds to histomorphometry measures of osteoid volume or thickness, and mineralization degree that corresponds to bone mineral density distribution. We identified the limitation of the previously developed model in predicting the mineralization delay observed in the situations of hypophosphatemia and hypocalcemia and proposed a model adaptation that predicts these outcomes. The resulting mathematical model can be used for in silico testing of hypotheses regarding the role of different physicochemical, molecular, or cellular factors in causing a specific disruption in mineralization dynamics.