Anna Fahlgren

Tartrate-resistant acid phosphatase (TRAP/ACP5), primarily known as an osteoclast marker, has emerged as a critical regulator of skeletal integrity, regulating sex-specific bone growth, and bone’s response to mechanical load in young adult male mice. In this study, we investigated the sex-specific roles of TRAP in bone structure and response to mechanical stimuli in old (19-month-old) wild-type (WT) and TRAP-deficient (TRAP ^-/- ) mice using micro-computed tomography, serum bone turnover markers, in vivo axial mechanical loading, and in vitro mechanotransduction assays. Our findings revealed that TRAP ^-/- mice of both sexes maintained shorter tibiae than WT mice independent of sex. Notably, male, but not female, TRAP ^-/- mice have increased trabecular bone volume fraction and cortical bone area compared to WT, indicative of disrupted bone remodelling processes in male mice. Interestingly, TRAP-deficiency substantially impaired the anabolic bone response to mechanical loading, affecting both trabecular and cortical compartments in both sexes, indicating that when challenged, TRAP is important for bone formation also in female mice. Mechanical stimulation in vitro of hematopoietic progenitor cells from WT and TRAP ^-/- mice revealed that the increased ATP-release in response to mechanical stimulation was only disrupted in male mice, while mechanically induced increase in osteoclast formation was inhibited in TRAP ^-/- mice of both sexes. These results highlight the importance of TRAP in maintaining trabecular architecture and cortical bone in male mice and underscore its critical function in mediating adaptive responses to mechanical loading of both sexes, during aging. Future investigations should focus on elucidation of TRAP-dependent pathways as potential therapeutic targets to counteract age-related deficits in bone adaptation and remodelling.

Low‐intensity loading maintains or increases bone mass, whereas lack of mechanical loading and high‐intensity loading decreases bone mass, possibly via the release of extracellular vesicles by mechanosensitive bone cells. How different loading intensities alter the biological effect of these vesicles is not fully understood. Dynamic fluid shear stress at low intensity (0.7 ± 0.3 Pa, 5 Hz) or high intensity (2.9 ± 0.2 Pa, 1 Hz) was used on mouse hematopoietic progenitor cells for 2 min in the presence or absence of chemical compounds that inhibit release or biogenesis of extracellular vesicles. We used a Receptor activator of nuclear factor kappa‐Β ligand‐induced osteoclastogenesis assay to evaluate the biological effect of different fractions of extracellular vesicles obtained through centrifugation of medium from hematopoietic stem cells. Osteoclast formation was reduced by microvesicles (10 000× g ) obtained after low‐intensity loading and induced by exosomes (100 000× g ) obtained after high‐intensity loading. These osteoclast‐modulating effects could be diminished or eliminated by depletion of extracellular vesicles from the conditioned medium, inhibition of general extracellular vesicle release, inhibition of microvesicle biogenesis (low intensity), inhibition of ESCRT‐independent exosome biogenesis (high intensity), as well as by inhibition of dynamin‐dependent vesicle uptake in osteoclast progenitor cells. Taken together, the intensity of mechanical loading affects the release of extracellular vesicles and change their osteoclast‐modulating effect.

Background: Bacillus Calmette-Guérin (BCG) immunotherapy is the standard-of-care adjuvant therapy for non-muscle-invasive bladder cancer in patients at considerable risk of disease recurrence. Although its exact mechanism of action is unknown, BCG significantly reduces this risk in responding patients but is mainly associated with toxic side-effects in those facing treatment resistance. Methods that allow the identification of BCG responders are, therefore, urgently needed. Methods: Fluorescently labelled UM-UC-3 cells and dissociated patient tumor samples were used to establish zebrafish tumor xenograft (ZTX) models. Changes in the relative primary tumor size and cell dissemination to the tail were evaluated via fluorescence microscopy at three days post-implantation. The data were compared to the treatment outcomes of the corresponding patients. Toxicity was evaluated based on gross morphological evaluation of the treated zebrafish larvae. Results: BCG-induced toxicity was avoided by removing the water-soluble fraction of the BCG formulation prior to use. BCG treatment via co-injection with the tumor cells resulted in significant and dose-dependent primary tumor size regression. Heat-inactivation of BCG decreased this effect, while intravenous BCG injections were ineffective. ZTX models were successfully established for six of six patients based on TUR-B biopsies. In two of these models, significant tumor regression was observed, which, in both cases, corresponded to the treatment response in the patients. Conclusions: The observed BCG-related anti-tumor effect indicates that ZTX models might predict the BCG response and thereby improve treatment planning. More experiments and clinical studies are needed, however, to elucidate the BCG mechanism and estimate the predictive value.

The alternative sigma factor RpoN (σ ⁵⁴ ), which is widely distributed in eubacteria, has been implicated in controlling gene expression of importance for numerous functions including virulence. Proper responses to host environments are crucial for bacteria to establish infection, and regulatory mechanisms involved are therefore of high interest for development of future therapeutics. Little is known about the function of RpoN in the intestinal pathogen Y. pseudotuberculosis , and we therefore investigated its regulatory role in this pathogen. This regulator was indeed found to be critical for establishment of infection in mice, likely involving its requirement for motility and biofilm formation. The RpoN regulon involved both activating and suppressive effects on gene expression which could be confirmed with mutagenesis of identified binding sites. This is the first study of its kind of RpoN in Y. pseudotuberculosis , revealing complex regulation of gene expression involving both productive and silent effects of its binding to DNA, providing important information about RpoN regulation in enterobacteria.

Background and purpose — Insufficient initial fixation or early micromotion of an implant is associated with a thin layer of fibrous tissue at the peri-implant interface. It is unknown if bone loss is induced by the fibrous tissue interface acting as an active biological membrane, or as a membrane that will produce supraphysiologic fluid flow conditions during gait, which activates the mechanosensitive osteocytes to mediate osteoclast differentiation. We investigated whether mechanically induced osteolysis is dependent on the fibrous tissue interface as a biologically active scaffold, or if it merely acts as a conduit for fluid flow, affecting the mechanosensitive osteocytes in the peri-prosthetic bone. Methods — Using a rat model of mechanically instability-induced aseptic loosening, we assessed whether the induction of osteoclast differentiation was dependent on the presence of a peri-implant fibrous interface. We analyzed the amount of osteoclast differentiation, osteocyte apoptosis, pro-resorptive cytokine expression and bone loss using immunohistochemistry, mRNA expression and micro-CT. Results — Osteoclast differentiation and bone loss were induced by mechanical instability but were not affected by the presence of the fibrous tissue membrane or associated with osteocyte apoptosis. There was no increased mRNA expression of any of the cytokines in the fibrous tissue membrane compared with the peri-implant bone. Interpretation — Our data show that the fibrous tissue membrane in the interface plays a minor role in inducing bone loss. This indicates that the peri-implant bone adjacent to loose bone implants might play an important role for osteoclast differentiation.

Cyclin‐dependent kinase 8 (CDK8) is a mediator complex‐associated transcriptional regulator that acts depending on context and cell type. While primarily under investigation as potential cancer therapeutics, some inhibitors of CDK8—and its paralog CDK19—have been reported to affect the osteoblast lineage and bone formation. This study investigated the effects of two selective CDK8/19 inhibitors on osteoclastogenesis and osteoblasts in vitro, and further evaluated how local treatment with a CDK8/19 inhibitor affects cancellous bone healing in rats. CDK8/19 inhibitors did not alter the proliferation of neither mouse bone marrow–derived macrophages (BMMs) nor primary mouse osteoblasts. Receptor activator of nuclear factor κΒ (NF‐κB) ligand (RANKL)‐induced osteoclastogenesis from mouse BMMs was suppressed markedly by inhibition of CDK8/19, concomitant with reduced tartrate‐resistant acid phosphatase (TRAP) activity and C‐terminal telopeptide of type I collagen levels. This was accompanied by downregulation of PU.1, RANK, NF‐κB, nuclear factor of activated T‐cells 1 (NFATc1), dendritic cell‐specific transmembrane protein (DC‐STAMP), TRAP, and cathepsin K in RANKL‐stimulated BMMs. Downregulating RANK and its downstream signaling in osteoclast precursors enforce CDK8/19 inhibitors as anticatabolic agents to impede excessive osteoclastogenesis. In mouse primary osteoblasts, CDK8/19 inhibition did not affect differentiation but enhanced osteoblast mineralization by promoting alkaline phosphatase activity and downregulating osteopontin, a negative regulator of mineralization. In rat tibiae, a CDK8/19 inhibitor administered locally promoted cancellous bone regeneration. Our data indicate that inhibitors of CDK8/19 have the potential to develop into therapeutics to restrict osteolysis and enhance bone regeneration.

Mechanical instability of bone implants stimulate osteoclast differentiation and peri‐implant bone loss, leading to prosthetic loosening. It is unclear which cells at the periprosthetic interface transduce mechanical signals into a biochemical response, and subsequently facilitate bone loss. We hypothesized that mechanical overloading of hematopoietic bone marrow progenitor cells, which are located near to the inserted bone implants, stimulates the release of osteoclast‐inducing soluble factors. Using a novel in vitro model to apply mechanical overloading, we found that hematopoietic progenitor cells released adenosine triphosphate (ATP) after only 2 min of mechanical loading. The released ATP interacts with its specific receptor P2X7 to stimulate the release of unknown soluble factors that inhibit (physiological loading) or promote (supraphysiological loading) the differentiation of multinucleated osteoclasts derived from bone marrow cultures. Inhibition of ATP‐receptor P2X7 by Brilliant Blue G completely abolished the overloading‐induced stimulation of osteoclast formation. Likewise, stimulation of P2X7 receptor on hematopoietic cells by BzATP enhanced the release of osteoclastogenesis‐stimulating signaling molecules to a similar extent as supraphysiological loading. Supraphysiological loading affected neither gene expression of inflammatory markers involved in aseptic implant loosening (e.g., interleukin‐1β (IL‐1β), IL‐6, tumor necrosis factor‐α, and PTGES2) nor expression of the osteoclast modulators receptor activator of nuclear factor κ‐Β ligand and osteoprotegerin. Our findings suggest that murine hematopoietic progenitor cells are a potential key player in local mechanical loading‐induced bone implant loosening via the ATP/P2X7‐axis. Our approach identifies potential therapeutic targets to prevent prosthetic loosening.

We aimed to develop an in vitro model for bone implant loosening, allowing analysis of biophysical and biological parameters contributing to mechanical instability‐induced osteoclast differentiation and peri‐implant bone loss. MLO‐Y4‐osteocytes were mechanically stimulated for 1 h by fluid shear stress using regimes simulating: (i) supraphysiological loading in the peri‐prosthetic interface (2.9 ± 2.9 Pa, 1 Hz, square wave); (ii) physiologic loading in the cortical bone (0.7 ± 0.7 Pa, 5 Hz, sinusoidal wave); and (iii) stress shielding. Cellular morphological parameters, membrane‐bound RANKL expression, gene expression influencing osteoclast differentiation, nitric oxide release and caspase 3/7‐activity were determined. Either Mouse bone marrow cells were cultured on top of loaded osteocytes or osteocyte‐conditioned medium was added to bone marrow cells. Osteoclast differentiation was assessed after 6 days. We found that osteocytes subjected to supraphysiological loading showed similar morphology and caspase 3/7‐activity compared to simulated physiological loading or stress shielding. Supraphysiological stimulation of osteocytes enhanced osteoclast differentiation by 1.9‐fold compared to physiological loading when cell‐to‐cell contact was permitted. In addition, it enhanced the number of osteoclasts using conditioned medium by 1.7‐fold, membrane‐bound RANKL by 3.3‐fold, and nitric oxide production by 3.2‐fold. The stimulatory effect of supraphysiological loading on membrane‐bound RANKL and nitric oxide production was higher than that achieved by stress shielding. In conclusion, the in vitro model developed recapitulated the catabolic biological situation in the peri‐prosthetic interface during instability that is associated with osteoclast differentiation and enhanced RANKL expression. The model thus provides a platform for pre‐clinical testing of pharmacological interventions with potential to stop instability‐induced bone implant loosening. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1425–1434, 2018.

Currently, there are no medications available to treat aseptic loosening of orthopedic implants. Using osteoprotegerin fusion protein (OPG‐Fc), we previously blocked instability‐induced osteoclast differentiation and peri‐prosthetic osteolysis. Wnt/β‐catenin signaling, which regulates OPG secretion from osteoblasts, also modulates the bone tissue response to mechanical loading. We hypothesized that activating Wnt/β‐catenin signaling by inhibiting glycogen synthase kinase‐3β (GSK‐3β) would reduce instability‐induced bone loss through regulation of both osteoblast and osteoclast differentiation. We examined effects of GSK‐3β inhibition on regulation of RANKL and OPG in a rat model of mechanical instability‐induced peri‐implant osteolysis. The rats were treated daily with a GSK‐3β inhibitor, AR28 (20 mg/kg bw), for up to 5 days. Bone tissue and blood serum were assessed by qRT‐PCR, immunohistochemistry, and ELISA on days 3 and 5, and by micro‐CT on day 5. After 3 days of treatment with AR28, mRNA levels of β‐catenin, Runx2, Osterix, Col1α1, and ALP were increased leading to higher osteoblast numbers compared to vehicle‐treated animals. BMP‐2 and Wnt16 mRNA levels were downregulated by mechanical instability and this was rescued by GSK‐3β inhibition. Osteoclast numbers were decreased significantly after 3 days of GSK‐3β inhibition, which correlated with enhanced OPG mRNA expression. This was accompanied by decreased serum levels of TRAP5b on days 3 and 5. Treatment with AR28 upregulated osteoblast differentiation, while osteoclastogenesis was blunted, leading to increased bone mass by day 5. These data suggest that GSK‐3β inactivation suppresses osteolysis through regulating both osteoblast and osteoclast differentiation in a rat model of instability‐induced osteolysis.