Therapeutic avenues in bone repair: Harnessing an anabolic osteopeptide, PEPITEM, to boost bone growth and prevent bone loss.

The existing suite of therapies for bone diseases largely act to prevent further bone loss but fail to stimulate healthy bone formation and repair. We describe an endogenous osteopeptide (PEPITEM) with anabolic osteogenic activity, regulating bone remodeling in health and disease. PEPITEM acts directly on osteoblasts through NCAM-1 signaling to promote their maturation and formation of new bone, leading to enhanced trabecular bone growth and strength. Simultaneously, PEPITEM stimulates an inhibitory paracrine loop: promoting osteoblast release of the decoy receptor osteoprotegerin, which sequesters RANKL, thereby limiting osteoclast activity and bone resorption. In disease models, PEPITEM therapy halts osteoporosis-induced bone loss and arthritis-induced bone damage in mice and stimulates new bone formation in osteoblasts derived from patient samples. Thus, PEPITEM offers an alternative therapeutic option in the management of diseases with excessive bone loss, promoting an endogenous anabolic pathway to induce bone remodeling and redress the imbalance in bone turnover.

Preclinical characterization of ISB 1342, a CD38 × CD3 T-cell engager for relapsed/refractory multiple myeloma.

Although treatment of multiple myeloma (MM) with daratumumab significantly extends the patient's lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/r MM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high-affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38, including those with a lower sensitivity to daratumumab. In a killing assay where multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity toward MM cells compared with daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in 2 therapeutic mouse models, unlike daratumumab. Finally, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/r MM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently being developed in a phase 1 clinical study.

In vitro and in vivo effects of zoledronic acid on senescence and senescence-associated secretory phenotype markers.

In addition to reducing fracture risk, zoledronic acid has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronic acid could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronic acid killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronic acid or vehicle for 8 weeks, zoledronic acid significantly reduced circulating SASP factors, including CCL7, IL-1ß, TNFRSF1A, and TGFß1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronic acid demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronic acid, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronic acid significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronic acid has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo. These data point to the need for additional studies testing zoledronic acid and/or other bisphosphonate derivatives for senotherapeutic efficacy.

In vitro and in vivo effects of zoledronate on senescence and senescence-associated secretory phenotype markers.

In addition to reducing fracture risk, zoledronate has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronate could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronate killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronate or vehicle for 8 weeks, zoledronate significantly reduced circulating SASP factors, including CCL7, IL-1ß, TNFRSF1A, and TGFß1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronate demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronate, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronate significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronate has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo . These data point to the need for additional studies testing zoledronate and/or other bisphosphonate derivatives for senotherapeutic efficacy.

Biomimetic hydrogel blanket for conserving and recovering intrinsic cell properties.

BACKGROUND: Cells in the human body experience different growth environments and conditions, such as compressive pressure and oxygen concentrations, depending on the type and location of the tissue. Thus, a culture device that emulates the environment inside the body is required to study cells outside the body. METHODS: A blanket-type cell culture device (Direct Contact Pressing: DCP) was fabricated with an alginate-based hydrogel. Changes in cell morphology due to DCP pressure were observed using a phase contrast microscope. The changes in the oxygen permeability and pressure according to the hydrogel concentration of DCP were analyzed. To compare the effects of DCP with normal or artificial hypoxic cultures, cells were divided based on the culture technique: normal culture, DCP culture device, and artificial hypoxic environment. Changes in phenotype, genes, and glycosaminoglycan amounts according to each environment were evaluated. Based on this, the mechanism of each culture environment on the intrinsic properties of conserving chondrocytes was suggested. RESULTS: Chondrocytes live under pressure from the surrounding collagen tissue and experience a hypoxic environment because collagen inhibits oxygen permeability. By culturing the chondrocytes in a DCP environment, the capability of DCP to produce a low-oxygen and physical pressure environment was verified. When human primary chondrocytes, which require pressure and a low-oxygen environment during culture to maintain their innate properties, were cultured using the hydrogel blanket, the original shapes and properties of the chondrocytes were maintained. The intrinsic properties could be recovered even in aged cells that had lost their original cell properties. CONCLUSIONS: A DCP culture method using a biomimetic hydrogel blanket provides cells with an adjustable physical pressure and a low-oxygen environment. Through this technique, we could maintain the original cellular phenotypes and intrinsic properties of human primary chondrocytes. The results of this study can be applied to other cells that require special pressure and oxygen concentration control to maintain their intrinsic properties. Additionally, this technique has the potential to be applied to the re-differentiation of cells that have lost their original properties.

Metabolic profiling of prostate cancer in skeletal microenvironments identifies G6PD as a key mediator of growth and survival.

The spread of cancer to bone is invariably fatal, with complex cross-talk between tumor cells and the bone microenvironment responsible for driving disease progression. By combining in silico analysis of patient datasets with metabolomic profiling of prostate cancer cells cultured with bone cells, we demonstrate the changing energy requirements of prostate cancer cells in the bone microenvironment, identifying the pentose phosphate pathway (PPP) as elevated in prostate cancer bone metastasis, with increased expression of the PPP rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD) associated with a reduction in progression-free survival. Genetic and pharmacologic manipulation demonstrates that G6PD inhibition reduces prostate cancer growth and migration, associated with changes in cellular redox state and increased chemosensitivity. Genetic blockade of G6PD in vivo results in reduction of tumor growth within bone. In summary, we demonstrate the metabolic plasticity of prostate cancer cells in the bone microenvironment, identifying the PPP and G6PD as metabolic targets for the treatment of prostate cancer bone metastasis.

The antidiabetic drug metformin acts on the bone microenvironment to promote myeloma cell adhesion to preosteoblasts and increase myeloma tumour burden in vivo.

Multiple myeloma is a haematological malignancy that is dependent upon interactions within the bone microenvironment to drive tumour growth and osteolytic bone disease. Metformin is an anti-diabetic drug that has attracted attention due to its direct antitumor effects, including anti-myeloma properties. However, the impact of the bone microenvironment on the response to metformin in myeloma is unknown. We have employed in vitro and in vivo models to dissect out the direct effects of metformin in bone and the subsequent indirect myeloma response. We demonstrate how metformin treatment of preosteoblasts increases myeloma cell attachment. Metformin-treated preosteoblasts increased osteopontin (OPN) expression that upon silencing, reduced subsequent myeloma cell adherence. Proliferation markers were reduced in myeloma cells cocultured with metformin-treated preosteoblasts. In vivo, mice were treated with metformin for 4 weeks prior to inoculation of 5TGM1 myeloma cells. Metformin-pretreated mice had an increase in tumour burden, associated with an increase in osteolytic bone lesions and elevated OPN expression in the bone marrow. Collectively, we show that metformin increases OPN expression in preosteoblasts, increasing myeloma cell adherence. In vivo, this translates to an unexpected indirect pro-tumourigenic effect of metformin, highlighting the importance of the interdependence between myeloma cells and cells of the bone microenvironment.

On/off switchable physical stimuli regulate the future direction of adherent cellular fate.

The utilization of cell-manipulating techniques reveals information about biological behaviors suited to address a wide range of questions in the field of life sciences. Here, we introduced an on/off switchable physical stimuli technique that offers precise stimuli for reversible cell patterning to allow regulation of the future direction of adherent cellular behavior by leveraging enzymatically degradable alginate hydrogels with defined chemistry and topography. As a proof of concept, targeted muscle cells adherent to TCP exhibited a reshaped structure when the hydrogel-based physical stimuli were applied. This simple tool offers easy manipulation of adherent cells to reshape their morphology and to influence future direction depending on the characteristics of the hydrogel without limitations of time and space. The findings from this study are broadly applicable to investigations into the relationships between cells and physiological extracellular matrix environments as well as has potential to open new horizons for regenerative medicine with manipulated cells.

A black heart: Aortic valve ochronosis secondary to alkaptonuria causing aortic stenosis.

Alkaptonuria is a rare autosomal recessive genetic disorder where an accumulation of homogentisic acid in the tissues leads to ochronosis-a pathological dark pigmentation. It can affect various tissues and the weight bearing joints of the body, leading to degenerative arthropathy. On the rare occasion, it causes cardiac manifestations. We describe a case of aortic valve stenosis due to ochronosis secondary to alkaptonuria requiring aortic valve replacement.

Adiponectin signalling in bone homeostasis, with age and in disease.

Adiponectin is the most abundant circulating adipokine and is primarily involved in glucose metabolism and insulin resistance. Within the bone, osteoblasts and osteoclasts express the adiponectin receptors, however, there are conflicting reports on the effects of adiponectin on bone formation and turnover. Many studies have shown a pro-osteogenic role for adiponectin in in vivo murine models and in vitro: with increased osteoblast differentiation and activity, alongside lower levels of osteoclastogenesis. However, human studies often demonstrate an inverse relationship between adiponectin concentration and bone activity. Moreover, the presence of multiple isoforms of adiponectin and multiple receptor subtypes has the potential to lead to more complex signalling and functional consequences. As such, we still do not fully understand the importance of the adiponectin signalling pathway in regulating bone homeostasis and repair in health, with age and in disease. In this review, we explore our current understanding of adiponectin bioactivity in the bone; the significance of its different isoforms; and how adiponectin biology is altered in disease. Ultimately, furthering our understanding of adiponectin regulation of bone biology is key to developing pharmacological and non-pharmacological (lifestyle) interventions that target adiponectin signalling to boost bone growth and repair in healthy ageing, following injury or in disease.

Biodegradable Magnesium Alloys Promote Angio-Osteogenesis to Enhance Bone Repair.

Biodegradable metallic materials represent a potential step-change technology that may revolutionize the treatment of broken bones. Implants made with biodegradable metals are significantly stronger than their polymer counterparts and fully biodegradable in vivo, removing the need for secondary surgery or long-term complications. Here, it is shown how clinically approved Mg alloy promotes improved bone repair using an integrated state of the art fetal mouse metatarsal assay coupled with in vivo preclinical studies, second harmonic generation, secretome array analysis, perfusion bioreactor, and high-resolution 3D confocal imaging of vasculature within skeletal tissue, to reveal a vascular-mediated pro-osteogenic mechanism controlling enhanced tissue regeneration. The optimized mechanical properties and corrosion rate of the Mg alloy lead to a controlled release of metallic Mg, Ca, and Zn ions at a rate that facilitates both angiogenesis and coupled osteogenesis for better bone healing, without causing adverse effects at the implantation site. The findings from this study support ongoing development and refinement of biodegradable metal systems to act as crucial portal technologies with significant potential to improve many clinical applications.

SIRT1 directly activates autophagy in human chondrocytes.

Osteoarthritis (OA) is the most common form of arthritis worldwide with no effective treatment. Ageing is the primary risk factor for OA. We sought to investigate if there is a distinct and functional convergence of ageing-related mechanisms SIRT1 and autophagy in chondrocytes. Our results show that, levels of SIRT1 are decreased in human normal aged and OA cartilage compared with young cartilage. Moreover, silencing SIRT1 in chondrocytes lead to decreased expression of chondrogenic markers but did not alter the expression of catabolic proteases. In contrast, activation of SIRT1 increased autophagy in chondrocytes by the deacetylation of lysine residues on crucial autophagy proteins (Beclin1, ATG5, ATG7, LC3). This activation was shown to be mTOR/ULK1 independent. Our results indicate that maintenance of autophagy in chondrocytes by SIRT1 is essential for preserving cartilage integrity throughout life and therefore is a target for drug intervention to protect against OA.

Modeling the Human Bone-Tumor Niche: Reducing and Replacing the Need for Animal Data.

Bone is the most common site for cancer metastasis. Understanding the interactions within the complex, heterogeneous bone-tumor microenvironment is essential for the development of new therapeutics. Various animal models of tumor-induced bone disease are routinely used to provide valuable information on the relationship between cancer cells and the skeleton. However, new model systems exist that offer an alternative approach to the use of animals and might more accurately reveal the cellular interactions occurring within the human bone-tumor niche. This review highlights replacement models that mimic the bone microenvironment and where cancer metastases and tumor growth might be assessed alongside bone turnover. Such culture models include the use of calcified regions of animal tissue and scaffolds made from bone mineral hydroxyapatite, synthetic polymers that can be manipulated during manufacture to create structures resembling trabecular bone surfaces, gel composites that can be modified for stiffness and porosity to resemble conditions in the tumor-bone microenvironment. Possibly the most accurate model system involves the use of fresh human bone samples, which can be cultured ex vivo in the presence of human tumor cells and demonstrate similar cancer cell-bone cell interactions as described in vivo. In addition, the use of mathematical modeling and computational biology approaches provide an alternative to preliminary animal testing. The use of such models offers the capacity to mimic significant elements of the human bone-tumor environment, and complement, refine, or replace the use of preclinical models. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Opportunities and Challenges in Functional Genomics Research in Osteoporosis: Report From a Workshop Held by the Causes Working Group of the Osteoporosis and Bone Research Academy of the Royal Osteoporosis Society on October 5th 2020.

The discovery that sclerostin is the defective protein underlying the rare heritable bone mass disorder, sclerosteosis, ultimately led to development of anti-sclerostin antibodies as a new treatment for osteoporosis. In the era of large scale GWAS, many additional genetic signals associated with bone mass and related traits have since been reported. However, how best to interrogate these signals in order to identify the underlying gene responsible for these genetic associations, a prerequisite for identifying drug targets for further treatments, remains a challenge. The resources available for supporting functional genomics research continues to expand, exemplified by "multi-omics" database resources, with improved availability of datasets derived from bone tissues. These databases provide information about potential molecular mediators such as mRNA expression, protein expression, and DNA methylation levels, which can be interrogated to map genetic signals to specific genes based on identification of causal pathways between the genetic signal and the phenotype being studied. Functional evaluation of potential causative genes has been facilitated by characterization of the "osteocyte signature", by broad phenotyping of knockout mice with deletions of over 7,000 genes, in which more detailed skeletal phenotyping is currently being undertaken, and by development of zebrafish as a highly efficient additional in vivo model for functional studies of the skeleton. Looking to the future, this expanding repertoire of tools offers the hope of accurately defining the major genetic signals which contribute to osteoporosis. This may in turn lead to the identification of additional therapeutic targets, and ultimately new treatments for osteoporosis.

How to do a simplified Dacron sleeve technique of aortic anastomosis.

Step-by-step diagram demonstrating technique of Dacron sleeve anastomosis for thoracic aorta.

Multiple myeloma increases nerve growth factor and other pain-related markers through interactions with the bone microenvironment.

Interactions between multiple myeloma (MM) and bone marrow (BM) are well documented to support tumour growth, yet the cellular mechanisms underlying pain in MM are poorly understood. We have used in vivo murine models of MM to show significant induction of nerve growth factor (NGF) by the tumour-bearing bone microenvironment, alongside other known pain-related characteristics such as spinal glial cell activation and reduced locomotion. NGF was not expressed by MM cells, yet bone stromal cells such as osteoblasts expressed and upregulated NGF when cultured with MM cells, or MM-related factors such as TNF-α. Adiponectin is a known MM-suppressive BM-derived factor, and we show that TNF-α-mediated NGF induction is suppressed by adiponectin-directed therapeutics such as AdipoRON and L-4F, as well as NF-κB signalling inhibitor BMS-345541. Our study reveals a further mechanism by which cellular interactions within the tumour-bone microenvironment contribute to disease, by promoting pain-related properties, and suggests a novel direction for analgesic development.

Transcriptomic profiling of the myeloma bone-lining niche reveals BMP signalling inhibition to improve bone disease.

Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis causing skeletal damage and pain. Mechanisms underlying myeloma-induced bone destruction are poorly understood and current therapies do not restore lost bone mass. Using transcriptomic profiling of isolated bone lining cell subtypes from a murine myeloma model, we find that bone morphogenetic protein (BMP) signalling is upregulated in stromal progenitor cells. BMP signalling has not previously been reported to be dysregulated in myeloma bone disease. Inhibition of BMP signalling in vivo using either a small molecule BMP receptor antagonist or a solubilized BMPR1a-FC receptor ligand trap prevents trabecular and cortical bone volume loss caused by myeloma, without increasing tumour burden. BMP inhibition directly reduces osteoclastogenesis, increases osteoblasts and bone formation, and suppresses bone marrow sclerostin levels. In summary we describe a novel role for the BMP pathway in myeloma-induced bone disease that can be therapeutically targeted.

Small Animal Video Tracking for Activity and Path Analysis Using a Novel Open-Source Multi-Platform Application (AnimApp).

Experimental biological model system outcomes such as altered animal movement capability or behaviour are difficult to quantify manually. Existing automatic movement tracking devices can be expensive and imposing upon the typical environment of the animal model. We have developed a novel multiplatform, free-to-use open-source application based on OpenCV, called AnimApp. Our results show that AnimApp can reliably and reproducibly track movement of small animals such as rodents or insects, and quantify parameters of action including distance and speed in order to detect activity changes arising from handling, environment enrichment, or temperature alteration. This system offers an accurate and reproducible experimental approach with potential for simple, fast and flexible analysis of movement and behaviour in a wide range of model systems.

Spermidine restores dysregulated autophagy and polyamine synthesis in aged and osteoarthritic chondrocytes via EP300.

Ageing is the primary risk factor for osteoarthritis (OA). A decline in the ageing-associated process of autophagy is suggested as a potential contributor to OA development. Polyamines such as spermidine decrease during ageing, contributing to impaired autophagy and reduced cellular function. However, the role of polyamines and their effect on the regulatory mechanism governing autophagy in aged and arthritic cartilage tissue has not been established. Elucidating if polyamine regulation of autophagy is impaired during ageing and OA in chondrocytes may lead to improved treatment approaches to protect against cartilage degradation. Our results indicate that polyamine synthesis was decreased in aged and OA cartilage, along with reduced autophagy activity, evidenced by decreased autophagy-related gene and protein expression and autophagosome formation. Importantly, spermidine treatment increased the expression of the acetyltransferase EP300, which binds to crucial autophagy proteins, Beclin1 and LC3, and elevates chondrocyte autophagy. Our data indicate spermidine prevents the ageing- and OA-related decrease in autophagy and may protect against OA development.