Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Methods For Studying Osteoenergetics And Metabolism

ARTICLES DISCUSSED:
Shen, L., Karner, C. M. Evaluation of amino acid consumption in cultured bone cells and isolated bone shafts. Journal of Visualized ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

Maine Medical Center Research Institute

Vanderbilt University Medical Center

Vanderbilt University

Marrow adipose tissue (MAT) is a recently identified endocrine organ capable of modulating a host of responses. Given its intimate proximity to the bone microenvironment, the impact marrow adipocytes exert on bone has attracted much interest and scientific inquiry. Although many questions and controversies remain about marrow adipocytes, multiple conditions/disease states in which alterations occur have provided clues about their function. The consensus is that MAT is associated inversely with bone density and quality. While further investigation is warranted, MAT has clearly been demonstrated as an active dynamic depot that contributes to bone turnover and overall metabolic homeostasis.

Bone-Fat Interaction.

Menopause is associated with bone loss and enhanced visceral adiposity. A polyclonal antibody that targets the β-subunit of the pituitary hormone follicle-stimulating hormone (Fsh) increases bone mass in mice. Here, we report that this antibody sharply reduces adipose tissue in wild-type mice, phenocopying genetic haploinsufficiency for the Fsh receptor gene Fshr. The antibody also causes profound beiging, increases cellular mitochondrial density, activates brown adipose tissue and enhances thermogenesis. These actions result from the specific binding of the antibody to the β-subunit of Fsh to block its action. Our studies uncover opportunities for simultaneously treating obesity and osteoporosis.

Blocking FSH induces thermogenic adipose tissue and reduces body fat.

Bone formation is an osteoblast-specific process characterized by high energy demands due to the secretion of matrix proteins and mineralization vesicles. While glucose has been reported as the principle fuel source for osteoblasts, recent evidence supports the tenet that osteoblasts can utilize fatty acids as well. Although the ability to accumulate lipid droplets has been demonstrated in many cell types, there has been little evidence that osteoblasts possess this characteristic. The current study provides evidence that osteoblastogenesis is associated with lipid droplet accumulation capable of supplying energy substrates (fatty acids) required for the differentiation process. Understanding the role of fatty acids in metabolic programming of the osteoblast may lead to novel approaches to increase bone formation and ultimately bone mass.

Intracellular lipid droplets support osteoblast function.

Because of heavy energy demands to maintain bone homeostasis, the skeletal system is closely tied to whole-body metabolism via neuronal and hormonal mediators. Glucose, amino acids, and fatty acids are the chief fuel sources for bone resident cells during its remodeling. Lipids, which can be mobilized from intracellular depots in the bone marrow, can be a potent source of fatty acids. Thus, while it has been suggested that adipocytes in the bone marrow act as "filler" and are detrimental to skeletal homeostasis, we propose that marrow lipids are, in fact, essential for proper bone functioning. As such, we examine the prevailing evidence regarding the storage, use, and export of lipids within the skeletal niche, including from both in vitro and in vivo model systems. We also highlight the numerous challenges that remain to fully appreciate the relationship of lipid turnover to skeletal homeostasis.

Lipids in the Bone Marrow: An Evolving Perspective.

Lysosomal acid lipase (LAL) is essential for cholesteryl ester (CE) and triacylglycerol (TAG) hydrolysis in the lysosome. Clinically, an autosomal recessive LIPA mutation causes LAL deficiency (LALD), previously described as Wolman Disease or Cholesteryl Ester Storage Disease (CESD). LAL-D is associated with ectopic lipid accumulation in the liver, small intestine, spleen, adrenal glands, and blood. Considering the importance of unesterified cholesterol and fatty acids in bone metabolism, we hypothesized that LAL is essential for bone formation, and ultimately, skeletal health. To investigate the role of LAL in skeletal homeostasis, we used LAL-deficient () mice, in vitro osteoblast cultures, and novel clinical data from LAL-D patients. Both male and female LAL mice demonstarted lower trabecular and cortical bone parameters , which translated to reduced biomechanical properties. Further histological analyses revealed that LAL mice had fewer osteoblasts, with no change in osteoclast or marrow adipocyte numbers. In studying the cell-autonomous role of LAL, we observed impaired differentiation of LAL calvarial osteoblasts and in bone marrow stromal cells treated with the LAL inhibitor lalistat. Consistent with LAL's role in other tissues, lalistat resulted in profound lipid puncta accumulation and an altered intracellular lipid profile. Finally, we analyzed a large de-identified national insurance database (i.e. 2016/2017 Optum Clinformatics®) which revealed that adults (≥18 years) with CESD (n = 3076) had a higher odds ratio (OR = 1.21; 95% CI = 1.03-1.41) of all-cause fracture at any location compared to adults without CESD (n = 13.7 M) after adjusting for demographic variables and osteoporosis. These data demonstrate that alterations in LAL have significant clinical implications related to fracture risk and that LAL's modulation of lipid metabolism is a critical for osteoblast function.

Loss of function of lysosomal acid lipase (LAL) profoundly impacts osteoblastogenesis and increases fracture risk in humans.

Skeletal remodeling is essential for proper maintenance of adult bone mass, and due to its heavy energetic demands this process is closely tied to whole body metabolic. Thus, bone formation by the osteoblast, bone resorption by the osteoclast, and mechano-sensing by the osteocyte, are highly coupled processes that are essential for bone turnover. When one experiences a disruption in these processes, over time increased skeletal fragility and fracture can result. In addition to these primary cells, secondary cells within the skeletal niche are suspected to directly coordinate bone health as well. The bone marrow compartment provides a unique microenvironment in which communication occurs between white blood cells, red blood cells, platelets, and immune cells, in addition to classic bone cells (osteoblasts, osteoclasts, and osteocytes) that can both directly and indirectly impact skeletal homeostasis. One such cell population that has attracted much attention and scientific inquiry in the past decade are bone marrow adipocytes (BMAdipo) which can be found interspersed throughout the marrow compartment, and collectively are often referred to as bone marrow adipose tissue (BMAT). Although our understanding of BMAT has advanced significantly in the past decade, many questions remain relative to their lineage and function. The current collection presents recent studies and the up-to-date understanding of bone marrow adipocytes, along with future clinical perspectives related to this unique depot.

Bone marrow adipocytes - Good, bad, or just different?

Bone marrow adipose tissue: New insights and clinical correlates from Best Practices.

Skeletal homeostasis is an exquisitely regulated process most directly influenced by bone resorbing osteoclasts, bone forming osteoblasts, and the mechano-sensing osteocytes. These cells work together to constantly remodel bone as a mechanism to prevent from skeletal fragility. As such, when an individual experiences a disconnect in these tightly coupled processes, fracture incidence increases, such as during ageing, gonadal hormone deficiency, weightlessness, and diabetes. While therapeutic options have significantly aided in the treatment of low bone mineral density (BMD) or osteoporosis, limited options remain for anabolic or bone forming agents. Therefore, it is of interest to continue to understand how osteoblasts regulate their metabolism to support the energy expensive process of bone formation.

Untargeted metabolomics in primary murine bone marrow stromal cells reveals distinct profile throughout osteoblast differentiation.

Late in the nineteenth century, it was theorized that a circulating product produced by the parathyroid glands could negatively impact skeletal homeostasis. A century later, intermittent administration of that protein, namely parathyroid hormone (PTH), was approved by the FDA and EMA as the first anabolic agent to treat osteoporosis. Yet, several unanswered but important questions remain about the skeletal actions of PTH.

Parathyroid hormone (PTH) regulation of metabolic homeostasis: An old dog teaches us new tricks.

Bone is a highly dynamic tissue that undergoes continuous remodeling by bone resorbing osteoclasts and bone forming osteoblasts, a process regulated in large part by osteocytes. Dysregulation of these coupled catabolic and anabolic processes as in the case of menopause, type 2 diabetes mellitus, anorexia nervosa, and chronic kidney disease is known to increase fracture risk. Recent advances in the field of bone cell metabolism and bioenergetics have revealed that maintenance of the skeleton places a high energy demand on these cells involved in bone remodeling. These new insights highlight the reason that bone tissue is the beneficiary of a substantial proportion of cardiac output and post-prandial chylomicron remnants and requires a rich supply of nutrients. Studies designed for the specific purpose of investigating the impact of dietary modifications on bone homeostasis or that alter diet composition and food intake to produce the model can be found throughout the literature; however, confounding dietary factors are often overlooked in some of the preclinical models. This review will examine some of the common pre-clinical models used to study skeletal biology and its pathologies and the subsequent impact of various dietary factors on these model systems. Furthermore, the review will include how inadvertent effects of some of these dietary components can influence bone cell function and study outcomes.

Elizabeth Rendina-Ruedy

Maine Medical Center Research Institute

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Methods For Studying Osteoenergetics And Metabolism

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics