The Ex Vivo Organ Culture of Bone.

The ex vivo organ culture of bone provides many of the advantages of both the whole organism and isolated cell strategies and can deliver valuable insight into the network of processes and activities that are fundamental to bone and cartilage biology. Through maintaining the bone and/or cartilage cells in their native environment, this model system provides the investigator with a powerful experimental protocol to address specific facets of skeletal growth and development. In this chapter, we outline the basic protocols and possible readouts of organ culture models to replicate; (a) linear bone growth (murine metatarsal culture model), (b) bone and cartilage metabolism (murine femoral head culture model), (c) bone response to mechanical stimulation (bovine trabecular core culture model), and (d) bone resorption and formation (murine calvaria culture model).

Mechanically induced Ca2+ oscillations in osteocytes release extracellular vesicles and enhance bone formation.

The vast osteocytic network is believed to orchestrate bone metabolic activity in response to mechanical stimuli through production of sclerostin, RANKL, and osteoprotegerin (OPG). However, the mechanisms of osteocyte mechanotransduction remain poorly understood. We've previously shown that osteocyte mechanosensitivity is encoded through unique intracellular calcium (Ca2+) dynamics. Here, by simultaneously monitoring Ca2+ and actin dynamics in single cells exposed to fluid shear flow, we detected actin network contractions immediately upon onset of flow-induced Ca2+ transients, which were facilitated by smooth muscle myosin and further confirmed in native osteocytes ex vivo. Actomyosin contractions have been linked to the secretion of extracellular vesicles (EVs), and our studies demonstrate that mechanical stimulation upregulates EV production in osteocytes through immunostaining for the secretory vesicle marker Lysosomal-associated membrane protein 1 (LAMP1) and quantifying EV release in conditioned medium, both of which are blunted when Ca2+ signaling was inhibited by neomycin. Axial tibia compression was used to induce anabolic bone formation responses in mice, revealing upregulated LAMP1 and expected downregulation of sclerostin in vivo. This load-related increase in LAMP1 expression was inhibited in neomycin-injected mice compared to vehicle. Micro-computed tomography revealed significant load-related increases in both trabecular bone volume fraction and cortical thickness after two weeks of loading, which were blunted by neomycin treatment. In summary, we found mechanical stimulation of osteocytes activates Ca2+-dependent contractions and enhances the production and release of EVs containing bone regulatory proteins. Further, blocking Ca2+ signaling significantly attenuates adaptation to mechanical loading in vivo, suggesting a critical role for Ca2+-mediated signaling in bone adaptation.

Models of ex vivo explant cultures: applications in bone research.

Ex vivo explant culture models are powerful tools in bone research. They allow investigation of bone and cartilage responses to specific stimuli in a controlled manner that closely mimics the in vivo processes. Because of limitations in obtaining healthy human bone samples the explant growth of animal tissue serves as a platform to study the complex physico-chemical properties of the bone. Moreover, these models enable preserving important cell-cell and cell-matrix interactions in order to better understand the behaviour of cells in their natural three-dimensional environment. Thus, the use of bone ex vivo explant cultures can frequently be of more physiological relevance than the use of two-dimensional primary cells grown in vitro. Here, we describe isolation and ex vivo growth of different animal bone explant models including metatarsals, femoral heads, calvaria, mandibular slices and trabecular cores. We also describe how these explants are utilised to study bone development, cartilage and bone metabolism, cancer-induced bone diseases, stem cell-driven bone repair and mechanoadaptation. These techniques can be directly used to understand mechanisms linked with bone physiology or bone-associated diseases.

T-Type voltage-sensitive calcium channels mediate mechanically-induced intracellular calcium oscillations in osteocytes by regulating endoplasmic reticulum calcium dynamics.

One of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular calcium (Ca(2+)), and osteocytes in particular exhibit robust oscillations in Ca(2+) when subjected to loading. Previous studies implicate roles for both the endoplasmic reticulum (ER) and T-Type voltage-sensitive calcium channels (VSCC) in these responses, but their interactions or relative contributions have not been studied. By observing Ca(2+) dynamics in the cytosol (Ca(2+)cyt) and the ER (Ca(2+)ER), the focus of this study was to explore the role of the ER and T-Type channels in Ca(2+) signaling in bone cells. We demonstrate that inhibition of T-Type VSCC in osteocytes significantly reduces the number of Ca(2+)cyt responses and affects Ca(2+)ER depletion dynamics. Simultaneous observation of Ca(2+) exchange among these spaces revealed high synchrony between rises in Ca(2+)cyt and depressions in Ca(2+)ER, and this synchrony was significantly reduced by challenging T-Type VSCC. We further confirmed that this effect was mediated directly through the ER and not through store-operated Ca(2+) entry (SOCE) pathways. Taken together, our data suggests that T-Type VSCC facilitate the recovery of Ca(2+)ER in osteocytes to sustain mechanically-induced Ca(2+) oscillations, uncovering a new mechanism underlying the behavior of osteocytes as mechanosensors.

Experimental studies of bone mechanoadaptation: bridging in vitro and in vivo studies with multiscale systems.

Despite advancements in technology and science over the last century, the mechanisms underlying Wolff's law-bone structure adaptation in response to physical stimuli-remain poorly understood, limiting the ability to effectively treat and prevent skeletal diseases. A challenge to overcome in the study of the underlying mechanisms of this principle is the multiscale nature of mechanoadaptation. While there exist in silico systems that are capable of studying across these scales, experimental studies are typically limited to interpretation at a single dimension or time point. For instance, studies of single-cell responses to defined physical stimuli offer only a limited prediction of the whole bone response, while overlapping pathways or compensatory mechanisms complicate the ability to isolate critical targets in a whole animal model. Thus, there exists a need to develop experimental systems capable of bridging traditional experimental approaches and informing existing multiscale theoretical models. The purpose of this article is to review the process of mechanoadaptation and inherent challenges in studying its underlying mechanisms, discuss the limitations of traditional experimental systems in capturing the many facets of this process and highlight three multiscale experimental systems which bridge traditional approaches and cover relatively understudied time and length scales in bone adaptation.

Cellular and Molecular Bioengineering: A Tipping Point.

In January of 2011, the Biomedical Engineering Society (BMES) and the Society for Physical Regulation in Biology and Medicine (SPRBM) held its inaugural Cellular and Molecular Bioengineering (CMBE) conference. The CMBE conference assembled worldwide leaders in the field of CMBE and held a very successful Round Table discussion among leaders. One of the action items was to collectively construct a white paper regarding the future of CMBE. Thus, the goal of this report is to emphasize the impact of CMBE as an emerging field, identify critical gaps in research that may be answered by the expertise of CMBE, and provide perspectives on enabling CMBE to address challenges in improving human health. Our goal is to provide constructive guidelines in shaping the future of CMBE.