As microfluidic technology continues to advance, in vitro models have been increasingly utilized to recapitulate complex biological microenvironments. While these systems offer a number of advantages over traditional cell culture methods, they require the ability to precisely manipulate small quantities of fluid. However, commercially produced equipment is often cost-prohibitive for smaller research groups with limited resources. Here, we present the development of an accurate, cost-effective, on-chip pumping system that can generate oscillating fluid flow within a custom-designed microfluidic device. The multilayer chip contains two cell culturing chambers that are separated by a thin permeable membrane. In the upper chamber, an oscillating sinusoidal fluid flow can be induced to mechanically stimulate cells with fluid shear stress. The pump consists of two 3D printed linear actuators that are controlled by an Arduino Mega 2560 microcontroller with custom-written software. To demonstrate the versatility of our system, we verified that a range of physiologically relevant levels of shear stress can be induced at multiple frequencies. Our results show that a maximum shear stress of at least ± 8 dyn/cm 2 can be induced at frequency of 1 Hz. This corresponds to a maximum volumetric flow rate of approximately 250 µl/min across an area of 0.33 mm 2 . Further, we have demonstrated that while flow is induced in the upper cell culturing chamber, the lower chamber remains in a quasi-static state. This allows for selective mechanical stimulation of cells in the upper chamber, while simultaneously allowing for real-time soluble signaling with unstimulated cells in the lower chamber.

Our lab has developed a lab-on-a-chip platform for bone remodeling that enables long-term culturing of bone cells out to 7 weeks and serves as a foundation toward a multicellular organ-on-a-chip system. Here, we optimized culturing protocols for osteoblasts, osteoclasts and osteocytes within the lab-on-a-chip and performed functional activity assays for quantifying bone formation and resorption. We analyzed cell seeding densities, feeding schedules and time in culture as a basis for optimizing culturing protocols. Further, we addressed concerns of sterility, cytotoxicity and leakage during the extended culture period within the polydimethylsiloxane chip. This system provides a method for quantifying the soluble effects of mechanically stimulated osteocytes on bone remodeling (formation/resorption).

The purpose of this paper is to emphasize the need for more complex bone remodeling platforms that allow for investigations of intricate multicellular interactions that regulate this process. We discuss the efforts we have taken to develop lab-on-a-chip systems for bone remodeling and the motivation for pursuing more advanced multicellular models. Further, we discuss mathematical modeling opportunities that will allow experimental results to extend beyond the set laboratory conditions. We advocate for the development of an agent-based model comprised of multiple cellular automata of each bone cell type. In total, this work requires a combination of techniques from bone biology, microfluidics, cell mechanobiology, mechanics, and mathematical modeling. Thus, significant advancements within the field will require a collective contribution from a variety of research laboratories.

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Marnie M Saunders*1, Sharon L Truesdell1, Estee L George1, James H Holda2 and Christopher Van Vranken1 Author Affiliations 1Department of Biomedical Engineering, The University of Akron, USA 2Department of Biology, The University of Akron, USA Received: November 02, 2019 | Published: November 19, 2019 Corresponding author: Marnie M Saunders, Department of Biomedical Engineering, The University of Akron, Akron, OH 44325- 0302, USA DOI: 10.26717/BJSTR.2019.23.003838

Bisphosphonate-related osteonecrosis of the jaw is a disease appearing after tooth removal in patients undergoing bisphosphonate treatment for metastasizing cancers and osteoporosis. The complexity of the condition requires a multicellular model to address the net effects of two key risk factors: mechanical trauma (pathologic overload) and inflammation. In this work, a system comprised of a polydimethylsiloxane chip and mechanical loading device is used to expose bisphosphonate-treated osteocytes to mechanical trauma. Specifically, osteocytes are treated with the potent nitrogen-containing bisphosphonate, zoledronic acid, and exposed to short-term pathologic overload via substrate stretch. During bone remodeling, osteocyte apoptosis plays a role in attracting pre-osteoclasts to sites of damage; as such, lactate dehydrogenase activity, cell death and protein expression are evaluated as functions of load. Additionally, the effects of osteocyte soluble factors on osteoclast and osteoblast functional activity are quantified. Osteoclast activity and bone resorption are quantified in the presence and absence of inflammatory components, lipopolysaccharide and interferon gamma. Results suggest that inflammation associated with bacterial infection may hinder bone resorption by osteoclasts. In addition, osteocytes may respond to overload by altering expression of soluble signals that act on osteoblasts to attenuate bone formation. These findings give insight into the multicellular interactions implicated in bisphosphonate-related osteonecrosis of the jaw.

This manuscript details the design, fabrication, characterization, and application of a 3D printed loading device for the investigation of cellular mechanotransduction pathways activated by matrix deformation. The device, which works as a screw jack, applies out-of-plane substrate distention to a thin polymer membrane via platen displacement. Load induces a strain gradient on the top surface of the membrane where cells are cultured. A high performance poly-lactic acid 3D filament was used for printing, resulting in a compact, cost-effective device that is fully autoclavable and compatible with standard laboratory incubators. The device was customized to accommodate a loadable polydimethylsiloxane chip developed in our lab for culturing MLO-Y4 osteocytes; however, the design can be easily adapted to load any mechanosensitive cells grown on an elastomeric membrane. Using finite element analysis, we demonstrated that the device can generate a range of strains to induce a variety of responses by the osteocytes. Cell viability data demonstrated that these ranges had the ability to engender load-induced apoptotic differences.

In terms of accurate and precise mechanical testing, machines run the continuum. Whereas commercial platforms offer excellent accuracy, they can be cost-prohibitive, often priced in the $100,000 - $200,000 price range. At the other extreme are stand-alone manual devices that often lack repeatability and accuracy (e.g., a manual crank device). However, if a single use is indicated, it is over-engineering to design and machine something overly elaborate. Nonetheless, there are occasions where machines are designed and built in-house to accomplish a motion not attainable with the existing machines in the laboratory. Described in detail here is one such device. It is a loading platform that enables pure uniaxial loading. Standard loading machines typically are biaxial in that linear loading occurs along the axis and rotary loading occurs about the axis. During testing with these machines, a load is applied to one end of the specimen while the other end remains fixed. These systems are not capable of conducting pure axial testing in which tension/compression is applied equally to the specimen ends. The platform developed in this paper enables the equal and opposite loading of specimens. While it can be used for compression, here the focus is on its use in pure tensile loading. The device incorporates commercial load cells and actuators (movers) and, as is the case with machines built in-house, a frame is machined to hold the commercial parts and fixtures for testing.

Researchers have been using lab-on-a-chip systems to isolate factors for study, simulate laboratory analysis and model cellular, tissue and organ level processes. The technology is increasing rapidly, but the bone field has been slow to keep pace. Novel models are needed that have the power and flexibility to investigate the elegant and synchronous multicellular interactions that occur in normal bone turnover and in disease states in which remodeling is implicated. By removing temporal and spatial limitations and enabling quantification of functional outcomes, the platforms should provide unique environments that are more biomimetic than single cell type systems while minimizing complex systemic effects of in vivo models. This manuscript details the development and characterization of lab-on-a-chip platforms for stimulating osteocytes and quantifying bone remodeling. Our platforms provide the foundation for a model that can be used to investigate remodeling interactions as a whole or as a standard mechanotransduction tool by which isolated activity can be quantified as a function of load.

ABSTRACTFunctional studies of skeletal anatomy are predicated on the fundamental assumption that form will follow function. For instance, previous studies have shown that the femora of specialized leaping primates are more robust than those of more generalized primate quadrupeds. Are such differences solely a plastic response to differential loading patterns during postnatal life, or might they also reflect more canalized developmental mechanisms present at birth? Here, we show that perinatalLemur catta, an arboreal/terrestrial quadruped, have less robust femora than perinatalPropithecus coquereli, a closely related species specialized for vertical clinging and leaping (a highly unusual locomotor mode in which the hindlimbs are used to launch the animal between vertical tree trunks). These results suggest that functional differences in long bone cross‐sectional dimensions are manifest at birth, belying simple interpretations of adult postcranial form as a direct record of loading patterns during postnatal life. Despite these significant differences in bone robusticity, we find that hindlimb bone mineralization, material properties, and measures of whole‐bone strength generally overlap in perinatalL. cattaandP. coquereli, indicating little differentiation in postcranial maturity at birth despite known differences in the pace of craniodental development between the species. In a broader perspective, our results likely reflect evolution acting during prenatal ontogeny. Even though primates are notable for relatively prolonged gestation and postnatal parental care, neonates are not buffered from selection, perhaps especially in the unpredictable and volatile environment of Madagascar. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 303:250–264, 2020. © 2018 American Association for Anatomy

Bone remodeling is an elegantly orchestrated process by which osteocytes, osteoblasts and osteoclasts function as a syncytium to maintain or modify bone. On the microscopic level, bone consists of cells that create, destroy and monitor the bone matrix. These cells interact in a coordinated manner to maintain a tightly regulated homeostasis. It is this regulation that is responsible for the observed increase in bone gain in the dominant arm of a tennis player and the observed increase in bone loss associated with spaceflight and osteoporosis. The manner in which these cells interact to bring about a change in bone quality and quantity has yet to be fully elucidated. But efforts to understand the multicellular complexity can ultimately lead to eradication of metabolic bone diseases such as osteoporosis and improved implant longevity. Experimentally validated mathematical models that simulate functional activity and offer eventual predictive capabilities offer tremendous potential in understanding multicellular bone remodeling. Here we undertake the initial challenge to develop a mathematical model of bone formation validated with in vitro data obtained from osteoblastic bone cells induced to mineralize and quantified at 26 days of culture. A cellular automata model was constructed to simulate the in vitro characterization. Permutation tests were performed to compare the distribution of the mineralization in the cultures and the distribution of the mineralization in the mathematical models. The results of the permutation test show the distribution of mineralization from the characterization and mathematical model come from the same probability distribution, therefore validating the cellular automata model.

Bone remodeling is a continual process in which old bone is resorbed by osteoclasts and new bone is formed by osteoblasts, providing a mechanism for bones' ability to adapt to changes in its mechanical environment. While the role of osteoblasts and osteoclasts in bone remodeling is well understood, the cellular regulation of bone remodeling is unclear. One theory is that osteocytes, found within bone, play an important role in controlling the bone remodeling response. Osteocytes possess gap junctions, narrow channels that extend between nearby cells and allow communication between cells via the transfer of small molecules and ions. This work investigated the potential role of gap junctional intercellular communication in bone remodeling by exposing osteocyte-like MLO-Y4 cells to mechanical strains and quantifying the expression of soluble factors, including sclerostin, a protein closely associated with bone remodeling. The soluble factors and sclerostin expression were further examined after inhibiting gap junctional intercellular communication to study the impact of the communication. At supraphysiologic strains, the inhibition of gap junctional intercellular communication led to increases in sclerostin expression relative to cells in which communication was present, indicating that the communication may play a significant role in regulating bone remodeling.

The field of mechanobiology aims to understand the role the mechanical environment plays in directing cell and tissue development, function and disease. The empirical aspect of the field requires the development of accurate, reproducible and reliable loading platforms that can apply microprecision mechanical load. In this study we designed, fabricated and characterized a pure uniaxial loading platform capable of testing small synthetic and organic specimens along a horizontal axis. The major motivation for platform development was in stimulating bone cells seeded on elastomeric substrates and soft tissue loading. The biological uses required the development of culturing fixtures and environmental chamber. The device utilizes commercial microactuators, load cells and a rail/carriage block system. Following fabrication, acceptable performance was verified by suture tensile testing.

Bone remodeling is a process in which bone is resorbed by osteoclasts and formed by osteoblasts. This is normally a paired process, although it can be disrupted by changes in mechanical load. One theory is that osteocytes play a key role in the cellular regulation of this process. Mechanotransduction studies, which investigate how cells convert mechanical stimuli into biophysical effects and cellular activity, offer one way to investigate this theory. Mechanotransduction work is commonly done by applying an isolated mechanical load to cells grown in vitro, and quantifying the response. While in vitro work does not fully replicate the natural environment, it does allow the study of isolated factors. In this study, a mechanical loading platform was designed, fabricated, and characterized for bone mechanotransduction studies. This platform was designed to tent cell-seeded substrates from below, loading using out of plane distension. This introduced a nonuniform strain profile, enabling the study of cells cultured under identical conditions and variable strains as a function of substrate location. An alphanumerically gridded polydimethylsiloxane well substrate was designed and fabricated for cellular loading experiments. Following initial characterization, a study was run to quantify the cellular activity of osteocyte-like MLO-Y4 cells as a function of strain field. The results indicated that regions with lower strains led to an increase in cellular activity while higher strains led to a reduction in cellular activity. This demonstrated that cells could be exposed to mechanically-induced microdamage using the microloading platform.

The data to complete these assignments is provided and can be downloaded: Suture data for Assignment 1; bending data for Assignment 2; torsion data for Assignment 3.

Mechanical testing is a useful tool in the field of biomechanics. Classic biomechanics employs mechanical testing for a variety of purposes. For instance, testing may be used to determine the mechanic