A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling.

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.

Cellular considerations for optimizing bone cell culture and remodeling in a lab-on-a-chip platform.

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).

Getting into Shape: Limb Bone Strength in Perinatal Lemur catta and Propithecus coquereli.

Functional 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 perinatal Lemur catta, an arboreal/terrestrial quadruped, have less robust femora than perinatal Propithecus 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 perinatal L. catta and P. 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.

The effects of mechanically loaded osteocytes and inflammation on bone remodeling in a bisphosphonate-induced environment.

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.

Bone remodeling platforms: Understanding the need for multicellular lab-on-a-chip systems and predictive agent-based models.

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.

Application of Design Aspects in Uniaxial Loading Machine Development.

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.

Bisphosphonate-related osteonecrosis of the jaw: a mechanobiology perspective.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a dramatic disintegration of the jaw that affects patients treated with bisphosphonates (BPs) for diseases characterized by bone loss. These diseases are often metastasizing cancers (like multiple myeloma, breast cancer and prostate cancer (Aragon-Ching et al., 2009)) as well as osteoporosis. BRONJ is incompletely understood, although it is believed to arise from a defect in bone remodeling-the intricate process by which sensory osteocytes signal to osteoclasts and osteoblasts to resorb and form bone in response to stimuli. Further, tooth extraction and infection have been overwhelmingly linked to BRONJ (Ikebe, 2013). Because bone cells are highly networked, the importance of multicellular interactions and mechanotransduction during the onset of these risk factors cannot be overstated. As such, this perspective addresses current research on the effects of BPs, mechanical load and inflammation on bone remodeling and on development of BRONJ. Our investigation has led us to conclude that improved in vitro systems capable of adequately recapitulating multicellular communication and incorporating effects of osteocyte mechanosensing on bone resorption and formation are needed to elucidate the mechanism(s) by which BRONJ ensues.

Lab-on-a-chip platforms for quantification of multicellular interactions in bone remodeling.

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.

A cellular automata model of bone formation.

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.

Design, fabrication and characterization of a pure uniaxial microloading system for biologic testing.

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.

Lower limb direct skeletal attachment. A Yucatan micropig pilot study.

Regardless of the type of prosthetic lower limb, successful ambulation requires proper prosthetic attachment. To help alleviate many of the problems associated with prosthetic attachment, direct skeletal attachment (DSA) has been proposed as an alternative to conventional sockets. The purpose of the current study was to evaluate the feasibility of lower limb DSA in a micropig model and to develop a systematic approach to the development and analysis of DSA systems. The DSA device consisted of two stages. The load-carrying stage embedded in the bone canal was designed using bone remodeling theory in conjunction with finite element analysis to approximate implant-induced remodeling and stabilization out to 36 months postimplantation. The skin-interfacing stage was designed to maintain an immutable infection barrier where the prosthesis exited the body. Following successful design, fabrication, and benchtop evaluation, the device was surgically implanted in a Yucatan micropig. The animal trial was successful out to 10 weeks and revealed potential flaws in the surgical protocol related to thermal necrosis. However, no signs of infection were present at the time of implant retrieval. While results of this pilot study support the feasibility of a DSA approach to prosthetic limb attachment, additional animal trials are necessary to prove long-term viability.

Osteocyte Characterization on Polydimethylsiloxane Substrates for Microsystems Applications.

In the body, osteocytes reside in lacunae, lenticular shaped cavities within mineralized bone. These cells are linked to each other and surface-residing osteoblasts via physical channels known as gap junctions. It has been suggested that osteocytes sense mechanical load applied to bone and relay that signal to osteoclasts and osteoblasts. Current in vitro and in vivo models of mechanotransduction face temporal and spatial barriers. Recent advances in polydimethylsiloxane (PDMS) based microfabrication techniques may be able to overcome some of these hurdles. However, before the bone research field can effectively utilize microsystems techniques, fundamental groundwork must be completed. This study characterized the behaviour of osteocytes on PDMS coated with collagen type I (CTI) and provides the framework for bone cell mechanotransduction studies using microsystems. The goal was to determine whether osteocytes were adversely affected by the substrate material by comparing their behaviour to a standard glass substrate. In addition, optimal culture conditions and time points for growing osteocytes on PDMS substrates were determined. Results of this study suggested that use of PDMS does not adversely affect osteocyte behaviour. Furthermore, the results demonstrated that osteocytes should be cultured for no less than 72 hours prior to experimentation to allow the establishment and maintenance of phenotypic characteristics. These results completed essential groundwork necessary for further studies regarding osteocytes in microsystems modelling utilizing PDMS.

Temporary anchorage device insertion variables: effects on retention.

OBJECTIVE: To quantify the influence of temporary anchorage device (TAD) insertion variables on implant retention. MATERIALS AND METHODS: Three hundred thirty TADs from three companies were placed in synthetic bone replicas at variable depths and angulations and compared. Clinically relevant forces were applied to the TADs until failure of retention occurred. RESULTS: In all three implants, increased insertion depth increased implant retention. As the distance from the abutment head to the cortical plate increased, the retention of all three implants decreased. A significantly greater force to fail was required for a 90 degrees insertion angle than for 45 degrees or 135 degrees insertion angles. No significant difference was found between the 45 degrees and 135 degrees insertion angles. A significant reduction in force to fail occurred when comparing 90 degrees and 45 degrees oblique insertion angles. CONCLUSIONS: Increasing penetration depth of TADs results in greater retention. Increased abutment head distance from cortical plate leads to decreased retention. Placement of TADs at 90 degrees to the cortical plate is the most retentive insertion angle. Insertion at an oblique angle from the line of force reduces retention of TADs.

Biomimetic bone mechanotransduction modeling in neonatal rat femur organ cultures: structural verification of proof of concept.

The goal of this work was to develop and validate a whole bone organ culture model to be utilized in biomimetic mechanotransduction research. Femurs harvested from 2-day-old neonatal rat pups were maintained in culture for 1 week post-harvest and assessed for growth and viability. For stimulation studies, femurs were physiologically stimulated for 350 cycles 24 h post-harvest then maintained in culture for 1 week at which time structural tests were conducted. Comparing 1 and 8 days in culture, bones grew significantly in size over the 7-day culture period. In addition, histology supported adequate diffusion and organ viability at 2 weeks in culture. For stimulation studies, 350 cycles of physiologic loading 24 h post-harvest resulted in increased bone strength over the 7-day culture period. In this work, structural proof of concept was established for the use of whole bone organ cultures as mechanotransduction models. Specifically, this work established that these cultures grow and remain viable in culture, are adequately nourished via diffusion and are capable of responding to a brief bout of mechanical stimulation with an increase in strength.

BioGlue and Dermabond save time, leak less, and are not mechanically inferior to two-layer and modified one-layer vasovasostomy.

OBJECTIVE: To compare operative time, patency, and integrity of glue-assisted versus suture-only vasovasostomies. DESIGN: A Medline search revealed no vasovasostomy studies testing tissue adhesives other than fibrin. We compare glue-reinforced to suture-only vasovasostomies. SETTING: An academic medical center. PATIENT(S): None. INTERVENTION(S): Using bull vas deferens, we performed: [1] two-layer anastomoses, [2] modified one-layer anatomoses, and [3] Bioglue, Dermabond, or CoSeal-reinforced anastomoses supported by three transmural sutures. MAIN OUTCOME MEASURE(S): Operative times were recorded, patency verified, and microscopic dissection performed to rule out luminal glue intravasation. Destructive mechanical testing was then completed with statistical comparison of load to failure, displacement to failure, and linear stiffness. RESULT(S): Operative time was greatest for two-layer anastomoses and significantly reduced for all three glue-reinforced three-suture anastomoses. All techniques were patent and free of glue intravasation. BioGlue and Dermabond demonstrated greater integrity than all other techniques. Mechanically, BioGlue and Dermabond were superior to both the unreinforced three stitch and CoSeal groups and were capable of resisting higher loads before failure. CONCLUSION(S): Glue-reinforced anastomoses are significantly less time consuming than traditional techniques. BioGlue and Dermabond have greater mechanical integrity and may be superior to both CoSeal and the sutured techniques.

Biomechanical analysis of in situ single versus double screw fixation in a nonreduced slipped capital femoral epiphysis model.

Slipped capital femoral epiphyses (SCFE) were created in 24 pairs of immature bovine femurs. In 17 pairs of femurs, the slip was left nonreduced (one-third diameter of physis), and in 7 pairs, the slip was reduced. Stabilization of the slips was with either 1 or 2 threaded 6.5-mm screws in a compression mode. The specimens were subjected to shear or torsional loading forces to failure, with the goal of trying to reproduce clinical conditions of in situ screw fixation for acute or unstable SCFE. In the nonreduced model, double-screw fixation was 312% stiffer than single-screw fixation under torsional loading. In the reduced model, double-screw fixation was 137% stiffer than single-screw fixation under torsional loading. The increased rotational stability of double-screw fixation under torsional loading conditions may justify its use in in situ stabilization of acute or unstable SCFE.

Modulation of connexin43 alters expression of osteoblastic differentiation markers.

Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43(-)). hFOB/Cx43(-) cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43(-) cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43(-) cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor alpha1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43(-). Osteopontin mRNA levels were increased in hFOB/Cx43(-) relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43(-) and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells.

Breast cancer metastatic potential: correlation with increased heterotypic gap junctional intercellular communication between breast cancer cells and osteoblastic cells.

The breast cancer metastasis-suppressor gene BRMS1 is downregulated in metastatic breast cancer cells. Previous reports have shown restoration of gap junctional intercellular communication (GJIC) in the metastatic human breast carcinoma cell line MDA-MB-435 (435) transfected with BRMS1 cDNA. Metastasis, to a large extent in most breast cancers, occurs to bone. However, the reason for this preferential metastasis is not known. We explored cell-to-cell communication between 435 carcinoma cells and a human osteoblastic cell line, hFOB1.19, to determine whether carcinoma cells can form gap junctions with bone cells and to explore the role of these heterotypic gap junctions and the BRMS1 gene in breast cancer metastasis to bone. 435 cells displayed greater cell-to-cell communication with hFOB 1.19 cells than with themselves. Transfection of BRMS1 into 435 cells increased homotypic gap junctional communication but did not significantly affect heterotypic communication with hFOBs. However, heterotypic communication of BRMS1 transfectants with hFOB cells was reduced relative to homotypic communication. In contrast, parental 435 cells displayed greater heterotypic communication with hFOBs relative to homotypic communication. Our results suggest that there are differences in the relative homotypic and heterotypic GJIC of metastasis-capable and -suppressed cell lines.

Effect of COX-2-specific inhibition on fracture-healing in the rat femur.

BACKGROUND: Nonsteroidal anti-inflammatory medications have been shown to delay fracture-healing. COX-2-specific inhibitors such as celecoxib have recently been approved for human use. Our goal was to determine, mechanically, histologically, morphologically, and radiographically, whether COX-2-specific inhibition affects bone-healing. METHODS: A nondisplaced unilateral fracture was created in the right femur of fifty-seven adult male rats. Rats were given no drug, indomethacin (1 mg/kg/day), or celecoxib (3 mg/kg/day) daily, starting on postoperative day 1. Fractures were analyzed at four, eight, and twelve weeks after creation of the fracture. Callus and bridging bone formation was assessed radiographically. The amounts of fibrous tissue, cartilage, woven bone, and mature bone formation were determined histologically. Morphological changes were assessed to determine fibrous healing, callus formation, and bone-remodeling. Callus strength and stiffness were assessed biomechanically with three-point bending tests. RESULTS: At four weeks, only the indomethacin group showed biomechanical and radiographic evidence of delayed healing. Although femora from rats treated with celecoxib appeared to have more fibrous tissue than those from untreated rats at four and eight weeks, radiographic signs of callus formation, mechanical strength, and stiffness did not differ significantly between the groups. By twelve weeks, there were no significant differences among the three groups. CONCLUSIONS: Postoperative administration of celecoxib, a COX-2-specific inhibitor, did not delay healing as seen at twelve weeks following fracture in adult rat femora. At four and eight weeks, fibrous healing predominated in the celecoxib group as compared with the findings in the untreated group; however, mechanical strength and radiographic signs of healing were not significantly inhibited. CLINICAL RELEVANCE: Many orthopaedists rely on narcotic analgesia for postfracture and postoperative pain, despite deleterious side effects and morbidity. Traditional nonsteroidal anti-inflammatory medications have been shown to delay fracture union. This effect may be smaller with COX-2-specific inhibitors.