In vitro osteocytic microdamage and viability quantification using a microloading platform.

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.

Impact of Gap Junctional Intercellular Communication on MLO-Y4 Sclerostin and Soluble Factor Expression.

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.

Development of a cost-effective torsional unit for rodent long bone assessment.

Thorough mechanical testing of rodent bones requires an understanding of bone behavior in a variety of loading modes including tension, compression, bending and shear. While these tests are easily conducted with single axis mechanical testing machines, it may also be desirable to determine torsional properties of bone. Although higher-end materials testing machines will enable torsional and/or rotational testing, simpler, less expensive systems rarely offer these capabilities. In this work, we illustrate the development of a torsional system that uses a simple rack and pinion concept to deliver a rotary motion to bones given the linear motion of a testing machine. As the bone field becomes increasingly interdisciplinary, more biologists and non-test engineers need cost-effective mechanical testing capabilities and the torsional system described here has proven to be more than adequate for standard biomechanical testing requirements. Furthermore, given the small-scale size of rodent long bones, a series of potting/testing fixtures were developed that enabled preparation and handling of the specimens without incurring damage to the bone shafts. Once fabricated the system was used to destructively load mice humeri and femurs and quantify torsional properties.

Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions.

The strong correlation between a bone's architectural properties and the mechanical forces that it experiences has long been attributed to the existence of a cell that not only detects mechanical load but also structurally adapts the bone matrix to counter it. One of the most likely cellular candidates for such a "mechanostat" is the osteocyte, which resides within the mineralized bone matrix and is perfectly situated to detect mechanically induced signals. However, as osteocytes can neither form nor resorb bone, it has been hypothesized that they orchestrate mechanically induced bone remodeling by coordinating the actions of cells residing on the bone surface, such as osteoblasts. To investigate this hypothesis, we developed a novel osteocyte-osteoblast coculture model that mimics in vivo systems by permitting us to expose osteocytes to physiological levels of fluid shear while shielding osteoblasts from it. Our results show that osteocytes exposed to a fluid shear rate of 4.4 dyn/cm(2) rapidly increase the alkaline phosphatase activity of the shielded osteoblasts and that osteocytic-osteoblastic physical contact is a prerequisite. Furthermore, both functional gap junctional intercellular communication and the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 signaling pathway are essential components in the osteoblastic response to osteocyte communicated mechanical signals. By utilizing other nonosteocytic coculture models, we also show that the ability to mediate osteoblastic alkaline phosphatase levels in response to the application of fluid shear is a phenomena unique to osteocytes and is not reproduced by other mesenchymal cell types.

Mechanical stimulation effects on functional end effectors in osteoblastic MG-63 cells.

Receptor activator of Nf-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been implicated in bone metabolism. Specifically, the balance of these factors in conjunction with receptor activator of Nf-kappaB (RANK) is believed to be key in determining the rate of osteoclastogenesis and the net outcome of bone formation/resorption. While it is well accepted that mechanical loading in vivo affects bone formation/resorption and that alterations in the responsiveness of bone cells to mechanical loading have been implicated in metabolic bone diseases, the effect of in vitro mechanical loading on osteoblastic production of OPG and RANKL has not been extensively studied. Thus, in the current study, we developed an in vitro model to load human osteoblasts and studied levels of OPG, RANKL, PGE(2) and macrophage colony stimulating factor (M-CSF). We hypothesized that stimulating osteoblastic cells would increase the release of soluble OPG relative to RANKL favoring a bone-forming (and resorption-inhibiting) event. To accomplish this, we developed a small-scale loading machine that imparts via bending, well-defined substrate deformation to bone cells cultured on artificial substrates. Following 2h of loading and a 1h incubation period, media was collected and levels of soluble OPG, RANKL, PGE(2) and M-CSF were quantified using ELISA and western blotting. We found that mechanical loading significantly increased soluble OPG levels relative to RANKL at this 3h time point. Levels of soluble and cellular RANKL detected were not significantly affected by mechanical stimulation. The relative shift in abundance of OPG over RANKL associated with applied mechanical stimulation suggests the soluble OPG:RANKL ratio may be important in load-induced coupling mechanisms of bone cells.

Expansion anchors for use in anterior cruciate ligament (ACL) reconstruction: establishing proof of concept in a benchtop analysis.

The current method for graft fixation in bone tendon-bone anterior cruciate ligament (ACL) reconstruction is the interference screw. Although this method of fixation provides for adequate graft fixation with respect to strength, intraoperative placement is difficult and the failure rate is high. To address these concerns, we have designed and fabricated prototype expansion anchors that could be expanded to anchor the graft in the bone tunnel. As a first step in assessing the validity of this concept, in the current work, we demonstrate that these systems are of comparable fixation strength (biomechanical pullout testing) to the standard interference screw, are smaller at the time of insertion and thus provide for increased visibility and ease of placement. The increased visibility should result in better placement and reduced failure rates. The increased ease of placement should result in significant savings in decreased OR time.

Development of a cost-effective loading machine for biomechanical evaluation of mouse transgenic models.

Recent advances in molecular biology have enabled the widespread use of transgenic mouse models. Whether these transgenics present with a skeletal phenotype is often of interest, particularly to orthopedic researchers. Unfortunately, the expense of commercial mechanical testing systems often impedes their routine use in biology laboratories. In this article, the development of a small-scale, relatively inexpensive loading machine, that is ideal for the biomechanical bend testing of mouse long bones, is detailed and the system in transgenic mouse tibiae testing is utilized.

Fluid flow-induced prostaglandin E2 response of osteoblastic ROS 17/2.8 cells is gap junction-mediated and independent of cytosolic calcium.

It has been well demonstrated that bone adapts to mechanical loading. To accomplish this at the cellular level, bone cells must be responsive to mechanical loading (mechanoresponsive). This can occur via such mechanisms as direct cell deformation or signal transduction via complex pathways involving chemotransport, hormone response, and/or gene expression, to name a few. Mechanotransduction is the process by which a bone cell senses a biophysical signal and elicits a response. While it has been demonstrated that bone cells can respond to a wide variety of biophysical signals including fluid flow, stretch, and magnetic fields, the exact pathways and mechanisms involved are not clearly understood. We postulated that gap junctions may play an important role in bone cell responsiveness. Gap junctions (GJ) are membrane-spanning channels that physically link cells and support the transport of small molecules and ions in the process of gap junctional intercellular communication (GJIC). In this study we examined the role of GJ and GJIC in mechanically stimulated osteoblastic cells. Following fluid flow stimulation, we quantified prostaglandin E(2) (PGE(2)) (oscillatory flow) and cytosolic calcium (Ca(2+)) (oscillatory and steady flow) responses in ROS 17/2.8 cells and a derivative of these cells expressing antisense cDNA for the gap junction protein connexin 43 (RCx16) possessing significantly different levels of GJIC. We found that the ROS17/2.8 cells possessing increased GJIC also exhibited increased PGE(2) release to the supernatant following oscillatory fluid flow stimulation in comparison to coupling-decreased RCx16 cells. Interestingly, we found that neither osteoblastic cell line responded to oscillatory or steady fluid flow stimulation with an increase in Ca(2+). Thus, our results suggest that GJ and GJIC may be important in the mechanotransduction mechanisms by which PGE(2) is mechanically induced in osteoblastic cells independent of Ca(2+).

Gap junctions and fluid flow response in MC3T3-E1 cells.

In the current study, we examined the role of gap junctions in oscillatory fluid flow-induced changes in intracellular Ca(2+) concentration and prostaglandin release in osteoblastic cells. This work was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Our results demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E(2) (PGE(2)) release, whereas cells with diminished gap junctional communication do not. Furthermore, we found that cytosolic Ca(2+) (Ca) response was unaltered by the disruption in gap junctional communication and was not significantly different among the cell lines. Thus our results suggest that gap junctions contribute to the PGE(2) but not to the Ca response to oscillatory fluid flow. These findings implicate gap junctional intercellular communication (GJIC) in bone cell ensemble responsiveness to oscillatory fluid flow and suggest that gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.

Breast cancer metastatic potential correlates with a breakdown in homospecific and heterospecific gap junctional intercellular communication.

Breast cancer progresses toward increasingly malignant behavior in tumorigenic and metastatic stages. In the series of events in the metastatic stage, tumor cells leave the primary tumor in breast and travel to distant sites where they establish secondary tumors, or metastases. In this report, we demonstrate that cell-cell communication via gap junctions is restored in the metastatic human breast carcinoma cell line MDA-MB-435 when it is transfected with breast metastasis suppressor 1 (BRMS1) cDNA. Furthermore, the expression profile of connexins (Cxs), the protein subunits of gap junctions, changes. Specifically, the expression of BRMS1 in MDA-MB-435 cells increases Cx43 expression and reduces Cx32 expression, resulting in a gap junction phenotype more similar to normal breast tissue. Taken together, these results suggest that gap junctional communication and the Cx expression profile may contribute to the metastatic potential of these breast cancer cells.

Effects of interference fit screw length on tibial tunnel fixation for anterior cruciate ligament reconstruction.

Graft-tunnel mismatch during arthroscopically assisted anterior cruciate ligament reconstruction using the central-third patellar tendon results in less than 20 mm of bone plug remaining in the tibial tunnel. We decided to evaluate the strength of bone plug fixation using interference fit screws that were less than 20 mm in length. Biomechanical testing was performed on 48 porcine hindquarters using 9-mm diameter interference fit screws that measured 12.5, 15, and 20 mm in length. No significant difference was noted between the different-length screws for insertion torque, divergence, stiffness, displacement, or load to failure. We believe, therefore, that comparable graft fixation can be achieved in the tibial tunnel using 9-mm diameter interference fit screws that are less than 20 mm long, and that these shorter screws may be useful in cases of graft-tunnel mismatch.

Analysis of mechanisms underlying BRMS1 suppression of metastasis.

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

An improved enzyme linked immunosorbent assay (ELISA) for the detection of porcine serum antibodies against Mycoplasma hyopneumoniae.

An ELISA for the detection of serum antibodies to Mycoplasma hypopneumoniae in pigs and based on a 43 kDa purified protein derived from the cytoplasmic membrane of M. hyopneumoniae strain J is described. This ELISA (MHPP ELISA) was compared with another recently described (Auspharm ELISA, Sheldrake and Romalis 1992) that is based on column-purified sonicated proteins of strain J. Using sample to negative ELISA ratios of 3 and 4 as cutoffs for inconclusive and positive reactors respectively (compared to 2 and 3 for the Auspharm ELISA), the two tests had high specificity (MHPP 99.6%; Auspharm 100%) in 280 SPF pigs. In 176 pigs from commercial herds with endemic M. hyopneumoniae, the MHPP ELISA showed a higher sensitivity than the Auspharm ELISA in both high lung score (LS > or = 5) (85.5% vs. 69.9%) and low lung score (0 < LS < 5) (57.9% vs. 49%) pigs when the positive cutoff for each test was selected. The sensitivity when the inconclusive cutoff was selected was similar in both tests (85%; 85.7%) when low and high lung score pigs were pooled. Altough the MHPP also gave more positive reactors in 36 pigs from M. hyopneumoniae-infected herds with no lung pathology at slaughter than the Auspharm ELISA (11 vs. 4), the total number of inconclusive and positive reactors in these pigs was similar for both tests (18 vs. 14). The MHPP ELISA gave significantly higher ELISA ratios in infected pigs (up to 17.9) than the Auspharm ELISA (up to 9), and earlier seroconversion in naturally-infected (6-8 weeks vs. 9-10 weeks) and experimentally-infected pigs (2-4 weeks vs. 4-6 weeks post infection).

Serum and mucosal antibody responses against Mycoplasma hyopneumoniae following intraperitoneal vaccination and challenge of pigs with M hyopneumoniae.

Pigs were immunised intraperitoneally when six weeks old and again at about 10 weeks old with killed Mycoplasma hyopneumoniae antigen prepared in an oil adjuvant. The pigs were challenged with live M hyopneumoniae (Beaufort strain) at between 11 and 15 weeks old. Antigen specific antibody levels for both IgG and IgA classes in serum and respiratory tract secretion were monitored over time. In serum anti-M hyopneumoniae antibody was detected shortly after the second intraperitoneal vaccination and was largely IgG. In respiratory tract secretion the response was observed after challenge, and was primarily IgA. Anti-M hyopneumoniae antibody-containing cells and their immunoglobulin class specificity were monitored in lung and tracheal lamina propria. In lung the majority of anti-M hyopneumoniae-containing cells were IgG, whereas in the tracheal lamina propria the majority were IgA. These results are discussed in terms of the use of intraperitoneal vaccination for the control of M hyopneumoniae infection.

Intraperitoneal vaccination of pigs to control Mycoplasma hyopneumoniae.

In a field trial at a commercial piggery 22 pigs were vaccinated intraperitoneally, at 30 days and 60 days old, with formalin-killed Mycoplasma hyopneumoniae plus adjuvant. Two other groups of the same size, one receiving a ration containing antibiotics, were not vaccinated. The mean enzyme-linked immunosorbent assay (ELISA) ratio of the vaccinated group increased significantly (P less than 0.001) after 30 days, and continued to rise until day 156 of life. In the other two groups the ELISA ratio did not increase significantly until day 115. The vaccinated pigs in the 30-day period after the first vaccination grew significantly (P less than 0.01) more slowly than the controls but between days 60 and 144 they grew significantly faster (P less than 0.05) than the two other groups, thus compensating for their previous, relative loss. At slaughter (at 163 days old), the mean weight was similar in all groups. The mean score for pneumonia at slaughter for the vaccinated, medicated and control groups was 2.6 (+/- 4.3), 9.4 (+/- 14.0) and 10.5 (+/- 12.4), respectively. The proportion of M hyopneumoniae-affected lungs (4.8 per cent) in vaccinated pigs, as judged by immunofluorescence, was significantly less (P less than 0.001) than the control groups (40 per cent). Thus, intraperitoneal vaccination with killed M hyopneumoniae plus adjuvant might control mycoplasmal pneumonia in commercial piggeries.

Origin of antibody in porcine bile after intraperitoneal immunisation.

Pigs were immunised intraperitoneally with ovalbumin (OVA) in Freund's complete adjuvant and killed between 52 and 71 days later. Sera, bile and spleen and liver tissue were collected at slaughter. IgG and IgA OVA antibody in bile and serum were detected by ELISA, and IgG and IgA anti-OVA containing cells (AOCC) in tissue were observed using double fluorochrome labelling techniques. The results indicated few IgA AOCC in spleen or liver, but an elevated IgA OVA response in bile compared to serum relative to IgG. The results indicate selective transport of IgA OVA antibody from serum to bile relative to IgG.

Serum antibody response to Mycoplasma hyopneumoniae measured by enzyme-linked immunosorbent assay after experimental and natural infection of pigs.

Studies were conducted under experimental and field conditions to determine the effect of infection with M. hyopneumoniae on the immune response in serum as measured by ELISA. Following intratracheal challenge or contact exposure, serologically negative pigs derived from mycoplasma-free piggeries developed an immune response within 10 days. This response continued to rise for a further 50 days. In a field study in a commercial piggery, no animals (0/44) were observed to have M. hyopneumoniae antibodies at day 86 of life. However between day 86 and day 144, 97.7% (42/43) animals sero-converted. These results are discussed in terms of infection spread, particularly in the grower/finisher shed.