Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study

OBJECTIVE: Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas. METHODS: Hypertrophic chondrocytes were isolated from embryonic chick sterna and seeded onto rectangular Gelfoam sponges. Seeded sponges were subjected to various levels of cyclic uniaxial tensile strains at 1 Hz with the computer-controlled Bio-Stretch system. Strain distribution across the sponge was quantified by digital image analysis. After mechanical loading, sponges were cut and the end and center regions were separated according to construct strain distribution. Total RNA was extracted from the cells harvested from these regions, and real-time quantitative RT-PCR was performed to quantify mRNA levels for type X collagen and a housing-keeping gene 18S RNA. RESULTS: Chondrocytes distributed in high (9%) local strain areas produced more than two times type X collagen mRNA compared to the those under no load conditions, while chondrocytes located in low (2.5%) local strain areas had no appreciable difference in type X collagen mRNA production in comparison to non-loaded samples. Increasing local strains above 2.5%, either in the center or end regions of the sponge, resulted in increased expression of Col X mRNA by chondrocytes in that region. CONCLUSION: These findings suggest that the threshold of chondrocyte sensitivity to inducing type X collagen mRNA production is more than 2.5% local strain, and that increased local strains above the threshold results in an increase of Col X mRNA expression. Such quantitative analysis has important implications for our understanding of mechanosensitivity of cartilage and mechanical regulation of chondrocyte gene expression

Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes

Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load‐bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β‐glycyrrhetinic acid, we also demonstrated that this osteocyte‐related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial‐derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain–related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity

High-resolution 3 T MRI of traumatic and degenerative triangular fibrocartilage complex (TFCC) abnormalities using Palmer and Outerbridge classifications

To investigate the usefulness of high-resolution 3 T magnetic resonance imaging (MRI) for the evaluation of traumatic and degenerative triangular fibrocartilage complex (TFCC) abnormalities among three groups: patients presenting with wrist pain who were (a) younger than age 50 years or (b) age 50 or older (PT<50 and PT≥50, respectively), and (c) asymptomatic controls who were younger than age 50 years (AC).High-resolution 3 T MRI was evaluated retrospectively in 96 patients, including 47 PT<50, 38 PT≥50, and 11 AC. Two board-certified radiologists reviewed the MRI images independently. MRI features of TFCC injury were analysed according to the Palmer classification, and cartilage degeneration around the TFCC was evaluated using the Outerbridge classification. Differences in MRI findings among these groups were detected using chi-square test. Cohen's kappa was calculated to assess interobserver and intra-observer reliability.The incidence of Palmer class 1A, 1C and 1D traumatic TFCC injury was significantly (p<0.05) higher in PT≥50 than in PT<50 (class 1A: 47.4% versus 27.7%, class 1C: 31.6% versus 12.8%, and class 1D: 21.1% versus 2.1%). Likewise, MRI findings of TFCC degeneration were observed more frequently in PT≥50 than in PT<50 (p<0.01). Outerbridge grade 2 or higher cartilage degeneration was significantly (p<0.01) more frequently seen in PT≥50 than in PT<50 (55.3% versus 17% in the lunate, 28.9% versus 4.3% in the triquetrum, 73.7% versus 12.8% in the ulna).High-resolution wrist MRI at 3 T enables detailed evaluation of TFCC traumatic injury and degenerative changes using the Palmer and Outerbridge classifications, with good or excellent interobserver and intra-observer reliability

Aggressive treatment of metastatic squamous cell carcinoma of the rectum to the liver: a case report and a brief review of the literature

BACKGROUND: Rectal squamous cell carcinoma (SCC) is a rare tumor. The incidence of this malignancy has been reported to be 0.25 to 1 per 1000 colorectal carcinomas. From a review of the English literature 55 cases of SCC of the rectum have been published. In this study we report a rectal metastatic SCC to the liver, discussing the efficacy of aggressive adjuvant and neo-adjuvant therapies on survival and prognosis. CASE PRESENTATION: A 39-year-old female patient with a pure SCC of the rectum diagnosed endoscopically is presented. The patient underwent initially neoadjuvant chemo-radiotherapy and then abdominoperineal resection with concomitant bilateral oophorectomy and hysterectomy, followed by adjuvant chemo-radiotherapy. Five months after the initial operation liver metastasis was demonstrated and a liver resection was carried out, followed by adjuvant chemotherapy. Eighteen months after the initial operation the patient is alive. CONCLUSION: Although prognosis of rectal SCC is worse than that of adenocarcinoma, an aggressive therapeutic approach with surgery as the primary treatment, followed by combined neo- and adjuvant chemo-radiotherapy, may be necessary in order to improve survival and prognosis

Mechanical Induction of PGE2 in Osteocytes Blocks Glucocorticoid-Induced Apoptosis Through Both the β-Catenin and PKA Pathways

Glucocorticoids are known to induce osteocyte apoptosis, whereas mechanical loading has been shown to sustain osteocyte viability. Here we show that mechanical loading in the form of fluid-flow shear stress blocks dexamethasone-induced apoptosis of osteocyte-like cells (MLO-Y4). Prostaglandin E2 (PGE2), a rapidly induced signaling molecule produced by osteocytes, was shown to be protective against dexamethasone-induced apoptosis, whereas indomethacin reversed the antiapoptotic effects of shear stress. This protective effect of shear stress was mediated through EP2 and EP4 receptors, leading to activation of the cAMP/protein kinase A signaling pathway. Activation of phosphatidylinositol 3-kinase, an inhibitor of glycogen synthesis kinase 3, also occurred, leading to the nuclear translocation of β-catenin, an important signal transducer of the Wnt signaling pathway. Both shear stress and prostaglandin increased the phosphorylation of glycogen synthesis kinase 3 α/β. Lithium chloride, an activator of the Wnt pathway, also was protective against glucocorticoid-induced apoptosis. Whereas it is known that mechanical loading increases cyclooxygenase-2 and EP2 receptor expression and prostaglandin production, dexamethasone was shown to inhibit expression of these components of the prostaglandin pathway and to reduce β-catenin protein expression. β-catenin siRNA knockdown experiments abrogated the protective effects of PGE2, confirming the central role of β-catenin in mediating the protection against dexamethasone-induced cell death. Our data support a central role for PGE2 acting through the cAMP/PKA and β-catenin signaling pathways in the protection of osteocyte apoptosis by fluid-flow shear stress. © 2010 American Society for Bone and Mineral Research

Microfluidic Enhancement of Intramedullary Pressure Increases Interstitial Fluid Flow and Inhibits Bone Loss in Hindlimb Suspended Mice

Interstitial fluid flow (IFF) has been widely hypothesized to mediate skeletal adaptation to mechanical loading. Although a large body of in vitro evidence has demonstrated that fluid flow stimulates osteogenic and antiresorptive responses in bone cells, there is much less in vivo evidence that IFF mediates loading-induced skeletal adaptation. This is due in large part to the challenges associated with decoupling IFF from matrix strain. In this study we describe a novel microfluidic system for generating dynamic intramedullary pressure (ImP) and IFF within the femurs of alert mice. By quantifying fluorescence recovery after photobleaching (FRAP) within individual lacunae, we show that microfluidic generation of dynamic ImP significantly increases IFF within the lacunocanalicular system. In addition, we demonstrate that dynamic pressure loading of the intramedullary compartment for 3 minutes per day significantly eliminates losses in trabecular and cortical bone mineral density in hindlimb suspended mice, enhances trabecular and cortical structural integrity, and increases endosteal bone formation rate. Unlike previously developed modalities for enhancing IFF in vivo, this is the first model that allows direct and dynamic modulation of ImP and skeletal IFF within mice. Given the large number of genetic tools for manipulating the mouse genome, this model is expected to serve as a powerful investigative tool in elucidating the role of IFF in skeletal adaptation to mechanical loading and molecular mechanisms mediating this process. © 2010 American Society for Bone and Mineral Research

Inhibition of Osteoclastogenesis by Mechanically Loaded Osteocytes: Involvement of MEPE

In regions of high bone loading, the mechanoresponsive osteocytes inhibit osteoclastic bone resorption by producing signaling molecules. One possible candidate is matrix extracellular phosphoglycoprotein (MEPE) because acidic serine- and aspartate-rich MEPE-associated motif peptides upregulate osteoprotegerin (OPG) gene expression, a negative regulator of osteoclastogenesis. These peptides are cleaved from MEPE when relatively more MEPE than PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) is present. We investigated whether mechanical loading of osteocytes affects osteocyte-stimulated osteoclastogenesis by involvement of MEPE. MLO-Y4 osteocytes were mechanically loaded by 1-h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) or kept under static control conditions. Recombinant MEPE (0.05, 0.5, or 5 μg/ml) was added to some static cultures. Mouse bone marrow cells were seeded on top of the osteocytes to determine osteoclastogenesis. Gene expression of MEPE, PHEX, receptor activator of nuclear factor kappa-B ligand (RANKL), and OPG by osteocytes was determined after PFF. Osteocytes supported osteoclast formation under static control conditions. Both PFF and recombinant MEPE inhibited osteocyte-stimulated osteoclastogenesis. PFF upregulated MEPE gene expression by 2.5-fold, but not PHEX expression. PFF decreased the RANKL/OPG ratio at 1-h PFF treatment. Our data suggest that mechanical loading induces changes in gene expression by osteocytes, which likely contributes to the inhibition of osteoclastogenesis after mechanical loading of bone. Because mechanical loading upregulated gene expression of MEPE but not PHEX, possibly resulting in the upregulation of OPG gene expression, we speculate that MEPE is a soluble factor involved in the inhibition of osteoclastogenesis by osteocytes

A conceptually new treatment approach for relapsed glioblastoma: Coordinated undermining of survival paths with nine repurposed drugs (CUSP9) by the International Initiative for Accelerated Improvement of Glioblastoma Care.

To improve prognosis in recurrent glioblastoma we developed a treatment protocol based on a combination of drugs not traditionally thought of as cytotoxic chemotherapy agents but that have a robust history of being well-tolerated and are already marketed and used for other non-cancer indications. Focus was on adding drugs which met these criteria: a) were pharmacologically well characterized, b) had low likelihood of adding to patient side effect burden, c) had evidence for interfering with a recognized, well-characterized growth promoting element of glioblastoma, and d) were coordinated, as an ensemble had reasonable likelihood of concerted activity against key biological features of glioblastoma growth. We found nine drugs meeting these criteria and propose adding them to continuous low dose temozolomide, a currently accepted treatment for relapsed glioblastoma, in patients with recurrent disease after primary treatment with the Stupp Protocol. The nine adjuvant drug regimen, Coordinated Undermining of Survival Paths, CUSP9, then are aprepitant, artesunate, auranofin, captopril, copper gluconate, disulfiram, ketoconazole, nelfinavir, sertraline, to be added to continuous low dose temozolomide. We discuss each drug in turn and the specific rationale for use- how each drug is expected to retard glioblastoma growth and undermine glioblastoma's compensatory mechanisms engaged during temozolomide treatment. The risks of pharmacological interactions and why we believe this drug mix will increase both quality of life and overall survival are reviewed