Evaluation of absorbable mesh fixation devices at various deployment angles.

BACKGROUND: Hernia repair failure may occur due to suboptimal mesh fixation by mechanical constructs before mesh integration. Construct design and acute penetration angle may alter mesh-tissue fixation strength. We compared acute fixation strengths of absorbable fixation devices at various deployment angles, directions of loading, and construct orientations. METHODS: Porcine abdominal walls were sectioned. Constructs were deployed at 30°, 45°, 60°, and 90° angles to fix mesh to the tissue specimens. Lap-shear testing was performed in upward, downward, and lateral directions in relation to the abdominal wall cranial-caudal axis to evaluate fixation. Absorbatack™ (AT), SorbaFix™ (SF), and SecureStrap™ in vertical (SSV) and horizontal (SSH) orientations in relation to the abdominal wall cranial-caudal axis were tested. Ten tests were performed for each combination of device, angle, and loading direction. Failure types and strength data were recorded. ANOVA with Tukey-Kramer adjustments for multiple comparisons and χ (2) tests were performed as appropriate (p < 0.05 considered significant). RESULTS: At 30°, SSH and SSV had greater fixation strengths (12.95, 12.98 N, respectively) than SF (5.70 N; p = 0.0057, p = 0.0053, respectively). At 45°, mean fixation strength of SSH was significantly greater than SF (18.14, 11.40 N; p = 0.0002). No differences in strength were identified at 60° or 90°. No differences in strength were noted between SSV and SSH with different directions of loading. No differences were noted between SS and AT at any angle. Immediate failure was associated with SF (p < 0.0001) and the 30° tacking angle (p < 0.01). CONCLUSIONS: Mesh-tissue fixation was stronger at acute deployment angles with SS compared to SF constructs. The 30° angle and the SF device were associated with increased immediate failures. Varying construct and loading direction did not generate statistically significant differences in the fixation strength of absorbable fixation devices in this study.

Frequent mutations in the RPL22 gene and its clinical and functional implications.

OBJECTIVE: To determine the frequency and spectrum of mutations in RPL22 a gene identified by The Cancer Genome Atlas (TCGA) as mutated in endometrioid endometrial cancer, and determine the relationship between RPL22 defects and clinicopathologic features. METHODS: Direct sequencing of the entire coding region of the RPL22 cDNA and exons 2/4 was performed in tumors with/without microsatellite instability (MSI). RPL22 expression was assessed by immunofluorescence microscopy in the KLE, RL952 and AN3CA cell lines, wildtype, heterozygous and homozygous mutants, respectively. Relationships between RPL22 mutation and clinicopathological features were assessed using Chi-squared analysis and Student's t test. Progression-free survival (PFS) was calculated from the date of diagnosis to the date of recurrence. RESULTS: A single nucleotide deletion in an A8 coding repeat was identified in exon 2 of the RPL22 gene in 116/226 (52%) of MSI-high tumors. No mutations were identified in MSI-stable tumors. Only 2% of the tumors expressed a homozygous A deletion. RPL22 mutation was not associated with stage, grade, race and lymphovascular space invasion. Women whose tumors harbored RPL22 mutations were significantly older (67 vs. 63years, p=0.005). There was no difference in PFS between patients with the wildtype and mutant genotypes. CONCLUSIONS: RPL22 is frequently mutated in MSI-high endometrioid endometrial cancers. The A8 mutation identified was not reported in the whole exome sequences analyzed by the TCGA. The demonstration of frequent mutation in RPL22 may point to a limitation of the exome capture and next generation sequencing analysis methods for some mononucleotide string mutations. Functional assessment of the RPL22 knockdown may be warranted.

Lithium chloride and inhibition of glycogen synthase kinase 3ß as a potential therapy for serous ovarian cancer.

OBJECTIVE: Lithium chloride (LiCl) has been shown to demonstrate anticancer properties at supratherapeutic doses. This study was designed to determine whether LiCl, as a single agent or in combination with cytotoxic agents, reduces ovarian cancer cell growth and metabolic activity at clinically achievable levels. METHODS: We studied the effects of LiCl on 2 high-grade serous ovarian cancer cell lines, SKOV3 and OVCA 433, and primary cultures developed from ascitic fluid collected from patients with metastatic high-grade serous ovarian cancer. We assessed proliferation and metabolism using cell cycle analysis, MTT assays, and cellular proliferation and clonogenic potential assays. RESULTS: Treatment with 1 mM LiCl had no effect on the cell cycle distribution or metabolic activity of the SKOV3 and OVCA 433 cell lines. Combination treatment with cisplatin or paclitaxel led to statistically significant decreases in metabolic activity in the OVCA 433 cell line and 50% of cultures investigated. The decreased metabolic activity was not, however, associated with decreased cell growth or clonogenic potential. CONCLUSIONS: Combination treatment with LiCl and cytotoxic agents at physiologically achievable drug concentrations reduces ovarian cancer cell metabolism but does not appear to affect cellular proliferation. The potential for combined lithium/cytoxic therapies appears to be limited based on our analysis of both established cell lines and short-term ovarian cancer cultures.

Hydrogel Encapsulation of Genome-Engineered Stem Cells for Long-Term Self-Regulating Anti-Cytokine Therapy.

Biologic therapies have revolutionized treatment options for rheumatoid arthritis (RA) but their continuous administration at high doses may lead to adverse events. Thus, the development of improved drug delivery systems that can sense and respond commensurately to disease flares represents an unmet medical need. Toward this end, we generated induced pluripotent stem cells (iPSCs) that express interleukin-1 receptor antagonist (IL-1Ra, an inhibitor of IL-1) in a feedback-controlled manner driven by the macrophage chemoattractant protein-1 (Ccl2) promoter. Cells were seeded in agarose hydrogel constructs made from 3D printed molds that can be injected subcutaneously via a blunt needle, thus simplifying implantation of the constructs, and the translational potential. We demonstrated that the subcutaneously injected agarose hydrogels containing genome-edited Ccl2-IL1Ra iPSCs showed significant therapeutic efficacy in the K/BxN model of inflammatory arthritis, with nearly complete abolishment of disease severity in the front paws. These implants also exhibited improved implant longevity as compared to the previous studies using 3D woven scaffolds, which require surgical implantation. This minimally invasive cell-based drug delivery strategy may be adapted for the treatment of other autoimmune or chronic diseases, potentially accelerating translation to the clinic.

The suture pullout characteristics of human and porcine linea alba.

There is a substantial prevalence of post-operative incisional hernia for both laparoscopic and laparotomy procedures, but there have been few attempts at quantifying abdominal wound closure methodology in the literature. One method to ascertain a more robust method of wound closure is the identification of the influence of suture placement parameters on suture pullout force. Current surgical practice involves a recommended bite depth and bite separation of 10mm, but the evidence base for this is not clear. In this paper, the suture pullout characteristics of both porcine and human linea alba were investigated to ascertain a suture placement protocol for surgical wound closure. Uniaxial suture pullout force testing on fresh frozen porcine and human linea alba samples was performed using standard materials testing machines. The influence of the number of suture loops, the bite depth and the bite separation of the sutures and the orientation of the sutures with respect to the principal fibre direction in the linea alba were assessed. Results showed a clearly identifiable relationship between pullout force of the suture, bite separation and bite depth, with low suture separation and high suture depth as optimal parameters for increasing pullout force. Resistance to pullout could be improved by as much as 290% when optimizing test conditions. Both human and porcine tissue were observed to exhibit very similar pullout force characteristics, corroborating the use of a porcine model for investigations into wound closure methodology. Orientation of suture application was also found to significantly affect the magnitude of suture pullout, with suturing applied longitudinally across a transverse defect resulting in higher pullout forces for small suture bite separations. Although further assessment in an environment more representative of in vivo conditions is required, these findings indicate that increasing the bite depth and reducing the bite separation with respect to the current surgical recommendations may reduce the risk of post-operative incisional hernia.

Multi-directional mechanical analysis of synthetic scaffolds for hernia repair.

Hernias remain one of the most common ailments to affect men and women worldwide. Surgical mesh materials were first used to reinforce hernia defects during surgery in the late 1950s (Laker, n.d.). Today, there are well over 50 prosthetic meshes available for hernia repair (Brown and Finch, 2010; Bryan et al., 2014; Hope and El-hayek, 2014). With the multitude of available options, surgeons are faced with the challenging task of optimizing mesh selection for each patient. If the mechanics of the mesh are not compatible with the surrounding tissue, mismatch can occur, which can lead to complications such as mesh failure and/or hernia recurrence. Unfortunately, many aspects of synthetic mesh mechanics remain poorly described. Therefore, the purpose of this study was to provide a more complete mechanical analysis of a variety of commercially available prosthetic meshes for hernia repair, including evaluation of meshes in a variety of orientations. Twenty different meshes were subjected to biaxial tensile tests at both 90° and 45° orientations, and results were analyzed for relative strength, strain behavior, and anisotropy. Peak tension and strain values varied dramatically across all mesh types for all directions, ranging between 4.08 and 25.74N/cm and -5% to 10% strain. Anisotropy ratios for the evaluated meshes ranged from 0.33 to 1.89, demonstrating a wide range in relative direction-dependence of mesh mechanics. While further study of prosthetic meshes and better characterization of properties of the human abdominal wall are needed, results of this study provide valuable data that may aid clinicians in optimizing mesh selection for specific patients and repair conditions.

Uniaxial and biaxial tensile stress-stretch response of human linea alba.

There are few studies on the stress-stretch behaviour of human linea alba, yet understanding the mechanics of this tissue is important for developing better methods of abdominal wound closure. Published data focuses mainly on porcine linea alba and for human tissue there are conflicting results and no bi-axial data available. This variability is likely due to challenges with the physical dimensions of the tissue and differences in experimental methodology. This study focussed on the tensile mechanical characterisation of the human linea alba using uniaxial and equi-load biaxial testing performed using image-based strain measurement methods. Thirteen freshly frozen human cadaveric abdominal walls were obtained and used to prepare 7 samples in both the transverse and longitudinal directions for uniaxial testing, and 13 square samples for bi-axial testing. The results showed significant anisotropy and for the equi-load biaxial tests the deformation was heavily biased in the longitudinal direction. In comparison with similar tests on porcine tissue from a previous study, it was found that the response of porcine linea alba to uniaxial loading is similar to that of human linea alba, with no statistically significant differences observed. Under biaxial loading human and porcine linea showed no statistical significance in the difference between their means in the transverse direction. However, a significant difference was observed in the longitudinal direction, and further study of the respective tissue structures is needed to better understand this result. These results provide the first data on the biaxial tensile properties of human linea alba and can aid in an improved assessment of wound closure mechanics.

Pore size and pore shape--but not mesh density--alter the mechanical strength of tissue ingrowth and host tissue response to synthetic mesh materials in a porcine model of ventral hernia repair.

BACKGROUND: Over 100 types of soft tissue repair materials are commercially available for hernia repair applications. These materials vary in characteristics such as mesh density, pore size, and pore shape. It is difficult to determine the impact of a single variable of interest due to other compounding variables in a particular design. Thus, the current study utilized prototype meshes designed to evaluate each of these mesh parameters individually. METHODS: Five prototype meshes composed of planar, monofilament polyethylene terephthalate (PET) were evaluated in this study. The meshes were designed to focus on three key parameters, namely mesh density, pore size, and pore shape. The prototype meshes were implanted in the preperitoneal, retrorectus space in a porcine model of ventral incisional hernia repair, and tissue ingrowth characteristics were evaluated after 90 days. Mesh-tissue composite specimens were obtained from each repair site and evaluated via T-peel mechanical testing. Force-displacement data for each T-peel test were analyzed and five characteristics of tissue ingrowth reported: peak force (fp), critical force (fc), fracture energy (Γc), work (W), and work density (Wden). Hematoxylin and eosin (H&E) stained sections of explanted mesh-tissue composites were also assessed for characteristics of tissue response including cellular infiltration, cell types, inflammatory response, extracellular matrix deposition, neovascularization, and fibrosis, with a composite score assigned to represent overall tissue response. RESULTS: The medium-weight, very large pore, hexagonal (MWVLH) mesh performed significantly better than the light-weight, medium pore, diamond (LWMD) mesh for all parameters evaluated (fp, fc, Γc, W, Wden) and trended toward better results than the medium-weight, medium pore, diamond (MWMD) mesh for the majority of the parameters evaluated. When the data for the five meshes was grouped to evaluate mesh density, pore size, and pore shape, differences were more pronounced. No significant differences were observed with respect to mesh density, however significant improvement in mechanical strength of tissue ingrowth occurred as pore size increased from medium to very large. In addition, the hexagonal pores resulted in the strongest tissue ingrowth, followed by the square pores, and finally the diamond pores. Scores for several histological parameters were significantly different for these prototype meshes. For example, the MWVLH mesh showed significantly greater tissue ingrowth by neovascularization histological score than MWMD and MWVLS meshes (p<0.05) and significantly less fibrosis than LWMD and MWVLS meshes (p<0.05). CONCLUSION: Pore shape and pore size significantly altered the mechanical strength of tissue ingrowth and host-site integration in a porcine model of ventral hernia repair, while mesh density had no effect.

Biaxial analysis of synthetic scaffolds for hernia repair demonstrates variability in mechanical anisotropy, non-linearity and hysteresis.

BACKGROUND: Over the past 60 years, the soft tissue repair market has grown to include over 50 types of hernia repair materials. Surgeons typically implant these materials in the orientation that provides maximum overlap of the mesh over the defect, with little regard for mechanical properties of the mesh material. If the characteristics of the meshes were better understood, an appropriate material could be identified for each patient, and meshes could be placed to optimize integration with neighboring tissue and avoid the mechanical mis-match that can lead to impaired graft fixation. The purpose of this study was to fully characterize and compare the mechanical properties of thirteen types of hernia repair materials via planar biaxial tensile testing. METHODS: Equibiaxial (i.e., equal simultaneous loading in both directions) and strip biaxial (i.e., loading in one direction with the other direction held fixed) tests were utilized as physiologically relevant loading regimes. After applying a 0.1N pre-load on each arm, samples were subjected to equibiaxial cyclic loading using a triangular waveform to 2.5mm displacement on each arm at 0.1Hz for 10 cycles. Samples were then subjected to two strip biaxial tests (using the same cyclic loading protocol), where extension was applied along a single axis with the other axis held fixed. RESULTS: The thirteen evaluated mesh types exhibited a wide range of mechanical properties. Some were nearly isotropic (C-QUR™, DUALMESH(®), PHYSIOMESH™, and PROCEED(®)), while others were highly anisotropic (Ventralight™ ST, Bard™ Mesh, and Bard™ Soft Mesh). Some displayed nearly linear behavior (Bard™ Mesh), while others were non-linear with a long toe region followed by a sharp rise in tension (INFINIT(®)). These materials are currently utilized in clinical settings as if they are uniform and interchangeable, and clearly this is not the case. The mechanical properties most advantageous for successful hernia repairs are currently only vaguely described in the clinical literature. The characteristics of the human abdominal wall must be extensively characterized to provide a thorough understanding of the tissue being reinforced/replaced by these meshes. A better understanding of these mechanical differences would enable matching of patient characteristics to a specific mesh with the properties best suited to that particular repair.

Geminin regulates the transcriptional and epigenetic status of neuronal fate-promoting genes during mammalian neurogenesis.

Regulating the transition from lineage-restricted progenitors to terminally differentiated cells is a central aspect of nervous system development. Here, we investigated the role of the nucleoprotein geminin in regulating neurogenesis at a mechanistic level during both Xenopus primary neurogenesis and mammalian neuronal differentiation in vitro. The latter work utilized neural cells derived from embryonic stem and embryonal carcinoma cells in vitro and neural stem cells from mouse forebrain. In all of these contexts, geminin antagonized the ability of neural basic helix-loop-helix (bHLH) transcription factors to activate transcriptional programs promoting neurogenesis. Furthermore, geminin promoted a bivalent chromatin state, characterized by the presence of both activating and repressive histone modifications, at genes encoding transcription factors that promote neurogenesis. This epigenetic state restrains the expression of genes that regulate commitment of undifferentiated stem and neuronal precursor cells to neuronal lineages. However, maintaining geminin at high levels was not sufficient to prevent terminal neuronal differentiation. Therefore, these data support a model whereby geminin promotes the neuronal precursor cell state by modulating both the epigenetic status and expression of genes encoding neurogenesis-promoting factors. Additional developmental signals acting in these cells can then control their transition toward terminal neuronal or glial differentiation during mammalian neurogenesis.

Aberrant methylation of the X-linked ribosomal S6 kinase RPS6KA6 (RSK4) in endometrial cancers.

PURPOSE: Effective treatments for advanced endometrial cancer are lacking. Novel therapies that target specific pathways hold promise for better treatment outcomes with less toxicity. Mutation activation of the FGFR2/RAS/ERK pathway is important in endometrial tumorigenesis. RPS6KA6 (RSK4) is a putative tumor suppressor gene and is a target of the ERK signaling pathway. We explored the role of RSK4 in endometrial cancer. EXPERIMENTAL DESIGN: We showed that RSK4 is expressed in normal endometrial tissue and is absent or much reduced in endometrial cancer. On the basis of previous reports on methylation in other cancers, we hypothesized that the absence of RSK4 transcript is associated with epigenetic silencing rather than mutation. We determined the methylation and expression status of RSK4 in primary endometrial cancers and cell lines and the effects of treatment with a demethylating agent. The relationship between RSK4 methylation and clinicopathologic features was assessed. RESULTS: RSK4 is frequently hypermethylated in endometrial cancer cells lines and in primary endometrial cancer compared with normal endometrial tissue. RSK4 methylation was significantly associated with tumor grade, with higher grade tumors having lower levels of methylation (P = 0.03). RSK4 methylation levels were not associated with other clinical variables. We did find that RSK4 methylation was significantly correlated with expression in primary endometrial tumors and in cell lines. Reactivation of RSK4 by 5-azacytidine was successfully performed showing 8- to more than 1,200-fold increases in transcript levels. CONCLUSION: RSK4 appears to be epigenetically silenced in endometrial cancer as evidenced by hypermethylation. Its role as a suppressor in endometrial cancer, however, remains uncertain.

Subcellular translocation signals regulate Geminin activity during embryonic development.

BACKGROUND INFORMATION: Geminin (Gem) is a protein with roles in regulating both the fidelity of DNA replication and cell fate during embryonic development. The distribution of Gem is predominantly nuclear in cells undergoing the cell cycle. Previous studies have demonstrated that Gem performs multiple activities in the nucleus and that regulation of Gem activation requires nuclear import in at least one context. In the present study, we defined structural and mechanistic features underlying subcellular localization of Gem and tested whether regulation of the subcellular localization of Gem has an impact on its activity in cell fate specification during embryonic development. RESULTS: We determined that nuclear localization of Gem is dependent on a bipartite NLS (nuclear localization signal) in the N-terminus of Xenopus Gem protein. This bipartite motif mapped to a Gem N-terminal region previously shown to regulate neural cell fate acquisition. Microinjection into Xenopus embryos demonstrated that import-deficient Gem was incapable of modulating ectodermal cell fate, but that this activity was rescued by fusion to a heterologous NLS. Cross-species comparison of Gem protein sequences revealed that the Xenopus bipartite signal is conserved in many non-mammalian vertebrates, but not in mammalian species assessed. Instead, we found that human Gem employs an alternative N-terminal motif to regulate the protein's nuclear localization. Finally, we found that additional mechanisms contributed to regulating the subcellular localization of Gem. These included a link to Crm1-dependent nuclear export and the observation that Cdt1, a protein in the pre-replication complex, could also mediate nuclear import of Gem. CONCLUSIONS: We have defined new structural and regulatory features of Gem, and showed that the activity of Gem in regulating cell fate, in addition to its cell-cycle-regulatory activity, requires control of its subcellular localization. Our data suggest that rather than being constitutively nuclear, Gem may undergo nucleocytoplasmic shuttling through several mechanisms involving distinct protein motifs. The use of multiple mechanisms for modulating Gem subcellular localization is congruent with observations that Gem levels and activity must be stringently controlled during cell-cycle progression and embryonic development.