Collective epithelial cell sheet adhesion and migration on polyelectrolyte multilayers with uniform and gradients of compliance.

Polyelectrolyte multilayers (PEMUs) are tunable thin films that could serve as coatings for biomedical implants. PEMUs built layer by layer with the polyanion poly(acrylic acid) (PAA) modified with a photosensitive 4-(2-hydroxyethoxy) benzophenone (PAABp) group and the polycation poly(allylamine hydrochloride) (PAH) are mechanically tunable by UV irradiation, which forms covalent bonds between the layers and increases PEMU stiffness. PAH-terminated PEMUs (PAH-PEMUs) that were uncrosslinked, UV-crosslinked to a uniform stiffness, or UV-crosslinked with an edge mask or through a neutral density optical gradient filter to form continuous compliance gradients were used to investigate how differences in PEMU stiffness affect the adhesion and migration of epithelial cell sheets from scales of the fish Poecilia sphenops (Black Molly) and Carassius auratus (Comet Goldfish). During the progressive collective cell migration, the edge cells (also known as 'leader' cells) in the sheets on softer uncrosslinked PEMUs and less crosslinked regions of the gradient formed more actin filaments and vinculin-containing adherens junctions and focal adhesions than formed in the sheet cells on stiffer PEMUs or glass. During sheet migration, the ratio of edge cell to internal cell (also known as 'follower' cells) motilities were greater on the softer PEMUs than on the stiffer PEMUs or glass, causing tension to develop across the sheet and periods of retraction, during which the edge cells lost adhesion to the substrate and regions of the sheet retracted toward the more adherent internal cell region. These retraction events were inhibited by the myosin II inhibitor Blebbistatin, which reduced the motility velocity ratios to those for sheets on the stiffer PEMUs. Blebbistatin also caused disassembly of actin filaments, reorganization of focal adhesions, increased cell spreading at the leading edge, as well as loss of edge cell-cell connections in epithelial cell sheets on all surfaces. Interestingly, cells throughout the interior region of the sheets on uncrosslinked PEMUs retained their actin and vinculin organization at adherens junctions after treatment with Blebbistatin. Like Blebbistatin, a Rho-kinase (ROCK) inhibitor, Y27632, promoted loss of cell-cell connections between edge cells, whereas a Rac1 inhibitor, NSC23766, primarily altered the lamellipodial protrusion in edge cells. Compliance gradient PAH-PEMUs promoted durotaxis of the cell sheets but not of individual keratocytes, demonstrating durotaxis, like plithotaxis, is an emergent property of cell sheet organization.

Quasi-Spherical Cell Clusters Induced by a Polyelectrolyte Multilayer.

Fibroblasts cultured on polyelectrolyte multilayers, PEMUs, made from poly(diallyldimethylammonium), PDADMA, and poly(styrene sulfonate), PSS, showed a variety of attachment modes, depending on the charge of the last layer and deposition conditions. PEMUs terminated with PDADMA (cationic) were cytotoxic when built in 1.0 M NaCl but cytophilic when built in 0.15 M NaCl. Cells adhered poorly to all PSS-capped (anionic) films. PEMUs built in 0.15 M NaCl but terminated with a layer of PSS in 1.0 M NaCl induced most cells to form spherical clusters after about 48 h of culture. These clusters still interrogated the surface, and when they were replated on control tissue culture plastic, cells emerged with close to 100% viability. Differences between the various surfaces were probed in an effort to identify the mechanism responsible for this unusual behavior, which did not follow accepted correlations between substrate stiffness and cell adhesion. No significant differences in roughness or wetting were observed between cluster-inducing PSS-capped multilayers and those that did not produce clusters. When the surface charge was assayed with radiolabeled ions a strong increase in negative surface charge was revealed. Viewing the multilayer as a zwitterionic solid and comparing its surface charge density to that of a cell membrane yields similarities that suggest a mechanism for preventing protein adhesion to the surface, a necessary step in the integrin-mediated mechanotransduction properties of a cell.

Human mesenchymal stem cell osteoblast differentiation, ECM deposition, and biomineralization on PAH/PAA polyelectrolyte multilayers.

Polyelectrolyte multilayer (PEMU) coatings built layer by layer with alternating pairs of polyelectrolytes can be tuned to improve cell interactions with surfaces and may be useful as biocompatible coatings to improve fixation between implants and tissues. Here, we show that human mesenchymal stromal cells (hMSCs) induced with bone differentiation medium (BDM) to become osteoblasts biomineralize crosslinked PEMUs built with the polycation poly(allylamine hydrochloride) (PAH) and the polyanion poly(acrylic acid) (PAA). Degrees of hMSC osteoblast differentiation and surface biomineralization on the smooth PAH-terminated PEMUs (PAH-PEMUs) and microstructured PAA-terminated PEMUs (PAA-PEMUs) reflect differences in cell-deposited extracellular matrix (ECM). BDM-induced hMSCs expressed higher levels of the early osteoblast differentiation marker alkaline phosphatase and collagen 1 (COL1) sooner on PAA-PEMUs than on PAH-PEMUs. Cells on both types of PEMUs proceeded to express the later stage osteoblast differentiation marker bone sialoprotein (BSP), but the BDM-induced cells organized a more amorphous Collagen I and denser BSP localization on PAA-PEMUs than on PAH-PEMUs. These ECM properties correlated with greater biomineralization on the PAA-PEMUs than on PAH-PEMUs. Together, these results confirm the suitability of PAH/PAA PEMUs as a substrate for hMSC osteogenesis and highlight the importance of substrate effects on ECM organization and BSP presentation on biomineralization.

Arthroscopically Assisted Treatment of Navicular Osteochondral Defect Using Flowable Collagen, Iliac Crest Bone Marrow Aspirate and Fibrin Glue: A Case Report.

UNLABELLED: A 32-year-old male recreational athlete presented with activity-related chronic dorsal midfoot pain. Conservative treatment, including a prolonged period of immobilization, physical therapy, nonsteroidal anti-inflammatory drugs, and use of a bone stimulator, failed to resolve his symptoms. Computed tomography and magnetic resonance imaging demonstrated a cystic appearing focus within the navicular in conjunction with a osteochondral lesion within the proximal articular surface of the navicular. This case report presents an arthroscopically assisted treatment of a navicular osteochondral lesion using curettage and backfilling with fibrin glue, flowable collagen, and autogenous bone grafting. LEVELS OF EVIDENCE: Therapeutic, Level IV.

Tibial tunnel placement accuracy during anterior cruciate ligament reconstruction: independent femoral versus transtibial femoral tunnel drilling techniques.

PURPOSE: This study aimed to compare the accuracy of tibial tunnel placement using independent femoral (IF) versus transtibial (TT) techniques. METHODS: Ten matched pairs of cadaveric knees were randomized so that one knee in the pair underwent arthroscopic TT drilling of the femoral tunnel and the other underwent IF drilling through an accessory medial portal. For both techniques, an attempt was made to place the femoral and tibial tunnels as close to the center of the respective anterior cruciate ligament (ACL) footprints as possible. Preoperative and postoperative computed tomography using a technique optimized for ligament evaluation allowed comparison of the anatomic ACL tibial footprint to the tibial tunnel aperture. The percentage of tunnel aperture contained within the native footprint, as well as the distance from the center of the tunnel aperture to the center of the footprint, was measured. Additionally, graft obliquity relative to the tibial plateau was evaluated in the sagittal plane. RESULTS: The percentage of tibial tunnel aperture contained within the native footprint averaged 71.6% ± 17.2% versus 52.1% ± 23.4% (P = .04) in the IF and TT groups, respectively. The distance from the center of the footprint to the center of the tibial tunnel aperture was 3.50 ± 1.6 mm and 4.40 ± 1.7 mm (P = .27) in the IF and TT groups, respectively. TT drilling placed 6 of 10 tunnels posterior to the center of the footprint versus 3 of 10 tunnels in IF drilling. The graft obliquity angles were 54.8° in TT specimens and 47.5° in IF specimens (P = .09). CONCLUSIONS: This study adds to the literature suggesting that TT drilling with an 8-mm reamer has deleterious effects on tibial tunnel aperture and position. IF drilling, which does not involve repeated reaming of the tibial tunnel, is associated with the placement of a higher percentage of the tunnel aperture within the native tibial footprint. There was not a significant difference between the IF and TT techniques in their ability to place the center of the tibial aperture near the center of the footprint or in graft obliquity. CLINICAL RELEVANCE: ACL reconstruction has continued to evolve in an attempt to restore the functional anatomy and biomechanical behavior of the knee. Tibial tunnel characteristics-such as location, aperture topography, and tunnel obliquity-are important factors to consider in ACL reconstruction. This study compares tibial tunnels after IF and TT techniques.

Decellularized ECM effects on human mesenchymal stem cell stemness and differentiation.

Microenvironment extracellular matrices (ECMs) influence cell adhesion, proliferation and differentiation. The ECMs of different microenvironments have distinctive compositions and architectures. This investigation addresses effects ECMs deposited by a variety of cell types and decellularized with a cold-EDTA protocol have on multipotent human mesenchymal stromal/stem cell (hMSC) behavior and differentiation. The cold-EDTA protocol removes intact cells from ECM, with minimal ECM damage and contamination. The decellularized ECMs deposited by cultured hMSCs, osteogenic hMSCs, and two smooth muscle cell (SMC) lines were tested for distinctive effects on the behavior and differentiation of early passage ('naïve') hMSC plated and cultured on the decellularized ECMs. Uninduced hMSC decellularized ECM enhanced naïve hMSC proliferation and cell motility while maintaining stemness. Decellularized ECM deposited by osteogenic hMSCs early in the differentiation process stimulated naïve hMSCs osteogenesis and substrate biomineralization in the absence of added dexamethasone, but this osteogenic induction potential was lower in ECMs decellularized later in the osteogenic hMSC differentiation process. Decellularized ECMs deposited by two smooth muscle cell lines induced naïve hMSCs to become smooth muscle cell-like with distinctive phenotypic characteristics of contractile and synthetic smooth muscle cells. This investigation demonstrates a useful approach for obtaining functional cell-deposited ECM and highlights the importance of ECM specificity in influencing stem cell behavior.

Transtibial Tunnel Placement in Posterior Cruciate Ligament Reconstruction: How It Relates to the Anatomic Footprint.

BACKGROUND: It is common to place the posterior cruciate ligament (PCL) tibial tunnel with a transtibial technique using a guide that attempts to place the center of the tunnel 1 to 1.5 cm distal to the tibiofemoral joint. It is unknown how well this technique will re-create the native tibial footprint of the PCL. PURPOSE: To evaluate the accuracy of tibial tunnel placement using a transtibial technique. STUDY DESIGN: Controlled laboratory study. METHODS: Ten cadaveric knees from 10 donors underwent arthroscopic transtibial drilling of the tibial tunnel with use of a posteromedial portal for visualization. The transtibial guide was rested flush against the tibial spines to allow for the guide to be as distal as possible, which was between 1 and 1.5 cm distal to the tibiofemoral joint line. Using this technique, an attempt was made to place the tibial tunnels as close to the center of the PCL footprint as possible. All knees underwent computed tomography both pre- and postoperatively with a previously reported technique optimized for ligament evaluation. This allowed comparison of the anatomic PCL tibial footprint to the tibial tunnel aperture. The percentage of tunnel aperture contained within the native footprint as well as the distance from the center of the tunnel aperture to the center of the footprint was measured. RESULTS: The percentage of tunnel aperture contained within the native footprint was 45.9% ± 23.1%. The distance from the center of the tibial tunnel aperture to the center of the tibial PCL footprint was 6.4 ± 2.3 mm. The tunnels were almost always (9/10) distal (or inferior) to the native footprint and either slightly lateral (5/10) or centered (5/10) in a medial to lateral direction. CONCLUSION: This study demonstrates that using the transtibial drilling technique in the tibia for PCL reconstruction places approximately half of the tibial tunnel aperture within the tibial footprint. Generally, the tunnel is distal to the footprint. CLINICAL RELEVANCE: Consideration should be given to the fact that, using this transtibial technique, the tibial tunnel aperture is generally not placed in the center of the footprint. This may not be a negative issue, however, since there are other potential advantages from distal tunnel placement.

Patterned friction and cell attachment on schizophobic polyelectrolyte surfaces.

A series of copolyelectrolytes with randomly positioned fluorinated (hydrophobic) and zwitterionic (hydrophilic) repeat units was synthesized and used to assemble multilayers. Regular layer-by-layer growth was observed for polymers with a charge density as low as 6%. The hydrophobicity of these "schizophobic" surfaces increased with increasing fluorine content. Polymer-on-polymer stamping was used to create patterned areas of low and high friction, probed by lateral force microscopy using a modified hydrophobic tip. "Contractile" A7r5 smooth muscle cells adhered to the fluorinated surfaces, but the introduction of zwitterion functionality induced a motile, less firmly attached morphology consistent with the "synthetic" motile phenotype of this cell line. In contrast with cells well adhered (on fluorinated) or completely nonadhering (on zwitterionic) films, incorporation of closely spaced repeat units with strongly contrasting hydrophobicity appears to generate intermediate cell adhesion behavior.

Inhibition by new glucocorticoid antedrugs [16α, 17α-d] isoxazoline and [16α, 17α-d]-3'-hydroxy-iminoformyl isoxazoline derivatives of chemotaxis and CCL26, CCL11, IL-8, and RANTES secretion.

The underlying inflammation present in chronic airway diseases is orchestrated by increased secretion of CC and CXC chemokines that selectively recruit the leukocyte populations into the pulmonary system. Human chemokines, eotaxins (CCL11 and CCL26), RANTES, and interleukin (IL)-8, are dramatically upregulated through G-protein receptors in cell inflammation, including human asthma. In previous studies, a series of new glucocorticoid antedrugs (GCAs) were synthesized as derivatives of isoxazoline and oxime, and their pharmacological properties based on the antedrug concepts were evaluated. Utilizing both human airway epithelium (HAE) and eosinophil (EOS) cell culture models, we carried out studies to test the hypothesis that new GCA cell treatment would ameliorate Th-1/Th-2-driven secretion of these asthmatic biomarkers, eotaxins (CCL11 and CCL26), RANTES, and IL-8 chemokines, that would in turn decrease recruitment, proliferation, and activation of EOS cells. Results demonstrate that isoxazoline and oxime derivatives exhibit concentration-dependent inhibition, and specifically the compound No. 7 decreases significantly the secretion of eotaxins, RANTES, and IL-8 in cytokine-stimulated HAE cells. It was shown that EOS proliferation and activation were reduced considerably, and cell apoptosis occurred when exposed to nonfluorinated isoxazoline derivatives. These results provide evidence that concentration and structural manipulation of GCAs could increase the anti-inflammatory potency in treatment of chronic diseases, including asthma.

CT pulmonary angiography after total joint arthroplasty: overdiagnosis and iatrogenic harm?

BACKGROUND: CT pulmonary angiography (CTPA) has become widely adopted to detect pulmonary embolism (PE) after total joint arthroplasty (TJA). CTPA is a sensitive tool, which has the ability to detect emboli that may be clinically insignificant. This may lead to iatrogenic harm from overtreatment. QUESTIONS/PURPOSES: The purpose of this study was to assess changing prevalence, mortality, treatment complications, and resource consumption associated with PE after TJA before and after the introduction of CTPA. METHODS: The Nationwide Inpatient Sample was used to identify 2,335,248 patients undergoing TJA from 1993 to 1998 before the introduction of CTPA and 6,321,671 patients from 1999 to 2008 after the introduction of CTPA. Bivariate and multivariate regression analysis was performed to compare changing prevalence of PE, mortality, potential treatment complications of anticoagulation, length of stay, and total charges before and after the introduction of CTPA in patients with PE. RESULTS: In-hospital diagnosis of PE after TJA increased (p < 0.001) from an average of 0.27% to 0.37% after the introduction of CTPA. All-cause mortality in patients with a diagnosis of PE decreased (p < 0.001) from 11.5% to 4.6% (odds ratio, 2.3; 95% confidence interval, 2.1-2.6) after the introduction of CTPA. Overall, PE was associated with increased (p < 0.001) risks for hematoma/seroma, postoperative infection, gastrointestinal bleed, and drug-related thrombocytopenia, although the prevalence of these complications has decreased after 1998 (p < 0.001). Length of stay doubled for patients with PE (both before and after CTPA) and total charges increased over 69% in both study periods for these patients. CONCLUSIONS: Adoption of CTPA appears to be associated with an increase in the diagnosis of PE after TJA and an associated decrease in case-fatality. Although CTPA may improve our ability to diagnose PE and possibly reduce mortality, the observed decrease in case-fatality could also be explained by the overdiagnosis of clinically unimportant emboli. The diagnosis of PE was strongly associated with potential iatrogenic harm from anticoagulation and increased length of stay and hospital charges in this study, emphasizing the importance of further investigation to define the role of CTPA in the diagnosis and treatment of PE after TJA.

Cell durotaxis on polyelectrolyte multilayers with photogenerated gradients of modulus.

Behaviors of rat aortic smooth muscle (A7r5) and human osteosarcoma (U2OS) cells on photo-cross-linked polyelectrolyte multilayers (PEMUs) with uniform, or gradients of, moduli were investigated. The PEMUs were built layer-by-layer with the polycation poly(allylamine hydrochloride) (PAH) and a polyanion poly(acrylic acid) (PAA) that was modified with photoreactive 4-(2-hydroxyethoxy) benzophenone (PAABp). PEMUs with different uniform and gradients of modulus were generated by varying the time of uniform ultraviolet light exposure and by exposure through optical density gradient filters. Analysis of adhesion, morphology, cytoskeletal organization, and motility of the cells on the PEMUs revealed that A7r5 cells established a polarized orientation toward increasing modulus on shallow modulus gradients (approximately 4.7 MPa mm(-1)) and durotaxed toward stiffer regions on steeper gradients (approximately 55 MPa mm(-1)). In contrast, U2OS cells exhibited little orientation or durotaxis on modulus gradients. These results demonstrate the utility of photo-cross-linked PEMUs to direct vascular and osteoblast cell behavior, a potential application for PEMU coatings on biomedical implants.

Anatomic femoral tunnels in posterior cruciate ligament reconstruction: inside-out versus outside-in drilling.

BACKGROUND: During posterior cruciate ligament (PCL) reconstruction, the placement and orientation of the femoral tunnel is critical to postoperative PCL function. PURPOSE: To compare the ability of outside-in (OI) versus inside-out (IO) femoral tunnel drilling in placing the femoral tunnel aperture within the anatomic femoral footprint of the PCL, and to evaluate the orientation of the tunnels within the medial femoral condyle. STUDY DESIGN: Controlled laboratory study. METHODS: Ten matched pairs of cadaver knees were randomized such that within each pair, 1 knee underwent arthroscopic OI drilling and the other underwent IO drilling. All knees underwent computed tomography (CT) both pre- and postoperatively with a technique optimized for ligament evaluation (80 keV with maximum mAs). Commercially available third-party software was used to fuse the pre- and postoperative CT scans, allowing comparison of the PCL footprint to the drilled tunnel. The percentage of tunnel aperture contained within the native footprint, as well as the distance from the center of the tunnel aperture to the center of the footprint, were measured. In addition, the orientation of the tunnels in the coronal and axial planes was evaluated. RESULTS: The OI technique placed 70.4% ± 23.7% of the tunnel within the native femoral footprint compared with 79.8% ± 16.7% for the IO technique (P = .32). The OI technique placed the center of the femoral tunnel 4.9 ± 2.2 mm from the center of the native footprint compared to 5.3 ± 2.0 mm for the IO technique (P = .65). The femoral tunnel angle in the coronal plane was 21.0° ± 9.9° for the OI technique and 37.0° ± 10.3° for the IO technique (P = .002). The tunnel angle in the axial plane was 27.3° ± 4.8° for the OI technique and 39.1° ± 11.5° for the IO technique (P = .01). CONCLUSION: This study demonstrates no difference in the ability of the OI and IO techniques to place the femoral tunnel within the PCL femoral footprint during PCL reconstruction. With the technique parameters used in this study, the IO technique created femoral tunnels with a more vertical and anterior orientation than the OI technique. CLINICAL RELEVANCE: Either technique can be used to place the femoral tunnel within the anatomic footprint. Consideration should be given to tunnel orientation following each technique, and what effect it has on graft bending angles, as these characteristics may affect graft strain and, ultimately, graft failure. In this regard, the IO technique likely produces gentler graft bending angles.

Cytotoxicity of free versus multilayered polyelectrolytes.

The cytotoxicity of polyelectrolytes commonly employed for layer-by-layer deposition of polyelectrolyte multilayers (PEMUs) was assessed using rat smooth muscle A7r5 and human osteosarcoma U-2 OS cells. Cell growth, viability, and metabolic assays were used to compare the responses of both cell lines to poly(acrylic acid), PAA, and poly(allylamine hydrochloride), PAH, in solution at concentrations up to 10 mM and to varying thicknesses of (PAA/PAH) PEMUs. Cytotoxicity correlated with increasing concentration of solution polyelectrolytes for both cell types and was greater for the positively charged PAH than for the negatively charged PAA. While metabolism and proliferation of both cell types was slower on PEMUs than on tissue culture plastic, little evidence for direct toxicity on cells was observed. In fact, evidence for more extensive adhesion and cytoskeletal organization was observed with PAH-terminated PEMUs. Differences in cell activity and viability on different thickness PEMU surfaces resulted primarily from differences in attachment for these adhesion-dependent cell lines.

Human coronary artery smooth muscle cell responses to bioactive polyelectrolyte multilayer interfaces.

Under normal physiological conditions, mature human coronary artery smooth muscle cells (hCASMCs) exhibit a "contractile" phenotype marked by low rates of proliferation and protein synthesis, but these cells possess the remarkable ability to dedifferentiate into a "synthetic" phenotype when stimulated by conditions of pathologic stress. A variety of polyelectrolyte multilayer (PEMU) films are shown here to exhibit bioactive properties that induce distinct responses from cultured hCASMCs. Surfaces terminated with Nafion or poly(styrenesulfonic acid) (PSS) induce changes in the expression and organization of intracellular proteins, while a hydrophilic, zwitterionic copolymer of acrylic acid and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-PAEDAPS) is resistant to cell attachment and suppresses the formation of key cytoskeletal components. Differential expression of heat shock protein 90 and actin is observed, in terms of both their magnitude and cellular localization, and distinct cytoplasmic patterns of vimentin are seen. The ionophore A23187 induces contraction in confluent hCASMC cultures on Nafion-terminated surfaces. These results demonstrate that PEMU coatings exert direct effects on the cytoskeletal organization of attaching hCASMCs, impeding growth in some cases, inducing changes consistent with phenotypic modulation in others, and suggesting potential utility for PEMU surfaces as a coating for coronary artery stents and other implantable medical devices.

Growth/differentiation factor-5 modulates the synthesis and expression of extracellular matrix and cell-adhesion-related molecules of rat Achilles tendon fibroblasts.

This study was designed to examine the cellular and molecular response of tendon fibroblasts to growth/differentiation factor-5 (GDF-5). Rat Achilles tendon fibroblasts (ATFs) were treated in culture with varying concentrations of GDF-5 (0-1000 ng/ml) over varying periods of time (0-12 days). Cell proliferation, evaluated through use of a standard MTT colorimetric assay, confirmed that GDF-5 stimulates ATF proliferation in a concentration- and time-dependent fashion. Temporal and concentration analysis revealed that GDF-5 increases total DNA, glycosaminoglycan (GAG), and hydroxyproline (HYP) content. Ratios of HYP/DNA and GAG/DNA increased with increasing concentrations of GDF-5 (0-1000 ng/ml). Expression of the following 12 extracellular matrix (ECM) and cell-adhesion-related genes was assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR): collagen I (col I), collagen III (col III), matrix metalloproteinases (MMP)-3 and -13, aggrecan, tissue inhibitor of matrix metalloproteinase (TIMP)-2, syndecan-4, N-cadherin, tenascin-C, biglycan, versican, and decorin. RT-PCR data revealed an increase in the expression of col I, col III, MMP-3, MMP-13, TIMP-2, syndecan-4, N-cadherin, tenascin-C, and aggrecan genes by day 6. A statistically significant decrease in TIMP-2 and MMP-13 was observed on day 12. Decorin expression was depressed at all time points in cells treated with GDF-5. There was no significant change in biglycan expression in ATFs supplemented with GDF-5. These findings suggest that GDF-5 induces cellular proliferation and ECM synthesis as well as expression of ECM and cell-adhesion-related genes in ATFs. This study further defines the influence of GDF-5 on rat ATFs through its action on the expression of genes that are associated with tendon ECM.

Smooth muscle cell phenotype modulation and contraction on native and cross-linked polyelectrolyte multilayers.

Smooth muscle cells convert between a motile, proliferative "synthetic" phenotype and a sessile, "contractile" phenotype. The ability to manipulate the phenotype of aortic smooth muscle cells with thin biocompatible polyelectrolyte multilayers (PEMUs) with common surface chemical characteristics but varying stiffness was investigated. The stiffness of (PAH/PAA) PEMUs was varied by heating to form covalent amide bond cross-links between the layers. Atomic force microscopy (AFM) showed that cross-linked PEMUs were thinner than those that were not cross-linked. AFM nanoindentation demonstrated that the Young's modulus ranged from 6 MPa for hydrated native PEMUs to more than 8 GPa for maximally cross-linked PEMUs. Rat aortic A7r5 smooth muscle cells cultured on native PEMUs exhibited morphology and motility of synthetic cells and expression of the synthetic phenotype markers vimentin, tropomyosin 4, and nonmuscle myosin heavy chain IIB (nmMHCIIB). In comparison, cells cultured on maximally cross-linked PEMUs exhibited the phenotype markers calponin, smooth muscle myosin heavy chain (smMHC), myocardin, transgelin, and smooth muscle alpha-actin (smActin) that are characteristic of the smooth muscle "contractile" phenotype. Consistent with those cells being "contractile", A7r5 cells grown on cross-linked PEMUs produced contractile force when stimulated with a Ca(2+) ionophore.

Smooth muscle titin Zq domain interaction with the smooth muscle alpha-actinin central rod.

Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells contain the actin filament-cross-linking protein alpha-actinin. In striated muscle Z-disks, alpha-actinin interacts with N-terminal domains of titin to provide a structural linkage crucial for the integrity of the sarcomere. We previously discovered a long titin isoform, originally smitin, hereafter sm-titin, in smooth muscle and demonstrated that native sm-titin interacts with C-terminal EF hand region and central rod R2-R3 spectrin-like repeat region sites in alpha-actinin. Reverse transcription-PCR analysis of RNA from human adult smooth muscles and cultured rat smooth muscle cells and Western blot analysis with a domain-specific antibody presented here revealed that sm-titin contains the titin gene-encoded Zq domain that may bind to the alpha-actinin R2-R3 central rod domain as well as Z-repeat domains that bind to the EF hand region. We investigated whether the sm-titin Zq domain binds to alpha-actinin R2 and R3 spectrin repeat-like domain loops that lie in proximity with two-fold symmetry on the surface of the central rod. Mutations in alpha-actinin R2 and R3 domain loop residues decreased interaction with expressed sm-titin Zq domain in glutathione S-transferase pull-down and solid phase binding assays. Alanine mutation of a region of the Zq domain with high propensity for alpha-helix formation decreased apparent Zq domain dimer formation and decreased Zq interaction with the alpha-actinin R2-R3 region in surface plasmon resonance assays. We present a model in which two sm-titin Zq domains interact with each other and with the two R2-R3 sites in the alpha-actinin central rod.

Implications of lumbar plexus anatomy for removal of total disc replacements through a posterior approach.

STUDY DESIGN: An anatomic study in which the lumbar plexuses of 14 embalmed cadavers were dissected bilaterally and measured using a posterior approach. OBJECTIVE: To determine the cephalocaudal (root-to-root) distances and the mediolateral (root-to-tether) distances within the lumbar plexus and determine the feasibility for removal of a lumbar total disc replacement (TDR) through these anatomic spaces using a posterior approach. SUMMARY OF BACKGROUND DATA: Currently, lumbar TDRs are implanted primarily through an anterior retroperitoneal or transperitoneal approach. However, revision surgeries through these approaches can be complicated by significant adhesions, with potential injuries to intra- and retroperitoneal contents. Advancements in accessing anterior column structures through a posterior lumbar extracavitary approach suggest that posterior removal of TDRs may be an alternative. Unlike the thoracic extracavitary approach in which ligation of the thoracic nerve rarely leaves significant morbidity, the lumbar extracavitary approach cannot rely on the analogous ligation of the lumbar root to achieve access. Therefore, feasibility of the lumbar extracavitary approach depends on the presence of sufficient anatomic space between the tethered nerves of the lumbar plexus. METHODS: Fourteen adult cadavers (5 M/9F) were dissected through a posterior approach to expose the lumbar plexus bilaterally. The root-to-root distances at levels L2-S1 and corresponding root-to-tether distances at levels L3-L5 were measured bilaterally. RESULTS: Root-to-root distance was smallest at the male L5-S1 interval (11.7 +/- standard deviations 4.1 mm). Root-to-tether distance was smallest at the female L5 (43.1 +/- standard deviations 8.4 mm). These plexus measurements compare favorably with the CHARITE TDR components, in which the thickest sliding core is 11.0 mm in height and the largest endplate is 42.0 mm in width. CONCLUSION: This anatomic study suggests that posterior TDR removal is possible in the lumbar spine without undue risk to the surrounding nervous structures.

Molecular identification and localization of cellular titin, a novel titin isoform in the fibroblast stress fiber.

We previously discovered a large titin-like protein-c-titin-in chicken epithelial brush border and human blood platelet extracts that binds alpha-actinin and organizes arrays of myosin II bipolar filaments in vitro. RT-PCR analysis of total RNA from human megakaryoblastic (CHRF-288-11) and mouse fibroblast (3T3) nonmuscle cells reveal sequences identical to known titin gene exon sequences that encode parts of the Z-line, I-band, PEVK domain, A-band, and M-line regions of striated muscle titins. In the nonmuscle cells, these sequences are differentially spliced in patterns not reported for any striated muscle titin isoform. Rabbit polyclonal antibodies raised against expressed protein fragments encoded by the Z-repeat and kinase domain regions react with the c-titin band in Western blot analysis of platelet extracts and immunoprecipitate c-titin in whole platelet extracts. Immunofluorescent localization demonstrates that the majority of the c-titin colocalizes with alpha-actinin and actin in 3T3 and Indian Muntjac deer skin fibroblast stress fibers. Our results suggest that differential expression of titin gene exons in nonmuscle cells yields multiple novel isoforms of the protein c-titin that are associated with the actin stress fiber structures.

Fibronectin and cell attachment to cell and protein resistant polyelectrolyte surfaces.

Culture of A7r5 smooth muscle cells on a polyelectrolyte multilayer film (PEMU) can influence various cell properties, including adhesion, motility, and cytoskeletal organization, that are modulated by the extracellular matrix (ECM) in vivo. ECM contribution to cell behavior on PEMUs was investigated by determining the amount of fibronectin (FN) bound to charged and hydrophobic PEMUs by optical waveguide lightmode spectroscopy and immunofluorescence microscopy. FN bound best to poly(allylamine hydrochloride) (PAH)-terminated and Nafion-terminated PEMUs. FN bound poorly to PEMUs terminated with a copolymer of poly(acrylic acid) (PAA) and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-AEDAPS). Cells adhered and spread well on the Nafion-terminated PEMU surfaces. In contrast, cells spread less and migrated more on both FN-coated and uncoated PAH-terminated PEMU surfaces. Both cells and FN interacted much better with Nafion than with PAA-co-PAEDAPS in a micropatterned PEMU. These results indicate that A7r5 cell adhesion, spreading, and motility on PEMUs can be independent of FN binding to the surfaces.