Pharmacokinetics of intravenous and transdermal fentanyl in alpacas.

The purpose of the study was to determine pharmacokinetics of fentanyl after intravenous (i.v.) and transdermal (t.d.) administration to six adult alpacas. Fentanyl was administered i.v. (2 µg/kg) or t.d. (nominal dose: 2 µg kg-1  hr-1 ). Plasma concentrations were determined using liquid chromatography-mass spectrometry. Heart rate and respiratory rate were assessed. Extrapolated, zero-time plasma fentanyl concentrations were 6.0 ng/ml (1.7-14.6 ng/ml) after i.v. administration, total plasma clearance was 1.10 L hr-1  kg-1 (0.75-1.40 L hr-1  kg-1 ), volumes of distribution were 0.30 L/kg (0.10-0.99 L/kg), 1.10 L/kg (0.70-2.96 L/kg) and 1.5 L/kg (0.8-3.5 L/kg) for V1 , V2 , and Vss , respectively. Elimination half-life was 1.2 hr (0.5-4.3 hr). Mean residence time (range) after i.v. dosing was 1.30 hr (0.65-4.00 hr). After t.d. fentanyl administration, maximum plasma fentanyl concentration was 1.20 ng/ml (0.72-3.00 ng/ml), which occurred at 25 hr (8-48 hr) after patch placement. The area under the plasma fentanyl concentration-vs-time curve (extrapolated to infinity) after t.d. fentanyl was 61 ng*hr/ml (49-93 ng*hr/ml). The dose-normalized bioavailability of fentanyl from t.d. fentanyl in alpacas was 35.5% (27-64%). Fentanyl absorption from the t.d. fentanyl patch into the central compartment occurred at a rate of approximately 50 µg/hr (29-81 µg/hr) between 8 and 72 hr after patch placement.

The use of sedatives, analgesic and anaesthetic drugs in the horse: an electronic survey of members of the American Association of Equine Practitioners (AAEP).

REASONS FOR PERFORMING STUDY: To determine the sedative, analgesic and anaesthetic drugs and techniques that are used by equine veterinarians. HYPOTHESIS OR OBJECTIVES: To provide equine veterinarians with information concerning veterinary use of anaesthetic techniques, a reflection of the collective experiences of the profession. METHODS: A survey was conducted of those members of the American Association of Equine Practitioners (AAEP) with an electronic mail address on file with the organisation using proprietary, web-based software. The survey was comprised of 30 questions divided into 8 sections: nonsteroidal anti-inflammatory drugs; local anaesthesia; alternative techniques; standing chemical restraint; epidural anaesthesia; short-term anaesthesia; long-term anaesthesia; and a place for the respondent to make comments. RESULTS: The response rate was 13.8% (952/6911) AAEP member veterinarians primarily use phenylbutazone and flunixin as anti-inflammatory drugs, and lidocaine and mepivacaine for local anaesthesia. Combinations of drugs are preferred for standing chemical restraint. While many veterinarians frequently utilise short-term anaesthesia, longer anaesthesia is less frequently performed. CONCLUSIONS: Most AAEP member veterinarians use sedatives in combination to provide standing chemical restraint. Extra-label use of drugs is a core component of current equine sedation and anaesthetic practice. POTENTIAL RELEVANCE: Equine veterinarians can compare their choices of anaesthetic drugs with others practising equine medicine and surgery and may be stimulated to investigate alternative methods of providing comfort to horses.

Effect of intravenous lidocaine administration on laminar inflammation in the black walnut extract model of laminitis.

REASONS FOR PERFORMING STUDY: Laminitis is a serious complication of horses suffering from sepsis/endotoxaemia-related events. Laminitis in horses and organ injury in human sepsis are both reported to involve inflammatory injury to the laminae/organs including early activation of endothelium and leucocytes leading to emigration of neutrophils into the tissue interstitium. In the black walnut extract (BWE) model, systemic inflammatory events coincide with marked increase in laminar mRNA concentrations of inflammatory genes including proinflammatory cytokines (i.e. IL-1beta, IL-6), COX-2, chemokines (i.e. IL-8) and endothelial adhesion molecules (i.e. ICAM-1 and E-selectin). In models of human sepsis, i.v. lidocaine has been reported to decrease leucocyte and endothelial activation, and the expression of proinflammatory cytokines and chemokines. OBJECTIVES: To evaluate the effect of i.v. lidocaine therapy on the inflammatory processes documented to occur in the BWE model of laminitis. METHODS: Twelve horses were administered BWE and treated immediately with either lidocaine (1.3 mg/kg bwt bolus, followed by 0.05 mg/kg bwt/min CRI, n=6) or saline (n=6) for 10 h. At 10 h post BWE administration, laminar samples were obtained under general anaesthesia for assessment of proinflammatory gene expression (using RT-qPCR) and leucocyte emigration (via CD13 immunohistochemistry). At 0, 3 and 10 h post BWE administration, skin samples were obtained for assessment of leucocyte emigration (via calprotectin immunohistochemistry). RESULTS: No significant differences between groups were noted for inflammatory gene mRNA concentrations (IL-1beta, IL-6, IL-8, COX-2) or for number of leucocytes present within the laminar interstitium or skin dermis. Increased (P<0.05) laminar E-selectin mRNA concentrations were present in the LD group (vs. SAL group). CONCLUSIONS: Continuous administration of i.v. lidocaine does not inhibit inflammatory events in either the laminae or skin in the horse administered black walnut extract. POTENTIAL RELEVANCE: This work questions the use of continuous i.v. administration of lidocaine as an effective anti-inflammatory therapy for systemic inflammation.

An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3-mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation.

The synthetic peptide Gly-Arg-Gly-Asp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts. Well-defined adhesion substrates were prepared with GRGDY between 10(-3) fmol/cm2 and 10(4) fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized. The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 10(4) fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin alpha V beta 3 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface-bound GRGDY, while fibronectin receptor antiserum specific for alpha 5 beta 1 did not. Furthermore, alpha V beta 3 was observed to cluster into focal contacts in spread cells, but alpha 5 beta 1 did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for alpha V beta 3-mediated cellular spreading, while 140 nm was required for alpha V beta 3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts; these spacings represent much fewer ligands than were previously thought to be required.

Pharmacokinetics of detomidine administered to horses at rest and after maximal exercise.

REASON FOR PERFORMING STUDY: Increased doses of detomidine are required to produce sedation in horses after maximal exercise compared to calm or resting horses. OBJECTIVES: To determine if the pharmacokinetics of detomidine in Thoroughbred horses are different when the drug is given during recuperation from a brief period of maximal exercise compared to administration at rest. METHODS: Six Thoroughbred horses were preconditioned by exercising them on a treadmill. Each horse ran a simulated race at a treadmill speed that caused it to exercise at 120% of its maximal oxygen consumption. One minute after the end of exercise, horses were treated with detomidine. Each horse was treated with the same dose of detomidine on a second occasion a minimum of 14 days later while standing in a stocks. Samples of heparinised blood were obtained at various time points on both occasions. Plasma detomidine concentrations were determined by liquid chromatography-mass spectrometry. The plasma concentration vs. time data were analysed by nonlinear regression analysis. RESULTS: Median back-extrapolated time zero plasma concentration was significantly lower and median plasma half-life and median mean residence time were significantly longer when detomidine was administered after exercise compared to administration at rest. Median volume of distribution was significantly higher after exercise but median plasma clearance was not different between the 2 administrations. CONCLUSIONS AND POTENTIAL RELEVANCE: Detomidine i.v. is more widely distributed when administered to horses immediately after exercise compared to administration at rest resulting in lower peak plasma concentrations and a slower rate of elimination. The dose requirement to produce an equivalent effect may be higher in horses after exercise than in resting horses and less frequent subsequent doses may be required to produce a sustained effect.

A bioactive injectable bulking material; a potential therapeutic approach for stress urinary incontinence.

Stress urinary incontinence (SUI) is a life changing condition, affecting 20 million women worldwide. In this study, we developed a bioactive, injectable bulking agent that consists of Permacol™ (Medtronic, Switzerland) and recombinant insulin like growth factor-1 conjugated fibrin micro-beads (fib_rIGF-1) for its bulk stability and capacity to induce muscle regeneration. Therefore, Permacol™ formulations were injected in the submucosal space of rabbit bladders. The ability of a bulking material to form a stable and muscle-inducing bulk represents for us a promising therapeutic approach to achieve a long-lasting treatment for SUI. The fib_rIGF-1 showed no adverse effect on human smooth muscle cell metabolic activity and viability in vitro based on AlamarBlue assays and Live/Dead staining. Three months after injection of fib_rIGF-1 together with Permacol™ into the rabbit bladder wall, we observed a smooth muscle tissue like formation within the injected materials. Positive staining for alpha smooth muscle actin, calponin, and caldesmon demonstrated a contractile phenotype of the newly formed smooth muscle tissue. Moreover, the fib_rIGF-1 treated group also improved the neovascularization at the injection site, confirmed by CD31 positive staining compared to bulks made of PermacolTM only. The results of this study encourage us to further develop this injectable, bioactive bulking material towards a future therapeutic approach for a minimal invasive and long-lasting treatment of SUI.

Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.

New generations of synthetic biomaterials are being developed at a rapid pace for use as three-dimensional extracellular microenvironments to mimic the regulatory characteristics of natural extracellular matrices (ECMs) and ECM-bound growth factors, both for therapeutic applications and basic biological studies. Recent advances include nanofibrillar networks formed by self-assembly of small building blocks, artificial ECM networks from protein polymers or peptide-conjugated synthetic polymers that present bioactive ligands and respond to cell-secreted signals to enable proteolytic remodeling. These materials have already found application in differentiating stem cells into neurons, repairing bone and inducing angiogenesis. Although modern synthetic biomaterials represent oversimplified mimics of natural ECMs lacking the essential natural temporal and spatial complexity, a growing symbiosis of materials engineering and cell biology may ultimately result in synthetic materials that contain the necessary signals to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis.

MICROPATTERNING OF GOLD SUBSTRATES BASED ON POLY(PROPYLENE SULFIDE-BL-ETHYLENE GLYCOL), (PPS-PEG) BACKGROUND PASSIVATION AND THE MOLECULAR-ASSEMBLY PATTERNING BY LIFT-OFF (MAPL) TECHNIQUE.

Poly(propylene sulfide-bl-ethylene glycol (PPS-PEG) is an amphiphilic block copolymer that spontaneously adsorbs onto gold from solution. This results in the formation of a stable polymeric layer that renders the surface protein resistant when an appropriate architecture is chosen. The established molecular assembly patterning by lift-off (MAPL) technique can convert a prestructured resist film into a pattern of biointeractive chemistry and a noninteractive background. Employing the MAPL technique, we produced a micron-scale PPS-PEG pattern on a gold substrate, and then characterized the patterned structure with Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) and Atomic Force Microscopy (AFM). Subsequent exposure of the PPS-PEG/gold pattern to protein adsorption (full human serum) was monitored in situ; SPR-imaging (i-SPR) shows a selective adsorption of proteins on gold, but not on PPS-PEG areas. Analysis shows a reduction of serum adsorption up to 93% on the PPS-PEG areas as compared to gold, in good agreement with previous analysis of homogenously adsorbed PPS-PEG on gold. MAPL patterning of PPS-PEG block copolymers is straightforward, versatile and reproducible, and may be incorporated into biosensor-based surface analysis methods.

Part II: Fibroblasts preferentially migrate in the direction of principal strain.

A growing body of evidence suggests that the sensory information from the cytoskeleton and integrins may be responsible for guiding migration during mechano- and haptotaxis. However, the dual function of these subcellular structures as mechano-sensors and -actuators is only partially understood. Using a new cell chamber described in the preceding companion paper (Ref to part I, Raeber et al. 2007a) we investigated the migration response of adhesion-dependent fibroblasts embedded 3-dimensionally within synthetic protease-sensitive poly(ethylene glycol) hydrogels to stepwise and cyclic mechanical loads. To that end, we developed a spatially and temporally resolved migration analysis technique capable of providing estimates of statistical cell migration parameters along and perpendicular to the main strain direction. Fibroblasts reoriented themselves in the direction of principal strain, increased their proteolytic migration activity and moved preferentially parallel to the principal strain axis. These results point to a possible correlation between planes of iso-strain and migration direction.

Part I: A novel in-vitro system for simultaneous mechanical stimulation and time-lapse microscopy in 3D.

To investigate the migration response of cells to changes in their biophysical environment, a novel uniaxial cell stimulation device (UCSD) has been designed and tested. The device is capable of applying very precise user-defined static or dynamic mechanical stimuli in a physiologically relevant strain window (up to 50%) and frequency bandwidth (up to 2 Hz) to cells residing in a three-dimensional (3D) environment while single-cell migration is simultaneously measured by time-lapse microscopy. The system is an advancement over uniaxial loading devices reported to date in that it allows temporal and spatial quantification of migration as a function of the micromechanical environment. We make use of the favorable physical and biological properties of poly(ethylene glycol) hydrogels as model matrix and present a method for fabricating cell-containing hydrogel constructs. The 3D strain field within these constructs is modeled by finite element analysis. Fibroblasts reversibly altered their morphology and orientation in response to the strain field. In the succeeding companion paper we then exploit the system to analyze fibroblast motility induced by different stimulation regimes (refer to part II).

Fiber density of collagen grafts impacts rabbit urethral regeneration.

There is a need for efficient and "off-the-shelf" grafts in urethral reconstructive surgery. Currently available surgical techniques require harvesting of grafts from autologous sites, with increased risk of surgical complications and added patient discomfort. Therefore, a cost-effective and cell-free graft with adequate regenerative potential has a great chance to be translated into clinical practice. Tubular cell-free collagen grafts were prepared by varying the collagen density and fiber distribution, thereby creating a polarized low fiber density collagen graft (LD-graft). A uniform, high fiber density collagen graft (HD-graft) was engineered as a control. These two grafts were implanted to bridge a 2 cm long iatrogenic urethral defect in a rabbit model. Histology revealed that rabbits implanted with the LD-graft had a better smooth muscle regeneration compared to the HD-graft. The overall functional outcome assessed by contrast voiding cystourethrography showed patency of the urethra in 90% for the LD-graft and in 66.6% for the HD-graft. Functional regeneration of the rabbit implanted with the LD-graft could further be demonstrated by successful mating, resulting in healthy offspring. In conclusion, cell-free low-density polarized collagen grafts show better urethral regeneration than high-density collagen grafts.

Microfluidic production of bioactive fibrin micro-beads embedded in crosslinked collagen used as an injectable bulking agent for urinary incontinence treatment.

Endoscopic injection of bulking agents has been widely used to treat urinary incontinence, often due to urethral sphincter complex insufficiency. The aim of the study was to develop a novel injectable bioactive collagen-fibrin bulking agent restoring long-term continence by functional muscle tissue regeneration. Fibrin micro-beads were engineered using a droplet microfluidic system. They had an average diameter of 140 µm and recombinant fibrin-binding insulin-like growth factor-1 (α2PI1-8-MMP-IGF-1) was covalently conjugated to the beads. A plasmin fibrin degradation assay showed that 72.5% of the initial amount of α2PI1-8-MMP-IGF-1 loaded into the micro-beads was retained within the fibrin micro-beads. In vitro, the growth factor modified fibrin micro-beads enhanced cell attachment and the migration of human urinary tract smooth muscle cells, however, no change of the cellular metabolic activity was seen. These bioactive micro-beads were mixed with genipin-crosslinked homogenized collagen, acting as a carrier. The collagen concentration, the degree of crosslinking, and the mechanical behavior of this bioactive collagen-fibrin injectable were comparable to reference samples. This novel injectable showed no burst release of the growth factor, had a positive effect on cell behavior and may therefore induce smooth muscle regeneration in vivo, necessary for the functional treatment of stress and other urinary incontinences. STATEMENT OF SIGNIFICANCE: Urinary incontinence is involuntary urine leakage, resulting from a deficient function of the sphincter muscle complex. Yet there is no functional cure for this devastating condition using current treatment options. Applied physical and surgical therapies have limited success. In this study, a novel bioactive injectable bulking agent, triggering new muscle regeneration at the injection site, has been evaluated. This injectable consists of cross-linked collagen and fibrin micro-beads, functionalized with bound insulin-like growth factor-1 (α2PI1-8-MMP-IGF-1). These bioactive fibrin micro-beads induced human smooth muscle cell migration in vitro. Thus, this injectable bulking agent is apt to be a good candidate for regeneration of urethral sphincter muscle, ensuring a long-lasting treatment for urinary incontinence.

Mechanisms of 3-D migration and matrix remodeling of fibroblasts within artificial ECMs.

The elucidation of molecular cell-extracellular matrix (ECM) interactions regulating tissue dynamics necessitates straightforward model systems that can dissect the associated physiological complexity into a smaller number of distinct interactions. Here we employ a previously developed artificial ECM model system to study dynamic cell-matrix interactions involved in proteolytic three-dimensional (3-D) migration and matrix remodeling at the level of single cells. Quantitative time-lapse microscopy of primary human fibroblasts exposed to exogenous physiological matrix metalloproteinase (MMP) inhibitors revealed that 3-D migration is dependent on cell seeding density and occurred via highly localized MMP- and tissue inhibitor of metalloproteinases-2-dependent processes. Stimulation of cells by tumor necrosis factor alpha led to a striking augmentation in fibroblast migration that was accompanied by induction of alphaVbeta3 integrin expression. In long-term cultures, extensive localized cellular matrix remodeling resulted in the morphogenesis of single cells into interconnected multicellular networks. Therefore, these tailor-made artificial ECMs can replicate complex 3-D cell-matrix interactions involved in tissue development and regeneration, an important step in the design of next-generation synthetic biomaterials for tissue engineering.

Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension.

Fibrin plays an important role in wound healing and regeneration, and enjoys widespread use in surgery and tissue engineering. The enzymatic activity of Factor XIIIa was employed to covalently incorporate exogenous bioactive peptides within fibrin during coagulation. Fibrin gels were formed with incorporated peptides from laminin and N-cadherin alone and in combination at concentrations up to 8.2 mol peptide per mole of fibrinogen. Neurite extension in vitro was enhanced when gels were augmented with exogenous peptide, with the maximal improvement reaching 75%. When this particular fibrin derivative was evaluated in rats in the repair of the severed dorsal root within polymeric tubes, the number of regenerated axons was enhanced by 85% relative to animals treated with tubes filled with unmodified fibrin. These results demonstrate that it is possible to enhance the biological activity of fibrin by enzymatically incorporating exogenous oligopeptide domains of morphoregulatory proteins.

Thermally-induced glass formation from hydrogel nanoparticles.

Amphiphilic hydrogel nanoparticles, composed of covalently cross-linked Pluronic F127 and PEG, exhibit a temperature- and concentration-dependent gelation in water which is interpreted as a colloidal glass formation. The possible applications of these phenomena in biomaterials and controlled release are also discussed.

Antagonism of detomidine sedation in the horse using intravenous tolazoline or atipamezole.

REASONS FOR PERFORMING STUDY: The ability to shorten the duration of sedation would potentially improve safety and utility of detomidine. OBJECTIVES: To determine the effects of tolazoline and atipamezole after detomidine sedation. HYPOTHESIS: Administration of tolazoline or atipamezole would not affect detomidine sedation. METHODS: In a randomised, placebo-controlled, double-blind, descriptive study, detomidine (0.02 mg/kg bwt i.v.) was administered to 6 mature horses on 4 separate occasions. Twenty-five mins later, each horse received one of 4 treatments: Group 1 saline (0.9% i.v.) as a placebo control; Group 2 atipamezole (0.05 mg/kg bwt i.v.); Group 3 atipamezole (0.1 mg/kg bwt i.v.); and Group 4 tolazoline (4.0 mg/kg bwt i.v.). Sedation, muscle relaxation and ataxia were scored by 3 independent observers at 9 time points. Horses were led through an obstacle course at 7 time points. Course completion time was recorded and the ability of the horse to traverse the course was scored by 3 independent observers. Horses were videotaped before, during and after each trip through the obstacle course. RESULTS: Atipamezole and tolazoline administration incompletely antagonised the effects of detomidine, but the time course to recovery was shortened. CONCLUSIONS AND POTENTIAL RELEVANCE: Single bolus administration of atipamezole or tolazoline produced partial reversal of detomidine sedation and may be useful for minimising detomidine sedation.

IGF-1-containing multi-layered collagen-fibrin hybrid scaffolds for bladder tissue engineering.

UNLABELLED: Clinical success of bladder reconstructive procedures could be promoted by the availability of functional biomaterials. In this study, we have developed a multi-layered scaffold consisting of a bioactive fibrin layer laminated between two collagen sheets all having undergone plastic compression. With this construct we performed bladder augmentation in a nude rat model after partial bladder excision and evaluated the morphological and functional behavior of the implant. The fibrin was functionalized with a recombinant human insulin-like growth factor-1 (IGF-1) variant that covalently binds fibrin during polymerization and has a matrix metalloproteinase-cleavage insert to enable cell-mediated release. The purified IGF-1 variant showed similar bioactivity in vitro compared to commercially available wild type (wt) IGF-1, inducing receptor phosphorylation and induction of human smooth muscle cell proliferation. In vivo, the multi-layered bioactive collagen-fibrin scaffolds loaded with the IGF-1 variant triggered dose-dependent functional host smooth muscle cell invasion and bundle formation with re-urothelialization 4weeks after surgery in a rat model. STATEMENT OF SIGNIFICANCE: The design of new bio-functional scaffolds that can be employed for bladder reconstructive procedures is a growing focus in the field of tissue engineering. In this study, a fibrin binding form of human insulin-like growth factor-1 (IGF-1) was produced and used to functionalize a multi-layered collagen-fibrin scaffold consisting of bioactive fibrin layer, sandwiched between two collagen gels. An effective dosage of our IGF-1 variant was successfully determined via a nude rat bladder model, which may play a critical role in estimating its therapeutic dosage in clinical trials. Thus, this new bioactive scaffold may offer an advanced approach to accelerate bladder regeneration.

The selective modulation of endothelial cell mobility on RGD peptide containing surfaces by YIGSR peptides.

The ability of the biomimetic peptides YIGSR, PHSRN and RGD to selectively affect adhesion and migration of human microvascular endothelial cells (MVEC) and vascular smooth muscle cells (HVSMC) was evaluated. Cell mobility was quantified by time-lapse video microscopy of single cells migrating on peptide modified surfaces. Polyethylene glycol (PEG) hydrogels modified with YIGSR or PHSRN allowed only limited adhesion and no spreading of MVEC and HVSMC. However, when these peptides were individually combined with the strong cell binding peptide RGD in PEG hydrogels, the YIGSR peptide was found to selectively enhance the migration of MVEC by 25% over that of MVEC on RGD alone (p<0.05). No corresponding effect was observed for HVSMC. This suggests that the desired response of specific cell types to tissue engineering scaffolds could be optimized through a combinatory approach to the use of biomimetic peptides.

3D morphology of cell cultures: a quantitative approach using micrometer synchrotron light tomography.

Current issues in both tissue engineering and cell biology deal with cell behavior extensively in 3D. Here, we explore synchrotron radiation micro-computed tomography as a tool for morphological characterization of such 3D cellular constructs, providing micrometer resolution in soft and hard tissues. Novel image processing techniques allowed quantification of local and global cell distributions, cell density, adhesive cell culture surface, and scaffold geometry. For proof of concept, we applied this technique to characterize the morphology of two cell cultures of different phenotypes, namely human dermal fibroblasts and mouse calvarial osteoblast-like cells, both seeded on a polymer multifilament yarn. From 3D visualizations in these case studies, we saw that the fibroblasts spanned between the yarn filaments and in this way encapsulated the yarn, whereas the osteoblast-like cells lined the filament surfaces and did not span between them. Differences found in cell distribution as a function of distance to the median yarn axis and the closest filament surface, respectively, quantified these qualitative impressions gained from 3D visualizations. Moreover, the volume-normalized adhesive surface differed by one order of magnitude between the two phenotypes. Our approach allows quantitative correlation of local scaffold geometry and cell morphology. It can be used to investigate the influence of cell phenotype as well as various biochemical agents on tissue engineering constructs and the behavior of cells in culture.

Self-assembly and steric stabilization at heterogeneous, biological surfaces using adsorbing block copolymers.

BACKGROUND: We present the synthesis, characterization and initial structure-function analysis of a new class of bioactive agent that allows the application of techniques from colloid science to biological surfaces. Stable colloidal suspensions can be generated by immobilizing a dense brush of soluble polymer at the colloidal surface, creating a zone protected against the adhesion of approaching particles, a phenomenon termed polymeric steric stabilization. This is often accomplished for aqueous colloidal dispersions using adsorbing block copolymers. We demonstrate that water-soluble block copolymers can be designed to adsorb onto heterogeneous biological surfaces and block cell-cell and cell-surface adhesion, using polymer compositions and architectures that are quite different from surfactants used for stabilizing nonbiological colloidal dispersions. RESULTS: Comb copolymers were synthesized having polycationic backbones (poly-L-lysine, PLL), serving as the anchor for binding to the net negatively charged biological surfaces, grafted with water-soluble polynonionic chains (polyethylene glycol, PEG), to block biological recognition, producing PLL-graft-PEG copolymers. Specific copolymers were found to sterically stabilize red blood cells from lectin-induced hemagglutination and fibroblasts from adhesion to fibronectin-coated surfaces. The polymer design principles, which appear to be unique for adsorption to heterogeneous biological surfaces, require the use of very high molecular weight comb copolymers, perhaps because anionic sites are non-uniformly distributed on biological surfaces, and the ability of larger copolymers to span between highly anionic sites. CONCLUSIONS: Water-soluble copolymers were produced that can block recognition at biological surfaces, on the basis of nonspecific physicochemical phenomena rather than specific biochemical interactions.