Versatile wedge-based system for the construction of unidirectional collagen scaffolds by directional freezing: practical and theoretical considerations.

Aligned unidirectional collagen scaffolds may aid regeneration of those tissues where alignment of cells and extracellular matrix is essential, as for instance in cartilage, nerve bundles, and skeletal muscle. Pores can be introduced by ice crystal formation followed by freeze-drying, the pore architecture reflecting the ice crystal morphology. In this study we developed a wedge-based system allowing the production of a wide range of collagen scaffolds with unidirectional pores by directional freezing. Insoluble type I collagen suspensions were frozen using a custom-made wedge system, facilitating the formation of a horizontal as well as a vertical temperature gradient and providing a controlled solidification area for ice dendrites. The system permitted the growth of aligned unidirectional ice crystals over a large distance (>2.5 cm), an insulator prolonging the freezing process and facilitating the construction of crack-free scaffolds. Unidirectional collagen scaffolds with tunable pore sizes and pore morphologies were constructed by varying freezing rates and suspension media. The versatility of the system was indicated by the construction of unidirectional scaffolds from albumin, poly(vinyl alcohol) (a synthetic polymer), and collagen-polymer blends producing hybrid scaffolds. Macroscopic observations, temperature measurements, and scanning electron microscopy indicated that directed horizontal ice dendrite formation, vertical ice crystal nucleation, and evolutionary selection were the basis of the aligned unidirectional ice crystal growth and, hence, the aligned unidirectional pore structure. In conclusion, a simple, highly adjustable freezing system has been developed allowing the construction of large (hybrid) bioscaffolds with tunable unidirectional pore architecture.

Preparation and characterization of injectable fibrillar type I collagen and evaluation for pseudoaneurysm treatment in a pig model.

OBJECTIVE: Despite the efficacy of collagen in femoral artery pseudoaneurysm treatment, as reported in one patient study, its use has not yet gained wide acceptance in clinical practice. In this particular study, the collagen was not described in detail. To further investigate the potential of collagen preparations, we prepared and characterized highly purified injectable fibrillar type I collagen and evaluated its use for femoral artery pseudoaneurysm (PSA) treatment in vivo using a pig model. METHODS: Purified fibrillar type I collagen was characterized using electron microscopy. The effect of three different sterilization procedures, ie, hydrogen peroxide gas plasma (H2O2), ethylene oxide gas (EtO), and gamma irradiation, was studied on both SDS-PAGE and platelet aggregation. Different collagen injectables were prepared (3%, 4%, and 5%) and tested using an injection force test applying a 21-gauge needle. To evaluate the network characteristics of the injectable collagen, the collagen was suspended in phosphate buffered saline (PBS) at 37°C and studied both macroscopically and electron microscopically. To determine whether the collagen induced hemostasis in vivo, a pig PSA model was used applying a 4% EtO sterilized collagen injectable, and evaluation by angiography and routine histology. RESULTS: Electron microscopy of the purified type I collagen revealed intact fibrils with a distinct striated pattern and a length<300 µm. Both SDS-PAGE and platelet aggregation analysis of the sterilized collagen indicated no major differences between EtO and H2O2 sterilization, although gamma-irradiated collagen showed degradation products. Both 3% and 4% (w/v) collagen suspensions were acceptable with respect to the force used (<50 N). The 4% suspension was selected as the preferred injectable collagen, which formed a dense network under physiologic conditions. Testing the collagen in vivo (n=5), the angiograms revealed that the PSA partly or completely coagulated. Histology confirmed the network formation, which was surrounded by thrombus. CONCLUSIONS: Collagen injectables were prepared and EtO sterilized without major loss of structural integrity and platelet activity. In vivo, the injectable collagen formed a dense network and triggered (partial) local hemostasis. Although optimization is needed, an injectable collagen may be used as a therapeutic agent for femoral PSA treatment.

An animal model for femoral artery pseudoaneurysms.

PURPOSE: To prepare a porcine model for femoral artery pseudoaneurysm via a one-step surgical procedure without the need for microsurgery. MATERIALS AND METHODS: This pseudoaneurysm model involves the preparation of an arteriovenous shunt between the femoral artery and femoral vein in which approximately 2 cm of the vein is segmented by proximal and distal closure with the use of ligatures. The femoral pseudoaneurysm models were evaluated by angiography, Doppler auscultation, and histologic examination. RESULTS: In seven of eight pigs, angiography and Doppler auscultation showed that the pseudoaneurysm models were open and that there was communication between the pseudoaneurysm model and the femoral artery. The mean length (+/-SD) of the pseudoaneurysm model was 1.9 cm +/- 0.3 (n= 7), with a neck region of 4 mm. Histologic analysis confirmed that pseudoaneurysm models were open and no thrombi were observed. CONCLUSIONS: The principal advantages of this model are the location of the pseudoaneurysm model, the short period of clamping, and the controllable size. The pig pseudoaneurysm model is straightforward and reproducible, and may serve as a useful tool in the evaluation of interventional strategies for treatment of pseudoaneurysms.

Fetal abdominal wall repair with a collagen biomatrix in an experimental sheep model for gastroschisis.

We evaluated the regeneration of the abdominal wall using a dual-layer collagen biomatrix, and the protective effect on the bowel of fetal abdominal wall repair in a fetal sheep model for gastroschisis. In 14 fetal lambs, the abdominal wall was opened at 79 days' gestation, creating a gastroschisis. In group 1, the gastroschisis was left uncovered. In group 2, the bowel was repositioned, and the defect was closed by suturing a collagen biomatrix into the abdominal wall. A cesarean section was performed at 140 days' gestation, and macroscopic and histological evaluation was performed. In the five lambs with a gastroschisis, the eviscerated part of the bowel was coalescent, showed extensive adhesions, and was covered by fibrous peel. In group 2, the abdominal wall had closed, with a firm connection to the native abdominal wall. The biomatrix was largely degraded and replaced by connective tissue with collagen and fibroblasts, neovascularisation, and scattered muscle cells. Minor or no adhesions of the bowel and no peel formation were observed. Abdominal wall tissue replacement using a collagen biomatrix was feasible in fetal lambs, resulting in a closed abdominal wall at birth. Immediate closure of the gastroschisis strongly diminished or prevented bowel adhesions and peel formation.

Fetal bladder wall regeneration with a collagen biomatrix and histological evaluation of bladder exstrophy in a fetal sheep model.

OBJECTIVES: To evaluate histological changes in an animal model for bladder exstrophy and fetal repair of the bladder defect with a molecular-defined dual-layer collagen biomatrix to induce fetal bladder wall regeneration. METHODS: In 12 fetal lambs the abdominal wall and bladder were opened by a midline incision at 79 days' gestation. In 6 of these lambs an uncorrected bladder exstrophy was created by suturing the edges of the opened bladder to the abdominal wall (group 1). The other 6 lambs served as a repair group, where a dual-layer collagen biomatrix was sutured into the bladder wall and the abdominal wall was closed (group 2). A caesarean section was performed at 140 days' gestation, followed by macroscopic and histological examination. RESULTS: Group 1 showed inflammatory and maturational changes in the mucosa, submucosa and detrusor muscle of all the bladders. In group 2, bladder regeneration was observed, with urothelial coverage, ingrowth of fibroblasts and smooth muscle cells, deposition of collagen, neovascularization and nerve fibre formation. This tissue replaced the collagen biomatrix. No structural changes of the bladder were seen in group 2. CONCLUSIONS: The animal model, as in group 1, for bladder exstrophy shows remarkable histological resemblance with the naturally occurring anomaly in humans. This model can be used to develop new methods to salvage or regenerate bladder tissue in bladder exstrophy patients. Fetal bladder wall regeneration with a collagen biomatrix is feasible in this model, resulting in renewed formation of urothelium, blood vessels, nerve fibres, ingrowth of smooth muscle cells and salvage of the native bladder.

Fatty-acid-binding protein from the flight muscle of Locusta migratoria: evolutionary variations in fatty acid binding.

Intracellular lipid-binding proteins have evolved from a common ancestral gene with the appearance of mitochondrial oxidation, to guarantee, for example, transport of fatty acids through the aqueous cytosol to their site of utilization. The mammalian forms of these lipid carriers are structurally well-characterized and have been categorized, on the basis of sequence similarities and several typical ligand-binding features, into four subfamilies. Only a single complex structure of an invertebrate fatty-acid-binding protein (FABP) has been reported to date, which reveals a unique ligand-binding arrangement yet unknown in vertebrate FABPs. In the present study, the structure of a second invertebrate FABP (locust muscle) complexed with a fatty acid has been determined on the basis of intermolecular NOE connectivities between the protein and the uniformly (13)C-enriched oleate ligand. The resulting ligand conformation, although resembling the closely related mammalian heart- and adipocyte-type FABPs, is characterized by certain binding features that differ significantly from the typical hairpin-turn ligand shapes of the latter forms. This is primarily due to an alanine-to-leucine substitution in locust FABPs that produces a steric hindrance for ligand binding. A comparison with an FABP from tobacco hornworm larvae furthermore demonstrates that certain amino acid substitutions that appear to be specific for invertebrates decidedly influence the binding arrangement inside the protein cavity. Hence, as a result of these evolutionary variations, invertebrate FABPs may display a much greater diversity in intracellular lipid binding than observed for the mammalian transport proteins, thus possibly providing new insights for the design of modified lipid carriers.

Reversal of lidocaine with epinephrine epidural anesthesia using epidural saline washout.

BACKGROUND AND OBJECTIVES: Prolonged motor and sensory block following epidural anesthesia can be associated with extended postoperative care unit stays and patient dissatisfaction. Previous studies have demonstrated a more rapid motor recovery following the administration of epidural crystalloids in patients who had received plain bupivacaine and lidocaine epidural anesthesia. However, epinephrine is commonly added to local anesthetics to improve the quality and prolong the duration of the epidural block. The objective of this study was to determine the relationship of 0.9% NaCl epidural catheter flush volume (i.e., washout) to the recovery of motor and sensory block in patients undergoing 2% lidocaine with epinephrine epidural anesthesia. METHODS: A prospective, randomized, double-blind study design was utilized. Thirty-three subjects scheduled for elective gynecologic or obstetrical surgical procedures underwent epidural anesthesia using 2% lidocaine with epinephrine (1:200,000). A T4 dermatome level of analgesia, determined by toothpick prick, was maintained intraoperatively. Following surgery, subjects were randomized to 1 of 3 treatment groups. Group 1 (control, n = 11) received no epidural 0.9% NaCl (normal saline [NS]) postoperatively. Group 2 (15 mL NS x 1, n = 10) received an epidural bolus of 15 mL NS. Group 3 (15 mL NS x 2, n = 12) received an epidural bolus of 15 mL NS postoperatively and a second 15-mL NS bolus 15 minutes later. Assessment of motor and sensory block was performed at 15-minute intervals until complete motor and sensory recovery. RESULTS: Times to partial and full motor and sensory recovery were significantly faster in the epidural NS groups than in the control group. Full motor recovery was more rapid in subjects receiving two 15-mL NS epidural NS boluses (30 mL total) compared with those receiving a single 15-mL NS bolus (108 +/- 9 min v 136 +/- 13 min) and significantly faster than control group subjects (153 +/- 14 min). Both NS x 1 and NS x 2 epidural bolus groups experienced significantly reduced times to complete sensory recovery when compared with the control group (NS x 1 = 154 +/- 13 min, NS x 2 = 153 +/- 9 min, control 195 +/- 14 min). CONCLUSIONS: A more rapid recovery of motor and sensory block in patients undergoing 2% lidocaine with epinephrine epidural anesthesia can be achieved with the use of 30 mL NS epidural washout. Reg Anesth Pain Med 2001;26:246-251.

A preliminary examination of genetic variation in a peripheral population of Blanding's turtle, Emydoidea blandingii.

Random amplified polymorphic DNA (RAPD) was used to compare the Nova Scotia population of Blanding's turtle (Emydoidea blandingii) with several populations from the species' main range. The Nova Scotia population is believed to have been isolated from the main range for 4000-8000 years. Cluster analysis using a neighbour-joining algorithm produced a dendrogram showing the Nova Scotia population clustering separately from those populations in the main range. Analysis of molecular variance shows 34.28% of total variance to be accounted for between the Nova Scotia population and populations in the main range. While this study is preliminary, the results suggest that the Nova Scotia population of Blanding's turtle may be important to the maintenance of genetic diversity in the species.