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Polyethylene terephthalate (PET) mesh is one of the most commonly used synthetic biomaterials for tension-free hernia repair. In an effort to improve the biocompatibility of PET mesh, gold nanoparticles (AuNP) in various concentrations were conjugated to the PET surface to develop PET-AuNP scaffolds. These novel scaffolds were characterized with Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) to assess the addition of functional groups, presence of AuNPs, and thermal stability of the modified PET mesh, respectively. The biocompatibility of the PET-AuNP scaffolds was evaluated through in vitro cell culture assays. The cellularity of cells exposed to the PET-AuNP scaffolds, as well as the scaffolds' ability to reduce reactive oxygen species, was assessed using L929 murine fibroblasts. Antimicrobial properties of AuNPs conjugated to PET mesh were tested against the bacteria Pseudomonas aeruginosa. Results from the FT-IR showed presence of COOH groups while SEM displayed bonding of AuNPs to the PET surface. DSC results indicated that the PET more than likely did not undergo any detrimental degradation due to the surface modification. Results from the in vitro studies showed that AuNPs, in optimal concentrations (1× concentrations), enhanced cellularity, reduced ROS, and reduced bacteria adhesion to PET. These studies demonstrated enhanced biocompatibility of the AuNP conjugated PET mesh over pristine PET mesh., (Copyright © 2011 Wiley Periodicals, Inc.)

As one of the most common proteins found in the human body, collagen is regarded as biocompatible and has many properties making it ideal for soft-tissue repair applications. However, collagen matrices fabricated from purified forms of collagen are notoriously weak and easily degraded by the body. The extracellular matrix of many tissues including human dermis, porcine dermis, and porcine small intestine submucosa are often utilized instead, and several of these scaffolds are crosslinked. Crosslinking has been shown to improve the mechanical properties of collagenous tissues and increase their resistance to degradation. In this study we investigated two novel "bionanocomposite" materials in which either gold nanoparticles or silicon carbide nanowires were crosslinked to a porcine tendon. Scanning electron micrographs confirmed that the nanomaterials were successfully crosslinked to the tissues. A collagenase assay, tensile testing, flow cytometry, and bioreactor studies were also performed to further characterize the properties of these novel materials. The results of these studies indicated that crosslinking porcine diaphragm tissues with nanomaterials resulted in scaffolds with improved resistance to enzymatic degradation and appropriate biocompatibility characteristics, thus warranting further study of these materials for soft tissue repair and tissue engineering applications., (Copyright © 2011 Wiley Periodicals, Inc.)

Carbon nanotubes (CNT) possess many unique electrical and mechanical properties that make them useful for a variety of industrial and biomedical applications. They are especially attractive materials for biomedical applications since their dimensions are similar to components of the extracellular matrix. In this study, amine-functionalized single-walled carbon nanotubes were crosslinked to an acellular porcine diaphragm tendon. The resulting bionanocomposite scaffolds were subjected to a number of materials characterization techniques including a collagenase assay, uniaxial tensile testing, modulated differential scanning calorimetry, and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to determine whether the properties of the original extracellular matrix were altered by the treatment processes. A variety of SWCNT concentrations were investigated. While none of the conditions investigated resulted in bionanocomposites with significantly improved physicochemical properties, no detrimental effects were observed due to any of the processing steps. Future studies should be performed to determine if carbon nanotubes can influence cellular adhesion and function in order to promote rapid integration and remodeling., (Copyright © 2011 Wiley Periodicals, Inc.)

Two novel, bionanocomposite scaffolds were evaluated in a rodent model over the course of three months to determine whether these scaffolds possessed adequate biocompatibility characteristics to warrant further evaluation as possible tissue reconstruction scaffolds. These bionanocomposite scaffolds were comprised of amine-functionalized gold nanoparticles (AuNP) or silicon carbide nanowires (SiCNW) crosslinked to an acellular porcine diaphragm tendon. It was hypothesized that the addition of nanomaterials to the porcine tendon would also improve its biocompatibility by imparting a nanostructured surface. As early as seven days after implantation, both types of bionanocomposite scaffolds displayed evidence of granulation tissue and the beginning of scaffold remodeling with new collagen deposited by the host, and by ninety-seven days the bionanocomposite scaffolds were completely remodeled with no evidence of any adverse host tissue reaction or scar tissue formation. The AuNP bionanocomposite scaffolds exhibited accelerated scaffold remodeling compared to the SiCNW scaffolds., (Copyright © 2010 Wiley Periodicals, Inc.)

Extracellular matrix (ECM) materials are currently utilized for soft tissue repair applications such as vascular grafts, tendon reconstruction, and hernia repair. These materials are derived from tissues such as human dermis and porcine small intestine submucosa, which must be rendered acellular to prevent disease transmission and decrease the risk of an immune response. The ideal decellularization technique removes cells and cellular remnants, but leaves the original collagen architecture intact. The tissue utilized in this study was the central tendon of the porcine diaphragm, which had not been previously investigated for soft tissue repair. Several treatments were investigated during this study including peracetic acid, TritonX-100, sodium dodecyl sulfate, and tri(n-butyl) phosphate (TnBP). Of the decellularization treatments investigated, only 1% TnBP was effective in removing cell nuclei while leaving the structure and composition of the tissue intact. Overall, the resulting acellular tissue scaffold retained the ECM composition, strength, resistance to enzymatic degradation, and biocompatibility of the original tissue, making 1% TnBP an acceptable decellularization treatment for porcine diaphragm tendon., (Copyright © 2010 Wiley Periodicals, Inc.)

This study utilized spectral and thermal analysis of explanted hernia mesh materials to determine material inertness and elucidate reasons for hernia mesh explantation. Composite mesh materials, comprised of polypropylene (PP) and expanded polytetrafluoroethylene (ePTFE) mesh surrounded by a polyethylene terephthalate (PET) ring, were explanted from humans. Scanning electron microscopy (SEM) was conducted to visually observe material defects while attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to find chemical signs of surface degradation. Modulated differential scanning calorimetry (MDSC) and thermogravimetric analysis (TGA) gave thermal stability profiles that showed changes in heat of fusion and rate of percent weight loss, respectively. ATR-FTIR scans showed higher carbonyl peak areas as compared to pristine for 91% and 55% of ePTFE and PP explants, respectively. Ninety-one percent of ePTFE explants also exhibited higher C--H stretch peak areas. Seventy-three percent of ePTFE explants had higher heats of fusion while 64% of PP explants had lower heats of fusion with respect to their corresponding pristines. Only 9% of PET explants exhibited a lower heat of fusion than pristine. Seventy-three percent of ePTFE explants, 73% of PP explants, and only 18% of PET explants showed a decreased rate of percent weight loss as compared to pristine. The majority of the PP and ePTFE mesh explants demonstrated oxidation and crosslinking, respectively, while the PET ring exhibited breakdown at the sites of high stress. The results showed that all three materials exhibited varied degrees of chemical degradation suggesting that a lack of inertness in vivo contributes to hernia mesh failure.

Optical sensors have proven to be a useful method in identifying explosive devices by recognizing vapors of explosive compounds that become airborne and emanate from the device. To detect high explosive compounds such as TNT, a molecularly imprinted polymer (MIP) sensing mechanism was developed. This mechanism consists of MIP microparticles prepared using methacrylic acid as the functional monomer. The MIP microparticles are then combined with fluorescent quantum dots via a simple cross-linking procedure. The result is a highly robust optical sensing scheme that is capable of functioning in an array of environmental conditions. To study the sensing mechanisms's ability to detect nitroaromatic analytes, the fluorescent-labeled MIP particles were tested for their performance in detecting aqueous 2,4-dinitrotoluene (DNT), a nitroaromatic molecule very similar to TNT, as well as TNT itself. These preliminary data indicate that the system is capable of detecting nitroaromatic compounds in solution with high sensitivity, achieving lower limits of detection of 30.1 and 40.7 microM for DNT and TNT, respectively. The detection mechanism also acted rapidly, with response times as low as 1 min for TNT. Due to the results of this study, it can be concluded that the fluorescent-labeled MIP system is a feasible method for detecting high explosives, with the potential for future use in detecting vapors from explosive devices.

Nanoporous materials can provide significant benefits to the field of biosensors. Their size and porous structure makes them an ideal tool for improving sensor performance. This study characterized a novel ultra low index of refraction nanoporous organosilicate (NPO) material for use as an optical platform for fluorescence-based optical biosensors. While serving as the low index cladding material, the novel coating based on organosilicate nanoparticles also provides an opportunity for a high surface area coating that can be utilized for immobilizing biological probes. Biological molecules were immobilized onto NPO, which was spin-coated on silicon and glass substrates. The biological molecule was composed of Protein A conjugated to AlexaFluor 546 fluorophore and then immobilized onto the NPO substrate via silanization. Sample analysis consisted of spectrofluorometry, FT-IR spectroscopy, scanning electron microscopy, contact angle measurement and ellipsometry. The results showed the presence of emission peaks at 574 nm, indicating that the immobilization of Protein A to the NPO material is possible. When compared to Si and glass substrates not coated with NPO, the results showed a 100X and 10X increase in packing density with the NPO coated films respectively. Ellipsometric analysis, FT-IR, contact angle, and SEM imaging of the surface immobilized NPO films suggested that while the surface modifications did induce some damage, it did not incur significant changes to its unique characteristics, i.e., pore structure, wettability and index of refraction. It was concluded that NPO films would be a viable sensor substrate to enhance sensitivity and improve sensor performance.

Regional anesthesia is preferred over general anesthesia for many surgical procedures; however, challenges associated with poor image guidance limit its widespread acceptance as a viable alternative. In B-mode ultrasound images, the current standard for guidance, nerves can be difficult to visualize due to their similar acoustic impedance with surrounding tissues and needles must be aligned within the imaging plane at limited angles of approach that can impede successful peripheral nerve anesthesia. These challenges lead to inadequate regional anesthesia, necessitating intraoperative interventions, and can cause complications, including hemorrhage, intraneural injections and even nerve paralysis. ARFI imaging utilizes acoustic radiation force to generate images that portray relative tissue stiffness differences. Peripheral nerves are typically surrounded by many different tissue types (e.g., muscle, fat and fascia) that provide a mechanical basis for improved image contrast using ARFI imaging over conventional B-mode images. ARFI images of peripheral nerves and needles have been generated in cadaveric specimens and in humans in vivo. Contrast improvements of >600% have been achieved for distal sciatic nerve structures. The brachial plexus has been visualized with improved contrast over B-mode images in vivo during saline injection and ARFI images can delineate nerve bundle substructures to aid injection guidance. Physiologic motion during ARFI imaging of nerves near arterial structures has been successfully suppressed using ECG-triggered image acquisition and motion filters. This work demonstrates the feasibility of using ARFI imaging to improve the visualization of peripheral nerves during regional anesthesia procedures.

A capillary-based optical biosensor has been developed to detect calpastatin, an indicator of meat tenderness. Longissimus muscle samples (n=11) were extracted from beef carcasses at 0 and 48h post-mortem. These samples were assayed for calpastatin by traditional laboratory methods and with a newly developed capillary tube biosensor as well as for Warner-Bratzler shear force (WBSF) and crude protein and the responses were compared. Additionally, the response from the capillary-based biosensor was compared to a previously developed optical fiber biosensor. When the 0 and 48h sampling periods were combined, the capillary tube biosensor was moderately accurate in predicting calpastatin activity (R(2)=0.6058). There was less variation in the 0h capillary tube biosensor compared to the 0h pre-column (P=0.006) and post-column optical fiber biosensors (P=0.047), therefore the capillary tube biosensor is a more precise system of measurement. This research further advances the development of a calpastatin biosensor and makes online assessment one step closer to reality.

An optical fiber biosensor to detect calpastatin has been investigated as a preliminary step in developing tenderness detection instrumentation. Longissimus dorsi samples were taken from beef carcasses (n=21) at 0, 24, 36 and 48h postmortem. Muscle homogenates were assayed for calpastatin activity using traditional methods and an optical fiber biosensor. Warner-Bratzler shear force was also performed on a steak from each carcass at 14d postmortem. Results demonstrated that the measurements with highest correlation between traditional calpastatin assays and optical biosensor readings were taken at 48h postmortem (r=0.597, P< or =0.01), suggesting that this is the best time for use of this biosensor in an on-line grading system. This research further advances the development of a calpastatin biosensor and would be useful in laboratory determination of the presence of biologically active calpastatin concentrations.

Hernia repair with prosthetic mesh significantly decreases the rate of recurrence compared with traditional, primary suture repair by reducing the tension on the edges of the wound. However, there are several complications associated with the use of mesh that may be due to the chronic inflammatory reaction to the mesh or a loss of compliance after degradation of the material. Mesh contraction and migration can also occur, sometimes resulting in a recurrent hernia. Based on the chemical structure of the polypropylene mesh material and the physiological conditions to which it is subjected, it is possible that oxidation is responsible for these changes in material properties. Oxidation would result in surface cracking, decreased melting temperature, loss of mass, and reduced compliance of the material. The objective of this study was to identify physiochemical changes in the surface and bulk properties of explanted polypropylene hernia meshes compared to pristine polypropylene mesh materials. Several characterization techniques were utilized, including scanning electron microscopy, differential scanning calorimetry, thermogravimetric analysis, and compliance testing. Overall, the results supported our hypothesis that oxidation is involved with the degradation of polypropylene hernia mesh materials.

Although polypropylene has been used as a hernia repair material for nearly 50 years, very little science has been applied to studying the body's effect on this material. It is possible that oxidation of mesh occurs as a result of the chemical structure of polypropylene and the physiological conditions to which it is subjected; this leads to embrittlement of the material, impaired abdominal movement, and chronic pain. It is also possible that lightweight polypropylene meshes undergo less oxidation due to a reduced inflammatory reaction. The objective of this study was to characterize explanted hernia meshes using techniques such as scanning electron microscopy, differential scanning calorimetry, thermogravimetric analysis, and compliance testing to determine whether the mesh density of polypropylene affects the oxidative degradation of the material. The hypothesis was that heavyweight polypropylene would incite a more intense inflammatory response than lightweight polypropylene and thus undergo greater oxidative degradation. Overall, the results support this theory.

Chronic infection of a prosthetic mesh implant is a severe complication of ventral hernia repair, and mesh explantation is usually required in these cases. Biologic mesh implants have a possible role in ventral hernia repair in this setting. Here we present a case of chronic mesh infection following ventral hernia repair and the use of a biologic mesh to repair the existing defect following explantation of the infected mesh. Analysis of the explant material demonstrated possible oxidative degradation of the original polypropylene. A review of the literature follows.

Background: Development of implantable biosensors for disease detection is challenging because of poor biocompatibility of synthetic materials. A possible solution involves engineering interface materials that promote selfassembly and adhesion of autologous cells on sensor surfaces. Crosslinked type-I collagen is an acceptable material for developing engineered basement membranes. In this study, we used functionalized gold nanoparticles as the crosslinking agent. Functionalized nanoparticles provide sites for crosslinking collagen as well as sites to deliver signaling compounds that direct selfassembly and reduce inflammation. The goal of this study was to obtain a quantitative parameter to objectively determine the presence of crosslinks., Methods: We analyzed TEM images of collagen fibrils by two methods: Run length analysis and topology analysis after medial axis transform., Results: Run length analysis showed a significant reduction of the interfibril spaces in the presence of nanoparticles (change of 40%, P < 0.05), whereas the fibril thickness remained unchanged. In the topological network, the number of elements, number of branches and number of sides increased significantly in the presence of nanoparticles (P < 0.05). Other parameters, especially the number of loops showed only a minimal and nonsignificant change. We chose a ratiometric parameter of the number of branches normalized by the number of loops to achieve independence from gross fibril density. This parameter is lower by a factor of 2.8 in the presence of nanoparticles (P < 0.05)., Conclusion: The numerical parameters presented herein allow not only to quantify fibril mesh complexity and crosslinking, but also to help quantitatively compare cell growth and adhesion on collagen matrices of different degree of crosslinking in further studies.

A biosensor that is portable and permits on-site analysis of samples would significantly reduce the large economical burden of food products recalls. A fiber optic portable biosensor utilizing the principle of fluorescence resonance energy transfer (FRET) was developed for fast detection of Salmonella typhimurium (S. typhimurium) in ground pork samples. Labeled antibody-protein G complexes were formed via the incubation of anti-Salmonella antibodies labeled with FRET donor fluorophores (Alexa Fluor 546) and protein G (PG) labeled with FRET acceptor fluorophores (Alexa Fluor 594). Utilizing silanization, the labeled antibodies-PG complexes were then immobilized on decladded, tapered silica fiber cores to form the evanescent wave-sensing region. The biosensors were tested in two different solutions: (1) PBS doped with S. typhimurium and (2) homogenized pork sample with S. typhimurium. The fiber probes tested in a S. typhimurium doped phosphate buffered solution demonstrated the feasibility of the biosensor for detecting S. typhimurium as well as determined the optimal packing density of the labeled antibody-PG complexes on the surface of fibers. The results showed that a packing density of 0.033 mg/ml produced the lowest limit of detection of 10(3)cells/ml with 8.2% change in fluorescence. The fiber probes placed in homogenized pork samples inoculated with S. typhimurium showed a limit of detection of 10(5)CFU/g with a 6.67% in fluorescence within a 5-min response time. These results showed that the FRET-based fiber optic biosensor can become a useful analytical tool for detection of S. typhimurium in real food samples.

Unlabelled: Historically, general anesthesia has been the "gold standard" for surgeons and patients when major hip surgery is being done. The recent introductions of improved techniques and catheters for continuous peripheral nerve blocks have made regional anesthesia more attractive to patients and surgeons. We focus on current trends and future directions in perioperative pain management for major orthopaedic procedures done on the hip. The use of epidural or spinal anesthesia during major hip surgery has been linked to a reduced risk of perioperative complications like deep venous thrombosis, less deterioration of cerebral and pulmonary functions in patients who are at high risk for complications, and overall reduced blood loss. In addition, continuous peripheral nerve blocks showed effective and safe postoperative pain control, allowing for lower opioids consumption, improved and earlier rehabilitation, and high patient satisfaction. Accurate patient selection and patient education are fundamental for the success of any regional anesthesia technique. Modern regional anesthesia for major hip surgery includes the use of a single shot and continuous epidural injections, single-shot and continuous spinal injection, continuous lumbar plexus blockade, and continuous peripheral blockade of the femoral and sciatic nerves. Continuous peripheral nerve blocks represent an adjunctive, effective, and safe technique for postoperative pain control after total hip arthroplasty. Future directions in postoperative pain control include the creation of a comprehensive system that supervises the use of continuous peripheral nerve blocks outside the acute inpatient setting for few days following the surgical procedure., Level of Evidence: Therapeutic study, Level V (expert opinion). See the Guidelines for Authors for a complete description of levels of evidence.

Background and Objectives: Continuous lumbar plexus infusion of local anesthetic after total knee arthroplasty has been shown to improve analgesia and early recovery as compared with patient-controlled analgesia (PCA) morphine. Any benefit of an infusion over a single-injection lumbar plexus block has not been directly shown however., Methods: In a double-blind, randomized, controlled trial, 32 patients undergoing total knee arthroplasty were randomly allocated to 1 of 2 groups: 0.1% levobupivacaine infusion or saline infusion. Preoperatively, all patients received a lumbar plexus block with 25 mL 0.5% levobupivacaine using a posterior approach with a catheter left in situ, a sciatic nerve block with 15 mL 0.5% levobupivacaine, and a spinal anesthetic. At the end of surgery, 0.1% levobupivacaine or saline was infused into the catheter at 10 mL/h for 48 hours. All patients also received PCA morphine. The primary endpoint was morphine use from the PCA machine. Secondary endpoints included pain scores, day of first postoperative mobilization, and nausea., Results: Patients receiving the levobupivacaine infusion used significantly less morphine than those receiving saline (19 mg [interquartile range (IQR) 8.5-29.5] vs 32 mg [IQR 23.5-53.0], P = .04) and also mobilized earlier postoperatively (day 1 or 2 [levobupivacaine] vs day 2 or 3 [saline], P = .001). Pain scores were similar., Conclusion: Postoperative infusion of local anesthetic around the lumbar plexus reduces morphine requirement and improves early recovery after total knee arthroplasty as compared with a single-injection block.

Objectives: To test the interrater reliability of a standardized method to analyze knee bone mineral density (BMD) using dual-energy x-ray absorptiometry (DXA); to compare spine, hip, and knee BMD of people with spinal cord injury (SCI) with able-bodied controls; and to determine the relation between hip BMD and knee BMD in SCI and able-bodied subjects., Design: Criterion standard and masked comparison., Setting: Primary care university hospital., Participants: A convenience sample of 11 subjects with complete SCI was age and sex matched with 11 able-bodied control subjects., Interventions: Not applicable., Main Outcome Measures: Four raters analyzed regions of interest according to operational definitions recently developed to standardize the analysis of BMD of the knee. Subjects with chronic SCI and matched controls underwent conventional DXA scans of the spine and hips and "less conventional" scans of the distal femurs and proximal tibias. The relation between hip and knee BMD was analyzed., Results: The knee measurements were highly reliable (femur intraclass correlation coefficient model 2,1 [ICC(2,1)]=.98; tibia ICC(2,1)=.89). Subjects with SCI had lower BMD values than controls at all hip and knee sites (P<.05). Lumbar spine BMD did not differ between groups. Hip BMD was moderately predictive of distal femur BMD (R2=.67), but less correlated with the proximal tibia (R2=.38)., Conclusions: Knee BMD can be reliably analyzed using DXA with this protocol. Subjects with SCI have diminished knee and hip BMD. Low hip BMD is associated with low distal femur BMD.

We have been investigating a fluorescence dual binding biosensor to detect calpastatin. Calpastatin is a protein found in meat and it is a regulator of meat tenderness. The ability to accurately predict the calpastatin concentration of beef with a biological sensor at the time of grading would lead to a more accurate assessment of the overall palatability of beef when it reaches the consumer. Meat can then be labeled as tender or tough, which would greatly enhance meat processors' ability to grade meat, allowing them to recover lost revenue. The biosensor technique utilized the chemical transduction principle of fluorescence resonance energy transfer (FRET). FRET requires the use of two fluorophores, termed a donor and acceptor. In this study, the donor fluorophore was conjugated to the protein, mu-calpain, while the acceptor fluorophore was conjugated to a monoclonal antibody. The results showed that in the presence of calpastatin, the labeled mu-calpain and antibody would bind to calpastatin, reducing the distance between the two proteins and eliciting a measurable change in fluorescence. The FRET dual binding technique was tested in heated and unheated meat extract, and a limit of detection for calpastatin was 120 ng/ml in diluted heated meat extract with no significant response in the unheated meat extract. Stable response times were achieved within 5 min. The proof-of-principle of utilizing a FRET dual binding technique to detect calpastatin in heated meat extract has been established.

Dystonia is a neurological movement disorder characterised by sustained muscle contractions, producing twisting and repetitive movements or abnormal posture. It is often accompanied by chronic pain. Specialist nurses now treat and support many patients.

A novel optical biosensor technique is being developed for the early detection of myocardial infarction by utilizing the distance-dependent chemical transduction method of fluorescence resonance energy transfer (FRET). The FRET process requires two fluorophores termed the donor and the acceptor. When in close proximity, the donor absorbs energy from the excitation source and non-radiatively transfers the energy to the acceptor, which in turn emits fluorescent energy. This distance-dependent property was utilized to detect conformational changes when antibodies combine with their respective antigens. The fluorophores were conjugated to an antibody-Protein A complex and then immobilized via silanization to the distal ends of optical fibers. Three different antibody-Protein A complexes were immobilized: generic IgG, cardiac Troponin T (cTnT), and cardiac Troponin I (cTnI). Results showed that upon the addition of the specific antigens, the antibodies underwent a conformational change, reducing the distance between the FRET fluorophores. The generic IgG responded to 233 nM antigens, whereas the cTnT biosensor had a limit of detection of 75 nM, and the cTnI biosensors had a limit of detection of 94 nM.

We report three patients undergoing cervical spine surgery who required awake fiberoptic intubation, and in whom sedation was provided using a dexmedetomidine infusion. Dexmedetomidine was used to provide a moderate level of sedation without causing respiratory distress or hemodynamic instability during fiberoptic intubation. Conditions for intubation were acceptable in all three patients after co-administration of topical anesthesia. Dexmedetomidine may serve as a useful adjunct for this procedure. The anesthetic management and anesthetic implications of using dexmedetomidine infusions for awake fiberoptic intubation are discussed.

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.

Objectives: To describe the onset of phantom leg pain in an amputee with the performance of a lumbar plexus block and the subsequent alleviation after the performance of a sciatic nerve block., Case Report: A 72-year-old American Society of Anesthesiologists physical status III woman presented for left total hip arthroplasty. Her history was significant for a left below the knee amputation. Since the amputation she had suffered from intermittent phantom leg pain. A lumbar plexus block was performed for postoperative pain management. After the lumbar plexus block, the patient experienced severe pain radiating to the left phantom foot. Because of the severity of the phantom pain, a sciatic nerve block was performed. The phantom leg pain resolved within 5 minutes. The intraoperative care under general anesthesia was uneventful. After surgery the patient had continued blockade in both nerve distributions with excellent analgesia. Full recovery of the lumbar plexus and sciatic nerve function was present on the first postoperative day., Conclusion: The temporal relationship between the onset of the phantom leg pain and the lumbar plexus block suggests a causal relationship. In this case, it appears that ongoing peripheral input from the lumbar plexus may have been sufficient for the tonic inhibition of phantom pain in the sciatic distribution. The immediate reactivation of the phantom pain and its subsequent relief suggests dynamic processing of peripheral inputs by central neurons, which apparently is rapid and reversible in some cases of phantom pain.

Accurate identification of surface landmarks is essential for the successful performance of peripheral nerve blocks. The variability between experienced and inexperienced practitioners in identifying anatomical landmarks has not been studied previously. Anaesthetists were asked to identify the point of needle insertion for posterior lumbar plexus and sciatic nerve blocks on a volunteer using a standard textbook description. The chosen point for needle insertion was described in terms of X and Y co-ordinates, measured in millimetres, from a zero reference point marked on a volunteer's back. Fifteen experienced and 22 inexperienced anaesthetists took part in the study. The lumbar plexus block mean [range] values for the X, Y co-ordinates were 80 [62-108], 66 [46-86] and 92 [49-150], 62 [0-131] in the experienced and inexperienced groups, respectively. The sciatic nerve block X, Y co-ordinates were 77 [62-99], 70 [49-89] and 68 [29-116], 62 [26-93] in the experienced and inexperienced groups, respectively. The variance for the point of needle insertion was significantly greater in the inexperienced group (p <0.01) for both the lumbar plexus and sciatic nerve blocks. We conclude that with increasing experience, there is decreased variability in determining the point of needle insertion using anatomical landmarks.

Background and Objectives: Central nervous system and cardiac toxicity following the administration of local anesthetics is a recognized complication of regional anesthesia. Levobupivacaine, the pure S(-) enantiomer of bupivacaine, was developed to improve the cardiac safety profile of bupivacaine. We describe 2 cases of grand mal seizures following accidental intravascular injection of levobupivacaine., Case Report: Two patients presenting for elective orthopedic surgery of the lower limb underwent blockade of the lumbar plexus via the posterior approach. Immediately after the administration of levobupivacaine 0.5% with epinephrine 2.5 microgram/mL, the patients developed grand mal seizures, despite negative aspiration for blood and no clinical signs of intravenous epinephrine administration. The seizures were successfully treated with sodium thiopental in addition to succinylcholine in 1 patient. Neither patient developed signs of cardiovascular toxicity. Both patients were treated preoperatively with beta-adrenergic antagonist medications, which may have masked the cardiovascular signs of the unintentional intravascular administration of levobupivacaine with epinephrine., Conclusions: Although levobupivacaine may have a safer cardiac toxicity profile than racemic bupivacaine, if adequate amounts of levobupivacaine reach the circulation, it will result in convulsions. Plasma concentrations sufficient to result in central nervous system toxicity did not produce manifestations of cardiac toxicity in these 2 patients.

In this paper, we present a potential biomolecular patterning method, laser-guided direct writing guidance (LGDW), which may be utilized to deposit organic and bioactive particles for biosensor arrays. The instrumentation and operation of the LGDW system is introduced and the system settings used to achieve deposition are reported. The biomolecule, avidin, was deposited onto a substrate using LGDW to evaluate the possible damage from the laser on the biomolecules. The functionality of avidin after laser-based guidance was examined by exposing the deposited avidin molecules to its ligand, biotin. The results show some avidin retained its affinity to biotin after LGDW demonstrating little damage to the biomolecules.

The ability to provide an extended duration of analgesia of days following a single injection without the need for catheters, pumps and infusion systems would be a great benefit in acute and chronic pain. Exciting progress is being made in our ability to combine local anaesthetics with liposomes and polymer microspheres. These interesting formulations in animal models have allowed up to 4 days of analgesia. Their use clinically will be a great advance which could possibly occur in the near future.

The development of a dual receptor detection method for enhanced biosensor monitoring was investigated by analyzing potential fluorescent resonance energy transfer (FRET) pairs. The dual receptor scheme requires the integration of a chemical transducer system with two unique protein receptors that bind to a single biological agent. The two receptors are tagged with special molecular groups (donors and acceptors fluorophores) while the chemical transduction system relies on the well-known mechanisms of FRET. During the binding event, the two FRET labeled receptors dock at the binding sites on the surface of the biological agent. The resulting close proximity of the two fluorophores upon binding will initiate the energy transfer resulting in fluorescence. The paper focuses on the analysis and optimization of the chemical transduction system. A variety of FRET fluorophore pairs were tested in a spectrofluorimeter and promising FRET pairs were then tagged to the protein, avidin and its ligand, biotin. Due to their affinities, the FRET-tagged biomolecules combine in solution, resulting in a stable, fluorescent signal from the acceptor FRET dye with a simultaneous decrease in fluorescent signal from the donor FRET dye. The results indicate that the selected FRET pairs can be utilized in the development of dual receptor sensors.

Background and Objectives: Continuous peripheral nerve block (CPNB) can provide surgical anesthesia, prolonged postoperative analgesia, and acceptable side effects. Despite these advantages, CPNB is not in widespread use. Recently a new CPNB catheter system (Contiplex, B. Braun, Bethlehem, PA) was developed based on an insulated Tuohy needle, which allows for injection of local anesthetic and catheter insertion without disconnection or needle movement. At present, no clinical studies exist describing this system., Methods: Data were prospectively gathered for 1 year from 228 patients in an ambulatory surgery center. All CPNB were performed using the Contiplex system to provide anesthesia and postoperative analgesia. CPNB were performed using 5 upper and lower extremity techniques. Postsurgery local anesthetic was infused and at 24 hours, a rebolus of local anesthetic was performed. The CPNB catheter was removed and patients were examined for loss of sensation. Patients were then discharged., Results: Initial peripheral block was successful in 94% of patients. Failed nerve block requiring general anesthesia occurred in 6%. The catheter was patent and functional in 90% of patients at 24 hours, and 8% of patients required more than 10 mg of intravenous morphine by 24 hours postsurgery. In the postanesthesia care unit (PACU), only 4 patients (1.7%) required treatment for nausea. At 24 hours and 7 days postsurgery, no patient reported a dysesthesia., Conclusions: CPNB using the insulated Tuohy catheter system offered acceptable anesthesia and prolonged pain relief postsurgery. There were few side effects. Reg Anesth Pain Med 2001;26:209-214.

Purpose: Major reconstructive surgery of the knee traditionally requires an extended hospital stay for pain management. Continuous peripheral nerve blockade is an alternative method of pain control but is seldom used in the ambulatory setting. This case illustrates the use of lumbar plexus and sciatic nerve peripheral catheters for major knee surgery using intermittent bolus dosing for outpatient analgesia., Clinical Features: A 20-yr-old male presented for multi-ligamentous knee reconstruction (posterior collateral ligament and revision anterior collateral ligament and lateral collateral ligament). Anesthesia was managed with a lumbar plexus and a sciatic nerve peripheral catheter and a light general anesthetic. Post-operative analgesia was provided with a 12-hr infusion of 0.2% ropivacaine in an over night recovery care centre. Subsequent catheter dosing was performed as an outpatient, twice a day using 0.2% ropivacaine, 10 ml in each catheter (four injections total). This provided 96 hr of analgesia and low supplemental opioid use., Conclusion: The use of a lumbar plexus and sciatic nerve peripheral catheter offered an alternative to conventional pain control that worked well in the ambulatory setting. By providing prolonged unilateral lower limb analgesia, extensive knee surgery was performed that would normally require a hospital stay for pain control. Using a bolus dosing method the risk of local anesthetic complications occurring outside of the hospital with a continuous infusion was minimized.

Continuous interscalene brachial plexus blockade traditionally requires a hospital stay for local anesthetic infusion, and achieving consistent catheter insertion may be difficult. Incorporating long-acting pain relief from a continuous peripheral nerve block, with a reliable method of catheter insertion, and a self-contained infusion system would be a valuable asset for short-stay care. We compared the efficacy of single injection interscalene brachial plexus blockade to a continuous peripheral nerve block, with an insulated Tuohy system and a disposable infusion pump. Forty adult patients scheduled for open rotator cuff repair were entered in this randomized, double-blinded, placebo-controlled study. Patients received an interscalene brachial plexus blockade and a continuous peripheral nerve catheter as their primary anesthetic and then, were assigned to receive one of two different postoperative infusions: either 0.2% ropivacaine at 10 mL/h via a disposable infusion pump or normal saline at 10 mL/h via a disposable infusion pump (n = 18-20 per group). Visual analog pain scores and postoperative morphine consumption were measured for 24 h. The ropivacaine group showed less pain than the placebo group (P: = 0.0001) between 12 and 24 h after the initial injection of local anesthetic. In addition, initial interscalene blockade was successful in all patients and all redosed catheters were functional after 24 h with the continuous catheter insertion system. We conclude that it is possible to achieve a high rate of successful catheter placement and analgesia by using the continuous catheter insertion system and a disposable infusion pump in the ambulatory setting. This method of analgesia may offer improved pain relief after outpatient rotator cuff repair.

This study assessed the effect of intravenous alcohol infusions on psychomotor impairment and compared it with that of alcohol administered orally. Comparisons were made between three European drink-driving limits of blood alcohol concentration (BAC) (20, 50 and 80 mg 100 ml-1) and an oral dose of alcohol 0.75 mg kg-1. Twelve volunteers, aged 22-34 yr, were recruited. At targets of 20, 50 and and 80 mg 100 ml-1, the mean (SD) BAC was 22.1 (3.7), 51.5 (3.3) and 80.5 (4.2) mg 100 ml-1, respectively. The peak BAC following an oral dose of alcohol 0.75 mg kg-1 ranged from 19 to 68 mg 100 ml-1. In psychomotor testing, choice reaction time deteriorated with increasing BAC and showed significant differences between baseline and the 50 (P < 0.05) and 80 mg 100 ml-1 (P < 0.01) conditions. Dual-task secondary reaction time deteriorated with increasing BAC and showed a statistically significant difference between all groups and baseline (oral and 20 mg groups, P < 0.05; 50 and 80 mg groups, P < 0.01). Dual-task tracking in the 50 and 80 mg groups was significantly different from baseline (P < 0.05 and P < 0.01, respectively). Oral dosing resulted in widely variable BACs, making it difficult to assess psychomotor impairment reliably. An intravenous infusion enables the BAC to be maintained within a narrow range. This allows precision when investigating the effects of alcohol on psychomotor performance.

We studied psychomotor performance in 10 healthy volunteers during recovery after a target-controlled infusion of propofol. Choice reaction time, dual task tracking with secondary reaction time and a within-list recognition task were assessed at target blood propofol concentrations of 0.8, 0.4 and 0.2 microgram ml-1. Performance was impaired most at the highest blood propofol concentration (choice reaction time increased by a mean of 247 ms and secondary reaction time by a mean of 178 ms). Choice reaction time and dual task tracking with secondary reaction time were the most sensitive and reliable methods of assessment (significant difference from baseline (P < 0.05) at a propofol concentration of 0.2 microgram ml-1 with choice and secondary reaction time testing). Within-list recognition assessment of memory was not sufficiently sensitive at very low propofol concentrations. The impairment in choice and secondary reaction time with a blood propofol concentration of 0.2 microgram ml-1 was less than that observed with a blood alcohol concentration of 50 mg 100 ml-1 and no greater than that observed with a blood alcohol concentration of 20 mg 100 ml-1 in a previous study involving healthy volunteers.

We investigated the safety of a patient-maintained system that allows individuals to operate a target-controlled infusion of propofol to achieve sedation. Ten healthy volunteers were recruited and instructed to try to anaesthetize themselves with the system. A target-controlled infusion of propofol was set to deliver a target propofol concentration of 1 microgram ml-1, and the subjects allowed to increase the target in increments of 0.2 microgram ml-1 by pressing a control button twice in 1 s. There was a lockout time of 2 min and a maximum permitted target concentration of 3 micrograms ml-1. Heart rate and pulse oximetry oxygen saturation (SpO2) were monitored continuously, and non-invasive arterial pressure, ventilatory frequencies and sedation scores were measured every 5 min. Sedation was continued until the subject stopped pressing the button. A keyword was then read for the individual to remember and sedation discontinued. There were no instances of significant decrease of SpO2 or loss of airway control. Maximum target blood concentration of propofol recorded ranged from 1.4 to 3 micrograms ml-1. Two subjects became oversedated, one of whom was unrousable with loss of eyelash reflex. No subject could recall the keyword, although one recognized it from a list of 10 words. We conclude that the patient-maintained sedation system described could not be guaranteed to produce only conscious sedation in all patients, and that close clinical supervision by an anaesthetist would still be required for safe operation.

This article describes fiber optic and electrochemical pH sensors that could become part of a therapeutic arsenal to quickly and aggressively treat stroke victims as well as people who have suffered brain trauma. The fiber optic sensor design was based on the immobilization of a pH-sensitive dye, seminaphthorhodamine-1 carboxylate (SNARF-1C), onto the end of a 125-microm-diameter silica optical fiber using the sol-gel method. A miniature bench-top fluorimeter system was developed for use with the optical fiber to obtain pH measurements. The electrochemical sensor was based on sputter-coated iridium oxide thin films. Linear and reproducible responses for both sensors were obtained in human blood with pH varying between 6.8 ato 8.0, which encompasses the clinically relevant range. In vivo studies were also performed and results indicated that both types of sensors tracked pH with very little drift.

A fiber optic biosensor for the detection of fibrinolytic products produced during lysis of "soft" blood clots is described. The biosensor was constructed to be selective toward D dimer antigens, which form from the dissolution of cross-linked fibrin clots. The presence of D dimer antigens above a threshold level is a clinical diagnostic used to determine the presence of such occlusions following a stroke. Fluorescein-labeled D dimer antibodies are immobilized on the tip of an optical fiber by dip coating from a silica sol-gel solution. When D dimer antigens combine with the antibodies, fluorescence intensity decreases. The response of the sensor was examined in phosphate buffered saline, human plasma, and blood. Calibration plots for the sensor were obtained in the clinically significant D dimer concentration range from 0.54 microgram/ml to 6 micrograms/ml. Changes in spectroscopic properties as the sol-gel encapsulated tagged antibodies aged were examined; a decrease in fluorescence intensity with age was noted. The D dimer antibodies remain viable for at least 4 weeks while encapsulated in the sol-gel network when stored at 4 degrees C in PBS solution. This novel sensor is being developed for use with other catheter-based microtools to treat stroke resulting from occlusion in the vascular system.

Background and Objective: The interaction of surgical optical fibers with tissue has been studied. STUDY DESIGN/METHODS AND MATERIALS: Fibers (600 microns) were lased in chicken and beef tissue using a Nd:YAG laser from 5 to 50 W in both cw and pulsed modes., Results: With longer lasing and higher power, larger transmission loss and degradation (burn-in) of the fiber tip, occurred. This degradation converts the Nd:YAG laser power to heat and leads to further energy loss. During contact lasing, tissue and blood adhere to the fiber tip surface limiting laser transmission, desiccating, and eventually destroying adhering tissues. Such tissue residues create high power densities and temperatures at the tip, which then cause a variety of degradation processes to be initiated., Conclusion: "Burned-in" fibers do not photocoagulate; rather they incise tissue. With continued lasing, thermal shock, chemical, and mechanical breakdown of the fiber leads to failure of the fiber tip and the spalling of glass fragments into the tissue bed.

Two techniques for the preparation of boar spermatozoa for in vitro fertilization were studied: a simple washing procedure and centrifugation on a discontinuous Percoll gradient. Their respective effects on motility of spermatozoa were analysed by computer-assisted sperm analysis. The Percoll density gradient technique selected spermatozoa with significantly (P < 0.0001) enhanced motility and movement characteristics. In vitro matured oocytes inseminated with spermatozoa prepared by Percoll gradient centrifugation had significantly (P < 0.0001) greater cleavage rates than did oocytes inseminated with washed spermatozoa. This increased penetration ability was not due to an increased proportion of acrosome-reacted spermatozoa. Transmission electron microscopy revealed no unique ultrastructural differences between the spermatozoa from either preparation. Spermatozoa prepared by Percoll gradient centrifugation are recommended for insemination and studies of porcine in vitro fertilization.

Study Hypothesis: Tetracaine will enhance cocaine toxicity., Study Population: Two hundred forty female Swiss albino mice weighing 27 to 45 g., Methods: Intraperitoneal injections of tetracaine and cocaine were given to groups of ten mice each in a controlled, blinded fashion. Either tetracaine or an equal volume of normal saline was given five minutes before one of six incremental doses of cocaine, ranging from 60 to 110 mg/kg. The experiment was repeated twice using two different doses of tetracaine: either an LD10 (40 mg/kg determined from preliminary studies), or one-twentieth of the dose of cocaine (which approximates the ratio used in tetracaine, epinephrine, and cocaine). Lethality was recorded at 24 hours. Lethality between groups was compared with a Wilcoxon sign-rank test., Results: Tetracaine reduced cocaine lethality at all doses. This reduction in lethality was statistically significant at both tetracaine doses (P < .05)., Conclusion: In the mouse, pretreatment with tetracaine significantly decreases cocaine lethality.

The combination of tetracaine, epinephrine, and cocaine has gained wide acceptance as a topical anesthetic agent for use on pediatric dermal lacerations in the ED. This is despite the fact that the optimal dose and formulation have yet to be determined. TAC can be applied painlessly to wounds and is about as effective as lidocaine infiltration for anesthetizing pediatric facial and scalp lacerations. It is relatively ineffective on lacerations located elsewhere or in adults. The most commonly used TAC solution contains high concentrations of cocaine, tetracaine, and epinephrine, drugs that individually can cause serious toxicity if absorbed in sufficient amounts. In addition, the three components of TAC may interact to potentiate their intrinsic toxicities. The deliberate and inadvertent application of TAC to mucous membranes has caused status epilepticus and two pediatric deaths. The risk of toxicity from misapplication of TAC is heightened because TAC is most effective and therefore most widely used on pediatric facial and scalp lacerations. Cocaine is also absorbed after TAC is applied to dermal lacerations and may cause toxicity by this route. Until additional research is performed to establish the minimum effective dose of TAC and its potential toxicity and until FDA approval is granted, we do not think that it can be recommended as the drug of choice for pediatric facial and scalp lacerations in the ED. If TAC is administered, a maximum dose of 2 to 3 mL of the "half-strength" formula proposed by Bonadio and Wagner should be used, and application should be performed by medical personnel, using a soaked gauze or cotton ball. Care should be taken to make sure none of the solution comes in contact with mucous membranes, and TAC should not be applied to lacerations involving the vermillion border of the lip or the lip itself. Close medical monitoring of the patient is essential to detect signs of toxicity. Research on other topical agents such as tetracaine with epinephrine is also needed. Although anesthetizing wounds painlessly remains a worthy goal, exposing patients to added and unknown risks and increasing the cost of health care is unacceptable.

The objectives of this study were to investigate whether estradiol treatment during lactation modifies 1) the patterns of endogenous LH, FSH, and prolactin (PRL) release; 2) the sensitivity of the pituitary to exogenous injections of LHRH; and 3) the responsiveness of the ovarian follicles to gonadotropin. Plasma LH, FSH, and PRL were determined in samples taken repeatedly from 18 sows on Days 24-27 of lactation. Ovaries were then recovered, and follicular development was assessed by measuring the follicular diameter (FFD) and follicular fluid estradiol-17 beta concentration (FFE) of the ten largest follicles dissected from each ovary. Sows were randomly allocated to one of four treatments: 1) Group C (4 sows) received no treatment; 2) Group LHRH (5 sows) received 800 ng of LHRH every 2 h throughout the sampling period; 3) Group E2 (4 sows) received subcutaneous implants containing estradiol-17 beta 24 h after start of sampling; 4) Group LHRH + E2 (5 sows) were administered a combination of LHRH and estradiol-17 beta implants. Between-animal variability for plasma LH, FSH, and PRL was considerable. LH concentration and LH pulse frequency increased (p less than 0.05) after LHRH treatment in the LHRH and LHRH + E2 groups; however, an acute inhibition of LH secretion was observed in the latter group immediately after estradiol implant application. In the absence of LHRH treatment, estradiol caused chronic inhibition of LH secretion. Follicular development was greater in the LHRH and LHRH + E2 groups compared to the C and E2 groups (p less than 0.05 for both FFD and FFE).(ABSTRACT TRUNCATED AT 250 WORDS)

Ovaries were recovered from groups of naturally cyclic pigs (N = 5) on each of Days 16, 18, 20 and 21 of the oestrous cycle. Follicular diameter, follicular fluid volume and concentrations of oestradiol, testosterone and progesterone, and granulosa cell number were determined in all follicles greater than or equal to 2 mm in diameter (n = 511). In alternate follicles either granulosa cell aromatase activity and theca testosterone content or 125I-labelled hCG binding to granulosa and theca were determined. The mean total number of follicles recovered per animal decreased as the follicular phase progressed and a strong positive relationship (P less than 0.001) existed between follicular diameter and volume on all days. The number of granulosa cells recovered per follicle was variable, and not related to oestrogenic activity of the follicles. Mean follicular fluid oestradiol, testosterone and 125I-labelled hCG binding all increased until Day 20 and decreased on Day 21, whereas mean theca testosterone content, 125I-labelled hCG binding to theca tissue and aromatase were all maximal on Day 21. On Days 20 and 21 a subset of 14-16 large follicles was readily distinguishable from the remaining smaller, less oestrogenically active population in each animal. Yet, consistently within these subsets there was a difference in follicular diameter of approximately 2.0 mm and also a considerable range of biochemical development even among follicles of equal size. These results indicate asynchrony at the time of recruitment and selection among follicles destined to ovulate and suggest that heterogeneity continues into the immediate preovulatory period.

Exogenous gonadotrophin (eg. PMSG) treatment results in follicular growth in most reproductive states, and suggests that lack of FSH and LH-like activity is a likely cause of arrested-follicular development in the pig. Endogenous changes in gonadotrophins between reproductive and non-reproductive states are consistent with this concept. However, changes in the pattern of LH secretion, rather than clearly defined changes in FSH release, appear to be the effective trigger for follicular recruitment. In other situations in vivo, follicular growth is initiated in the absence of demonstrable changes in LH or FSH secretion, suggesting that other hormones interact to sensitize the ovary to gonadotrophins. Such effects have been reported for insulin, cortisol, thyroxine and IGF-1 in vitro, and the steroid environment of follicles also affects gonadotrophin receptors and receptor-mediated mechanisms. Once initiated, the regulation of follicle maturation also involves interactions between gonadotrophins and steroids, as well as other intra-ovarian regulators. Co-operation exists between theca and granulosa in the synthesis of steroids and androgen precursor availability is an important regulator of the oestrogenic activity of follicles. Considerable heterogeneity exists in the morphological and biochemical maturity of preovulatory follicles and inter-follicular regulation via endocrine and paracrine mechanisms may be an essential component of the selection process.