Gold and Hydroxyapatite Nano-Composite Scaffolds for Anterior Cruciate Ligament Reconstruction: In Vitro Characterization.

Current anterior cruciate ligament (ACL) graft replacement materials often fail due to the lack of biological integration. While many newly developed extracellular matrix based scaffolds show good biocompatibility they often do not entice cellular remodeling and the rebuilding of a functional ligament. We have proposed the conjugation of gold nanoparticles (AuNP) and hydroxyapatite nanoparticles (nano-HAp) to acellular tissue to enhance cell attachment and proliferation while maintaining an improved degradation resistance and open microstructure. We are the first to investigate the double conjugation of AuNP and nano-HAp onto decellularized tissue to improve the tissue remodeling response. Decellularized porcine diaphragm was crosslinked with two types of nano-HAp and amine-functionalized AuNP with 1-ethyl-3-(3-dimethlaminopropyl) carbodiimide (EDC) crosslinker. Scaffolds were characterized using electron microscopy, differential scanning calorimetry, and fibroblast assays. Results demonstrated that scaffolds with nano-HAp have increased thermal stability at low levels of crosslinking. The open microstructure of the scaffold was not compromised allowing for cell migration while still providing increased degradation resistance. The addition of < 200 nm nano-HAp decreased cell viability compared to scaffolds without nanoparticles, but the addition of AuNP to scaffolds showed enhanced cell viability in the presence of < 200 nm nano-HAp. The addition of < 40 nm nano-HAp showed an increase in cell viability compared to scaffolds crosslinked without nanoparticles. It is concluded that attaching AuNP and < 40nm nano-HAp to extracellular matrices may improve overall properties.

An in vivo study of a gold nanocomposite biomaterial for vascular repair.

Currently vascular repairs are treated using synthetic or biologic patches, however these patches have an array of complications, including calcification, rupture, re-stenosis, and intimal hyperplasia. An active patch material composed of decellularized tissue conjugated to gold nanoparticles (AuNPs) was developed and the long term biocompatibility and cellular integration was investigated. Porcine abdominal aortic tissue was decellularized and conjugated with 100 nm gold nanoparticles (AuNP). These patches were placed over a longitudinal arteriotomy of the thoracic aorta in six pigs. The animals were monitored for six months. Gross, histological, and immunohistochemical analyses of the patches were performed after euthanasia. Grossly there was minimal scar tissue with the patches still visible on the outer surface of the vessel. The inner lumen was smooth with a seamless transition from patch to native tissue. Histology demonstrated infiltration of host cells into the patch material. The immunohistochemical results demonstrated an endothelial cell layer forming over the patch within the vessel. Smooth muscle cells were repopulating the biomaterial in all animals. These results demonstrated that the AuNP biomaterial patch integrated well with the host tissue and did not failed over the six month implantation time.

Materials characterization and histological analysis of explanted polypropylene, PTFE, and PET hernia meshes from an individual patient.

During its tenure in vivo, synthetic mesh materials are exposed to foreign body responses, which can alter physicochemical properties of the material. Three different synthetic meshes comprised of polypropylene, expanded polytetrafluoroethylene (ePTFE), and polyethylene terephthalate (PET) materials were explanted from a single patient providing an opportunity to compare physicochemical changes between three different mesh materials in the same host. Results from infrared spectroscopy demonstrated significant oxidation in polypropylene mesh while ePTFE and PET showed slight chemical changes that may be caused by adherent scar tissue. Differential scanning calorimetry results showed a significant decrease in the heat of enthalpy and melt temperature in the polypropylene mesh while the ePTFE and PET showed little change. The presence of giant cells and plasma cells surrounding the ePTFE and PET were indicative of an active foreign body response. Scanning electron micrographs and photo micrographs displayed tissue entrapment and distortion of all three mesh materials.

A comparative study of the remodeling and integration of a novel AuNP-tissue scaffold and commercial tissue scaffolds in a porcine model.

The extracellular matrices of a variety of human and animal tissues have been utilized as scaffold materials for soft tissue applications including hernia repair, dermal grafts, and tendon, ligament, and cartilage reconstruction. While these biological scaffolds are expected to demonstrate superior tissue integration, there is very little evidence documenting the properties and behavior of these materials in vivo. This in vivo study investigated four biological scaffolds: two commercially available (a moderately crosslinked scaffold and a noncrosslinked scaffold) and two novel porcine diaphragm biological scaffolds (one with and one without the incorporation of gold nanoparticles). The scaffolds were implanted in a porcine model and evaluated over 1, 3, and 6 months. The moderately crosslinked scaffolds demonstrated the least cellular infiltration and evidence of fibrosis. The noncrosslinked scaffolds demonstrated the greatest cellular infiltration, but these scaffolds were delaminated and exhibited a rapid loss of integrity. The porcine diaphragm scaffolds with and without nanoparticles showed evidence of tissue remodeling and cellular infiltration, with no evidence of encapsulation. While there were no significant differences in the performance of the two novel scaffolds, the gold nanoparticle scaffold typically exhibited higher cellular infiltration. This study demonstrated the potential biocompatibility of a gold nanoparticle-tissue scaffold.

Conjugation of gold nanoparticles to polypropylene mesh for enhanced biocompatibility.

Polypropylene mesh materials have been utilized in hernia surgery for over 40 years. However, they are prone to degradation due to the body's aggressive foreign body reaction, which may cause pain or complications, forcing mesh removal from the patient. To mitigate these complications, gold nanomaterials were attached to polypropylene mesh in order to improve cellular response. Pristine samples of polypropylene mesh were exposed to hydrogen peroxide/cobalt chloride solutions to induce formation of surface carboxyl functional groups. Gold nanoparticles were covalently linked to the mesh. Scanning electron microscopy confirmed the presence of gold nanoparticles. Differential scanning calorimetry and mechanical testing confirmed that the polypropylene did not undergo any significantly detrimental changes in physicochemical properties. A WST-1 cell culture study showed an increase in cellularity on the gold nanoparticle-polypropylene mesh as compared to pristine mesh. This study showed that biocompatibility of polypropylene mesh may be improved via the conjugation of gold nanoparticles.

Method of preparing a decellularized porcine tendon using tributyl phosphate.

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.

Assessment of the biocompatibility of two novel, bionanocomposite scaffolds in a rodent model.

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.

Enhanced efficiency of a capillary-based biosensor over an optical fiber biosensor for detecting calpastatin.

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.

Correlation between a novel calpastatin biosensor and traditional calpastatin assay techniques.

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.

Characterization of heavyweight and lightweight polypropylene prosthetic mesh explants from a single patient.

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.

Materials characterization of explanted polypropylene hernia meshes.

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.

Use of porcine dermal collagen as a prosthetic mesh in a contaminated field for ventral hernia repair: a case report.

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.

Variability in determination of point of needle insertion in peripheral nerve blocks: a comparison of experienced and inexperienced anaesthetists.

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.

Development of dual receptor biosensors: an analysis of FRET pairs.

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.

Ambulatory surgery for multi-ligament knee reconstruction with continuous dual catheter peripheral nerve blockade.

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 peripheral nerve block for ambulatory surgery.

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.

Blood alcohol concentration and psychomotor effects.

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.

Blood propofol concentration and psychomotor effects on driving skills.

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.

Interscalene brachial plexus block with a continuous catheter insertion system and a disposable infusion pump.

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.

Safety of patient-maintained propofol sedation using a target-controlled system in 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.