A pilot study of an anti-MRSA bio-engineered lacteal complex (anti-MRSA BLC) in a murine septicemia model.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen of humans and other animals, causing septicemia, abcessation, toxemia, and other infectious diseases. Refined bioengineered lacteal complex (BLC), made specifically against MRSA, is a novel complex of low molecular weight immunogenic and antimicrobial molecules. It was evaluated in vivo using a mouse model of MRSA-induced peritonitis. Intraperitoneal dosing of anti-MRSA BLC demonstrated a therapeutic effect (83% survival) against an intraperitoneal MRSA challenge that caused 100% mortality in untreated animals. Anti-MRSA BLC is a promising therapeutic modality for MRSA infection.

An instrumented scaffold can monitor loading in the knee joint.

No technique has been consistently successful in the repair of large focal defects in cartilage, particularly in older patients. Tissue-engineered cartilage grown on synthetic scaffolds with appropriate mechanical properties will provide an implant, which could be used to treat this problem. A means of monitoring loads and pressures acting on cartilage, at the defect site, will provide information needed to understand integration and survival of engineered tissues. It will also provide a means of evaluating rehabilitation protocols. A "sensate" scaffold with calibrated strain sensors attached to its surface, combined with a subminiature radio transmitter, was developed and utilized to measure loads and pressures during gait. In an animal study utilizing six dogs, peak loads of 120N and peak pressures of 11 MPa were measured during relaxed gait. Ingrowth into the scaffold characterized after 6 months in vivo indicated that it was well anchored and bone formation was continuing. Cartilage tissue formation was noted at the edges of the defect at the joint-scaffold interfaces. This suggested that native cartilage integration in future formulations of this scaffold configured with engineered cartilage will be a possibility.

TGF-beta1-enhanced TCP-coated sensate scaffolds can detect bone bonding.

Porous polybutylene terephthalate (PBT) scaffold systems were tested as orthopedic implants to determine whether these scaffolds could be used to detect strain transfer following bone growth into the scaffold. Three types of scaffold systems were tested: porous PBT scaffolds, porous PBT scaffolds with a thin beta-tricalcium phosphate coating (LC-PBT), and porous PBT scaffolds with the TCP coating vacuum packed into the scaffold pores (VI-PBT). In addition, the effect of applying TGF-beta1 to scaffolds as an enhancement was examined. The scaffolds were placed onto the femora of rats and left in vivo for 4 months. The amount of bone ingrowth and the strain transfer through various scaffolds was evaluated by using scanning electron microscopy, histology, histomorphometry, and cantilever bend testing. The VI-PBT scaffold showed the highest and most consistent degree of mechanical interaction between bone and scaffold, providing strain transfers of 68.5% (+/-20.6) and 79.2% (+/-8.7) of control scaffolds in tension and compression, respectively. The strain transfer through the VI-PBT scaffold decreased to 29.1% (+/-24.3) and 30.4% (+/-25.8) in tension and compression when used with TGF-beta1. TGF-beta1 enhancement increased the strain transfer through LC-PBT scaffolds in compression from 9.4% (+/-8.7) to 49.7% (+/-31.0). The significant changes in mechanical strain transfer through LC-PBT and VI-PBT scaffolds correlated with changes in bone ingrowth fraction, which was increased by 39.6% in LC-PBT scaffolds and was decreased 21.3% in VI-PBT scaffolds after TGF-beta1 enhancement. Overall, the results indicate that strain transfer through TCP-coated PBT scaffolds correlate with bone ingrowth after implantation, making these instrumented scaffolds useful for monitoring bone growth by monitoring strain transfer.

Surface enhancements accelerate bone bonding to CPC-coated strain gauges.

Calcium phosphate ceramic (CPC)-coated strain gauges have been used for in vivo bone strain measurements for up to 18 weeks, but they require 6 to 9 weeks for sufficient bonding. Osteogenic protein-1 (OP-1), PepTite (a proprietary ligand), calcium sulfate dihydrate (CSD), transforming growth factor beta-1 (TGF-beta1 ), and an endothelial cell layer with and without TGF-beta1 were used as surface enhancements to accelerate bone-to-CPC bonding. Young male Sprague-Dawley rats were implanted with unenhanced and enhanced CPC-coated gauges. Animals were allowed normal activity for 3 weeks and then calcein labeled. Femurs were explanted following euthanasia. A gauge was attached with cyanoacrylate to the opposite femur in the same position as the CPC-coated gauge. Bones were cantilever-loaded to assess strain transfer. They were sectioned and stained with mineralized bone stain (MIBS) and examined with transmitted and ultraviolet light. Mechanical testing indicated increased sensing accuracy for TGF-beta1 and OP-1 enhancements to 105 +/- 14% and 92 +/- 12% versus 52 +/- 44% for the unenhanced gauges. The PepTite and the endothelial-cell-layer-enhanced gauges showed lower sensing accuracy, and histology revealed a vascular layer near CPC particles. TGF-beta1 increased bone formation when used prior to endothelial cell sodding. CSD prevented strain transfer to the femur. TGF-beta1 and OP-1 surface enhancements produced accurate in vivo strain sensing on the rat femur after 3 weeks.

In vivo strain measurements collected using calcium phosphate ceramic-bonded strain gauges.

Identification of the strains and the strain changes caused by implants is critical to the understanding of bone remodeling and can identify design changes needed to prevent bone loss near orthopedic implants. Calcium phosphate ceramic (CPC) coated strain gauges have been developed to allow long-term in vivo strain measurements. Previously used cyanoacrylate-bonded gauges have uncharacterizable sensing accuracy because the adhesive is resorbed from the instant it is placed in vivo. In this study CPC-coated strain gauges were used to measure physiologically "normal" bone strains collected from the proximal femora of dogs at a series of gait speeds and the postmortem sensing accuracy of the gauges was evaluated. Three male dogs were surgically implanted with up to six wired CPC-coated strain gauges placed around the circumference of their proximal femora. The dogs were trained to run on a treadmill, and in vivo strain measurements were collected following a 12-week period. The animals were tetracyline labeled and then euthanized and their femora explanted. Gauges were attached with cyanoacrylate to the intact contralateral control femora in the same position as the CPC-coated gauges on the test femora. Both femora were tested in cantilever bending to assess the functionality of the gauges and quality of the CPC-bone bond. After testing, all bones were embedded, sectioned, and ground. Sections from each femur were stained with mineralized bone stain and examined with transmitted and ultraviolet light to assess bone formation. Additional sections were examined with backscattered electron microscopy to confirm bone bonding to coatings. Wired gauges attached with the CPC coatings measured strain patterns during gait at several treadmill speeds. Patterns were similar and peak strains the same over a 2-week period. Mechanical testing showed bonding of CPC-coated gauges, and histologic examination showed intimate contact between gauge coatings and bone surfaces. Further development of CPC-bonded strain gauges is expected to result in a measurement system that provides ease of placement, and consistent longer term bone strain measurements with characterizable accuracy.

Evaluation of factors affecting bonding rate of calcium phosphate ceramic coatings for in vivo strain gauge attachment.

The aim of this study was to compare the bone-bonding rates of eight calcium phosphate ceramic (CPC) coatings attached to strain gauges, alone and in conjunction with an OP1 device (Creative BioMolecules, Hopkinton, MA) and autologous concentrated pericyte cells. These coatings were studied to develop faster bone bonding to long-term in vivo strain sensors. Characterization of the CPC powders using electron microscopy and X-ray diffraction showed that they had shapes ranging from spherical to rocklike and properties ranging from highly crystalline to amorphous. CPC coated gauges were placed on the femora of young male dogs during aseptic surgery and were initially held in place using resorbable sutures. Test groups were euthanized after 3, 9, and 12 weeks. Both femora of the dogs were explanted and cantilever loaded. Response of the implanted hydroxyapatite (HA) coated gauges were compared to the response of bench-top glued sets of gauges (controls) attached to the contralateral femur and reported as a percentage of the control values. One CPC coating type showed an average response of 30% of controls after 3 weeks, four showed average responses higher than 75% after 9 weeks, and three showed averages higher than 82% after 12 weeks in vivo. Amorphous CPC coatings bonded more quickly than crystalline ones and particle shape had less effect than crystal structure on bonding rates. When either OP1 or autologous concentrated pericyte cells were placed on selected CPC coated gauge surfaces, the CPC5 coated gauges bonded best after 3 weeks with a response of 59%. After the same time period in vivo, CPC3 and CPC7 provided responses of 40 and 16%, respectively. Comparison of a soluble calcium-coated CPC with an uncoated one that had identical crystal structure and similar particle shape indicated that the calcium coating slowed bone bonding substantially in the young dog model. Optical microscopy of stained undecalcified bone sections and backscattered electron imaging indicated bone formation at all bone-HA interfaces and an increase in the number of areas of bone remodeling adjacent to the gauge at all time periods. Gross bone remodeling due to strain gauge placement was only observed near the distalmost cell-seeded strain gauges. Selection of the type of coating and enhancement system can accelerate bone bonding to strain sensors but must be tailored to the bone of the model in which it is being used. Augmentation of CPC coatings with cells or OP1 resulted in variable enhancement of the bonding rate and depended on the CPC and the enhancement system.

Conductive hearing loss in bighorn sheep.

In January 1993 we simulated a conductive hearing loss in three Mexican bighorn sheep (Ovis canadensis mexicana) by placing bone wax or saline solution in their ear canals. Our objective was to test whether lesions of the external auditory canal caused by psoroptic mites (Psoroptes ovis) may lead to conductive hearing loss in bighorn sheep. We assessed the effects of these manipulations using the auditory brainstem response test. Placing saline solution in the external auditory canal, which loads the tympanic membrane, had a more dramatic effect on the auditory brainstem response than did bone wax. We propose that decreased hearing sensitivity or alterations in resonance characteristics of the external auditory canal, due to psoroptic scabies lesions, may make bighorn sheep more susceptible to predation.

Load transfer through a hydroxyapatite-coated canine hip implant.

Strain transfer near hydroxyapatite (HA) coated canine hip implants was examined using simulated anatomical loading based on in vivo strain measurements. Strain changes near implants relative to intact control values were in excess of 100% for transverse and principal strains for zero time period (immediate postimplant) specimens. They were generally smaller (100% or less) for axial, transverse, and principal minimums in the same locations for 4 months postimplantation specimens. Cortical bone loss occurred in all implanted femora. The most extensive loss, up to 47%, occurred adjacent to the proximal section of the implant. Extensive trabecular bone formation, over 300% in some regions of each femur, was noted in all implanted femora. Backscattered electron imaging along the HA-coated sections of the implants showed extensive bone bonded to the coating. NOrmal light and UV light micrographs showed direct bone apposition to the implant surfaces and extensive bone formation in all test animals. Microscopy revealed no evidence of any soft tissue layer between the implant and bone. Bone was typically found in direct contact with the implant surface. Histomorphometry indicated that bone formation rates in the implanted femora were elevated, up to 850%, relative to controls. Fewer formation sites were noted on the posterior and lateral (in two cases zero sites). Mineral apposition rates (MAR) from two of the dogs were slightly elevated (from 110-113%) in the implanted femora relative to controls and depressed (to about 83% of controls) in a third.

Hydroxyapatite-coated strain gauges for long-term in vivo bone strain measurements.

The aim of this study was to examine the bonding process between hydroxyapatite-coated strain gauges and bone in order to continue development of a long term in vivo strain measurement device. Two types of commercially available hydroxyapatite (HA) particles were applied to the sensing surface of uniaxial strain gauges using a polysulfone solution as an adhesive. Characterization by scanning electron microscopy and x-ray diffraction (XRD) was used to determine materials property differences between the two powders. Interfacial strengths between the HA coatings and the strain gauges were tested and found comparable to interfacial strength obtained by a plasma sprayed HA coating on the surface of a titanium implant. Gauges were surgically placed on the periosteal surface of greyhound femora. Three groups of dogs were implanted with gauges for periods of 3, 6, and 12 weeks using cyanoacrylate, resorbable sutures, and cable ties to initially hold the gauge against the surface of the bone. Following euthanasia, the femora of the dogs were explanted and subjected to cantilever loading. Response of the implanted HA-coated gauges were compared to a control set that had been freshly glued onto the contralateral femur. Full response, that is, 100% of the strain measurement with respect to the control, was obtained after 12 weeks in vivo. Attachment of HA-coated gauges with circumferential suture showed bonding, while HA-coated gauges attached with cyanoacrylate did not bond to bone. After mechanical testing, femora were embedded in polymethylmethacrylate, cut, ground, and polished. Sections were stained using mineralized bone stain (MIBS) and optical microscopy was performed using transmitted and fluorescent light to allow analysis of remodeling occurring in the region of the strain gauges. Bone formation occurred at the HA surface of sutured gauges, and a fibrous tissue layer developed between the bone and HA coating when the tissue adhesive was used to initially bond the gauge. Fluorescence microscopy indicated an increase in the number of areas of bone remodeling adjacent to the gauge but a normal rate of remodeling of 0.93 +/- 0.07 mum/day was observed. No gross bone remodeling due to strain gauge placement was observed. Backscattered electron imaging (BSE) indicated new bone apposition at all time periods.

Passive protection of segmented swine colonic loops against swine dysentery.

Swine-ligated loops were used to demonstrate passive protection against swine dysentery. Loops inoculated with immune sera containing complement and with homologous Treponema hyodysenteriae were normal at necropsy. Loops inoculated with heat-inactivated immune sera and heterologous T hyodysenteriae were not protected. Loops inoculated with heat-inactivated immune sera and homologous T hyodysenteriae were partially protected. Positive control loops inoculated with isolate B204 (88%) or B234 (44%) T hyodysenteriae and normal sera developed lesions typical of swine dysentery, whereas negative control loops inoculated with nonexposed sera only were normal.

Regional hyperthermia by magnetic induction in a beagle dog model: analysis of thermal dosimetry.

We have investigated magnetic induction heating techniques for achieving normal tissue hyperthermia in a beagle dog model to clarify the physics and physiology of "regional heating," to develop an animal model of regional heating in humans, and to develop a method of rapid regional heating in dogs for a normal visceral tissue toxicity study. Heating was done with a concentric coil or a coaxial pair of coils applied to the abdominal region, and with or without surface cooling blankets in each case. Thermometers were placed at multiple visceral and subcutaneous sites including an intraarterial thermocouple at the aortic arch level. With either electrode arrangement and no surface cooling, whole-body hyperthermia ( WBH ) at 42 degrees C was produced within 30 to 55 min with 250 W applied power; the 42 degrees C state could be maintained with 40 to 60 W of power. Thermal gradients in these cases reflected nonuniform power deposition superimposed upon arterial temperature elevation. With surface cooling blankets added, systemic heating was significantly reduced, and temperature gradients again reflected the nonuniform power deposition. Regional heating in a dog produces WBH unless sufficient surface cooling is used to provide a heat dissipation rate balancing the heat absorption rate; this latter case best models the use of inductive techniques in humans. The coaxial pair of coils, without surface cooling, produced rapid WBH and the visceral temperature maximum and minimum were within Tesoph + 0.21 degrees C and Tesoph - 0.07 degrees C, respectively (95% confidence index; Tesoph = esophageal temperature). This is an appropriate technique for the proposed toxicity study.

A standardized animal model for evaluation of hemostatic effectiveness of various materials.

Two anatomic sites of bleeding injuries were selected in dogs to evaluate the hemostatic effectiveness and handling properties of various hemostatic materials. Split-thickness skin excisions inflict a wound of 90 cm2 area with regular spotted bleeding, which allows gravimetric quantitation of blood loss and of ease of removal of the agent. Excision of a wedge of the spleen 0.5 cm deep and 4 cm wide induced reproducible and moderate bleeding in pentobarbital-anesthesized dogs. Three to four similar injuries could be inflicted in the same spleen with comparable blood flow. In this model of bleeding injury only the highly effective hemostatic agents based on collagen and with a sheet-like structure successfully stopped the bleeding within 3 minutes. Powder-like collagen was effective only when pressed against the wound. Gelatin sponge or oxidized cellulose materials were less effective.

The lateral nasal gland of dog: its structure and secretory content.

Lateral nasal glands of 12-15 weeks old pups are immature. Gland acini of pups and mature dogs contain numerous electron-lucent vacuoles with basophilic, electron-dense cores. The vacuoles contain both acid and neutral glycoproteins, sialated glycoproteins being the dominant acidic moiety. Lateral and basal cytoplasmic extensions of the acinar cytoplasm greatly increase the cell surface are. Electron-dense granules, smaller and less numerous than the lucent vacuoles of acini, occur in intercalated duct cells. Secretions collected form the excretory duct of the lateral nasal gland from conscious dogs contain 235 mg protein/100 ml secretory fluid. Immunoglobulin A accounts for 14% of the secretory protein. Total sialic acid content is 0.024% of the secretion by weight.

Attempts to produce salivary cysts in the dog.

Attempts were made to create salivary cysts by ligation of the mandibular salivary duct at the angle of the mandible and near the frenulum of the tongue, by rupture of the mandibular duct at the angle of the mandible and near the frenulum of the tongue, and by direct trauma to the mandibular salivary gland. Cysts were not formed as a result of the experimental procedures, but observations are made on the results obtained.

Suppurative otitis media in the rabbit: prevalence, pathology, and microbiology.

The middle ears of 2001 young and 583 adult rabbits being slaughtered for human consumption were examined. Otitis media was found in 87 (4%) of the young rabbits, and in 188 (32%) of the adult rabbits. The condition was bilateral in 61% of the affected young rabbits and 70% of the affected adults. Pasteurella multocida was isolated from 60 (98%) of 61 affected young rabbits. The gross and microscopic lesions and secondary complications were similar to those described in children. Rupture of the tympanic membrane was observed in both affected young and affected adult rabbits.