Femoral and sciatic nerve blockades and incision site infiltration in rabbits undergoing stifle joint arthrotomy.

This study was designed to determine whether perineural injections of local anaesthetics decreases intraoperative nociception and improves postoperative analgesia in New Zealand White rabbits undergoing experimental stifle arthrotomy. All animals were anaesthetized using isoflurane and received morphine intramuscularly. The sciatic and femoral nerves of the leg to be operated on were located using a nerve stimulator (1 Hz, 0.5 mA). Rabbits were assigned to a treatment group (LB; n = 12) or a placebo group (P; n = 12) in a randomized blinded fashion. Group LB received lidocaine 2% (1 mg/kg) combined with bupivacaine 0.5% (0.25 mg/kg) injections around the sciatic and femoral nerves (0.1 mL/kg total volume per site) and subcutaneous infiltration of the incision site with lidocaine 1% (1.25 mg/kg). Group P received the same volume of 0.9% NaCl. Rabbits in group P required higher doses of intraoperative fentanyl and propofol to reduce heart rate and suppress increase in systolic blood pressure, and maintain an adequate anaesthetic plane. Interventional analgesia (buprenorphine and carprofen) was required significantly earlier in rabbits in group P (2 and 6 h after the first nerve blockade, respectively) based on assessment of their pain-related behaviour and range of motion. Using a visual analogue scale (0 mm= no pain to 100 mm= maximal possible pain), rabbits in group LB were judged to show significantly less pain compared with rabbits in group P (14 ± 10 mm and 37 ± 25 mm, respectively) 2 h after nerve blockade. In conclusion, this technique of perineural analgesia combined with incision site infiltration reduced intraoperative fentanyl requirements and improved postoperative analgesia in rabbits.

Augmentation of bone defect healing using a new biocomposite scaffold: an in vivo study in sheep.

Previous studies support resorbable biocomposites made of poly(L-lactic acid) (PLA) and beta-tricalcium phosphate (TCP) produced by supercritical gas foaming as a suitable scaffold for tissue engineering. The present study was undertaken to demonstrate the biocompatibility and osteoconductive properties of such a scaffold in a large animal cancellous bone model. The biocomposite (PLA/TCP) was compared with a currently used beta-TCP bone substitute (ChronOS, Dr. Robert Mathys Foundation), representing a positive control, and empty defects, representing a negative control. Ten defects were created in sheep cancellous bone, three in the distal femur and two in the proximal tibia of each hind limb, with diameters of 5 mm and depths of 15 mm. New bone in-growth (osteoconductivity) and biocompatibility were evaluated using microcomputed tomography and histology at 2, 4 and 12 months after surgery. The in vivo study was validated by the positive control (good bone formation with ChronOS) and the negative control (no healing with the empty defect). A major finding of this study was incorporation of the biocomposite in bone after 12 months. Bone in-growth was observed in the biocomposite scaffold, including its central part. Despite initial fibrous tissue formation observed at 2 and 4 months, but not at 12 months, this initial fibrous tissue does not preclude long-term application of the biocomposite, as demonstrated by its osteointegration after 12 months, as well as the absence of chronic or long-term inflammation at this time point.

Surface polishing positively influences ease of plate and screw removal.

Difficulties removing temporary fracture fixation devices due to excessive bony on-growth results in extended surgical time leading to excessive blood loss, debris contamination and potentially refracture. Commercially available locking plates and screws are manufactured for clinics with a micro-rough surface, which contributes to the excessive bony on-growth reported. We have applied polishing technology to commercially pure titanium locking compression plates (LCP) and titanium-6%aluminium-7%niobium (TAN) plates and screws to assess if it can alleviate problems with strong bony overgrowth. Samples were implanted for 6, 12 and 18 months in a bilateral sheep tibia non fracture model and assessed for screw removal torque, percentage of bone contact and tissue-material response. Both electropolishing (p=0.001) and paste polishing (p=0.010) of TAN screws significantly reduced the mean torque required for removal compared to their micro-rough counterparts. This was accompanied by a trend for a lower percentage of bone contact for polished screws. This difference in bone contact was significant for paste polished TAN screws (p<0.001 parallel but not electropolished TAN screws (p=0.066). Ex vivo, soft tissue removal was much easier (approximately five minutes) for polished constructs, which was difficult and at least four times longer for standard micro-rough constructs. We suggest that polishing of locked plate/screw systems will improve ease of removal and reduce implant related removal complications encountered due to excessive strong bony on-growth while maintaining biocompatibility and implant stability. Future studies aim to assess the potential of this technology in the next level of complication, a fracture model.

Tissue reaction to implants of different metals: a study using guide wires in cannulated screws.

Cannulated screws, along with guide wires, are typically used for surgical fracture treatment in cancellous bone. Breakage or bending deformation of the guide wire is a clinical concern. Mechanically superior guide wires made of Co-Cr alloys such as MP35N and L605 may reduce the occurrence of mechanical failures when used in combination with conventional (316L stainless steel) cannulated screws. However the possibility of galvanic or crevice corrosion and adverse tissue reaction, exists when using dissimilar materials, particularly in the event that a guide wire breaks, and remains in situ. Therefore, we designed an experiment to determine the tissue reaction to such an in vivo environment. Implant devices were designed to replicate a clinical situation where dissimilar metals can form a galvanic couple. Histological and SEM analyses were used to evaluate tissue response and corrosion of the implants. In this experiment, no adverse in vivo effects were detected from the use of dissimilar materials in a model of a broken guide wire in a cannulated screw.

An in vivo evaluation of surface polishing of TAN intermedullary nails for ease of removal.

Fractures of the tibia and femoral diaphysis are commonly repaired by intra-medullary (IM) nailing. Currently IM nails are available in either electropolished stainless steel (SS) or in Titanium-Aluminium-Niobium (TAN). After healing, removal of the nails still is common but removal of TAN IM nails often has complications whereas SS IM nails of the same design are less often associated with problems. We believe the differences in removal are due to the ability of TAN to promote strong bone on-growth. We have previously shown in vivo that polishing cortical screws reduces removal torque and the percentage of bone-implant contact. Therefore, we postulate that bony on-growth onto IM nails can be reduced by means of surface polishing, for ease of removal. Here we aim to compare the pull-out forces for removal of standard TAN (TAN-S) compared to experimental paste polished TAN (TAN-PP) IM nails from a bilateral non-fracture sheep tibia model after 12 months implantation. Histological analysis was also performed to assess tissue on-growth to the nails. We show that polishing significantly reduces (p=0.05) the extraction force required for TAN IM nail removal. This effect in part is attributable to the distinct tissue-material reaction produced. For TAN-S nails direct bone contact was observed while for TAN-PP nails a fibrous tissue interface was noted. Since TAN is preferred over SS for IM nailing due to superior biocompatibility and mechanical properties, we believe these findings could be used to recommend changes to current surface technologies of intramedullary nails to reduce complications seen with nail removal especially in rapidly growing bone in children.

Regeneration of large bone defects in sheep using bone marrow stromal cells.

Bone repair was addressed in a critical-sized defect model in sheep, combining a ceramic biomaterial and mesenchymal progenitor cells. The defects in the tibial mid-diaphysis were treated with autologous bone or with a silicon-stabilized tricalcium phosphate biomaterial, implemented or not by the addition of expanded bone marrow stromal cells. An internal locking compression plate and an external fixator were applied for stabilization. Radiographies were taken during the 8 months follow-up: the pixel grey levels of the lesion areas were determined to evaluate the repair process radiologically. Microradiography, histology and vascular density tests were performed. The autologous bone-treated group performed best, as assessed radiologically, within 20-24 weeks after surgery. Very limited healing was detected in the other experimental group: a partial bone deposition occurred at the periphery of the bony stumps only in the cell-seeded scaffolds. Interestingly, this effect ended within 20-24 weeks, as for the autologous bone, suggesting similar kinetics of the repair processes involved. Moreover, bone deposition was located where a significant reduction of the ceramic scaffold was detected. Faxitron microradiography and histology data confirmed these results. Vascular density analysis evidenced that cell-seeded scaffolds supported an increased vascular ingrowth. Thus, the interactions with the proper microenvironment and the oxygen and nutrient supply in the inner part of the constructs seem fundamental to initiate scaffold substitution and to improve cell performance in tissue-engineered approaches to bone repair.

Animal models for implant biomaterial research in bone: a review.

Development of an optimal interface between bone and orthopaedic and dental implants has taken place for many years. In order to determine whether a newly developed implant material conforms to the requirements of biocompatibility, mechanical stability and safety, it must undergo rigorous testing both in vitro and in vivo. Results from in vitro studies can be difficult to extrapolate to the in vivo situation. For this reason the use of animal models is often an essential step in the testing of orthopaedic and dental implants prior to clinical use in humans. This review discusses some of the more commonly available and frequently used animal models such as the dog, sheep, goat, pig and rabbit models for the evaluation of bone-implant interactions. Factors for consideration when choosing an animal model and implant design are discussed. Various bone specific features are discussed including the usage of the species, bone macrostructure and microstructure and bone composition and remodelling, with emphasis being placed on the similarity between the animal model and the human clinical situation. While the rabbit was the most commonly used of the species discussed in this review, it is clear that this species showed the least similarities to human bone. There were only minor differences in bone composition between the various species and humans. The pig demonstrated a good likeness with human bone however difficulties may be encountered in relation to their size and ease of handling. In this respect the dog and sheep/goat show more promise as animal models for the testing of bone implant materials. While no species fulfils all of the requirements of an ideal model, an understanding of the differences in bone architecture and remodelling between the species is likely to assist in the selection of a suitable species for a defined research question.

Engineering of bone using bone marrow stromal cells and a silicon-stabilized tricalcium phosphate bioceramic: evidence for a coupling between bone formation and scaffold resorption.

Resorbable porous ceramic constructs, based on silicon-stabilized tricalcium phosphate, were implanted in critical-size defects of sheep tibias, either alone or after seeding with bone marrow stromal cells (BMSC). Only BMSC-loaded ceramics displayed a progressive scaffold resorption, coincident with new bone deposition. To investigate the coupled mechanisms of bone formation and scaffold resorption, X-ray computed microtomography (muCT) with synchrotron radiation was performed on BMSC-seeded ceramic cubes. These were analyzed before and after implantation in immunodeficient mice for 2 or 6 months. With increasing implantation time, scaffold thickness significantly decreased while bone thickness increased. The muCT data evidenced that all scaffolds showed a uniform density distribution before implantation. Areas of different segregated densities were instead observed, in the same scaffolds, once seeded with cells and implanted in vivo. A detailed muX-ray diffraction analysis revealed that only in the contact areas between deposited bone and scaffold, the TCP component of the biomaterial decreased much faster than the HA component. This event did not occur at areas away from the bone surface, highlighting coupling and cell-dependency of the resorption and matrix deposition mechanisms. Moreover, in scaffolds implanted without cells, both the ceramic density and the TCP:HA ratio remained unchanged with respect to the pre-implantation analysis.

Effect of heel elevation on forelimb conformation in horses.

OBJECTIVE: To determine the conformational changes in the distal forelimb of horses following heel elevation of 15 degrees and greater. An experimental study with repeated, within-horse measurements. PROCEDURE: Five clinically normal, mixed-breed horses were used to determine distal forelimb conformation following heel elevation from 0 to 45 degrees in 15 degree increments. Data were also compared to conformation of the limb in a Kimzey splint. Conformation was determined using lateral to medial radiographic projections. Conformation parameters assessed included measurement of apparent lengths of digital flexor tendons from the origin of their accessory ligaments to the points of insertion, and the joint angles of the distal limb. RESULTS: For angles of heel elevation from 15 to 45 degrees, the degree of joint angulation increased (increasing flexion) with heel elevation for the metacarpophalangeal, and proximal and distal interphalangeal joints. Conversely, the measured apparent lengths of the digital flexor tendons in the distal limb, decreased. CONCLUSION: The data supports the practice of elevating the heel (greater than 15 degrees) for conditions in which decreased fetlock extension may be desired such as with laceration or injury to the digital flexor tendons. Further study is required to determine whether heel elevation greater than 15 degrees reduces in vivo digital flexor tendon tension and also to ensure that the marked flexion of the distal interphalangeal joint with greater heel elevation is not detrimental over a prolonged period that may be required for the rehabilitation of flexor tendon injuries in the horse.

Management of obstructive urolithiasis and concurrent urethral rupture in a goat.

A 4-month-old, male castrated Alpine White goat presented to the University of Guelph, Veterinary Teaching Hospital, with urolithiasis and a ruptured urethra. A tube cystostomy was performed using an indwelling Foley catheter. Postoperatively, the goat remained in good health, and was discharged with the cystostomy catheter in situ. Five weeks following surgery, the goat was noted to pass urine through its urethra. Seven weeks following surgery, the catheter became dislodged and the goat was observed to urinate normally. Six months following surgery, the goat was apparently healthy and urinating normally. Tube cystostomy may be a useful therapeutic option in the management of small ruminants with obstructive urolithiasis with concurrent rupture of the urethra, especially for cases in which treatment cost is a limiting factor.

An investigation of 2 techniques for optimizing joint surface congruency using multiple cylindrical osteochondral autografts.

PURPOSE: To compare 2 techniques for optimizing joint congruency for miniature osteochondral autografting in the knee: intrinsic postoperative forces acting on overdrilled autografts protruding from the femur versus alignment by a surgeon at the time of grafting. TYPE OF STUDY: Controlled animal model experiment. METHODS: A full-thickness cartilage defect was created on the weight-bearing surface of the medial femoral condyle of 13 mature sheep. Three 4.5 x 10 mm cylindrical autografts were inserted into 14-mm deep recipient holes such that the grafts were held in place by side-wall friction alone. One treatment group received grafts that were delivered flush with the surrounding cartilage and the second group received grafts that were left 2-mm proud of the joint surface. RESULTS: Three months postoperatively, the proud grafts had been repositioned by weight bearing but perigraft fissuring and fibroplasia, and subchondral cavitations were serious complications. It is suspected that these complications were caused by excessive motion between the graft and recipient site in the proud grafts. CONCLUSIONS: Grafts should be delivered flush with the joint surface when performing osteochondral transfers to avoid graft micromotion and the consequent interference with graft integration and function.

Treatment of a granuloma caused by Halicephalobus gingivalis in a horse.

A 12-year-old Arabian gelding with a granuloma caused by the nematode Halicephalobus gingivalis caudal and dorsal to the left orbit was examined. There was no evidence of dissemination of the nematode to the kidneys or the CNS, and the horse was treated with ivermectin (1.2 mg/kg [0.55 mg/lb] of body weight, p.o., every 2 weeks for 3 treatments). The granuloma was surgically debulked 2 days after the first dose of ivermectin. The granuloma resolved with no evidence of nematode infection after 18 months. Halicephalobus gingivalis is a ubiquitous saprophytic nematode that has been reported to infect humans and horses. The nematode may form granulomas in the integument or may disseminate to various organs with a tropism for the CNS and kidneys. Once clinical signs of CNS involvement develop, the disease is rapidly fatal.

Surgical repair of a ruptured cranial cruciate ligament in a dromedary camel.

A 14-year-old dromedary camel was examined because of lameness of the left hind limb of 3.5 months'duration. The camel injured the stifle joint when it slipped while reclining into sternal recumbency. Radiography did not reveal major abnormalities that could account for the lameness, but nuclear scintigraphy revealed increased radionuclide uptake in the left stifle joint. Intra-articular injection of anesthetic further localized the injury to the area of the left stifle joints, and arthroscopy of that joint revealed rupture of the cranial cruciate ligament. Surgical repair of the ligament was attempted, using an autogenous graft obtained from the tensor fascia lata muscle. Outcome for the camel initially was considered good on the basis of a substantial improvement in attitude, apparent increase in comfort, and decrease in lameness. The tendency for the camel to remain in sternal recumbency after surgery increased the number and severity of postoperative complications. The camel was doing well 9 months after surgery; however, it was euthanatized 16 months after surgery because of a 4-month episode of lameness. Necropsy revealed chronic osteoarthritis and bilateral rupture of the cranial cruciate ligaments.

Content and distribution of macro- and micro-elements in the body of pasture-fed young horses.

OBJECTIVE: To determine the content and distribution of Na, K, Ca, P, Mg, S, Cu, Mn, Fe and Zn in the body of pasture-fed young horses and then use a factorial model to calculate the dietary mineral requirements for growth. PROCEDURE: Twenty-one foals were killed at about 150 days of age and the organs, soft tissues, skin and bones and a sample of muscle were dissected out and weighted. The mineral concentrations of elements in all soft tissues and bones were measured by inductively coupled emission spectrometry. The total mineral element composition associated with a tissue was determined from the weight of tissue and its mineral element concentration. RESULTS: Expressed as a percent of total body mineral elements, muscle contained 20% Na, 78% K, 32% Mg, 62% Cu, 36% Mn and 57% Zn, bone contained 47% Na, 99% Ca, 81% P, 62% Mg, 30% Mn and 28% Zn while the organs accounted for a smaller percentage ranging from 0.06% for Ca to 26% for Fe. In liver Cu accounted for 9.2% of total body Cu. Each kilogram of empty body weight was associated with 1.0 g Na, 2.5 g K, 17.1 g Ca, 10.1 g P, 0.4 g Mg, 1.1 mg Cu, 0.39 mg Mn, 52.5 mg Fe and 21.4 mg Zn. CONCLUSION: The mineral element content of body weight gain is a component used in the factorial model to determine dietary mineral element requirements for growth. The calculated dietary mineral requirements, expressed per kg dry matter, for a 200 kg horse gaining 1.0 kg/day were 1.0 g Na, 2.1 g K, 4.6 g Ca, 3.5 g P, 0.7 g Mg, 4.5 mg Cu and 25 mg Zn.

Concentrations of macro- and micro-elements in the milk of pasture-fed thoroughbred mares.

OBJECTIVE: To determine the changes in Ca, P, Mg, Na, K, S, Cu, Fe and Zn concentrations of milk during the lactation in pasture-fed Thoroughbred mares and then calculate the dietary mineral requirements of the sucking foal and the lactating mare. PROCEDURE: Milk was sampled on days 1, 3, 7, 14, 21, 28 and at various times between 55 to 65, 85 to 95 and 135 to 150 days after parturition from 21 pasture-fed mares. The concentrations of macro- and micro-elements in the milk were determined by inductively coupled plasma emission spectrometry. RESULTS: Concentrations (mg/L) of these elements were highest in colostrum (Mg 302, Na 561, K 955, S 1035, Cu 0.76, Fe 0.79 and Zn 5.5) except for Ca (1245) and P (895), which where highest on day 7. The mean milk mineral element concentrations (mg/L) over days 55 to 150 were Ca 843, P 543, Mg 47, Na 120, K 590, S 219, Cu 0.19, Fe 0.34 and Zn 2.1. The mean plasma element concentrations (mg/L) over the same period were Ca 120, P 77.1, Mg 17.0, Na 3110, K 168, S 983, Cu 1.1, Fe 1.5 and Zn 0.49. Concentration gradients between plasma and milk were observed and, in the case of Ca, P, Mg, K and Zn, their concentrations in milk were greater than those in plasma, while a reverse situation was observed for Na, S, Cu and Fe. CONCLUSION: With the exception of Ca and P, the highest concentrations of mineral elements were observed in the colostrum. The nursing foal should have access to good pasture or creep feed, because the intakes of Ca, P and Cu from milk may be less than optimum to meet the daily mineral element requirements.

Effect of copper supplementation on copper status of pregnant mares and foals.

Pregnant Thoroughbred mares (n = 21) were grazed on tall fescue pasture containing 4.4-8.6 mg Cu/kg dry matter (DM). Twelve mares were supplemented with approximately 0.5 mg Cu/kg liveweight (LW)/day as copper sulphate for the final 13-25 weeks gestation. Plasma copper concentration declined during the third trimester for all mares and was not affected by copper supplementation. Liver copper concentration of mares and foals was determined when the foals were 4-10-days-old. Copper supplementation of the mares tended to increase mare liver copper concentration (P<0.08) and increased foal liver copper concentration (P<0.01). There was a significant relationship between mare and foal liver copper concentration (P<0.001, r2 = 0.7). Increasing the copper intake of mares is effective in enhancing the copper status of foals.

Effect of copper supplementation on the copper status of pasture-fed young Thoroughbreds.

The effect of copper supplementation of pasture fed mares and foals on the copper status of the foals, in terms of plasma, soft tissue and bone copper concentrations and caeruloplasmin activity, was investigated. Twenty-one Thoroughbred foals from either control mares (n = 9), or copper-supplemented mares (n = 12) were divided randomly into control (pasture only, n = 10) or supplemented (pasture and oral copper sulphate, n = 11) groups. The pasture diet was grazed by all animals, and contained 4.4-8.6 mg Cu/kg dry matter (DM). The copper supplement for the mares contained copper sulphate equivalent to 0.5 mg Cu/kg liveweight (LW)/day. This daily dose was converted to allow administration as a thrice weekly dose (i.e. multiplied by 7/3) which was given for 13-25 weeks prior to foaling. The supplemented foals, also dosed 3 times a week, received 0.2 mg Cu/kg LW/day at age 21 days, which was increased to 0.5 mg Cu/kg LW/day at 49 days and was continued at this level until euthanasia at 150 days. Foal plasma copper concentration and caeruloplasmin activity increased from birth to 21 days post partum and then plateaued at a concentration similar to the mare, but the rise in these indices was not affected by copper supplementation of the mare or foal. Copper supplementation of the foal increased foal liver copper concentration at 150 days (P<0.03). Copper intake of diets containing approximately 8-28 mg Cu/kg DM is well reflected by liver copper concentration, but is poorly reflected by bone, other soft tissue copper concentrations and circulating copper status indices.

Effect of copper supplementation on the evidence of developmental orthopaedic disease in pasture-fed New Zealand Thoroughbreds.

The effect of copper status on the evidence of bone and cartilage lesions was investigated in 21 Thoroughbred foals. The foals and their dams were grazed on pasture containing 4.4-8.6 mg Cu/kg dry matter (DM). Four treatment groups were created by randomly allocating mares and their foals to either copper supplemented (0.5 mg Cu/kg liveweight (LW)/day), or control (pasture only) groups. This experimental design allowed the effect of copper supplementation of mare and foal to be examined independently. Parameters of bone and cartilage development were assessed in the foals both in vivo, and at post mortem at approximately age 150 days. Mare copper supplementation significantly (P<0.01) decreased radiographic indices of physitis in the distal third metatarsal bone of the foals at 150 days, and the prevalence of articular cartilage lesions (P<0.05). Articular cartilage lesions were minor in all foals, with no evidence of clinical developmental orthopaedic disease (DOD) in vivo, with the exception of minor radiographic changes assessed at post mortem. Copper supplementation of the foal had no effect on any of the bone and cartilage parameters. Copper supplementation of the mares did not abolish DOD in the growing foals, emphasising the probable multifactorial nature of this condition. However, mare supplementation may be a useful treatment regime on a farm where the incidence and severity of DOD are of concern.

Liver biopsy techniques for adult horses and neonatal foals to assess copper status.

OBJECTIVE: To evaluate standing, percutaneous, ultrasound-guided, transthoracic liver biopsy in mares, and transabdominal laparoscopically-guided, liver biopsy under general anaesthesia in foals, as techniques for obtaining tissue for assessment of copper status. The techniques were evaluated with respect to ease of use and effect on the animal. PROCEDURE: Twenty of 24 Thoroughbred mares and 21 of their foals were biopsied. The animals were part of a larger study of the effect of copper supplementation on copper status and the prevalence of developmental orthopaedic disease. Livers were also collected from unrelated horses and sampled to investigate the variability in the distribution of copper in liver tissue. RESULT: The biopsy technique caused no lasting effect on the mares, but there was an increased risk of viscus penetration associated with taking multiple biopsy cores. The use of ultrasonography to scan the target area for the liver identified four cases that were not appropriate candidates for liver biopsy, because of large intestine being located in the biopsy area. In the foals there were no serious postoperative adverse effects, nor was there any evidence of problems caused by the procedure when the abdomen was examined post-mortem at 5 months of age. In livers collected to investigate the variability of copper concentration, copper appeared to be relatively evenly distributed through the liver. CONCLUSION: Standing, percutaneous, ultrasound-guided, transthoracic liver biopsy in mares, and transabdominal, laparoscopically-guided, liver biopsy under general anaesthesia in foals are convenient procedures for obtaining liver tissue for assessing copper status in horses. The use of ultrasound to identify liver tissue is recommended, especially in older mares.