The role of the nucleus pulposus in intervertebral disc recovery: Towards improved specifications for nucleus replacement devices.

Nucleus replacement devices (NRDs) have potential to treat degenerated or herniated intervertebral discs (IVDs). However, IVD height loss is a post-treatment complication. IVD height recovery involves the nucleus pulposus (NP), but the mechanism of this in response to physiological loads is not fully elucidated. This study aimed to characterise the non-linear recovery behaviour of the IVD in intact, post-nuclectomy, and post-NRD treatment states, under physiological loading. 36 bovine IVDs (12 intact, 12 post-nuclectomy, 12 post-treatment) underwent creep-recovery protocols simulating Sitting, Walking or Running, followed by 12 h of recovery. A rheological model decoupled the fluid-independent (elastic, fast) and fluid-dependent (slow) recovery phases. In post-nuclectomy and post-treatment groups, nuclectomy efficiency (ratio of NP removed to remaining NP) was quantified following post-test sectioning. Relative to intact, post-nuclectomy recovery significantly decreased in Sitting (-0.3 ± 0.4 mm, p < 0.05) and Walking (-0.6 ± 0.3 mm, p < 0.001) coupled with significant decreases to the slow response (p < 0.05). Post-nuclectomy, the fast and slow responses negatively correlated with nuclectomy efficiency (p < 0.05). In all protocols, the post-treatment group performed significantly worse in recovery (-0.5 ± 0.3 mm, p < 0.01) and the slow response (p < 0.05). Results suggest the NP mainly facilitates slow-phase recovery, linearly dependent on the amount of NP present. Failure of this NRD to recover is attributed to poor fluid imbibition. Additionally, unconfined NRD performance cannot be extrapolated to the in vitro response. This knowledge informs NRD design criteria to provide high osmotic pressure, and encourages testing standards to incorporate long-term recovery protocols.

In vitro investigations of Staphylococcus aureus biofilms in physiological fluids suggest that current antibiotic delivery systems may be limited.

Orthopaedic surgical site infections, especially when a hardware is involved, are associated with biofilm formation. Clinical strategies for biofilm eradication still fall short. The present study used a novel animal model of long-bone fixation with vancomycin- or gentamicin-controlled release and measured the levels of antibiotic achieved at the site of release and in the surrounding tissue. Then, using fluids that contain serum proteins (synovial fluid or diluted serum), the levels of vancomycin or gentamicin required to substantially reduce colonising bacteria were measured in a model representative of either prophylaxis or established biofilms. In the in vivo model, while the levels immediately adjacent to the antibiotic release system were up to 50× the minimal inhibitory concentration in the first 24 h, they rapidly dropped. At peripheral sites, values never reached these levels. In the in vitro experiments, Staphylococcus aureus biofilms formed in serum or in synovial fluid showed a 5-10 fold increase in antibiotic tolerance. Importantly, concentrations required were much higher than those achieved in the local delivery systems. Finally, the study determined that the staged addition of vancomycin and gentamicin was not more efficacious than simultaneous vancomycin and gentamicin administration when using planktonic bacteria. On the other hand, for biofilms, the staged addition seemed more efficacious than adding the antibiotics simultaneously. Overall, data showed that the antibiotics' concentrations near the implant in the animal model fall short of the concentrations required to eradicate biofilms formed in either synovial fluid or serum.

A large animal model that recapitulates the spectrum of human intervertebral disc degeneration.

OBJECTIVE: The objective of this study was to establish a large animal model that recapitulates the spectrum of intervertebral disc degeneration that occurs in humans and which is suitable for pre-clinical evaluation of a wide range of experimental therapeutics. DESIGN: Degeneration was induced in the lumbar intervertebral discs of large frame goats by either intradiscal injection of chondroitinase ABC (ChABC) over a range of dosages (0.1U, 1U or 5U) or subtotal nucleotomy. Radiographs were used to assess disc height changes over 12 weeks. Degenerative changes to the discs and endplates were assessed via magnetic resonance imaging (MRI), semi-quantitative histological grading, microcomputed tomography (µCT), and measurement of disc biomechanical properties. RESULTS: Degenerative changes were observed for all interventions that ranged from mild (0.1U ChABC) to moderate (1U ChABC and nucleotomy) to severe (5U ChABC). All groups showed progressive reductions in disc height over 12 weeks. Histological scores were significantly increased in the 1U and 5U ChABC groups. Reductions in T2 and T1ρ, and increased Pfirrmann grade were observed on MRI. Resorption and remodeling of the cortical boney endplate adjacent to ChABC-injected discs also occurred. Spine segment range of motion (ROM) was greater and compressive modulus was lower in 1U ChABC and nucleotomy discs compared to intact. CONCLUSIONS: A large animal model of disc degeneration was established that recapitulates the spectrum of structural, compositional and biomechanical features of human disc degeneration. This model may serve as a robust platform for evaluating the efficacy of therapeutics targeted towards varying degrees of disc degeneration.

Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors.

Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3ß2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved ß-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, ß2, and ß4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4ß2 and α3ß2-nAChRs (IC50 ~0.17 and >3 µM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 µM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3ß2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3ß2-nAChRs.

Arthroscopy of the normal cadaveric ovine femorotibial joint: a systematic approach to the cranial and caudal compartments.

OBJECTIVES: Preclinical studies using large animal models play an intergral part in translational research. For this study, our objectives were: to develop and validate arthroscopic approaches to four compartments of the stifle joint as determined via the gross and arthroscopic anatomy of the cranial and caudal aspects of the joint. METHODS: Cadaveric hindlimbs (n = 39) were harvested from mature ewes. The anatomy was examined by tissue dissection (n = 6), transverse sections (n = 4), and computed tomography (n = 4). The joint was arthroscopically explored in 25 hindlimbs. RESULTS: A cranio-medial portal was created medial to the patellar ligament. The cranio-lateral portal was made medial to the extensor digitorum longus tendon. The medial femoral condyle was visible, as well as the cranial cruciate ligament, caudal cruciate ligament and both menisci with the intermeniscal ligament. Valgus stress improved visibility of the caudal horn of the medial meniscus and tibial plateau. To explore the caudal compartments, a portal was created 1 cm proximal to the most caudal aspect of the tibial condyle. Both femoral condyles, menisci, caudal cruciate ligament, the popliteal tendon and the menisco-femoral ligament were visible. The common peroneal nerve and popliteal artery and vein are vulnerable structures to injury during arthroscopy. CLINICAL SIGNIFICANCE: The arthroscopic approach developed in this research is ideal to evaluate the ovine stifle joint.

Molecular engineering of an orthopaedic implant: from bench to bedside.

The use of metallic implants has revolutionised the practice of orthopaedic surgery. While the safety and biocompatibility of these devices are excellent, a small percentage becomes infected. These infections are due to the formation of a biofilm that harbours bacteria encased in a complex extracellular matrix. The matrix serves as a barrier to immune surveillance as well as limiting the biocidal effects of systemic and local antibiotics. The objective of the review is to describe a novel approach to controlling implant infection using an antibiotic that is linked to titanium through a self-assembled monolayer of siloxy amines. We show that the hybrid-engineered surface is stable, biocompatible and resists colonisation by bacterial species most commonly associated with implant-related infections. Studies with rodent bone infection models suggest that the engineered titanium surface prevents bone infection. Results of a very recent investigation utilising a sheep model of infection indicate that the titanium-tethered antibiotic controls infection without compromising bone formation and remodelling. From all of these perspectives, the tethered antibiotic holds promise of providing a novel and practical approach to reducing implant-associated infections.

Intravenous technetium-99m labelled PEG-liposomes in horses: a safety and biodistribution study.

REASONS FOR PERFORMING STUDY: Liposomes are phospholipid nanoparticles that extravasate at sites of increased vascular permeability. They have potential in equine medicine for targeted drug delivery and diagnostic imaging of infectious, inflammatory and neoplastic lesions. OBJECTIVES: This study evaluates the safety and biodistribution of i.v. polyethyleneglycol(PEG) liposomes in normal horses. METHODS: PEG-liposomes were prepared by the film hydration method and labelled using (99m) Tc-hexamethyl-propylene-amine-oxime. A single dose of 0.24 µmol/kg bwt (99m) Tc-PEG-liposomes and 2.4 µmol/kg bwt unlabelled PEG-liposomes was administered to 10 conscious horses via i.v. infusion at a rate of 6 µmol/min for the first 15 min and 60 µmol/min thereafter. Clinical parameters, haematology, plasma biochemistry and serum complement activity were monitored serially. Scintigraphic imaging was performed at 1, 12 and 21 h post infusion (PI). Six horses were subjected to euthanasia at 24 h PI. The percentage injected dose per kilogram of tissue was calculated for multiple organs. Results were analysed using repeated measures ANOVA. RESULTS: Horses did not demonstrate adverse reactions during or after liposome infusion. There was a significant elevation in heart rate and respiratory rate at 20 and 25 min PI. No significant complement consumption was detected, although a trend for decreased total haemolytic complement values at 20 min PI was present. Scintigraphic studies revealed a prolonged vascular phase that lasted to 21 h PI, with a reproducible pattern of organ distribution. Biodistribution studies revealed the highest concentrations of radiopharmaceutical within the lung, kidney, liver and spleen. CONCLUSIONS: Intravenous liposome administration appears to be safe in horses. When administered in combination with PEG-liposomes, (99m) Tc-PEG-liposomes have long circulating characteristics and a reproducible pattern of organ distribution in horses. POTENTIAL RELEVANCE: Radiolabelled liposomes may be useful for detecting infection, inflammation and neoplasia in the horse. Liposomes have significant potential for targeted drug delivery in the horse. This study establishes the scintigraphic findings and tissue distribution of 99mTc-PEG-liposomes after i.v. administration in healthy horses.

Preclinical animal models in single site cartilage defect testing: a systematic review.

OBJECTIVE: Review the literature for single site cartilage defect research and evaluate the respective strengths and weaknesses of different preclinical animal models. METHOD: A literature search for animal models evaluating single site cartilage defects was performed. Variables tabulated and analyzed included animal species, age and number, defect depth and diameter and study duration. Cluster analyses were then used to separate animals with only distal femoral defects into similar groups based on defect dimensions. Representative human studies were included allowing comparison of common clinical lesions to animal models. The suitability of each species for single site cartilage defect research and its relevance to clinical human practice is then discussed. RESULTS: One hundred thirteen studies relating to single site cartilage defects were reviewed. Cluster analysis included 101 studies and placed the murine, laprine, ovine, canine, porcine and caprine models in group 1. Group 2 contained ovine, canine, porcine, caprine and equine models. Group 3 contained only equine models and humans. Species in each group are similar with regard to defect dimensions. Some species occur in multiple groups reflecting utilization of a variety defect sizes. We report and discuss factors to be considered when selecting a preclinical animal model for single site cartilage defect research. DISCUSSION: Standardization of study design and outcome parameters would help to compare different studies evaluating various novel therapeutic concepts. Comparison to the human clinical counterpart during study design may help increase the predictive value of preclinical research using animal models and improve the process of developing efficacious therapies.

Conservative management of 17 horses with nonarticular fractures of the tibial tuberosity.

REASONS FOR PERFORMING STUDY: Fractures of the tibial tuberosity (FTT) are caused by direct trauma, and are the second most commonly reported injury in event horses with stifle trauma. Conservative management of horses with FTT has been advocated, but results and prognosis for this method of therapy are unknown. OBJECTIVES: To report and review the findings of a retrospective study of 17 horses admitted to a veterinary teaching hospital from 1986-2001 with nonarticular FTT that received conservative management. METHODS: Subject details, aetiology of the accident, limb affected, degree of lameness at time of admission, size and degree of displacement of the fracture fragment, complications such as comminution of the fracture fragment or damage to soft tissue structures within the affected stifle, and treatment recommendations were obtained from medical records. Owners and trainers were contacted regarding the horse's return to athletic use. The follow-up period consisted of 11-154 months. RESULTS: Two horses were reportedly sound, but unable to return to competition for unrelated reasons. Of the horses that completed the rehabilitation period, 12/15 (80%) returned to athletic use at the same level as before the injury. Three horses were diagnosed with damage to soft tissue supporting structures of the affected stifle and could not return to their former level of competition. CONCLUSIONS: Concurrent soft tissue damage, diagnosed at the time of the initial injury, was statistically significant in precluding horses from returning to athletic careers. All other variables were found to have no effect upon outcome. POTENTIAL CLINICAL RELEVANCE: This retrospective study suggests that the conservative management of nonarticular FTT is a viable treatment modality in managing athletic equine patients presenting with these fractures.

Proximal interphalangeal arthrodesis in 22 horses.

The purpose of this study was to evaluate a new method of internal fixation technique for pastern arthrodesis. Pastern arthrodeses are performed commonly in horses with chronic osteoarthritis of the pastern joint or, in cases of acute traumatic injury to the pastern, in which the weightbearing bony column must be restored. Chronic osteoarthritis of the pastern is a frequent cause of lameness in the equine athlete and is evidenced by chronic lameness localised to the pastern joint, and supported radiographically by periosteal proliferation and loss of joint space. Nonsurgical and surgical treatments have both been described in the literature. Complications following pastern arthrodesis have been reported on several occasions and appear to focus on excessive periarticular exostoses and increased time in a cast due to prolonged time to bony fusion. The hospital records of horses presenting for pastern arthrodesis to the Rood and Riddle Equine Hospital in Lexington, Kentucky, were reviewed and 22 met criteria for inclusion in the study. Horses with chronic osteoarthritis of the proximal interphalangeal joint or horses with an acute traumatic injury to the pastern undergoing pastern arthrodesis with one of the following techniques were included in the study. Horses with severe comminution of the middle phalanx were excluded. Three 5.5 mm cortical bone screws placed in lag fashion alone or in combination with a 4 or 3 hole dynamic compression plate affixed with 4.5 mm cortical bone screws were compared. A lower limb fibreglass cast was applied in all cases. Period in cast, time to return to intended use, complications encountered and outcome were evaluated. Seven of the 8 hindlimbs treated with the combination technique became sound. Three out of 6 of the front limbs treated with the combination technique became sound. Four of the 5 horses with hindlimbs, and one of the 2 with front limbs, treated with screws only returned to their intended use. The type of internal fixation did not appear to influence the overall number of horses returning to the intended level of performance. The period spent in cast and the time to return to soundness were decreased in horses operated on using the combination technique. We concluded that, in the immediate postoperative period, the combination of the parallel screw technique with a dorsally-applied dynamic compression plate provides the most stable and secure fixation, minimising motion, expediting bone remodelling and therefore favouring rapid fusion of that joint.