Matrix metalloproteinase-based photodynamic molecular beacons for targeted destruction of bone metastases in vivo.

The metastatic spread of cancer from the primary site or organ is one of its most devastating aspects, being responsible for up to 90% of cancer-associated mortality. Bone is one of the common sites of metastatic spread, including the vertebrae. Regardless of the treatment strategy, the clinical goals for patients with vertebral metastases are to improve the quality of life by preventing neurologic decline, to achieve durable pain relief and enhance local tumor control. However, in part due to the close proximity of the spinal cord, current treatment options are limited. We propose a novel therapeutic strategy with the use of photodynamic molecular beacons (PMBs) for targeted destruction of spinal metastases, particularly to de-bulk lesions as an adjuvant to vertebroplasty or kyphoplasty in order to mechanically stabilize weak or fractured vertebrae. The PDT efficacy of a matrix metalloproteinase-specific PMB is reported in a metstatic model that recapitulates the clinical features of tumor growth within the bone. We demonstrate that not only does tumor cell destruction occur but also the killing of bone stromal cells. The potential of PMB-PDT to destroy metastatic tumors, disrupt the osteolytic cycle and better preserve critical organs with an increased therapeutic window compared with conventional photosensitizers is demonstrated.

Gene delivery to the spinal cord using MRI-guided focused ultrasound.

Non-invasive gene delivery across the blood-spinal cord barrier (BSCB) remains a challenge for treatment of spinal cord injury and disease. Here, we demonstrate the use of magnetic resonance image-guided focused ultrasound (MRIgFUS) to mediate non-surgical gene delivery to the spinal cord using self-complementary adeno-associated virus serotype 9 (scAAV9). scAAV9 encoding green fluorescent protein (GFP) was injected intravenously in rats at three dosages: 4 × 10(8), 2 × 10(9) and 7 × 10(9) vector genomes per gram (VG g(-1)). MRIgFUS allowed for transient, targeted permeabilization of the BSCB through the interaction of focused ultrasound (FUS) with systemically injected Definity lipid-shelled microbubbles. Viral delivery at 2 × 10(9) and 7 × 10(9) VG g(-1) leads to robust GFP expression in FUS-targeted regions of the spinal cord. At a dose of 2 × 10(9) VG g(-1), GFP expression was found in 36% of oligodendrocytes, and in 87% of neurons in FUS-treated areas. FUS applications to the spinal cord could address a long-term goal of gene therapy: delivering vectors from the circulation to diseased areas in a non-invasive manner.

Local treatment of mixed osteolytic/osteoblastic spinal metastases: is photodynamic therapy effective?

The widespread use of systemic and local therapies aimed at spinal metastatic lesions secondary to breast cancer has increased the incidence of mixed osteolytic/osteoblastic patterns of bony disease. The complex structure of these lesions requires novel therapeutic approaches to both reduce tumor burden and restore structural stability. In photodynamic therapy (PDT), a minimally invasive approach can be used to employ light to activate a photosensitizing agent that preferentially accumulates in tumor tissue, leading to cell toxicity and death. Previous work in an osteolytic rat model (MT-1) demonstrated that PDT effectively ablates tumor and improves vertebral structural properties. The aim of this study was to assess the efficacy of PDT in a rat model of mixed osteolytic/osteoblastic spinal metastases. Mixed spinal metastases were generated through intracardiac injection of Ace-1 canine prostate cancer cells into female athymic rats (day 0). A single PDT treatment was applied to lumbar vertebra L2 of tumor-bearing and healthy control rats (day 14). PDT-treated and untreated control rats were euthanized and excised spines imaged with µCT to assess bone quality (day 21). Spines were mechanically tested or histologically processed to assess mechanical integrity, tumor burden, and remodelling properties. Untreated tumor-bearing vertebrae showed large areas of osteolysis and areas of immature, new bone formation. The overall bone quality resulting from these lesions consisted of decreased structural properties but without a significant reduction in mechanical integrity. PDT was shown to significantly decrease tumor burden and osteoclastic activity, thereby improving vertebral bone structural properties. While non-tumor-bearing vertebrae exhibited significantly more new bone formation following PDT, the already heightened level of new bone formation in the mixed tumor-bearing vertebrae was not further increased. As such, the effect of PDT on mixed metastases may be more influenced by suppression of osteoclastic resorption as opposed to the triggering of new bone formation.

Intraoperative cone-beam CT spatial priors for diffuse optical fluorescence tomography.

A hybrid system for intraoperative cone-beam CT (CBCT) imaging and continuous-wave fluorescence tomography (FT) has been developed using an image-guidance framework. Intraoperative CBCT images with sub-millimeter spatial resolution are acquired with a flat-panel C-Arm. Tetrahedral meshes are generated from CBCT for finite element method implementation of diffuse optical tomography (NIRFAST). Structural data from CBCT is incorporated directly into the optical reconstruction process using Laplacian-type regularization ('soft spatial priors'). Experiments were performed using an in-house optical system designed for indocyanine green (ICG) fluorescence. A dynamic non-contact geometry was achieved using a stereoscopic optical tracker for real-time localization of a laser diode and CCD camera. Source and detector positions were projected onto the boundary elements of the tissue mesh using algorithms for ray-triangle intersection and camera lens calibration. Simulation studies showed the capabilities of a soft-prior approach, even in the presence of segmentation uncertainties. Experiments with ICG targets embedded in liquid phantoms determined the improvements in the quantification of the fluorophore yield, with errors of 85% and <20% for no priors and spatial priors, respectively. Similar results were observed with the ICG target embedded in ex vivo porcine loin, with errors of 52% and 12%, respectively. A proof-of-principal animal study was performed in a VX2-tumor in vivo rabbit model using liposomal nanoparticles co-encapsulating contrast for CT (iohexol) and fluorescence (ICG) imaging. Fusion of CBCT and FT reconstructions demonstrated concurrent anatomical and functional delineations of contrast enhancement around the periphery of the buccal tumor. These developments motivate future clinical translation of the FT system into an ongoing CBCT-guided head and neck surgery trial.

Mosaicplasty with photooxidized, mushroom shaped, bovine, osteochondral xenografts in experimental sheep.

The goal was to study the performance of mushroom shaped, photooxidized, osteochondral grafts in mosaicplasty focusing on graft stability and survival. Mushroom shaped, photooxidized grafts (6 mm for the cartilaginous head of the mushroom, 3 mm for the stem) were implanted in the medial femoral condyle of 10 sheep. Four transplants were inserted per condyle in an overlapping fashion using the pressfit technique (n=40 grafts in 10 condyles). The grafts were followed for 6 and 12 months. Semi-quantitative evaluation of graft performance was performed using a validated score system. All grafts were mechanically stable at 6 and 12 months with one exception, where the mushroom head broke off. The formation of cystic lesions in the subchondral bone area was minimal. Repopulation of the old photooxidized cartilage was noticed with cells invading the matrix from the subchondral bone area and also from the pannus on the surface. Fusion between host and graft cartilage was observed in some of the grafts at 12 months, while remodeling of the calcified cartilage zone and tidemark was noticed in all grafts. Results scored significantly better for the 6 months compared to the 12 months group if cartilage surface integrity was compared (p<0.05). In all other variables no significant differences were found between groups. Despite moderate graft recession in the 12 months group partial fusion of grafts and functional results were satisfactory. The photooxidized mushroom shaped osteochondral transplants may be a suitable type of graft for functional results in cartilage resurfacing if stable anchorage of the grafts can be achieved.

Tumor regression induced by intratumoral injection of DNA coding for human interleukin 12 into melanoma metastases in gray horses.

Preclinical studies investigating new therapeutic principles against melanoma are presently being carried out in mouse models; however, these are not optimal. Here we describe a novel animal model using gray horses. These animals spontaneously develop metastatic melanoma that resembles human disease and is thus highly relevant for preclinical studies testing new immunotherapy protocols. We found that injection of plasmid DNA coding for the human cytokine interleukin 12 into established metastases induced significant regression in all 12 treated lesions in a total of 7 horses. Complete disappearance was observed in one treated lesion, with no recurrence after 6 months. No adverse events have been observed in any of the animals during and after treatment. These results demonstrate the effectiveness and safety of interleukin 12 encoding plasmid DNA therapy against established metastatic disease in a large animal model and serve as a basis for a clinical trial.

Comparative analysis of equine lymphocyte subsets in whole blood and gradient-purified samples.

In the present study, two methods of lymphocyte preparation, whole blood lysis and Ficoll-Paque separation, prior to FACS analysis were compared. The comparison was done with single and dual-colour staining techniques. Monoclonal antibodies (mAb) against eCD4, eCD5, eCD8 and eMHC class II were used. There was no significant difference in the results obtained by these two methods.

The role of neutrophil chemotactic cytokines in the pathogenesis of equine chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is defined as a chronic obstructive inflammatory disease affecting the small airways associated with hay dust exposure (Lowell, F.C., 1964. Observation on heaves. An asthma like syndrome in the horse, J. Allergy 35, 322-330). The disease corresponds histopathologically to a chronic bronchiolitis (Gerber, H., 1973. Chronic pulmonary disease in the horse, Equine Vet. J. 5, 26-33; Winder, N.C., Grünig, G., Hermann, M., Howald, B., von Fellenberg, R., 1989. Comparison of respiratory secretion cytology and pulmonary histology in horses, J. Vet. Med., A36, 32-38) and is mainly characterized by the presence of neutrophil granulocytes in the small bronchioles. Around 12-50% of all horses in Europe and the northern United States suffer from this disease (Mc Pherson, E.A., Lawson, G.H.K., Murphy, J.R., Nicholson, J.M., Fraser, J.A., Breeze, R.G., Pirie, H.M., 1978. Chronic obstructive pulmonary disease (COPD): Identification of affected horses, Eq. Vet. J. 10, 47-53; Larson, V.L., Busch, R.H., 1985. Equine tracheobronchial lavage: Comparison of lavage cytologic features in horses with chronic obstructive pulmonary disease, Am. J. Vet. Res., 46, 144-146; Bracher, V., von Fellenberg, R., Winder, N.C., Grünig, G., 1991. An investigation of the incidence of chronic obstructive pulmonary disease (COPD) in random populations of swiss horses, Equine Vet. J. 23, 136-141). The number of neutrophils in the bronchoalveolar lavage (BAL) and in tracheobronchial secretions (TBS) correlates with the severity of the disease. The present study is focused on the mechanisms which lead to the infiltration of neutrophil granulocytes in the lung of horses. We found that: (1). A strong chemotactic activity in the BAL fluid is associated with high levels of dust exposition. (2). In vitro stimulated alveolar macrophages have impaired phagocytosis efficiency and secrete two chemo-attractants specific for neutrophil granulocytes: Interleukin-8 (IL-8) (Wuyts, A., Proost, P., Put, W., Lenaerts, J.-P., Paemen, L., van Damme, J., 1994. Leucocyte recruitment by monocyte chemotactic proteins (MCPs) secreted by human phagocytes, J. Immunol. Meth. 174, 237-247) and macrophage inflammatory protein-2 (MIP-2) (Wolpe, S.D., Sherry, B., Juers, D., Davatelis, G. Yurt, R.W., Cerami, A., Identification and characterisation of macrophage inflammatory protein-2, Proc. Natl. Acad. Sci. USA 86, 612-616; Tekamp-Olson, P., Gallegos, C., Bauer, D., 1990. Cloning and characterisation of cDNAs for murine macrophage inflammatory protein-2 and its human homologues, J. Exp. Med., 172, 911-927; Driscoll, K.E., 1994. Macrophage inflammatory proteins: Biology and role in pulmonary inflammation. Exp. Lung Res., 20, 473-490). This is associated with the appearance of chemotactic activity in the supernatant. These data confirmed our working hypothesis that bronchiolar neutrophilia may be the consequence of a (over)stimulation of pulmonary macrophages leading to expression of cytokines chemotactic for neutrophil granulocytes.

Cytokine mRNA levels in isolated feline monocytes.

Real-time PCR systems were developed to quantitate cytokine expression in short-time cultivated feline monocytes. Feline-specific interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) primers as well as TaqMan probes were designed and were adapted to a quantitative PCR system which had been previously established for feline IL-10 and IL-12 p40. Quantitative analysis of cytokine messenger RNA (mRNA) transcription based on the comparison of the cytokine with the housekeeping gene feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing universally expressed mRNA. GAPDH mRNA was readily detectable in cDNA prepared from short-time cultivated peripheral blood monocytes. Cytokine mRNA was demonstrated in all samples at variable amounts. IL-1beta and TNF-alpha mRNA was constitutively expressed whereas IL-6, IL-10 and IL-12 p40 mRNA was generally expressed at a lower level and was occasionally not detected. There was a great variability of cytokine production between individual cats and at different time points in the same cat.

Long term in-vivo studies of a photo-oxidized bovine osteochondral transplant in sheep.

BACKGROUND: Articular cartilage has limited capacity to repair. Defects greater than 3 mm heal with formation of inferior fibrous cartilage. Therefore, many attempts have been made to find the ideal graft for larger cartilage lesions. Different grafts, such as untreated or cryopreserved osteochondral transplants, have been used with variable success. METHODS: Photo-oxidized osteochondral grafts were implanted in both femoral condyles of one ovine knee. Untreated xenogeneic and autogeneic grafts served as controls. Three groups of 8 sheep each were formed and they were sacrificed 6, 12 or 18 months after surgery. RESULTS: The macroscopic evaluation of the condyle and graft showed a well-maintained cartilage surface in most grafts at all time points. However, the host cartilage matrix deteriorated considerably in all xenogeneic, most autogeneic and fewer of the photo-oxidized grafts at 12 and 18 months, respectively. The blue colour of the photo-oxidized grafts resulting from the process of photo-oxidation was visible in all grafts at 6 months, had diminished at 12 months and had completely disappeared at 18 months after surgery. Histologically a loss of matrix staining was almost never noticed in untreated xenografts, transiently at 6 months in photo-oxidized grafts and increased at 12 and 18 months. Fusion between graft and host cartilage could be seen in photo-oxidized grafts at 12 and 18 months, but was never seen in autografts and xenografts. CONCLUSIONS: The photo-oxidation of osteochondral grafts and its use as transplant appears to have a beneficial effect on cartilage and bone remodelling. Osteochondral grafts pre-treated with photo-oxidation may be considered for articular cartilage replacement and therefore may delay artificial joint replacements in human patients.

Upregulation of mRNA of interleukin-1 and -6 in subchondral cystic lesions of four horses.

This study investigated the potential association of interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in subchondral cystic lesions (SCL) in horses. With the technique of in situ hybridisation in paraffin sections of fibrous tissue of SCL and quantitative real-time PCR in fresh frozen fibrous tissue and undecalcified bone sections of SCL embedded in acrylic resin, upregulation of mRNA of both cytokines could be demonstrated. mRNA of IL-1beta was upregulated at the periphery of the cystic lesion adjacent to normal bone, whereas IL-6 mRNA was upregulated within the fibrous tissue found within the centre of the SCL. It was concluded that both cytokines are associated in pathological bone resorption observed in SCL and, in combination with increased production of prostaglandin E2, may be responsible for the slow healing, maintenance or further expansion of the cystic lesions.

Spontaneous production of nitric oxide (NO), prostaglandin (PGE2) and neutral metalloproteinases (NMPs) in media of explant cultures of equine synovial membrane and articular cartilage from normal and osteoarthritic joints.

Nitric oxide (NO), prostaglandin E2 (PGE2), and the activity of neutral metalloproteinases (NMPs) were measured in conditioned media of equine synovial membrane and articular cartilage explant cultures from horses with normal joints (n = 7) and from horses affected with moderate (n = 7) or severe osteoarthritis (n = 14) as judged by macroscopic appearance. Normal articular cartilage appeared glossy and bluish-white, was of normal thickness and showed no evidence of discolouration, fibrillation or other cartilage discontinuity. Slight discolouration and fibrillation or minor clefts of the cartilage were considered as moderate OA, whereas erosions of articular cartilage down to the subchondral bone were considered as cases of severe OA. Explant cultures of equine synovial membrane and articular cartilage released the local mediators, NO and PGE2, as well as detectable levels of NMP activity into culture media. Concentrations of NO were higher in articular cartilage explants compared to synovial membrane explants, whereas concentrations of PGE2 were higher in synovial membrane explants. The NMPs with collagenolytic activities were similar in both explant cultures, whereas gelatinolytic activities were higher in synovial membrane explant cultures and caseinolytic activities were generally higher in articular cartilage explant cultures. Furthermore it was shown that concentrations or enzyme activities increased according to the severity of disease of the joints. Concentrations for NO, collagenolytic and gelatinolytic NMPs were relatively stable, whereas PGE2 and caseinolytic NMP concentrations increased over time in culture.

Clinical comparison of preanaesthetic intramuscular medetomidine and dexmedetomidine in domestic sheep.

Medetomidine and its active d-enantiomer, dexmedetomidine, are highly selective alpha-2 agonists with potent sedative, anaesthetic-sparing and analgesic effects. These properties make them an ideal pre-anaesthetic medication for noxious surgical procedures. However, sheep can develop adverse hypoxaemic effects after intravenous alpha-2 agonists. Objective of the present study was to compare intramuscular injection of medetomidine or dexmedetomidine at equipotent doses as preanaesthetic medication prior to isoflurane anaesthesia in sheep. Nineteen healthy, adult, non-pregnant, female sheep of various breeds were used. The study was carried out as a randomised, blind trial. Group A received 15 micrograms/kg bwt dexmedetomidine and group B received 30 micrograms/kg bwt medetomidine intramuscularly (i.m.) 30 minutes prior to induction of anaesthesia. Anaesthesia was induced with ketamine (2.0 mg/kg bwt i.v.) and maintained with isoflurane in 100% oxygen. End expired anaesthetic concentration (FEiso), respiratory frequency (fR), direct arterial blood pressures and heart rates (HR) were measured. Arterial blood samples were taken at intervals. Data were averaged over time (sum of measurements/number of measurements) and tested for differences between groups by independent t-tests, and ANOVA for repeated measures followed by Bonferroni corrected t-tests. There were no differences in demographic data between the groups. Duration of anaesthesia [A: 170 (42) minutes, B: 144 (33) minutes] and duration of surgery [A: 92 (32) minutes, B: 85 (31) minutes] were similar in both groups. Average FEiso concentrations required to maintain a surgical plane of anaesthesia were not significantly different between groups [A: 0.82 (0.14)%; B: 1.00 (0.25)%]. Mean average fR, did not differ between groups [A: 31 (14), B: 37 (15)]. Heart rates were significantly lower in group B over the course of the anaesthesia. Mean arterial blood pressures (MAP) were not significantly different between the groups. The PaO2 was less variable in group A than in group B. Individual minimum values were 19.1 kPa and 7.9 kPa in group A and B, respectively. There were no significant differences in PaCO2 and paH between the groups and over time. In conclusion, intramuscular application of dexmedetomidine at 15 micrograms/kg bwt and medetomidine at 30 micrograms/kg bwt prior to isoflurane anaesthesia induced similar changes in clinically monitored cardiorespiratory parameters. The observed differences (heart rates, PaO2) between dexmedetomidine and medetomidine at the chosen dose relationship can be considered clinically not significant. At the chosen dose rates individual animals responded with a transient drop in blood oxygenation, therefore careful monitoring is required. In addition, in compromised sheep medetomidine and dexmedetomidine should be used carefully.

In vitro studies of a photo-oxidized bovine articular cartilage.

Bovine articular cartilage was photo-oxidized and cultured with native articular bovine cartilage and synovial membrane to study the interaction between these tissues mimicking the physiological situation in the joint. The photo-oxidation was applied as a pretreatment of cartilage for future use in cartilage resurfacing procedures in joints. Properties of the transplant were assessed by testing the production of local mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and neutral metalloproteinase activities under normal conditions and after stimulation with various stimulants representative of inflammatory changes in pathophysiological conditions. Unlike normal cartilage photo-oxidized cartilage did not release significant amounts of NO and PGE2 and showed less gelatinolytic and caseinolytic activity compared to native bovine articular cartilage. Enzyme activity of the combined cultures was at a level intermediate between that of photo-oxidized cartilage and native cartilage cultures alone. In contrast to normal cartilage, living chondrocytes were not visible in photo-oxidized cartilage using live/dead staining. These results indicate, that the photo-oxidized cartilage may have a beneficial effect on adjacent native host cartilage and therefore be a suitable transplant for use in in vivo experiments.

The use of photooxidized, mushroom-structured osteochondral grafts for cartilage resurfacing--a comparison to photooxidized cylindrical grafts in an experimental study in sheep.

OBJECTIVE: This article addresses the problem of structural design with osteochondral grafts used for cartilage resurfacing. METHODS: Photooxidized cylindrical or mushroom-shaped grafts were surgically implanted in the weight bearing area of the medial and lateral femoral condyles of eight sheep (condyles: N=8/group). Both types of photooxidized grafts contained no viable chondrocytes at the time of implantation. Results were evaluated at 2 and 6 months after surgical implantation of the grafts. Qualitative and quantitative evaluation of the subchondral bone area was performed using plastic embedded sections of non-decalcified bone and cartilage specimens and placing emphasis on graft anchorage, cyst-like lesions at the base of the cartilage junction and at the base of the graft in the subchondral bone region. Cartilage morphology was studied qualitatively focusing on viability of the graft and adjacent host cartilage, while a score system was developed for semi-quantitative evaluation of the overall articular cartilage performance. The semiquantitative scores and histomorphometrical measurements were subjected to statistical analysis using a factorial analysis of variance (ANOVA-test). RESULTS: The photooxidized mushroom-shaped grafts developed less fibrous tissue and cyst-like lesions in the subchondral bone area at 2 and 6 months compared to the cylindrical grafts. Areas of endochondral ossification and bone remodeling were noticeable in the mushroom structured grafts at 2 months, and also bone remodeling was more complete at 6 months than with the cylindrical grafts. Increased numbers of cells were seen in the basal remodeling zones of both graft types increased from the 2 months to the 6 months specimens, but mushroom structured grafts showed better results. In both graft types, however, the midzone of the cartilage matrix was still acellular at 6 months. Cells from the subchondral bone area started to penetrate the calcified cartilage zone and tide mark at 2 months and repopulated the old photooxidized cartilage matrix already at 6 months after implantation. Cartilage repopulation was dependent on a stable subchondral bone area in both types of grafts. Matrix degradation of the adjacent host cartilage was minimal at 2 and 6 months. At 6 months a junction between host and graft cartilage was already noticed in some of the mushroom-shaped grafts. CONCLUSION: This study confirmed the importance of the subchondral bone area for osteochondral graft survival. In addition it demonstrated that the structure of the graft influences considerably the architecture of the subchondral bone, and with this the possibility for the repopulation of the old cartilage matrix including the junction between the host and graft cartilage matrix.

Pharmacokinetics and sedative effects of intramuscular medetomidine in domestic sheep.

Intramuscular (i.m.) administration of medetomidine (MED) may avoid the severe pressor effects caused by peripheral actions of MED associated with intravenous (i.v.) dosing. The purpose of this study was to determine the pharmacokinetics, the time course of sedation and occurence of hypoxaemia after i.m. administration of MED in domestic sheep. The MED was injected i.m. at a dose of 30 micro g/kg in nine domestic sheep. Blood was sampled at 0, 5, 10, 20, 30, 40, 60, 120, 180, 240, 360 and 600 min after MED. Sedation was assessed and arterial blood samples were taken before and 35 min after MED application. Mean (SD) pharmacokinetic parameters of i.m. MED were: absorption half-life: 13.2 (7.5) min; terminal half-life: 32.7 (14.9) min; time to peak concentration: 29.2 (8.9) min; peak concentration: 4.98 (1.89) ng/mL; volume of distribution: 3.9 (2.4) l/kg; total body clearance: 81.0 (21.5) mL/(min kg). Peak sedation occurred between 30 and 40 min after injection of MED. The degree of sedation correlated with individual plasma concentrations (rS: 0.926). One animal became hypoxaemic (PaO2 = 54.1 mmHg).

Fibrous tissue of subchondral cystic lesions in horses produce local mediators and neutral metalloproteinases and cause bone resorption in vitro.

OBJECTIVES: To define the release of nitric oxide (NO), prostaglandin E2 (PGE2), and the neutral metalloproteinases (NMPs) in horses with subchondral cystic lesions (SCL) and to study bone resorption triggered by conditioned media of fibrous tissue of SCL in vitro. STUDY DESIGN: Equine explant cultures of fibrous tissue of SCL, and synovial membrane and articular cartilage of normal horses and horses affected with moderate and severe osteoarthritis were performed. NO, PGE2, and NMP concentrations of media samples were measured, and osteoclast formation and activation was studied in vitro. ANIMALS: Experiment 1: 32 horses with SCL (n = 8), normal joints (7), and joints with moderate (7) and severe (10) osteoarthritis (OA). Experiment 2: 22 horses with SCL (n = 3), normal joints (7), and chip fractures (12). Experiment 3: Conditioned media of fibrous tissue from 3 horses with SCL of the medial femoral condyle (n = 1), distal metacarpal bone (1), and tarsal bone (1). METHODS: Determinations of local mediator concentrations were made with the Griess assay for NO and an enzyme immunoassay kit for PGE2 concentrations in biological fluids. Enzyme activities were assessed with radiolabeled substrates indicating collagenolytic, gelatinolytic, and caseinolytic activities. The resorption pit assay was used to assess osteoclast recruitment and activity. RESULTS: Fibrous tissue of SCL produced NO, PGE2, and NMPs. Of all the variables measured, PGE2 concentrations were the highest in cystic tissue of SCL compared with synovial membrane and articular cartilage from normal joints and joints with chip fractures, indicating that this mediator may play an important role in pathological bone resorption associated with SCL. These findings were supported by the observation that conditioned media of SCL tissue were capable of recruiting osteoclasts and increasing their activity. CONCLUSION: Fibrous tissue of SCL released NO, PGE2, and NMPs into the culture media. It is suspected that intralesional fibrous tissue may play an active role in the pathological process of bone resorption occurring in SCL in horses and may be partly responsible for the maintenance, slow healing rate, and expansion of these lesions. CLINICAL RELEVANCE: Understanding the pathogenesis of SCL will help to establish successful therapy in horses affected with SCL.

Quantitative real-time PCR for equine cytokine mRNA in nondecalcified bone tissue embedded in methyl methacrylate.

Specific amplification and quantitation of nucleic acid sequences by the polymerase chain reaction (PCR) has been extensively used for the detection of viral infection and gene expression. Although successful amplification of DNA and RNA sequences extracted from paraffin embedded tissue have been described, there are presently no reports available regarding RNA analysis from bone and calcified tissues embedded in hydrophobic acrylic resin. Here we describe a general method for quantitation of specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and embedded in a hydrophobic acrylic resin. Total RNA was extracted from defined regions of single 50 microm sawed sections. These RNA preparations are suitable for quantitative PCR analysis of mRNA of different cytokines. In addition, the universally expressed housekeeping GAPDH mRNA proved to be useful as an amplification control and to correct for the degree of RNA degradation, which may vary considerably among samples. Reverse transcribed mRNA was amplified and quantitated in Real-Time PCR using a fluorescein labeled internal TaqMan probe.

Changes in subchondral bone in cartilage resurfacing--an experimental study in sheep using different types of osteochondral grafts.

OBJECTIVE: This article addresses the subchondral bone integrity in cartilage resurfacing by comparing fresh, untreated auto-, xeno-, and photooxidized osteochondral allo- and xenografts. Photooxidation was expected to improve mechanical stability of the osteochondral grafts through an improved linkage of the collagen fibers within the bone matrix. DESIGN: Untreated auto- and xenografts and with photooxidation pretreated allo- and xenografts were surgically implanted in femoral condyles of sheep (n=40). After 2, 6, 12 and 18 months results were evaluated histologically using non-decalcified bone embedded in acrylic resin. Qualitative evaluation was performed with emphasis on bone matrix, biomechanical stability of graft anchorage, formation of cystic lesions, and bone resorption and formation. Quantitative evaluation of the total subchondral bone area was conducted histomorphometrically. Statistical analysis (factorial ANOVA test) was used to compare differences between groups with respect to the percentage of bone matrix and fibrous tissue per section. RESULTS: Subchondral bone resorption was fastest in untreated, fresh autografts, followed by photooxidized allografts, untreated, fresh xenografts and last pretreated photooxidized xenografts. Cystic lesions were seen in all types of grafts, but were most pronounced at 6 months in autografts and least in photooxidized grafts. Cyst-like lesions had subsided substantially in the untreated auto- and photooxidized xenografts, if no graft dislocation occurred during the healing period. Mononuclear cell infiltration and an increase in the presence of multinuclear cells were observed at 2 months, mostly in untreated autografts, followed by photooxidized allo- and untreated xenografts. They were much higher in numbers compared to photooxidized grafts, at least in the early specimens at 2 months. Graft stability was linked to the rate of bone resorption. CONCLUSION: Substantial resorption of the subchondral bone, involving the development of cyst-like lesions, lead to dislocation and finally to cartilage matrix degradation of the grafts. The process of photooxidation decreased the speed of bone resorption in osteochondral grafts and, thus, improved graft stability and cartilage survival. These results suggest that the remodeling of the subchondral bone of the host and the graft within the first 6 months is an important factor in graft stability and overall results of cartilage resurfacing.