Characterisation of subchondral bone repair following transplantation of bioreactor-manufactured autologous osteochondral graft in a sheep model.

To date, no single approach to the treatment of osteochondral defects has resulted in satisfactory long-term outcomes, especially in a young and active human population. Emerging innovative tissue engineering strategies, including the use of composite scaffolds, novel cell sources and bioreactors, have shown promising results. However, these techniques need to be validated in translational animal models before they can be implemented in clinical practice. The aim of the present study was to analyse morphological and microarchitectural parameters during subchondral bone repair following transplantation of bioreactor-manufactured autologous osteochondral grafts in a sheep model. Animals were divided into 4 treatment groups: nasal chondrocyte (NC) autologous osteochondral grafts, articular chondrocyte (AC) autologous osteochondral grafts, cell-free scaffolds (CFS) and empty defects (EDs). After 6 weeks, 3 months and 12 months, bone remodelling was assessed by histology and micro-computed tomography (µCT). Although gradual remodelling and subchondral bone repair were seen in all groups across the time points, the best results were observed in the NC group. This was evidenced by the extent of new tissue formation and its best integration into the surrounding tissue in the NC group at all time points. This also suggested that nasal septum chondrocyte-seeded grafts adapted well to the biomechanical conditions of the loaded joint surface.

Articular cartilage repair by genetically modified bone marrow aspirate in sheep.

Bone marrow presents an attractive option for the treatment of articular cartilage defects as it is readily accessible, it contains mesenchymal progenitor cells that can undergo chondrogenic differentiation and, once coagulated, it provides a natural scaffold that contains the cells within the defect. This study was performed to test whether an abbreviated ex vivo protocol using vector-laden, coagulated bone marrow aspirates for gene delivery to cartilage defects may be feasible for clinical application. Ovine autologous bone marrow was transduced with adenoviral vectors containing cDNA for green fluorescent protein or transforming growth factor (TGF)-beta1. The marrow was allowed to clot forming a gene plug and implanted into partial-thickness defects created on the medial condyle. At 6 months, the quality of articular cartilage repair was evaluated using histological, biochemical and biomechanical parameters. Assessment of repair showed that the groups treated with constructs transplantation contained more cartilage-like tissue than untreated controls. Improved cartilage repair was observed in groups treated with unmodified bone marrow plugs and Ad.TGF-beta1-transduced plugs, but the repaired tissue from TGF-treated defects showed significantly higher amounts of collagen II (P<0.001). The results confirmed that the proposed method is fairly straightforward technique for application in clinical settings. Genetically modified bone marrow clots are sufficient to facilitate articular cartilage repair of partial-thickness defects in vivo. Further studies should focus on selection of transgene combinations that promote more natural healing.

Feline high-rise syndrome: 119 cases (1998-2001).

High-rise syndrome was diagnosed in 119 cats over a 4-year period. 59.6% of cats were younger than one year, and the average height of the fall was four stories. High-rise syndrome was more frequent during the warmer period of the year. 96.5% of the presented cats, survived after the fall. 46.2% of cats had fractured limbs; 38.5% of fractures were of the forelimb, 61.5% of the hindlimb. The tibia was fractured most often (36.4%), followed by the femur (23.6%). 78.6% of femoral fractures were distal. The mean age of patients with femoral fractures was 9.1 months, and with tibial fractures 29.2 months. Thoracic trauma was diagnosed in 33.6% of cats. Pneumothorax was diagnosed in 20% of cats, and pulmonary contusions in 13.4%. Falls from the seventh or higher stories, are associated with more severe injuries and with a higher incidence of thoracic trauma.

Possibility of blocking n. ischiadicus within foramen ischiadicum majus in sheep.

The blocking of the ischiadic nerve within foramen ischiadicus majus of 8 sheep was performed with 6-12 ml 2% xylocain from three different places: 1. From the middle of a line joining the top of processus spinosus of the 1st sacral vertebra and the peak of trochanter major. The needle pierces vertically through the skin, gluteal fascia and m. gluteus medius till foramen ischiadicum majus. 2. Along the most prominent part of crista iliaca on the gluteal surface of ala ossis ilium along the medial side of well expressed linea glutea. The needle pierces by an angle of 45 degrees through the skin, gluteal fascia and m. gluteus medius till the nerve itself. When the nerve is touched the animal makes a jerk. 3. Along the caudal part of tuber sacrale with the needle orientated caudo-ventro-laterally through the skin, gluteal fascia and m. gluteus medius toward foramen ischiadicum majus. The most appropriate approach for blocking is along crista glutea on the gluteal surface of ala assis ilii. In this approach the success is complete. The symptoms of the blocking appear immediately after the application. The complete akinesia of the limb appears 15 minutes after the application of 6 ml, and 5-10 minutes after being applied 12 ml of 2% xylocain. The blocking symptoms disappear within 90-100 or 120-140 minutes. Beside the blocking of n. ischiadicus, a simultaneous blocking of n. gluteus cranialis, n. caudalis and n. cutaneus femoris caudalis as well was performed within foramen ischiadicus majus.

Incidence of foreign-body-induced ileus in dogs.

While playing or simply because of avidity, dogs may ingest a variety of foreign bodies. Ingested foreign bodies, which are not stopped in the mouth or oesophagus, enter into the stomach. Once a foreign body has passed through the pylorus, jejunum and ileum appear to be the most common sites of the small intestine obstruction. The records of 103 cases, treated at the Clinic for Surgery, Orthopaedics and Ophthalmology of the Veterinarian Faculty, University of Zagreb from January 1981 till December 1998 were analysed. The analysis included the incidence of ileus caused by foreign bodies and the distribution of patients by sex, age, breed, duration of illness, site of obstruction, types of foreign bodies and the interrelation between these parameters. The results of our research show that the number of patients with foreign body induced ileus is increasing. Males ingested foreign bodies more often than females. Foreign body induced ileus was more frequently found in animals below 2 years of age. Foreign bodies were mostly ingested by mongrels, but also by popular dog breeds such as Dobermanns, Poodles, Cocker Spaniels and Rottweiler. Most of these ileus cases were found in March and October and the predominant clinical signs included anorexia, dehydration, abdominal tenderness and absence of defecation. The most common site of small intestine obstruction by foreign bodies was the jejunum, and the most effective treatment was enterotomy. Dogs mostly ingested stones, plastic and rubber objects. The treatment was more successful in dogs below 2 years of age. Patients that died post-surgically, died mostly the first day after surgery.

Regeneration of articular cartilage chondral defects by osteogenic protein-1 (bone morphogenetic protein-7) in sheep.

The efficacy of osteogenic protein-1 (OP-1; BMP-7) in regeneration of articular cartilage was examined by creating knee chondral defects in sheep. With a specially designed instrument in both knees, two 10 mm (diameter) chondral defects were created: one in the trochlea and the other on the femoral condyle. The recombinant BMP was delivered via an extra-articulary positioned mini-osmotic pump, which was fixed to the femoral diaphysis above the knee joint, and connected by a polyethylene tubing to the articular space. Prior to use, the compatibility of OP-1 with mini-osmotic pumps was tested in vitro by measuring aggregation/precipitation and modification of the released protein by size exclusion and reversed phase HPLC. The average amount of aggregation was 15% and about 5% of OP-1 was modified. However, the biological activity of OP-1 released from pumps over a period of 2 weeks at 37 degrees C was equal to ROS cell assay OP-1 standard. Following surgery, a total of 55 microg (low dose) or 170 microg (high dose) OP-1 in acetate buffer (pH 4.5) was slowly released from the pump over a period of 2 weeks. The pumps connected to control knees were filled with acetate buffer as a vehicle. Twelve animals were operated, six of which were treated with the low OP-1 dose, and six with the high OP-1 dose. Three sheep of each group were killed either at 3 or 6 months following surgery, based on arthroscopical evaluation. The chondral defects in the control knees remained empty during the observation period. At 3 months following surgery, defects treated with both OP-1 doses were filled with connective tissue and cartilage. At 6 months following surgery, both doses of OP-1 stimulated regeneration in treated knees. The boundaries between new and old cartilage were well fused and mechanically resisted animals' weight bearing. The regenerated cartilage was rich in proteoglycans and type II collagen, as demonstrated by toluidine blue staining and immunohistochemistry. No signs of endochondral bone formation above the bony tidemark were observed. We suggest that a recombinant bone morphogenctic protein stimulates ingrowth of mesenchymal cells into the chondral defects which then transform into newly formed articular cartilage-like tissue.

Biological repair of thyroid cartilage defects by osteogenic protein-1 (bone morphogenetic protein-7) in dog.

The efficacy of human recombinant osteogenic protein-1 (OP-1; bone morphogenetic protein-7) in regeneration of dog larynx was examined by treating thyroid cartilage defects (1.5 cm2) in dogs with thyroid allografts covered with host perichondrium or fascia. Prior to implantation allografts were frozen, thawed and demineralized. The treatment groups were as follows: I--Allograft control implant (n = 3); II--Implants coated with 500 micrograms OP-1 (n = 4); III--Implants coated with 100 micrograms OP-1 (n = 3); IV--Implants coated with 500 micrograms OP-1 and covered with neck fascia (n = 3); and V--Implants extracted with 1 M NaCl and guanidine hydrochloride, and coated with 500 micrograms OP-1 (n = 4). Dogs were sacrificed four months following surgery. Each larynx was removed, carefully dissected and a three-dimensional reconstruction of the defect area was performed on serial sections. The results revealed that the implants of control dogs remained intact with no apparent reduction in size and new tissue formation. OP-1 enriched thyroid allografts, dose dependently induced bone, cartilage and ligament-like structures comprising up to 80% of the total regenerated defect area. Boundaries of the defects healed by formation of new bone when bone resided within the old thyroid cartilage layers. Old cartilage not containing bone within its layers healed by complete integration with newly formed cartilage. Both new bone and cartilage were embedded into layers of new ligament-like tissue which expressed specific morphologic and molecular markers. The three newly formed tissues were tightly connected into a "bone-cartilage-ligament continuum" of tissues, suggesting that OP-1 served as a multiple tissue morphogen in this specific microenvironment.