Corticosteroids are prescribed for the treatment of many medical conditions and their adverse effects on bone, including steroid-associated osteoporosis and osteonecrosis, are well documented. Core decompression is performed to treat osteonecrosis, but the results are variable. As steroids may affect bone turnover, this study was designed to investigate bone healing within a bone tunnel after core decompression in an experimental model of steroid-associated osteonecrosis. A total of five 28-week-old New Zealand rabbits were used to establish a model of steroid-induced osteonecrosis and another five rabbits served as controls. Two weeks after the induction of osteonecrosis, core decompression was performed by creating a bone tunnel 3 mm in diameter in both distal femora of each rabbit in both the experimental osteonecrosis and control groups. An in vivo micro-CT scanner was used to monitor healing within the bone tunnel at four, eight and 12 weeks postoperatively. At week 12, the animals were killed for histological and biomechanical analysis. In the osteonecrosis group all measurements of bone healing and maturation were lower compared with the control group. Impaired osteogenesis and remodelling within the bone tunnel was demonstrated in the steroid-induced osteonecrosis, accompanied by inferior mechanical properties of the bone. We have confirmed impaired bone healing in a model of bone defects in rabbits with pulsed administration of corticosteroids. This finding may be important in the development of strategies for treatment to improve the prognosis of fracture healing or the repair of bone defects in patients receiving steroid treatment

Objectives. Recent studies have shown that systemic injection of rapamycin can prevent the development of osteoarthritis (OA)-like changes in human chondrocytes and reduce the severity of experimental OA. However, the systemic injection of rapamycin leads to many side effects. The purpose of this study was to determine the effects of intra-articular injection of Torin 1, which as a specific inhibitor of mTOR which can cause induction of autophagy, is similar to rapamycin, on articular cartilage degeneration in a rabbit osteoarthritis model and to investigate the mechanism of Torin 1’s effects on experimental OA. Methods. Collagenase (type II) was injected twice into both knees of three-month-old rabbits to induce OA, combined with two intra–articular injections of Torin 1 (400 nM). Degeneration of articular cartilage was evaluated by histology using the Mankin scoring system at eight weeks after injection. Chondrocyte degeneration and autophagosomes were observed by transmission electron microscopy. Matrix metallopeptidase-13 (MMP-13) and vascular endothelial growth factor (VEGF) expression were analysed by quantitative RT-PCR (qPCR).Beclin-1 and light chain 3 (LC3) expression were examined by Western blotting. Results. Intra-articular injection of Torin 1 significantly reduced degeneration of the articular cartilage after induction of OA. Autophagosomes andBeclin-1 and LC3 expression were increased in the chondrocytes from Torin 1-treated rabbits. Torin 1 treatment also reduced MMP-13 and VEGF expression at eight weeks after collagenase injection. Conclusion. Our results demonstrate that intra-articular injection of Torin 1 reduces degeneration of articular cartilage in collagenase-induced OA, at least partially by autophagy activation, suggesting a novel therapeutic approach for preventing cartilage degeneration and treating OA. Cite this article: N-T. Cheng, A. Guo, Y-P. Cui. Intra-articular injection of Torin 1 reduces degeneration of articular cartilage in a rabbit osteoarthritis model. Bone Joint Res 2016;5:218–224. DOI: 10.1302/2046-3758.56.BJR-2015-0001

Objectives. The purpose of this study was to evaluate chronological changes in the collagen-type composition at tendon–bone interface during tendon–bone healing and to clarify the continuity between Sharpey-like fibres and inner fibres of the tendon. Methods. Male white rabbits were used to create an extra-articular bone–tendon graft model by grafting the extensor digitorum longus into a bone tunnel. Three rabbits were killed at two, four, eight, 12 and 26 weeks post-operatively. Elastica van Gieson staining was used to colour 5 µm coronal sections, which were examined under optical and polarised light microscopy. Immunostaining for type I, II and III collagen was also performed. Results. Sharpey-like fibres comprised of type III collagen in the early phase were gradually replaced by type I collagen from 12 weeks onwards, until continuity between the Sharpey-like fibres and inner fibres of the tendon was achieved by 26 weeks. Conclusions. Even in rabbits, which heal faster than humans, an observation period of at least 12 to 26 weeks is required, because the collagen-type composition of the Sharpey-like fibre bone–tendon connection may have insufficient pullout strength during this period. These results suggest that caution is necessary when permitting post-operative activity in humans who have undergone intra-bone tunnel grafts

Objectives. Sustained intra-articular delivery of pharmacological agents is an attractive modality but requires use of a safe carrier that would not induce cartilage damage or fibrosis. Collagen scaffolds are widely available and could be used intra-articularly, but no investigation has looked at the safety of collagen scaffolds within synovial joints. The aim of this study was to determine the safety of collagen scaffold implantation in a validated in vivo animal model of knee arthrofibrosis. Materials and Methods. A total of 96 rabbits were randomly and equally assigned to four different groups: arthrotomy alone; arthrotomy and collagen scaffold placement; contracture surgery; and contracture surgery and collagen scaffold placement. Animals were killed in equal numbers at 72 hours, two weeks, eight weeks, and 24 weeks. Joint contracture was measured, and cartilage and synovial samples underwent histological analysis. Results. Animals that underwent arthrotomy had equivalent joint contractures regardless of scaffold implantation (-13.9° versus -10.9°, equivalence limit 15°). Animals that underwent surgery to induce contracture did not demonstrate equivalent joint contractures with (41.8°) or without (53.9°) collagen scaffold implantation. Chondral damage occurred in similar rates with (11 of 48) and without (nine of 48) scaffold implantation. No significant difference in synovitis was noted between groups. Absorption of the collagen scaffold occurred within eight weeks in all animals. Conclusion. Our data suggest that intra-articular implantation of a collagen sponge does not induce synovitis or cartilage damage. Implantation in a native joint does not seem to induce contracture. Implantation of the collagen sponge in a rabbit knee model of contracture may decrease the severity of the contracture. Cite this article: J. A. Walker, T. J. Ewald, E. Lewallen, A. Van Wijnen, A. D. Hanssen, B. F. Morrey, M. E. Morrey, M. P. Abdel, J. Sanchez-Sotelo. Intra-articular implantation of collagen scaffold carriers is safe in both native and arthrofibrotic rabbit knee joints. Bone Joint Res 2016;6:162–171. DOI: 10.1302/2046-3758.63.BJR-2016-0193

Growth plates taken from five- to 20-week-old Japanese white rabbits were immunostained for c-Myc protein. This was localised both in the proliferating zone and upper hypertrophic zone at five weeks, whereas after ten weeks it was found mostly in the lower hypertrophic zone. The proliferating chondrocytes tended to show nuclear staining and the hypertrophic cells cytoplasmic staining, although the terminal hypertrophic chondrocytes sometimes expressed the protein in their nuclei. In the younger rabbits, c-Myc co-localised with proliferating cell nuclear antigen, whereas in the hypertrophic zone of older rabbits, it was present in some chondrocytes the nuclei of which also contained DNA breaks. Our study suggests that, in the rabbit growth plate, c-Myc is associated with different cellular processes, depending on the age and the developmental stage of the chondrocytes

We produced large full-thickness articular cartilage defects in 33 rabbits in order to evaluate the effect of joint distraction and autologous culture-expanded bone-marrow-derived mesenchymal cell transplantation (ACBMT) at 12 weeks. After fixing the knee on a hinged external fixator, we resected the entire surface of the tibial plateau. We studied three groups: 1) with and without joint distraction; 2) with joint distraction and collagen gel, and 3) with joint distraction and ACBMT and collagen gel. The histological scores were significantly higher in the groups with ACBMT collagen gel (p < 0.05). The area of regenerated soft tissue was smaller in the group allowed to bear weight (p < 0.05). These findings suggest that the repair of large defects of cartilage can be enhanced by joint distraction, collagen gel and ACBMT

In a rabbit model we investigated the efficacy of a silk fibroin/hydroxyapatite (SF/HA) composite on the repair of a segmental bone defect. Four types of porous SF/HA composites (SF/HA-1, SF/HA-2, SF/HA-3, SF/HA-4) with different material ratios, pore sizes, porosity and additives were implanted subcutaneously into Sprague-Dawley rats to observe biodegradation. SF/HA-3, which had characteristics more suitable for a bone substitite based on strength and resorption was selected as a scaffold and co-cultured with rabbit bone-marrow stromal cells (BMSCs). A segmental bone defect was created in the rabbit radius. The animals were randomised into group 1 (SF/HA-3 combined with BMSCs implanted into the bone defect), group 2 (SF/HA implanted alone) and group 3 (nothing implanted). They were killed at four, eight and 12 weeks for visual, radiological and histological study. The bone defects had complete union for group 1 and partial union in group 2, 12 weeks after operation. There was no formation of new bone in group 3. We conclude that SF/HA-3 combined with BMSCs supports bone healing and offers potential as a bone-graft substitute

Objectives. The aim of this experimental study on New Zealand’s white rabbits was to investigate the transplantation of autogenous growth plate cells in order to treat the injured growth plate. They were assessed in terms of measurements of radiological tibial varus and histological characteristics. . Methods. An experimental model of plate growth medial partial resection of the tibia in 14 New Zealand white rabbits was created. During this surgical procedure the plate growth cells were collected and cultured. While the second surgery was being performed, the autologous cultured growth plate cells were grafted at the right tibia, whereas the left tibia was used as a control group. . Results. Histological examinations showed that the grafted right tibia presented the regular shape of the plate growth with hypertrophic maturation, chondrocyte columniation and endochondral calcification. Radiological study shows that the mean tibial deformity at the left angle was 20.29° (6.25 to 33) and 7.21° (5 to 10) in the right angle. . Conclusion. This study has demonstrated that grafting of autogenous cultured growth plate cells into a defect of the medial aspect of the proximal tibial physis can prevent bone bridge formation, growth arrest and the development of varus deformity. Cite this article: Bone Joint Res 2014;3:310–16

We investigated the effects of low-intensity pulsed ultrasound on distraction osteogenesis in a rabbit model. Callotasis of the right tibia was performed in 70 male Japanese white rabbits using mini-external fixators. In the first part of the study in 64 animals using normal distraction (waiting period seven days; distraction rate 0.5 mm/12 hours; distraction period ten days), we evaluated the distraction site by radiography, measurement of the bone mineral density (BMD), mechanical testing, and histology. In the second part in six rabbits using fast distraction (waiting period 0 days; distraction rate 1.5 mm/12 hours; distraction period seven days) the site was evaluated radiologically. Half of the animals (35) had received ultrasound to their right leg (30mW/cm. 2. ) for 20 minutes daily after ceasing distraction (ultrasound group), while rigid fixation only was maintained in the other half (control group). With normal distraction, the hard callus area, as shown by radiography, the BMD, and the findings on mechanical testing, were significantly greater in those receiving ultrasound than in the control group. Histological analysis showed no tissue damage attributable to exposure to ultrasound. With fast distraction, immature bone regeneration was observed radiologically in the control group, while bone maturation was achieved in the ultrasound group. We conclude that ultrasound can accelerate bone maturation in distraction osteogenesis in rabbits, even in states of poor callotasis

The feasibility of bone transport with bone substitute and the factors which are essential for a successful bone transport are unknown. We studied six groups of 12 Japanese white rabbits. Groups A to D received cylindrical autologous bone segments and groups E and F hydroxyapatite prostheses. The periosteum was preserved in group A so that its segments had a blood supply, cells, proteins and scaffold. Group B had no blood supply. Group C had proteins and scaffold and group D had only scaffold. Group E received hydroxyapatite loaded with recombinant human bone morphogenetic protein-2 and group F had hydroxyapatite alone. Distraction osteogenesis occurred in groups A to C and E which had osteo-conductive transport segments loaded with osteo-inductive proteins. We conclude that scaffold and proteins are essential for successful bone transport, and that bone substitute can be used to regenerate bone

In 16 mature New Zealand white rabbits mesenchymal stem cells were aspirated from the bone marrow, cultured in monolayer and implanted on to a full-thickness osteochondral defect artificially made on the patellar groove of the same rabbit. A further 13 rabbits served as a control group. The rabbits were killed after 14 weeks. Healing of the defect was investigated histologically using haematoxylin and eosin and Safranin-O staining and with immunohistochemical staining for type-II collagen. We also used a reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA of type-I and type-II collagen. The semiquantitative histological scores were significantly higher in the experimental group than in the control group (p < 0.05). In the experimental group immunohistochemical staining on newly formed cartilage was more intense for type-II collagen in the matrix and RT-PCR from regenerated cartilage detected mRNA for type-II collagen in mature chondrocytes. These findings suggest that repair of cartilage defects can be enhanced by the implantation of cultured mesenchymal stem cells

We attempted to repair full-thickness defects in the articular cartilage of the trochlear groove of the femur in 30 rabbit knee joints using allogenic cultured chondrocytes embedded in a collagen gel. The repaired tissues were examined at 2, 4, 8, 12 and 24 weeks after operation using histological and histochemical methods. The articular defect filling index measurement was derived from safranin-O stained sections. Apoptotic cellular fractions were derived from analysis of apoptosis in situ using TUNEL staining, and was confirmed using caspase-3 staining along with quantification of the total cellularity. The mean articular defect filling index decreased with time. After 24 weeks it was 0.7 (. sd. 0.10), which was significantly lower than the measurements obtained earlier (p < 0.01). The highest mean percentage of apoptotic cells were observed at 12 weeks, although the total cellularity decreased with time. Because apoptotic cell death may play a role in delamination after chondrocyte transplantation, anti-apoptotic gene therapy may protect transplanted chondrocytes from apoptosis

We studied bone-tendon healing using immunohistochemical methods in a rabbit model. Reconstruction of the anterior cruciate ligament was undertaken using semitendinosus tendon in 20 rabbits. Immunohistochemical evaluations were performed at one, two, four and eight weeks after the operation. The expression of CD31, RAM-11, VEGF, b-FGF, S-100 protein and collagen I, II and III in the bone-tendon interface was very similar to that in the endochondral ossification. Some of the type-III collagen in the outer layer of the graft, which was deposited at a very early phase after the operation, was believed to have matured into Sharpey-like fibres. However, remodelling of the tendon grafted into the bone tunnel was significantly delayed when compared with this ossification process. To promote healing, we believe that it is necessary to accelerate remodelling of the tendon, simultaneously with the augmentation of the ossification

We have investigated in vitro the release kinetics and bioactivity of fibroblast growth factor-2 (FGF-2) released from a carrier of fibrin sealant. In order to evaluate the effects of the FGF-2 delivery mechanism on the repair of articular cartilage, full-thickness cylindrical defects, 5 mm in diameter and 4 mm in depth, which were too large to undergo spontaneous repair, were created in the femoral trochlea of rabbit knees. These defects were then filled with the sealant. Approximately 50% of the FGF-2 was released from the sealant within 24 hours while its original bioactivity was maintained. The implantation of the fibrin sealant incorporating FGF-2 successfully induced healing of the surface with hyaline cartilage and concomitant repair of the subchondral bone at eight weeks after the creation of the defect. Our findings suggest that this delivery method for FGF-2 may be useful for promoting regenerative repair of full-thickness defects of articular cartilage in humans

Bone apatite contains carbonate and is therefore not pure hydroxyapatite. We have successfully developed sintered carbonate apatite (CA) with a concentration of carbonate of 6 weight% and have evaluated its osteoconductive and bioresorption characteristics. Cylindrical porous sintered CA and sintered hydroxyapatite (HA) measuring 4 × 4 mm with a porosity of 20% were implanted into surgically-created bone defects in the knees of rabbits. The animals were killed after 1, 3, 6 and 12 months. The defects were evaluated by microfocus CT and histology. Bone growth into and around both materials increased. Newly-formed bone was placed in direct contact with both. Osteoclast-like cells resorbed only CA, and were coupled with osteoblasts. The porosity of sintered CA increased, indicating bioresorption, whereas that of sintered HA did not increase. Our findings indicate that sintered CA may be useful as a bioresorbable bone substitute

We carried out limb lengthening in rabbits and then transplanted osteoblast-like cells derived from the tibial periosteum to the centres of distracted callus immediately after distraction had been terminated. Two weeks later the transaxial area ratio at the centre of the distracted callus and the bone mineral density (BMD) were significantly higher in the transplanted group, by 21% and 42%, respectively, than in the non-injected group or the group injected with physiological saline (p < 0.05). Callus BMD as a percentage of density in uninvolved bone was also significantly higher in the transplanted group (p < 0.05) than in the other two groups, by 27% and 20% in the second and fourth weeks, respectively (p < 0.05). Mechanically, the callus in the transplanted group tended to be stronger as shown by the three-point bending test although the difference in fracture strength was not statistically significant. Our results show that transplantation of osteoblast-like cells promotes maturity of the distracted callus as observed at the second and fourth weeks after lengthening. The method appears promising as a means of shortening the consolidation period of callus distraction and decreasing complications during limb lengthening with an external fixator

In a study combining tissue mechanics and fracture morphology for the first time, we examined the ruptured surfaces of anterior cruciate ligaments of rabbits and related their appearance to the initial loading conditions. Sixteen specimens were stretched to failure at rates of displacement of 10 and 500 mm/min. We used video images to study the changes which occurred during the fracture process and SEM to examine the appearance of the ruptured surfaces. The surfaces of ligaments tested at 10 mm/min had more pulled-out collagen fibres and the fibres had more pronounced waviness compared with those tested at 500 mm/min. We have shown that the macroscopic appearance of ruptured ligaments can be related to their microscopic appearance and that it is possible to deduce whether failure was by gradual tearing of the fibres or catastrophic failure

We aimed to determine whether extracorporeal shock waves of varying intensity would damage the intact tendo Achillis and paratenon in a rabbit model. We used 42 female New Zealand white rabbits randomly divided into four groups as follows: group a received 1000 shock-wave impulses of an energy flux density of 0.08 mJ/mm. 2. , group b 1000 impulses of 0.28 mJ/mm. 2. , group c 1000 impulses of 0.60 mJ/mm. 2. , and group d was a control group. Sonographic and histological evaluation showed no changes in group a, and transient swelling of the tendon with a minor inflammatory reaction in group b. Group c had formation of paratendinous fluid with a significant increase in the anteroposterior diameter of the tendon. In this group there were marked histological changes with increased eosin staining, fibrinoid necrosis, fibrosis in the paratenon and infiltration of inflammatory cells. We conclude that there are dose-dependent changes in the tendon and paratenon after extracorporeal shock-wave therapy and that energy flux densities of over 0.28 mJ/mm. 2. should not be used clinically in the treatment of tendon disorders

Chondrocytes of the growth plate are generally assumed to undergo apoptosis, but the mechanisms which induce this cell death are not known. The Fas receptor is a mediator of the apoptotic signal in some systems. We studied its expression in situ in growth plates of rabbits aged from five to 20 weeks. In addition, we investigated the immunolocalisation in the growth plates of the bone proteins, osteonectin and osteocalcin, and the changes in their expression with age. The Fas-positive chondrocytes were found mostly in the hypertrophic zone, as were the osteonectin-positive and osteocalcin-positive cells. The percentage of Fas-positive cells increased with age whereas little change was found in the number of osteonectin-positive and osteocalcin-positive chondrocytes. Many of the Fas-positive chondrocytes were also TUNEL-positive. This strongly suggests that apoptosis in the growth plate is mediated through the Fas system. Double immunostaining for osteocalcin and Fas showed that not all hypertrophic chondrocytes were of the same cell type. Some chondrocytes stained for osteocalcin only, others for Fas only, while some were positive for both

We have examined the process of fusion of the intertransverse processes and bone graft in the rabbit by in situ hybridisation and evaluated the spatial and temporal expression of genes encoding pro-α1 (I) collagen (COL1A1), pro-α1 (II) collagen (COL2A1) and pro-α1 (X) collagen (COL10A1). Beginning at two weeks after operation, osteogenesis and chondrogenesis occurred around the transverse process and the grafted bone at the central portion of the area of the fusion mass. Osteoblasts and osteocytes at the newly-formed woven bone expressed COL1A1. At the cartilage, most chondrocytes expressed COL2A1 and some hypertrophic chondrocytes COL10A1. In some regions, co-expression of COL1A1 and COL2A1 was observed. At four weeks, such expressions for COL1A1, COL2A1 and COL10A1 became prominent at the area of the fusion mass. From four to six weeks, bone remodelling progressed from the area of the transverse processes towards the central zone. Osteoblasts lining the trabeculae expressed a strong signal for COL1A1. At the central portion of the area of the fusion mass, endochondral ossification progressed and chondrocytes expressed COL2A1 and COL10A1. Our findings show that the fusion process begins with the synthesis of collagens around the transverse processes and around the grafted bone independently. Various spatial and temporal osteogenic and chondrogenic responses, including intramembranous, endochondral and transchondroid bone formation, progress after bone grafting at the intertransverse processes. Bone formation through cartilage may play an important role in posterolateral spinal fusion