Aim. Debridement, Antibiotics, Irrigation, and implant Retention (DAIR) is a surgical treatment protocol suitable for some patients with fracture related infection (FRI). Clinically relevant pre-clinical models of DAIR are scarce and none have been developed in large animals. Therefore, this project aimed to develop a large animal model for FRI including a DAIR approach and compare outcomes after 2 or 5 weeks of infection. Method. Swiss Alpine sheep (n=8), (2–6 years, 50–80 kg) were included in this study. This study was approved by cantonal Ethical authorities in Chur, Switzerland. A 2 mm osteotomy was created in the tibia and fixed with a 10-hole 5.5 mm steel plate. Subsequently, 2.5 mL of saline solution containing 10. 6. CFU/mL of Staphylococcus aureus MSSA (ATCC 25923) was added over the plate. Sheep were observed for 2 (n=3) or 5 weeks (n=5) until revision surgery, during which visibly infected or necrotic tissues were removed, and the wound flushed with saline. All samples were collected for bacterial quantification. After revision surgery, the sheep were treated systemically for 2 weeks with flucloxacillin and for 4 weeks with rifampicin and cotrimoxazole. After 2 further weeks off antibiotics, the animals were euthanized. Bacteriological culture was performed at the end of the study. Bone cores were isolated from the osteotomy site and processed for Giemsa & Eosin and Brown and Brenn staining. A radiographical examination was performed every second week. Results. Bacteriological evaluation of the retrieved samples during revision surgery showed no significant difference between the 2 vs 5 weeks infection periods in term of total CFU counts. At the end of the study, radiographical examination showed callus formation over the osteotomy site in both groups, although the osteotomy was not completely healed in either group. At euthanasia, the 2 weeks infection group showed a higher soft tissue burden compared to the 5 weeks group, whereby the infection in the 5 weeks group was primarily located in the bone and bone marrow. Conclusions. The large animal model of FRI and DAIR was successfully established. Bacteriological outcomes highlight that the increasing duration of the infection does not change the outcome but the location of the infection from a predominantly soft tissue infection to a deeper bone and intramedullary (IM) channel infection. The debridement of the IM channel could potentially reduce the infection burden by eliminating those bacteria not easily reached by systemic antibiotics, though is not practical using conventional techniques

Aims. The “2 to 10% strain rule” for fracture healing has been widely interpreted to mean that interfragmentary strain greater than 10% predisposes a fracture to nonunion. This interpretation focuses on the gap-closing strain (axial micromotion divided by gap size), ignoring the region around the gap where osteogenesis typically initiates. The aim of this study was to measure gap-closing and 3D interfragmentary strains in plated ovine osteotomies and associate local strain conditions with callus mineralization. Methods. MicroCT scans of eight female sheep with plated mid-shaft tibial osteotomies were used to create image-based finite element models. Virtual mechanical testing was used to compute postoperative gap-closing and 3D continuum strains representing compression (volumetric strain) and shear deformation (distortional strain). Callus mineralization was measured in zones in and around the osteotomy gap. Results. Gap-closing strains averaged 51% (mean) at the far cortex. Peak compressive volumetric strain averaged 32% and only a small tissue volume (average 0.3 cm. 3. ) within the gap experienced compressive strains > 10%. Distortional strains were much higher and more widespread, peaking at a mean of 115%, with a mean of 3.3 cm. 3. of tissue in and around the osteotomy experiencing distortional strains > 10%. Callus mineralization initiated outside the high-strain gap and was significantly lower within the fracture gap compared to around it at nine weeks. Conclusion. Ovine osteotomies can heal with high gap strains (> 10%) dominated by shear conditions. High gap strain appears to be a transient local limiter of osteogenesis, not a global inhibitor of secondary fracture repair. Cite this article: Bone Joint Res 2025;14(1):5–15

Biphasic calcium phosphate (BCP) with a characteristic needle-shaped submicron surface topography (MagnetOs) has attracted much attention due to its unique bone-forming ability which is essential for repairing critical-size bone defects such as those found in the posterolateral spine. Previous in vitro and ex-vivo data performed by van Dijk LA and Yuan H demonstrated that these specific surface characteristics drive a favorable response from the innate immune system. This study aimed to evaluate and compare the in vivo performance of three commercially-available synthetic bone grafts, (1) i-FACTOR Putty. ®. , (2) OssDsign. ®. Catalyst Putty and (3) FIBERGRAFT. ®. BG Matrix, with that of a novel synthetic bone graft in a clinically-relevant instrumented sheep posterolateral lumbar spine fusion (PLF) model. The novel synthetic bone graft comprised of BCP granules with a needle-shaped submicron surface topography (MagnetOs) embedded in a highly porous and fibrillar collagen matrix (MagnetOs Flex Matrix). Four synthetic bone grafts were implanted as standalone in an instrumented sheep PLF model for 12 weeks (n=3 bilateral levels per group; levels L2/3 & L4/5), after which spinal fusion was determined by manual palpation, radiograph and µCT imaging (based on the Lenke scale), range-of-motion mechanical testing, and histological and histomorphological evaluation. Radiographic fusion assessment determined bilateral robust bone bridging (Lenke scale A) in 3/3 levels for MagnetOs Flex Matrix compared to 1/3 for all other groups. For µCT, bilateral fusion (Lenke scale A) was found in 2/3 levels for MagnetOs Flex Matrix, compared to 0/3 for i-FACTOR Putty. ®. , 1/3 for OssDsign. ®. Catalyst Putty and 0/3 for FIBERGRAFT. ®. BG Matrix. Fusion assessment for MagnetOs Flex Matrix was further substantiated by histology which revealed significant graft resorption complemented by abundant bone tissue and continuous bony bridging between vertebral transverse processes resulting in bilateral spinal fusion in 3/3 implants. These results show that MagnetOs Flex Matrix achieved better fusion rates compared to three commercially-available synthetic bone grafts when used as a standalone in a clinically-relevant instrumented sheep PLF model

Introduction. Low back pain (LBP) is a worldwide leading cause of disability. This preclinical study evaluated the safety of a combined advanced therapy medicinal product developed during the European iPSpine project (#825925) consisting of mesendoderm progenitor cells (MEPC), derived from human induced pluripotent stem cells, in combination with a synthetic poly(N-isopropylacrylamide) hydrogel (NPgel) in an ovine intervertebral disc degeneration (IDD) model. Method. IDD was induced through nucleotomy in 4 adult sheep, 5 lumbar discs each (n=20). After 5 weeks, 3 alternating discs were treated with NPgel (n=6) or NPgel+MEPC (n=6). Before sacrifice, animals were subjected to: MRI of lumbar spines (disc height and Pfirmann grading); blood sampling (hematological, biochemical, metabolic and lymphocyte/monocytes immunological). After 3 months the sheep were sacrificed. The spines were processed for: macroscopic morphology (Thompson grading), microscopic morphology (Histological grading), and glycosaminoglycan content (GAG, DMMB Assay). Furthermore, at sacrifice biodistribution of human MEPC was assessed by Alu-sequences quantification (qPCR) from three tissue samples of heart, liver, spleen, brain, lungs, and kidneys, and PBMCs collected to assess activation of systemic immune cells. To each evaluation, appropriate statistical analysis was applied. Result. Flow cytometry showed no induction of systemic activation of T cells or monocytes. Alu quantification did not give detection of any cells in any organ. Disc height index was slightly increased in discs treated with NPgel+MEPC. Pfirmann's and Thompson's classification showed that treatment with NPgel or NPgel+MEPC gave no adverse reactions. Histological grading showed similar degeneration in vertebrae treated with NPgel+MEPC or with NPgel alone. The amount of GAG was significantly increased in the nucleus pulposus following treatment with NPgel+MEPC compared to NPgel alone, in which a decrease was observed compared to untreated discs in both nucleus pulposus and annulus fibrosus. Conclusion. This study showed the safety of both NPgel+MEPC and NPgel treatments

Chronic rotator cuff tears are a major problem especially in the elderly population. Refixation is associated with high re-rupture rates. Therefore new implants or healing methods are needed. For a control of success biomechanical characteristics of native as well as treated tendons are of particular importance. Currently, tensile tests with static material testing machines are the most common technique for the biomechanical characterization of tendons. Resulting values are the maximum force (Fmax), stiffness and the Young´s modulus. However, no information is given about the allocation of strains over the tendon area. In addition, the determination of Fmax results in tissue destruction thus foreclosing further evaluation like histology. The Digital Image Correlation (DIC) is a contact-free non-destructive optical measuring method which gives information about distribution of strains by tracking the areal shift of an applied speckle pattern. The needed speckle pattern has to have a high contrast, a homogeneous distribution and a good adhesion to the surface. The method is established for the characterization of construction materials [1] to detect e.g. weak points. The present study examined if DIC is applicable for the complementary biomechanical evaluation of the sheep infraspinatus tendon. Fine ground powder extracted from a printer cartridge was chosen as a starting point. Preliminary to the in vitro experiments, the powder was applied on sheets with different methods: brushing, blowing, sieving and stamping. Stamping showed best results and was used for further in vitro tests on cadaveric native tendons (n=5). First, the toner powder was transferred to coarse-grained abrasive paper using a brush and stamped on the tendon surface. Afterwards DIC analysis was performed. For the in vivo tests, the left infraspinatus tendon of two German black-headed Mutton Sheep was detached and then refixed with bone anchors, the right tendon was used as native control (authorization: AZ 33.19-42502-04-17/2739). 12 weeks after surgery the animals were euthanized, the shoulders were explanted and DIC measurement performed. The speckle pattern could be applied adequately on the smooth tendon surfaces of native tendons. All specimens could be analyzed by DIC with sufficient correlation coefficients. The highest displacements were measured in the peripheral areas, whereas the central part of the tendon showed a low displacement. Repaired left tendons showed obvious differences already macroscopically. The tendons were thicker and showed inhomogeneous surfaces. Application of the toner powder by stamping was distinctly more complicated, DIC analysis could not produce sufficient correlation coefficients. In summary, transfer of DIC to native infraspinatus tendons of sheep was successful and can be further transferred to other animal and human tendons. However, irregular surfaces in tendon scar tissues affect the application of an adequate speckle pattern with a stamp technique. Therefore, further modifications are necessary. This research project has been supported by the German Research Foundation “Graded Implants FOR 2180 – tendon- and bone junctions” WE 4262/6-1

Summary. Timing for the application and use of fentanyl patches for pre-emptive analgesia and sedation is crucial to obtain good clinical outcomes. Placement and timing is important to maximise clinical effect and apparent levels of analgesia. Introduction. The use of sheep as preclinical models for the investigation of orthopaedic conditions is gaining momentum, the control of their pain is a significant ethical issue. The daily need for injecting non-steroidal anti-inflammatory drugs (NSAIDs) and/or the shorter acting opioids increases the demand for handling post-operatively which can increase animal distress and risk of human injury. NSAIDs can have a negative effect on bone healing, complicating results. Opioid analgesics have no impact on bone healing. Fentanyl patches have become another option for use in pain management. Pre-emptive analgesia helps reduce the demand on post-operative analgesic use. Fentanyl has the added benefit of producing mild sedation. This study evaluated the pharmacokinetics of fentanyl patches in sheep in an effort to maximise pre and post-surgical analgesia. Methods. Eight sheep were divided into 2 groups of 4. Both groups had a 100µg/kg/hr fentanyl patch (Durogesic – Janssen, Sydney, Australia) applied to the clipped and cleaned skin of the antebrachium and were held in place with a light bandage. (A dose rate range of 1- 1.6µg/kg/hr was achieved). Group 1 had a second patch applied after 72 hours and group 2 had a second patch applied after 24 hours. Blood samples were taken at 0, 3, 6, 12, 24, 36, 48 and 72 hours post patch application. The blood was immediately spun down and the serum drawn off and frozen. Serum levels for fentanyl were measured using commercial ELISA kits and read using a spectrophotometer. Animal behaviour throughout the study was observed and recorded by trained staff (CC, JR). Results. Six hours after the patch application, the sheep were relaxed and easily approachable. They stood calmly while blood was being drawn. This behaviour remained up to the 48 hour time point at which time cornering them in their pen became marginally more difficult, however they still stood calmly for the blood collection. By 72 hours, all sheep co-operation had dissipated. Peak blood levels of Fentanyl were reached by 12 hours post patch application. These levels were maintained with a relatively flat drug plateau for the prescribed 72 hours post application. No difference was found in the peak drug levels post application of the second patch between the two groups. There was no second higher peak in blood levels attained. Discussion. This study quantified the drug absorption and elimination curves of fentanyl using a controlled application method in an effort to better apply and manage post-surgical analgesia in sheep used for orthopaedic studies. The results indicate that the application of fentanyl for pre-emptive surgical analgesia can be applied for the full duration of 72 hours prior to the application of the second patch at the time of surgery. No benefit regarding analgesia appears to be gained from changing the first patch after 24 hours as peak serum levels are not affected. However for peak sedation the second patch can be applied anytime from 6 to 48 hours. This analgesic regime is beneficial to the animal as well as its handling and management

Objectives. We sought to determine if a durable bilayer implant composed of trabecular metal with autologous periosteum on top would be suitable to reconstitute large osteochondral defects. This design would allow for secure implant fixation, subsequent integration and remodeling. Materials and Methods. Adult sheep were randomly assigned to one of three groups (n = 8/group): 1. trabecular metal/periosteal graft (TMPG), 2. trabecular metal (TM), 3. empty defect (ED). Cartilage and bone healing were assessed macroscopically, biochemically (type II collagen, sulfated glycosaminoglycan (sGAG) and double-stranded DNA (dsDNA) content) and histologically. Results. At 16 weeks post-operatively, histological scores amongst treatment groups were not statistically different (TMPG: overall 12.7, cartilage 8.6, bone 4.1; TM: overall 14.2, cartilage 9.5, bone 4.9; ED: overall 13.6, cartilage 9.1, bone 4.5). Metal scaffolds were incorporated into the surrounding bone, both in TM and TMPG. The sGAG yield was lower in the neo-cartilage regions compared with the articular cartilage (AC) controls (TMPG 20.8/AC 39.5, TM 25.6/AC 33.3, ED 32.2/AC 40.2 µg sGAG/1 mg respectively), with statistical significance being achieved for the TMPG group (p < 0.05). Hypercellularity of the neo-cartilage was found in TM and ED, as the dsDNA content was significantly higher (p < 0.05) compared with contralateral AC controls (TM 126.7/AC 71.1, ED 99.3/AC 62.8 ng dsDNA/1 mg). The highest type II collagen content was found in neo-cartilage after TM compared with TMPG and ED (TM 60%/TMPG 40%/ED 39%). Inter-treatment differences were not significant. Conclusions. TM is a highly suitable material for the reconstitution of osseous defects. TM enables excellent bony ingrowth and fast integration. However, combined with autologous periosteum, such a biocomposite failed to promote satisfactory neo-cartilage formation. Cite this article: E. H. Mrosek, H-W. Chung, J. S. Fitzsimmons, S. W. O’Driscoll, G. G. Reinholz, J. C. Schagemann. Porous tantalum biocomposites for osteochondral defect repair: A follow-up study in a sheep model. Bone Joint J 2016;5:403–411. DOI: 10.1302/2046-3758.59.BJR-2016-0070.R1

The purpose of the study was to assess the incorporation of defatted, and deproteinated bovine cancellous bone in a sheep bone graft model. Cylindrical defects were created in the femoral condyles of 12 sheep using custom-made trephines. The defect was filled with a cylinder of prepared bovine bone. The removed cylinder of bone was implanted into a defect created in the opposite femoral condlyle. Fluorochrome bone labels were administered over an 8-week period and the sheep sacrificed at 10 weeks. Undecalcified thin bone sections were viewed with a fluorescent microscope. ln one sheep there was a technical problem leading to unsatisfactory histology. All other sheep showed similar histology. The autograft incorporated rapidly with the graft showing a rim of reactive bone and the graft itself showing rapid laying down of new bone on its surface. The xenograft showed a similar reactive rim of new bone with deposition of new bone throughout the graft and resorption of the graft material. This study demonstrates that specially prepared bovine cancellous bone can act as a scaffold for the depostion of new bone in a sheep model. The role of this material in humans is to be evaluated

Introduction Magnetic resonance imaging (MRI) is a valid investigation for the diagnosis of intervertebral disc disease, including infection, but it is expensive and difficult to access conveniently for research studies on live animals under anaesthesia. The aim of this study was to compare the MRI signal changes in spines from living and recently deceased sheep. Methods MRI was conducted on the lumbar spines of six adult sheep from research studies investigating bacterial discitis, chemical discitis and disc degeneration resulting from annular incision. The sheep was anaesthetised and the lumbar spines were imaged with a Siemens Magneton Vision MRI (Numaris VB33G software) using T1 SGE, T1 FSE, STIR and T2 FSE sequences. The sheep were killed with an intravenous overdose of barbiturate and identical images were obtained commencing five minutes post mortem. Results For each of the disease processes under consideration the MRI contrast relationships were maintained between all of the anatomical structures of interest. The post-mortem images provided improved clarity, particularly in the STIR and T2-weighted sequences, due to the absence of pulsation from the CSF and aorta, as well as the absence of respiratory artefact. Discussion The MRI appearances of spinal tissues following death correlate well with those in the live animal confirming the validity of this method of investigation. This is particularly relevant for optimisation of a busy clinical resource for research purposes

Purpose: To determine if an adenovirus vector expressing BMP-7 can alter the progression of post-traumatic osteoarthritis. Method: Preliminary dose-response studies were done in ovine metacarpal-phalangeal joints using 10^9, 10^10, and 10^11 virus particles (VP). In-vitro transfection efficiency studies were done using ovine synovial cells, chondrocytes and HEK293 cells. In-vivo studies were conducted in 16 sheep that underwent surgery to create bilateral contusive impact injuries to the medial femoral condyle. One week later 10^9 VP were injected into one joint of each sheep, while four sheep remained untreated bilateral controls. Three months later the sheep were sacrificed for assessments including histological scoring, cartilage glycosaminoglycan assays, and immunostaining for Col2 3/4 short collagen fragments that are generated by metalloproteinases during OA progression. Results: Transfection with 10^9 VP produced slightly longer expression than higher concentrations of VP. HEK293 cells expressed BMP-7 quickly but synoviocytes and chondrocytes expressed this protein at 48 and 96 hours. Knee joints that received Ad5-BMP-7 produced up to 2.5 ng of BMP-7 between day seven and 21. These joints had reduced cartilage degneration at the injury sites and less centrifugal progression of OA across the femoral condyle. Histological scores were reduced as was Col2 C3/4 short immunostaining. Conclusion: BMP-7 has a homeostatic role in cartilage and can be used therapeutically. 1. Ad5-BMP-7 transfection of synovial tissue produced sufficient BMP-7 to stop the progression of degenerative changes after trauma that would usually lead to OA. Adenoviral vectors can create inflammation and neutralizing antibodies but these complications were minimized by using a low (10^9) dose. Human trials using similar vectors are ongoing and the outcome of these will determine whether gene therapy will become a useful tool when patients are at risk of post-traumatic OA

Scientific Background: Adult articular cartilage, critical to proper joint function, has minimal self-repair ability. No adequate repair technique exists for large defects. Cryopreservation which is a process of deep-freezing of cells and tissues, enables the preservation of a high proportion of cells when the tissue is thawed and implanted. Aim: To evaluate a novel method for cryopreservation of articular cartilage in the form of osteochondral sheep cylinders. Materials and Methods: Osteochondral cylinders, 9mm diameter x 15mm length, were drilled from fresh cadaver sheep knee condyles. A bank of harvested cryo-preserved osteochondral cylinders was created. 17 sheep were used for transplantation. The thawed cylinders (allografts) were transplanted into the medial femoral condyle of the knee while the lateral femoral condyle received the fresh cylinder autograft as a control. The sheep were followed up for one year, following which in-vitro studies were performed to assess for articular cartilage viability. Results: Clinically, histologically and radiologically there was almost 100% incorporation of allogenic osteochondral cylinders involving most of the weight-bearing area of the sheep knees. Conclusion: Osteochondral cylinders can be successfully frozen and then transplanted into sheep knees with regeneration of knee function. Successful cryopreservation of human cartilage will be a major breakthrough in the treatment of cartilage lesions

Summary Statement. An autologous thrombin activated 3-fold PRP, mixed with a biphasic calcium phosphate at a 1mL:1cc ratio, is beneficial for early bone healing in older age sheep. Introduction. The management of bone defects continues to present challenges. Upon activation, platelets secrete an array of growth factors that contribute to bone regeneration. Therefore, combining platelet rich plasma (PRP) with bone graft substitutes has the potential to reduce or replace the reliance on autograft. The simple, autologous nature of PRP has encouraged its use. However, this enthusiasm has failed to consistently translate to clinical expediency. Lack of standardisation and improper use may contribute to the conflicting outcomes reported within both pre-clinical and clinical investigations. This study investigates the potential of PRP for bone augmentation in an older age sheep model. Specifically, PRP dose is controlled to provide clearer indications for its clinical use. Methods. Eighty 11mm diameter defects of 20mm in depth were created in the cancellous bone within the epiphyseal region of the medial proximal tibia and distal femur of twenty five-year-old sheep. The defects were treated with three doses of an autologous thrombin activated PRP combined with a biphasic calcium phosphate (BCP). Activated platelet poor plasma (PPP) and the BCP alone provided reference groups, while the autograft and empty defects served as controls. All animals were sacrificed at four weeks post-operatively for radiographic assessment, micro-computed tomography quantification, histological assessment, histomorphometric quantification of new bone area and bone ingrowth, and weekly fluorochrome bone label quantification. TGF-β1 concentrations were quantified using enzyme-linked immunosorbent assays. Results. The PRP had a 2.9-fold (0.4) increase in platelet concentration, a 0.57-fold (0.09) decrease in leukocytes, and a 0.65-fold (0.11) decrease in fibrinogen. After activation, the PRP had an 8.9-fold (1.5) increase in TGF-β1 serum concentration above baseline. Eleven (11) mm diameter cancellous bone defects in the hind legs of five-year-old sheep do not spontaneously heal within four weeks. PRP dose had a significant effect on the radiographic grade. The highest dose of PRP treatment had a significantly greater micro-CT BV/TV over the BCP alone (PRP: 30.6±1.8%; BCP: 24.5±0.1%). All doses of PRP treatment were significantly greater than the BCP alone for both the histomorphometric new bone area (PRP: 14.5±1.3%; BCP: 9.7±1.5%) and bone ingrowth depth (PRP: 2288±210µm; BCP:1151±268µm). From week two onwards, PRP had a significant effect on the weekly bone ingrowth over BCP, however, autograft had the greatest amount of fluorescently labelled bone within the first three weeks. PRP has a significant effect on the shape and density of osteoblasts within the central region of the defect compared to the BCP alone, however, was not significantly different to autograft. TGF-β1 appeared a better predictor of healing outcomes than platelet concentration, however both had relatively weak correlations (r<.324). Conclusion. PRP induces new bone formation with a dose dependant response at four weeks when used with a biphasic calcium phosphate in older age sheep

Aim. The treatment of chronic orthopedic device-related infection (ODRI) often requires multiple surgeries and prolonged antibiotic therapy. In a two-stage exchange procedure, the treatment protocol includes device removal and placement of an antibiotic-loaded bone cement spacer to achieve high local antibiotic concentrations. At the second stage, further surgery is required to remove the spacer and replace it with the definitive device. We have recently developed a thermo-responsive hyaluronan hydrogel (THH) that may be loaded with antibiotics and used as delivery system. Since the material is bio-resorbable, it does not require surgical removal and may therefore be suitable for use as treatment strategy in a single-stage exchange. This aim of this study was to evaluate gentamicin sulphate (Genta)-loaded THH (THH-Genta) for treating a chronic Staphylococcus aureus ODRI in sheep using a single-stage procedure. Methods. Twelve Swiss-alpine sheep received an IM tibia nail and an inoculation of a gentamicin-sensitive clinical strain of Staphylococcus aureus. After letting a chronic infection develop for 8 weeks, a revision procedure was performed: the implant was removed, the IM canal debrided and biopsies were taken for culture. The IM canal was then filled with 25ml THH-Genta (1% Genta) or left empty (control group) prior to the implantation of a sterile nail. An ultrafiltration probe was placed within the IM cavity to collect extracellular fluid and determine local antibiotic levels for 10 days. Both groups received systemic amoxicillin and clavulanic acid for 2 weeks, followed by 2 weeks without treatment for antibiotic washout. At euthanasia, IM nail, bone marrow, bone and tissue samples were harvested for quantitative bacteriology. Results. All sheep were infected at revision surgery as confirmed by cultures of biopsies and sonication of the IM nail. Local Genta concentrations ranged on average from 830µg/ml postoperatively to below 5µg/ml after 8 days. At euthanasia, S. aureus was detected in 5/5 IM nails, 5/5 bone marrow samples, and 8/25 superficial soft tissue samples in the control group (one control sheep was excluded for having a superinfection). In the THH-Genta group, S. aureus was cultured from 0/6 IM nails, 1/6 bone marrow samples, and 1/30 superficial soft tissue samples. Conclusions. The THH showed a Genta release pattern that started with high local concentrations and decreased to low concentrations within 10 days. Local Genta delivery by THH combined with systemic antibiotics significantly reduced infection rates whereas systemic therapy alone was unable to eradicate infection in any animal

INTRODUCTION:. To avoid the early onset of osteoarthritis after partial meniscectomy an effective replacement of injured meniscal tissue would be desirable. The present study investigates the behaviour of a new silk derived scaffold supplied by Orthox Ltd. (Abingdon, UK) in an in vivo sheep model. METHODS:. The scaffolds where derived from silk fibres by processing into an open porous matrix. Nine sheep (4 ± 1 years) underwent partial meniscectomy at the anterior horn of the medial meniscus followed by implantation of a scaffold. The unoperated contralateral stifle joint served as control. After six months the animals were sacrificed and the joints inspected for inflammation. The Young's modulus of the tibial cartilage, meniscus and scaffold was determined by indentation or confined compression tests. All tissues were fixed in formaldehyde for histology. The data were analysed by a Wilcoxon and Mann-Whitney-U-test. RESULTS:. The sheep were free of lameness 4 days p.o. The macroscopic analysis of the genual region and of the synovial membrane showed no signs of inflammation. This was confirmed by histological sections of synovial membrane, meniscus and scaffold. In histology, amorphous material, some fibroblast-like cell clusters and connective tissue formation was visible inside the pores of the scaffold. There were no statistically significant differences between the Young's moduli of the three measuring points in the operated and unoperated stifle joints. The meniscal tissue showed a higher modulus than the scaffolds. The scaffold's modulus significantly increased after three months implantation. DISCUSSION & CONCLUSIONS:. The presented silk scaffold withstood the loads occurring during the six months implantation period. It showed promising properties concerning biocompatibility and cartilage protection and its mechanical properties started to approach those of meniscal tissue

The aim of this study was to determine the function of the meniscofemoral ligament in the cranio-caudal and rotatory laxity of the ovine stifle. Twenty fresh cadaveric ovine stifles were harvested from fully mature sheep, average weight 25kg. The joint was denuded of its muscular attachments leaving the capsule, including the patella and patellar tendon undisturbed. The femur and tibia were divided 10 cm from the joint line, positioned in cylindrical pots, and secured in polymethylmethacrylate bone cement. The stifles were tested in a four-degree-of-freedom rig positioned in an Instron materials testing machine. This allowed unconstrained coupled tibial rotations and translations during application of cranial (anterior) and caudal (posterior) draw forces. Forces up to a maximum of 100Nm were applied in the anterior and posterior directions, and the resultant translations were measured. These parameters were assessed at 30, 60, 90, and 110 degrees of flexion in ten intact stifles. Similar measurements were carried out after division of the caudal (posterior) cruciate ligament, followed by division of the meniscofemoral ligament. The sequence of division was reversed for a further ten stifles. Division of the meniscofemoral ligament resulted in an 18–38% increase in posterior translation at all angles of flexion, both in the intact and in the caudal cruciate ligament-deficient stifle (p< 0.05). There was no significant increase in anterior translation. This effect was largest with the joint relatively extended (at 30°). Division of the meniscofemoral ligament also resulted in a 5–32% increase in internal rotation of the tibia after application of a 6Nm torque in the caudal cruciate-deficient knee. This was significant at 30° and 110° flexion (p< 0.05). The meniscofemoral ligament is a significant secondary restraint in resisting the posterior draw and internal tibial rotation in the sheep stifle joint. This is the first study demonstrating a functional role for this structure in any animal. Its counterpart in the human is the posterior meniscofemoral ligament of Wrisberg. Several studies have demonstrated similarities between the sheep stifle and the human knee. Confirmation of a similar role for the ligament of Wrisberg in the human knee would have a significant bearing on the prognosis and management of the posterior cruciate ligament injured knee

Introduction: Pin loosening is a common complication associated with external fixation. Various attempts such as coating the pins with calcium phosphates, have been made to modify the pin surface in order to reduce pin loosening and provide good fixation. Animal and clinical studies showed that fixation using tapered external fixation pins coated with hydroxyapatite is superior to standard pins. However, there is no data on cylindrical pins either fully or partially coated with HA. A partial coating could be a solution to optimize pin fixation with the advantage of easier removal compared to fully coated pins. Our purpose was to compare standard and partially coated cylindrical Apex pins implanted in a sheep model at 2 and 6 weeks. As controls we included fully coated tapered pins. Materials/Methods: Five groups of pins were studied. Group A included standard cylindrical Apex pins implanted in sheep which were euthanized 2 weeks after surgery; Group B included partially coated Apex pins implanted in sheep which were euthanized 2 weeks after surgery; Group C included tapered pins fully coated with HA implanted in sheep which were euthanized 2 weeks after surgery; Group D included Standard Apex pins implanted in sheep which were euthanized 6 weeks after surgery; Group E included partially coated Apex pins implanted in sheep which were euthanized 6 weeks after surgery. With the tapered pins, full contact between the coated surface and both cortices was obtained. After pin implantation, a unilateral external fixator was mounted onto the pins (Stryker carbon blue monotube Ø 20/250mm). A 5mm long removal osteotomy was performed in the mid-part of the tibial diaphysis, so that 3 pins were situated above the gap and 3 below to ensure load transfer through the bone-pin interface. Extraction torque and tibial torque resistance and histological analyses were obtained after pin removal. Results: At 2 weeks, mean insertion torque was significantly higher for group C compared to group A (p = 0.03). Mean extraction torque was statistically higher for group B compared to group A (p = 0.001). A statistically significant difference was found in the Pettine index (p = 0.03) between groups A and B. At 6 weeks, no differences in extraction torque were seen. Discussion: We believe that the partial application of the coating reduced the potential for osteointegration and the ultimate fixation of the coated Apex pins

Introduction: Vertebral compression fractures are common in osteoporosis, resulting in spinal deformities, severe back pain and decreased mobility. Vertebroplasty and kyphoplasty procedures aim to restore the integrity of the deformed vertebral body by injection of biocompatible cement. To date, there have been no long-term studies of the bone-cement interaction in this setting. A reliable large animal model of vertebral osteoporosis would be useful to fully characterise the disease process, to assess potential treatment regimens and to investigate the biocompatibility of bone cements used in kyphoplasty and vertebroplasty. The aim of this pilot study was to develop such a model with ovariectomy, low calcium diet and continuous steroid treatment. Methods: To induce osteoporosis, ten lactating ewes (mean age 8 years) were ovariectomised, injected weekly with 9 mg dexamethasone (Dexafort, Intervet, Australia) and fed low calcium diet. Weekly serum samples were taken to quantify generalised bone resorption (Type 1 collagen C-telopeptide [CTX], ‚-Cross Laps assay, Roche Diagnostics, Australia). Dual-energy X-ray absorptiometry (DEXA, Hologic QDR 1000+, USA) was used to monitor bone mineral density (BMD) in the lumbar spine (L3-L6) after 0, 2, 4, 6 and 9 months of treatment. At each time interval two sheep were killed by barbiturate injection. The entire lumbar spine (L1-L6) was processed for histology, quantitative histomorphometry, mechanical testing and micro-CT (computed tomography). Results: CTX levels increased rapidly after two months (p< 0.05). Baseline BMD in the lumbar spine (0.87±0.06 g/cm2) decreased by 16.9±3.8% or 2.72 standard deviations (p< 0.001) after nine months of treatment. Structural parameters of cancellous bone also showed osteoporotic change. Trabecular bone volume of L2, L3 and L6 vertebrae (pooled) progressively decreased from 24.9±1.2% at two months to 16.5±0.47% at nine months (p< 0.05). Trabecular thickness decreased from 0.14±0.01mm to 0.09±0.01mm, (p< 0.05) and trabecular spacing increased from 0.42±0.03mm to 0.47±0.02mm in the same period. The compressive load at which the L1 vertebrae failed decreased by 39.4% after 9 months. Discussion: This pilot study has demonstrated by DEXA, cancellous bone histomorphometry and mechanical testing, significant bone loss in the sheep lumbar spine up to nine months after ovariectomy and continuous steroid treatment. Assuming that the baseline BMD is representative of mature sheep, the changes in the lumbar spine could be interpreted as osteoporotic. Vertebral bone loss did not reach levels that would result in fracture. However, further work is underway using higher steroid doses to accelerate bone loss. This experimental model will be used to assess aspects of osteoporosis in general and vertebral augmentation procedures in particular

Introduction: Modern imaging techniques have demonstrated that up to 28% of patients with spinal cord injury develop syringomyelia. Cyst formation and enlargement are thought to be related to abnormalities of cerebrospinal fluid hydrodynamics, however the exact mechanism and route of entry into the spinal cord remain incompletely understood. Previous work in rats has demonstrated that experimental post-traumatic syrinxes occur more reliably and are larger when the excitotoxic injury is combined with arachnoiditis produced by subarachnoid kaolin injection. A sheep model of post-traumatic syringomyelia (P.T.S.) has been characterised and studies of cerebrospinal fluid dynamics are currently being undertaken. The aim of this study was to assess the effect of focal subarachnoid space blockage on spinal fluid pressures and flow. Methods: Arachnoiditis was induced in 5 sheep by injection of 1.5 mls of kaolin in the subarachnoid space (SAS) of upper thoracic spinal cord. The animals were left for 6–8 weeks before C.S.F. studies were undertaken. In another 5 sheep, a ligature was passed around the spinal cord to simulate an acute blockage of the subarachnoid space. Fluid-coupled monitors were used to measure blood pressure, central venous pressure and subarachnoid pressure (1cm rostral and 1 cm caudal to the arachnoiditis or ligature). Fiberoptic monitors were used to measure intracranial pressure. In the ligature group, subarachnoid pressures were also measured prior to tying the ligature to obliterate the SAS and served as baseline control pressures. The effects of Valsalva and Queckenstedt manoeuvres on SAS pressures were examined in both groups. CSF flow was studied at 0 and 10 minutes after injection of the CSF tracer horseradish peroxidase (HRP). Vibratome sections of the spinal cord were processed using tetramethylbenzidine and sections examined under light microscopy. Results: The mean SAS pressure rostral to the arachnoiditis was found to be greater than the mean caudal SAS pressure by 1.7 mmHg. In the ligature group, the difference was 0.9 mmHg, being higher in the caudal SAS. Queckenstedt manoeuvre exaggerated this difference to 3 mmHg in the Kaolin group and 4 mmHg in the ligature group. The effect of Valsalva was much less marked in both groups. Perivascular spaces were enlarged in most cases of arachnoiditis and HRP was seen to stain these spaces and the central canal within 10 minutes. Discussion: Post-traumatic syrinxes are usually juxtaposed to the injury site with 80% occurring rostral, 4% caudal and 15%in both directions. The finding of a higher subarachnoid pressure rostral to the injury site may help explain this phenomenon. We hypothesize that a reduction of compliance in subarachnoid space increases the pulse pressure and hence increases perivascular flow of C.S.F. contributing to the formation and enlargement of PTS. We are currently investigating this hypothesis by measuring subarachnoid space compliance directly in the sheep model of arachnoiditis described above

INTRODUCTION: Modern imaging techniques have demonstrated that up to 28% of patients with spinal cord injury develop syringomyelia. Cyst formation and enlargement are thought to be related to abnormalities of cerebrospinal fluid hydrodynamics, however the exact mechanism and route of entry into the spinal cord remain incompletely understood. Previous work in rats has demonstrated that experimental post-traumatic syrinxes occur more reliably and are larger when the excitotoxic injury is combined with arachnoiditis produced by subarachnoid kaolin injection. A sheep model of post-traumatic syringomyelia (P.T.S.) has been characterised and studies of cerebrospinal fluid dynamics are currently being undertaken. The aim of this study was to assess the effect of focal subarachnoid space blockage on spinal fluid pressures and flow. METHODS: Arachnoiditis was induced in five sheep by injection of 1.5 mls of kaolin in the subarachnoid space (SAS) of upper thoracic spinal cord. The animals were left for 6–8 weeks before C.S.F. studies were undertaken. In another five sheep, a ligature was passed around the spinal cord to simulate an acute blockage of the subarachnoid space. Fluid-coupled monitors were used to measure blood pressure, central venous pressure and subarachnoid pressure (1 cm rostral and 1 cm caudal to the arachnoiditis or ligature). Fiberoptic monitors were used to measure intracranial pressure. In the ligature group, subarachnoid pressures were also measured prior to tying the ligature to obliterate the SAS and served as baseline control pressures. The effects of Valsalva and Queckenstedt manoeuvres on SAS pressures were examined in both groups. CSF flow was studied at 0 and 10 minutes after injection of the CSF tracer horseradish peroxidase (HRP). Vibratome sections of the spinal cord were processed using tetramethylbenzidine and sections examined under light microscopy. RESULTS: The mean SAS pressure rostral to the arachnoiditis was found to be greater than the mean caudal SAS pressure by 1.7 mmHg. In the ligature group, the difference was 0.9 mmHg, being higher in the caudal SAS. Queckenstedt manoeuvre exaggerated this difference to 3 mmHg in the Kaolin group and 4 mmHg in the ligature group. The effect of Valsalva was much less marked in both groups. Perivascular spaces were enlarged in most cases of arachnoiditis and HRP was seen to stain these spaces and the central canal within 10 minutes. DISCUSSION: Post-traumatic syrinxes are usually juxtaposed to the injury site with 80% occurring rostral, 4% caudal and 15% in both directions. The finding of a higher subarachnoid pressure rostral to the injury site may help explain this phenomenon. We hypothesise that a reduction of compliance in subarachnoid space increases the pulse pressure and hence increases peri-vascular flow of C.S.F. contributing to the formation and enlargement of PTS. We are currently investigating this hypothesis by measuring subarachnoid space compliance directly in the sheep model of arachnoiditis described above

Short Summary. The present study demonstrated the feasibility of culturing a large number of standardised granular MSC-containing constructs in a packed bed/column bioreactor that can produce sheep MSC-containing constructs to repair critical-size bone defects in sheep model. Introduction. Endogenous tissue regeneration mechanisms do not suffice to repair large segmental long-bone defects. Although autologous bone graft remains the gold standard for bone repair, the pertinent surgical technique is limited. Tissue constructs composed of MSCs seeded onto biocompatible scaffolds have been proposed for repairing bone defects and have been established in clinically-relevant animal models. Producing tissue constructs for healing bone defects of clinically-relevant volume requires a large number of cells to heal an approximately 3 cm segmental bone defect. For this reason, a major challenge is to expand cells from a bone marrow aspirate to a much larger, and sufficient, number of MSCs. In this respect, bioreactor systems which provide a reproducible and well-controlled three-dimensional (3D) environment suitable for either production of multiple or large size tissue constructs are attractive approaches to expand MSCs and obtain MSC-containing constructs of clinical grade. In these bioreactor systems, MSCs loaded onto scaffolds are exposed to fluid flow, a condition that provides both enhanced access to oxygen and nutrients as well as fluid-flow-driven mechanical stimulation to cells. The present study was to evaluate bioreactor containing autologous MSCs loaded on coral scaffolds to repair critical-size bone defects in sheep model. Materials and Methods. Animals: 12 two-year-old, female Pre-Alpes sheep were used and reared in accordance with the European Committee for Care. Three-dimensional, porous scaffolds (each 3×3×3 mm) of natural coral exoskeleton were used as substrates for cell attachment. The packed bed/column bioreactor set-up used in the present study was composed of a vertical column filled with MSC-containing constructs. Sheep MSCs were isolated from sheep bone marrow. MSCs were seeded on scaffolds and cultured overnight under standard cell-culture condition. MSC-containing constructs were r placed into the perfusion bioreactor and were either exposed to a perfusion medium flow rate of 10 mL/min for 7 continuous days. Osteoperiosteal segmental (25 mm) defects were made in the left metatarsal bone of 12 sheep. The defect was either filled with coral scaffolds alone (Group 1; five sheep); or filled with coral scaffolds loaded with MSCs (Group 2; five sheep); or filled with autologous bone graft (Group 3; 2 sheep). Results. At 6 month after implantation, radiographs showed resorption of the coral scaffold in all animals but this process was not complete and not the same in all animal. At 6 month radiographs showed more bone formation in group 2 than in group 1. New bone formation volume in each defect was assessed by micro-computed tomography. Volume of bone healing was higher in group 2 than group 1. Discussion. The potential of MSC-containing constructs in a bioreactor for repairing long segmental critical-sized bone defects in sheep was investigated. In one animal of the group 2 the volume of new bone formation was 2066 mm3 and was similar to the bone volume of group 3 (2300 mm3). Our results may have important implications in bone tissue engineering. We observed that the bone tissue regenerationosteogenic ability of bone constructs processed in bioreactor approached the bone autografts