Meniscal tears are the most common knee injuries, occurring in acute ruptures or in chronic degenerative conditions. Meniscectomy and meniscal repair are two surgical treatment options. Meniscectomy is easier, faster, and the patient can return to their normal activities earlier. However, this procedure has long-term consequences in the development of degenerative changes in the knee, potentially leading to knee replacement. On the other hand, meniscal repair can offer prolonged benefits to the patients, but it is difficult to perform and requires longer rehabilitation. Sutures are used for meniscal repairs, but they have limitations. They induce tissue damage when passing through the meniscus. Furthermore, under dynamic loading of the knee, they can cause tissue shearing and potentially lead to meniscal repair failure. Our team has developed a new technology of resistant adhesive hydrogels to coat the suture used to repair meniscal tissue. The objective of this study is to biomechanically compare two suture types on bovine menisci specimens: 1) pristine sutures and 2) gel adhesive puncture sealing (GAPS) sutures, on a repaired radial tear under cyclic tensile testing. Five bovine knees were dissected to retrieve the menisci. On the 10 menisci, a complete radial tear was performed. They were separated in two groups and repaired using either pristine (2-0 Vicryl) or GAPS (2-0 Vicryl coated with adhesive hydrogels) with a single stitch and five knots. The repaired menisci were clamped on an Instron machine. The specimens were cyclically preconditioned between one and 10 newtons for 10 cycles and then cyclically loaded for 500 cycles between five and 25 newtons at a frequency of 0.16 Hz. The gap formed between the edges of the tear after 500 cycles was then measured using an electronic measurement device. The suture loop before and after testing was also measured to ensure that there was no suture elongation or loosening of the knot. The groups were compared statistically using Mann-Whitney tests for nonparametric data. The level of significance was set to 0.05. The mean gap formation of the pristine sutures was 5.61 mm (SD = 2.097) after 500 cycles of tensile testing and 2.38 mm (SD = 0.176) for the GAPS sutures. Comparing both groups, the gap formed with the coated sutures was significantly smaller (p = 0.009) than with pristine sutures. The length of the loop was equal before and after loading. Further investigation of tissue damage indicated that the gap was formed by suture filament cutting into the meniscal tissue. The long-term objective of this research is to design a meniscal repair toolbox from which the surgeon can adapt his procedure for each meniscal tear. This preliminary experimentation on bovine menisci is promising because the new GAPS sutures seem to keep the edges of the meniscal tear together better than pristine sutures, with hopes of a clinical correlation with enhanced meniscal healing

Meniscal tears are the most common knee injuries, occurring in acute ruptures or in chronic degenerative conditions. Meniscectomy and meniscal repair are two surgical treatment options. Meniscectomy is easier, faster, and the patient can return to their normal activities earlier. However, this procedure has long-term consequences in the development of degenerative changes in the knee, potentially leading to knee replacement. On the other hand, meniscal repair can offer prolonged benefits to the patients, but it is difficult to perform and requires longer rehabilitation. Sutures are used for meniscal repairs, but they have limitations. They induce tissue damage when passing through the meniscus. Furthermore, under dynamic loading of the knee, they can cause tissue shearing and potentially lead to meniscal repair failure. Our team has developed a new technology of resistant adhesive hydrogels to coat the suture used to repair meniscal tissue. The objective of this study is to biomechanically compare two suture types on bovine menisci specimens: 1) pristine sutures and 2) gel adhesive puncture sealing (GAPS) sutures, on a repaired radial tear under cyclic tensile testing. Five bovine knees were dissected to retrieve the menisci. On the 10 menisci, a complete radial tear was performed. They were separated in two groups and repaired using either pristine (2-0 Vicryl) or GAPS (2-0 Vicryl coated with adhesive hydrogels) with a single stitch and five knots. The repaired menisci were clamped on an Instron machine. The specimens were cyclically preconditioned between one and 10 newtons for 10 cycles and then cyclically loaded for 500 cycles between five and 25 newtons at a frequency of 0.16 Hz. The gap formed between the edges of the tear after 500 cycles was then measured using an electronic measurement device. The suture loop before and after testing was also measured to ensure that there was no suture elongation or loosening of the knot. The groups were compared statistically using Mann-Whitney tests for nonparametric data. The level of significance was set to 0.05. The mean gap formation of the pristine sutures was 5.61 mm (SD = 2.097) after 500 cycles of tensile testing and 2.38 mm (SD = 0.176) for the GAPS sutures. Comparing both groups, the gap formed with the coated sutures was significantly smaller (p = 0.009) than with pristine sutures. The length of the loop was equal before and after loading. Further investigation of tissue damage indicated that the gap was formed by suture filament cutting into the meniscal tissue. The long-term objective of this research is to design a meniscal repair toolbox from which the surgeon can adapt his procedure for each meniscal tear. This preliminary experimentation on bovine menisci is promising because the new GAPS sutures seem to keep the edges of the meniscal tear together better than pristine sutures, with hopes of a clinical correlation with enhanced meniscal healing

Mincing cartilage with commercially available shavers is increasingly used for treating focal cartilage defects. This study aimed to compare the impact of mincing bovine articular cartilage using different shaver blades on chondrocyte viability. Bovine articular cartilage was harvested using a scalpel or three different shaver blades (2.5 mm, 3.5 mm, or 4.2 mm) from a commercially available shaver. The cartilage obtained with a scalpel was minced into fragments smaller than 1 mm. 3. All four conditions were cultivated in a culture medium for seven days. After Day 1 and Day 7, metabolic activity, RNA isolation, and gene expression of anabolic (COL2A1, ACAN) and catabolic genes (MMP1, MMP13), Live/Dead staining and visualization using confocal microscopy, and flow cytometric characterization of minced cartilage chondrocytes were measured. The study found that mincing cartilage with shavers significantly reduced metabolic activity after one and seven days compared to scalpel mincing (p<0.001). Gene expression of anabolic genes was reduced, while catabolic genes were increased after day 7 in all shaver conditions. The MMP13/COL2A1 ratio was also increased in all shaver conditions. Confocal microscopy revealed a thin line of dead cells at the lesion site with viable cells below for the scalpel mincing and a higher number of dead cells diffusely distributed in the shaver conditions. After seven days, there was a significant decrease in viable cells in the shaver conditions compared to scalpel mincing (p<0.05). Flow cytometric characterization revealed fewer intact cells and proportionally more dead cells in all shaver conditions compared to the scalpel mincing. Mincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing. This indicates that mincing cartilage with a shaver should be considered a matrix rather than a cell therapy. Further experimental and clinical studies are required to standardize the mincing process with a shaver. Acknowledgements: This study received unrestricted funding from KARL STORZ SE & Co. KG

Introduction: Seven patients underwent successful revision total knee replacement for aseptic loosening. Bovine bone graft was used to reconstruct bony defects in all. Materials and methods: This is a retrospective review. Between April 2000 and March 2003, bovine bone (Tutobone™, Wescott-Medical, UK) was used in 7 revision arthroplasty cases (4 right knees & 3 left). There were 5 males and 2 females. The average age was 70.4 years. All revisions were carried out for aseptic loosening of the prostheses associated with massive osteolysis and bone loss. The bone defects on the tibia and femur were as follows: (Obtained from operative records. Classified according to Anderson Orthopaedic Research Institute classification). Type I. Type IIA. Type IIB. Type III. TIBIA. 3. 1. 2. 1. FEMUR. 2. 3. 2. 0. The tibial defects were corrected by impaction grafting and femoral condyle defects were corrected by using bovine bone as bulk grafts. Semi-constrained constrained stemmed cemented modular knee prostheses (TC3, Depuy) were used in all. Clinical outcomes were recorded by the Oxford Knee Score. Serial radiographs were evaluated for graft density, integration, implant loosening, alignment and subsidence. Results: At recent follow-up, radiographs showed good graft integration, no loosening, and no subsidence of the implant and good prostheses alignment. The average Oxford Knee Score was 20.4. Conclusion & discussion: Bovine bone substitute is an alternative. The bone defects in these patients were successfully reconstructed with bovine bone. It is an osteo-conductive matrix with intact type-I collagen that provides mechanical stability. It is also cost effective. Early results are encouraging but long-tem follow-up is needed

Miniscrew implants (MSIs) are widely used to provide absolute anchorage for the orthodontic treatment. However, the application of MSIs is limited by the relatively high failure rate (22.86%). In this study, we wished to investigate the effects of amorphous and crystalline biomimetic calcium phosphate coating on the surfaces of MSIs with or without the incorporated BSA for the osteointegration process with an aim to facilitate the early loading of MSIs. Amorphous and crystalline coatings were prepared on titanium mini-pin implants. Characterizations of coatings were examined by Scanning electron microscopy (SEM), Confocal laser-scanning dual-channel-fluorescence microscopy (CLSM) and Fourier-transform infrared spectroscopy (FTIR). The loading and release kinetics of bovine serum albumin (BSA) were evaluated by Enzyme linked immunosorbent assay (ELISA). Activity of alkaline phosphate (ALP) was measured by using the primary osteoblasts. In vivo, a model of metaphyseal tibial implantation in rats was used (n=6 rats per group). We had 6 different groups: no coating no BSA, no coating but with surface adsorption of BSA and incorporation of BSA in the biomimetic coating in the amorphous and crystalline coatings. Time points were 3 days, 1, 2 and 4 weeks. Histological and histomorphometric analysis were performed and the bone to implant contact (BIC) of each group was compared. In vitro, the incorporation of BSA changed the crystalline coating from sharp plates into curly plates, and the crystalline coating showed slow-release profile. The incorporation of BSA in crystalline coating significantly decreased the activity of ALP in vitro. In vivo study, the earliest significant increase of BIC appeared in crystalline coating group at one week. The crystalline coating can serve as a carrier and slow release system for the bioactive agent and accelerate osteoconductivity at early stage in vivo. The presence of BSA is not favorable for the early establishment of osteointegration

More than 250,000 people are suffering from Anterior Cruciate Ligament (ACL) related injuries each year in the US, with a cost of $17–25K/patient. There is an unmet clinical demand for improving grafts/scaffolds to provide biological integration in addition to mechanical support. Currently, no data is available for the utilization of fibrous scaffolds with bimodal distribution for ACL regeneration. The novelty in this study is that it proposes for the first time to investigate the collagen fibril diameter distribution in healthy and injured bovine ACL tissue, and utilization of such structure for scaffold design. Objectives are 1) developing a bovine ACL tear model and measuring the collagen fibril diameter distribution of both healthy and injured ACL tissues, and 2) fabricating scaffolds to mimic the structural properties of healthy and injured ACL tissue. Bovine ACL tissues (1–3 years old) were harvested and characterized for their fibril diameter distribution using Transmission Electron Microscopy (TEM) and biomechanical properties under tension. The electrospun polycaprolactone (PCL) scaffolds were characterized using SEM and mechanical testing. Healthy and injured ACL fibril diameter, and that of PCL scaffolds representing healthy and injured ACL are compared using unpaired student t-test. The proposed fibrous scaffold design represents a significant departure from the conventional unimodal approach, and is expected to have significant contribution to ACL regeneration. These discoveries will serve as the foundation for the development of biomimetic tissue engineering substrates aimed at promoting biological graft fixation

Introduction: Human autologous chondrocyte transfer requires a small biopsy of articular cartilage (300–500 mg wet weight) obtained by arthroscopy from the patient’s knee joint. Chondrocytes are isolated and seeded at low density in monolayer culture to increase cell number. A common problem with this technique is that chondrocytes lose their phenotype by reverting to a fibroblast phenotype and synthesize a different matrix. Collagen type II and aggrecans are unique to hyaline cartilage-matrix. They form an extensive three-dimensional network of extracellular matrix in which other cell adhesion and growth factor molecules are integrated. It has been shown that a three dimension environment coupled with growth factors are important for the maintenance of the chondrocyte phenotype. Although cells cultured in alginate beads maintain their phenotype they do not proliferate well. Aim of study: To develop and optimise a bovine chondrocyte culture system as a model for optimising human chondrocyte proliferation without dedifferentiation and their future transplantation. The optimum cell density determined for bovine chondrocyte cultures was used for human chondrocyte cultures. Cell proliferation and matrix synthesis of cultured human chondrocytes from normal as well as damaged knee joint articular cartilage obtained from debridement arthroscopy was investigated. Methods: Bovine chondrocytes were seeded in collagen type I gels at various densities ranging from 104 to 106 cells/ml to obtain the minimal cell density required in a collagen gel culture system in which chondrocytes can proliferate and yet retain their unique phenotype. The media were supplemented with either bovine foetal calf serum (FCS) or a combination of three growth factors (3GFs), TGF-b1 + IGF-I + b-FGF. Cells and matrix were analysed on day 7, 14, and 21 of culture. Cell proliferation was determined by the trypan blue exclusion test. Cell morphology and matrix present were evaluated with both light and electron microscopy. A collagen type II specific antibody coupled with FITC conjugate was used to detect type II collagen neo-deposit in relation to the seeded type I collagen gels. The newly synthesised matrix was monitored after labelling cells with 35S-sulphate and 3H-proline. The collagen type was determined by SDS-PAGE Fluorography. Analysis of morphology and matrix synthesis was performed as. Results: Cell proliferation: Bovine chondrocytes cultured in collagen type I gels at low density proliferated up to 40 fold after 3 weeks while high density cultures proliferated only about 3 fold. There was no significant difference in cell numbers at day 21 in cultures supplemented with FCS or 3GFs. Therefore all human chondrocyte cultures were cultured at low density. Preliminary results from human chondrocyte cultures were obtained from 4 patients aged 59+19. After 4 weeks, human chondrocytes cultured at low density supplemented with FCS proliferated up to 10 fold in monolayer culture and up to 4 fold in collagen type I gels. Morphology: At all cell densities, the majority of bovine chondrocytes in the gels remained rounded while some cells near the surface of the gels were elongated. Human chondrocytes cultured at low density also demonstrated similar morphology. Matrix synthesis: For bovine chondrocyte culture, after 2 weeks in culture more than 70% of 35S-sulphate and 3H-proline incorporated matrix. Conclusions: This study has shown that bovine chondrocytes cultured at low density in collagen type I gels proliferated better than at high density and retained their phenotype. This low-density bovine chondrocyte culture model is applicable to human chondrocyte culture in vitro. Preliminary results shows that human chondrocytes obtained from patients aged 39–72 can proliferate both in gels and monolayer. Age of chondrocytes and growth factors may affect the growth of cells. This model system needs to be further investigated in normal human chondrocytes

Introduction and Objective. Low back pain (LBP) is a major cause of long-term disability in adults worldwide and it is frequently attributed to intervertebral disc (IVD) degeneration. So far, no consensus has been reached regarding appropriate treatment and LBP management outcomes remain disappointing. Spine unloading or traction protocols are common non-surgical approaches to treat LBP. These treatments are widely used and result in pain relief, decreased disability or reduced need for surgery. However, the underlying mechanisms -namely, the IVD unloading mechanobiology- have not yet been studied. The aim of this first study was to assess the feasibility of IVD unloading in a large animal organ culture set-up and evaluate its impact on mechanobiology. Materials and Methods. Bovine tail discs (diameter 16.1 mm ± 1.2 mm), including the endplates, were isolated and prepared for culture. Beside the day0 sample that was processed directly, three other discs were cultured for 3 days and processed on day4. One disc was loaded in the bioreactor according to a previously established physiological (compressive) loading protocol (2h/day, 0.2Hz). The two other discs were embedded in biocompatible resin, leaving the cartilage endplate free to permit nutrient diffusion, and fitted in the traction holder; one of these discs was kept in free swelling conditions, whereas the second was submitted to cyclic traction loading (2h/day, 0.2Hz) corresponding to 30% of the animal body weight corrected for organ culture. Results. The cell viability assessed on lactate dehydrogenase and ethidium homodimer stained histological slides was not different between the three cultured discs. This means that the disc viability was not affected neither by the embedding, nor by the traction itself. Compared to the physiologically loaded disc, the gene expression of COL1, COL2 and ACAN was higher in the nucleus pulposus and inner annulus fibrosus of the traction treated disc. In the outer annulus fibrosus of this disc TAGLN and MKX were higher expressed upon traction than in the physiologically loaded disc. Conclusions. Based on these preliminary data, we can conclude that large animal organ culture allows effective unloading of the disc, while preserving cell viability and modulating cellular gene expression responses. This sets the ground for future experiments and opens the door to an evidence-based improvement of clinical spine traction protocols and LBP management overall

Purpose: To develop an improved understanding of the in vivo behavior of intervertebral disc (IVD) cells for determining the phenotype of a differentiated stem cell in tissue engineering applications. Methods: Nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from adult bovine tails while notochordal cells were extracted from fetal bovine intervertebral disc. Ten million cells (of each cell type) in 500 & #61549;l of DMEM were then injected subcutaneously in C57Bl/6 mice. After 2 weeks, the mice were sacrificed and the specimens harvested. They were examined grossly, histologically and by scanning electronic microscopy (SEM) for the evidence of IVD-like structure formation. Proteoglycan was assessed by the GAG assay and PCR for analysis of gene expression. Control tissue (from bovine NP and AF) were directly fixed in glutaraldehyde, without any isolation technique and examined in SEM. Results: After 2 weeks, SEM examination of specimens from AF and NP closely resembled normal bovine AF and NP. Of special interest here was the finding that some mice injected with cells from the AF developed an organized arrangement of parallel collagen fibres while NP cells injected mice had an amorphous structure with few collagen fibers. The GAG assay showed pro-teoglycan content for each samples, ranging from 3.8 microg to 26 microg. The morphology of the specimens retrieved from notochordal cells injected mice were also amorphous punctuated with thin collagen fibrils. Conclusions: This study demonstrates that subcutaneous injection of bovine disc cells in mice can result in formation of disc structures similar to those of the bovine IVD. We believe that the cellular communication of the bovine disc cells is maintained in the mouse leading to architectural organization of the collagen fibers with the mouse as a source of nutrients. This technology may be useful in determining the phenotype of a differentiated stem cell for tissue engineering of IVD

Fragmentation of SLRPs, including decorin, biglycan, lumican, keratocan and fibromodulin, has been shown to occur in osteoarthritic articular cartilage. We have previously shown an increased expression of lumican and keratocan, in osteoarthritic articular cartilage. The long-term aim of this project is to develop ELISAs for the detection of SLRP metabolites, and validate these potential biomarkers with synovial fluid and serum samples from a large cohort of normal and osteoarthritic patients. Initially, we aimed to determine whether SLRPs could be detected in synovial fluid and whether they were post-translationally modified with glycosaminoglycan (GAG) attachments; and whether bovine nasal cartilage (BNC) would be a plentiful source of native SLRP for ELISA development. Proteoglycans were extracted from BNC in guanidine hydrochloride. BNC extract and bovine synovial fluid was separated on an associative CsCl gradient. BNC CsCl cuts containing sulphated GAG were further purified using anion exchange chromatography. SLRPs in each fraction were detected using Western Blotting. Human recombinant lumican was expressed in Chinese hamster ovary (CHO) cells. Monoclonal antibodies that recognise epitopes on the core protein of human and bovine lumican and decorin were purified from hybridoma media using Protein G and Protein A affinity chromatography respectively. Monoclonal antibody activity against native and recombinant SLRPs was then determined using a direct ELISA. Preliminary tests showed that bovine synovial fluid contains keratocan and lumican with GAG attachments. BNC is a good source of post-translationally modified decorin, keratocan and biglycan but lumican was present predominantly without GAG attachments. Human recombinant lumican was successfully expressed with GAG attachments by CHO cells. Initial tests showed that the mAb against decorin was able to detect native decorin, with GAG attachments, in direct ELISA conditions. We have identified a plentiful source of native SLRP and begun ELISA development to ascertain whether these proteoglycans are potential biomarkers of OA

Few studies have investigated the direct effect of bacteria and their products on articular cartilage chondrocytes ex vivo. An ex vivo model that allows the analysis of chondrocytes in situ would therefore be an important and exciting area of future research. It was hypothesised that a bovine cartilage explant model of septic arthritis would be an ideal model for providing fundamental information on the basic cellular mechanisms of cartilage destruction and chondrocyte death induced by bacterial infection uncomplicated by the immune response. A fresh metacarpophalangeal joint from an abattoir slaughtered 3-year-old cow was skinned, rinsed in water and opened under sterile conditions. The cartilage explants were harvested using surgical scalpels and placed into a total of three tissue culture bottles (2 explants per bottle) containing 10ml Dulbecco's Modified Eagle Medium (DMEM). 50ml of a knee aspirate from a patient with septic arthritis, containing Group B streptococci (GBS), was added to bottle 1, 50ml of a negative knee aspirate was added to bottle 2 and 50ml DMEM to bottle 3. The explants were incubated at 37°C for 24 hours. They were then stained with the fluorescent probes Chloromethylfluorescein Di-acetate (CMFDA) and Propidium Iodide and analysed using a Confocal Scanning Laser Microscope. Cell counts to assess percentage cell death were performed using Velocity 4 software. There was strikingly more cell death observed at 24 hours in the cartilage explant exposed to bacteria in comparison to the non-infected controls. The percentage chondrocyte death was 43% in the presence of GBS, 0.8% in the presence of the negative aspirate and 0.2% in the presence of the DMEM control. Although this is a very preliminary pilot study, it demonstrates an extremely rapid effect on the cartilage. Future bovine explant studies of septic arthritis will therefore be feasible and achievable

Introduction. Sclerostin has been implicated in mechanotransduction in bone and recent data show a lack of response to loading in the sclerostin transgenic mouse. Sclerostin, the protein product of the SOST gene, is an attractive therapeutic target for low bone mass conditions, including osteoporosis. It is expressed exclusively by mature osteocytes in bone and we have shown that sclerostin targets pre-osteocytes/osteocytes to regulate bone mineralization and osteoclast activity, as well as inducing catabolic gene expression in osteocytes themselves and promoting osteocyte-mediated bone loss (osteocytic osteolysis). The aim of this study was to examine the direct effects of sclerostin on anabolic responses to loading in bone ex vivo. Methods. 10 × 5mm bovine sternum trabecular bone cores were perfused with osteogenic media at 37°C for up to 3 weeks in individual bone culture chambers. The cores were divided into 3 groups; a) mechanically loaded (300 cycles, 4000 μstrain, 1 Hz/day), b) identical loading regime with continuous perfusion of 50 ng/ml recombinant human sclerostin and c) unloaded controls. Loading was accomplished using a second-generation Zetos™ bone loading system. Daily measurements of bone stiffness (Young's modulus), media pH and ionic calcium concentrations were made. Histomorphometric assessment, including fluorochrome labelling analysis, was made of resin-embedded, non-decalcified samples at the end of the experiment. Gene expression in the bovine bone was examined by real-time RT-PCR. Results. Bovine bone cores showed a steady increase in Young's modulus with daily application of mechanical loading. This increase in stiffness was blocked by the co-addition of sclerostin. Sclerostin also induced bone acidification and a net release of bone calcium, indicated by the decrease in media pH and the relative increase in ionic calcium concentrations in the presence of sclerostin. Sclerostin also completely abrogated loading-induced calcium/calcein uptake. Sclerostin induced an increase in the expression of the bone resorption genes, tartrate resistant acid phosphatase (TRAP), carbonic anhydrase and cathepsin K and induced the release of β-CTX. Histological examination revealed a significant increase in the size of the osteocyte lacunae in sclerostin-treated bone cores, suggesting a role for osteocytic osteolysis in this effect. Discussion/Conclusion. The observation that sclerostin abrogated the loading-induced increase in bone stiffness constitutes direct evidence for a negative effect of sclerostin on the anabolic response to mechanical loading. Our findings may be explained in part by the observation that sclerostin negatively controls mineralization by late osteoblasts and pre-osteocytes (1). It is also possible that osteocytes themselves are capable of releasing bone mineral in response to sclerostin. This study demonstrates that sclerostin directly antagonises the anabolic effects of mechanical loading in the absence of external (circulating, neural, hormonal) influences. The mechanisms, by which sclerostin exerts these effects, warrant further study

Recent evidence indicates that link N can stimulate synthesis of proteoglycans and collagen by adult (2–4 years old) bovine disc tails. Here we sought to determine the effect of link N on the accumulation of disc matrix proteins from young (eight to twenty month old) bovine tails. We show that degradation products of link protein generated by matrix metalloproteinases cannot “feed-back” and stimulate matrix assembly of the disc matrix from young bovine tails but may have a regulatory role in cell proliferation. Link N may have value only in stimulating the growth and regeneration of the old damaged intervertebral disc. To date, there have been no reports on the effect of the amino terminal peptide of link protein (DHLSD-NYTLDHDRAIH) (link N) on disc cells from young (eight to twenty month old) bovine coccygeal discs. Link N is produced when removed by proteolysis from the N-terminus of the link protein of cartilage proteoglycan aggregates. We recently showed that link N can act directly on disc cells from adult (two to four years old) bovine discs to stimulate matrix production (. J Cell Biochem. , . 2003. ; . 88. :. 1202. –13. ). To examine whether link N can play a role in maintaining the matrix integrity of young bovine disc cells. Nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from fresh grade I discs from young steers, and cultured in pellets at 1 million cell per tube in 1 ml of DMEM-high glucose supplemented with 1% 100X Pen-Strep, 1% ITS, 1 mg/ml BSA, and 50 μg/ml ascorbic acid. Cell pellets were digested and then analysed for sulfated glycosaminoglycan, type II collagen, percent denatured type II collagen, type IX collagen, and DNA content, using specific assays. A concentration of 100 ng/ml link N significantly increased the DNA content of AF cells. However, link N had no significant effect on proteoglycan, type II and type IX collagen accumulation. This study demonstrates that link N at a concentration of 10 ng/ml and 100 ng/ml cannot stimulate matrix production but may increase cell division in young bovine disc tails

Formation of micro-cracks occurs in bone due to daily activities. Through a mechanism of self-repair, these micro-cracks are detected, and the damaged areas are restored, avoiding further propagation. The Scissors Model suggests that the osteocyte processes that cross the micro-cracks break as consequence of the cyclic displacements of the micro-crack faces, due to fatigue, and this triggers the remodelling processes. A fresh bovine tibia bone was cut in sections oriented 20° from the transversal direction. The cortical bone was sliced using a circular saw and shaped to the dimensions: 20 × 10 × 1 (mm) and the surfaces were polished. µCT images were obtained from all the samples (μCT 40, Scanco Medical, Brüttisellen, Switzerland). From the DICOM files, the geometries were reconstructed and meshed using tetrahedrons, in ICEM CFD. The Elasticity Modulus (E) was determined in Bonemat, by applying an empirical relationship Elasticity-Density from the literature. The parts were then imported into ANSYS APDL to simulate micro-crack propagation in bone. This model will be validated with further experimental work where the micro-crack will be initiated in the prepared samples and propagated due to fatigue loading, and the osteocyte processes will be visualized in the Scanning Electron Microscope (SEM). This investigation aims to study how cyclic loading in bones and failure of osteocyte processes can trigger target the mechanism of bone remodelling. The resulting model can later contribute for the investigation of treatments for bone diseases such as osteoporosis and the response of bone to the presence of orthopaedic implants

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

Articular cartilage has very poor repair potential, however it has an extraordinary capacity to withstand physiological mechanical loads in an intact joint. The nature and extent of chondrocyte death in articular cartilage following many forms of injury (trephine, scalpel, osteotome, sutures and drilling) has been characterised, but the ability to bear mechanical injury from iatrogenic surgical interventions is still unknown. A standard arthroscopic probe was moved at varying physiological pressures along the articular cartilage of joint before staining with fluorescent dyes to allow live/dead cell imaging using laser confocal scanning microscopy and imaging software, Image J. Bovine metatarsal phalangeal joints and fresh human cadaveric femoral condyles were used. The probe caused statistically significant chondrocyte death in bovine cartilage (p=0.02). Mild pressure 5% cell death, moderate (standard arthroscopic technique pressure) 22% and severe pressure 38%. A similar result was seen in human tissue with 24% cell death at moderate pressure compared to a control (p=0.0699). The widely assumed benign arthroscopic probe produces significant cell death in articular cartilage when used at standard operating pressures

Bone cement reaches high temperatures while polymerising. Bone has been shown to be sensitive to thermal injury with osteonecrosis reported after one minute at 47°C. Necrosis during cementing might compromise the bone-cement interface. Some surgeons fill the joint cavity with irrigation fluid to provide a heatsink during cementing, but this has not been supported by research. We used a model acetabulum in a bovine humerus to allow measurement of bone temperatures in cementing. Models were prepared with a 50mm diameter acetabulum and three temperature probe holes. Four warmed models were cemented with Palacos RG using a standard mixing system and a 10mm UMHWPE disc to represent an acetabular component. Two of the acetabular models were filled with room temperature water to provide a heatsink. An electronic probe measured temperature at 5 second intervals from the moment of cementing. In the models with no heatsink, peak temperature was 40.3°C. The highest temperature rise was 7.5°C. In the models with a heatsink, there was a mean fall of 4.4°C. These results suggest that using a heatsink while cementing prostheses may reduce the peak bone temperature

Staphylococcus aureus is the most common bacterial isolate in septic arthritis. From studies on isolated cartilage cells, the ‘pore-forming’ alpha and gamma toxins are considered the most virulent factors. However, understanding the response of in situ chondrocytes is important in order to identify new treatments to reduce the extent of cartilage damage during, and following, episodes of septic arthritis. Animal models can give useful information; however the interpretation of data can be complex because of the strong immune response. Thus, to clarify the role of S. aureus toxins on in situ chondrocytes we have developed a bovine cartilage explant model. Metacarpophalangeal joints, from 3-year-old cows, were opened under sterile conditions within 6hrs of slaughter and cartilage explants harvested. Explants were placed into flasks containing Dulbecco's Modified Eagle Medium (DMEM). Aspirates from a patient with septic arthritis of the hip, containing S. aureus, were compared to negative aspirates (no bacterial growth) from a patient with an inflamed knee joint (controls). The explants were incubated at 37 degrees Celsius and stained after 18, 24 and 40hrs with the fluorescent probes chloromethylfluorescein di-acetate and propidium iodide (10 micromolar each) to label living chondrocytes green and dead cells red respectively. Following imaging of cartilage by confocal laser scanning microscopy, the percentage cell death at each time point was obtained using Volocity 4 software. There was no detectable change in chondrocyte viability (<1% cell death) over 40hrs incubation with the negative aspirate. However, for the aspirate from a patient positive for S. aureus, there was a rapid increase in cell death between 18 and 24hrs (0.2 +/− 0.3% to 23 +/− 5% cell death respectively) and almost complete cell death at 40hrs (80 +/− 12%; data are means +/− s.d; n=4). These results show that a strain of S. aureus capable of manifesting clinical disease exerts a potent effect on in situ chondrocytes. In the absence of an immune response, chondrocyte death was purely the result of the bacteria and their products. This bovine cartilage explant model could therefore be useful for studying the effects of S. aureus on chondrocyte behaviour and, ultimately, cartilage integrity

Animal models have shown that artificially induced temporomandibular joint (TMJ) disc displacement or perforation affect histology and biochemistry of joint cartilage, leading to osteoarthritic changes. However, it is still unclear whether TMJ disc cartilage fails simply due to wear or is degraded by a biological response to mechanical loading. In order to gain insight into TMJ cartilage mechanobiology, a system reproducing the dynamic TMJ compression effects on live tissues was developed. Bovine nasal septum (BNS) cartilage was chosen as a convenient tissue model. However, little information is available in the literature on its material properties. Aim of this study was to determine BNS material properties using a viscoelastic model and verify its suitability as model for TMJ disc cartilage. Cartilage samples were harvested from the central part of BNSs of young, healthy animals. Stress-relaxation tests in unconfined compression were performed on cylindrical plugs samples, obtained by means of biopsy punches. A 10% strain (strain rate 0.01 mm/s) was applied and held for 30 minutes. Stress was estimated from the compressive force data and the initial cross-sectional area. Experimental data were fit to a mathematical model in MATLAB. Experimental results show a highly viscoelastic behavior of the BNS, with a maximum average stress of 0.73 ± 0.14 MPa and relaxed stress of 0.21 ± 0.03 MPa. The numerical model shows good correspondence to the experimental data (R2=0.96). The average values for the instantaneous and relaxed elastic moduli are E0= 7.72 MPa and ER= 2.30 MPa, in the same order of magnitude as the TMJ disc. We conclude that bovine nasal septum can be modeled as viscoelastic tissue and can be used as a first approximation to study mechanobiology of the TMJ disc

Testing potential therapeutics in the regeneration of the disc requires the use of model systems. Although several animal models have been developed to test intervertebral disc (IVD) regeneration, application becomes costly when used as a screening method. The bovine IVD organ culture system offers an inexpensive alternative, however, in the current paradigm, the bony vertebrae is removed to allow for nutrient diffusion to disc cells. This provides limitations on the conditions and strategies one can employ in investigating IVD regeneration and mechanisms in degenerative disc disease (i.e. complex loading). Although one method has been attempted to extend the survival of bovine vertebrae containing IVDs (vIVD) cell viability declined after two weeks in culture. Our goal was to develop and validate a long-term organ culture model with vertebral bone, which could be used subsequently for studying biological repair of disc degeneration and biomechanics. Preparation of vIVDs: Bovine IVDs from the tails of 22–28-month-old steers were prepared for organ culture by parallel cuts through the adjacent vertebral bodies at 1cm from the endplates using an IsoMet®1000 Buehler precision sectioning saw. vIVDs were split into two groups: IVDs treated with PrimeGrowth Media kit (developed by Intervertech and licensed to Wisent Bioproducts) and IVDs with DMEM. The PrimeGrowth group was incubated for 1h in PrimeGrowth Isolation Medium (Cat# 319–511-EL) and the DMEM group for 1h in DMEM. After isolation, IVDs were washed in PrimeGrowth Neutralisation Medium (Cat# 319–512-CL) while the other IVDs were washed in DMEM. The discs isolated with PrimeGrowth and DMEM were cultured for up to 5 months in sterile vented 60 ml Leakbuster™ Specimen Containers in PrimeGrowth Culture Medium (Cat# 319–510-CL) and DMEM with no mechanical load applied. Live/Dead Assay: vIVDs cultured for 1 or 5 months were dissected and cell viability was assessed in different regions by confocal microscopy using Live/Dead® (Invitrogen) fluorescence assay. Glucose Diffusion: After one month of culture, vIVDs were incubated for 72h in diffusion medium containing PBS (1x), CaCl2 (1mM), MgCl2 (0.5mM), KCl2 (5mM), 0.1% BSA and 150µM 2-NDBG, a D-glucose fluorescent analogue. Discs were dissected and IVD tissues were incubated in guanidinium chloride extraction buffer. Extracts were measured for fluorescence. After 5 months in culture, vIVDs prepared with PrimeGrowth kit demonstrated approximately 95% cell viability in all regions of the disc. However, dramatic reductions (∼90%) in vIVD viability were measured in DMEM group after 1 month. vIVD viability was related to the amount of 2-NDBG incorporated into the disc tissue. We have developed a novel method for isolating IVDs with vertebral bone capable of long-term viability. This method may not only help in the discovery of novel therapeutics in disc regeneration, but could also advance our understanding on complex loading paradigms in disc degeneration