Tranexamic acid (TXA) is an anti-fibrinolytic medication commonly used to reduce peri-operative bleeding. Increasingly, topical administration as an intra-articular injection or peri-operative wash is being administered at concentrations between 10–100mg/ml. This study investigated effects of TXA on human periarticular tissues and primary cell cultures using clinically relevant concentrations. Tendon, synovium and cartilage obtained from routine orthopaedic surgeries were used ex vivo or cultured for in vitro studies using various concentrations of TXA. They were stained with 5-chloromethylfluorescein diacetate and propidium iodide and imaged using confocal microscopy to identify the proportion of live and dead cells. The in vitro effect of TXA on primary cultured tenocytes, synovial like fibroblast (FLS) cells and chondrocytes was investigated using cell viability assays (MTT), fluorescent microscopy and multi-protein apoptotic arrays for cell death. There was significant (p<0.01) increase in cell death in all tissue treated with 100mg/ml TXA, ex vivo. MTT assays revealed significant (p<0.05) decrease in cell viability following treatment with 50 or 100mg/ml of TXA within 4 hours of all cell types cultured in vitro. Additionally, there was significant (p<0.05) increase in cell apoptosis detected by fluorescent microscopy within 1 hour of exposure to TXA. Furthermore, multi-protein apoptotic arrays detected increased apoptotic proteins within 1 hour of TXA treatment in tenocytes and FLS cells. Our study provides evidence of TXA cytotoxicity to human peri-articular tissues ex vivo and in vitro at concentrations and durations of treatment routinely used in clinical environments. Clinicians should therefore show caution when considering use of topical TXA administration.
Bone cutting produces heat which macroscopically leads to charring and the formation of bone dust. As part of a project to design a novel bone cutting device, we studied the extent of histological thermal damage from bone cutting with different cutting blades. Three blades were used: a bone hacksaw made in the nineteenth century which was used for amputation, a sagittal saw blade made by Ortho Solutions, and a sagittal saw blade made by Stryker. Sheep femurs were harvested from recently euthanised animals and cuts were made with these three devices, producing ring-shaped bone specimens. Specimens were immediately stored in formaldehyde, decalcified, and stained with hematoxylin and eosin. The edge of the specimens was then photographed microscopically, and the images examined with the computer programme Axiovision (Carl Zeiss AG, Oberkochen, Germany). Visual examination allowed identification of live and dead osteocytes, and also to measure their depth from the surface. A minimal of 7 images was obtained per blade. The hacksaw specimens had the highest percentage of live osteocytes (n=214, 59.8%), and with the shortest average depth where live osteocytes were located (169μm, SD 78.15). In comparison, the percentage of live osteocytes for the Ortho Solutions (n=156, 17.4%) and Stryker (n=168, 29.5%) blades were much lower. The difference in average depths where live osteocytes were located was statistically significant between the three groups (p < 0.001). The average depths of dead osteocytes were shallowest for the Stryker (115μm, SD 67.56) and hacksaw (118.28 μm, SD 75.16) groups with no statistical difference between them. In conclusion the hacksaw appeared to produce the least thermal damage histologically during cutting. The results reflect a relationship between certain features in cutting blade designs and the extent of thermal damage. Future experiments to directly measure heat produced during cutting are planned.
Bone cutting produces heat which macroscopically leads to charring and the formation of bone dust. As part of a project to design a novel bone-cutting device, we studied the extent of histological thermal damage from different cutting blades. Three blades were used: a nineteenth century bone hacksaw, and modern sagittal saw blades manufactured by Ortho Solutions and Stryker. Sheep femurs were harvested from recently euthanised animals and cuts were made with these blades. Specimens were immediately stored in formaldehyde, decalcified, and stained with hematoxylin and eosin. The edge of the specimens was then photographed microscopically, and the images examined with Axiovision software (Carl Zeiss AG, Oberkochen, Germany). Visual examination allowed identification of live and dead osteocytes, and also to measure their depth from the surface. A minimal of 7 images was obtained per blade. The hacksaw specimens had the highest percentage of live osteocytes (n=214, 59.8%), and the shortest average depth where live osteocytes were located (169 μm, SD 78.15). In comparison, the percentage of live osteocytes for the Ortho Solutions (n=156, 17.4%) and Stryker (n=168, 29.5%) blades were much lower. The difference in average depths where live osteocytes were located was statistically significant between the three groups (p<0.001). In conclusion the hacksaw appeared to produce the least thermal damage histologically during cutting. The results reflect a relationship between certain features in cutting blade designs and the extent of thermal damage. Future experiments to monitor heat produced during cutting are planned.
Staphylococcus aureus is a highly virulent pathogen and implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections suggest that alpha-(Hla), beta-(Hlb) and gamma-(Hlg) toxins are key virulence factors, with the ‘pore-forming’ alpha-toxin considered the most potent. Here, we have assessed the influence of alpha-toxin alone on in situ chondrocyte viability. Osteochondral explants were harvested from the metacarpophalangeal joints of 3-year-old cows and cultured in Dulbecco's Modified Eagle's Medium. The flasks were then inoculated with isogenic ‘knockout’ strains of S. aureus: DU5946 (Hla+Hlb-Hlg-: alpha-toxin only strain) or DU1090 (Hla-Hlb+Hlg+: beta- and gamma-toxin only strain). Explants were incubated (37°C) and stained after 18, 24 and 40hrs with chloromethylfluorescein-di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Axial sections were imaged by confocal microscopy and the percentage cell death determined. Alpha-toxin-producing S. aureus caused 24.8+/−3.7% chondrocyte death at 18hrs and 44.6+/−7.2% death at 24hrs. At 40hrs, there was significantly more chondrocyte death (87.4+/−3.6%;p<0.001) compared to the alpha-toxin knockout strain, which was negligible (4.1+/−1.7%; means+/−SEM; N=4 independent experiments). In this in vitro bovine cartilage explant model, whereby the effects of defined toxins were determined in isolation of a complex host immune response, in situ chondrocyte viability was dramatically and exclusively reduced by alpha-toxin. This work forms the basis for developing a rational treatment to reduce the extent of cartilage destruction during an episode of septic arthritis. IDMS was supported by Orthopaedic Research UK and The Royal College of Surgeons of Edinburgh.
We have demonstrated that toxins produced by Staphylococcus aureus, a common infective agent in septic arthritis (SA), cause rapid in situ chondrocyte death. Here, we have compared the sensitivity of chondrocytes within the superficial and deep zones (SZ, DZ) of cartilage to the same toxins. Culture medium containing the toxins produced by S. aureus strain 8325-4, which include alpha-, beta-, and gamma-toxin, was prepared. Cartilage explants free of subchondral bone were taken from the metacarpophalangeal joints of 3-year-old cows, and incubated (37°C) with the toxins. Explants were stained after 6hrs with chloromethylfluorescein-di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Full-thickness coronal sections were imaged by confocal microscopy and the percentage cell death within the SZ (100μm from articular surface) and DZ (100μm from subchondral bone interface) determined. Both zones were incubated with the same toxin culture medium for the same time period. At 0hrs, chondrocytes within all zones were >98% viable. However, after incubation with toxin-containing culture medium for 6hrs, 71.9+/−11.2% of the SZ cells were dead compared to only 47.4+/−6.7% in the DZ (p=0.03;data are means+/−SEM;N=4). These results suggest that SZ chondrocytes are considerably more sensitive to S. aureus toxins than those within deeper zones. As SZ chondrocytes are close to the synovial fluid harbouring bacterial toxins, these data emphasise the need to remove bacteria and their products aggressively as part of the treatment of SA. IDMS was supported by Orthopaedic Research UK and The Royal College of Surgeons of Edinburgh.
The cartilage diseases such as osteoarthritis and chondral injuries are considered irreversible and the result of recent treatments remains not optimal. One of the reasons is due to the poor understanding of chondrocyte behaviours. To understand more about cartilage, we designed a series of novel experiments. First, a total joint of bovine metatarsophalanges was isolated as our novel model. We chose it because the configuration and the healing potential were similar to human, and many variables of large animal studies could be controlled in laboratory. The model not only provided a good ex vivo platform for cartilage researches but also connected in vitro cellular studies and in vivo animal studies. To mimic joint movement a special driving machine was designed. To characterise the novel model viabilities of chondrocytes and contents of sulphated glycosaminoglycan (GAGs) in extracellular matrixes were measured every seven days. The preliminary results revealed the viabilities of chondrocytes remained above 80% alive in the middle zone after four-weeks culture. The GAGs contents decreased after this culturing period. The experiments still carry on going to compare the static and dynamic models which joint movement could be a determinative factor to the viability of chondrocytes. Cellular treatment is the recent mainstream for cartilage diseases. If advanced knowledge in chondrocyte behaviours could be obtained from this model, development of optimal treatment will be possible in the future.
Staphylococcus aureus is a highly virulent pathogen and is implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections have suggested that alpha (Hla), beta (Hlb) and gamma (Hlg) toxins are key virulence factors. In particular, the ‘pore-forming’ alpha toxin is believed to be most potent. In this study, we have assessed the influence of alpha toxin on in situ chondrocyte viability. Osteochondral explants were harvested from the metacarpophalangeal joints of 3-year-old cows and placed into flasks containing Dulbecco's Modified Eagle's Medium. The flasks were then inoculated with the following isogenic ‘knockout’ strains of S. aureus: DU5946 (Hla+Hlb-Hlg-) or DU1090 (Hla-Hlb+Hlg+). The explants were incubated (37°C) and stained after 18, 24 and 40hrs with chloromethylfluorescein di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Axial sections were imaged by confocal microscopy and the percentage cell death obtained using Volocity 4 software. The alpha toxin-producing S. aureus caused rapid cell death, with 24.8+/−3.7% at 18hrs and 44.6+/−7.2% at 24hrs. At 40hrs, there was significantly more chondrocyte death (87.4+/−3.6%; p<0.001) compared to the alpha toxin knockout strain (4.1+/−1.7%; means +/− SEM; n=4). In situ chondrocyte viability was significantly compromised by alpha toxin, with beta and gamma toxins having minimal effect. Further work will clarify the exact mechanism through which this important toxin induces chondrocyte death. Thereafter, it is hoped that targeted treatments can be developed to reduce the extent of cartilage destruction during, and after, an episode of septic arthritis.
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.
In some centres, serial bedside aspirations, in association with intravenous antibiotics, are still an accepted treatment for septic arthritis (Mathews, Postgraduate Medical Journal, 2008). However, there is a risk that bacterial products remain in the joint, even when the bacteria have been destroyed. We have conducted a study to ascertain whether bacterial products alone have an effect on in situ chondrocyte viability. A hip aspirate (25μl), containing Staphylococcus aureus, from a patient with septic arthritis was added to 5ml culture medium and incubated (37°C) for 48hrs. The solution was then centrifuged (3400g for 10mins) and the supernatant removed. Cartilage explants were harvested from a bovine metacarpophalangeal joint, placed into the bacterial supernatant and incubated at 37°C. Explants were removed at hourly intervals over a 6-hour period and stained with the fluorescent probes chloromethylfluorescein di-acetate (10μM) and propidium iodide (10μM) to label living chondrocytes green and dead cells red respectively. Following imaging of cartilage by confocal microscopy, the percentage cell death at each time point was obtained using Volocity 4 software. Chondrocyte death increased markedly with time: 0.04% at 2hrs, 28% at 4hrs and 39% at 6hrs. This study shows that bacterial products rapidly penetrate the cartilage matrix and have a damaging effect on in situ chondrocyte viability. Further work will clarify the contributions made by the various toxic components in the culture supernatant, but these data support the need to remove the bacteria and their products aggressively as part of the treatment of septic arthritis.
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.
In Scotland, the number of primary total knee replacements (TKRs) performed annually has been steadily increasing. Data from the Scottish Arthroplasty Project has recently demonstrated that the number of knee replacements performed annually has now outstripped the number of hip replacements. The price of the implant is fixed but the length of hospital stay (LOHS) is variable. An understanding of what currently influences LOHS may therefore be of paramount importance in order that we can influence some of these parameters, with resulting benefit to our patients as well as contributing significantly and favourably towards the health economics of this procedure. This study investigates the influence of intra- and post-operative variables on LOHS. All patients who underwent primary unilateral TKR in the region of Fife, Scotland, United Kingdom, during the period December 1994 to February 2007 were prospectively investigated. The following intra and postoperative details were recorded: length of operation, need for urinary catheterisation, patella resurfacing, lateral release, blood transfusion, the presence of superficial or deep infection, day 1 post-operative haemoglobin and haemoglobin drop (haemoglobin drop between admission haemoglobin and day 1 post-operative haemoglobin). The data was analysed using univariate and multiple linear regression statistical analysis. Data on LOHS was available from a total of 2105 primary unilateral TKRs. The median LOHS was 8.0 days. The highly significant intra and post-operative factors associated with an increased LOHS were lateral release, post-operative haemoglobin, blood transfusion, urinary catheterisation, deep and superficial infection. An awareness and understanding of these factors may enable us to influence them favourably with resulting reduction in the LOHS and, therefore, the associated costs.
There are many management solutions for the fixation of Periprosthetic fractures with intact stem of Hip and shoulder arthroplasties. The Bio Mechanics of single plate application are unlikely to be strong enough to commence mobilisation and its effectiveness against torsional strain with an osteoporotic bone quality is of concern. Double plate fixation as discussed at the last South African Orthopaedic Congress by Mr Floyd et al is another option but this again may have some biomechanical concerns and biological compromise at the fracture site due to periosteal stripping. Implant revision with a longer stem is a bigger surgical insult to a potentially frail group of patients with questionable bone quality. We report a short series of 16 peri-prosthetic fractures with intact stem that are managed with Zimmer cable plate fixation System. The results were very satisfactory and we consider this an attractive option to be considered in the management of this difficult presentation. This is a retrospective study. We present the results of 13 Periprosthetic Femoral Shaft fractures and 3 humeral periprosthetic fractures in 16 patients treated with cable plate fixation system. Majority of the patients were over 60 years with an ASA rating of 3–4. The procedures were performed in a district general hospital in the UK between August 2001 to December 2005. The patients presented with in 1–20 years following initial Arthroplasty. All the fractures were fixed with Zimmer cable plate fixation system. An 8 hole plate was most commonly used for femoral fractures through the lateral approach for TYPE 2 fractures. The proximal end of the plate was secured with 3–4 cable ties. Early partial weight bearing was encouraged. The majority of the patients were discharged within 12 weeks. Of the 3 humeral fractures union was achieved at 12 weeks in 2. There was 1 case of implant failure due to a further fracture noted in a manic depressive patient, who was not compliant. All proximal femoral fractures showed evidence of clinical and radiological union by 6 months. The majority (7/13) had united within 20 weeks. There were no complications noted. We recommend this effective method should seriously be considered in the management of this difficult and increasingly occurring complication in a frail population.
Proximal femoral fractures, whether it is due to meta-static destruction or periprosthetic fractures with loose femoral component with secondary osteolysis of the proximal femur in the elderly patient is a major task. We find the Cannulock hip system quite useful in tackling this issue. It offers various options for the management of this complex pathology. We present the results of 11 Cannulock Hip Arthroplasty performed in 10 patients (Age Range 55–92). 6 out of 11 patients was noted to have metastatic destruction of proximal femur including the head and neck down to lesser trochanter. Ca of Bronchus and Breast with multiple bony metastsis were responsible for these cases. 4/11 had loose femoral component with type 2 periprosthetic fractures. 1 out of 11 had failed DCS fixation for Reverse oblique fracture. The procedures were done in a district general Hospital in the UK between August 2001–Jan 2006. The patients were mostly ASA 4. The Cannulock Hip system offered the simplicity of a Hemiarthroplasty with an advantage of Intramedullary nailing option. This has the option of fitting standard Bipolar Head or 22 mm metallic head in case of Peri Prosthetic fracture where the acetabular component is intact. Long stem with HA coating and standard options for cemented stem insertion. The long stem with a bow enables easy insertion with distal locking facility. In our study all the patients were excellent with both clinical and readilogical out come, however sadly 1 patient died with in 3 months of surgery. 5/6 patients with metastatic bone tumour were discharged at the mean of 8 weeks with no clinical concerns. 4 patients with femoral stem revisions and 1 patient with failed DCS were discharged at a mean of 4 months. We find the Cannulock hip arthroplasty system quite versatile in the management of these complex injuries.
