Objectives. Thermal stability is a key property in determining the suitability of an antibiotic agent for local application in the treatment of orthopaedic infections. Despite the fact that long-term therapy is a stated goal of novel local delivery carriers, data describing thermal stability over a long period are scarce, and studies that avoid interference from specific carrier materials are absent from the orthopaedic literature. Methods. In this study, a total of 38 frequently used antibiotic agents were maintained at 37°C in saline solution, and degradation and antibacterial activity assessed over six weeks. The impact of an initial supplementary heat exposure mimicking exothermically curing bone cement was also tested as this material is commonly used as a local delivery vehicle. Antibiotic degradation was assessed by liquid chromatography coupled to mass spectrometry, or by immunoassays, as appropriate. Antibacterial activity over time was determined by the Kirby-Bauer disk diffusion assay. Results. The heat exposure mimicking curing bone cement had minimal effect on stability for most antibiotics, except for gentamicin which experienced approximately 25% degradation as measured by immunoassay. Beta-lactam antibiotics were found to degrade quite rapidly at 37°C regardless of whether there was an initial heat exposure. Excellent long-term stability was observed for aminoglycosides, glycopeptides, tetracyclines and quinolones under both conditions. Conclusions. This study provides a valuable dataset for orthopaedic surgeons considering local application of antibiotics, and for material scientists looking to develop next-generation controlled or extended-release antibiotic carriers. Cite this article: E. Samara, T. F. Moriarty, L. A. Decosterd, R. G. Richards, E. Gautier, P. Wahl. Antibiotic stability over six weeks in aqueous solution at body temperature with and without heat treatment that mimics the curing of bone cement. Bone Joint J 2017;6:296–306. DOI: 10.1302/2046-3758.65.BJR-2017-0276.R1

Introduction. Collagen is the predominant component of extracellular matrix in various connective tissues and makes up to 25% to 35% of the whole protein content in animal bodies. Type II collagen was first introduced from chicken sternal cartilage and presents supportive function in cartilaginous tissue. Since type II collagen is the major component of cartilage in joint, this study is aiming to determine an optimal type II collagen material for the development of medical devices for articular cartilage regeneration. In order to make more effective use of underutilized food waste, type II collagens from mammalian tissue sources (porcine tracheal cartilage; auricular cartilage; articular cartilage) and marine tissue sources (cuckoo ray, blonde ray, thorn back ray, lesser spotted dogfish) were isolated through acid-pepsin digestion under 4°C and characterized by various biological, biochemical and biophysical analysis. Pepsin cleaves the telopeptide region of the collagen molecule and pepsin treated collagen extraction ensures higher collagen yield. Telopeptide-free collagen reveals cytocompatibility, biodegradability and lower toxicity. The number and size of collagen chains were revealed by SDS-polyacrylamide gel electrophoresis. Intermolecular crosslinking density was quantified by Ninhydrin assay. Thermal stability was tested by differential scanning calorimetry (DSC) and enzymatic degradation was assessed by collagenase assay. Human chondrocytes were seeded on to collagen sponges at a density of 30,000 cells per sponge. Cell morphology (DAPI/ Rhodamine Phalloidin), viability(LIVE/DEAD®), proliferation(PicoGreen®) and metabolic activity (alamarBlue®) were analysed. Quantitative morphometric analysis was carried out using ImageJ software. Conclusion. Porcine articular cartilage and cartilaginous fishes yield high purity type II collagen. Type II collagen isolated from cartilaginous fishes exhibited similar crosslinking density and thermal stability. Among various porcine cartilaginous tissues, articular cartilage was the most resistant to enzymatic degradation and female trachea exhibited the highest cross-linking density. The biological, biochemical and thermal properties of type II collagen are dependent on the tissue and gender from which the collagen was extracted

Porcine and fish by-products in particular are rich sources for collagen, which is the main component of the extracellular matrix (ECM). Although there are studies investigating different collagen derived from various tissue sources for the purpose of creating biomaterials, the comparison of biophysical, biochemical and biological properties of type II collagen isolated from cartilaginous tissues has yet to be assessed. In addition, it has been shown from previous studies that sex steroid hormones affect the collagen content in male and female animals, herein, type II collagens from male and female porcine cartilage were assessed in order to investigate gender effects on the property of collagen scaffolds. Moreover, type II collagen has a supportive role in articular cartilage in the knee joint. Therefore, the aim is to assess the properties of type II collagen scaffolds as a function of species, tissue and gender for cartilage regeneration. Type II collagen was extracted from male and female porcine trachea, auricular, articular cartilage and cartilaginous fish through acid-pepsin digestion at 4°C. SDS-PAGE was conducted to confirm the purity of extracted collagen. Collagen sponges were created via freeze-drying. Scaffold structure and pore size were evaluated by scanning electron microscopy (SEM). Thermal stability was assessed by differential scanning calorimetry (DSC). Sponges were seeded with human adipose derived stem cells to assess chondro-inductive potential of collagen sponges after 7, 14 and 21 days of culture. In conclusion, collagen sponges support the proliferation and differentiation of human adipose derived stem cells to different extents

Thermostability is a key property in determining the suitability of local delivery of antibiotics in the treatment of orthopaedic infections. Herein, we aimed to assess the thermal stability and antibacterial activity of ciprofloxacin, ceftriaxone, gentamycine and vancomycine in high temperature conditions. Using a standardized E-test method, minimally inhibited concentration of each antibiotic substance against Staphylococcus aureus cultures were determined. The solutions of antimicrobial drugs ciprofloxacin 2 mg/ml, ceftriaxone 200 mg/ml, gentamycine 40 mg/ml and vancomycine 200 mg/ml were diluted twofold in deionised water. Acquired solutions were divided into three aliquots. The first aliquot was held at 40°C for 30 min in a waterbath, the second and the third aliquots were exposed to 80 and 100°C for 30 min in hot-air sterilizer, respectively. The treated solutions were tested for residual activity against S. aureus using a standardized disk diffusion method. Mediums with untreated antibiotic solutions and S. aureus were used as control. Plates were incubated at 37°C, at which time zones of inhibition (ZoI) were measured to the nearest whole millimeter for 14 days. The investigation indicated that the temperature elevation impacted considerably on antimicrobial activity and antibiotic stability overall. The in vitro temperature-response curves showed that ZoI diameter decreases logarithmically with elevated temperatures. Gentamicin was the only drug that was found to be affected to some extent. Results from the study provides a valuable dataset for orthopaedic surgeons considering local application of antibiotics and methods of antibiotic impregnation