Objectives. Osteophytes are products of active endochondral and intramembranous ossification, and therefore could theoretically provide significant efficacy as bone grafts. In this study, we compared the bone mineralisation effectiveness of osteophytes and cancellous bone, including their effects on secretion of growth factors and anabolic effects on osteoblasts. Methods. Osteophytes and cancellous bone obtained from human patients were transplanted onto the calvaria of severe combined immunodeficient mice, with Calcein administered intra-peritoneally for fluorescent labelling of bone mineralisation. Conditioned media were prepared using osteophytes and cancellous bone, and growth factor concentration and effects of each graft on proliferation, differentiation and migration of osteoblastic cells were assessed using enzyme-linked immunosorbent assays, MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assays, quantitative real-time polymerase chain reaction, and migration assays. Results. After six weeks, the area of mineralisation was significantly higher for the transplanted osteophytes than for the cancellous bone (43803 μm. 2. , . sd. 14660 versus 9421 μm. 2. , . sd. 5032, p = 0.0184, one-way analysis of variance). Compared with cancellous bone, the conditioned medium prepared using osteophytes contained a significantly higher amounts of transforming growth factor (TGF)-β1 (471 pg/ml versus 333 pg/ml, p = 0.0001, Wilcoxon rank sum test), bone morphogenetic protein (BMP)-2 (47.75 pg/ml versus 32 pg/ml, p = 0.0214, Wilcoxon rank sum test) and insulin-like growth factor (IGF)-1 (314.5 pg/ml versus 191 pg/ml, p = 0.0418, Wilcoxon rank sum test). The stronger effects of osteophytes towards osteoblasts in terms of a higher proliferation rate, upregulation of gene expression of differentiation markers such as alpha-1 type-1 collagen and alkaline phosphate, and higher migration, compared with cancellous bone, was confirmed. Conclusion. We provide evidence of favourable features of osteophytes for bone mineralisation through a direct effect on osteoblasts. The acceleration in metabolic activity of the osteophyte provides justification for future studies evaluating the clinical use of osteophytes as autologous bone grafts. Cite this article: K. Ishihara, K. Okazaki, T. Akiyama, Y. Akasaki, Y. Nakashima.
Ceramic-on-ceramic (CoC) total hip arthroplasty (THA) can produce articular noise during the normal activities, generating discomfort to the patient. THA noise has to be investigated also as a potential predictor and a clinical sign of prosthetic failure. An observational study has been carried out to characterize the noise in CoC cementless THA, and to analyze the related factors. A total of 46 patients with noisy hip have been enrolled in 38 months, within the follow-up protocol normally applied for the early diagnosis of ceramic liner fracture [1]. Noise recording was based on a high-quality audible recorder (mod. LS 3, Olympus, Japan) and a portable ultrasonic transducer (mod USB AE 1ch, PAC, USA). The sensors for noise recording were applied to the hip of the patient during a sequence of repeatable motorial activities (forward and backward walking, squat, sit in a chair, flexion and extension of the leg). Sessions were also video-recorded to associate the noise emission to the specific movements. Each noise event was initially identified by the operator and therefore classified by comparison to the spectral characteristics (duration, intensity and frequency) of the main noise types. Number and spectral characteristics of noise events were obtained and correlated to the factors describing the clinical status of the patient, the surgical approach, the prosthetic device implanted. The study investigated also the noise as a sign of implant failure, by comparison with the total number of implants failed in the cohort during the study. We observed three types of noise with the main spectral characteristics in agreement to the literature: clicking, squeaking and popping. Among the identified types of noise, squeaking showed the longest duration and the highest amplitude. The 63% of hip presented the emission of just one type of noise, while the remaining a mix of types. The movement with the highest presence of noise was walking, followed by squat. Correlation was found between the noise type and the dimension of the ceramic head (p<0.001), with the sizes of 32 mm more affected by squeaking that the smaller one. Squeaking appeared before during the follow-up than the other types of noise. The 35% (16/46) of the noisy hips were revised during the study. Among the revised hips, the 81% (13/16) were affected by impingement and/or severe damage of the prosthetic components. The antiversion of the cup (p=0.008), the presence of debris in the synovial fluid (p=0.021) and the average frequency of squeaking (p=0.006) were significant predictors for the revision, but it has to be mentioned that the squeaking data was obtained on a small subset of revised patients. Ultrasonic analysis did not show significant correlations. The study presented and validated an experimental procedure to analyze noisy hips in clinical trials. Noise is confirmed to be a significant parameter in the follow-up evaluation of ceramic THA.
Periprosthetic osteolysis is the most common long-term complication of a total joint arthroplasty, often resulting in aseptic loosening of the implant. As we aim at developing a safe and minimally invasive implant refixation procedure, thorough characterisation of the properties of the periprosthetic tissue is needed. In this pilot study, the periprosthetic tissue of eleven patients undergoing hip revision surgery due to aseptic loosening was obtained. Histology, confocal microscopy, atomic force microscopy (AFM) and nanoindentation were performed to structurally and mechanically characterise the tissue. The study was approved by the Medical Ethical Committee of the Leiden University Medical Center.Background
Methods
Injury to the syndesmosis occurs in 10–13% of all operative ankle fractures and there is evidence that both incomplete treatment and malreduction of the syndesmosis can lead to poor clinical outcomes. Much attention has been given to post–operative malreduction documented by computer tomography (CT), however, there is limited data about the intact positioning and relative motion of the native syndesmosis. The aim of this study is to elucidate more detailed information on the position of the fibula in the syndesmosis during simulated weight–bearing in intact state, with sequential ligament sectioning and following two reconstructive techniques. Fourteen paired, fresh–frozen human cadaveric limbs were mounted in a weight–bearing simulation jig. CT scans were obtained under simulated foot–flat loading (75 N) and in single–legged stance (700 N), in five foot positions: neutral, 15° external rotation, 15° internal rotation, 20° dorsiflexion, and 20° plantarflexion. The elements of the syndesmosis and the deltoid ligament were sequentially sectioned. One limb of each pair was then reconstructed via one of two methods: Achilles autograft and peroneus longus ligamentoplasty. The specimens were rescanned in all 5 foot positions following each ligament resection and reconstruction. Measurements of fibular diastasis, rotation and anterior–posterior translation were performed on the axial cuts of the CT scans, 1 cm proximal to the roof of the plafond. Multiple measurements were made to define the position of the fibula in the incisura. Clinically relevant deformity patterns were produced. The deformity at the incisura was consistent with clinical injury, and the degree of displacement in all ligament states was dependent on the foot position. The most destructive state resulted in the most deformity at the syndesmosis. Differences between the intact and reconstructed states were found with all measurements, especially when the foot was in external rotation and dorsiflexion. There was no significant difference with direct comparison of the reconstructions. This study has detailed the motion of the fibula in the incisura and its variation with foot position. Neither reconstruction was clearly superior and both techniques had difficulty in the externally rotated and dorsiflexed foot positions. This study design can serve as a model for future ex–vivo testing of reconstructive techniques.
Tendon cells originate from yet poorly described precursor cells and develop in a particular “niche” close to vascular walls. Several factors have been described to determine this niche such as mechanical stimuli, oxygen tension, composition and structure of the extracellular matrix (ECM). Also, the vasculature is considered to play a crucial role for tendon cell development, yet evidence of how this is accomplished is lacking. In this study we therefore focussed on the endothelium of tendon vessels postulating the existence of a paracellular barrier. By electron microscopy, immunohistochemistry, and RT-PCR we investigated the presence of constituents making up such an endothelial barrier which we subsequently tested for its functionality by tracer injection. Moreover, we performed differentiation experiments into the adipogenic, chondrogenic and osteogenic lineage on tendon derived cells in the presence and absence of serum. Expression levels and activity of matrixmetalloproteinases (MMPs) were assessed by western blot and zymography.Introduction
Materials and Methods
Hyaline cartilage defects are a significant clinical problem for which a plethora of cartilage repair techniques are used. One such technique is cartilage replacement therapy using autologous chondrocyte or mesenchymal stem cell (MSC) implantation (ACI). Mesenchymal stem cells are increasingly being used for these types of repair technique because they are relatively easy to obtain and can be expanded to generate millions of cells. However, implanted MSCs can terminally differentiate and produce osteogenic tissue which is highly undesirable, also, MSCs generally only produce fibrocartilage which does not make biomechanically resilient repair tissue, an attribute that is crucial in high weight-bearing areas. Tissue-specific adult stem cells would be ideal candidates to fill the void, and as we have shown previously in animal model systems [Dowthwaite et al, 2004, J Cell Sci 117;889], they can be expanded to generate hundreds of millions of cells, produce hyaline cartilage and they have a restricted differential potential. Articular chondroprogenitors do not readily terminally differentiate down the osteogenic lineage. At present, research focused on isolating tissue-specific stem cells from articular cartilage has met with modest success. Our results demonstrate that using differential adhesion it is possible to easily isolate articular cartilage progenitor populations from human hyaline cartilage and that these cells can be subsequently expanded in vitro to a high population doubling whilst maintaining a normal karyotype. Articular cartilage progenitors maintain telomerase activity and telomere length that are a characteristic of progenitor/stem cells and differentiate to produce hyaline cartilage. In conclusion, we propose the identification and characterisation of a novel articular cartilage progenitor population, resident in human cartilage, which will greatly benefit future cell-based cartilage repair therapies.
Aim of the study was to evaluate if abrasion-arthroplasty (AAP) and abrasion-chondroplasty (ACP) leads to a release of mesenchymal stem cell (MSC) like cells from the bone marrow to the joint cavity where they probably differentiate into a chondrogenic phenotype. Cartilage demage is a sever problem in our aging society. About 5 million people only in Germany are affected. Osteoathritis is a degeneration of cartilage caused by aging or traumata 50 % of the people over 40 have signs of osteoarthritis. But the ability of self-regeneration of cartilage is strongly limited. There are different approaches to therapy osteoathritic lesions. Arthroscopic treatment of OA includes bone marrow stimulation technique such as abrasion arthroplasty (AAP) and microfracturing (MF). Beside the support of chondrocyte progenitor cells the environment is also important for the commitment to chondrocytes. Therefore insulin-like growth factor-1 (IGF-1) and transforming growth factor beta-1 (TGF-β1) are important factors during the regeneration process. In the present study we characterised the heamarthrosis and the released cells after AAP and its ability to differentiate into the chondrocyte lineage. Postoperative haemarthrosis was taken 5, 22 or 44 hours after surgery. 7.5 mg Dexamethasone (Corticosteroid) was administered into the knee joint to prevent postoperative inflammation. Mononuclear cells were isolated from haemarthrosis from the drainage bottle by ficoll density gradient centrifugation. The isolated cells were characterised using fluorescence-activated cell-sorting (FACS) analysis for characteristic markers of MSC such as CD 44, 73, 90, 105. After expanding cells were cultured in a pellet culture. After 3 weeks, histochemistry and immunohistochemistry against Sox9, collagen II and proteoglycan were performed. The release of IGF1, BMP4 and BMP7 was analysed in haemarthrosis serum by ELISA and Luminex technology.Introduction
Material and Methods
Injectable scaffolds which also deliver cells and bioactive molecules to augment bone healing overcome many of the limitations associated with current bone graft substitutes. The aim of this study was to develop and test a novel injectable scaffold that self-assembles isothermically in situ to form a biodegradable porous osteoconductive material, and to assess the viability of human mesenchymal stem cells (hMSC) seeded onto the scaffold. Rheological assessment was performed on three different molecular weights (Mw) of poly(lactic-co-glycolic acid) (PLGA) (26kDa, 53kDa and 92kDa) combined with differing ratios of polyethylene glycol (PEG) to control the temperature required for scaffold self-assembly. The strength (MPa) and stiffness (Young's Modulus) patterns of the scaffolds were assessed in compression. The cell viability, proliferation and distribution patterns of hMSCs seeded within the scaffold were assessed through various assays (Alamar Blue), confocal microscopy and micro-CT. The hMSC differentiation in osteogenic media was characterised by the identification of specific bone formation markers (e.g. alkaline phosphatase).Background/Study Aim
Methods
Metal and their alloys have been widely used as implantable materials and prostheses in orthopaedic surgery. However, concerns exist as the metal nanoparticles released from wear of the prostheses cause clinical complications and in some cases result in catastrophic host tissue responses. The mechanism of nanotoxicity and cellular responses to wear metal nanoparticles are largely unknown. The aim of this study was to characterise macrophage phagocytosed cobalt/chromium metal nanoparticles both in vitro and in vivo, and investigate the consequent cytotoxicity. Two types of macrophage cell lines, murine RAW246.7 and human THP-1s were used for in vitro study, and tissues retrieved from pseudotumour patients caused by metal-on-metal hip resurfacing (MoMHR) were used for ex vivo observation. Transmission electron microscopy (TEM), scanning electron microscopy (SEM) in combination with backscatter, energy-disperse X-ray spectrometer (EDS), focused ion beam (FIB) were employed to characterise phagocytosed metal nanoparticles. Alamar blue assay, cell viability assays in addition to confocal microscopy in combination with imaging analysis were employed to study the cytotoxiticy in vitro. The results showed that macrophages phagocytosed cobalt and chromium nanoparticles in vitro and the phagocytosed metal particles were confirmed by backscatter SEM+EDS and FIB+EDS. these particles were toxic to macrophages at a dose dependent manner. The analysis of retrieved tissue from revision of MoMHR showed that cobalt/chromium metal nanoparticles were observed exclusively in living macrophages and fragments of dead macrophages, but they were not seen within either live or dead fibroblasts. Dead fibroblasts were associated with dead and disintegrated macrophages and were not directly in contact with metal particles; chromium but not cobalt was the predominant component remaining in tissue. We conclude that as an important type of innate immune cells and phagocytes, macrophages play a key role in metal nanoparticles related cytotoxicity. Metal nanoparticles are taken up mainly by macrophages. They corrode in an acidic environment of the phagosomes. Cobalt that is more soluble than chromium may release inside macrophages to cause death of individual nanoparticle-overloaded macrophages. It is then released into the local environment and results in death of fibroblasts and is subsequently leached from the tissue.
Metal Injection Molding could provide cost saving of about 20–50% for implantable medical device manufacturing and hence healthcare public spending. Corrosion behaviour and biocompatibility of the new manufactured alloy were studied and showed similar behaviour compared to the traditional one. The growing trend for total joint arthroplasties could raise healthcare costs in the near future. Metal Injection Molding (MIM) is a near net shape manufacturing technology and allows the production of finite prosthesis components saving the machining step, and so resources, up to 20–50%. In order to apply such process to the production of actual devices, the bulk material have to show biocompatibility and corrosion behaviour similar to the traditional one. (ASTM F2083, ISO 21536) The aim of this work was to compare cast and forged CoCrMo alloy with the MIM one from the electrochemical point of view and cytocompatibility.Summary
Introduction
The chemistry, amount, morphology, and size distribution of wear debris from silicon nitride coatings generated in the bearing surface can potentially reduce the negative biological response and increase the longevity compared to conventional materials in joint replacements. Total hip implants have a high success rate at 15 years of implantation, but few survive over 25 years. At present, revisions are mostly due to aseptic loosening, believed to mainly be caused by the biological response to wear debris generated in the joint bearing. For the polymer liners the size of the wear debris determines the biological response, while for metal bearing surfaces a limitation is the metal ion release. When ceramics are used, the wear debris is in general small and mechanical factors may be the main cause for failure. A more recent, experimental alternative is to let the well-known metallic substrate serve as the soft, tough bulk, and additionally apply a hard and smooth ceramic coating. In this way a lower wear rate and reduced metal ion release could be obtained. Furthermore, the chosen composition, silicon nitride (SixNy), contains no detrimental ions, and silicon nitride debris has been shown to slowly dissolve in aqueous medium. Altogether, it can potentially increase the longevity of the implant. However, the debris from SixNy coatings has not yet been characterised. In this study, a wear model test was performed to generate wear debris from SixNy coatings. The debris was characterised using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) in combination with computational calculations.Summary Statement
Introduction
Mesenchymal stem cells from human semitendinosus and gracilis tendons (TSPCs) could be a promising MSCs resource for tissue-engineering application. In comparison to adipose-derived stem cells, TSPCs possess similar stem-cells properties and a higher chondrogenic differentiation potential. Mesenchymal stem cells (MSCs) isolated from bone marrow (BMSCs) or adipose tissue (ASCs) have been deeply characterised for their usefulness in musculoskeletal tissue regeneration. However, other potentially valuable MSCs sources have been recently proposed. The goal of this study was to isolate MSCs from human semitendinosus and gracilis tendons (TSPCs, tendon stem progenitor cells) and to compare their features with that of human ASCs.Summary
Introduction
To determine the reliability, reproducibility, variability and validity of the Osteoarthritis Research Society International (OARSI) Osteoarthritis Cartilage Histopathology (OACH) system and Mankin Histopathology – Histochemical Grading System (HHGS) when applied to the characterisation of the osteoarthritic human knee. Kellgren-Lawrence and Line Drawing Atlas (LDA) radiology scores clinically graded the knees of ten patients undergoing total knee arthroplasty due to osteoarthritis. The tibial plateaux were scored using the Modified Collins (MC) and Société Française d'Arthroscopie (SFA). Three observers, twice scored, using both the OACH and HHGS systems across a single complete medial and lateral tibial plateau transect taken to include the region with the most severe OA lesion. Intra and inter-observer reliability, reproducibility, variability and validity were quantified, and the correlation between the two histopathology scoring systems was calculated.Objective
Method
One of the most common pathogen causing musculoskeletal infections is Summary
Introduction
Intra-articular screw fixation is indicated for internal fixation of large osteochondral fragments secondary to trauma or osteochondritis dissecans. During surgery, orthopaedic drills are used to prepare a hole through which the screw can pass. Previous work has shown that mechanical injury to articular cartilage results in a zone of cell death adjacent to the traumatised articular cartilage (1). Here, we characterise and quantify the margin of in situ chondrocyte death surrounding drill holes and screws (standard cortical and headless compression designs) placed in mature bovine articular cartilage to model the orthopaedic procedure. Drill holes (1mm) were made through the articular cartilage and bone of intact bovine metacarpophalangeal joints obtained from 3-yr old cows within 12hrs of slaughter. Osteochondral explants (∼1cm square and 2-3mm thick) encompassing the drilled holes in articular cartilage and subchondral bone were harvested using a chisel. Explants were then incubated in Dulbecco's modified Eagle's medium for 45mins with CMFDA (5-chloromethylfluorescein diacetate) and PI (propidium iodide; both at 10micromolar) to identify/quantify living and dead in situ chondrocytes respectively in a consecutive series of axial optical sections using confocal scanning laser microscopy (CLSM). The drill holes through cartilage appeared to have clearly defined edges with no macroscopic evidence of cartilage splitting. However visualisation of fluorescently-labelled in situ chondrocytes by CLSM demonstrated clear cell death around the periphery of the drilled hole which was 166±19 micrometers in width. This increased with a larger diameter (1.5mm) drill to 450±151 micrometers (all data are means±s.e.m.; n=3). Preliminary experiments indicated that the margin of chondrocyte death around a 1.5mm hole was dramatically increased further by the insertion of screws into pre-drilled holes. These results suggest that the mechanical trauma associated with cartilage drilling and the insertion of intra-articular screws occurs with marked death of in situ chondrocytes extending into normal cartilage beyond the area occupied by the screw. As chondrocytes are not replaced in mature cartilage, their loss around the hole/screw will mean that the extracellular matrix is not maintained, inevitably leading to cartilage failure.
Iliac crest bone marrow aspirate (ICBMA) is frequently cited as the ‘gold-standard’ source of MSCs. MSCs have been shown to reside within the intramedullary (IM) cavities of long-bones [Nelea, 2005] however a comparative assessment with ICBMA has not yet been performed and the phenotype of the latter compartment MSCs remains undefined in their native environment. Aspiration of the IM cavities of 6 patients' femurs with matched ICBMA was performed. The long-bone-fatty-bone-marrow (LBFBM) was filtered (70μm) to separate liquid and solid fractions and the solid fraction was briefly (60min, 37oC) digested with collagenase. MSC enumeration was performed using the colony-forming-unit-fibroblast (CFU-F) assay and quantification of cells with the CD45low CD271+ phenotype by flow-cytometry. [Jones 2002, Buhring 2007] MSCs were cultured and standard expansion media and passage 2 cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages.Introduction
Methods
Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis.INTRODUCTION
MATERIALS AND METHODS
Despite poly(vinyl alcohol) (PVA) hydrogel-based drug delivery systems have been extensively studied in the last years, so far there is no research investigating hydrogels in microspherical shape. In the present study, hydrogels for drug delivery systems were obtained from different formulations of poly(vinyl alcohol), poly(acrylic acid), ciprofloxacin and hydroxyapatite (Hap) aqueous solutions and shaped into spheres through dripping the solution into liquid nitrogen at extremely low temperatures. Hydrogels were then strengthened by freeze-thaw cycles.
The architecture within which cells reside is key to mediating their specific functions within the body. In this study, we use melt electrospinning writing (MEW), a recently developed 3D printing technology unique in its ability to generate ECM like fibres and control their deposition, to fabricate cell micro-environments with various fibrous architectures to study their effect on human stem cell behaviour. We designed, built and optimised a MEW apparatus and used it to fabricate four different platform designs of 10.4±2μm fibre diameter, with angles between fibres on adjacent layers of 90°, 45°, 10° and R (random).
Adipose-derived mesenchymal stem cells (ADMSCs) are a promising strategy for orthopaedic applications, particularly in bone repair. Human ADMSCs were cultured in medium supplemented with HPL, Hplasma and a combination of HPL and Hplasma (HPL+Hplasma). Characteristics of these ADMSCs, including osteogenesis, were evaluated in comparison with those cultured in fetal bovine serum (FBS).Objectives
Methods
