Low back pain resulting from Interertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect. However, their behaviour in the harsh degenerate environment is unknown. Porcine NC cells (pNCs), and Human NP cells from degenerate IVDs were cultured in alginate beads to maintain phenotype. Cells were cultured alone or in combination, or co-stimulated with notochordal cell condition media (NCCM), in media to mimic the healthy and degenerate disc environment, together with controls for up to 1 week. Following culture viability, qPCR and proteomic analysis using Digiwest was performed. A small increase in pNC cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in pNCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Human NP cells cultured with NCCM, showed a decrease in IL-8 production compared to human NP cells alone when cultured in healthy media. However, gene expression analysis (ACAN, VEGF, MMP3 and IL-1β) demonstrated no significant difference between NP only and NP+NCCM groups. Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The potential use of NC cell sources for regenerative therapies can then be translated to investigate the potential use of iPSCs differentiated into NC cells as a regenerative cell source.
Low back pain is strongly associated with degeneration of the intervertebral disc (IVD). During degeneration, altered matrix synthesis and increased matrix degradation, together with accompanied cell loss is seen particularly in the nucleus pulposus (NP). It has been proposed that notochordal (NC) cells, embryonic precursors for the cells within the NP, could be utilized for mediating IVD regeneration. However, injectable biomaterials are likely to be required to support their phenotype and viability within the degenerate IVD. Therefore, viability and phenotype of NC cells were analysed and compared within biomaterial carriers subjected to physiological oxygen conditions over a four-week period were investigated. Porcine NC cells were incorporated into three injectable hydrogels: NPgel (a L-pNIPAM-co-DMAc hydrogel), NPgel with decellularized NC-matrix powder (dNCM) and Albugel (an albumin/ hyaluronan hydrogel). The NCs and biomaterials constructs were cultured for up to four weeks under 5% oxygen (n=3 biological repeats). Histological, immunohistochemical and glycosaminoglycans (GAG) analysis were performed to investigate NC viability, phenotype and extracellular matrix synthesis and deposition. Histological analysis revealed that NCs survive in the biomaterials after four weeks and maintained cell clustering in NPgel, Albugel and dNCM/NPgel with maintenance of morphology and low caspase 3 staining. NPgel and Albugel maintained NC cell markers (brachyury and cytokeratin 8/18/19) and extracellular matrix (collagen type II and aggrecan). Whilst Brachyury and Cytokeratin were decreased in dNCM/NPgel biomaterials, Aggrecan and Collagen type II was seen in acellular and NC containing dNCM/NPgel materials. NC containing constructs excreted more GAGs over the four weeks than the acellular controls. NC cells maintain their phenotype and characteristic features in vitro when encapsulated into biomaterials. NC cells and biomaterial construct could potentially become a therapy to treat and regenerate the IVD.
Low back pain resulting from Intervertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect, however, their behaviour in the harsh degenerate environment is unknown. Thus, we aimed to investigate and compare their physiological behaviour in Porcine NC cells were encapsulated in 3D alginate beads to maintain their phenotype then cultured in media to mimic the healthy and degenerate disc environment, together with control NC media for 1 week. Following which viability using PI and Calcein AM, RNA extraction and RT-PCR for NC cell markers, anabolic and catabolic genes analysed. Proteomic analysis was also performed using Digiwest technology.Backgrounds and aim
Methodology
Low back pain is strongly associated with degeneration of the intervertebral disc (IVD). During degeneration, altered matrix synthesis and increased matrix degradation, together with accompanied cell loss is seen particularly in the nucleus pulposus (NP). It has been proposed that notochordal (NC) cells, embryonic precursors for the cells within the NP, could be utilized for mediating IVD regeneration. However, injectable biomaterials are likely to be required to support their phenotype and viability within the degenerate IVD. Therefore, viability and phenotype of NC cells were analysed and compared within biomaterial carriers subjected to physiological oxygen conditions over a four-week period were investigated. Porcine NC cells were incorporated into three injectable hydrogels: NPgel (a L-pNIPAM-co-DMAc hydrogel), NPgel with decellularized NC-matrix powder (dNCM) and Albugel (an albumin/ hyaluronan hydrogel). The NCs and biomaterials constructs were cultured for up to four weeks under 5% oxygen (n=3 biological repeats). Histological, immunohistochemical and glycosaminoglycans (GAG) analysis were performed to investigate NC viability, phenotype and extracellular matrix synthesis and deposition.Objectives
Methodology
Intervertebral disc (IVD) degeneration accompanying with low back pain is a serious worldwide problem. Even though, surgical treatments are available for pain relief, there is an urgent need to establish enduring cell-based remedies. Notochordal (NC) cells as the ancestor of nucleus pulposus (NP) cells in human IVD are a promising therapeutic target. It has been reported that the loss of NC cells after childhood could promote the onset of disc degeneration. Thus, we firstly, aimed to optimise the culture of NC cells in vitro without using the FCS in alginate (3D) culture systems, secondly, investigate their behaviour in healthy and degenerate niche and lastly, co-culture these cells with degenerated NP cells to assess their regeneration potentials. Porcine NC cells were extracted using pronase treatment followed by overnight digestion in 0.01% collagenase II. After extraction, cells were culture in 1.2% alginate beads (gold standard 3D culture) in either low glucose DMEM or αMEM medium. Cells were harvested after 24 hours, 1 week and 2 weeks for gene expression analysis and formalin fixed paraffin embedding. Quantitative Real-Time PCR and Immuno-staining were performed for analysis of NC markers (KRT18, FOXA2 and T) and COL I as a negative marker. Next, NC cells were cultured in healthy and degenerate medium to assess their viability and behaviour.Introduction and Objective
Materials and Methods
The advent of EOS imaging has offered clinicians the opportunity to image the whole skeleton in the anatomical standing position with a smaller radiation dose than standard spine roentgenograms. It is known as the fifth modality of imaging. Current NICE guidelines do not recommend EOS scans over x-rays citing: “The evidence indicated insufficient patient benefit in terms of radiation dose reduction and increased throughput to justify its cost”. We retrospectively reviewed 103 adult and 103 paediatric EOS scans of standing whole spines including shoulders and pelvis for those undergoing investigation for spinal deformity in a tertiary spinal centre in the UK. We matched this against a retrospective control group of 103 adults and 103 children who underwent traditional roentgenograms whole spine imaging at the same centre during the same timeframe. We aimed to compare the average radiation dose of AP and lateral images between the two modalities. We utilised a validated lifetime risk of cancer calculator (Background
Methods
Heterotopic ossification is the formation of lamellar bone in soft tissues and is a common complication of high-energy combat injury. This disabling condition can cause pain, joint ankylosis, and skin ulceration in the residua of amputees. This project is aimed at developing a novel treatment to dissolve hydroxyapatite in heterotopic ossification and prevent the crystallisation of this this mineral at sites of ectopic bone formation. Previously reported results demonstrated that hexametaphosphate could dissolve hydroxyapatite at physiological pH. Further work has been undertaken to investigate the mechanism of this dissolution and establish a means of temporal control of action. In addition, physicochemical analyses of samples of human heterotopic ossification have yielded important insights into the nature of this pathological tissue. Techniques include mapped micro X-ray fluorescence, mapped Raman spectroscopy, scanning electron microscopy, and micro computed tomography. Formulation engineering work has begun in order to develop an appropriate delivery vehicle for this agent. This includes rheological testing and hexametaphosphate elution profiles. Finally, micro CT analysis has shown that hexametaphosphate is able to dissolve human heterotopic ossification tissue. In summary, this work has moved us closer towards our goal of a novel injectable agent for the treatment and prevention of heterotopic ossification.
Bone is a hierarchically structured hard tissue that consists of approximately 70 wt% low-crystallinity hydroxyapatite. Intricate tubular channels, such as Haversian canals, Volkman's canals, and canaliculi are a preserved feature of bone microstructure. These structures provide pathways for vasculature and facilitate cell-to-cell communication processes, together supporting viability of cellular components and aiding in remodeling processes. Unfortunately, many commercial bone augmentation materials consist of highly crystalline phases that are absent of the structuring present within the native tissue they are replacing. This work reports on a the development of a novel bone augmentation material that is able to generate biologically analogous tubular calcium phosphate mineral structures from hydrogel-based spheres that can be packed into defects similar to those encountered in vivo. Calcium loaded spheres were made by adding 5 wt% agar powder to 1 M calcium nitrate solutions, before heating the mixture to 80–90 oC and feeding droplets of gel into a reservoir of liquid nitrogen. Deposition of tubular mineral was initiated by exposure to ammonium phosphate solutions at concentrations between 500 mM and 1 M, and was characterized by micro-XRF mapping, XRD and SEM techniques. For an ex vivo model, human bone tissue was collected from patients undergoing elective knee replacement surgery. The United Kingdom National Research Ethics Service (East of Scotland Research Ethics Service) provided ethical approval (11/ES/1044). The augmented defect of the model was characterised by micro-XRF mapping and micro-CT techniques.Background
Experimental
We reproduced a frequently cited study performed at our University Hospital that was published in the British Medical Journal in 1981 assessing the extent of “snow and ice” fractures during the winter period. As per the original study, four days of snow and ice were identified as well as two control periods when snow and ice wasn't recorded; four days within the same year, with a similar amount of sunshine hours, and four days one calendar year later. The distribution of fractures according to age and sex in addition to the anatomical location were examined in relation to the presence of snow and ice as well as comparisons with the index study 33 years ago.Background
Methods
Calcium orthophosphates, such as hydroxyapatite (Ca5(PO4)3OH) (HA), have long been employed as bone graft materials. Recent work has suggested that calcium pyrophosphate (Ca2P2O7) (CaPy) may strongly stimulate bone deposition. In this study we compare calcium orthophosphate and pyrophosphate precipitates as suitable bone regeneration materials. As well as HA, two forms of pyrophosphate precipitate were compared in this work: amorphous calcium pyrophosphate (amCaPy) and star particle calcium pyrophosphate (stCaPy). Briefly, 0.15M Na4P2O7·10H2O and 0.3M Ca2Cl·2H2O solutions of equivalent volume were combined and left to age before performing a series of filtration and re-suspension steps upon the precipitate. Drying yielded amCaPy powder. stAmPy was produced by the same procedure however the pH of the starting solutions were altered to pH7 before combination.Background
Methods
At first-stage revision surgery for infection of total knee arthroplasties, antibiotic-impregnated cement spacers are frequently implanted. Two types of cement spacers are commonly used, “static” and “articulating” cement spacers. Advocates of cement spacers state that they deliver high doses of antibiotics locally, increase patient comfort, allow mobility and provide joint stability. They also minimize contracture of collateral ligaments, thereby facilitating re-implantation of a definitive prosthesis at a later stage. The use of these cement spacers, however, are not without significant complications, including patella tendon injuries. We describe a series of three patients who sustained patella tendon injuries in infected total knee arthroplasties following the use of a static cement spacer at first-stage knee revision. The patella tendon injuries resulted in significant compromise to wound healing and knee stability requiring multiple surgeries. The mid-term function was poor with an Oxford score at 24 months ranging from 12–20 Based on our experience, we advise caution in the use of static cement spacer blocks. If they are to be used, we recommend that they should be keyed in the bone to prevent patella tendon injuries.
Honey has been used as a topical antiseptic for at least 5,000 years. SurgiHoney is a CE licensed sterile product, which has been proven to be non-toxic and effective when used topically in the treatment of chronically infected wounds. The key difference from other medical grade honey is the broad spectrum antimicrobial characteristics with activity against Gram +ve, Gram –ve and multi-resistant organisms. Its novel role against the bacterial bioburden and biofilm associated with periprosthetic infections around total knee arthroplasties (TKA's) is therefore considered. SurgiHoney was used as an implant coating immediately prior to wound closure after implantation of salvage endoprosthesis for multiply revised, infected TKA's undergoing staged reconstruction. We report a consecutive series of multi-revised, infected revision TKA's where SurgiHoney was used as an active antimicrobial coating. We discuss its intra-operative application and early clinical outcomes. The use of Surgihoney as a novel anti-microbial is established in the management of complex wound infections. This is the first reported use of SurgiHoney as a deep, implant coating in the salvage of prosthetic joint infection.
Joint degeneration may make a total knee arthroplasty (TKA) a requirement for pain relief and function. However, the presence of ipsilateral limb osteomyelitis (OM) makes surgical management extremely challenging. We report the experience of a high volume revision knee surgeon managing ipsilateral limb multi resistant OM and the outcome of subsequent TKA. Four consecutive patients were identified who had either ipsilateral femoral or tibial chronic osteomyelitis treated prior to undergoing TKA. Surgery to eradicate the osteomyelitis involved a Lautenbach compartmental debridement, and where necessary, healing by secondary intention. The decision to proceed to a TKA was based on history, clinical examination and radiological findings of advanced osteoarthritic change. The patients had a mean age of 50 years. They had a background of multi-organism OM and underwent single-stage TKAs at an average of 63 months following eradication of the underlying OM. Three patients did well but had complications associated with poor skin and soft tissues, and abnormal bone anatomy. One patient developed an infection and following a re-revision had an arthrodesis. The results for the four cases are summarised in Table 1. We have highlighted that patients with ipsilateral limb multi resistant OM are a difficult cohort to manage.
A common step to revision surgery for infected total knee replacement (TKR) is a thorough debridement. Whilst surgical and mechanical debridement are established as the gold standard, we investigate a novel adjuvant chemical debridement using an Acetic Acid (AA) soak that seeks to create a hostile environment for organisms, further degradation of biofilm and death of the bacteria. We report the first orthopaedic in vivo series using AA soak as an intra-operative chemical debridement agent for treating infected TKR's. We also investigate the in vitro efficacy of AA against bacteria isolated from infected TKR's. A prospective single surgeon consecutive series of patients with infected TKR were treated according to a standard debridement protocol. Patients in the series received sequential debridement of surgical, mechanical and finally chemical debridement with a 10 minute 3% AA soak. In parallel, we isolated, cultured and identified bacteria from infected TKR's and assessed the in vitro efficacy of AA. Susceptibility testing was performed with AA solutions of different concentrations as well as with a control of a gentamicin sulphate disc. The effect of AA on the pH of tryptone soya was also monitored in an attempt to understand its potential mechanism of action. Physiological responses during the AA soak were unremarkable. Intraoperatively, there were no tachycardic or arrythmic responses, any increase in respiratory rate or changes in blood pressure. This was also the case when the tourniquet was released. In addition, during the post-operative period no increase in analgesic requirements or wound complications was noted. Wound and soft tissue healing was excellent and there have not been any early recurrent infections at mean of 18 months follow up. In vitro, zones of inhibition were formed on less than 40% of the organisms, demonstrating that AA was not directly bactericidal against the majority of the clinical isolates. However, when cultured in a bacterial suspension, AA completely inhibited the growth of the isolates at concentrations as low as 0.19%v/v. This study has shown that the use of 3% AA soak, as part of a debridement protocol, is safe. Whilst the exact mechanism of action of acetic acid is yet to be determined, we have demonstrated that concentrations as low as 0.19%v/v in solution in vitro is sufficient to completely inhibit bacterial growth from infected TKR's.
Calcium phosphate (CaP) particles have attracted great interest as transfection reagents, yet little is known about their mechanism of internalisation. We report live cell time-course tracking of CaP particles during internalisation and the influence of Ca:P ratio on transfection efficiency. Relatively recent work has seen calcium phosphate (CaP) salts used for the delivery of biological materials into cells in the form of peptides, polymers and DNA sequences. Calcium phosphate salts have a critical safety advantage over other vectors such as viruses in that they pose no risk of pathogenicity due to mutation and show no apparent cytotoxicity. Previous work within the group showed that Ca:P ratio influenced the transfection efficiency, but the fate of the particles on internalisation is yet unknown. The difficulty in tracking the particles can be related to the visual similarity to granulation within the cells. Using a surface modification method that enables the fluorescent labeling of silicon-substituted hydroxyapatite (SiHA) particles, we have tracked the internalisation of the particles to understand their mechanism of entry and how particle composition may influence transfection efficiency.Summary Statement
Introduction
Total Knee Arthroplasty (TKA) aims to deliver relief from pain and restore normal function. Unfortunately, a significant cohort of patients report poor outcomes. Synovial fluid metabolite concentrations at surgery predict outcome of TKA, assessed by a validated measure.Introduction
Hypothesis
Osteoarthritis (OA) represents a leading cause of disability and a growing burden on healthcare budgets. OA is particularly vexing for young, active patients who have failed less invasive therapies but are not yet candidates for arthroplasty. Often, patients suffering in this wide therapeutic gap face a debilitating spiral of disease progression, increasing pain, and decreasing activity until they become suitable arthroplasty patients. An implantable load absorber was evaluated for the treatment of medial knee OA in this patient population. Joint overload has been cited as a contributor to OA onset or progression. In response, the KineSpring® System (Moximed, Inc, USA) has been designed to reduce the load acting on the knee. The absorber is implanted in the subcutaneous tissue without violating the joint capsule, thus preserving the option of future arthroplasty. The implant is particularly useful for young, active patients, given the reversibility of the procedure and the preservation of normal flexibility and range of motion. The KineSpring System was implanted in 55 patients, with the longest duration exceeding two years. The treated group had medial knee OA, included younger OA sufferers (range 31–68 years), with a mean BMI > 30kg/m2. Acute implant success, adverse events, and clinical outcomes using validated patient reported outcomes tools were recorded at baseline, post-op, 2 and 6 weeks, and 3, 6, 12 and 24 months post-op. All patients were successfully implanted with a mean procedure time of 76.4 min (range 54–153 minutes). Mean hospital length of stay was 1.7 days (range 1–3 days), and patients recovered rapidly, achieving full weight bearing within 1–2 wks and normal range of motion by 6 weeks. Most patients experienced pain relief and functional improvement with 85% (35/41) reporting none or mild pain on the WOMAC pain subscale and 90% (37/41) reporting functional impairment as none on mild on the WOMAC function subscale at the latest follow-up visit (mean 9.3 ± 3.5 months). Clinically meaningful and statistically significant pain reduction and functional improvement were noted with baseline WOMAC pain scores (0–100 scale) improving from 42.4 to 16.1 (p<0.001) and WOMAC function (0–100 scale) improving from 42.0 to 14.7 (p<0.001) at latest follow-up. Patients reported satisfaction with the implant and its appearance.Introduction
Methods and Results
Unique progenitor cells have been identified recently and successfully cultured in vitro from human articular cartilage. These cells are able to maintain chondrogenic potential upon extensive expansion. In this study, we have developed a sheep, ex-vivo model of cartilage damage and repair, using these progenitor cells. This study addresses the question can such a model be used to determine factors required for progenitor cell proliferation, differentiation and integration of matrix onto bone. The hypothesis was that sheep allogenic cartilage derived progenitor cells could regenerate artificially damaged sheep articular cartilage in an osteochondral culture model. Progenitor cells were derived from ovine articular cartilage using a differential adhesion assay to fibronectin and expanded clonally. These clonal cells were marked with lentiviral vectors derived from the Human Immunodeficiency Virus-1. When a self-inactivating lentiviral vector encoding a ubiquitous phosphoglycerate kinase promoter, driving a Green Fluorescent Protein (GFP) reporter gene, was used to transduce these cells, up to 80% of these progenitor cells expressed GFP. Normal sheep medial femoral condyles containing about 2mm thick sub-condral bone were obtained and 4mm circular defects created on the cartilage surface using a biopsy punch. Condyles were cultured for two weeks in vitro with GFP labelled progenitor cells within a fibrin glue scaffold (Tisseel Lyo) and matrix production (collagen) as determined by spatially offset Raman spectroscopy and immunohistochemistry was demonstrated. Progenitor cells were able to proliferate and differentiate into collagen producing cells. Such an ex-vivo model system is an effective tool for the analysis of cartilage repair from various sources of stem cells. These ex-vivo experiments and variations on defect type, size, titration of scaffold and progenitor cell numbers requirements can further be used as a basis for screening prior to in vivo experiments.
High tibial Osteotomy (HTO) realigns the forces in the knee to slow the progression of osteoarthritis. This study relates the changes in knee joint biomechanics during level gait to glutamate signalling in the subchondral bone of patients pre and post HTO. Glutamate transmits mechanical signals in bone and activates glutamate receptors to influence inflammation, degeneration and nociception in arthritic joints. Thus glutamate signalling is a mechanism whereby mechanical load can directly modulate joint pathology and pain. 3D motion analysis was used to assess level gait prior to HTO (n=5) and postoperatively (n=2). A biomechanical model of each subject was created in Visual3D (C-motion. Inc) and used for biomechanical analysis. Gene expression was analysed by RT-PCR from bone cores from anterior and posterior drill holes, subdivided according to medial or lateral proximal tibia from HTO patients (n=5).BACKGROUND
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.
