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Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_16 | Pages 54 - 54
17 Nov 2023
Bishop M Zaffagnini S Grassi A Fabbro GD Smyrl G Roberts S MacLeod A
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Abstract

Background

Distal femoral osteotomy is an established successful procedure which can delay the progression of arthritis and the need for knee arthroplasty. The surgery, however, is complex and lengthy and consequently it is generally the preserve of highly experienced specialists and thus not widely offered. Patient specific instrumentation is known to reduce procedural complexity, time, and surgeons’ anxiety levels1 in proximal tibial osteotomy procedures. This study evaluated a novel patient specific distal femoral osteotomy procedure (Orthoscape, Bath, UK) which aimed to use custom-made implants and instrumentation to provide a precision correction while also simplifying the procedure so that more surgeons would be comfortable offering the procedure.

Presenting problem

Three patients (n=3) with early-stage knee arthritis presented with valgus malalignment, the source of which was predominantly located within the distal femur, rather than intraarticular. Using conventional techniques and instrumentation, distal femoral knee osteotomy cases typically require 1.5–2 hours surgery time. The use of bi-planar osteotomy cuts have been shown to improve intraoperative stability as well as bone healing times2. This normally also increases surgical complexity; however, multiple cutting slots can be easily incorporated into patient specific instrumentation.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_9 | Pages 76 - 76
17 Apr 2023
Hulme C Roberts S Gallagher P Jermin P Wright K
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Stratification is required to ensure that only those patients likely to benefit, receive Autologous Chondrocyte Implantation (ACI); ideally by assessing a biomarker in the blood. This study aimed to assess differences in the plasma proteome of individuals who respond well or poorly to ACI.

Isobaric tag for relative and absolute quantitation (ITRAQ) mass spectrometry and label-free proteomics analyses were performed in tandem as described previously by our group (Hulme et al., 2017; 2018; 2021) using plasma collected from ACI responders (n=10) compared with non-responders (n=10) at each stage of surgery (Stage I, cartilage harvest and Stage II, cell implantation).

iTRAQ using pooled plasma detected 16 proteins that were differentially abundant at baseline in ACI responders compared with non-responders (n=10) (≥±2.0 fold; p<0.05). Responders demonstrated a mean Lysholm (patient reported functional score from 0–100) improvement of 33±13 and non-responders a mean worsening of −13±13 points. The most pronounced plasma proteome shift was seen in response to Stage I surgery in ACI non-responders, with 48 proteins being differentially abundant between the two surgical procedures. We have previously noted this marked shift in response to initial surgery in the SF of ACI non-responders, several of these proteins were associated with the Acute Phase Response. One of these proteins, clusterin, could be confirmed in patients’ plasma using an independent immunoassay using individual samples. Label-free proteomic data from individual samples identified only cartilage acidic protein-1 (known to associate with osteoarthritis progression) to be significantly more abundant at Stage I in the plasma of non-responders.

This study indicates that proteins can be identified within the plasma that have potential use in ACI patient stratification. Further work is required to validate the findings of this discovery-phase work in larger ACI cohorts.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_9 | Pages 75 - 75
17 Apr 2023
Tierney L Kuiper J Williams M Roberts S Harrison P Gallacher P Jermin P Snow M Wright K
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The objectives of the study were to investigate demographic, injury and surgery/treatment-associated factors that could influence clinical outcome, following Autologous Chondrocyte Implantation (ACI) in a large, “real-world”, 20 year longitudinally collected clinical data set.

Multilevel modelling was conducted using R and 363 ACI procedures were suitable for model inclusion. All longitudinal post-operative Lysholm scores collected after ACI treatment and before a second procedure (such as knee arthroplasty but excluding minor procedures such as arthroscopy) were included. Any patients requiring a bone graft at the time of ACI were excluded. Potential predictors of ACI outcome explored were age at the time of ACI, gender, smoker status, pre-operative Lysholm score, time from surgery, defect location, number of defects, patch type, previous operations, undergoing parallel procedure(s) at the time of ACI, cell count prior to implantation and cell passage number.

The best fit model demonstrated that for every yearly increase in age at the time of surgery, Lysholm scores decreased by 0.2 at 1-year post-surgery. Additionally, for every point increase in pre-operative Lysholm score, post-operative Lysholm score at 1 year increased by 0.5. The number of cells implanted also impacted on Lysholm score at 1-year post-op with every point increase in log cell number resulting in a 5.3 lower score. In addition, those patients with a defect on the lateral femoral condyle (LFC), had on average Lysholm scores that were 6.3 points higher one year after surgery compared to medial femoral condyle (MFC) defects. Defect grade and location was shown to affect long term Lysholm scores, those with grade 3 and patella defects having on average higher scores compared to patients with grade 4 or trochlea defects.

Some of the predictors identified agree with previous reports, particularly that increased age, poorer pre-operative function and worse defect grades predicted poorer outcomes. Other findings were more novel, such as that a lower cell number implanted and that LFC defects were predicted to have higher Lysholm scores at 1 year and that patella lesions are associated with improved long-term outcomes cf. trochlea lesions.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_7 | Pages 59 - 59
4 Apr 2023
MacLeod A Roberts S Mandalia V Gill H
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Conventional proximal tibial osteotomy is a widely successful joint-preserving treatment for osteoarthritis; however, conventional procedures do not adequately control the posterior tibial slope (PTS). Alterations to PTS can affect knee instability, ligament tensioning, knee kinematics, muscle and joint contact forces as well as range of motion.

This study primarily aimed to provide a comprehensive investigation of the variables influencing PTS during high tibial osteotomy using a 3D surgical simulation approach. Secondly, it aimed to provide a simple means of implementing the findings in future 3D pre-operative planning and /or clinically.

The influence of two key variables: the gap opening angle and the hinge axis orientation on PTS was investigated using three independent approaches: (1) 3D computational simulation using CAD software to perform virtual osteotomy surgery and simulate the post-operative outcome. (2) Derivation of a closed-form mathematical solution using a generalised vector rotation approach (3) Clinical assessment of synthetically generated x-rays of osteoarthritis patients (n=28; REC reference: 17/HRA/0033, RD&E NHS, UK) for comparison against the theoretical/computational approaches.

The results from the computational and analytical assessments agreed precisely. For three different opening angles (6°, 9° and 12°) and 7 different hinge axis orientations (from −30° to 30°), the results obtained were identical. A simple analytical solution for the change in PTS, ΔPs, based on the hinge axis angle, α, and the osteotomy opening angle, θ, was derived:

ΔPs=sin-1(sin α sin θ)

The clinical assessment demonstrated that the absolute values of PTS, and changes resulting from various osteotomies, matched the results from the two relative prediction methods.

This study has demonstrated that PTS is impacted by the hinge axis angle and the extent of the osteotomy opening angle and provided computational evidence and analytical formula for general use.


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_16 | Pages 11 - 11
1 Dec 2021
Hulme C Gallacher P Jermin P Roberts S Wright K
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Abstract

Purpose

Stratification is required to ensure that only patients likely to benefit, receive Autologous Chondrocyte Implantation (ACI). At Stage I (SI), healthy cartilage is harvested from the joint and chondrocytes culture expanded before being implanted into a chondral/osteochondral defect at Stage II (SII). In ACI non-responders, there is a marked shift in the profile and abundance of proteins detectable in the synovial fluid (SF) at SII, many being associated with an acute phase response (APR). However, clinical biomarkers are easier to measure in blood than SF, so we have now performed this investigation in plasma.

Methods

Isobaric tag for relative and absolute quantitation mass-spectrometry was used to assess the proteome in plasma pooled from ACI responders (mean Lysholm improvement of 33, n=10) or non-responders (mean: −13 points, n=10), collected at SI or SII surgeries. Interactome networks were generated using STRING. Plasma proteome data were compared to matched SF data, previously analysed, to identify any proteins that changed across the fluids. Clusterin concentration was quantitated (ELISA; Biotechne).


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_16 | Pages 38 - 38
1 Dec 2021
Hopkins T Wright K Roberts S Jermin P Gallacher P Kuiper JH
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Abstract

Objectives

In the human knee, the cells of the articular cartilage (AC) and subchondral bone (SB) communicate via the secretion of biochemical factors. Chondrocyte-based AC repair strategies, such as articular chondrocyte implantation, are widely used but there has been little investigation into the communication between the native SB cells and the transplanted chondrocytes. We hypothesise that this communication depends on the health state of the SB and could influence the composition and quality of the repair cartilage.

Methods

An indirect co-culture model was developed using transwell inserts, representing a chondrocyte/scaffold-construct for repair of AC defects adjoining SB with varying degrees of degeneration. Donor-matched populations of human bone-marrow derived mesenchymal stromal cells (BM-MSCs) were isolated from the macroscopically and histologically best and worst osteochondral tissue, representing “healthy” and “unhealthy” SB. The BM-MSCs were co-cultured with normal chondrocytes suspended in agarose, with the two cell types separated by a porous membrane. After 0, 7, 14 and 21 days, chondrocyte-agarose scaffolds were assessed by gene expression and biochemical analyses.


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_2 | Pages 77 - 77
1 Mar 2021
Wang J Roberts S McCarthy H Tins B Gallacher P Richardson J Wright K
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Abstract

Objectives

Meniscus allograft and synthetic meniscus scaffold (Actifit®) transplantation have shown promising outcomes for symptoms relief in patients with meniscus deficient knees. Untreated chondral defects can place excessive load onto meniscus transplants and cause early graft failure. We hypothesised that combined ACI and allograft or synthetic meniscus replacement might provide a solution for meniscus deficient individuals with co-existing lesions in cartilage and meniscus.

Methods

We retrospectively collected data from 17 patients (16M, 1F, aged 40±9.26) who had ACI and meniscus allograft transplant (MAT), 8 patients (7M, 1F, aged 42±11) who underwent ACI and Actifit® meniscus scaffold replacement. Other baseline data included BMI, pre-operative procedures and cellular transplant data. Patients were assessed by pre-operative, one-year and last follow-up Lysholm score, one-year repair site biopsy, MRI evaluations.


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_2 | Pages 8 - 8
1 Mar 2021
Hulme CH Perry J Roberts S Gallacher P Jermin P Wright KT
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Abstract

Objectives

The ability to predict which patients will improve following routine surgeries aimed at preventing the progression of osteoarthritis is needed to aid patients being stratified to receive the most appropriate treatment. This study aimed to investigate the potential of a panel of biomarkers for predicting (prior to treatment) the clinical outcome following treatment with microfracture or osteotomy.

Methods

Proteins known to relate to OA severity, with predictive value in autologous cell implantation treatment or that had been identified in proteomic analyses (aggrecanase-1/ ADAMTS-4, cartilage oligomeric matrix protein (COMP), hyaluronic acid (HA), Lymphatic Vessel Endothelial Hyaluronan Receptor-1, matrix metalloproteinases-1 and −3, soluble CD14, S100 calcium binding protein A13 and 14-3-3 protein theta) were assessed in the synovial fluid (SF) of 19 and 13 patients prior to microfracture or osteotomy, respectively, using commercial immunoassays. Levels of COMP and HA were measured in the plasma of these patients. To find predictors of postoperative function, multiple linear regression analyses were performed.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_9 | Pages 67 - 67
1 May 2017
Bhattacharjee A McCarthy H Tins B Kuiper J Roberts S Richarson J
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Background

Structural and functional outcome of bone graft with first or second generation autologous chondrocyte implantation (ACI) in osteochondral defects has not been reported.

Methods

Seventeen patients (mean age of 27±7 years, range 17–40), twelve with osteochondritis dissecans (OD) (ICRS Grade 3 and 4) and five with isolated osteochondral defect (OCD) (ICRS Grade 4) were treated with a combined implantation of a unicortical autologous bone graft with ACI (the Osplug technique). Functional outcome was assessed with Lysholm scores. The repair site was evaluated with the Oswestry Arthroscopy Score (OAS), MOCART score and ICRS II histology score. Formation of subchondral lamina and lateral integration of the bone grafts were evaluated from MRI scans.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_16 | Pages 33 - 33
1 Oct 2016
Roberts S Salter D Ralston S
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TRIM32 is a candidate gene at the 9q33.1 genetic susceptibility locus for hip osteoarthritis (OA). Increased cartilage degradation typical of OA has previously been demonstrated in Trim32 knockout mice.

Our aim is to investigate the role of TRIM32 in human and murine articular tissue.

TRIM32 expression in human articular cartilage was examined by immunostaining. TRIM32 expression was compared in femoral head chondrocytes from patients with and without primary hip OA (n=6/group) and examined by Western blotting. Aggrecanolysis by femoral head explants from Trim32 knockout (T32KO) and wild-type (WT) mice was compared following stimulation with IL1α or retinoic acid (RA) and was assessed by DMMB assay (n=4/group). Expression of chondrocyte phenotype markers was measured by qPCR and compared between articular chondrocytes from WT and T32KO mice following catabolic (IL1α/TNFα) or anabolic (Oncostatin-M (OSM)/IGF1) stimulation.

TRIM32 expression was demonstrated in human articular cartilage; TRIM32 expression by chondrocytes was reduced in patients with hip OA (p=0.03). Greater aggrecanolysis occurred in cartilage explants from T32KO mice after treatment with no stimulation (p=0.03), IL1α (p=0.02), and RA (p=0.001). Unstimulated T32KO chondrocytes expressed reduced Col2a1 (p=8.53×10−5), and Sox9 (p=2.35×10−6). Upon IL1α treatment, T32KO chondrocytes expressed increased Col10a1 (p=0.0003). Upon anabolic stimulation, T32KO chondrocytes expressed increased Col2a1 (OSM: p=0.001; IGF: p=0.001), and reduced Sox9 (OSM: p=0.0002; IGF: p=0.0006).

These results indicate that altered TRIM32 expression in human articular tissue is associated with OA, and that Trim32 knockout results in increased cartilage degradation in murine femoral head explants. Predisposition to cartilage degeneration with reduced Trim32 expression may involve increased chondrocyte hypertrophy upon catabolic cytokine stimulation and dysregulation of Col2a1 and Sox9 expression upon anabolic stimulation.


Orthopaedic Proceedings
Vol. 97-B, Issue SUPP_3 | Pages 4 - 4
1 Apr 2015
Tanagho A Hatab S Roberts S Shewale S
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Introduction:

Antimicrobial resistance is an important patient safety issue. Antibiotic Stewardship is one of the key strategies in tackling this problem. We present our data over a two year period from October 2011 to December 2013.

Method:

A multidisciplinary, consultant led antibiotic ward round was implemented in October 2011. This involved the consultant orthopaedic surgeon, microbiologist, pharmacist and antibiotic prescription nurse. Data from the meetings was collected prospectively over a 118 week period using a standard data form.

The case notes, prescription kardex, laboratory results including microbiology data and clinical information of patients was available at the time of the Ward round. The indications for, choice of antibiotics, duration and further treatment plan were made and a note for the case notes was dictated immediately. Changes to prescriptions were also made at the time.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 240 - 240
1 Jul 2014
Bhattacharjee A Kuiper J Harrison P Roberts S Richardson J
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Summary

Randomised controlled study evaluating new bone formation in vivo in fracture non-unions by bone marrow derived stromal cells (BMSC). These cells do not show statistically significant new bone formation. Age of the patient during fracture, diabetes and doubling time had been observed to be correlated with fracture healing.

Introduction

Regenerating new bone by cell therapy could provide therapeutic options in many conditions such as fracture non-unions and osteo-chondral defect regeneration in advance OA. In this randomised controlled study we evaluated the efficacy of new bone formation by bone marrow derived stromal cells (BMSC) in patients with non-union.


Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_17 | Pages 27 - 27
1 Apr 2013
Owen SJ Roberts S Eisenstein S Jones P Sharp CA
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Background

Intervertebral disc cells exist in a challenging physiological environment. Disc degeneration occurs early in life implying that disc cells may no longer be able to maintain a functional tissue. We hypothesise that disc cells have a stress response different from most other cells because of the disc environment. We have compared the stress response of freshly isolated and cultured bovine nucleus pulposus (NP) cells with bovine dermal fibroblasts, representative of cells from a vascularised tissue.

Methods

Freshly isolated and passaged bovine NP cells and dermal fibroblasts were cultured for 3 days then subjected to either thermal stress at 45°C for 1h followed by recovery times of 6, 24 and 48h or nutrient stress involving culture without serum for 6, 24 and 48 h. At each time point, cell number and viability were assessed and heat shock protein 70 (Hsp70) measured in cell lysates by an enzyme-linked immunosorbent assay.


Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_13 | Pages 20 - 20
1 Mar 2013
Bhattacharjee A Menan C Wright K Roberts S Richardson JB
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The potential of cells derived from human umbilical cord(UC) for orthopaedic cell engineering is evaluated by dissecting the UC into four distinct anatomical structures – cord lining (CL), Wharton's Jelly (WJ), umbilical cord artery (UCA) and umbilical cord vein (UCV). Cells from individual anatomical layers were grown by explant culture technique for 21 days. Tri-lineage differentiation and growth kinetics of cells from each layer were compared. Flowcytometry was done according to ISCT criteria to ascertain their surface antigen expressions. Cells from all four layers differentiated into bone, cartilage and fat. Osteogenic and chondrogenic differentiation was variable for each type of cells. All cells expressed surface antigens characteristic of mesenchymal stem cells (MSC). These cells can form a potential cell source in cell engineering to produce bone and cartilage although individual cell type needs to be characterised from each anatomical layer of UC and identify the best cell type for cell engineering.


Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_4 | Pages 24 - 24
1 Jan 2013
Owen S Caterson B Roughley P Eisenstein S Roberts S
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Background

Proteoglycans (PGs) have long been known to be important to the functioning of the intervertebral disc. The most common PG is aggrecan, but there are also small leucine-rich proteoglycans (SLRPs) which constitute only a small percentage of the total PGs. However, they have many important functions, including organising the collagen, protecting it from degradation and attracting growth factors to the disc. We have examined how the core proteins of these molecules vary in intervertebral discs from patients with different pathologies.

Methods

Discs were obtained from patients with scoliosis (n=7, 19–53y), degenerative disc disease (DDD) (n=6, 35–51y) and herniations (n=5, 33–58y). Proteoglycans were extracted and the SLRPs (biglycan, decorin, fibromodulin, keratocan and lumican) were characterised via Western blotting following enzymatic digestion with chondroitinase ABC and keratanase.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVI | Pages 31 - 31
1 Aug 2012
McCarthy H Roberts S
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Autologous chondrocyte implantation (ACI) has been used for many years for the treatment of symptomatic defects in articular joints, predominantly the knee. Traditionally, cells were implanted behind a periosteal membrane, but in more recent times Chondrogide, a membrane consisting of porcine collagens I and III, has been used. There have been trials comparing the clinical outcome of these two groups of patients; in this study we compare the histological outcome using the two different patch types.

In a study of 100 patients having received ACI treatment of cartilage defects in the knee, 41 received Chondrogide (ACI-C) and 59 received periosteum (ACI-P). All of these patients had a post-operative biopsy taken at a mean of 16.9±9.2 months and 20.8±23.2 months for ACI-C and ACI-P respectively for histology using the ICRS II scoring system. Lysholm scores, a measure of knee function, were obtained pre- and post-operatively at the time of biopsy and statistical differences tested for via a Mann-Whitney U-test.

The mean age of the two groups at treatment was 37±8 and 35±10 years, the size of defect treated was 6.1±5.4 and 4.4±2.7 cm2 and the biopsy follow-up time was 50.6±22.2 and 81.2±34.8 months for ACI-C and ACI-P patients respectively. Both groups exhibited a significant improvement in Lysholm score from pre-operative to the time of biopsy (14.3±25.7; n=100), although there was no significant difference in improvement in Lysholm score between the two patch types. There was no significant difference between the histology score of the two groups, nor was the score found to correlate with the Lysholm score at that time. The individual components of the ICRS II score did not differ significantly with patch type (even for the surface architecture) apart from cellular morphology which was 6.5±3 and 8.2±1.6 for ACI-C and ACI-P respectively.

The histological quality of repair tissue formed with ACI-C differed little from that seen with ACI-P, despite the former group being biopsied ∼4 months sooner after treatment and being used to treat defects which were 39% larger. Hence Chondrogide appears just as suitable as periosteum for use as a patch in the procedure of ACI.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVI | Pages 122 - 122
1 Aug 2012
Kumar KS Murakibhavi V Roberts S Guerra-Pinto F Robinson E Harrison P Mangam D McCall I Richardson J
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Background

Autologous Chondrocyte Implantation (ACI) is a procedure which is gaining acceptance for the treatment of cartilage defects in the knee with good results and a long term durable outcome. Its use in other joints has been limited, mainly to the ankle. We aimed to assess the outcome of ACI in the treatment of chondral and osteochondral defects in the hip.

Methods

Fifteen patients underwent ACI for chondral or osteochondral defects in the femoral head with a follow up of upto 8 years (mean of 2 years) in our institution with a mean age of 37 years at the time of operation. Pre-operatively hip function was assessed by using the Harris Hip Score and MRI. Post-operatively these were repeated at 1 year and hip scores repeated annually. Failure was defined as a second ACI to the operated lesion or a conversion to a hip resurfacing or replacement.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVI | Pages 87 - 87
1 Aug 2012
Williams R Khan I Richardson K Nelson L Baird D Roberts S Dudia J Briggs T Fairclough J Archer C
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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.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XVIII | Pages 81 - 81
1 May 2012
Quasnichka H Kerr B Wright A Roberts S Hughes C Caterson B
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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.


The Journal of Bone & Joint Surgery British Volume
Vol. 81-B, Issue 6 | Pages 1064 - 1068
1 Nov 1999
Richardson JB Caterson B Evans EH Ashton BA Roberts S

Tissue engineering is an increasingly popular method of addressing pathological disorders of cartilage. Recent studies have demonstrated its clinical efficacy, but there is little information on the structural organisation and biochemical composition of the repair tissue and its relation to the adjacent normal tissue. We therefore analysed by polarised light microscopy and immunohistochemistry biopsies of repair tissue which had been taken 12 months after implantation of autologous chondrocytes in two patients with defects of articular cartilage.

Our findings showed zonal heterogeneity throughout the repair tissue. The deeper zone resembled hyaline-like articular cartilage whereas the upper zone was more fibrocartilaginous. The results indicate that within 12 months autologous chondrocyte implantation successfully produces replacement cartilage tissue, a major part of which resembles normal hyaline cartilage.