A comprehensive review of quantum bioreactor cell manufacture: Research and clinical applications.

The Quantum cell expansion system manufactured by Terumo-BCT is perhaps the most widely reported Good Manufacturing Practice-compliant bioreactor used for the expansion of adherent cell populations, both for research purposes and clinical cell-based therapies/trials. Although the system was originally designed for adherent cell expansion, more recently suspension cultures and extracellular vesicle manufacturing protocols have been published using the Quantum system. Cell therapy research and regenerative medicine in general is a rapidly expanding field and as such it is likely that the use of this system will become even more widespread and perhaps mandatory, for both research and development and in the clinic. The purpose of this review is to describe, compare and discuss the diverse range of research and clinical applications currently using the Quantum system, which to our knowledge has not previously been reviewed. In addition, current and future challenges will also be discussed.

Extracellular Vesicle Depletion Protocols of Foetal Bovine Serum Influence Umbilical Cord Mesenchymal Stromal Cell Phenotype, Immunomodulation, and Particle Release.

The immunomodulatory properties of MSCs can be recreated using their extracellular vesicles (EVs). Yet, the true capabilities of the MSC EVs cannot be distinguished from contaminating bovine EVs and protein derived from supplemental foetal bovine serum (FBS). FBS EV depletion protocols can minimise this, but vary in terms of depletion efficiency, which can negatively impact the cell phenotype. We explore the impact of FBS EV depletion strategies, including ultracentrifugation, ultrafiltration, and serum-free, on umbilical cord MSC characteristics. Whilst a greater depletion efficiency, seen in the ultrafiltration and serum-free strategies, did not impact the MSC markers or viability, the MSCs did become more fibroblastic, had slower proliferation, and showed inferior immunomodulatory capabilities. Upon MSC EV enrichment, more particles, with a greater particle/protein ratio, were isolated upon increasing the FBS depletion efficiency, except for serum-free, which showed a decreased particle number. Whilst all conditions showed the presence of EV-associated markers (CD9, CD63, and CD81), serum-free was shown to represent a higher proportion of these markers when normalised by total protein. Thus, we caution MSC EV researchers on the use of highly efficient EV depletion protocols, showing that it can impact the MSC phenotype, including their immunomodulatory properties, and stress the importance of testing in consideration to downstream objectives.

Investigation of the blood proteome in response to spinal cord injury in rodent models.

STUDY DESIGN: Explanatory and mechanistic study. OBJECTIVES: A better understanding of the 'whole-body' response following spinal cord injury (SCI) is needed to guide future research aimed at developing novel therapeutic interventions and identifying prognostic indicators for SCI. This study aimed to characterise the blood proteome following contusion or complete SCI compared to a sham injury in rat models. SETTING: United Kingdom. METHODS: Pooled blood samples from one and seven days after a contusion (serum; n = 5) or from 14 days and 112 days post-complete transection SCI (plasma; n = 8) and their sham-injured counterparts were subjected to independent iTRAQ nanoflow liquid chromatography tandem mass-spectrometry proteomic analyses. Pathway analyses of the proteins that were differentially abundant between SCI and their matched sham injured counterparts were completed to indicate biological pathways that may be changed in response to SCI. RESULTS: Eleven and 42 proteins were differentially abundant (≥±2.0 FC; p ≤ 0.05) between the contusion SCI and sham injured animals at 24 h and seven days post-injury, respectively. Seven and tweleve proteins were differentially abundant between complete and sham injured rats at 14 and 112 days post-injury, respectively. Acute-phase response signalling and Liver X Receptor/Retinoic X Receptor activation were identified as differentially regulated pathways in both models of SCI. CONCLUSIONS: We have utilised longitudinal preclinical SCI models to provide an insight into the blood proteome changes that result following SCI and to highlight a number of biological pathways of interest for future studies.

High content and high throughput screening to assess the angiogenic and neurogenic actions of mesenchymal stem cells in vitro.

Mesenchymal stem cells are universally regarded across many fields of medicine, as one of the most promising cell types for use in cell-based therapies. Although not yet fully understood, the therapeutic effects of these cells are largely attributed to the trophic actions of growth factors and cytokines present in the cell secretome. Specifically, the angiogenic and neurogenic properties of these cells make them attractive for the repair of vascularised and innervated tissues. In this study, we investigate the effect of mesenchymal stem cell conditioned media on in vitro assays of angiogenesis and nerve growth. We describe the use of two state of the art high content and high throughput cell analysis systems and compare them against manual analysis techniques. Mesenchymal stem cell secretomes stimulated angiogenesis and nerve growth in vitro in a donor dependant manner. Levels of neuroregulin, platelet-derived growth factor-AA and glial-derived neurotrophic factor, positively correlated with the observed angiogenic effects of these cells. High content and high throughput cell analysis systems such as the ones used in this study, may provide rapid screening tools to assist not only with patient selection but the identification of predictive therapeutic markers to support clinical outcome monitoring for patients treated with stem cell therapies.

Using nanopore sequencing to identify bacterial infection in joint replacements: a preliminary study.

This project investigates if third-generation genomic sequencing can be used to identify the species of bacteria causing prosthetic joint infections (PJIs) at the time of revision surgery. Samples of prosthetic fluid were taken during revision surgery from patients with known PJIs. Samples from revision surgeries from non-infected patients acted as negative controls. Genomic sequencing was performed using the MinION device and the rapid sequencing kit from Oxford Nanopore Technologies. Bioinformatic analysis pipelines to identify bacteria included Basic Local Alignment Search Tool, Kraken2 and MinION Detection Software, and the results were compared with standard of care microbiological cultures. Furthermore, there was an attempt to predict antibiotic resistance using computational tools including ResFinder, AMRFinderPlus and Comprehensive Antibiotic Resistance Database. Bacteria identified using microbiological cultures were successfully identified using bioinformatic analysis pipelines. Nanopore sequencing and genomic classification could be completed in the time it takes to perform joint revision surgery (2-3 h). Genomic sequencing in this study was not able to predict antibiotic resistance in this time frame, this is thought to be due to a short-read length and low read depth. It can be concluded that genomic sequencing can be useful to identify bacterial species in infected joint replacements. However, further work is required to investigate if it can be used to predict antibiotic resistance within clinically relevant timeframes.

Chondrocyte Isolation and Expansion.

Chondrocyte isolation requires a combination of enzymatic and mechanical digestion of cartilaginous tissues in order to release the chondrocytes. Extracted primary chondrocytes will then adhere to standard tissue culture plastics, typically in small clusters, over a period of a few days in monolayer culture. Chondrocyte populations are expanded in a basal medium containing serum, supplemented with ascorbic acid, antibiotics, and sometimes antifungal agents and growth factors. Here we describe the standard research grade and good manufacturing practice (GMP) protocols used for the isolation and expansion of chondrocytes by the Oswestry/Keele University Orthopaedic Research (OsKOR) group and John Charnley GMP and MHRA licensed laboratory, both based at the RJAH Orthopaedic Hospital, Oswestry, UK.

Genomic Sequencing to Diagnose Prosthetic Joint Infection in the Knee: A Case Report.

There is currently no "gold-standard" method to diagnose prosthetic joint infections (PJI), and the current practice of using microbiological cultures has many limitations. The identification of the bacterial species causing the infection is crucial to guide treatment; therefore, a robust method needs to be developed. Here, we attempt to use genomic sequencing with the MinION device from Oxford Nanopore Technologies to identify the species of bacteria causing PJI in a 61-year-old male. Genomic sequencing with the MinION presents an opportunity to produce species identification in real-time and at a smaller cost than current methods. By comparing results with standard hospital microbiological cultures, this study suggests that nanopore sequencing using the MinION could be a faster and more sensitive method to diagnose PJI than microbiological cultures.

Proteomic Analyses of Autologous Chondrocyte Implantation Plasma Highlight Cartilage Acidic Protein 1 as a Candidate for Preclinical Screening.

BACKGROUND: Stratification is required to ensure that only patients likely to benefit receive autologous chondrocyte implantation (ACI). It would be advantageous to identify biomarkers to predict ACI outcome that are measurable in blood, avoiding the need for an invasive synovial fluid harvest. PURPOSE: To assess if proteomic analyses can be used to identify novel candidate blood biomarkers in individuals who respond well or poorly to ACI. STUDY DESIGN: Controlled laboratory study. METHODS: Isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry was used to assess the proteome in plasma pooled from ACI responders (mean Lysholm improvement after ACI, 33; n = 10) or nonresponders (mean, -13; n = 10), collected at the time of surgery for cartilage harvest (stage 1) or implantation of culture-expanded chondrocytes (stage 2). An alternative proteomic method, label-free quantitation liquid chromatography-tandem mass spectrometry, was used to analyze plasma samples (majority matched to iTRAQ) individually. Differentially abundant proteins (±2.0-fold) were analyzed from both proteomic data sets, and markers of interest identified via pooled iTRAQ were validated via immunoassay of individual samples. RESULTS: Protein differences could be detected in the plasma preoperatively between ACI responders and nonresponders (16 proteins; ≥±2.0-fold change; P < .05) using iTRAQ proteomics. The most pronounced plasma proteome shift was evident in response to stage 1 surgery in ACI nonresponders, with 48 proteins being differentially abundant between the procedures. Label-free quantitation liquid chromatography-tandem mass spectrometry analysis of these same plasma samples (nonpooled) resulted in very few proteins being identified that were significantly differentially abundant. However, this work highlighted cartilage acidic protein 1 as being increased preoperatively in nonresponders as compared with responders. CONCLUSIONS: This study is the first to use proteomic techniques to profile the plasma of individuals treated with ACI. Despite iTRAQ analysis of pooled plasmas indicating that there are differences in the plasma proteome between responders and nonresponders to ACI, these findings were not replicated when assessed using an alternative nonpooled technique. This study highlights some of the difficulties in profiling the plasma proteome in an attempt to identify novel biomarkers. Regardless, cartilage acidic protein 1 has been identified as a protein candidate, which is detectable in plasma and can predict outcome to ACI before treatment. CLINICAL RELEVANCE: Candidate plasma protein biomarkers identified in this study have the potential to help determine which patients will be best suited to treatment with ACI.

Human vascularised synovium-on-a-chip: a mechanically stimulated, microfluidic model to investigate synovial inflammation and monocyte recruitment.

Healthy synovium is critical for joint homeostasis. Synovial inflammation (synovitis) is implicated in the onset, progression and symptomatic presentation of arthritic joint diseases such as rheumatoid arthritis and osteoarthritis. Thus, the synovium is a promising target for the development of novel, disease-modifying therapeutics. However, target exploration is hampered by a lack of good pre-clinical models that accurately replicate human physiology and that are developed in a way that allows for widespread uptake. The current study presents a multi-channel, microfluidic, organ-on-a-chip (OOAC) model, comprising a 3D configuration of the human synovium and its associated vasculature, with biomechanical and inflammatory stimulation, built upon a commercially available OOAC platform. Healthy human fibroblast-like synoviocytes (hFLS) were co-cultured with human umbilical vein endothelial cells (HUVECs) with appropriate matrix proteins, separated by a flexible, porous membrane. The model was developed within the Emulate organ-chip platform enabling the application of physiological biomechanical stimulation in the form of fluid shear and cyclic tensile strain. The hFLS exhibited characteristic morphology, cytoskeletal architecture and matrix protein deposition. Synovial inflammation was initiated through the addition of interleukin-1ß(IL-1ß) into the synovium channel resulting in the increased secretion of inflammatory and catabolic mediators, interleukin-6 (IL-6), prostaglandin E2 (PGE2), matrix metalloproteinase 1 (MMP-1), as well as the synovial fluid constituent protein, hyaluronan. Enhanced expression of the inflammatory marker, intercellular adhesion molecule-1 (ICAM-1), was observed in HUVECs in the vascular channel, accompanied by increased attachment of circulating monocytes. This vascularised human synovium-on-a-chip model recapitulates a number of the functional characteristics of both healthy and inflamed human synovium. Thus, this model offers the first human synovium organ-chip suitable for widespread adoption to understand synovial joint disease mechanisms, permit the identification of novel therapeutic targets and support pre-clinical testing of therapies.

The Upscale Manufacture of Chondrocytes for Allogeneic Cartilage Therapies.

Allogeneic chondrocyte therapies need to be developed to allow more individuals to be treated with a cell therapy for cartilage repair and to reduce the burden and cost of the current two-stage autologous procedures. Upscale manufacture of chondrocytes using a bioreactor could help provide an off-the-shelf allogeneic chondrocyte therapy with many doses being produced in a single manufacturing run. In this study, we assess a good manufacturing practice-compliant hollow-fiber bioreactor (Quantum®) for adult chondrocyte manufacture. Chondrocytes were isolated from knee arthroplasty-derived cartilage (n = 5) and expanded in media supplemented with 10% fetal bovine serum (FBS) or 5% human platelet lysate (hPL) on tissue culture plastic (TCP) for a single passage. hPL-supplemented cultures were then expanded in the Quantum bioreactor for a further passage. Matched, parallel cultures in hPL or FBS were maintained on TCP. Chondrocytes from all culture conditions were characterized in terms of growth kinetics, morphology, immunoprofile, chondrogenic potential (chondrocyte pellet assays), and single telomere length analysis. Quantum expansion of chondrocytes resulted in 86.4 ± 38.5 × 106 cells in 8.4 ± 1.5 days, following seeding of 10.2 ± 3.6 × 106 cells. This related to 3.0 ± 1.0 population doublings in the Quantum bioreactor, compared with 2.1 ± 0.6 and 1.3 ± 1.0 on TCP in hPL- and FBS-supplemented media, respectively. Quantum- and TCP-expanded cultures retained equivalent chondropotency and mesenchymal stromal cell marker immunoprofiles, with only the integrin marker, CD49a, decreasing following Quantum expansion. Quantum-expanded chondrocytes demonstrated equivalent chondrogenic potential (as assessed by ability to form and maintain chondrogenic pellets) with matched hPL TCP populations. hPL manufacture, however, led to reduced chondrogenic potential and increased cell surface positivity of integrins CD49b, CD49c, and CD51/61 compared with FBS cultures. Quantum expansion of chondrocytes did not result in shortened 17p telomere length when compared with matched TCP cultures. This study demonstrates that large numbers of adult chondrocytes can be manufactured in the Quantum hollow-fiber bioreactor. This rapid, upscale expansion does not alter chondrocyte phenotype when compared with matched TCP expansion. Therefore, the Quantum provides an attractive method of manufacturing chondrocytes for clinical use. Media supplementation with hPL for chondrocyte expansion may, however, be unfavorable in terms of retaining chondrogenic capacity.

Concise review: Bone marrow for the treatment of spinal cord injury: mechanisms and clinical applications.

Transplantation of bone marrow stem cells into spinal cord lesions enhances axonal regeneration and promotes functional recovery in animal studies. There are two types of adult bone marrow stem cell; hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs). The mechanisms by which HSCs and MSCs might promote spinal cord repair following transplantation have been extensively investigated. The objective of this review is to discuss these mechanisms; we briefly consider the controversial topic of HSC and MSC transdifferentiation into central nervous system cells but focus on the neurotrophic, tissue sparing, and reparative action of MSC grafts in the context of the spinal cord injury (SCI) milieu. We then discuss some of the specific issues related to the translation of HSC and MSC therapies for patients with SCI and present a comprehensive critique of the current bone marrow cell clinical trials for the treatment of SCI to date.

Phenotypic characterization of regional human meniscus progenitor cells.

Stimulating meniscus regeneration using meniscal progenitor cells has been suggested as a promising new strategy. However, there is a lack of studies which decisively identify and characterize progenitor cell populations in human meniscus tissues. In this study, donor-matched progenitor cells were isolated via selective fibronectin adhesion from the avascular and vascular regions of the meniscus and chondroprogenitors from articular cartilage (n = 5). The mixed populations of cells from these regions were obtained by standard isolation techniques for comparison. The colony formation efficacy of avascular progenitors, vascular progenitors and chondroprogenitors was monitored using Cell-IQ® live cell imaging. Proliferation rates of progenitors were compared with their mixed population counterparts. Cell surface markers indicative of mesenchymal stromal cells profile and progenitor markers were characterized by flow cytometry in all populations. The fibrochondrogenic capacity was assessed via fibrochondrogenic differentiation and measuring GAG/DNA content and morphology. All meniscal progenitor and chondroprogenitor populations showed superior colony forming efficacy and faster proliferation rates compare to their mixed populations. Progenitor populations showed significantly higher positivity for CD49b and CD49c compared to their mixed population counterparts and chondroprogenitors had a higher positivity level of CD166 compared to mixed chondrocytes. GAG/DNA analysis demonstrated that progenitor cells generally produced more GAG than mixed populations. Our study demonstrates that the human meniscus contains meniscal progenitor populations in both the avascular and vascular regions. Meniscal progenitors derived from the vascular region exhibit enhanced proliferative and fibrochondrogenic characteristics compared to those from the avascular region; this may associate with the enhanced meniscal healing potential in the vascular region. These findings build on the body of evidence which suggests that meniscal progenitors represent an attractive cell therapy strategy for meniscal regeneration.

The management of talar osteochondral lesions - Current concepts.

Osteochondral lesions of the talus (OLTs) are a common complication following trauma, involving both the articular cartilage and the underlying subchondral bone, with variable aetiologies and often presenting with non-specific symptoms. Diagnosis of OLTs requires a combination of clinical assessment and imaging and despite many different treatment options, there is no generalised consensus regarding which option is the most effective. Left untreated, OLTs risk progressing to osteoarthritis. Acute non-displaced OLTs can be treated non-operatively. However, OLTs refractory to non-surgical care for three to six months may be suitable for surgical care. In these cases, conservative treatments are often unsuccessful, particularly for larger and more severe defects and so the majority require surgical intervention. Although bone marrow stimulation techniques remain the "gold standard" for lesions <150 mm2, there still requires a need for better long term clinical data and cost-benefit analyses compared with other treatment options. Biological attempts at either regenerating or replacing the articular cartilage are however demonstrating some promising results, but each with their own advantages and disadvantages. In this review, we summarise the clinical management of OLTs and present the current concepts of different treatment regimes.

Cell therapy for cartilage repair.

Regenerative medicine, using cells as therapeutic agents for the repair or regeneration of tissues and organs, offers great hope for the future of medicine. Cell therapy for treating defects in articular cartilage has been an exemplar of translating this technology to the clinic, but it is not without its challenges. These include applying regulations, which were designed for pharmaceutical agents, to living cells. In addition, using autologous cells as the therapeutic agent brings additional costs and logistical challenges compared with using allogeneic cells. The main cell types used in treating chondral or osteochondral defects in joints to date are chondrocytes and mesenchymal stromal cells derived from various sources such as bone marrow, adipose tissue or umbilical cord. This review discusses some of their biology and pre-clinical studies before describing the most pertinent clinical trials in this area.

An In Vitro System to Study the Effect of Subchondral Bone Health on Articular Cartilage Repair in Humans.

Chondrocyte-based cartilage repair strategies, such as articular chondrocyte implantation, are widely used, but few studies addressed the communication between native subchondral bone cells and the transplanted chondrocytes. An indirect co-culture model was developed, representing a chondrocyte/scaffold-construct repair of a cartilage defect adjoining bone, where the bone could have varying degrees of degeneration. Human BM-MSCs were isolated from two areas of subchondral bone in each of five osteochondral tissue specimens from five patients undergoing knee arthroplasty. These two areas underlaid the macroscopically and histologically best and worst cartilage, representing early and late-stage OA, respectively. 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, and the abundance of selected proteins in conditioned media was assessed by ELISA. Co-culture with late-OA BM-MSCs resulted in a reduction in GAG deposition and a decreased expression of genes encoding matrix-specific proteins (COL2A1 and ACAN), compared to culturing with early OA BM-MSCs. The concentration of TGF-ß1 was significantly higher in the early OA conditioned media. The results of this study have clinical implications for cartilage repair, suggesting that the health of the subchondral bone may influence the outcomes of chondrocyte-based repair strategies.

Routinely Measured Hematological Markers Can Help to Predict American Spinal Injury Association Impairment Scale Scores after Spinal Cord Injury.

Neurological outcomes following spinal cord injury (SCI) are currently difficult to predict. While the initial American Spinal Injury Association Impairment Scale (AIS) grade can give an estimate of outcome, the high remaining degree of uncertainty has stoked recent interest in biomarkers for SCI. This study aimed to assess the prognostic value of routinely measured blood biomarkers by developing prognostic models of AIS scores at discharge and 12 months post-injury. Routine blood and clinical data were collected from SCI patients (n = 417), and blood measures that had been assessed in less than 50% of patients were excluded. Outcome neurology was obtained from AIS and Spinal Cord Independence Measure III (SCIM-III) scores at discharge and 12 months post-injury, with motor (AIS) and sensory (AIS, touch and prick) abilities being assessed individually. Linear regression models with and without elastic net penalization were created for all outcome measures. Blood measures associated with liver function, such as alanine transaminase, were found to add value to predictions of SCIM-III at discharge and 12 months post-injury. Further, components of a total blood count, including hemoglobin, were found to add value to predictions of AIS motor and sensory scores at discharge and 12 months post-injury. These findings corroborate the results of our previous preliminary study and thus provide further evidence that routine blood measures can add prognostic value in SCI and that markers of liver function are of particular interest.

Human Mesenchymal Stromal Cells Enhance Cartilage Healing in a Murine Joint Surface Injury Model.

Human umbilical cord (hUC)- or bone marrow (hBM)-derived mesenchymal stromal cells (MSCs) were evaluated as an allogeneic source of cells for cartilage repair. We aimed to determine if they could enhance healing of chondral defects with or without the recruitment of endogenous cells. hMSCs were applied into a focal joint surface injury in knees of adult mice expressing tdTomato fluorescent protein in cells descending from Gdf5-expressing embryonic joint interzone cells. Three experimental groups were used: (i) hUC-MSCs, (ii) hBM-MSCs and (iii) PBS (vehicle) without cells. Cartilage repair was assessed after 8 weeks and tdTomato-expressing cells were detected by immunostaining. Plasma levels of pro-inflammatory mediators and other markers were measured by electrochemiluminescence. Both hUC-MSC (n = 14, p = 0.009) and hBM-MSC (n = 13, p = 0.006) treatment groups had significantly improved cartilage repair compared to controls (n = 18). While hMSCs were not detectable in the repair tissue at 8 weeks post-implantation, increased endogenous Gdf5-lineage cells were detected in repair tissue of hUC-MSC-treated mice. This xenogeneic study indicates that hMSCs enhance intrinsic cartilage repair mechanisms in mice. Hence, hMSCs, particularly the more proliferative hUC-MSCs, could represent an attractive allogeneic cell population for treating patients with chondral defects and perhaps prevent the onset and progression of osteoarthritis.

Identification of Candidate Synovial Fluid Biomarkers for the Prediction of Patient Outcome After Microfracture or Osteotomy.

BACKGROUND: Biomarkers are needed to predict clinical outcomes for microfracture and osteotomy surgeries to ensure patients can be better stratified to receive the most appropriate treatment. PURPOSE: To identify novel biomarker candidates and to investigate the potential of a panel of protein biomarkers for the prediction of clinical outcome after treatment with microfracture or osteotomy. STUDY DESIGN: Descriptive laboratory study. METHODS: To identify novel candidate biomarker proteins, we used label-free quantitation after liquid chromatography-tandem mass spectrometry of dynamic range-compressed synovial fluids (SFs) from individuals who responded excellently or poorly (based on change in Lysholm score) to microfracture (n = 6) or osteotomy (n = 7). Biomarkers that were identified in this proteomic analysis or that relate to osteoarthritis (OA) severity or have predictive value in another early OA therapy (autologous cell implantation) were measured in the SF of 19 and 13 patients before microfracture or osteotomy, respectively, using commercial immunoassays, and were normalized to urea. These were aggrecanase-1 (ADAMTS-4), cartilage oligomeric matrix protein (COMP), hyaluronan (HA), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), matrix metalloproteinase 1 and 3, soluble CD14, S100 calcium binding protein A13, and 14-3-3 protein theta (YWHAQ). Levels of COMP and HA were also measured in the plasma of these patients. To find predictors of postoperative function, multivariable regression analyses were performed. RESULTS: Proteomic analyses highlighted YWHAQ and LYVE-1 as being differentially abundant between the clinical responders/improvers and nonresponders after microfracture. A linear regression model after backward variable selection could relate preoperative concentrations of SF proteins (HA, YWHAQ, LYVE-1), activity of ADAMTS-4, and patient demographic characteristics (smoker status and sex) with Lysholm score 12 months after microfracture. Further, a generalized linear model with elastic net penalization indicated that lower preoperative activity of ADAMTS-4 in SF, being a nonsmoker, and being younger at the time of operation were indicative of a higher postoperative Lysholm score (improved joint function) after osteotomy surgery. CONCLUSION: We have identified biomarkers and generated regression models with the potential to predict clinical outcome in patients treated with microfracture or osteotomy of the knee. CLINICAL RELEVANCE: Candidate protein biomarkers identified in this study have the potential to help determine which patients will be best suited to treatment with microfracture or osteotomy.

Evaluating patient perspectives on participating in scientific research and clinical trials for the treatment of spinal cord injury.

A questionnaire was developed to evaluate patients' perspective on research aimed at improving functions and overcoming complications associated with spinal cord injury (SCI). The first three sections were based on published and validated assessment tools. The final section was developed to assess participant perspectives on research for SCI. One thousand patients were approached, of which 159 participated. Fifty-eight percent of participants were satisfied with their 'life as a whole'. Two factors could be generated that reflected the variance in the data regarding participants' life with a SCI: "Psychosocial and physical wellbeing" and "Independent living". The majority of participants stated they would be involved in research (86%) or clinical trials (77%). However, the likelihood of participation dropped when potential risks of the research/trials were explained. Which participants would be willing to participate in research could not be predicted based on the severity of their injury, their psychosocial and physical wellbeing or their independent living. Despite participant establishment of a life with SCI, our data indicates that individuals strive for improvements in function. Participant willingness to be included in research studies is noteworthy and scientists and clinicians are encouraged to involve more patients in all aspects of their research.

Characterization of regional meniscal cell and chondrocyte phenotypes and chondrogenic differentiation with histological analysis in osteoarthritic donor-matched tissues.

Meniscus degeneration is closely related to the progression of knee osteoarthritis (OA). However, there is currently a lack of quantitative and objective metrics to assess OA meniscal cell phenotypes. In this study we investigated the phenotypic markers and chondrogenic potency of avascular and vascular meniscal cells and chondrocytes from medial OA knee joints (n = 10). Flow cytometry results showed that a significantly greater percentage of meniscal cells were positive for CD49b, CD49c and CD166 compared to donor-matched chondrocytes after 14 days in monolayer culture. The integrins, CD49b and CD29, were expressed at a significantly higher level on avascular meniscal cells derived from tissues with a more degenerated inner border than non-degenerate menisci, suggesting that the integrin family may play an important role in meniscus OA pathology. Collagen fibres arranged in a "tree-like" formation within the meniscus appeared to have less blood vessels associated with them in the vascular region of the most degenerate menisci, which may indicate that such structures are involved in the pathological process. We have demonstrated that meniscal cells derived from the lateral meniscus in medial OA patients have chondrogenic capacity in vitro and hence could represent a potential cell source to consider for meniscus tissue engineering.