This study investigated the effect of hUC-MSCs on osteoarthritis (OA) progression in a xenogeneic model. Male, 10 week-old C57BL/6 mice underwent sham surgery (n = 15) or partial medial meniscectomy (PMM; n = 76). 5x105 hUC-MSCs (from 3 donors: D1, D2 and D3) were phenotyped via RT-qPCR and immunoprofiling their response to inflammatory stimuli. They were injected into the mouse joints 3 and 6 weeks post-surgery, harvesting joints at 8 and 12 weeks post-surgery, respectively. A no cell ‘control’ group was also used (n = 29). All knee joints were assessed via micro-computed tomography (μCT) and histology and 10 plasma markers were analysed at 12 weeks. PMM resulted in cartilage loss and osteophyte formation resembling human OA at both time-points. Injection of one donor's hUC-MSCs into the joint significantly reduced the loss of joint space at 12 weeks post-operatively compared with the PMM control. This ‘effective’ population of MSCs up-regulated the genes, IDO and TSG6, when stimulated with inflammatory cytokines, more than those from the other two donors. No evidence of an inflammatory response to the injected cells in any animals, either histologically or with plasma biomarkers, arose. Beneficial change in a PMM joint was seen with only one hUC-MSC population, perhaps indicating that cell therapy is not appropriate for severely osteoarthritic joints. However, none of the implanted cells appeared to elicit an inflammatory response at the time-points studied. The variability of UC donors suggests some populations may be more therapeutic than others and donor characterisation is essential in developing allogeneic cell therapies.

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.

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 × 10 ⁶ cells in 8.4 ± 1.5 days, following seeding of 10.2 ± 3.6 × 10 ⁶ 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.

Background: Treatment decisions for cartilage defects are often based on lesion size. Magnetic resonance imaging (MRI) is widely used to diagnose cartilage defects noninvasively; however, their size estimated from MRI may differ from defect sizes measured during arthrotomy, especially after debridement to healthy cartilage if undergoing autologous chondrocyte implantation. Purpose/Hypothesis: The purpose of this study was to evaluate the reliability of 2 methods to assess knee cartilage defect size on preoperative MRI and determine their accuracy in predicting postdebridement defect sizes recorded during arthrotomy. It was hypothesized that defect size would be predicted more accurately by the total area of abnormal articular cartilage rather than the area of full-thickness cartilage loss as identified on MRI. Study Design: Cohort study (diagnosis); Level of evidence, 3. Methods: This study included 64 patients (mean age, 41.8 ± 9.6 years) who underwent autologous cell therapy. Each patient received a 3-T MRI at 6.1 ± 3.0 weeks before cell implantation. Three raters, a radiologist, a surgeon, and a scientist, measured (1) the full-thickness cartilage defect area and (2) the total predicted abnormal cartilage area, identified by an abnormal signal on MRI. Interrater reliability was assessed using the intraclass correlation coefficient (ICC). Actual pre- and postdebridement defect sizes were obtained from intraoperative surgical notes. Postdebridement surgical measurements were considered the clinical reference standard and were compared with the radiologist’s MRI measurements. Results: Eighty-seven defects were assessed, located on the lateral (n = 8) and medial (n = 26) femoral condyle, trochlea (n = 17), and patella (n = 36). The interrater reliability of the cartilage defect measurements on MRI was good to excellent for the full-thickness cartilage defect area (ICC = 0.74) and the total predicted abnormal cartilage area (ICC = 0.78). The median full-thickness cartilage defect area on MRI underestimated the median postdebridement defect area by 78.3%, whereas the total predicted abnormal cartilage area measurement underestimated the postdebridement defect area by 14.3%. Conclusion: Measuring the full-thickness cartilage defect area on MRI underestimated the area to treat, whereas measuring the total abnormal area provided a better estimate of the actual defect size for treatment.

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.

Inflammation of the synovium (synovitis) is implicated in the onset, progression and clinical manifestation of osteoarthritis (OA), although its prevalence at different stages of the disease has yet to be definitively established. Synovial macrophages play a central role in synovitis and can demonstrate pro- and anti-inflammatory phenotypes. The pervasiveness and variation in phenotypic identity of macrophages in early- and late-OA synovia is unclear. In the present study we investigated the frequency and severity of synovitis and assessed macrophage phenotypes in synovia from patients with high risk of developing PTOA (deemed early-OA) or late-OA. Synovial samples were collected from patients undergoing cell therapy treatment for early-OA or arthroplasty for late-OA. Synovitis was assessed using a semi-quantitative, histological scoring system. Macrophage abundance and phenotypic characteristics were assessed by immunohistochemistry and image analysis. Study parameters were compared between the early- and late-OA groups and correlated with demographic and clinical information. Synovitis was more prevalent and generally more severe in early-OA synovia compared to late-OA synovia (effect size; d ​= ​0.76). There were more macrophages overall (d ​= ​1.04), with more demonstrating markers characteristic of a pro-inflammatory (M1) phenotype (d ​= ​0.86), in the early-OA cohort. Synovitis severity was significantly correlated with the total number of macrophages (ρ ​= ​0.47), and with the presence of both M1 (ρ ​= ​0.65) and M2 (ρ ​= ​0.49) macrophage markers (M2 typically considered to indicate an anti-inflammatory or wound-healing phenotype). Our data suggest that synovial inflammation may play a greater role in the early stages of OA than in end-stage disease, and is at least partly mediated by synovial macrophages.