T. Hopkins (Oswestry, GB)
Robert Jones and Agnes Hunt Orthopaedic Hospital Spinal StudiesPresenter Of 3 Presentations
P007 - In Vitro Modelling of Human Cartilage: Comparing Traditional Culture Methods with Pregenerate Organ-on-a-Chip System
Abstract
Purpose
In vitro modelling of human cartilage is commonly used to understand injury and disease in the tissue and to test treatment options. Organ-on-a-chip (OOAC) is an emerging modelling technology, incorporating cell culture chambers and microfluidics, that seeks to improve current modelling capabilities by increasing physiological relevance. Here, we assess the Pregenerate OOAC system to investigate human chondrocyte responses to pro-inflammatory stimuli cf. traditional chondrocyte culture techniques.
Methods and Materials
The Pregenerate OOAC consists of a central cell chamber (volume: 7.5µl) connected to a medium channel with an inlet at either end; six of such culture systems are contained on one chip (Figure 1A). Chondrocytes were isolated from macroscopically intact cartilage obtained during knee arthroplasty (n=5) and culture-expanded in monolayer to passage 2. Chondrocytes were encapsulated in Fibrin hydrogel and 2,200 chondrocytes loaded into each cell chamber of the microfluidic chips. Parallel cultures were set in high-density pellets (200k cells/pellet) or in monolayer (5000 cells/cm2). After 21 days, cultures were stimulated with a pro-inflammatory cocktail (50ng/ml TNF-α, 10ng/ml IL-1β and 25ng/ml IFN-γ) for 24 hours prior to assessing gene expression levels of COL1A1, COL2A1, COL10, ACAN, SOX9, MMP1, MMP3, MMP13 and ADAMTS-5.
Results
Fluorescence microscopy demonstrated evenly distributed chondrocytes within the culture chamber (Figure 1B). Pro-inflammatory stimulation down-regulated COL1A1, COL2A1, ACAN and SOX-9 in monolayer and pellet cultures only; COL10 was down-regulated in monolayer and pellet cultures but up-regulated in the Pregenerate chip. MMP3 and MMP13 were up-regulated in all three culture systems. ADAMTS-5 was down-regulated in the chips only. There was no change in expression of MMP1 in any of the culture systems.
Conclusion
These results demonstrate distinct differences in human chondrocyte responses to pro-inflammatory stimuli in the Pregenerate microfluidic chip cf. traditional culture conditions. Further work is needed to investigate these differences and which system is more synonymous with in vivo behaviour.
P024 - Macroscopic Assessment of Articular Cartilage Quality Correlates with Histological Assessment of Subchondral Bone Health
Abstract
Purpose
In the human knee, the articular cartilage (AC) and subchondral bone (SB) form a biomechanically and biochemically functional osteochondral unit. Macroscopic cartilage assessment by arthroscopy is commonly used to determine AC lesion severity prior to surgery. We previously reported that the health of the SB could influence outcomes following cell-based therapy for AC repair, but macroscopic assessment gives no indication of SB health. Our aim was to assess correlation between the macroscopic and histological grading systems.
Methods and Materials
Osteochondral tissue samples were collected from patients undergoing total knee replacement surgery (TKR). AC was graded macroscopically using the ICRS Cartilage Lesion Classification System (grade 0 (normal) – grade 4 (severely abnormal)). Two osteochondral fragments, one with the lowest grade (most normal) and one with the highest grade (most abnormal) cartilage, were obtained for each TKR patient. Sections were stained with haematoxylin & eosin and safranin O/fast green before histological assessment. AC was graded using the OARSI OA Cartilage Histopathology Assessment System (grade 0 (normal) – grade 6 (deformation)). SB was graded using the SB Histological Grading System (grade 0 (early OA) – grade 3 (late-stage OA)). The relationship between scores was assessed using Kendall’s rank correlation coefficient (Kendall’s tau).
Results
Sixteen TKR patients (6 female, 10 male; 68.2±6.7SD years-old), were included, resulting in the generation of 32 paired osteochondral fragments with the most normal and most abnormal cartilage for each patient. The ICRS score demonstrated moderate-to-strong correlation with both the AC and SB histological scores (Table 1, Figure 1). The AC and SB histological scores were also strongly correlated (Table 1).
Conclusion
Our results suggest that macroscopic cartilage assessment, using the ICRS scoring system, may give an indication of histologically assessed AC and SB health. This study provides further evidence for the close association between the AC and SB in patients with established knee osteoarthritis.
P025 - Severity of Synovitis and Macrophage Phenotypes in Knee Cartilage Defect and Osteoarthritis Cohorts
Abstract
Purpose
Growing evidence suggests that the synovium is important in the pathogenesis of osteoarthritis (OA). Synovial macrophages demonstrate considerable plasticity in mediating joint inflammation and OA progression. In this study we examined the synovia from patients with either cartilage defects or end-stage OA in the knee.
Methods and Materials
Synovial tissues were obtained from patients undergoing cell therapy to treat cartilage defects (at time of cell implantation, ~3 weeks post cell harvest) or arthroplasty surgery. Haematoxylin and eosin-stained sections of synovial tissue were assessed for synovitis using a histological scoring system (Krenn score). Pan-macrophage (CD68) and polarisation markers (CD86:M1 and CD206:M2) were assessed by immunohistochemical staining and subsequent semi-quantitative analysis.
Results
Samples of synovium were collected during surgery from 15 cell therapy (6 female, 9 male; 39.2±2.18SEM years) and 15 arthroplasty patients (8 female, 7 male; 67.3±2.80SEM years). Mean synovitis scores were significantly higher in the cell therapy group cf. the arthroplasty group (Table 1). The mean intensity of CD68, CD86 and CD206 immunostaining was also significantly higher in the cell therapy group cf. the arthroplasty group. The ratio of CD86 to CD206 was significantly higher in the cartilage injury synovia cf. OA synovia. Linear regression analysis of the full dataset indicated that CD86 intensity was a significant predictor of synovitis severity (cor.coeff.=0.052, p=0.016, 95% CI [0.036-0.234]).
Conclusion
Increased synovitis, abundance of macrophages (CD68, CD86 and CD206 positive) and increased CD86:CD206 staining ratio (indicative of M1 favoured polarisation) in synovia from patients undergoing cell therapy for cartilage defects is suggestive of a macrophage-mediated, pro-inflammatory immune response. Whether this response relates to the harvest stage of cell therapy or cartilage defect pathology warrants further investigation. Conversely, the reduced abundance of macrophages in arthroplasty synovia suggests a reduced ability of the synovium to mount an immune response at the end-stage of OA, indicative of organ-level failure within the joint.
Presenter Of 3 Presentations
P007 - In Vitro Modelling of Human Cartilage: Comparing Traditional Culture Methods with Pregenerate Organ-on-a-Chip System
Abstract
Purpose
In vitro modelling of human cartilage is commonly used to understand injury and disease in the tissue and to test treatment options. Organ-on-a-chip (OOAC) is an emerging modelling technology, incorporating cell culture chambers and microfluidics, that seeks to improve current modelling capabilities by increasing physiological relevance. Here, we assess the Pregenerate OOAC system to investigate human chondrocyte responses to pro-inflammatory stimuli cf. traditional chondrocyte culture techniques.
Methods and Materials
The Pregenerate OOAC consists of a central cell chamber (volume: 7.5µl) connected to a medium channel with an inlet at either end; six of such culture systems are contained on one chip (Figure 1A). Chondrocytes were isolated from macroscopically intact cartilage obtained during knee arthroplasty (n=5) and culture-expanded in monolayer to passage 2. Chondrocytes were encapsulated in Fibrin hydrogel and 2,200 chondrocytes loaded into each cell chamber of the microfluidic chips. Parallel cultures were set in high-density pellets (200k cells/pellet) or in monolayer (5000 cells/cm2). After 21 days, cultures were stimulated with a pro-inflammatory cocktail (50ng/ml TNF-α, 10ng/ml IL-1β and 25ng/ml IFN-γ) for 24 hours prior to assessing gene expression levels of COL1A1, COL2A1, COL10, ACAN, SOX9, MMP1, MMP3, MMP13 and ADAMTS-5.
Results
Fluorescence microscopy demonstrated evenly distributed chondrocytes within the culture chamber (Figure 1B). Pro-inflammatory stimulation down-regulated COL1A1, COL2A1, ACAN and SOX-9 in monolayer and pellet cultures only; COL10 was down-regulated in monolayer and pellet cultures but up-regulated in the Pregenerate chip. MMP3 and MMP13 were up-regulated in all three culture systems. ADAMTS-5 was down-regulated in the chips only. There was no change in expression of MMP1 in any of the culture systems.
Conclusion
These results demonstrate distinct differences in human chondrocyte responses to pro-inflammatory stimuli in the Pregenerate microfluidic chip cf. traditional culture conditions. Further work is needed to investigate these differences and which system is more synonymous with in vivo behaviour.
P024 - Macroscopic Assessment of Articular Cartilage Quality Correlates with Histological Assessment of Subchondral Bone Health
Abstract
Purpose
In the human knee, the articular cartilage (AC) and subchondral bone (SB) form a biomechanically and biochemically functional osteochondral unit. Macroscopic cartilage assessment by arthroscopy is commonly used to determine AC lesion severity prior to surgery. We previously reported that the health of the SB could influence outcomes following cell-based therapy for AC repair, but macroscopic assessment gives no indication of SB health. Our aim was to assess correlation between the macroscopic and histological grading systems.
Methods and Materials
Osteochondral tissue samples were collected from patients undergoing total knee replacement surgery (TKR). AC was graded macroscopically using the ICRS Cartilage Lesion Classification System (grade 0 (normal) – grade 4 (severely abnormal)). Two osteochondral fragments, one with the lowest grade (most normal) and one with the highest grade (most abnormal) cartilage, were obtained for each TKR patient. Sections were stained with haematoxylin & eosin and safranin O/fast green before histological assessment. AC was graded using the OARSI OA Cartilage Histopathology Assessment System (grade 0 (normal) – grade 6 (deformation)). SB was graded using the SB Histological Grading System (grade 0 (early OA) – grade 3 (late-stage OA)). The relationship between scores was assessed using Kendall’s rank correlation coefficient (Kendall’s tau).
Results
Sixteen TKR patients (6 female, 10 male; 68.2±6.7SD years-old), were included, resulting in the generation of 32 paired osteochondral fragments with the most normal and most abnormal cartilage for each patient. The ICRS score demonstrated moderate-to-strong correlation with both the AC and SB histological scores (Table 1, Figure 1). The AC and SB histological scores were also strongly correlated (Table 1).
Conclusion
Our results suggest that macroscopic cartilage assessment, using the ICRS scoring system, may give an indication of histologically assessed AC and SB health. This study provides further evidence for the close association between the AC and SB in patients with established knee osteoarthritis.
P025 - Severity of Synovitis and Macrophage Phenotypes in Knee Cartilage Defect and Osteoarthritis Cohorts
Abstract
Purpose
Growing evidence suggests that the synovium is important in the pathogenesis of osteoarthritis (OA). Synovial macrophages demonstrate considerable plasticity in mediating joint inflammation and OA progression. In this study we examined the synovia from patients with either cartilage defects or end-stage OA in the knee.
Methods and Materials
Synovial tissues were obtained from patients undergoing cell therapy to treat cartilage defects (at time of cell implantation, ~3 weeks post cell harvest) or arthroplasty surgery. Haematoxylin and eosin-stained sections of synovial tissue were assessed for synovitis using a histological scoring system (Krenn score). Pan-macrophage (CD68) and polarisation markers (CD86:M1 and CD206:M2) were assessed by immunohistochemical staining and subsequent semi-quantitative analysis.
Results
Samples of synovium were collected during surgery from 15 cell therapy (6 female, 9 male; 39.2±2.18SEM years) and 15 arthroplasty patients (8 female, 7 male; 67.3±2.80SEM years). Mean synovitis scores were significantly higher in the cell therapy group cf. the arthroplasty group (Table 1). The mean intensity of CD68, CD86 and CD206 immunostaining was also significantly higher in the cell therapy group cf. the arthroplasty group. The ratio of CD86 to CD206 was significantly higher in the cartilage injury synovia cf. OA synovia. Linear regression analysis of the full dataset indicated that CD86 intensity was a significant predictor of synovitis severity (cor.coeff.=0.052, p=0.016, 95% CI [0.036-0.234]).
Conclusion
Increased synovitis, abundance of macrophages (CD68, CD86 and CD206 positive) and increased CD86:CD206 staining ratio (indicative of M1 favoured polarisation) in synovia from patients undergoing cell therapy for cartilage defects is suggestive of a macrophage-mediated, pro-inflammatory immune response. Whether this response relates to the harvest stage of cell therapy or cartilage defect pathology warrants further investigation. Conversely, the reduced abundance of macrophages in arthroplasty synovia suggests a reduced ability of the synovium to mount an immune response at the end-stage of OA, indicative of organ-level failure within the joint.