We collected 42 samples from 3 different clinics (clinic A: n = 8, clinic B: n = 9, clinic C = 25). The clinically relevant information related to these patients are reported in Fig. 1. Briefly, patients (28% female and 72% male) with a mean age of 32 years (range 18 to 60 years) mostly (56%) had a healthy body weight range (body mass index (BMI): 18.5–25.0 kg/m2). The patients experienced their knee injury from 3 months to up 9 years prior to the intervention. The time of symptom onset ranged between less than six months (14%), six to twelve months (8%) or more than 12 months (78%). The lesions were located either in single compartments (50% patella, 26% condyle, 15% trochlea) or multiple compartments (9%) and generally of severe grade (International Cartilage Repair Society (ICRS) macroscopic score 3–4).
Characterisation of the properties of cartilage and chondrocytes from central and peripheral regions of the cartilage lesion
For a certain number of the collected cartilage samples, we could not perform the full set of characterisations for example due to their limited sizes (see Table 1). Overall, our results (reported in Table 1) showed the following trends: (i) the cartilage samples were highly viable and had heterogeneous properties, (ii) cartilage biopsy quality was the parameter that mainly differed between the peripheral and central samples, while (iv) the inter-clinical variability was relatively low (Table 1). A detailed description of the results is presented in the following sections.
Representative Safranin O pictures of cartilage samples are displayed in Fig. 2A. While similar fractions of peripheral and central cartilage samples had intermediate quality (grade 1 and 2), large differences were observed in the percentage of cartilage samples with grade 0 (5% peripheral vs 32% central) and grade 3 (32% peripheral vs 2% central) (Fig. 2A). Grading of the peripheral cartilage specimens was 1.9-fold higher (p = 0.001) than that of central samples. Inter-clinically, no significant differences in cartilage quality were observed, even if the differences in the quality between peripheral and central sample varied to some extent (histological grading peripheral vs central were 1.9-fold (p = 0.13), 4.2fold (p = 0.006) and 1.5- fold (p = 0.095) respectively for clinic A, B and C) (Fig. 2B).
A more in depth characterisation was performed to assess the presence and expression of additional cartilage makers as well as cartilage-degrading markers on tissues with different histological grades. Immunohistochemical results showed that good quality tissues (grade 2–3) contained more type II collagen and less type I collagen than bad quality cartilage (grade 0–1). Instead, MMP13 was almost solely detected in bad quality cartilage tissues. Also, at gene expression level, a trend towards higher mRNA expression of aggrecan and type II collagen mRNA, but lower versican and MMP13 expression by cells from good quality tissues was demonstrated (Additional file 3). These results indicate the more degenerative status of the bad vs good quality tissues.
Large inter-donor variability in the cellularity was observed (from less than 0.5 to more than 10 million cells/gram of tissue) so that no statistically significant differences (overall and between clinics) in this parameter could be observed (Fig. 2C).
Cell viability was generally high (ranging from 73–100%) in the collected samples. However, statistically significant higher percentage of viable cells were counted in peripheral as compared to central samples (98.6% vs 96.8%, p = 0.006). No statistically significant difference in this parameter was observed between clinics (Fig. 2D).
Proliferation rates of cells were variable (ranging from 0.06 to 0.45 number of doublings per day), therefore no statistically significant differences in this parameter were observed between peripheral and central chondrocytes and among clinics (Fig. 2E).
Chondrogenic capacity of expanded chondrocytes was assessed by culturing the cells in micromass pellets in medium containing TGFβ. Cartilaginous extracellular matrix deposition, visualised by Safranin O staining, demonstrated that peripheral and central chondrocytes exhibit a variable capacity to form cartilaginous tissue. The quality of the tissue was scored using the grading system Bern Score (BS). We observed that both peripheral and central samples generated tissues with bad (BS < 3.0), averaged (BS: 3.0–6.0) and good (BS: >6.0) cartilaginous quality (Fig. 2F). However, a higher percentage of peripheral (vs central) chondrocytes produced pellets falling in the latter, good, category (60% vs 40% respectively). Consequently, BS was higher in tissues generated by peripheral vs central chondrocytes (6.5 ± 0.5 vs 5.2 ± 0.4, p = 0.037). No other statistically significant inter-clinical difference in this parameter was observed (Fig. 2G). Biochemical analyses of the pellets demonstrated a correlation between the BS and the GAG/DNA contents (Fig. 2H). RT-PCR analyses revealed no statistically significant differences in the expression of COL1, COL2 and aggrecan among the groups (data not shown).
Characterisation of the properties of chondrocytes isolated from cartilage tissues of different inflammatory status.
We then investigated whether the aforementioned properties of cartilage tissues/chondrocytes differed in samples derived from joints with different degrees of joint inflammation. For this purpose, synovial tissues were collected from the same joints from which cartilage samples were harvested. The majority of the collected synovium had inflammatory/degenerated appearances. Based on the expression level of IL6 and IL8, however, two inflammatory groups could be defined: high inflammation and low inflammation (differences in the expression levels of IL6 and IL8 in these two groups were 131.7-fold (p < 0.001) and 1081.3-fold (p < 0.001), respectively). Expression levels of IL6 and IL8 in the high and low inflammation groups were comparable to those measured in synoviocytes isolated from osteoarthritic patients (Kellgren and Lawrence grade 2–3) or healthy patients (results kindly provided by Prof. G. Lisignoli, University of Bologna, IT). Biopsies in the high and low inflammation categories were derived from patients with similar age (30 ± 9 vs 33 ± 12 years), time of symptom onset (mainly > 12 months) and severity of cartilage damage (mainly ICRS score 4). Hematoxylin & Eosin staining of the synovial tissues demonstrated the presence of variable amounts of fibroblastic and adipocytic cells in both groups. Instead, inflammatory cells were reproducibly more present in the synovial tissues of the high inflammation group (Fig. 3B). Immonohistochemical analyses showed the presence of IL6 positive areas in the synovium of the high inflammation group (50% of the analysed samples), while no detectable IL6 staining was observed in any synovium of the low inflammation group (Fig. 3C).
No statistically significant differences in any of the investigated parameters between peripheral and central samples were observed in the low inflammation group, probably due to the limited amounts of samples analysed in this group (n = 7, corresponding to 18% of the total). Instead, in the high inflammation group (n = 27), cartilage quality (1.8 ± 0.2 vs 1.0 ± 0.2, p = 0.009) and cell viability (99.5%±0.3% vs 96.8%±1.0%, p = 0.003) were higher in the peripheral vs central samples (Fig. 3C). In addition, we observed a trend towards a higher proliferation capacity (proliferation rate: 0.24 ± 0.03 vs 0.20 ± 0.03, p = 0.120) and lower post-expansion chondrogenic capacity (BS: 6.3 ± 0.6 vs 5.1 ± 0.6, p = 0.099) between central vs peripheral chondrocytes (Fig. 3D).
Characterisation of the properties of chondrocytes isolated from cartilage tissues of different quality.
Considering that the majority of the cartilage samples derived from high inflammatory joints, we decided to only use samples of this group for additional investigations aimed at assessing whether and to which extent chondrocyte phenotype and properties vary according to the quality of the tissue from which the cells were derived.
RT-PCR analyses showed that the phenotype of central chondrocytes did not differ when derived from bad (grade 0–1) or good (grade 2–3) quality cartilage as shown by similar level of the mRNA ratios for aggrecan/versican (Agg/Ver) and type II collagen/type I collagen (COL2/COL1). Contrarily, in peripheral chondrocytes significantly higher mRNA ratios of Agg/Ver (5.2-fold, p = 0.032) and COL2/COL1 (169.6-fold, p = 0.016) were observed for cells from the good vs low bad quality cartilage tissue. Interestingly, peripheral chondrocytes from the good – but not bad – quality cartilage tissues had higher values of both ratios (22.4fold, p = 0.016 and 121.5-fold, p = 0.032, respectively) than the corresponding central cells (Fig. 4A and Table 2). For the central chondrocytes, expression levels of MMP13 and ADAMTS5 also remained unaffected irrespective of the quality of the starting tissue, while these factors were higher expressed by peripheral chondrocytes of bad vs good quality cartilage (31.3-fold, p = 0.008 and 2.1-fold, p = 0.056, respectively). Despite both deriving from good quality starting tissue, the expression of MMP13 and ADMTS5 was, respectively, 19.3-fold (p = 0.056) and 3.0-fold (p = 0.111) higher in central chondrocytes as compared to peripheral chondrocytes (Fig. 4A and Table 2). Noteworthy, despite these detected lower expression levels in peripheral chondrocytes (vs central), they expressed the mRNA of these factors at a higher level than reference chondrocytes from uninjured healthy cartilage and at levels more similar to OA chondrocytes (Fig. 4A).
Among the different properties investigated, cell viability was observed to not significantly differ between chondrocytes (both peripheral and central) in bad vs good quality cartilage tissues, even if trends towards a lower percent of viable cells were seen between central vs peripheral chondrocytes in the bad quality cartilage tissues (Fig. 4B and Table 2). Proliferation rates of central chondrocytes did not differ in cartilage tissues of different quality. Instead, peripheral chondrocytes exhibited a reduced proliferation capacity (1.7-fold, p = 0.041) in tissue with good (vs bad) quality, so that in these good quality tissues this parameter was lower as compared to that of the central chondrocytes (1.5-fold, p = 0.073) (Fig. 4B and Table 2). The post-expansion chondrogenic capacity of central and peripheral chondrocytes was observed not to significantly differ among tissues with different qualities. However, in bad quality cartilage tissue peripheral chondrocytes were observed to have a superior post-expansion differentiation capacity as compared to central chondrocytes (p = 0.047) (Fig. 4B and Table 2).