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Primary culture of chondrocytes after collagenase IA or II treatment of articular cartilage from elderly patients undergoing arthroplasty

Liuliu Xiong
Liuliu Xiong
, 
Meng Cui
Meng Cui
, 
Ziye Zhou
Ziye Zhou
, 
Minchen Wu
Minchen Wu
, 
Quanming Wang
Quanming Wang
, 
Haiyan Song
Haiyan Song
 und 
Lei Ding
Lei Ding
   | 30. Apr. 2021
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Article Category: Technical report
Online veröffentlicht: 30. Apr. 2021
Seitenbereich: 91 - 99
DOI: https://doi.org/10.2478/abm-2021-0011
Schlüsselwörter
© 2021 Liuliu Xiong et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1

Comparison of cell yield between 3 chondrocyte isolation methods. Chondrocytes were isolated from elderly human articular cartilage using 3 methods. We divided cartilage slices in 3 groups: Group I, digested sequentially with pronase E and collagenase IA; Group II, digested with collagenase II only; or Group III, digested sequentially with pronase E and collagenase II. After digestion, total cell number was counted and mean cell yield (No. of chondrocytes per mg wet weight of cartilage) is plotted. We compared the difference in cell yield between Groups I and II with a 2-tailed t test assuming unequal variances. Error bars represent SD. SD, standard deviation.
Comparison of cell yield between 3 chondrocyte isolation methods. Chondrocytes were isolated from elderly human articular cartilage using 3 methods. We divided cartilage slices in 3 groups: Group I, digested sequentially with pronase E and collagenase IA; Group II, digested with collagenase II only; or Group III, digested sequentially with pronase E and collagenase II. After digestion, total cell number was counted and mean cell yield (No. of chondrocytes per mg wet weight of cartilage) is plotted. We compared the difference in cell yield between Groups I and II with a 2-tailed t test assuming unequal variances. Error bars represent SD. SD, standard deviation.

Figure 2

Viability of chondrocytes isolated from 11 cartilage specimens using pronase E and collagenase IA. Cartilage specimens obtained from 11 patients were first incubated with 0.4% pronase E and then with 0.02% collagenase IA. The viability of chondrocytes isolated from each patient is plotted. The long dashed line indicates the mean level of chondrocyte viability (84%) computed from 11 specimens. The short dashed lines indicate one standard deviation from the mean (8%).
Viability of chondrocytes isolated from 11 cartilage specimens using pronase E and collagenase IA. Cartilage specimens obtained from 11 patients were first incubated with 0.4% pronase E and then with 0.02% collagenase IA. The viability of chondrocytes isolated from each patient is plotted. The long dashed line indicates the mean level of chondrocyte viability (84%) computed from 11 specimens. The short dashed lines indicate one standard deviation from the mean (8%).

Figure 3

Representative light microscopic images of monolayer human chondrocytes. (A) Passage 0 human chondrocytes isolated with pronase E and collagenase IA seeded at 2.5 × 105 cells per cm2 and visualized at day 2 post-seeding (4× objective lens, scale bar 200 μm) and (B) detail of the insert in the black square as imaged with a 10× objective (scale bar 50μm). (C) Passage 0 chondrocytes at day 10 post-seeding imaged with a 4× objective (scale bar 200 μm). (D) Passage 1 chondrocytes at day 1 post-seeding imaged with a 10× objective (scale bar 200 μm).
Representative light microscopic images of monolayer human chondrocytes. (A) Passage 0 human chondrocytes isolated with pronase E and collagenase IA seeded at 2.5 × 105 cells per cm2 and visualized at day 2 post-seeding (4× objective lens, scale bar 200 μm) and (B) detail of the insert in the black square as imaged with a 10× objective (scale bar 50μm). (C) Passage 0 chondrocytes at day 10 post-seeding imaged with a 4× objective (scale bar 200 μm). (D) Passage 1 chondrocytes at day 1 post-seeding imaged with a 10× objective (scale bar 200 μm).

Donor patient characteristics and wet weights of cartilage harvested

Patient characteristic No. of patients % of total patients Mean yield of wet cartilage ± SD (mg)
Age (y) 50–59 1 6 3430
60–69 6 33 3760 ± 1489
70–79 8 44 4211 ± 1716
80–89 3 17 2740 ± 1221
Sex Male 6 33 3708 ± 1035
Female 12 67 3804 ± 1781
Diagnosis Femoral neck fracture 11 61 3245 ± 1137
Femoral head necrosis 4 22 3253 ± 765
Hip osteoarthritis 1 6 6450
Knee osteoarthritis 2 11 6370

Donor patient characteristics and chondrocyte yield for each treatment group

Treatment Characteristic No. of patients Chondrocyte yield (mean cells ±SD)
Group I (n = 11): pronase E + collagenase IA Age (y) 60–69 4 2940 ± 629
70–79 6 2390 ± 1050
80–89 1 2130
Sex Male 3 2514 ± 601
Female 8 2586 ± 992
Diagnosis Femoral neck fracture 6 2587 ± 1056
Femoral head necrosis 2 3002
Hip osteoarthritis 1 3030
Knee osteoarthritis 2 1837
Group II (n = 5): collagenase II Age (y) 50–59 1 2729
60–69 1 747
70–79 1 669
80–89 2 789
Sex Male 2 1702
Female 3 773 ± 119
Diagnosis Femoral neck fracture 3 749 ± 133
Femoral head necrosis 2 1738
Group III (n = 2): pronase E + collagenase II Age (y) 60–69 1 1291
70–79 1 170
Sex Male 1 1291
Female 1 170
Diagnosis Femoral neck fracture 2 730

Donor patient characteristics and wet weight of harvested cartilage for each treatment group

Group No. (cartilage treatment; sample size) Patient No. Age (y) Sex Diagnosis Wet weight of cartilage (mg) (mean ± SD)
I (Pronase E + Collagenase IA; n = 11) 1 76 F Left femoral neck fracture 4396 ± 1546
2 62 M Right femoral head necrosis
3 62 F Right femoral head necrosis
4 81 F Right femoral neck fracture
5 77 M Left femoral neck fracture
6 62 F Left femoral neck fracture
7 61 F Right knee osteoarthritis
8 74 M Left femoral neck fracture
9 75 F Left knee osteoarthritis
10 70 F Right hip osteoarthritis
11 79 F Left femoral neck fracture
II (Collagenase II; n = 5) 12 83 F Left femoral neck fracture 2854 ± 876.5
13 67 F Right femoral head necrosis
14 55 M Right femoral head necrosis
15 72 F Right femoral neck fracture
16 87 M Left femoral neck fracture
III (Pronase E + Collagenase II; n = 2) 17 64 M Left femoral neck fracture 2640
18 72 F Left femoral neck fracture
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