1. More cartilage specimens were harvested from younger or male patients or tibial plateau than ones from older or female patients or femoral head.
As summarized in Table 1, four of the 24 enrolled patients, accounting for 17% of the cohort, were in the 50–59, 70–79, and 90–99 age groups, respectively. Six of the enrolled patients, accounting for 25% of the cohort, were in the 60–69 and 80–89 age groups, respectively. Among those age groups, the most cartilage specimens were harvested from the youngest group, 50–59 years old, which were 11,129 ± 3,823 mg per patient in average. The yield of cartilage specimens decreased with the increase of patients’ age.
Seventy-nine percent of the enrolled patients were women, which was 3 times more than men. The average yield of cartilage specimens per male patient was 11,247 ± 4,777 mg. This value was much higher than that harvested from female patients, which was 6,958 ± 2,939 mg per patient.
Two thirds of cartilage specimens were harvested from femoral head of patients diagnosed with femoral neck fracture while only one third from tibial plateau of patients with late stage OA. The average wet weight of cartilage specimens from the femoral head was 6,067 ± 2,034 mg per patient, which was less than that from tibial plateau (11,420 ± 3,869 mg per patient).
2. Without pre-treatment of pronase E, cartilage slices could not be completely digested by collagenase IA.
When cartilage slices were not pre-treated with pronase E, even after 16 hours of incubation with 0.02% collagenase IA, around half of cartilage slices still remained undigested (Fig. 1A). However, when cartilage slices were firstly incubated with 0.4% pronase E for 90 min and then with 0.02% collagenase IA for 16 hours, cartilage slices were barely visible to the naked eye (Fig. 1B).
3. Without pre-treatment of pronase E, cartilage slices yield significantly lower number of chondrocytes.
When cartilage slices were not treated with pronase E prior to collagenase IA incubation, the average yield of chondrocytes was 1,895 ± 688 chondrocytes per mg wet cartilage. However, when cartilage slices were firstly incubated with 0.4% pronase E and then with 0.02% collagenase IA, the average yield of chondrocytes increased to 3,399 ± 1,637 chondrocytes per mg wet cartilage. This increase of chondrocyte yield was statistically significant (P = 0.0067) (Fig. 2). Chondrocyte yield in subgroups divided by age, sex, and diagnosis of patients was detailed in Supplemental table 2.
Supplemental table 2. Chondrocyte yield in each enzymatic treatment group and subgroups divided based on patient characteristics.
Enzymatic treatment (N) | Characteristics of patients | # of patients | Chondrocyte yield (# of cells/mg wet cartilage) (Mean ± SD) |
Pronase E + collagenase IA (N = 19): | Age (yrs) | 50–59 | 3 | 3,519 ± 1,500 |
60–69 | 3 | 3,005 ± 1,960 |
70–79 | 4 | 4,342 ± 1,579 |
80–89 | 6 | 2,828 ± 2,077 |
90–99 | 3 | 3,555 ± 671 |
Sex | Male | 4 | 3,802 ± 1,577 |
Female | 15 | 3,291 ± 1,689 |
Diagnosis | Femoral neck fracture | 13 | 3,072 ± 1,742 |
Late stage knee OA | 6 | 4,107 ± 1,218 |
Collagenase IA only (N = 5): | Age (yrs) | 50–59 | 1 | 1,269 |
60–69 | 3 | 1,893 ± 743 |
90–99 | 1 | 2,526 |
Sex | Male | 1 | 2,750 |
Female | 4 | 1,681 ± 571 |
Diagnosis | Femoral neck fracture | 3 | 1,762 ± 671 |
Late stage knee OA | 2 | 2,094 ± 928 |
3. Chondrocytes isolated from cartilage slices with pronase E pre-treatment showed higher viability than those from cartilage slices without the pre-treatment.
With pronase E pre-treatment, the percentage of viable chondrocytes ranged from 91–98%. Chondrocytes with lowest viability (91%) were isolated from a 74 years old female patient and chondrocytes with highest viability (98%) were from a 73 years old female patient, respectively. In average, the viability of chondrocytes isolated from cartilage slices with pronase E pre-treatment followed by collagenase IA treatment was 94% ± 2%. In contrast, the average chondrocyte viability dropped to 86% ± 6% when cartilage slices were not pre-treated with pronase E. This viability decrease was statistically significant (P = 0.03) (Fig. 3).
4. Chondrocytes isolated from cartilage slices with pronase E pre-treatment grew in monolayers while chondrocytes from cartilage slices without pronase E pre-treatment could not grow in the same plane in a 2-D culturing system and showed increasing cell death with time.
After being seeded at 2.5 × 105 cells/cm2 inside a 12-well culture plate, chondrocytes isolated from cartilage slices with pronase E pre-treatment grew in monolayer manner and reached confluency at Day 3 post-seeding. Typical morphology of articular chondrocytes that is either rounded or polygonal was observed when examined with a light microscope (Fig. 4A-C). However, chondrocytes isolated from cartilage slices without pronase E pre-treatment could not fully attach to culture plates to form a monolayer even after being seeded for 5 days. Moreover, tissue debris and floated cells were apparent and seemed to increase with time (Fig. 4D-F).
5. Articular chondrocyte phenotype of cells isolated from cartilage slices with pronase E pre-treatment was determined by expression ratio of collagen type II to I.
At day 5 post-seeding, monolayer chondrocytes isolated from cartilage slices treated with 0.4% pronase E and then 0.02% collagenase IA were lysed and examined for expression of collagen type I and II relative to 18S rRNA. The average ratio of collagen type II to I mRNA expression in those cells was 13.2 ± 7.5 (Fig. 5). This ratio indicated that the phenotype of those cells was articular chondrocytes which are the only cell type in human tissues expresses collagen type II and the expression level is way above collagen type I.