A. Design of experiment
Fig. 3. Design of experiment
B. Analysis method Chapter I
1) The gross and SEM images of cell-derived ECM scaffold 2) Characterization of cell-derived ECM scaffold
3) The distribution and morphology of seeded chondrocytes on the scaffolds 4) The gross morphology of the neocartilage tissue
5) The volume and compressive strength measurement 6) Chemical analysis for neocartilage tissue
7) The histological assay with Safranin-O staining 8) The collagen type II immunostaining
Chapter II
1) The gross morphology of neocartilage tissue 2) The volume of neocartilage tissue
3) The histological assay with Safranin-O and Alcian-blue staining 4) The RT-PCR analysis
5) Chemical analysis for neocartilage tissue 6) The collagen type II immunostaining 7) The western blot analysis
8) The compressive strength measurement
Chapter III
1) The gross finding of the cartilage defect with implants 2) The Safranin-O staining of repaired cartilage on defects 3) The ICRS histological score
4) The collagen type II immunostaining of repaired cartilage
C. Preparation of cell-derived ECM scaffold
The construction of cell-derived ECM scaffold was described in our previous study (Jin et al 2007). Briefly, articular chondrocytes were acquired from the knees of 2-3 week-old porcine using a collagenase digestion method (Min et al 1998). The isolated chondrocytes were cultured at a density of 1.9 x 105 cells/cm2 in 100 mm dishes using
Dulbecco’s Modified Egle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics-antimycotics and 50 µg/ml L-ascorbic acid for 3-4 days.
The medium was then removed and the cell layer containing the ECM components was carefully detached from the bottom using 0.05% trypsin-EDTA (Gibco; Grand Island, NY, U.S.A.) and a wide-bore pipette. The cell/ECM membrane was transferred individually in a 50 ml conical tube containing 30 ml DMEM and 5% FBS. The tube was then centrifuged at 600 g for 20 mins to consolidate the membrane into a pellet-type construct. It was then incubated overnight at 37℃ and transferred to a six-well culture plate for extended culture. The constructs were allowed to grow into the cartilage-like tissue for 3 weeks with the culture medium (5 ml) changed three times a week (Park et al 2006). After 3 weeks, the neocartilage constructs were washed in phosphate-buffered saline (PBS) and subjected to the three cycles of freeze (-20°C) and thawing (room temperature) at 12 hr intervals. The constructs were then stored at -20°C
for 1 day and freeze-dried for 48 hrs at -56°C under a pressure of 5 mTorr. A cylindrical form of cell-derived ECM scaffold was obtained by cutting the constructs using a biopsy punch (6 mm in diameter) and trimming the uncut area at the top and bottom layers by less than 1 mm using a clean razor blade (Fig. 4).
D. Characterization of cell-derived ECM scaffold
After fabrication, the color and size of the cell-derived ECM scaffolds were observed by gross images. The micro-structure of the ECM scaffold was analyzed by the images
obtained using a scanning electron microscope (SEM). Briefly, the cell-derived ECM scaffolds were fixed at 4℃ in 2.5% glutaraldehyde prepared in 0.1 M PBS for 2 hr and washed twice in PBS for 1 hr each. Then the fixed samples were dehydrated in a series of ethanol solutions (from 70% to 100%) and cut into pieces of 1~2 mm in size using a razor blade. The cross-sections were coated with gold under 50 mTorr and at 5 mA for 50 sec using a sputter coater (Sanyu Denshi, Tokyo, Japan). The samples were observed with a SEM (JSM-6400Fs; JEOL, Tokyo, Japan) operated at voltage of 5 kV. The cell-derived ECM scaffolds were analyzed by mercury intrusion porosimeter using an AutoPore II 9220 (Micromeritics Co. Ltd., USA) to determine pore size distributions, specific pore area, median pore diameter and porosity. The tensile strength of the cell-derived ECM scaffold (n=5, 6 mm in length, 3 mm in height and 3~5 mm in width, respectively) was measured at rupture point by Universal Testing Machine (Model H5K-T, H.T.E; Salfords, England) with free load of 0.01 N.
E. Seeding rabbit chondrocytes to the scaffold
The cell-derived ECM and PGA scaffolds (6 mm in diameter) were soaked in sterile 70% ethanol for l hr, washed several times in PBS, and immersed in DMEM overnight prior to cell seeding. The rabbit chondrocytes were obtained from New Zealand white rabbits (2 weeks old) and cultured in the same medium as above used for porcine chondrocytes (Min et al 1998). The cells were seeded at passage 1 dynamically in the scaffolds for 1.5 hr using a rotator at 3 x 106 cells/ml of density.
In chapter I: The chondrocytes-seeded scaffolds were cultured for 2 days, 1, 2, and 4 weeks in vitro for analysis with the medium changed 3 times a week. The distribution and morphology of chondrocytes in the scaffolds were observed by SEM as above at 1 day and 7 days culture. The PGA scaffold was used as control.
In chapter II: Specimens of the cell-ECM scaffold constructs were immersed briefly for 48 hrs in the culture media before the nude mice implantation. Under sterile conditions in a clean room, four constructs at a time subcutaneously were implanted in the backs of the eighteen nude mice. Six mice were sacrificed at each time point of 1, 2, and 3 weeks post-implantation. The cell-free scaffold was used as control.
In chapter III: The chondrocyte-seeded ECM scaffold was cultivated in 6-well plates for 2 days, 2, and 4 weeks in vitro before implantation. The empty group was used as control group.
F. The gross morphology and the volume changes of neocartilage tissues
The gross morphology was observed after culturing the chondrocytes-seeded cell-derived ECM and PGA scaffolds. The volume of neocartilage tissue was measured using a computer vision system developed in our laboratory (Choi et al 2006). Briefly, three images of anteroposterior, posteroanterior and lateral views of the neocartilage (256x256 pixels) were obtained on a white background and put into the computer vision system. The sequence of the image processing algorithm was divided into three stages
of image preparation, shape extraction, and surface area measurements. The volume was calculated using the base area, the topmost area, and the height.
G. Compressive strength of neocartilage tissue
The neocartilage tissues were subjected to the mechanical compressive strength test using a Universal Testing Machine (Model H5K-T, H.T.E; Salfords, England). The specimens (n=4) were cut into a uniform disk shape and then placed on a metal plate, where they were pressed at a crosshead speed of 1 mm/min at a free load of 0.001 N.
The individual compressive strengths were calculated at 10% strain point (Jin et al 2007).
H. Chemical assays of neocartilage tissue
The DNA, GAG and collagen contents were determined by digesting dried samples in a papain solution (5 mM L-cysteine, 100 mM Na2HPO4, 5 mM EDTA, and 125 µg/ml papain type III, pH 6.4) at 60°C for 24 hr, followed by centrifugation at 12,000 g
for 10 min. The supernatant was used for assays. The total DNA content was determined using quit-iT DNA assay Kit (Invitrogen Eugene, Oregon, USA,). The GAG content was measured by the dimethylmethylene blue (DMB) colorimetric assay (Shihabi and Dyer 1988) using chondroitin sulfate from the shark cartilage for a standard curve (Sigma, St Louis, MO, USA). The collagen content was measured by the analysis for hydroxyproline (Reddy and Enwemeka 1996). The collagen concentration
was calculated by comparing the data with the optical density of a standard solution of bovine collagen (0~10 µg/ml of tracheal cartilage; Sigma Chemical Co, St Louis, MO, USA).
I. Histology and immunohistochemistry
The neocartilage tissues were fixed with 4% formalin for at least 24 hr. The tissues were then embedded in paraffin and sectioned by 4 µm in thickness. The sections were stained with Safranin O/Fast green or Alcian-Blue stain. For immunohistochemical analysis of type II collagen, the sections were treated with 3% H2O2 for 5 min and reacted with 0.15% Triton X-100 to increase the tissue permeability. Once nonspecific binding was blocked with 1% bovine serum albumin (BSA), the sections were incubated for 1 hr with mouse anti-rabbit collagen type II antibody (1:200; Chemicon, Temecula, CA). Then they were incubated sequentially with biotinylated secondary antibody (1:200) for 1 hr and peroxidase-conjugated streptavidin solution for 30 min (DAKO, Carpentaria, CA, USA) at room temperature. The sections were finally counterstained with Mayer’s hematoxylin (Sigma, St Louis, MO, USA) and mounted with a mount solution prior to microscopic observation (Nikon E600, Tokyo, Japan).
J. RT-PCR analysis
The total RNA from each neocartilage was extracted with Trizol reagent (Gibco;
Carlsbad, CA, U.S.A). Each RNA sample was reverse-transcribed with Superscript First
Standard Synthesis System (Gibco; Carlsbad, CA, U.S.A) to produce complementary DNAs (cDNAs). PCR reactions were preformed with specific primers for types I and II collagens, and cDNA samples. Glyceraldehyde-3-phsphatase dehydrogenase (GAPDH) was used as a housekeeping gene. The system was programmed to run for 35 cycles and the end products were separated with 1.5% agarose gel electrophoresis and visualized with ethidium bromide.
K. Western blot analysis
Synthesis of types II and I collagen in the neocartilage tissues was screened with Western blotting. Total proteins were extracted from the tissues with a lysis buffer of 40 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% Nonidet p-40 (NP-40), 2 µg/ml aprotinin,
2 µg/ml pestetin, 2 µg/ml leupetin, and 100 µg/ml phenylmethylsulfonyl fluoride (PMSF). Calibrated by the BCA method, equal amount of the proteins was loaded and separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were then transferred to a nitrocellulose membrane (Millipore; Bedford, MA, U.S.A.) with a transblot apparatus (Bio-Rad; Hercules, CA, U.S.A.). The blotting membrane was incubated first with a mouse anti-rabbit types II or I collagen monoclonal antibody (Chemicon; Temecula, CA, U.S.A.), diluted at 1:1000 ratio and then rinsed three times with Tris-buffered saline (TBS) containing 0.5%
Tween 20. This was followed by incubation with a secondary antibody, peroxidase-labeled sheep anti-mouse IgG (Lockland; Gilbertsville, PA, U.S.A.). This was
visualized with an ECL kit (Amersham; Buckinghamshire, UK)
L. Experimental design and surgery in rabbit
The experimental protocol was approved by the Institutional Animal Experiment Committee. Eighteen New Zealand white rabbits were used in the study with average weight of 3.0~3.5 kg. The surgical procedures were performed under general anesthesia with katamin and lumpun (ratio 3.5:1.5), including limb preparation and draping. Both knee joints were operated in the same surgery. An arthrotomy was made through a midline longitudinal incision on a medial parapatellar with the patella dislocated laterally to expose the femoral condyles. To create an osteochondral defect, a 5 mm drill was used at the patella groove. There were total 36 condyles that were assigned to four groups including untreated control (group 1) and experimental groups implanted with engineered cartilages cultured in vitro for 2 days, 2 weeks and 4 weeks (groups 2, 3 and 4, respectively). The implants were inserted in the defects and press-fixed without any covers or suture materials. At 1 and 3 months after surgery, the rabbits were euthanized by over-dose injection of Pentobarbital to retrieve the femoral condyles.
M. Histological scoring (ICRS score) in rabbit
To evaluate the quality of the repaired articular cartilage in the defects, the modified version of the histological grading scale was used (Wakitani et al 1994). The scale consists of seven categories and assigns a score ranging from 0 to 18 points (Table 1).
The parameters included such as cell morphology, matrix staining (Safranin-O), structural integrity, surface regularity, thickness of cartilage, regenerated subchondral bone and integration with adjacent cartilage.
Table. 1. ICRS histological scoring system
N. Statistical analysis
Statistical analysis of the experimental data was carried out using a one-way analysis of the variance (ANOVA) for multiple comparisons and a student t test (two-tail) for the pair wise comparisons. Statistical significance was assigned as *p<0.05, **p<0.01 and
***p<0.001, respectively.