ACL fibroblasts from skeletally IMMATURE, ADOLESCENT and ADULT pigs were used in this study. All ACL tissues were obtained from animals used in other IACUC-approved studies at Children’s Hospital, Boston. Five animals were used for each age group. ACL tissues obtained from each of the three age groups were placed into explant culture. Primary outgrowth cells were grown to confluence and passaged. Fourth passage cells were used for all assays. Cells from each of the three age groups were seeded on two types of 3D scaffold: (1) collagen hydrogel (COL) without PRP and (2) collagen – PRP composite (CPC), and thus 6 experimental groups (three age groups on each of two scaffolds) were established. Constructs from all the groups were cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM, Mediatech, Herndon, VA) containing 10% fetal bovine serum (FBS, Hyclone Inc, South Logan, UT) and 1% antibiotic/antimycotic solution (AB/AM, Mediatech, Herndon, VA) for 14 days. Scaffold contraction was observed at day 0, 1, 4, 7 and 14. DNA content, cellular metabolic activity and apoptosis within the scaffolds were measured at day 14 using DNA, MTT and TUNEL assays. Collagen expression in ACL cells was also assessed using quantitative real time polymerase chain reaction (RT-PCR).
In the Yucatan mini-pig model, physes close between 14 and 18 months. For this study, immature animals (mean±SD: 8±2.1 months), adolescent animals (mean±SD: 16±2.2 months), and adult animals aged 24 months and greater (mean±SD: 26.1±1.1 months) were used. The status of the physes were verified radiographically in all animals prior to tissue procurement and the immature animals were found to have open physes, the adolescent animals had closed tibial and femoral physes but an unfused tibial tubercle, and the adult animals had a physeal scar but no open physis. The ACL tissues from each animal were harvested from the knees using sterile technique. General anesthesia was induced. Both limbs were shaved, prepared with an iodine preparation, and sterilely draped. An incision 4 cm in length was made at the medial border of the patellar tendon using a No.15 blade. The medial retinaculum was divided at the patellar tendon border. The patella was gently retracted laterally and the fat pad was resected to expose the ACL. Biopsies of ACL tissue were taken from the middle third of the ligament for all of the retrievals. Care was taken to avoid the insertion sites. The ACL tissue was placed into phosphate-buffered saline (PBS) and brought to the sterile hood where it was sectioned into explants.
After harvest, explants were washed three times in 10% Antibiotic-Antimycotic solution (AB/AM) (Mediatech Inc., Herndon, VA) followed by three washes with sterile 1X Phosphate Buffer Saline (PBS) (EMD Chemicals, Gibbstown, NJ) and transferred onto 35 mm well plates with six wells per plate. The explants were allowed to adhere to the plates and then media was slowly added. Explants were cultured in complete media containing DMEM (Mediatech, Inc., Herndon, VA), 10% FBS (HyClone Inc., South Logan, UT) and 1% AB/AM (Mediatech, Inc., Herndon, VA). The explants were maintained in culture, and the media was changed two times per week. When the primary outgrowth cells were 80% confluent, they were trypsinized and frozen until all age groups had been collected for the experiment. Cells were frozen at 1 million cells per milliliter media solution in cryogenic vials and stored at −80°C until use. After defrosting, the cell solutions were seeded sterilely into T-75 flasks with a seeding density of 0.5×106 cells per flask. Complete media was added to complete the volume to 12ml. The cells were maintained in culture with medium changes two times per week until 80% confluence was achieved. Cells were passaged another time to further expand the number of cells. All cells used for this experiment were fourth passage.
Preparation of collagen hydrogel
Acid-soluble, Type I collagen slurry was made by sterilely harvesting bovine knee capsular tissue which was solubilized in an acidic solution as previously described (12
). Collagen content within the slurry was adjusted to 8 mg/ml and neutralized with 0.1M HEPES (Cellgro, Mediatech, Inc, Herndon, VA), 5X phosphate buffered saline (PBS) (HyClone Logan, Utah), and 7.5% sodium bicarbonate (Cambrex BioScience Walkersville, Inc., Walkersville, MD).
Preparation of Platelet-Rich Plasma (PRP)
PRP was prepared from 300mL of porcine blood taken from a single animal. This PRP preparation was used for all experiments. The whole blood was collected in a bag with 10% acid-citrate dextrose at Children’s Hospital (ARCH) in Boston. The blood was transferred into 15ml centrifuge tubes (10 ml per tube) which were then centrifuged for 6 minutes at 150g (GH 3.8 rotor, Beckman GS-6 Centrifuge, Fullerton, CA). The supernatant was aspirated and collected as PRP in a 50 ml tube. The platelet concentration in the PRP was 628×106/ml while in the systemic blood was 224×106/ml.
Construct preparation and cultivation
Cells were resuspended with PBS (collagen group) or PRP (CPC group). 8 mL of each cell suspension was mixed with 8 mL of the collagen hydrogel. The final cell density in the mixture was 5×105 cells/ml. For each group, aliquots of the hydrogel – cell mixture was delivered to 3cm long semi-cylindrical molds with a polyester mesh at each end to anchor the gels. Each construct was placed in culture, warmed in a humidified 5%CO2/37 °C incubator for 1 hour to achieve gelation, and then cultured with completed DMEM media. Media was changed every three days during the culture period. The constructs were assessed on day 14.
Digital pictures of the cultures were taken on days 0, 1, 4, 7 and 14, and construct “area” was measured using Image J software (NIH, Bethesda, MD). Contraction was measured for each construct as the percent decrease in area at day1, 4, 7 and 14 with respect to the time zero value.
DNA content of the constructs was determined by using Quant-iT™ PicoGreen dsDNA Assay Kit at day 14, with type 1 highly polymerized calf thymus DNA as a standard.
Cellular metabolic activity
The 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was performed to obtain the index of cellular metabolic activity. In brief, constructs were rinsed with PBS and incubated in a solution of 0.5 mg/mL MTT in DMEM for 4 h in a 5% CO2/37°C incubator. Constructs were then incubated in a solution of 0.1 N HCl in isopropyl alcohol for an additional 4 hours and the optical density of the resulting supernatant was measured at 570 nm using a microplate reader (Molecular Devices, Sunnyvale, CA).
Apoptosis was assessed using the terminal deoxynucleotidyl transferase biotin 2′-deoxyuridine 5′-triphosphate nick end labeling (TUNEL) assay. In brief, constructs cultured for 14 days were rinsed in PBS, fixed for 24 h in 10% neutral buffered formalin, embedded in paraffin, and sectioned to 6 μm. The sections were stained with a commercial available TUNEL kit (Roche, Indianapolis, IN) according to the manufacturer’s instructions. Subsequently, the sections were treated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes, Carlsbad, CA) to quantify the total number of nuclei. The apoptotic (TUNEL-positive) and total (DAPI-positive) cells were manually counted for the tested specimens from each group, and then the apoptotic cells were expressed as a percentage of the total cells.
Real time - PCR
Total RNA was extracted from constructs using an RNeasy mini kit (Qiagen, CA). Briefly, constructs that had been cultured for 14 days were rinsed in PBS, cut into small pieces, lysed with supplied buffer (Qiagen, USA), and transferred to RNeasy spin columns. RNA concentration and purity were determined at 260 and 280 nm, respectively. The RNA samples were reverse transcribed into cDNA using RETROscript Kit (Ambion, TX) following the supplier’s instructions. Real time - PCR was performed in ABI PRISM 7900 Sequence Detection System (Applied Biosystems, CA) using SYBRGreen PCR Master Mix Kit (Applied Biosystems, CA). Targeted genes were type I and type III procollagens (COL1A1 and COL3A1) and GAPDH was selected as a reference gene. The primer sequences of selected genes for real time - PCR were listed in . The transcript level of target genes normalized to GAPDH was calculated using the 2−ΔCt formula.
Real-Time PCR primer sequences
Descriptive data were summarized as mean ± standard deviation. Differences in group means between samples were assessed using a mixed model analysis of variance (ANOVA) strategy with age (Immature, Adolescent, Adult) and group (CPC, COL) set as fixed factors (37
). Multiple comparisons among each factor were performed with the least significant difference (LSD) procedure to control for comparison wise error rate (38), with values of p
<0.05 considered significant. Statistical analysis was performed using the SPSS statistical package (version 18.0, SPSS Inc./IBM, Chicago, IL).