Collection of Blood and Anterior Cruciate Ligament Tissue from the Patients
With the approval of our institutional ethics committee, we obtained informed consent from four ACL-deficient patients (two women and two men with a mean age of twenty-seven years [range, eighteen to thirty-five years]) who had sustained the injury an average of twelve months (range, nine to twenty-four months) previously. At the time of ACL reconstruction surgery, blood (54 mL) was drawn from the forearm vein of each patient into a 60-mL syringe and was treated with an anticoagulant, citrate dextrose solution (Boehringer Laboratories, Norristown, Pennsylvania). Removed ACL remnant tissue was used as a source of ACL cells in the following experiments.
Plasma Preparation
Collected blood was brought immediately to the laboratory, and both platelet-rich plasma and platelet-poor plasma were isolated with use of a platelet concentration system (Symphony; DePuy Spine, Raynham, Massachusetts). Briefly, the blood that had been treated with anticoagulant was separated into plasma and hemocyte (erythrocyte and leukocyte) fractions, and the plasma was separated into platelet-rich plasma (containing a high number of platelets) and platelet-poor plasma (containing few platelets) by means of continuous two-step sedimentation. Isolated platelet-poor plasma and platelet-rich plasma were clotted with a 10% thrombin solution (v/v, 1000 U/mL in 100-mM CaCl2) to yield a final thrombin concentration of 100 U/mL, followed by centrifugation (1500 g for five minutes) and separation into fibrin clots and soluble supernatants. These final soluble releasates from platelet-rich clot and platelet-poor clot were frozen and stored at —80°C until used.
Concentration of Growth Factors in the Plasma Releasates
The concentrations of each of the basic growth factors (PDGF-AB, TGF-ß1, VEGF, and EGF) in both platelet-poor clot releasate and platelet-rich clot releasate were determined with use of separate specific enzyme-linked immunosorbent assay kits according to the manufacturer's instructions (Quantikine immunoassay; R&D Systems, Minneapolis, Minnesota). These kits contain specific antibodies directed to the targeted human growth factors. Readings of color absorbance were made with use of a multiple plate reader (Corona Electric, Tokyo, Japan) with wavelength absorption at 530 nm subtracted from readings at a 450-nm wavelength.
Cell Isolation and Culture
Cells were isolated from remnant tissues obtained during ACL reconstruction surgery by means of sequential enzyme digestion with 0.2% Pronase (EMD Bioscience, La Jolla, California) for one hour and 0.025% collagenase P (Roche Applied Science, Indianapolis, Indiana) for sixteen hours at 37°C. After several washes in Dulbecco modified Eagle medium and Ham F-12 medium (DMEM/F12; Mediatech, Herndon, Virginia), the isolated cells were cultured in monolayer in complete media containing 10% fetal bovine serum (FBS; HyClone, Logan, Utah), 25 µg/mL ascorbic acid (Sigma-Aldrich, St. Louis, Missouri), 360 mg/mL L-glutamine (Mediatech), and 50 mg/mL gentamicin (Invitrogen, Carlsbad, California) at 37°C and 5% CO2 in air atmosphere. In all cases, the medium was changed every other day. Platelet-rich plasma and platelet-poor plasma releasates were applied to the ACL cells from the same patient autologously. The ACL cells were cultured in the same manner for each test.
Cell Culture Protocol for the Studies
The cells from the initial culture were used for a cell viability assay, whereas the ones from the second passage were used for collagen and gene-expression assays. After twenty-four to forty-eight hours of preculture in complete medium, the cells were placed in serum-free medium, which consisted of DMEM/F12 with supplements as described above, for twelve hours. The cells were then cultured in serum-free medium with either 5% fetal bovine serum, 5% platelet-poor clot releasate, 5% platelet-rich clot releasate, or 10% platelet-rich clot releasate in SFM (serum-free medium). The cultures were incubated for twenty-four hours, forty-eight hours, and four days for the cell viability assay and for seven days for the measurement of collagen content. ACL cells were also cultured in the medium with 5% fetal bovine serum, 5% platelet-poor clot releasate, or 5% platelet-rich clot releasate for forty-eight hours for gene expression assays. Because of the limited amounts of blood sample and ACL tissue collected from each patient, the 10% platelet-rich clot releasate cells were not assayed for gene expression. The medium was changed every other day for each group.
Cell Viability Testing
A WST-8 assay (Doujin Laboratories, Kumamoto, Japan) was used to quantify cell numbers after twenty-four hours, forty-eight hours, and four days in culture. Ten microliters of tetrazolium salt WST-8 was added to each well in a ninety-six-well plate, and light absorbance was detected with use of an EZS-ABS microplate reader (Iwaki, Tokyo, Japan) at 450 nm after a two-hour incubation. The amount of chromophore that is generated is directly proportional to the number of living cells.
Measurement of Collagen Content
After the incubation with the corresponding media for a week, the cell layer in a 100-mm culture plate was collected with use of a cell scraper and was digested with 0.05-M acetic acid including pepsin (1 mg/mL) (Wako, Osaka, Japan) at 4°C with rotation for three days. After pH was brought to 8.0 with NaOH, each sample was treated with pancreatic elastase (1 mg/mL in 1× TBS [Tris-buffered saline]) (Sigma-Aldrich) at 4°C for two days. The soluble collagen in the supernatant was precipitated with 2.7-M NaCl and then was dissolved again in 0.05-M acetic acid. To measure the concentration of soluble collagen, the Sircol assay (Biocolor, Belfast, Northern Ireland) was performed according to the manufacturer's protocol, followed by reading color absorbance at 540 nm with a micro-well plate colorimeter (Corona Electric). The collagen content was normalized by dividing by the DNA content of each sample as measured with use of the bisbenzimidazole fluorescent dye method (Hoechst 33258; Polysciences, Warrington, Pennsylvania).
Gene Expression of Collagen Types I and III
Following a forty-eight-hour culture in the corresponding media in culture flasks, the cells were collected with use of a cell scraper into micro-centrifuge tubes and were washed with saline solution twice. Total RNA was isolated with use of TRIzol (Invitrogen). The purified RNA was dissolved in DEPC (diethylpyrocarbonate)-treated water, and its concentration was measured with a spectrophotometer (JENWAY, Genova, United Kingdom). One microgram of total RNA was reverse-transcribed into complementary DNA (cDNA) with use of the SuperScript First-Strand cDNA Synthesis system (Invitrogen). All of the required incubations were done with use of a PCR (polymerase chain reaction) thermal cycler (TP600; Takara, Kyoto, Japan). Amplification of the target gene was carried out in triplicate with use of a 7500 Real Time PCR System (Applied Biosystems, Foster City, California) with a pair of primers for human type-I collagen a1 chain and human type-III collagen a1 chain (TaqMan Gene Expression Assays, Inventoried; Applied Biosystems) and a primer for human beta-actin (TaqMan Endogenous Control; Applied Biosystems). Thermal cycling and fluorescence detection with use of SYBR Green PCR Mix (Applied Biosystems), and the quantitative results of real-time PCR were assessed with a cycle threshold (Ct) value, which was normalized to the Ct value of the endogenous gene (beta-actin). The results then were calculated with use of ?Ct of the control (5% fetal bovine serum) as a calibrator.
Statistical Analysis
The experiment was performed in triplicate for all four donor samples. The results for collagen content were assessed by means of relative quantification with use of the fetal bovine serum group as the control group. The values were reported as the mean and the standard deviation (SD) of the results of the four separate cultures. The Kruskal-Wallis H test with the Dunnett test as a post hoc test was used to assess the effects of the treatments on cell viability, collagen content, and gene expression of type-I and III collagen. The level of significance was set at p < 0.05 for the comparison of 5% platelet-rich clot releasate versus 5% platelet-poor clot releasate and the comparison of 5% platelet-rich clot releasate versus 10% platelet-rich clot releasate.
Source of Funding
This work was supported by a research grant from DePuy Japan and in part by Grant-in-Aid for Scientific Research (B) 17390408 from the Japan Society for the Promotion of Science (JSPS). The funds were used to purchase cell culture medium and reagents. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.