Tissue Collection and T-LE Allograft Preparation:
Articular cartilage was obtained from human donors deemed eligible for tissue donation for research purposes. Fresh cartilage tissue samples and T-LE Allograft tissue was obtained from 36 to 39-year-old male and female research consented cadaveric human donors which were recovered within 72 hours of death, articular cartilage was harvested from uncompromised regions of weight-bearing joint surfaces. Once collected, all tissue samples were placed in a sterile container with Chondrocyte Growth Medium (CGM) (Cell Applications, San Diego, CA, USA) and used immediately or stored at 4°C until further processing.
To prepare the T-LE Allograft samples, articular cartilage was shaved to a 1mm thickness and punched into disks between 9-20mm in diameter, followed by a proprietary laser etching and cryopreservation process prior to being stored at -80°C for two years until further testing. For fresh articular cartilage, tissue was processed no more than 72h after death for analysis.
For viability testing, twelve T-LE Allografts cryopreserved for 2 years at -80°C and nine fresh articular cartilage specimens were digested in a collagenase digestion solution consisting of CGM, 1 mg/ml collagenase type I (MediaTech, Manassas, VA, USA) and 2 mg/ml collagenase type II (GibCO, Gaithersburg, MD, USA) overnight at standard conditions (37 ˚C; 5% CO2). The collected tissue digests were run through a 40-micron cell strainer to eliminate particulate debris and spun down at 500g for 10 minutes to produce a cell pellet. The cell concentrate produced was then stained with trypan blue, an exclusion dye, at a 1:1 ratio (Invitrogen, Carlsbad, CA, USA) and counted using an automated cell counter (Invitrogen, Carlsbad, CA, USA). Viability results were averaged for each sample and test group, with the standard deviation (SD) reported for each group.
For live/dead staining a 3mm biopsy punch was taken from a T-LE Allograft and placed into 0.5mL of Phosphate Buffered Saline (PBS) (Mediatech, Manassas, VA, USA), with 0.5µL of both calcein AM and ethidium homodimer-1 (Invitrogen, Carlsbad, CA, USA) and added to a 24 well plate, which was then wrapped in foil to protect from light and incubated for 30 minutes at room temperature. Samples were then imaged using a Nikon D-Eclipse C1 Laser Scanning Confocal Microscope (Nikon, Tokyo, Japan).
In Vitro Fixation Outgrowth Assay and Metabolic Testing:
To evaluate the capacity for T-LE Allografts to retain metabolic activity in the presence of a fixation method, an in vitro model was developed to monitor graft tissue outgrowth and metabolism over several time points. First, thin layers of TISSEEL (Baxter, Deerfield, IL, USA) fibrin glue, a standard method of graft fixation in a clinical setting (Supplementary Figure 1A), were placed on the bottoms of a 24 well culture plate followed by promptly placing the tissue graft on the TISSEEL surface and culturing for several week intervals . T-LE Allografts cryopreserved for 2 years at -80°C and fresh non-cryopreserved T-LE Allografts 14 days post date of death (stored at 4°C in media) were utilized to report cellular outgrowth from the graft into the TISSEEL layer in vitro. The metabolic activity of chondrocyte outgrowth and proliferation of the graft was assessed using PrestoBlue™ Dye (Life Technologies, Carlsbad, CA, USA) at 0, 3, 6, 9- and 12-weeks post-fixation. PrestoBlue dye was added at a 1:10 ratio and the samples were incubated for 3 hours under standard conditions. Chondrocytes used for the standard curve of the PrestoBlue assay were produced through a digestion of fresh cartilage tissue in a collagenase digestion solution overnight. These cells were cultured for at least one week and were used within 6 passages. A 100uL aliquot of each sample and known standard was placed in triplicate into a 96-well plate and read using a plate reader at 535 and 615nm. Fluorescent readings were recorded using a Synergy H1 Hybrid Reader (Biotek, Winooski, VT, USA) and quantified using the linear regression fit equation produced from the standard curve.
The viable cell count for each donor was averaged from multiple samples. The donors were then grouped based on preservation method, then the average of the fresh T-LE Allografts (3 donors) and the average of the cryopreserved T-LE Allografts (3 donors) were visualized in a bar graph. The standard deviation was calculated from all data points for each group and was used to calculate the standard error of the mean (SEM) which is shown as plus or minus error bars for each group at each time point.
Fixation Assay Histology and Immunofluorescent (IF) Imaging:
At 12-weeks culture in the fixation assay, explanted T-LE Allografts were fixed with 10% neutral buffered formalin (Richard Allen Scientific, Kalamazoo, MI, USA) for 48 hours, processed, embedded, and cut. The tissue sections were placed on glass histology slides and stained for Safranin-O (IHC World, Woodstock, MD, USA) and Hematoxylin & Eosin (H&E) (VWR, Radnor, PA, USA) according to the manufacturers protocol. The explanted samples were also stained for immunofluorescent imaging with primary-conjugated antibody Collagen-II FITC (Life Sciences Bio, Seattle, WA, USA) and primary-unconjugated antibody Aggrecan (R&D Systems, Minneapolis, MN, USA) samples were counterstained with a PE-conjugated secondary antibody Mouse Anti-Human IgM Fc Fragment (Novus Biologicals, Centennial, CO, USA). The fixed histology sections were blocked with 10% Fetal Bovine Serum (FBS) (Omega Scientific, Tarzana, CA, USA) in PBS for 1 hour. The primary antibodies were added to the samples at a concentration of 1:200 (Collagen II) and 1:100 (Aggrecan) in 1.5% FBS/PBS to incubate overnight at 4˚C and protected from light. The samples were washed and secondary antibody was added at a concentration of 1:100 in 1.5% FBS/PBS and incubated for 2 hours protected from light. The samples were then washed and imaged on a confocal microscope. Histology samples were imaged with a Nikon Eclipse Ci Microscope (Nikon, Tokyo, Japan). Immunofluorescent imaging was performed using a Nikon D-Eclipse C1 Laser Scanning Confocal Microscope (Nikon, Tokyo, Japan).
Growth Factor and Sulfated Glycosaminoglycan (sGAG) Testing:
T-LE Allografts cryopreserved for 2-years and fresh articular cartilage were incubated in CGM with 2% Antibiotic-Antimycotic (GibCO, Gaithersburg MD, USA) for 1 week under standard conditions. Following the cell culture, the cell media supernatant was collected and stored at -80°C for testing. Tissue was homogenized with Cell Lysis Buffer (RayBiotech, Norcross, GA, USA) and Protease Inhibitor Cocktail (Thermo Scientific, Waltham, MA, USA). Homogenized samples were sonicated and placed on ice for 2 hours. Homogenates were passed through a 100-micron cell strainer and centrifuged at 12,000g for 10 minutes. Tissue lysate was collected and stored at -80˚C until further use.
For assessing growth factors, tissue lysates and cell culture supernatants from T-LE Allograft and fresh articular cartilage tissue were analyzed for the activity of immunosorbent antibodies to detect and quantify the protein content of Bone Morphogenic Protein – 7 (BMP-7), Transforming Growth Factor – β1 (TGF-β1), Proteoglycan – 4 (PRG-4), and Basic Fibroblast Growth Factor (b-FGF). T-LE Allografts growth factor profiles were evaluated using ELISA kits (Blue Gene Biotech, Shanghai, China) for TGF-β1, (Wuhan Fine Biotech Co., Ltd. Wuhan, China) for BMP-7, (Cusabio, Houston, TX, USA) for PRG-4 and (R&D Systems, Minneapolis, MN, USA) for b-FGF. ELISAs were performed per the manufacturer’s protocol and the results were read at 450nm with a BioTek Synergy H1 Hybrid Reader. The data was normalized with the weight and volumes of the respective tissue homogenates and cell culture supernatants. For quantifying sGAG, cell culture supernatants were analyzed using a colorimetric assay which employs 1, 9-dimethylmethylene blue to detect sulfated-glycosaminoglycans by providing a label for the sulfated polysaccharides of the proteoglycan. The culture supernatants were analyzed using a BioVision Blyscan Detection kit (BioColor Life Sciences, Carrickfergus, UK). The manufacturer’s protocol for the Blyscan Detection Kit were followed and the absorbance was read at 656nm with a BioTek Synergy H1 Hybrid Reader. Standard curves were created for each assay and used to quantify the growth factors or matrix proteins in each sample, data was then analyzed per manufacturer’s instructions.
After measuring the absorbance for each assay, replicate samples were averaged, and protein concentrations were calculated from the absorbance reading using each assay’s standard curve. The following types of curves were populated for each of the assays: Linear Regression Formula (BMP-7, b-FGF, sGAG), 4-PL Curve (PRG4, the 4-PL curve was constructed using the BioTeK Synergy H1 software and the concentrations were computed from the software), and a Logarithmic Curve (TGF-β). Each sample’s growth factor concentration was normalized using the sample’s initial weight and buffer dilution. A Dixon Q test was used to identify and eliminate outliers (p ≤ 0.05) in each sample group. The concentration values were then plotted on boxplots, the sample number (N) of each group is shown below each boxplot. Due to availability of samples, not all donors could be analyzed for all assays. The p-values were calculated using a Student’s T-test, assuming unequal variance, comparing the mean of the fresh cartilage and the cryopreserved T-LE Allograft. For all analysis, a p-value ≤ 0.05 was considered significant.
BM-MSC Migration and Differentiation Co-Culture
Cryopreserved T-LE Allograft samples and fresh cartilage samples were co-cultured alongside bone marrow-derived mesenchymal stem cells (BM-MSC) to assess the allograft’s ability to signal BM-MSCs through the release of cytokines and growth factors (Supplemental Figure 1B, C). To extract BM-MSCs, vertebral bodies from a research consented donor were ground, washed, filtered, and concentrated to collect a cell pellet, which was then cryopreserved. Prior to use, the cryopreserved cell suspension was thawed in a 37˚C water bath. The cells were then lysed with ammonium chloride lysis buffer (BD Biosciences, San Jose, CA, USA) to remove red blood cells and filtered again through a 40-micron filter. The lysed and strained BM-MSCs were then cultured for the migration and differentiation assays.
For the migration assay, an 8.0µm Transwell® insert (Thermo Fischer Scientific, Waltham, MA, USA) was seeded with 100,000 BM-MSCs and a 10mm biopsy punch of the Cryopreserved T-LE Allograft or fresh cartilage was placed in the bottom of the well to avoid direct contact with the seeded cells. The cells and tissue were co-cultured for 24 hours under standard conditions in Dulbecco’s Modified Eagle Medium (DMEM) (Corning, Corning, NY, USA) supplemented with 2% Antibiotic-Antimycotic (GibCO, Gaithersburg, MD, USA). After 24 hours, the Transwell inserts were fixed with 10% neutral buffered formalin (Richard Allen Scientific, Kalamazoo, MI, USA), permeated with .01% Triton-X (Amresco Chemicals, Solon, OH, USA), unmigrated cells on the top side of the filter were wiped off with a cotton swab and the migrated cells were stained with May-Giemsa Stain (abcam, Cambridge, UK). The underside of the Transwell was imaged with a Leica DM IL LED (Leica Microsystems, Wetzlar, Germany) microscope for visual assessment of cellular migration.
For the differentiation assay, cryopreserved T-LE Allograft samples and fresh cartilage samples were also co-cultured alongside BM-MSC to assess the allograft’s ability to direct the MSCs into chondrogenic lineage. A 24-well plate was seeded with 100,000 BM-MSCs and incubated at standard conditions for 24 hours to allow for attachment. Following BM-MSC attachment, a 6mm biopsy punch of the Cryopreserved T-LE Allograft or fresh cartilage was placed into the 8.0µm Transwell insert (Thermo Fischer Scientific, Waltham, MA, USA) and placed into each well. The cells and tissue were co-cultured for one week under standard conditions in DMEM (Corning, Corning, NY, USA) supplemented with 2% Antibiotic-Antimycotic (GibCO, Gaithersburg, MD, USA), and 10% FBS (Omega Scientific, Tarzana, CA, USA). After 1 week, the plate was fixed with 10% neutral buffered formalin (Richard Allen Scientific, Kalamazoo, MI, USA) for 30 minutes, permeated with .01% Triton-X (Amresco Chemicals, Solon, OH, USA), stained with Alcian Blue (IHC World, Woodstock, MD, USA), and imaged with a Nikon Eclipse Ci Microscope (Nikon, Tokyo, Japan).
Immunogenicity Testing with Flow Cytometry:
Cryopreserved T-LE Allograft samples and samples from fresh cartilage donors were collected for immunogenicity testing. The tissue samples were digested overnight under standard conditions in a collagenase digestion solution. Digestions were spun in a centrifuge at 500g for 10 minutes, aspirated, and then resuspended in CGM. The cells were cultured under standard conditions in CGM until 80% confluency, after which the cells were trypsinized, counted, and diluted in a Fluorescently Activated Cell Sorting (FACS) buffer: 3% FBS (Omega Scientific, Tarzana, CA, USA), PBS (Mediatech, Manassas, VA, USA), 0.1% Sodium Azide (Sigma Aldrich, St. Louis, MO, USA). A concentration of 500,000 cells per 100ul were labeled with 10ul of the appropriate antibody or isotype control. The following labels were assessed: CD44-PE, CD44-PE Isotype Control, CD49a-Pacific Blue, CD49a-Pacific Blue Isotype Control, and CD45-FITC (BD Biosciences, San Jose, CA, USA). Samples were incubated for 30 minutes at 4˚C with the appropriate label and then spun in a centrifuge at 1500rpm for 10 minutes. The supernatants were decanted, and the samples were resuspended in 500µL of FACS buffer. Samples were run on the NovoCyte Flow Cytometer (ACEA Biosciences, Inc., San Diego CA, USA) with an overlay of the respective isotype control or negative control. A minimum of 100,000 events were collected for each specimen. The percent positive for each cluster of differentiation (CD) marker was calculated.
Quality control testing and compensation was performed on the NovoCyte Flow Cytometer prior to sample analysis. Debris was eliminated from the sample through FSC-H vs. SSC-H analysis of the samples. The FSC-H vs SSC-H plot also determined the size and granularity of the cells to ensure that the correct cell population was analyzed. The chondrocyte population for either the stained control or sample was gated using sequential gating methods for the specific expression marker, and a histogram was populated for the sample. The negative control samples were gated at 0.00% and the positive samples were measured as anything beyond that threshold on the X-axis. The sample and the isotype control were overlaid onto the same histogram. The percentage on the histogram represents the percent positive for that specific cluster of differentiation marker in the samples. A sample with a percentage of less than 1% was considered a negatable reading.
Supplementary Figure 1: Experimental design for In Vitro Fixation Model (A), Migration (B) and Differentiation (C) Assays.