3.1. Manufacturing of Atelocollagen Scaffolds
The collagen used in this study was derived from bovine knees (Research 87, Boylston, MA, USA). The tissue was sterilely harvested, minced, and solubilized in an acidic pepsin solution. The resulting slurry was frozen overnight at −80 °C and lyophilized under full vacuum at −35 °C for three days and re-hydrated with sterile water to a collagen concentration >10 mg/mL. The same reconstituted atelocollagen slurry was used in all experiments.
The atelocollagen scaffolds were then made by mixing the atelocollagen slurry with HEPES Buffer (Cellgro, Mediatech, Inc, Herndon, VA, USA), and neutralizing to a pH of 7.4 using 7.5% sodium bicarbonate (Cambrex BioScience Walkersville, Inc., Walkersville, MD, USA). The solution was transferred to a sterile 100 mm petri dish, and incubated for 20 min at 37 °C and 5% CO2 to allow for gelation. The resulting gel was frozen at −80 °C overnight and lyophilized.
3.2. Gelatin Scaffolds
Commercially available gelatin scaffolds (Surgifoam® sheets, Johnson and Johnson, Binghampton, NY, USA) of the same thickness of the atelocollagen sheet were obtained, and a sterile one centimeter diameter circular punch was used to cut out individual scaffolds from both materials. Unlike the atelocollagen scaffolds which were not cross-linked, the gelatin scaffolds were chemically cross-linked with an aldehyde during manufacturing.
3.3. Preparation of Fibrin Clot Scaffolds
Fibrin clots were prepared by adding a 500 units/mL thrombin NaCl solution (King Pharmaceuticals, Bristol, TN) to complete medium (Dulbecco’s Minimum Essential Medium (DMEM) (Cellgro, Mediatech Inc., Herndon, VA, USA) supplemented with 10% defined fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1% antibiotic-antimycotic solution (Cellgro, Mediatech Inc., Herndon, VA, USA), to create a 10 units/mL solution. 715 µL of the thrombin solution was transferred to each well of a 24-well plate. 285 µL of a 20 mg/mL fibrinogen solution (Sigma-Aldrich, Saint Louis, Missouri, Product#: F4753) was added to each thrombin aliquot. The plate was mixed on a shaker table at 60 oscillations/min for 30 seconds. The plate was put into an incubator (37 °C, 5% CO2) for 30 min to allow fibrin clot formation. A sterile one centimeter circular punch was then used to cut individual scaffolds.
3.4. Preparation of Blood Clot Scaffolds
A total of 300 mL of whole blood was drawn from two hematologically normal volunteers meeting all criteria of the American Association of Blood Banks (Food and Drug Administration, Center for Biologics Evaluation and Research). Blood was collected in 60 cc syringes with 10% acid-citrate dextrose as an anticoagulant at the Immune Disease Institute (Boston, MA, USA). 120 mL of blood was used for making blood clots while 180 mL was used for making platelet-rich plasma as described in the next section. A solution of 1000 Units thrombin/mL CaCl2 was used to clot the whole blood. 100 µL aliquots of the thrombin solution were placed into wells of a 24-well plate. 900 µL of whole blood was added, and the plate was placed on a shaker table at 60 oscillations/minute for 30 seconds, and then incubated (37 °C, 5% CO2) for 20 min to complete blood clot scaffold formation. A sterile one centimeter circular punch was then used to create uniform individual scaffolds.
3.5. Preparation of Enzyme Stock Solutions
The MMP-1 stock solution was made by combining 50 mg of MMP-1 (Worthington Biochemical Corporation, Lakewood, NJ, product number LS004214) with 200 mL of PBS, yielding a 250 µg/mL (72.2 units/mL) MMP-1 solution. Aliquots of the solution were then frozen at −80 °C in 50 cc tubes.
The elastase stock solution was made by combining 5 mg of elastase (Worthington Biochemical Corporation, Lakewood, NJ, product number LS006363) with 50 mL of PBS, yielding a 100 µg/mL (0.86 units/mL) solution. Aliquots of the solution were then frozen at −80 °C in 50 cc tubes.
The plasmin stock solution was made by combining 1.5 mg of plasmin (Sigma Aldrich Co., St. Louis, MO, USA, product number P1867) with 37.5 mL of ultrapure water to yield a 40 µg/mL plasmin (0.416 units/mL) solution. Aliquots of the solution were then frozen at −80 °C in 50 cc tubes.
3.6. Enzyme Degradation Experiment
Enzymatic degradation rate was determined by measuring the change in surface area of the scaffolds over time. Atelocollagen, gelatin, fibrin clot, and blood clot scaffolds were subjected to each of the enzyme stock solutions. Experiments were run in triplicate. We also observed the response of the scaffolds when exposed to the solvents used to make the enzyme stock solutions (PBS for MMP-1 and elastase; water for plasmin). The scaffolds were placed into wells of a 12-well plate and 1.0 mL of solution was added to each well. The solutions were changed after 12, 24, and 36 h. Digital images were taken with a Canon Rebel XT camera at 12 h, 24 h, and 48 h. Surface area measurements of the scaffolds were determined using the NIH Image-J software (v. 1.38×).
3.7. Cell Source
Human ACL explants were obtained from the knee using sterile technique during ACL reconstruction. After ligament harvest, explants were cultured in completed medium (Dulbecco’s modified Eagle medium or DMEM) containing 4.5 g/L glucose, 10% fetal bovine serum, and 1% AB/AM) which was changed two times per week. When primary outgrowth cells were 80% confluent, they were trypsinized, counted, and suspended in complete medium.
3.8. Measurement of Cell Proliferation in Scaffolds
Cell proliferation was determined by indirectly measuring the change in cell number in the scaffolds between two and ten days, using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Atelocollagen and gelatin scaffolds (n = 6, per experimental group and time point) were placed into the wells of 12-well plates. The scaffolds were seeded with 2.5 × 105 cells by placing 250 µL of the cell solution on top of each scaffold and incubating (37 °C, 5% CO2) for one hour to allow for absorption. After incubation, 0.75 mL of complete medium was added to each well and the plates were placed back into the incubator. Media was changed every other day.
At 2 and 10 days, the MTT working solution was prepared at a concentration of 1 mg/mL in complete medium from a sterile stock MTT solution (Sigma-Aldrich, St. Louis, MO, USA, Cat. #: M5655-500MG). The media was aspirated from each well and 1 mL of the working MTT solution was added to each well. The plates were incubated for 3 h (37 °C, 5% CO2). Subsequently, the excess MTT solution was removed and 1 mL of sterile 1× Phosphate Buffer Saline (EMD Chemicals, Gibbstown, NJ, USA, Cat. #: B10241-34) was added to each well, placed on a agitator table (Fisher Scientific Clinical Rotator, 100 rpm) and left to rinse at room temperature for 30 min. Rinses were repeated until the absorbance readings of the wash were less than 0.100. All PBS was then removed and each scaffold transferred into 3 ml centrifuge tubes. 1mL of a detergent containing 20% aqueous SDS/formamide (1:1 volume ratio) was added to each tube and incubated overnight in a 37 °C water bath. The tubes were vortexed for 5 seconds and then centrifuged for 5 min at 250 × g. Aliquots of the supernatant from each tube (200 µL) were then transferred into a 96-wellplate, and the absorbencies were measured at 562 nm. The cell proliferation assay was carried out after 2 and 10 days of culture, and cell proliferation was determined by calculating the change in absorbance from day 2 to day 10.
Measurement of the contraction of the atelocollagen and gelatin cell-seeded scaffolds was also performed using surface area measurements of the scaffolds at days 1, 2, 5, 6, 7 and 9. Photos of the scaffolds were taken and the area of each scaffold calculated using Image J. Contraction was recorded as the percent change in scaffold area compared to day 1.
3.9. Statistical Analysis
All statistical analysis was performed using SPSS version 16.0. For the enzyme degradation experiments, a mixed model repeated measures ANOVA was utilized. For the cellular proliferation experiments, a two way ANOVA was used with Fisher’s LSD post hoc test to detect significant differences between groups. Results were given as mean ± SD. For all experiments, a p-value < 0.05 was deemed statistically significant.