Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

The following procedures adhere to a protocol that was approved by the Institutional Review Board of University of California, Davis; consult with the local ethics board prior to beginning research.

1. Isolate Primary Fibroblasts from Human ACL Remnants

NOTE: Maintain sterility and perform all steps in a biological safety cabinet (BSC).

1. Obtain approval from the appropriate ethics review board for the collection and use of human tissues as described below.
2. Prepare 5x antibiotic/antimycotic (ABAM) solution by diluting 100x antibiotic/antimycotic solution in 1X phosphate-buffered saline (PBS)
3. Collect ACL tissue fragments in 5X ABAM solution in a 50 mL conical tube, store at 4 °C until the digestion step. Cut tissue into smaller fragments with a razor blade if necessary to a maximum size of 1 x 1 x 1 cm³.
   CAUTION: Comply with local biohazard regulations for proper use of biohazard material and decontamination and disposal of biohazard waste.
4. Prepare a sufficient volume of collagenase solution to submerge the tissue fragments. Dissolve collagenase type II (1 mg/mL) in high glucose Dulbecco's modified Eagle medium (DMEM) containing 1x penicillin/streptomycin, and 20% fetal bovine serum (FBS) and filter at 0.22 µm.
5. Rinse ACL tissue 3 times in PBS.
6. Digest tissue. Transfer ACL tissue to a new 50 mL conical tube and add a sufficient volume of collagenase solution to submerge the tissue. Incubate at 37 °C overnight (~17 h).
7. Before the digestion duration is complete, prepare growth media (GM) by supplementing DMEM high glucose media with 10% FBS and 100 U/mL penicillin.
8. 15 min before the digestion time is complete, briefly vortex the tissue-containing 50 mL tube 3 times every 5 min.
9. Using a 25 mL serological pipette, triturate the digested tissue vigorously to break the tissue up further and dislodge cells.
10. Centrifuge at 1,500 x g for 5 min. Aspirate the supernatant and resuspend pellet in 10 mL GM.
11. Repeat steps 1.8-1.9 three more times.
12. After the last centrifugation, resuspend the pellet in 5-10 mL GM. Use a small sample of the cell suspension to perform a cell count with a hemocytometer and assess cell viability using Trypan Blue.
13. Plate the cell suspension onto 15 cm tissue culture plates at a density of 3-4 x 10⁵ cells per plate.
14. Culture to 70% confluence in a sterile incubator maintained at 37 °C and 5% CO₂, changing the GM every three days. Use or store (see below) cells within 5 passages.
15. Freeze cells for future use.
    1. Trypsinize cells at 70% confluence as follows. Aspirate media and wash cells with PBS. Add enough pre-warmed (37 °C) 0.05% trypsin to just cover the bottom of tissue culture plates. Place plates in the culture incubator for ~5 min until cells are detached (verify cells are floating using a light microscope). Use a pipette to collect cells and dispense into a Falcon tube.
    2. Centrifuge to pellet cells, and resuspend cells in DMEM high glucose media containing 20% FBS and 10% dimethyl sulfoxide (DMSO). Aliquot cell suspension into cryovials and cool at -1 °C/min for at least 24 h. Store frozen cryovials in liquid nitrogen.

2. Prepare Silicone-coated Plates

1. Prepare 35 mm tissue culture plates: Remove lids and lay out open plates on a flat surface.
2. Mix silicone elastomer kit according to manufacturer's instructions.
3. Use a 10 mL syringe to dispense approximately 2 mL per 35 mm plate.
4. Allow silicone to cure at room temperature for 2-3 days.

3. Prepare Brushite Cement Anchors

1. In advance, prepare silicone reverse-molds containing cylindrical wells for anchor formation. Reverse molds can be made to the specifications of the desired anchor shape and size.
   1. Determine the desired height and diameter of the final anchor. This protocol uses a custom-made mold formed from craft silicone in a 35 mm tissue culture dish in which plastic cylinders of about 3.25 mm in diameter were placed, allowing for a final mold height of about 6.5 mm. Final anchor dimensions are approximately 3-3.5 mm in height and about 3.4 mm in diameter with 1.5 mm pin protruding from the bottom of the anchor.
   2. Add uncured silicone to a 35 mm dish in which the mold will be made. Place plastic cylinders accordingly.
      NOTE: The size of the plastic cylinders will determine the diameter of the final anchors. The placement of the plastic cylinders and the amount of silicone used can be modified to produce anchors of different heights. The thickness between the bottom of the wells in the mold and the bottom of the mold itself will determine how much of the pin can protrude from the bottom of the anchor allowing the anchor to later be pinned securely in the silicone-coated dish.
   3. After allowing the silicone to cure, remove the plastic cylinders and remove the mold from the 35 mm dish.
2. Prepare a 3.5 M orthophosphoric/100 mM citric acid solution. Dissolve 0.961 g citric acid in 11.5 mL orthophosphoric acid. Bring the volume of the solution up to 50 mL with MilliQ water. Store solution at room temperature and protect from light.
3. Prepare molds: Place one minutien pin in the center of each cylindrical well in the molds.
4. Combine β-tricalcium phosphate and orthophosphoric/citric acid solution at a 1 g/mL concentration in a plastic weigh boat on ice.
5. Mix the cement vigorously using a plastic cell scraper.
6. Triturate the cement to continue mixing and pipette mixture into the mold
7. Centrifuge the filled mold for 1 min at 2,250 x g.
8. Allow brushite cement anchors to set at room temperature overnight.
9. Remove anchors from the mold and pin two anchors 12 mm apart in each silicone-coated plate.
10. Sterilize pinned plates by spraying with 70% ethanol, filling both the plates and lids, and placing into a BSC. After at least 30 min, aspirate plates and replace lids, storing in the BSC until needed.

4. Obtain Human Serum

1. Ensure that approval by the appropriate ethics review board has been obtained for this protocol.
2. Ensure that written informed consent has been obtained from human subjects to participate in a given intervention (e.g., exercise, food or drug intervention) that will effect desired changes in serum. Here, we describe the collection at rest and 15 min after resistance exercise.
3. Using a trained phlebotomist, obtain a resting blood sample from a participant by venipuncture into an appropriate evacuated container.
4. Collect a post-exercise blood sample 15 min after having the participants engage in the desired exercise protocol. As previously described¹, use the resistance exercise protocol in this experiment to stimulate an endogenous biochemical response.
   1. Have participants perform five sets of leg press with one-minute rest between sets. Next, have the participants perform a set of knee extensions and a set of hamstring curls consecutively with no rest and then repeat the back-to-back exercises three times with 1 min rest between sets.
5. Allow blood to clot before centrifuging at 1,500 x g for 10 min. Under sterile conditions, transfer serum to sterile tubes for future media supplementation (serum stored at 4 °C) and biochemical analysis (a small aliquot of serum stored at -20 °C).

5. Form Engineered Ligaments

NOTE: In advance, expand primary fibroblasts and prepare silicone-coated plates with pinned brushite anchors.

1. Prepare reagents:
   1. Prepare thrombin. Dissolve bovine thrombin at 200 U/mL in DMEM high glucose media. Filter at 0.22 µm, aliquot, and store at -20 °C.
   2. Prepare fibrinogen. Dissolve bovine fibrinogen at 20 mg/mL in DMEM high glucose media. Incubate for 3-4 h in a 37 °C water bath, swirling every 30 min to aid dissolution. Filter at 0.22 µm (multiple filters may be needed), aliquot, and store at -20 °C.
   3. Prepare aprotinin. Dissolve aprotinin in 10 mg/mL in water. Filter at 0.22 µm, aliquot, and store at -20 °C.
   4. Prepare aminohexanoic acid. Dissolve aminohexanoic acid at 0.1g/mL in water. Filter at 0.22 µm, aliquot, and store at 4 °C.
   5. Prepare ascorbic acid. Dissolve ascorbic acid in DMEM high glucose media at a concentration of 50 mM. Filter at 0.22 µm and store at 4 °C.
   6. Prepare L-proline. Dissolve L-proline in PBS at a concentration of 50 mM. Filter at 0.22 µm and store at 4 °C.
   7. Prepare transformation growth factor-β1 (TGF-β1). Reconstitute TGF-β1 according to the manufacturer's directions at a concentration of 10 µg/mL. Aliquot and store at -20 °C.
2. Determine the number of constructs required for the experiment and ensure sufficient numbers of pinned plates are prepared. Both biological and technical replicates are recommended. In the study¹ highlighted here, use duplicate technical replicates and 12 biological replicates (serum from 12 individuals at rest and after exercise).
   NOTE: Perform the following steps under sterile conditions in a BSC.
3. Expand cells by culturing in 15 cm plates to 70% confluence. 2.5 x 10⁵ cells are required per construct.
4. Trypsinize cells and resuspend in GM at a concentration of 3.67 x 10⁵ cells/mL.
5. Generate a master mix for the number of constructs required: For 1 construct, the master mix contains 681 µL cell suspension (containing 2.5 x 10⁵ cells), 29 µL thrombin, 2 µL aprotinin, and 2 µL aminohexanoic acid.
6. After mixing the master mix well, add 714 µL to each pinned plate in a 'figure 8' pattern around the brushite cement anchors. Ensure that the master mix directly contacts the sides of the anchors.
7. Gently tap each plate to distribute the master mix evenly across the plate.
8. For one plate at a time, quickly add 286 µL fibrinogen in a dropwise fashion evenly over one plate and immediately slide the plate back and forth and side to side over the surface of the BSC to distribute the fibrinogen to form the cell-embedded fibrin gels. Proceed to the next plate.
9. Place the constructs in a sterile incubator maintained at 37 °C and 5% CO₂ and incubate for at least 15 min to allow polymerization of the fibrinogen.
10. Prepare sufficient feed media (FM) for 2 mL per construct. Supplement GM with 200 µM ascorbic acid, 50 µM proline, and 5 ng/mL TGF-β1.
11. Add 2 mL FM to cover each construct. Culture the constructs in a sterile incubator maintained at 37 °C and 5% CO₂ for a total of 14 days or to the desired endpoint, refreshing the media every second day with 2 mL FM

6. Tensile Testing Engineered Ligaments

NOTE: Tensile testing was performed using a custom-built tensile tester in a PBS bath; reverse-molded grips that are coupled to the force transducer hold brushite cement anchors in place during the test.

1. Determine the length and width of the ligament constructs using digital calipers; calculate the cross-sectional area of the tissue.
2. Unpin the ligament construct from the plate and place the anchors in the reverse molded grips, ensuring the construct is submerged in PBS.
3. Adjust the distance between the grips, setting the length of the construct to its initial length.
4. Begin the test: strain the construct to failure at a strain rate of 0.4 mm/s (or ~3%/s).
5. After completion of the test, process the tissue remnants for collagen content (see section 7).
6. From the resultant load-deformation data, calculate stress-strain data and quantify mechanical properties of interest; for example, maximal tensile load, ultimate tensile strength and modulus (i.e., elastic property over a linear region of the stress-strain curve).

7. Quantification of Collagen Content of Engineered Ligaments

1. Remove engineered ligaments from brushite cement anchors and dry at 120 °C for 25 min.
2. Determine the dry mass of constructs and place into individual 1.5 mL tubes. Dry constructs may be stored at room temperature until further processing.
3. To each construct, add 200 µL 6 M HCl. Boil at 120 °C in a heating block for 2 h in a fume hood.
   CAUTION: HCl is highly corrosive and acidic, the use of boil-proof/safe-lock tubes or other method of securing tubes is recommended.
4. Centrifuge the tubes briefly to collect liquid, and leave them uncapped to evaporate at 120 °C in a heating block for 1.5 h in a fume hood.
5. Resuspend the resultant pellet in 200 µL hydroxyproline buffer. Store at -20 °C until needed.
   1. Prepare hydroxyproline buffer. In 300 mL water, add 16.6 g citric acid, 4 mL acetic acid, 11.4 g NaOH and stir until dissolved. pH to 6-6.5 and bring volume up to 500 mL. Add 250 µL toluene as a preservative and store at 4 °C protected from light.
6. Prepare reagents.
   1. Prepare trans-4-Hydroxy-L-proline. Dissolve in water to make a 4 mg/mL solution.
   2. Prepare chloramine-T. Dissolve in water to make a 14.1 mg/mL solution.
   3. Prepare aldehyde-perchlorate. Dissolve 1.5 g 4-dimethylaminobenzaldehyde in 6 mL 1-propanol, 2.6 mL perchloric acid (CAUTION: corrosive, strong oxidizer, use appropriate precautions), and 0.5 mL water.
7. In a set of new 1.5 mL tubes, dilute a sample of each resuspended pellet in hydroxyproline buffer to a volume of 200 µL.
   NOTE: Dilutions may range from 1:4 to 1:50 of sample:buffer depending on the expected collagen content of the sample; thus some trial-and-error testing may be needed to determine a dilution factor that is appropriate for the given sample set (i.e., place the samples toward the middle of the standard curve).
8. Prepare hydroxyproline standards. Dilute hydroxyproline in hydroxyproline buffer (see section 7.5.1) to 80 µg/mL. Perform serial dilutions to make 6-8 200 µL standards between 0-20 µg/mL.
9. Add 150 µL 14.1 mg/mL Chloramine T solution to each standard and diluted sample. Vortex and incubate at room temperature for 20 min.
10. Add 150 µL aldehyde-perchlorate solution to each sample and diluted sample. Vortex and incubate in a heating block at 60 °C for 15 min. Dispose of the excess aldehyde-perchlorate solution as hazardous waste according to local regulations (contains perchloric acid).
11. Allow standards and samples to cool, before aliquoting 200 µL of each, in duplicate, into 96-well plates.
12. Read plate at 550 nm in a spectrophotometer. Dispose of plate and the remaining volume in 1.5 mL tubes as hazardous waste according to local regulations (contains perchloric acid).
13. Calculation of total collagen and collagen fraction.
    1. Convert the absorbance value for each sample to micrograms of hydroxyproline using the hydroxyproline standard curve.
    2. Multiply each well by 2.5 to calculate amount of hydroxyproline in the diluted sample. Recall that only 200 of the 500 µL total mixture (200 µL diluted sample + 150 µL chloramine T +150 µL AP solution) is added to each sample well.
    3. Multiply by dilution factor (Section 7.7) to calculate amount of hydroxyproline in original sample.
    4. Divide by 0.137 to calculate the amount of collagen (assumes that collagen contains 13.7% hydroxyproline²⁰).
       NOTE: Mammalian hydroxyproline abundance in collagen varies slightly across tissues and mammalian species; for example, pig and sheep Achilles tendon contain 13.5 and 13.7% (hydroxyproline mass/dry tissue mass), respectively²¹. Here, use 13.7% to estimate percent hydroxyproline in collagen, which is used to calculate the collagen content of a tissue sample using the following equation:
       
    5. Divide by the dry mass to calculate the collagen fraction and convert to a percentage.
       

8. Quantification of Molecular Endpoints

NOTE: In addition to primary outcomes of tensile testing and collagen content, molecular endpoints can be measured on 2D or 3D tissue to add mechanistic insight. Bioassays can be used to determine molecular endpoints (see the following section below for context The impact of the post-exercise serum milieu on in vitro ligament function).

1. For 3D tissue:
   1. Prepare constructs as per Step 5 above.
   2. After construct treatment/intervention, snap-freeze constructs in liquid nitrogen.
   3. Using a mortar and pestle cooled on dry ice, grind constructs into a powder. Continue at step 8.4/5).
2. For 2D tissue:
   1. Culture human ACL fibroblasts to confluence in a monolayer in six-well plates containing DMEM.
   2. Aspirate DMEM and apply treatment media according to your experimentation strategy (e.g., time course or dose response experiments).
   3. Aspirate treatment media and wash cells with PBS.
   4. Scrape cells to collect using an appropriate extraction buffer/reagent (see next).
3. Protein expression analysis: Use a cytosolic extraction buffer (e.g., 250 mM sucrose, 50 mM Tris pH 7.4, 5 mM MgCl2, and protease/phosphatase inhibitor cocktail) to obtain protein lysates. Perform protein concentration assay and continue analysis according to standard western blot procedures.
4. Gene expression analysis: Isolate total RNA using 500 µL RNA isolation reagent according to the manufacturer's instructions to obtain high-quality RNA. Perform reverse-transcription and real-time quantitative PCR analysis according to standard procedures.
5. DNA isolation: Isolate and quantify genomic DNA using DNA isolation reagent according to the manufacturer's instructions. Quantify DNA concentration by using a spectrophotometer to measure the sample absorbance at 260 nm.