The overall goal of this model is to determine the impact of exercise and nutrition on connective tissue synthesis and function. This technique uses human serum which is taken before and after exercise in order to treat 3D tissues, so that we can understand how exercise affects the function of ligaments and tendons. 3D tissue ensuring combines the benefits of well-characterized in vitro assays with the function, morphology and biochemical characteristics of in vivo tissues.
After preparing silicon-coated 35-millimeter plates in advance according to the text protocol, prepare anchor molds by placing one minutien pin in the center of each cylindrical well in the molds. Combine beta tricalcium phosphate and orthophosphoric citric acid solution at one to three grams per milliliter in a plastic weigh boat on ice. Use a plastic cell scraper to vigorously mix the cement.
Triturate the cement to continue mixing, and pipette the mixture into the molds, then centrifuge the filled mold at 2, 250 times g for five minutes. Following the centrifugation allow the brushite cement anchors to set at room temperature overnight. The following morning remove the anchors from the mold and pin two anchors, 12 millimeters apart in each silicon-coated plate.
Sterilize the pinned plates by spraying them with 70%ethanol, filling both the plates and lids, then place the plates into a biological safety cabinet, or BSC. After at least 30 minutes, aspirate the plates and allow them to try in the BSC. Replace the lids and store them in the BSC until needed.
Comply with local biohazard regulations for proper use and disposal of biohazardous material. Maintain sterility and perform construct formation steps in a biological safety cabinet. After preparing primary fibroblasts, reagents and silicon-coated plates with pinned brushite anchors, according to the text protocol, culture the cells in 15 centimeter plates to 70%confluence.
Next, prepare a master mix composed of the following, 681 microliters per construct of cell suspension, 29 microliters per construct of thrombin, two microliters per construct of aprotinin, and two microliters per construct of aminohexanoic acid. After mixing the solution well, add 714 microliters to each pinned plate in a figure-eight pattern around the brushite cement anchors, ensuring that the master mix directly contacts the sides of the anchors. Gently tap each plate to evenly distribute the mix.
The most critical step is forming the cell-seated fibrin gel. Ensure that the cell-seating master mix directly contacts the anchors, and tap the plate to distribute the mixture evenly. One plate at a time, add 286 microliters of fibrinogen in a drop-wise fashion evenly over the plate.
Immediately slide the plate back and forth and side to side to distribute the fibrinogen to form the cell-embedded fibrin gels, then proceed to the next plate. Place the constructs in a sterile incubator maintained at 37 degrees Celsius and 5%carbon dioxide, and incubate them for at least 15 minutes to allow polymerization of the fibrinogen. Prepare two milliliters per construct of feed medium by supplementing growth medium with 200 micromole of ascorbic acid, 50 micromole of Prolene, and five nanograms per milliliter of TGF beta 1.
Use two milliliters of feed medium to cover each construct, and culture the constructs in a sterile incubator maintained at 37 degrees Celsius and 5%carbon dioxide for a total of 14 days, or to the desired endpoint, refreshing the medium every second day with two milliliters of feed medium. To collect human serum, draw a resting blood sample according to the text protocol, then have participants perform a mode of exercise that fits your research goal. Shown here is a 30 minute bout of high-intensity resistance exercise.
Collect a blood sample 15 minutes after the exercise session, then after allowing the blood to clot, spin the sample at 1, 500 times g for 10 minutes. Under sterile conditions, transfer the serum to sterile tubes for media preparation. Following the preparation of ligament constructs, carry out tensile testing using a tensile tester in a PBS bath with reverse molded grips that are coupled to the force transducer.
That holds brushite cement anchors in place during the test. Using digital calipers, determine the length and with of the ligament constructs, and calculate the cross-sectional area of the tissue. Unpin the ligament construct from the plate, and place the anchors in the reverse molded grips, ensuring the construct is submerged in PBS, then adjust the distance between the grips, setting the length of the construct to its initial length.
Begin the test, and strain the construct to failure at a strain rate of 0.4 millimeters per second, or 3%per second. Following the test, process the tissue and carry out collagen quantification of the engineered ligaments according to the text protocol. After one to two days in culture, the ACL fibroblasts have attached to the fibrin gel, extended cell processes, and started to exert traction forces.
Tension is generated between the two anchor points, and the cells align parallel to this axis, and begin to deposit collagen. Shown here is a representative stress strain plot for a ligament tested to failure. In these experiments, human serum was isolated from pre and post-exercise blood samples and used in place of FBS to generate media enriched with the exercise-induced biochemical milieu.
As shown in this graph, after a 14-day culture period the ligament constructs demonstrated a significant increase in both maximal tensile load and collagen content in response to the post-exercise serum. Once mastered, ligament constructs can be made over the course of two days, and will be ready for tensile testing, collagen content quantification, and other biochemical analyses in two weeks. This translational in vitro in vivo model is part of a broader mission to understand how exercise improves the function of ligaments, tendons, and actually, almost every tissue in the body.
So this is a really robust and strong technique where we can determine the collagen content and connective tissue function following treatments with exercise, nutrition, loading, any types of growth factors, all types of different interventions that we can do in humans or animals.
