Orthopedic Tendon & Ligament Collagen Scaffold Final Report. Mario Rossi Anne Tucker Brian Ziola. 3 May 2017 Dr. Nasir Projects II

Transcription

1 Orthopedic Tendon & Ligament Collagen Scaffold Final Report Mario Rossi Anne Tucker Brian Ziola 3 May 2017 Dr. Nasir Projects II

2 Abstract We conducted background research in order to find a clinically relevant need. We met with researchers at Beaumont and Providence to discuss some clinically relevant needs. We developed three individual need statements and analyzed them through a need screening. From this, we were able to narrow our list to a single need. The need which we decided to move forward with was that there is a need for a similar biomechanical property, anatomically correct solution in tendon/ligament injury repair. Our solution to this problem uses a tissue engineered approach. Our design utilizes a scaffold made from collagen which mimics the biomechanical properties of the anatomical tendon/ligament. It will follow the same hierarchal structure seen in tendons/ligaments. Contrary to other tissue engineered approaches, our design will not include cells seeded on it. Instead, we plan to coat the scaffold with alginate which contains stromal derived factor 1α (SDF-1ɑ). It is a chemokine which is released in burst fashion at the site of an injury. The SDF-1 is used to recruit stem cells to the area. For our design, it will recruit stem cells to our scaffold after implantation. From there, the cells will attach to the scaffold, differentiate, and proliferate. The goals for our project are to design and create a scaffold that has similar biomechanical properties to that of anatomical tendons/ligaments. Along with that, the scaffold will recruit Mesenchymal Stem cells (MSC) in vitro and will promote cell differentiation and proliferation. promotes cell growth. It will be an alternative to grafting for implantation. Our design was constructed using an electrospinning process to achieve collagen fibers. The alginate with SDF-1 infused was dip coated onto the fiber. Once constructed, and throughout the design fabrication, we had to complete testing to ensure the function and biocompatibility of the design. We conducted a biocompatibility test and received results

3 showing that cells preferred our scaffold and could live on it for prolonged periods of time. We also conducted mechanical testing by tensile loading. The results were less than expected with an elastic modulus between 4-12 MPa and ultimate tensile strength between MPa. To ensure the release of SDF-1 from our scaffold, we conducted a release study and received good results. It was seen that 62% of the 24 hour payload was released within the first 4 hours, showing a burst release profile. Finally, to test the fully constructed design, we conducted a migration study. Results showed that MSC s migrated through the membrane and attached to our scaffold. This shows that the SDF-1 was released and attracted the cells to travel through the small pore membrane, which they otherwise would not do without enticement. Motivation and Background To determine a need, we conducted a lot of background research. We met with researchers from Beaumont and Providence to determine clinically relevant needs. They were good resources because they work closely with physicians and surgeons. They have a strong understanding for where a need exists. The researchers we met with at Beaumont included Dr. Tristan Maerz and Meagan Salisbury. The researcher we met with at Providence was Dr. Therese Bou-Akl. From these meetings, we were presented with several needs which could be met. After observing these needs from our meetings, we then conducted a needs screening to eliminate the needs which we thought were not something we should pursue. We were left with the need for a similar biomechanical property, anatomically correct solution in tendon/ligament injury repair. Literature research was conducted to understand our need better and what the current solutions are.

4 From our literature research, we were able to understand the disease state fundamentals for a tendon/ligament. Both, tendons and ligaments are important to the function of the body by distributing the stress that bones and muscles endure during activity. Tendon s are a connective tissue whose primary function is to connect muscle and bone (Kirkendall). Ligaments are extremely similar to tendons except that they connect bone to bone. Both tendons and ligaments are capable of resisting high tensile forces (Kirkendall). The anatomy of tendons are very similar to that of muscles. The primary unit of tendons are collagen fibers. These primary fibers bunch together to form bundles called subfascicles. These subfascicles then gather together to form secondary bundles called fascicles. Finally, these fascicles then form tertiary bundle groups which compose the tendon (Tendon Anatomy). Figure 1: Image of tendon anatomy form Encyclopaedia Britannica Ligaments and tendons primary function are that of a connective tissue capable of resisting large tensile forces. However, too much stress on the ligaments or tendons can result in catastrophic tears or ruptures. When this happens, the tendons and ligaments can no longer

5 perform their primary function of connecting bone to bone or muscle to bone. This can result in in symptoms such as reduced stability or inability to fully use a certain muscle. Other symptoms could be muscle atrophy, or loss in muscle mass. Without repairing these ligaments or tendons, the body will not be able to perform its full, normal functions. A tendon or ligament rupture occurs when the connective tissue fails and tears completely. Tendon ruptures and ligament tears usually occur due to direct trauma or excessive force on the tendon or ligament. This results in pain, swelling, and loss of muscle or joint function. Tears are most commonly associated with sports. They occur quite quickly but can be preluded by symptoms such as tendonitis. Some of the most common ligament injuries are ACL tears and lateral ankle ligament tears There are around 95,000 ACL ruptures every year in the United States and 23,000 occur a day in the United States, some of which result in torn ligaments. The most common tendon injury is Achilles tendon tears. From the years 2005 to 2011 there were 14,127 Achilles tendon tears in the US alone. After understanding the disease fundamentals, we conducted an analysis of the current solutions. The current solutions can be divided into 2 groups: surgical and non-surgical. The surgical group includes suturing and grafting. Either the two sections of the injured tendon can be sutured back together as one, or a graft from either the patient or a donor can replace the injured one. There are limitations to this approach where the tensile load is not distributed evenly. Also, when grafting there is a risk for immune rejection if taken from a donor or harvest site morbidity if taken from the patient. Non-surgical approaches include icing, pharmaceuticals, and physical therapy. The limitations to this approach are that they are temporary and are unable to repair complex tendon injuries. The non-surgical approach is usually administered first before

6 seeking out surgical repair because they are less invasive and cheaper. Figure 2 presents the process flow for both solution approaches. Figure 2: Existing solutions presented in a flowchart After understanding the current solutions, we conducted a market analysis for our identified need. We found that the current market size for orthopedic soft tissues is $5B. Along with that, a growth to $9.39B is expected by The orthopaedic soft tissue market includes: biceps tenodesis, lateral epicondylitis, gluteal tendon, rotator cuff, epicondylitis, achilles tendinosis, pelvic organ prolapse, gluteal tendon, cruciate ligaments, hip arthroscopy. All of these are associated with tendon/ligament injury and/or repair. It is found that, non-surgical repair is the preferred method. Preventative devices are typically used by athletes or someone who suffered a tendon/ligament injury in the past. They are less effective due to their low usage. Severe injuries can only be repaired by surgical methods. The patient will usually be treated with at-home methods and pharmaceuticals first. If this is not effective, they will add in physical therapy. And, if this is not enough, they will be given a surgical intervention. Hospitals,

7 insurance providers, and doctors like to start with the lower cost methods. Surgery is typically only chosen if necessary to repair the damage due to its cost and invasiveness. From our analysis, we identified the influential stakeholders as patients, athletes, orthopaedic surgeons, and insurance providers. From these stakeholders, we determined the decision makers to be both the surgeons and insurance providers. They were chosen due to their ability to influence the type of treatment the most. Figure 3 shows the current market landscape and where the gap exists. Figure 3: Analysis of soft tissue market The construction of a collagen based scaffold that has similar biomechanical properties has much relevance to the orthopedic soft tissue market. The current method of practice for tendon or ligament injury repair is to either take a tendon graft from another part of the body or to suture the existing tendon/ligament back together, or to use a graft from another person. There are limitations to these current treatment methods because suturing the tendon or ligament back together would make it not distribute tensile load evenly, using a graft from another part of the body cause donor site morbidity, and using a graft from another person has a risk for immune

8 rejection. The reason this approach to tendon/ligament is so important is that it does not use one of the existing methods of treatment, and therefore, does not have the same limitations. Instead this project offers an alternative approach in which the same collagen scaffold is used for everyone. The unique aspect of this scaffold is that there is an alginate layer that contains SDF-1, a chemokine that will attract mesenchymal stem cells to the injury location. This aspect means that no cells will have to be dealt with before implantation. That is, there is only the scaffold preparation that needs to take place. Once the scaffold is inserted it will attract the stem cells to the injury site where the cells will bind to the scaffold and start proliferating and differentiating. The broader aspect of this project is to create a tendon/ligament scaffold that will attract mesenchymal stem cells in-vivo to speed up the healing and recovery process. Methods Equipment and Testing To ensure our design was safe and completed the functions it was designed to do, we had to conduct several test and studies. The first test completed was the biocompatibility test. This was done by placing our cross-linked samples into PDMS wells. They were then seeded with fibroblast cells and left there for three hours. After that, they were transferred into cell culture dishes and were monitored for 3 weeks. Samples were taken during this 3 week period and measured using the spectrophotometer to calculate cell numbers. Confocal microscopy was also done to visualize cells on the surface of the scaffold. The reason for this test was to ensure the biocompatibility of our scaffold after all of the processing was completed. The next major test was the mechanical testing. This was done to understand the tensile strength of the scaffold. We did this by conducting tensile tests to failure on the ESEM mechanical testing setup. To do this, a

9 sample is placed between two moving clamps. As the clamps are pulled apart from each other, the sample is placed under tensile force until it breaks. Another major study was the SDF-1 release study. This study was done to determine the amount of SDF-1 released in vitro and to graph the release profile of the SDF-1 from the alginate coating. This was done by coating samples with the SDF-1 infused alginate and placing them in medium. Samples of the medium are taken at specific timepoints and are then placed into an ELISA kit which measures the amount of the SDF-1 in the samples. The kit is measured in the spectrophotometer to get values for the concentration of SDF-1 present. The final study conducted was the migration study. The study was done to determine if the stem cells would migrate to the scaffold by the release of the SDF-1 from the alginate coating. In order to create our scaffold and test our design, we had to use many pieces of equipment and conduct several tests. We utilized equipment to both fabricate our design as well as to characterize specific parts of our final design. To fabricate our scaffold, we had to lyophilize the concentrated collagen solution we received from Dr. Bou-Akl. This was done by placing the tubes of solution on the freeze dryer. Located in the cell biology research lab on campus. The freeze dryer works by removing the liquid from the solution under a vacuum and it leaves a lyophilized collagen matrix. Another piece of equipment essential to fabricate our scaffold was the electrospinner. The process works by creating a solution of collagen and inserting it into a syringe that will shoot it out towards a collector. In order to attract the solution to the collector, a potential gradient is created of 25kV. The distance between the syringe tip and the collector was 12mm, and the solution concentration was 8% W/V. After spinning, you are left with a thin sheet of collagen fibers which is the peeled and cross-linked to make them

10 stronger and easier to work with. To create our alginate coating, we needed to use benchtop lab equipment such as stir plates, beakers, graduated cylinders, scale, etc. The alginate solution was made by mixing bought alginate with distilled water. To cure the alginate, we used CaCl2 solution. This was created by dissolving the CaCl2 in distilled water. To work with the cells, several specialized pieces of equipment were used. The main one was the laminar flow culture hood. Another was the cell incubator. We also used the centrifuge, micropipettes, transfer tubes, petri dishes, ethanol solution, etc. To measure the cell numbers for the biocompatibility test, we used the spectrophotometer. We also had to use PDMS wells to seed the cells onto the scaffolds for the test. We also looked at the cells on the scaffold using the confocal microscope to get a visual of what was happening. To do the release study, we had to use the spectrophotometer along with an ELISA kit specific to the SDF-1 we were releasing. To complete the migration study, we used a dye kit along with the spectrophotometer to measure the number of cells. We also had to use a trans-well membrane plate in order to separate the cells from the scaffold to ensure the cells were required to migrate onto the scaffold surface by exerting energy. For mechanical testing, we began using the MTS machine located at Beaumont Hospital. We quickly realized that we could not get samples large enough to test using the machine, therefore we completed the rest of the mechanical testing inside the ESEM located on campus. The ESEM was also used for characterization of the effects of cross-linking along with characterization of the scaffold surface.

11 Analysis, Controls, and Results ESEM Characterization ESEM images were taken of the freshly spun electrospun collagen fibers. Figure 4 shows a high magnification image and low magnification image. The low magnification image shows speckles which are assumed to be small chunks of collagen. This suggests that all of the collagen was not dissolved into the solution during electrospinning. This would result in small chunks of collagen coming out of the syringe needle and ending up on the final sample. The high magnification image does a good job showing the depth of the fibers as well as the average diameter which was measured to be less than 1 micron. This varied slightly from sample to sample due to the collagen was slightly different each batch of rat tails that were used to extract the collagen from. Also from the high magnification image, it appears that there is a general trend of alignment. This is due to the collector being on a spinning drum. For our tests, this was turned all the way up (1400 rpm). The faster the speed the more aligned the fibers would be. Figure 4. Freshly electrospun collagen fibers. High magnification (left) low magnification (right)

12 The cross-linked collagen images near the same magnification can be seen below in Figure 5. The cross-linked collagen sample looks as though a piece of paper was crunched up. That is, the sheet of collagen seems to have crunched up onto itself. This makes sense because cross linking should bond the fibers together from various strands. This can be seen in the high magnification image. The fibers appear to have gotten thicker and also there seems to be a random bonding pattern that occurs making it more of mass of collagen than individual fibers. Although, there seems to be some porosity and depth to the cross-linked collagen. Figure 5. Cross-linked collagen samples. High magnification (left) and low magnification (right). The control used for this part of the experiment was comparing the before and after cross-linking images. This was used to see if there was in fact a difference in characteristic between the freshly electrospun collagen and the cross-linked collagen. This was used to verify if the cross-linking procedure actually did something to the fibers. Biocompatibility testing

13 The standard curve that was created to quantify the number of cells that were present on the scaffold during the biocompatibility test can be seen below in Figure 6. From the figure, it is clear that there is a linear correlation between the absorbance value that was received and the number of cells present. Figure 6. Biocompatibility standard curve The linear equation present on the graph was used to take the absorbance values that were received from the alamarblue assay on the collagen scaffolds with the fibroblasts seeded on them. The average number of cells were calculated at each time point: 1 day, 7 days, 14 days, and 21 days. Figure 7 below shows the biocompatibility results. At day 1 there is about 60 thousand cells present on each scaffold wand that value increases over the next two weeks. At day 21 the overall cell count decreases. This could be due to some of the cells died off because the media was not changed on a consistent basis or because as the fibroblasts prepare to excrete new extracellular matrix they slow down proliferation which would cause the decrease.

14 Figure 7. Biocompatibility test results The control which was used in this test was to use a well on the 24-well plate which contained no cells. AlamarBlue assay was put in this well and the same procedure was conducted as if it was a sample well. This was used as a background or what a absorbance reading of the assay that contained no cells should be. In addition to the biocompatibility quantification using the alamarblue assay, confocal microscopy using a live dead assay where Calcein AM and ethidium homodimer were used to have the live cells emit a wavelength of light at one color and the dead cells emit at another. The confocal image below, in Figure 8, shows a day 22 confocal image. This method was used to get an idea of how the cells attached to the scaffolds. There appears to be relatively few dead cells while a lot of alive cells are present. The live cells seem to follow the contours of the scaffold suggesting the are attached to the surface with less cells underneath the surface of the collagen scaffold. Further segment-by-segment analysis would be needed to determine how far the fibroblasts were able to penetrate into the scaffold.

15 Figure 8. Day 22 confocal microscopy image live cells (green) and dead cells (red). SDF-1 Release Study from Alginate For the release study, four collagen scaffolds were coated in alginate that contained an estimated 100 ng of SDF-1. The scaffolds were placed in HEPES release media and 200 ul of this release media was taken out and replaced with fresh media at the following time points: 20 minutes, 40 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours. The taken samples were then analyzed using an ELISA kit. Due to the amount of wells on the ELISA kit plate only three of the four samples were analyzed. As part of the protocol for the plate, a standard curve needed to be created with known amounts of SDF-1. The plate was read using a spectrophotometer. The standard curve can be seen below in Figure 9.

16 Figure 9. Standard curve for release study From this standard curve there is a clear linear relationship concentration and absorbance value. Using this, we were able to take the absorbance values that were received for the collagen sample wells and use the linear equation to calculate the amount of grams of SDF-1 that was in each sample. The average release curve for the three samples can be seen below in Figure 10. Figure 10. SDF-1 release profile from alginate coating From the release profile, 62 % of the 24 hour release amount is released in the first four hours. This is good because we were looking for a more burst release profile for the SDF-1. A better idea of the release profile would have been obtained if 48 hour and 72 hour timepoints were taken; however, due to the cost of the ELISA kit there was only room for one plate in the

17 budget. This meant that testing had to be stopped at 24 hours. This profile is nice because the initial dosage of SDF-1 can be tailored to allow for a specific amount to be released over various timepoints. The control that was used for this test was a background value that contained no SDF-1 was used as the control wells to see how no SDF-1 was read by the spectrophotometer. Mesenchymal Stem Cell Migration Study The mesenchymal migration study was conducted using 4 samples that were coated with alginate that contained an estimated 600 ng of SDF-1. In addition to the sample wells 1 ml of cell culture media was placed into each well. The same 600 ng dosage of SDF-1 was used for the positive control wells that contained only the dosage in 1 ml of cell culture media with no alginate or collagen scaffold. For the negative control well there was just 1 ml of cell culture media in the bottom well with no collagen, alginate, or SDF-1. Fluorescence values were taken using a spectrophotometer at 2, 4, 6, 24, and 48 hour time points. A standard curve, Figure 11, was created using a known amount of cells and measuring the fluorescence value that was given off. From the graph there is a linear relationship between increasing cell number and increasing fluorescence values. Figure 11. Standard curve for migration testing.

18 The standard curve data can be used to convert fluorescence values to a cell quantity value. However, because of the results and the high fluorescence values received for the migration testing, this was never done. The migration testing results can be seen below in Figure 12. Based on this data, a two sample t-test was conducted to see if the there was a significant difference between the values received for positive and negative controls. There was a significant difference (P<.01) between the two. The positive control group that contained SDF-1 was higher at all timepoints than the negative control group. At every timepoint, the sample fluorescence reading was lower than the negative and positive control wells. This could be due to the scaffold being on the bottom of the well which would result in a large void where no cells could get to the bottom of the plate. This would result in a much lower fluorescence value than wells without the collagen scaffold. After 48 hours, there was migration of the negative control group that was present. This could be due to insufficient attachment time. Figure 12. Migration testing results. The control used for this experiment was the negative control wells which were under the same experimental conditions just without the presence of SDF-1. This would be used to see if

19 the cells migrated due to SDF-1 or if they would naturally migrate even without the presence of SDF-1. Mechanical Testing The mechanical testing results can be seen below in Figure 13. The figure shows two samples that were tensile loaded until failure. From the figure, for sample 1, the ultimate tensile strength was just over 1.5 Mpa while for sample 2 it was just under 3 Mpa. The elastic modulus range that was calculated for the samples was 4 Mpa (sample 1) to 12 Mpa (the steeper part of sample 2). Two samples are shown but several were tested. The reason for this is due to the other samples did not have such a clear curve. That is, the individual fibers of the scaffold would pull apart without a clear failure point. The scaffold do not seem to be as mechanically strong as we would have like suggesting that further steps need to be taken to ensure the cross-linking process Figure 13. Mechanical testing results was done correctly or braiding of theses scaffolds together could be done to see how that would affect the mechanical properties. Further only very small cross sectional area samples were

20 tested. It would have been nice to test a bundle of several however due to limited resources this was not possible. Discussion The goal of this study was to create a biocompatible, mechanically strong collagen scaffold that would attract mesenchymal stem cells within the body in order to regrow a damaged tendon or ligament. Over the course of our research, several aspects of our initial design had to be altered due to the resources and time available. The amount of collagen we were able to extract and spin was not large enough to create the anatomically correct tendo structure we initially designed. Due to this we focused focused our attention on the functionality and biocompatibility of the collagen itself as well as the alginate and SDF-1 s ability to recruit stem cells. Our biocompatibility test showed promising results with cells able to grow and proliferate on the collagen scaffold. The alginate, SDF-1 release study showed that the SDF-1 was released in burst fashion as the alginate degraded as we had hoped. Unfortunately, due to the limited number of wells available in our ELISA kit, we were unable to view the completion of the release graph. For this reason it would be interesting to conduct further testing to see how the SDF-1 behaved past the 24 hour mark. We ran into some difficulty with the mechanical testing of the cross-linked collagen sheets. We initially attempted to test the collagen in a MTS machine. The MTS machine was too big for the collagen so we were unable to obtain measurable results. We then attempted to use the mechanical tester in the ESEM. However, the vacuum inside the ESEM dried up our samples. We were able to achieve measureable results by taking the mechanical tester out of the ESEM in order to keep our sample damp as we took the measurements. While our data was measureable, the elastic modulus and tensile strength of the

21 collagen was not any where near that of an actual tendon or ligament. This could be due to the cross-linking method used, the type of collagen used, or the size of the samples. Further testing would need to be completed in order to show if collagen is capable of demonstrating tensile strength similar to that of tendons or ligaments. We were able to conduct a migration study to show if SDF-1 could recruit cells to grow on the collagen scaffold. The results of this study were inconclusive. Further testing needs to be done in order to prove that SDF-1 can truly help to recruit cells to grow on the collagen scaffold. From our study, we were able to prove that cross-linked collagen fibers are biocompatible and a plausible material for a biological scaffold. We were also able to show that alginate can serve as a method the slow release of SDF-1. In future, testing needs to be done to prove that the scaffold is mechanically strong enough to be implanted in the body and that SDF-1 can indeed recruit cells to the scaffold in order to grow a new tendon. Timeline Figure 14: Timeline of project broken down by function

22 Grey cells depict the original allotted time for each function. The green cells represents the extensions needed for certain functions. It is noticed that nearly every function within the validation testing took much longer than originally anticipated.

23 Protocols Electrospinning Operating Procedure Goal: To electrospin collagen in order to create collagen scaffolds. Materials/Supplies: Collagen HFP Deionized water Syringe Electrospinner Procedures: Place 8% weight/volume dry collagen in small test tube Place 2.45 ml of HFP in tube with collagen Place 0.05 ml of deionized water in tube Vortex until the collagen is completely dissolved Place dissolved collagen in syringe on electrospinner Place collection plate opposite syringe Set voltage to around 25 kv and flow rate to around 1-2 ml per hour Spin until desired collagen thickness is achieved or all the collagen is used Cross-linking Operating Procedure Goal: To crosslink the collagen scaffolds to increase their mechanical strength and workability. Materials: Collagen fibers or sheets EDC

24 Acetone Deionized water Sodium phosphate Ethanol Test tubes Procedure: Dissolve 10 mm EDC in 90% acetone Submerge collagen in 90% acetone solution for 24 hours Place collagen in fresh 90% acetone solution for another 24 hours Wash in 50% acetone for 4 hours Wash in 0.1 M sodium Phosphate solution for 2 hours Rinse in deionized water and dry Sterilize in 70% Ethanol for 24 hours Goal: Biocompatibility Testing Operating Procedure To evaluate the proliferation of osteoblast-like cells on electrospun collagen nanofiber scaffolds Timepoints: 1 day 8 days 15 days Groups Crosslinked Fibers (n=6) Materials/supplies: Electrospun collagen nanofiber sheets Dulbecco s Modified Eagle Medium (DMEM) with high glucose, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin PBS 0.25% Trypsin-EDTA

25 Cells: PDMS mold AlamarBlue reagent Live/dead viability reagent Experimental: ~2,000,000 Standard curve: ~250,000 Total: ~2,250,000 Procedures: 1. Cell seeding a. Soak collagen nanofiber scaffolds (1cm long) in ethanol for 1 hour b. Wash scaffolds with DI Water 3 times, then aspirate c. UV sterilize collagen scaffolds and PDMS molds in cell culture hood overnight d. Soak collagen scaffolds in FBS, and incubate for 30 min e. Prepare cells at 100,000/ml concentration. f. Place each collagen scaffold into a well of PDMS mold. Seed 50,000 cells in 500 μl medium to each scaffold sample. 2. AlamarBlue assay a. After overnight incubation, transfer scaffold samples to a low attachment 24-well plate b. At each time point, use 6 samples from each group for alamarblue assay. c. Add 500 μl alamarblue stock reagent to 5 ml culture medium to make alamarblue working solution. d. Add 350 μl working solution to each well containing scaffold samples. Also add 350 μl working solution to an empty well to serve as negative control. e. Incubate 6 hours at 37 C on a shaker plate f. Pipet three 100 μl aliquots from each well into a 96-well plate. Use a spectrophotometer to measure fluorescence intensity and absorbance intensity (@570nm) of each well. g. Rinse samples gently with medium, and continue culture in 24-well plate. h. To make a standard curve, seed cells in a 24-well plate with 25,000, 50,000, 75,000, and 100,000 cells in 500 μl medium. After overnight incubation, add 350 μl alamarblue working solution to each well. 3. Live/dead viability assay a. At each time point, use one sample from each group for live/dead viability assay. b. Prepare 2 μm calcein AM and 4 μm EthD-1 staining solution by dissolving 1 μl calcein Am stock and 4 μl EthD-1 in 2 ml sterile PBS. Cover the tube containing the staining solution with aluminum foil. c. Aspirate medium from the well containing scaffold sample. Wash the scaffold with warm sterile PBS.

26 d. Add 150 μl staining solution to each well. e. Incubate for 15min in the dark. f. View the sample under confocal fluorescent microscope. 4. ESEM imaging a. At each timepoint, fix one sample from each group using 4% glutaraldehyde for 15 mins at room temperature. b. Transfer sample to PBS containing 30%, and store at 4 C. Mechanical Testing Operating Procedure 1. Double-click on the TestWork 4 icon on the computers desktop 2. Opening a test method a. Once logged in, the Open Method dialogue will appear b. Use the pull down arrow to find the specific method to run c. Click on Tensile Ultimate Failure 3. Calibrating the device a. Click on the file menu option Tools the highlight Calibrate b. The device Calibrate dialogue will appear. Select the channel the device is connected to, then select the correct device in the Devices window c. Click on calibrate 4. Meter reset a. TestWork 4 reassign the current value that is read to zero, zero or tare a data channel before running a test so that the test will begin with the value of zero b. Locate the meter for the channel on the test window c. Right-click within the meter and select zero channel d. Never zero or tare a load channel after inserting a test specimen into the fixture, doing so may result in a lower reading than it truly is 5. Choose a cell load a. Click on Edit, go to Method b. Choose Limit Detection, load limit, Default Value c. Change fail to 250N 6. Mount the specimen a. Place sample into a MTS clamp in the MTS machine b. Make sure the sample is lined up straight and not twisted c. Measure the distance between the two clamps (gauge length) with a caliper to make sure it is set to 1 cm 7. Starting test a. Click on the Green Arrow icon button to start the program b. You will be prompted to input data i. Fiber Gauge Length

27 ii. Pre-load Rate iii. Pre-load Stop Load iv. Test Speed c. You can get the parameters needed for your test using a program to calculate to calculate your input data (only need to input your mounted sample length) d. 1% strain/sec is used in this test e. Edit the appropriate information and click ok f. Execution of physical test will begin once all required inputs are edited 8. Save data a. Click review tab, choose test b. Highlight file export preview, click specimen c. Click file and then save d. Change to.csv file to be able to open it in Excel Goal: Migration Testing Operating Procedure To evaluate if Mesenchymal stem cells will be attracted to the SDF-1 that is released from that alginate coating of the collagen scaffolds. Timepoints: T 0 (24 hours after seeding) 2 hrs 4 hrs 6 hrs 24 hrs 48 hrs The cells will be fixed after this time point Groups: Control (n=3) Scaffolds coated with SDF-1 alginate mixture (n=6) Materials: Transwell Membrane system used Corning FluoroBlock inserts in a 24-well plate Cell Stain

28 Vybrant CFDA SE Cell Tracer Kit Cells required Experimental: ~2x10 6 Standard Curve: ~105,000 Total 2.105x10 6 Cell Culture: Culture Conditions: 37C, 5% CO 2 Culture Media Standard Rat MSC Media MSC Growth Media 90% DMEM containing glutamax Low glucose DMEM Glutamax-I supplement Add to media at a concentration of 4 mm (0.02 ml Glutamax per 1mL media) Glutamax original concentrations = 200 mm, dilute in media to 4 mm 10% FBS +1% Penicillin-Streptomycin with Fungizone Low Serum Rat MSC Media MSC growth media 95% low glucose DMEM containing glutamax 5% FBS +1% Penicillin-Streptomycin with Fungizone Protocol Isolate Rat BM mononuclear cells Culture cells for more than 3 days in standard rat MSC media at standard culture conditions Switch cells to low-serum media and culture for 24 hours Stain cells (Vybrant CFDA SE Cell Tracer Kit) Reagent Preparation Dissolve CFDA stock in 90 ul DMSO Generates a 10 mm stock solution Dilute with PBS to reach a working solution of 10 um Add 9.99 ml PBS to a 15 ml conical tube Add 10 ul of 10 mm CFDA stock solution Sonicate 5 min.

29 Detach cells from plates using 1 ml of accutase 7 minute incubation Add 2-3 ml of standard media and transfer top contents of the flask to a 15 ml conical tube Pellet via centrifugation and aspirate supernatant Re-suspend cells in stain working solution (~1 ml per million cells) Incubate 15 minutes at 37C Re-Pellet via centrifugation and aspirate supernatant Re-suspend in 1mL of fresh, pre-warmed media Incubate 30 min at 37C Re-pellet via centrifugation, aspirate supernatant, and resuspend in 1mL of low serum media Take cell count Seed stained cells into transwell inserts Seed 50,000 viable cells in 500 ul of media per well into the bottom well of transwell inserts. Incubate ~24 hours to ensure cell attachment Exchange media in the bottom well transwell chamber Add collagen scaffold to the lower chamber with the SDF-1 alginate coating in 1 ml of fresh media Measure the fluorescent intensity of each well Incubate cells at standard culture conditions At each timepoint Measure fluorescent intensity of each well using spectrophotometer at 492 nm for the excitation wavelength and 517 nm for the emission wavelength Return the plate to the incubator until the next timepoint At the final timepoint Measure fluorescent intensity as discussed above Fix cells by adding 300 ul and 1 ml of 4% glutaraldehyde to the top and bottom wells respectively Incubate for 15 minutes at room temperature Rinse with equal volumes of PBS Seal the plate and store in fridge for later The standard curve is done in a similar fashion as for the biocompatibility standard curve mentioned above.

30 Raw Data Biocompatibility Testing Table 1. Raw absorbance data for biocompatibility test Table 2. Raw cell count data with person who performed task

31 Release Study ELISA Table 3. Raw spectrophotometer data for ELISA kit SDF-1 Release results Migration Testing Table 4. Raw migration study data using spectrophotometer.

32 References 1. By Procedure (Rotator Cuff Repair, Epicondylitis, Achilles Tendinosis Repair, Pelvic Organ Prolapsed, Gluteal Tendon, Cruciate Ligaments Repair, Hip Arthroscopy, Biceps Tenodesis), By Injury Location (Knee, Shoulder, Hip, Small Joints), And Trend Analysis From 2013 To "Orthopedic Soft Tissue Repair Market Size, Share Report, 2024." Orthopedic Soft Tissue Repair Market Size, Share Report, N.p., Oct Web. 27 Oct Shearn, Jason T. et al. Tendon Tissue Engineering: Progress, Challenges, and Translation to the Clinic. Journal of musculoskeletal & neuronal interactions 11.2 (2011): Print. 3. "Research and Markets Adds Report: Orthopedic Soft Tissue Repair Market - Global Industry Analysis, Size, Share, Growth, Trends and Forecast, " PRNewswire. N.p., 25 Mar Web. 27 Oct Kirkendall, DT and Garrett, WE. Function and Biomechanics of Tenodons. Scandinavian Journal of Medicine and Science in Sports. 2(1997): Web. 24 Oct Tendon. Encyclopaedia Britannica. Encyclopaedia Britannica, Inc. 2 Feb Web. 25 Oct Renstrom, Per A.F.H. and Lynch, Scott A. Ankle Ligament Injuries. Brazilian Journal of Sports Medicine. 4.3 (1998). Web. 27 Oct Erickson, Brandon J. et al. Trends in the Management of Achilles Tendon Ruptures in the United States Medicare Population, Orthopedic Journal of Sports Medicine. 2.9 (2014). Web. 27 Oct