Suture Displacement Testing in Uniaxial Skin Loading Jeremy Chin BE 210-101 4/21/2007 BACKGROUND The skin is a highly organized multi-cellular organ whose tensile properties are greatly dictated by its fibrous sub-cellular components. Through uniaxial testing, the influence of structural properties, such as directionality, on mechanical characteristics can be analyzed to better understand failure properties in wound mechanics. Imaging techniques applied to such wound mechanics studies allow a quantitative measure of the mechanical responses, such as strain, of a material, such as stitches in skin, while preserving structural integrity during testing. Past experimentation revealed that original orientation of skin relative to the bone on which it lies affects its structural properties, such as elastic modulus and failure stress, with perpendicular orientations of chicken drumstick skin having higher properties than parallel orientations, though not significantly so (Table 1). Moreover, recent studies state that due to the constant perpendicular orientation to the wound, parallel orientation to loading, and lower force/area parallel to the load in each stitch, the interrupted vertical mattress stitch exhibits significantly greater mechanical properties, specifically lesser strain under loading, than the interrupted horizontal mattress stitch, in fabric (Table 2). In pull-out tests, the ultimate strength (1) and stiffness (2) of vertical mattress stitches was found to be greater than horizontal mattress stitches. Therefore, to better simulate realistic skin loading situations in surgical operations in which a straight wound is sutured, elements of both of these past two investigations will be combined to verify the conclusion reached in the previous suture technique comparison. Skin samples uniformly oriented so that they are loaded perpendicularly to the bone will be stitched together, equal numbers with each type of stitch, and uniformly loaded in an Instron 4444 located in front of a camera, such that skin and suture deformation, but not failure, occurs. The camera will be used to compare the stitch length both before and during loading, such that the average deformation can be determined – from this, suture strain can be calculated and t-tested between the horizontal and vertical mattress stitched groups. OBJECTIVE Again, it has previously been shown that fabric samples sutured with vertical mattress stitches will have significantly less strain, greater ultimate strength (1), and greater stiffness (2) under equal loading levels than horizontal mattress stitches, and that skin oriented perpendicularly to the bone has a greater elastic modulus and failure stress than skin in the parallel direction. Therefore, this experiment will again evaluate the quality of the vertical and horizontal mattress techniques, but instead of using fabric, will make use of real skin samples for better simulation of actual surgical situations in uniaxial loading conditions, since real skin has a much greater overall elasticity qualitatively compared to stiff fabric, which was used previously. Aside from being simply another test to verify previous results, this experiment can quantify whether the difference in suture performance is ultimately significant relative to skin deformation. EQUIPMENT Major Equipment • Instron 4444 with associated clamps, pneumatic system o The Instron is necessary to load the skin samples to a medium point between the no-load position and failure, such that the samples are stretched enough for suture length to change, but not be significantly affected by tearing. • Computers (2, one for Instron, one for camera) o The computers are necessary for the proper operation and readout of the Instron and the camera, as well as the measurement of suture length on the images taken. Lab Equipment • Calipers / rulers o The calipers, rulers, or similar measuring device are necessary for measuring skin sample dimensions before and after cutting to ensure uniform size. They are also needed to quantify thickness for the determination of cross-sectional area, and for calibration of the camera. • Cutting boards o The boards are needed as a firm surface on which to cut and stitch the skin. • Weight set o The weights are required to calibrate the Instron. Supplies • Needles o The needles are necessary for the quick and accurate stitching of the “wound” as skin samples are joined together. • Paper towels o The paper towels keep the skin moist (preventing it from drying out) before it is tested. • Scissors o The scissors are necessary for cutting packaging. • Sharpie pens o The pens are needed to mark the skin samples for identification and for analysis of skin deformation. • String (nylon suture), Coats & Clark Tex 26 continuous filament nylon 6.6 unbonded sewing thread or similar o The suture is needed to secure the samples together to simulate a repaired wound. Newly Purchased Equipment • Canon PowerShot A560 o The camera is necessary for high- resolution, speedy image capture before and during sample loading, with automatic zoom to fill the image with the sample. • ImageJ o This program is required for the measurement of suture and skin deformation. • Maxell AA Alkaline Battery Value Pack o These batteries are required to ensure power for the camera. • Perdue Chicken Drumsticks o These drumsticks are needed as a source of chicken skin. • Slik Tripod with 3-Way Panhead o This tripod is needed to mount the camera on a stable surface for consistent measuring. • X-Acto Knives, No. 1 o These knives are required to cut the chicken skin with speed and accuracy. PROPOSED METHODS AND ANALYSIS Estimated Total In-Lab Time: 5 hours General Protocol: • Calibrate Instron 4444. • Set up camera on tripod, zooming into testing region, and connect to computer. Ensure that camera has sufficient power, and replace batteries if necessary. • Place ruler in clamps and photograph; use ImageJ to determine conversion factor between pixels and inches / centimeters, as well as quantifying accuracy and precision. Establish a consistent edge-based measurement system for highest precision between samples. • Remove chicken skin from 12 drumsticks with X-Acto knives. • Cut each piece of skin into two 1 x 1.5 inch (2.54 x 3.81 cm) rectangles, with the longer dimension being directed perpendicular to the bone; wrap in paper towels to retain moisture. • Using the following suture techniques, use needles and string to stitch two rectangles (the two from each drumstick) together along the 1 inch (2.54 cm) shorter sides, repeating with each skin sample until there are a total of 12 samples, 6 of each type. o Interrupted horizontal mattress: insert suture 1 mm from wound, cross under wound perpendicular to the wound; bring up 1 mm on other side of wound and bring 1 mm over, parallel to wound; insert, cross under wound perpendicular to wound once more; bring up, tie across. o Interrupted vertical mattress: insert suture 1 mm from wound, cross under wound perpendicular to the wound; bring up 1 mm on other side of wound and bring 1-2 mm further parallel to wound; insert, reverse direction under previous suture to 2-3 mm beyond wound on the starting side. • Using the Sharpie pens, mark each sample for identification; also, draw a cross on each rectangle of skin (2 per sample) from the center of each edge across the center to the other side, for skin deformation quantitative analysis. • Place a sample into the Instron and determine the no-load position; using the height-limiting clamp on the frame, determine the clamp position of that no-load position, and then raise the clamp 2 cm (5 mm before average failure rate from past studies), as measured with the ruler. • At the no-load position, photograph each sample, then test each sample by stretching it to that 2 cm limit in the Instron, with a crosshead speed of 20 mm/min, and photographing it at that position. • Using images and calibration data, determine the skin and suture deformation using the ImageJ program to measure the change in the dimensions of the crosses drawn on the rectangles as well as the change in suture length, as well as any change in wound length. • Calculate strain of sutures, and associated skin stress / strain. • Run one-tailed, unequal variance t-tests between horizontal mattress sutures and vertical mattress sutures to determine if significantly higher strain occurred in tissues sutured with the vertical mattress technique. An equal variance test can be used if the difference between variances of the two groups is less than 5%. • Run two-tailed, unequal variance t-tests between the average suture deformations and the average skin deformations for horizontal and vertical mattress samples to determine the magnitude of skin deformation relative to suture deformation. Equal variance testing can be done if warranted by the aforementioned criterion. POTENTIAL PITFALLS / ALTERNATIVE ANALYSIS A major source of error may be the lack of uniformity in the sutures, since if more than one individual performs a particular suture technique, there are likely to be greater differences between samples than the random errors that a single individual would make. It is necessary to ensure that each suture begins and ends on approximately the same horizontal axis, and that each section of tissue overlaps equally. Thus, having one group member responsible for all stitching of a particular type can ensure the greatest likelihood of uniformity of samples, to prevent nonuniform loading on each suture once deformation occurs. Since experimental inconsistency can result from variations in the exact force with which the tissue samples are knotted and tied together, such that the force on each hole made by the sutures varied, use of the same length of string and ensuring a uniform length pulled through each knot for each stitch can help to reduce variations in tension. To facilitate the best determination of deformation, the averages of all stitch lengths before and during loading can be used instead of relying on just one shortest or longest stitch, which also helps compensate for the varying tension. Further error can stem from the inaccuracy of the ruler used for calibration, on which the millimeter markings may be longer than 1 pixel in the image taken. There may be inherent error as certain known lengths may display a range of pixel counts, rather than one value each time a measurement is taken. By using a camera with a resolution of 3072 x 2304 pixels, there is a maximum of 3072 pixels of height, if the camera is rotated, in which to quantify the deformation experienced by the samples. The high resolution maximizes the precision by which changes in stitch length and skin length / width can be determined, since each pixel represents a smaller length increment on the sample. Focus issues may also affect the system precision, since focus is achieved by adjusting the focal length of the camera lens and thus slightly shrinking the effective viewing area. Having an autofocus camera with a sophisticated processor minimizes the possibility of out-of-focus images, especially when mounted on a stable tripod, and aperture and shutter settings can be adjusted for best quality. Using a consistent measuring technique, such as going from the upper edge of a marking / suture / edge on the bottom to the lower edge of any marking on the top will also allow for an improvement in data precision. Although any one-view method will make it impossible to measure both horizontal mattress stitches’ horizontal and vertical stitch lengths simultaneously, as they are on opposite sides of the samples, the increased precision of the imaging system will allow for quantification of any deformation in the horizontal direction between samples, whether in the skin or sutures. By having a total of six samples of each type for each suture type per group, the sample size is likely to insulate against wide variation in skin properties, especially when comparing results with other groups’ data. BUDGET Purchases: Canon PowerShot A550 ($198.00) – Amazon.com (www.amazon.com) • Digic III processor, 7.1 Megapixel, 4X optical zoom, 4X digital zoom • Uses 2 AA batteries • Comes with ZoomBrowser EX camera control software ImageJ (free) – http://rsb.info.nih.gov/ij/index.html • Free, downloadable Java-based image analysis program Maxell AA Alkaline Battery Value Pack ($6.99) – Amazon.com (www.amazon.com) • 20 batteries per pack Perdue Chicken Drumsticks ($4.49 / package) – Amazon.com (www.amazon.com) • 6 chicken drumsticks per package Slik Tripod with 3-Way Panhead ($19.95) – Amazon.com (www.amazon.com) • Extendable, lockable tripod with maximum height of 59.5” X-Acto Knife, No. 1 ($4.99) – Amazon.com (www.amazon.com) • 5 No. 11 blades per package Each group performing the experiment will receive 2 packages of drumsticks. Total Costs: Item Quantity Price Per Unit Total Cost Canon PowerShot A560 1 $198.00 $198.00 ImageJ 1 free free Maxell Battery Value Pack 1 $6.99 $6.99 Perdue Chicken Drumsticks 40 $4.49 $179.60 Slik Tripod 1 $19.95 $19.95 X-Acto Knife 5 $4.99 $34.95 Total ------$457.45 REFERENCES 1. Asik M, Sener N, et al. “Strength of different meniscus suturing techniques.” Knee Surgery, Sports Traumatology, Arthroscopy. Volume 5, Number 2 / April, 1997. 80-83. 2. Becker R, Starke C, et al. “Biomechanical Properties Under Cyclic Loading of Seven Meniscus Repair Techniques.” Clinical Orthopaedics & Related Research. 400: 236-245, July 2002. APPENDIX Loading Direction Sample Failure Stress (N/mm2) Elastic Modulus (N/mm2) 1 1.57 8.79 2 0.57 4.24 Parallel 3 0.63 4.34 4 0.81 3.27 5 1.47 5.74 Mean 1.01 ± 0.48 5.28 ± 2.15 1 2.77 10.51 2 1.61 4.90 Perpendicular 3 1.04 7.79 4 2.11 10.33 5 1.02 4.55 Mean 1.71 ± 0.74 7.62 ± 2.85 Table 1 Skin sample data. One-tail t-testing of failure stress (parallel vs. perpendicular skin) had a p-value of 0.059, while one-tail t-testing of elastic moduli (parallel vs. perpendicular skin) had a p-value of 0.094. Trial Vertical Mattress Suture Strain (cm) Horizontal Mattress Suture Strain (cm) 1 0.291 0.334 2 0.208 0.436 3 0.181 0.508 4 0.142 0.561 Average 0.205 0.460 Std. Dev. 0.063 0.098 Variance 0.251 0.313 Table 2 Suture testing data. One-tailed, unequal variance t-testing of the strain provided a p- value between horizontal and vertical mattress stitches (for vertical suture strain < horizontal suture strain) of 0.00024.