Additional file 1 of Anterior cruciate ligament microfatigue damage detected by collagen autofluorescence in situ
Creators: name: Kim, Jinhee
name: Baek, So Young
name: Schlecht, Stephen H.
name: Beaulieu, Mélanie L.
name: Bussau, Lindsay
name: Chen, Junjie
name: Ashton-Miller, James A.
name: Wojtys, Edward M.
name: Banaszak Holl, Mark M.
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Space Science
subject: Physical Sciences not elsewhere classified
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Physiology
subject: Biotechnology
subject: Environmental Sciences not elsewhere classified
subject: Developmental Biology

description: Additional file 1: Table S1. Peak cumulative kinematic measures for each knee. Figure S1. Imaging processing steps of CLEM AF images for fiber orientation distribution and coherency analysis with Fiji and representative results. Left: ImageJ macro code for image processing steps. Right: vector field map shows orientation of vectors aligned with outlines of fibers where the longer length indicates higher coherency. Orientation map shows fiber orientation indicated by color legend (Scale bar: 100 μm). Figure S2. SHG and AF images of control and tested (100 cycles) paired knee specimens P2 and P3 using a benchtop CMM. Red dashed boxes (100 × 100 μm2) indicate regions of interest (ROI) used to quantify SHG and AF intensity. Figure S3. Changes in AF intensity and coherency as a function of total cycles in single knee specimens. a) Specimen S1, b) Specimen S2, c) Specimen S3, and d) Specimen S4. The AF intensity increases with increasing loading cycles while the coherency decreases, except for specimen S4. The color of circles indicates the progression of loading cycles, where the darker blue indicates higher total cycles. Figure S4. Single knee specimen changes in AF intensity and depth of image acquired as a function of total cycles using CLEM. a) Specimen S1, b) Specimen S2, c) Specimen S3, and d) Specimen S4. No significant trend is shown. Total cycles are normalized to indicate the end of the cycle as 1. Figure S5. Specimen S1 AR and CLEM AF image of ACL before load (a, c, e) and after 5 pre-load (b, d, f). Before load a) AR image showing CLEM probe placement for AF image capture at distal (green; c, d) and proximal (blue; e, f) regions of ACL. Midsubstance region was unable to be imaged due to tissue damage. Images brightness and contrast are adjusted for better visualization. Scalebar 100 μm. Figure S6. Specimen S2 AR and CLEM AF image of ACL before load (a, c, e, g) and after 5 pre-load and 4 cycles (b, d, f, h). Before load a) AR image showing CLEM probe placement for AF image capture at distal (green; c, d), midsubstance (red; e, f) and proximal (blue; g, h) regions of ACL. Images brightness and contrast are adjusted for better visualization. Scalebar 100 μm. Figure S7. Specimen S3 AR and CLEM AF image of ACL before load (a, c, g, k), after 5 pre-load (d, h, l), additional 33 cycles (e, i, m) and up to 45 cycles (b, f, j, n). Before load a) AR image showing CLEM probe placement for AF image capture at distal (green; c, d, e, f), midsubstance (red; g, h, i, j) and proximal (blue; k. l, m, n) regions of ACL. Images brightness and contrast are adjusted for better visualization. Scalebar 100 μm. Figure S8. Specimen S4 AR and CLEM AF image of ACL before load (a, c, g, k), after 5 pre-load and additional 33 cycles (d, h, l), up to 66 cycles (e, i, m) and up to 100 cycles (b, f, j, n). Before load a) AR image showing CLEM probe placement for AF image capture at distal (green; c, d, e, f), midsubstance (red; g, h, i, j) and proximal (blue; k. l, m, n) regions of ACL. Images brightness and contrast are adjusted for better visualization. Scalebar 100 μm. Figure S9. Effect of dehydration on tissue AF measured with CLEM and AFM-IR. AF images of ACL in the a) initial hydrated state b) dehydrated state subjected to N2 gas for 8 minutes c) rehydrated state with water d) post storage in 4 °C refrigerator for 24 hours. e) AF reported as percentage of initial state measurement remains steady from steps a – c however, increases 16% after preservation in the refrigerator for 24 hours. f) AFM-IR spectrum parallels the lack of effect of dehydration seen by a steady 1672 cm− 1/1740 cm− 1 ratio until the tissue is kept in the refrigerator which completely reduces the 1672 cm− 1 signal, leaving only the 1740 cm− 1 peak (red spectrum). Table S2. Tensile testing sequence parameters for femur – ACL – tibia complex (FATC) cadaveric ACL. Figure S10. Femur – ACL – Tibia complex (FATC) cyclic tensile test of ACL following sequence in Table S2. a) Cadaver knee with 45° flexion angle in the tensile test device. Femur was exercised up and down (yellow arrow) b) ACL with a partial tear (red dotted circle) at the proximal region during the 11th cycle of the 8th testing sequence with 200 N cyclic load at 20 mm/min. The three regions of AF image acquisition are proximal (P), midsubstance (M) and distal (D). c) The change in mean AF intensity of the image for the three regions exhibit the largest AF signal in the proximal region at the end of the testing sequence, corresponding to the hole. (standard error for each data point range was 20 – 35 not shown). d) CLEM AF images of ACLs during testing sequence number 2, 4, 6 and 8 (point of tear). Scale bar: 100 μm.