Michael Gillespie and Jonathan Wisco, Physiology and Developmental Biology
Due to the relatively poor fiber contrast of tendons in comparison with muscles, the anatomical, and therefore, functional relationship of muscle attachments to bones via tendons has not been well mapped. We were interested in mapping the 3D gross anatomical structure of tendons using a MicroScribe 3D digitizer (GoMeasure3D, Amherst, VA), with the goal to compare fibrous architecture between tendons of various types of muscles and muscle classes. We developed a stain made of blue dye and powdered sugar to increase fiber visibility, thus allowing for 3D digitization. This technique was applied to human cadaveric calcaneal tendon and quadriceps tendon.
Materials and Methodology
Our preliminary attempts to develop an effective stain were performed on chicken feet tendons. Two stains derived from various fine ground chili powders and one made from powdered sugar and blue dye were applied. One drop of water was added to each teaspoon of powder to create a thick paste that was then applied to the tendon. Gently rubbing the paste into the tendon ensured that the grains of powder penetrated into the ridges between individual fiber bundles. The remaining stain was then wiped off of the surface of the tendon fiber bundles, leaving clearly marked fiber boundaries. (Figure A).
Upon determining the best staining method, we applied it to a sample of human patellar and quadriceps tendon dissected from paraformaldehyde embalmed human cadaveric specimens obtained from the University of Utah Body Donor Program. Deep fascia tissue on the tendons was removed to reveal the fiber bundle surface. The staining procedure developed in preliminary attempts was then applied.
Cadaveric tendon samples were dissected and isolated, after which remaining deep fascia was removed and our novel staining method was applied. Coronal slabs of approximately 1 mm in thickness were cut, then stained with the aforementioned technique. The surface layer for each slab was digitized. We used a MicroScribe 3D digitizer to plot points along the surface of the slab according to the fibers that were visible from the stain. in space along the surface of a desired anatomical structure. A fine-tipped stylus, connected to the base of the digitizer by a rotating arm, was used to record the pathway of individual fiber bundles on tendon samples (Figure B). Two coronal slab surfaces of quadriceps tendon and three surfaces of patellar ligament were digitized. Data were imported into MAYA Autodesk for reconstruction and rendering.
Stains made from fine-ground chili powder somewhat increased fiber visibility. However, it was found that due to the finer grain powder and more pronounced color contrast, the blue stain achieved greater fiber visibility (Figure A).. The blue stain was also effective in staining remaining deep fascia, making it easier to locate and remove.
A MicroScribe 3D digitizer was used to collect fiber map data for two 1 mm coronal slabs of human patellar tendon and three 1mm coronal slabs of quadriceps tendon. All slabs were MicroScribed twice, once before applying our stain and once after. The mean number of fibers that were visualized during MicroScribing before staining was 12.8 ± 3.54. The mean number of fibers visualized after staining was 18 ± 4.05. A non-parametric Wilcoxon Signed Ranks Test on pre-stain fiber count versus post-stain fiber count revealed a significant increase in the number of fibers MicroScribed after staining (Z= -2.023, p= 0.043, two-tailed).
These types of data-driven 3D tendon fiber maps, made possible by our novel staining technique, open up the possibility for further investigation into the microstructure of tendons. Through observing the differences among various types of tendons, we can now look for relationships between microstructure and specific function. This study has implications for the advancement of biomechanical models, artificial reconstruction, and surgical repair of these tissues.