Abstract
Collagen is the primary structural protein in animals. Serving as nanoscale biological ropes, collagen fibrils are responsible for providing strength to a variety of connective tissues such as tendon, skin, and bone. Understanding structure-function relationships in collagenous tissues requires the ability to conduct a variety of mechanical experiments on single collagen fibrils. Though significant advances have been made, certain tests are not possible using the techniques currently available. In this report we present a new atomic force microscopy (AFM) based method for tensile manipulation and subsequent nanoscale structural assessment of single collagen fibrils. While the method documented here cannot currently capture force data during loading, it offers the great advantage of allowing structural assessment after subrupture loading. To demonstrate the utility of this technique, we describe the results of 23 tensile experiments in which collagen fibrils were loaded to varying levels of strain and subsequently imaged in both the hydrated and dehydrated states. We show that following a dehydration-rehydration cycle (necessary for sample preparation), fibrils experience an increase in height and decrease in radial modulus in response to one loading-unloading cycle to strain <5%. This change is not altered by a second cycle to strain >5%. In fibril segments that ruptured during their second loading cycle, we show that the fibril structure is affected away from the rupture site in the form of discrete permanent deformations. By comparing the severity of select damage sites in both hydrated and dehydrated conditions, we demonstrate that dehydration masks damage features, leading to an underestimate of the degree of structural disruption. Overall, the method shows promise as a powerful tool for the investigation of structure-function relationships in nanoscale fibrous materials.
Highlights
Collagen fibrils are the main load bearing element of all structural tissues in humans, and most animals
In this report we present a new technique for stretching single collagen fibrils in tension
In phosphate buffered saline (PBS), the remaining core of the tendon was splayed open with fine tipped glass rods and metal tweezers, releasing individual collagen fibrils into the PBS. 1 mL aliquots of the PBS/collagen fibril mixture were pippetted into glass dishes, which were left stationary for 30 minutes to allow the suspended fibrils to sink and adhere to the glass substrate
Summary
Tissue acquisition and collagen fibril extractionThe forelimb of a 24–36 month old steer was collected fresh from a slaughterhouse (Oulton farm, Nova Scotia, Canada), where the animal was killed for food. The forelimb was transferred on ice to the laboratory for dissection, where the common digital extensor tendon (CDET) was immediately removed and stored at −86°C. To extract individual collagen fibrils, the tendon was thawed and the epitenon removed using a razor blade. In phosphate buffered saline (PBS), the remaining core of the tendon was splayed open with fine tipped glass rods and metal tweezers, releasing individual collagen fibrils into the PBS. 1 mL aliquots of the PBS/collagen fibril mixture were pippetted into glass dishes, which were left stationary for 30 minutes to allow the suspended fibrils to sink and adhere to the glass substrate. The adhered fibrils were triplerinsed using deionized water and dried under compressed nitrogen. Glass dishes were stored in a dessicator for 5–7 days
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