Collagen Hybridizing Peptides Promote Collagen Fibril Growth In Vitro

Abstract

Recreating the structural and mechanical properties of native tissues in vitro presents significant challenges, particularly in mimicking the dense fibrillar network of extracellular matrixes such as skin and tendons. This study develops a reversible collagen film through cycling collagen self-assembly and disassembly, offering an innovative approach to address these challenges. We first generated an engineered collagen scaffold by applying plastic compression to the collagen hydrogel. The reversibility of the collagen assembly was explored by treating the scaffold with lactic acid, leading to its breakdown into an amorphous gel─a process termed defibrillogenesis. Subsequent immersion of this gel in phosphate buffer facilitated the reassembly of collagen into fibrils larger than those in the original scaffold yet with the D-banding pattern characteristic of collagen fibrils. Transfer learning of the mobileNetV2 convolutional neural network trained on atomic force microscope images of collagen nanoscale D-banding patterns was created with 99% training and testing accuracy. In addition, extensive external validation was performed, and the model achieved high robustness and generalization with unseen data sets. Further innovation was introduced by applying collagen hybridizing peptides, which significantly accelerated and directed the assembly of collagen fibrils, promoting a more organized and aligned fibrillar structure. This study not only demonstrates the feasibility of creating a reversible collagen film that closely mimics the density and structural properties of the native matrix but also highlights the potential of using collagen hybridizing peptides to control and enhance collagen fibrillogenesis. Our findings offer promising tissue engineering and regenerative medicine strategies by enabling precise manipulation of collagen structures in vitro.

Keywords: CHP; CMP; atomic force microscopy; collagen; fibrillogenesis; peptide.

Figure 1

Cycling collagen self-assembly process to create reversible collagen film. (a) Plastic compression on collagen hydrogel creates a high-density engineered collagen scaffold (ECS) with well-defined collagen fibrils. (b) Acetic acid degradation of ECS creates an amorphous collagen film. The photo on the left is ECS. The middle photo is acetic acid degraded collagen film before air-dry, and the right photo is air-dried AA = RCF. (c) Lactic acid degradation of ECS creates amorphous collagen film. The photo on the left is ECS. The middle photo is lactic acid degraded collagen film before air-dry, and the right photo is air-dried LA = RCF. (d) Submerge the acetic acid degraded collagen film in PBS results in fibril reformation. (e) Submerge the lactic acid degraded collagen film in PBS results in fibril reformation.

Figure 2

Utilizing convolutional neural network to recognize collagen D-banding pattern. (a) Training data set processing that split 50 AFM images of D-banding and 50 AFM images of no D-banding following a 10 × 10 grid, yielding 5000 AFM images of D-banding and 5000 images of no D-banding used in training. (b) Confusion matrix of the training results with MobileNetV2. True positive = 1, false positive = 0, true negative = 0.98, false negative = 0.02. (c) External validation results on the D-banding image that was recognized as all yes. (d) External validation result on no D-banding image that was recognized as all no. (e) External validation result on rat tail tendon image was recognized as all yes. (f) External validation result on acetic acid degraded reversible collagen film (AA-RCF) image that was recognized as all yes. (g) External validation result on lactic acid degraded reversible collagen film (LA-RCF) image that was recognized as all yes. (h) External validation result on partially degraded collagen scaffold using a low concentration of lactic acid image was recognized as all no. Circles show collagen D-banding but not 67 nm recognized as no. Green box = D-banding; red box = no D-banding.

Figure 3

Collagen hybridizing peptide (CHP) mediated collagen fibril growth in high-density collagen film. (a) Bright-field and fluorescent images of CHP-mediated collagen fibril growth, images taken after fibrils are fully formed and air-dried. (b) Time-lapse imaging of PBS-mediated (control) collagen fibril growth (in liquid) with initial growth at 1 h and full growth after 8 h. (c) Time-lapse imaging of CHP-mediated collagen fibril growth (in liquid) with initial growth at 10 min and total growth after 2 h. (d) Second harmonic generation imaging of PBS-mediated collagen fibril growth (left) showing nucleation site (circles) and fibril growth in all directions and CHP-mediated collagen fibril growth (right) showing nucleation site (circles) and more directional growth from nucleation site. Images taken after fibrils are fully formed and air-dried. (e) Fibril linearity measurements of PBS-mediated growth (left) and CHP-mediated growth (right); linear fibrils are highlighted in green. (f) Quantification of linear fibrils between PBS and CHP-mediated fibril growth, p < 0.0001, nonpaired t test.

Figure 4

Morphological features of PBS and CHP-mediated collagen fibril growth. (a) AFM images of PBS-mediated collagen fibril bundles; core areas are nucleation points with densely packed, randomly organized collagen fibrils. Fringe areas have smaller fibrils that are randomly organized. (b) AFM images of CHP-mediated collagen fibril bundles; core areas are nucleation points with densely packed, highly aligned collagen fibrils. Fringe areas have smaller fibrils that are randomly organized. (c) Individual single fibrils at the extremities of the fringe area. (d) ColD recognition results in CHP-mediate collagen fibril growth. Green boxes follow collagen fibrils with 67 nm D-banding pattern, and red boxes follow the background with lactic acid-degraded amorphous collagen. (e) Proposed CHP-binding mechanism: CHP hybridizing to denatured collagen triple helices but not intact collagen fibrils in tissue (top) and binding to intact and reversible collagen molecules in high-density collagen film (bottom).