# Therapeutically Induced Modulation of Collagen I-to-III Ratio Three Weeks After Rabbit Achilles Tendon Full Transection

## TL;DR

This study investigates how a growth factor-releasing implant affects collagen composition in healing rabbit tendons, using a new staining method to assess tissue repair.

## Contribution

The study introduces a novel implant for tendon healing and evaluates a cost-effective staining method for collagen analysis.

## Key findings

- PDGF-BB therapy increased collagen I-to-III ratios in healing tendon zones compared to control.
- Herovici staining was more effective than IHC for assessing collagen ratios in this model.
- Collagen maturation was observed distal to the implant, not directly at the implant interface.

## Abstract

Achilles tendon ruptures, frequently occurring in both athletes and the elderly, are characterized by a slow healing process. To accelerate repair, novel implant materials have been developed. We evaluated an implant designed to release a specific growth factor, using a rabbit Achilles tendon full-transection model, with the material placed around a conventional suture. To monitor healing progression, we assessed the collagen I-to-III ratio, as collagen III is typically deposited first and later replaced by collagen I. This study compared a growth factor-releasing implant against an empty control. In particular, we analyzed different local zones of the healing tendon. Additionally, we compared two staining methods for measuring the collagen ratio: a cost-effective, simultaneous staining method (Herovici) versus a more expensive dual-labeling approach (Immunohistochemistry). Although we found a weak correlation between the two methods, the Herovici method was more discriminative in this specific model, allowing for a better assessment of collagen I-to-III ratios across different zones of the healing tendon. Based on Herovici, there were higher collagen I-to-III ratios for the healing zone in the core of the tendon, as well as for the reactive zone near the implant for the platelet-derived growth factor-BB (PDGF-BB) therapy, compared to the empty control. In addition, the ratio was higher in the healing tendon zone compared to the zone within the implant pores.

During tendon healing, collagen III expression precedes that of collagen I. The collagen I-to-III ratio at a certain time point post-laceration serves as an indicator of the healing status. Consequently, it is crucial to understand how different therapeutic approaches to support tendon healing affect the collagen I-to-III ratio in the extracellular matrix of a healing tendon, particularly across distinct anatomical zones. We compared the impact of a platelet-derived growth factor-BB (PDGF-BB) treatment via controlled release from coaxially electrospun DegraPol® (Ab medica, Cerro Maggiore, Italy) hollow-fiber mesh with a treatment by the vehicle alone (no PDGF-BB) in the rabbit Achilles tendon full transection model and provide data on the collagen I-to-III ratio 3 weeks post-operation. For this purpose, we compared a dual-color Herovici staining to two single IHC labeling, for collagen I and collagen III, respectively. Herovici staining (HV) was expected to offer a more precise approach (pink-to-blue histogram) than the two separately labeled IHC stainings, both with chromogenic DAB labeling (red-to-green histogram), despite an anticipated positive correlation of the data assessed by these methods. Different zones were compared, i.e., native tendon tissue, reactive zone at interface to implant, hot zone within the core of the healing tendon and the zone within the scaffold, meaning the collagen deposited within the fibers of the implanted DegraPol® tube, respectively. The analysis revealed that the ratios obtained via HV correlated weakly with the ratios obtained by IHC. Based on HV, PDGF-BB therapy led to higher collagen I-to-III ratios in all zones, except for the zone within the scaffold pores, while IHC did not reveal significant differences. Notably, collagen I-to-III ratios were not higher in immediate proximity, but rather distal from the PDGF-BB releasing implant, specifically in the core of the healing tendon tissue. Hence, a PDGF-BB therapy is suggestive of greater collagen maturation in specific zones of the healing tendon.