# Chondroitinase Versus Papain Digestion Leads to Different Outcome for In Vitro Simulation of Degenerated Discs

## TL;DR

This study compares how two enzymes, chondroitinase and papain, affect disc degeneration models and hydrogel injection outcomes in bovine discs.

## Contribution

The study provides new insights into how chondroitinase and papain differently simulate disc degeneration and affect hydrogel distribution.

## Key findings

- Chondroitinase increases range of motion and neutral zone more than papain.
- Hydrogel injected after chondroitinase spreads in a cloud-like pattern, while papain results in a monolithic defect.
- Both enzymes allow more hydrogel injection but affect disc structure differently.

## Abstract

Biomaterials play an increasing role in intervertebral disc regeneration and require preclinical testing, typically performed using organ culture and in vitro models. Native human discs are limited, and animal models often fail to mimic human disc degeneration. Thus, enzymes like chondroitinase ABC (chABC) and papain are used to simulate degenerative tissue changes and enable biomaterial injection. In previous work, we characterized the biomechanical and morphological effects of papain, which forms cavities in the disc. In contrast, chABC does not form cavities, but its biomechanical effects remain insufficiently characterized. This study aims to evaluate the macroscopic and biomechanical effects of chABC—specifically, range of motion (ROM), neutral zone (NZ), and disc height—in a bovine organ culture model, and assess the distribution of an injected hydrogel, comparing the results to published papain data.

Four groups of fresh bovine tail segments were prepared (n ≥ 10) and three received injections of chABC, papain, or PBS, followed by 7 days of culture. For papain and PBS, published data were supplemented with new specimens. Complex simulated physiological loading was applied to diminish disc swelling. The maximum volume of a serum‐albumin‐hydrogel was injected into all four groups. ROM, NZ, and disc height were measured before and after enzyme treatment, loading, and injection. Post‐injection, microCT scans visualized material distribution within the discs.

ChABC increased ROM by up to 92.1%, NZ by up to 79.4%, and decreased disc height by 2.1 mm. Hydrogel injection decreased ROM and NZ but increased disc height in all groups while enzyme treatments allowed more hydrogel injection (0.6 mL for chABC). Exemplary scans showed cloud‐like hydrogel spread for chABC and a round‐shaped degradation defect for papain.

The findings indicate that chABC better simulates disc degeneration, whereas papain better models nucleotomies, and both enzymes preserve annulus integrity—providing valuable models for biomechanical testing.

Chondroitinase ABC and papain similarly alter disc biomechanics and increase injectable volume, but produce fundamentally different structural morphologies. Papain induces cavities, whereas chondroitinase preserves tissue integrity. Hydrogel distributions differ—cloud‐like for chABC, monolithic for papain—supporting chondroitinase as a degeneration and papain as a nucleotomy model.