# A scaffold-free cartilage construct fabricated using a bio 3D printer accelerates critical-size bone defect regeneration

## TL;DR

A new scaffold-free method using bio-3D printing and cartilage constructs accelerates healing of critical bone defects in rats.

## Contribution

This is the first study to regenerate long bone defects using scaffold-free cartilage from AT-MSCs by mimicking endochondral ossification.

## Key findings

- MSC-Ch group showed significantly higher bone volume ratios compared to other groups at 6 and 12 weeks.
- Histology revealed robust new bone formation in the MSC-Ch group, with bone bridging and minimal fibrosis.
- Undifferentiated MSCs and the Defect group showed limited healing, with fibrous and adipose tissue filling the defect.

## Abstract

Critical-size bone defects (CSD), often resulting from trauma or tumour resection, represent a challenging clinical condition that is difficult to treat. Although autologous bone grafting is a common treatment, limitations such as donor site morbidity necessitate the development of novel therapeutic strategies. Approaches that mimic endochondral ossification, the natural process of bone development and healing, are increasingly recognised for their bone regenerative potential. The combination of mesenchymal stem cells and scaffolds, used in many bone regeneration studies, has drawbacks, such as scaffold-derived complications including chronic inflammation and fibrosis. To overcome these issues, we used a bio-three-dimensional (3D) printer that enables the fabrication of scaffold-free 3D cellular constructs. This study aimed to establish a novel therapeutic strategy for CSD by generating scaffold-free cartilage constructs from rat adipose tissue-derived mesenchymal stromal cells (rAT-MSCs) and evaluating their regenerative potential.

Scaffold-free cellular constructs were fabricated using rAT-MSCs. Cartilage constructs were generated by chondrogenic induction. A 5-mm CSD was created in the diaphysis of the rat femur. Three experimental groups were established: a Defect group (n = 9), in which no material was implanted into the defect; an MSC group (n = 9), in which undifferentiated constructs were implanted into the defect; and an MSC-Ch (AT-MSC-derived chondrocyte) group (n = 9), in which cartilage constructs were implanted into the defect. Computed tomography (CT) and histological analyses were performed at 6 and 12 weeks post-implantation.

CT scans showed significantly higher bone volume/total volume ratios in the MSC-Ch group than in the Defect and MSC groups at 6 and 12 weeks (p < 0.01). Histologically, the MSC-Ch group exhibited robust formation of new cortical and cancellous bone, continuous with native bone margins, leading to bone bridging. In contrast, the Defect and MSC groups demonstrated new bone formation confined to the periphery of the defect, with the central regions predominantly occupied by adipose and fibrous tissues. Histological scoring supported these findings, with the MSC-Ch group achieving significantly higher scores than the Defect and MSC groups at both time points (p < 0.05).

Implantation of scaffold-free cartilage constructs derived from rAT-MSCs effectively promoted CSD healing. To the best of our knowledge, this is the first study to successfully regenerate a long bone CSD using AT-MSCs as the cell source by mimicking the endochondral ossification pathway. However, further studies using larger animal models are required to validate and translate these findings.

Fabricating scaffold-free cartilage from AT-MSCs with a bio-3D printer is a novel solution for bone regeneration, circumventing biomaterial-related complications. This reconstructive method shows promise for various bone defects, including trauma, revision arthroplasty, spinal pathologies, and periodontal disease.

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