Long-term in vivo integrity and safety of 3D-bioprinted cartilaginous constructs.

Peter Apelgren,Paul Gatenholm,Linnéa Stridh Orrhult,Kajsa Markstedt,Lars Kölby,Camilla Brantsing,Karin Säljö,Anders Lindahl,Matteo Amoroso

doi:10.1002/jbm.b.34687

Abstract

Long-term stability and biological safety are crucial for translation of 3D-bioprinting technology into clinical applications. Here, we addressed the long-term safety and stability issues associated with 3D-bioprinted constructs comprising a cellulose scaffold and human cells (chondrocytes and stem cells) over a period of 10 months in nude mice. Our findings showed that increasing unconfined compression strength over time significantly improved the mechanical stability of the cell-containing constructs relative to cell-free scaffolds. Additionally, the cell-free constructs exhibited a mean compressive stress and stiffness (compressive modulus) of 0.04 ± 0.05 MPa and 0.14 ± 0.18 MPa, respectively, whereas these values for the cell-containing constructs were 0.11 ± 0.08 MPa (p = .019) and 0.53 ± 0.59 MPa (p = .012), respectively. Moreover, histomorphologic analysis revealed that cartilage formed from the cell-containing constructs harbored an abundance of proliferating chondrocytes in clusters, and after 10 months, resembled native cartilage. Furthermore, extension of the experiment over the complete lifecycle of the animal model revealed no signs of ossification, fibrosis, necrosis, or implant-related tumor development in the 3D-bioprinted constructs. These findings confirm the in vivo biological safety and mechanical stability of 3D-bioprinted cartilaginous tissues and support their potential translation into clinical applications.

Highlights

To isolate human primary nasal chondrocytes, cartilage samples were sliced into 1 mm × 1 mm pieces, and after discarding the medium, transferred to pronase (Serva, Heidelberg, Germany) digestion solution (10 ml; 20 mg/ml [2%] pronase in DMEM supplemented with 1% P/S without fetal bovine serum (FBS))
Our results indicated that stromal vascular fraction (SVF)-derived stem cells exerted quantitatively similar trophic effects on chondrocyte proliferation to those observed using bone-marrow-derived mesenchymal stem cells (MSCs)
The cell count and mechanical data showed that both human primary nasal chondrocytes (hNCs)/MSCs and hNC/SVF promoted the in vivo formation of similar native-like cartilaginous tissue after 8 months

Summary

| MATERIALS AND METHODS

Human bone-marrow-derived MSCs originally obtained from female donors (age range: 18–30 years; Rooster Bio, Frederick, MD) were cultured under standard culture conditions (37C and 5% CO2) using an MSC high-performance media kit (Rooster Bio). To isolate human primary nasal chondrocytes (hNCs), cartilage samples were sliced into 1 mm × 1 mm pieces, and after discarding the medium, transferred to pronase (Serva, Heidelberg, Germany) digestion solution (10 ml; 20 mg/ml [2%] pronase in DMEM supplemented with 1% P/S without FBS). Upon reaching 80 to 90% confluence, cells were detached, counted, and resuspended in standard culture medium before mixing with nanofibrillated cellulose/alginate (NFC-A) bioink (CELLINK AB, Gothenburg, Sweden). The SVF pellet was resuspended in standard culture medium, and the number of cells was determined before printing. Constructs were cross-linked with 100 mM CaCl2 solution for 5 min and washed in standard culture medium, followed by implantation into nude mice within 1 hr after printing.

| RESULTS

Findings

| DISCUSSION

| CONCLUSIONS