A programmable 4-dimensional bio-Kirigami system could generate complex tissue curvatures while supporting cell survival and extracellular matrix formation, suggesting a potential approach for engineering tissues that progressively acquire structural stability during maturation.
In the experimental study, the researchers developed a bio-Kirigami system consisting of a deformable, cell-laden hydrogel attached to a relatively rigid outer hydrogel ring. Using sequential photolithography, they combined 2 hydrogels with different swelling, degradation, and mechanical properties to generate controlled shape transformations during culture. Initial experiments incorporated NIH3T3 fibroblasts to evaluate deformation and cell viability. Subsequent analyses used human mesenchymal stem cells to investigate cartilage-like tissue formation. The constructs were cultured under standard expansion or chondrogenic conditions for 21 days, followed by an additional 6 days after removal of the supporting framework.
The researchers assessed programmed shape transformation, cell viability, and structural stability following removal of the outer ring. Key outcomes included deformation height, glycosaminoglycan deposition, DNA content, and histologic evidence of cartilage-like extracellular matrix formation. Additional tests evaluated how cell density, hydrogel dimensions, and ultraviolet crosslinking duration influenced deformation.
During the initial 21-day culture period, human mesenchymal stem cell–laden constructs in both standard expansion and chondrogenic conditions developed dome-shaped curvatures. No statistically significant differences in deformation height or morphing rates were observed between the groups, indicating comparable initial shape transformations.
Following removal of the supporting hydrogel ring, the constructs demonstrated different structural responses. Those maintained in standard expansion medium gradually collapsed, whereas constructs cultured under chondrogenic conditions retained their dome-shaped architecture throughout theobservation period.
Biochemical analyses demonstrated greater glycosaminoglycan content in constructs cultured under chondrogenic conditions compared with controls. Histologic assessments revealed stronger Alcian blue staining and positive type II collagen staining, supporting cartilage-like extracellular matrix deposition. DNA content was also higher in the chondrogenic group, which was attributed to improved structural stability and reduced cell loss.
The researchers reported that a relatively high cell density of 40 million cells/mL was required to generate sufficient cartilage-like extracellular matrix to maintain the engineered curvature after removal of the supporting framework. They noted that matrix accumulation during tissue maturation contributed to the transition from externally supported deformation to tissue-supported structural stability.
Additional analyses demonstrated that the bio-Kirigami system could generate several programmed geometries, including crosses, double strips, domes, and more complex curved structures. Both cell-laden and cell-free constructs exhibited progressive deformation during the initial 7-day culture period, with final configurations consistent with computational simulations.
The researchers examined factors influencing shape transformation. Increasing the width of the deformable hydrogel component reduced deformation, whereas changes in cell density, hydrogel thickness, and ultraviolet crosslinking duration did not produce statistically significant differences in overall morphing capacity. Live/dead staining demonstrated predominantly viable cells throughout the 7-day culture period, supporting the system's compatibility with living cells during deformation.
The study had several limitations. The engineered constructs primarily underwent deformation along the positive vertical axis, limiting their ability to reproduce multidirectional tissue morphogenesis. Residual hydrogel materials could interfere with cellular interactions and extracellular matrix remodeling and potentially raise immunotoxicity concerns. The cartilage tests included relatively small sample sizes. Structural stability was evaluated for just 6 days after scaffold removal, and the researchers did not investigate implantation, long-term functional performance, or tissue regeneration in vivo.
The findings demonstrate the feasibility of engineering cell-laden hydrogel constructs that undergo programmed shape transformations and develop sufficient cartilage-like extracellular matrix to maintain complex curvatures after external support is removed. Further research is needed to improve deformation control, address residual scaffold materials, and determine the approach's applicability to tissue-specific regenerative medicine.
"The 4D bio-Kirigami constructs supported long-term cell viability, differentiation, and tissue maturation while maintaining structural integrity postexcision," wrote lead study author Kaelyn L. Gasvoda, of the University of Illinois Chicago, and colleagues.
The study authors declared no conflicts of interest.
Source: Cell Biomaterials