Toward Adipose Tissue Engineering Using Thiol-Norbornene Photo-Crosslinkable Gelatin Hydrogels.

Nowadays, breast implants, lipofilling, and microsurgical free tissue transfer are the most often applied procedures to repair soft tissue defects resulting from mastectomies/lumpectomies following breast cancer. Due to the drawbacks and limitations associated with these conventional clinical practices, there is a need for alternative reconstructive strategies. The development of biomimetic materials able to promote cell proliferation and adipogenic differentiation has gained increasing attention in the context of adipose reconstructive purposes. Herein, thiol-norbornene crosslinkable gelatin-based materials were developed and benchmarked to the current commonly applied methacryloyl-modified gelatin (GelMA) with different degrees of substitutions focussing on bottom-up tissue engineering. The developed hydrogels resulted in similar gel fractions, swelling, and in vitro biodegradation properties compared to the benchmark materials. Furthermore, the thiol-ene hydrogels exhibited mechanical properties closer to those of native fatty tissue compared to GelMA. The mechanical cues of the equimolar GelNB DS55% + GelSH DS75% composition resulted not only in similar biocompatibility but also, more importantly, in superior differentiation of the encapsulated cells into the adipogenic lineage, as compared to GelMA. It can be concluded that the photo-crosslinkable thiol-ene systems offer a promising strategy toward adipose tissue engineering through cell encapsulation compared to the benchmark GelMA.

Screening of two-photon activated photodynamic therapy sensitizers using a 3D osteosarcoma model.

Photodynamic therapy (PDT) involves a photosensitizing agent activated with light to induce cell death. Two-photon excited PDT (TPE-PDT) offers numerous benefits compared to traditional one-photon induced PDT, including an increased penetration depth and precision. However, the in vitro profiling and comparison of two-photon photosensitizers (PS) are still troublesome. Herein, we report the development of an in vitro screening platform of TPE-PS using a 3D osteosarcoma cell culture. The platform was tested using three different two-photon (2P) active compounds - a 2P sensitizer P2CK, a fluorescent dye Eosin Y, and a porphyrin derivative (TPP). Their 2P absorption cross-sections (σ2PA) were characterised using a fully automated z-scan setup. TPP exhibited a remarkably high σ2PA at 720 nm (8865 GM) and P2CK presented a high absorption at 850 nm (405 GM), while Eosin Y had the lowest 2P absorption at the studied wavelengths (<100 GM). The cellular uptake of PS visualized using confocal laser scanning microscopy showed that both TPP and P2CK were internalized by the cells, while Eosin Y stayed mainly in the surrounding media. The efficiency of the former two TPE-PS was quantified using the PrestoBlue metabolic assay, showing a significant reduction in cell viability after two-photon irradiation. The possibility of damage localization was demonstrated using a co-culture of adipose derived stem cells together with osteosarcoma spheroids showing no signs of damage to the surrounding healthy cells after TPE-PDT.

Heterocellular 3D scaffolds as biomimetic to recapitulate the tumor microenvironment of peritoneal metastases in vitro and in vivo.

Peritoneal metastasis is a major cause of death and preclinical models are urgently needed to enhance therapeutic progress. This study reports on a hybrid hydrogel-polylactic acid (PLA) scaffold that mimics the architecture of peritoneal metastases at the qualitative, quantitative and spatial level. Porous PLA scaffolds with controllable pore size, geometry and surface properties are functionalized by type I collagen hydrogel. Co-seeding of cancer-associated fibroblasts (CAF) increases cancer cell adhesion, recovery and exponential growth by in situ heterocellular spheroid formation. Scaffold implantation into the peritoneum allows long-term follow-up (>14 weeks) and results in a time-dependent increase in vascularization, which correlates with cancer cell colonization in vivo. CAF, endothelial cells, macrophages and cancer cells show spatial and quantitative aspects as similarly observed in patient-derived peritoneal metastases. CAF provide long-term secretion of complementary paracrine factors implicated in spheroid formation in vitro as well as in recruitment and organization of host cells in vivo. In conclusion, the multifaceted heterocellular interactions that occur within peritoneal metastases are reproduced in this tissue-engineered implantable scaffold model.