Cellular Interaction of Bone Marrow Mesenchymal Stem Cells with Polymer and Hydrogel 3D Microscaffold Templates

Abstract

Biomimicking biological niches of healthy tissues or tumors can be achieved by means of artificial microenvironments, where structural and mechanical properties are crucial parameters to promote tissue formation and recreate natural conditions. In this work, three-dimensional (3D) scaffolds based on woodpile structures were fabricated by two-photon polymerization (2PP) of different photosensitive polymers (IP-S and SZ2080) and hydrogels (PEGDA 700) using two different 2PP setups, a commercial one and a customized one. The structures’ properties were tuned to study the effect of scaffold dimensions (gap size) and their mechanical properties on the adhesion and proliferation of bone marrow mesenchymal stem cells (BM-MSCs), which can serve as a model for leukemic diseases, among other hematological applications. The woodpile structures feature gap sizes of 25, 50, and 100 μm and a fixed beam diameter of 25 μm, to systematically study the optimal cell colonization that promotes healthy cell growth and potential tissue formation. The characterization of the scaffolds involved scanning electron microscopy and mechanical nanoindenting, while their suitability for supporting cell growth was evaluated with live/dead cell assays and multistaining 3D confocal imaging. In the mechanical assays of the hydrogel material, we observed two different stiffness ranges depending on the indentation depth. Larger gap woodpile structures coated with fibronectin were identified as the most promising scaffolds for 3D BM-MSC cellular models, showing higher proliferation rates. The results indicate that both the design and the employed materials are suitable for further assays, where retaining the BM-MSC stemness and original features is crucial, including studies focused on BM disorders such as leukemia and others. Moreover, the combination of 3D scaffold geometry and materials holds great potential for the investigation of cellular behaviors in a co-culture setting, for example, mesenchymal and hematopoietic stem cells, to be further applied in medical research and pharmacological studies.

Two-Photon Polymerization of 2.5D and 3D Microstructures Fostering a Ramified Resting Phenotype in Primary Microglia

“…The most significant limitation of 2PP is the relatively long printing time hindering the upscaling of the technology ( Moroni et al, 2018 ). Multiple polymeric, hydrogel or composite materials were employed in combination with 2PP to fabricate micro- and nano-patterns as well as 3D structures for in vitro cellular studies involving neuroblastoma, glioblastoma, prostate cancer, murine cerebellar granule, chondrocytes, macrophages, neuronal and stem cells ( Marino et al, 2013 ; Accardo et al, 2017 , 2018 ; Turunen et al, 2017 ; Maciulaitis et al, 2019 ; Fendler et al, 2019 ; Babi et al, 2021 ; Bertels et al, 2021 ; Maciulaitis et al, 2021 ; Nouri-Goushki et al, 2021 ; Akolawala et al, 2022 ; Costa et al, 2022 ).…”

In Vitro and In Vivo Modeling of Normal and Leukemic Bone Marrow Niches: Cellular Senescence Contribution to Leukemia Induction and Progression

“…Comparisons between both 2D and 3D systems under the same experimental conditions have been made using bone-derived materials as scaffolds to adhere patient CML-derived MSC cells supporting leukemic cells; the Ph+ subpopulation was reduced during culture in both systems, but in 3D cultures, this reduction was slower and leukemic cells showed higher CFU rates and frequency of long-term culture-initiating cells after 2 or 5 weeks [34]. Moreover, 3D models are preferred in many cases due to their capability to support complex volumetric structures with minimal toxicity, favoring cell-biomaterial interactions and nutrient exchange [103].…”

“…Hence, several protocols for bioengineering 3D HSC niches have been established for mouse and human HSC and LSC expansion [4,[103][104][105][106][107][108][109][110][111]. A well-designed artificial niche must be adaptable and scalable, facilitate selective incorporation of a variety of biochemical signals and allow the use of high-throughput or single-cell techniques, in addition to overcoming biomechanical and biotransport restrictions [68].…”

“…A well-designed artificial niche must be adaptable and scalable, facilitate selective incorporation of a variety of biochemical signals and allow the use of high-throughput or single-cell techniques, in addition to overcoming biomechanical and biotransport restrictions [68]. A big step towards the modeling of the leukemic niche was the possibility to grow leukemic cells as spheroids [107,112,113], or their implantation into polymer matrixes [37,109,112] or inside porous scaffolds [103,105,111,114]. The use of bioreactors or microfluidic devices further improved nutrient supply and increased the number of parameters involved on the niche building [115][116][117][118][119].…”

“…Open-cell foam scaffolds made with low-density biomaterials with distinct levels of elasticity were also adopted as analogs of the trabecular bone and used with stromal cells to support HSC expansion [2,85]. Other 3D models rely on woodpile structures fabricated by two-photon polymerization of different photosensitive polymers or hydrogels; these scaffolds are adequate to study mechanical properties on the adhesion and proliferation of MSC [103]. Indeed, scaffold type, matrix stiffness, and architecture applied to HSC or LSC cultures differ greatly and, consequently, can dramatically influence cell fate, as described for MSC [91,92].…”