Ultrasound-Driven ex-vivo Engineering of Tissue-Guiding Hydrogels for Bone Regeneration
Acoustics
Biochemistry
Biophysics
Final Report Abstract
Despite rapid advancements in tissue engineering (TE) over 30 years, challenges persist, notably in accurately reconstructing damaged tissue's histoarchitecture. The acoustophoresis method, utilizing ultrasound-generated standing waves to organize cells in a medium, offers promise. With an in-situ polymerizing gel as the ultrasound medium, cells can be immobilized, initiating tissue-specific differentiation. Demand is high for novel 3-D constructs for tissue regeneration like bone and cartilage. In this project mesenchymal stromal cells (MSCs), pivotal in musculoskeletal TE, were arranged in parallel planes within a 3-D fibrin hydrogel using acoustophoresis. A Tissue Engineering Acoustophoretic (TEA) set-up was developed, employing specific acoustic parameters: 2.5 MHz frequency, 7.7 V peak-to-peak voltage, continuous wave mode. MSC metabolic activity surged post-acoustophoresis, aligning with randomly seeded cells' activity in non-patterned gels over a 21-day culture. Pro-osteogenic factors were introduced to 3-D constructs, cultured to confer bone-like properties. Expression of pro-osteogenic genes was elevated in patterned 3-D hydrogels versus non-patterned ones. Remarkably, MSCs patterned in TEA showed enhanced osteogenic marker expression, even without pro-osteogenic additives, suggesting support for osteogenesis via the layered cell organization. Alizarin red staining indicated increased calcium content in patterned gels, though not statistically significant (p=0.056). Furthermore, MSCs differentiating in patterned constructs exhibited thicker actin fibers compared to non-patterned gels, indicative of osteoblast differentiation. Fibronectin fibers, following actin fiber organization, were confirmed by exogenous fibronectin addition. MSC hydrogels patterned via TEA displayed unique ECM fiber organization, defining their distinct histoarchitecture. The TEA setup can be adapted for engineering other tissues and can be further extended by combinations of two or three transducers depending on the desired tissue structure.
Publications
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Pulsed ultrasound for bone regeneration – outcomes and hurdles in the clinical application: a systematic review. European Cells and Materials, 42, 281-311.
Puts, R.; Vico, L.; Beilfuß, N.; Shaka, M.; Padilla, F. & Raum, K.
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A Tissue Engineering Acoustophoretic (TEA) Set-up for the Enhanced Osteogenic Differentiation of Murine Mesenchymal Stromal Cells (mMSCs). International Journal of Molecular Sciences, 23(19), 11473.
Zhang, Hui; Beilfuss, Nirina; Zabarylo, Urszula; Raum, Kay & Puts, Regina
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Tissue Engineering Acoustophoretic (TEA) Set-Up Enhances Osteogenic Potential of Mesenchymal Stromal Cells in Anisotropic 3-D Constructs. Poster at International Ultrasonics Symposium 2022, Venice, Ital
Zhang, H.; Beilfuss, N.; Zabarylo, U.; Raum, K. & Puts, R.
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Focused Low-Intensity Pulsed Ultrasound (FLIPUS) Mitigates Apoptosis of MLO-Y4 Osteocyte-like Cells. Bioengineering, 10(3), 387.
Puts, Regina; Khaffaf, Aseel; Shaka, Maria; Zhang, Hui & Raum, Kay
