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Ultrasound-Derived Mechanical Stimulation of Alginate Hydrogels for Bone Repair: an In Vitro Study

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Abstract

Purpose

Cell therapy is a new and evolving treatment for large-scale bone defects. Here, we describe a novel approach that combines hydrogel-based cell therapy with low-intensity pulsed ultrasound (LIPUS), an FDA-approved treatment for fracture repair.

Methods

Bone marrow–derived stromal cells (BMSCs) were encapsulated in RGD-peptide-coupled alginate hydrogels and mechanically stimulated using LIPUS-derived acoustic radiation force (ARF).

Results

Mechanical analysis of alginate hydrogels revealed a dependence on either alginate concentration or cross-linker concentration, revealing different ways to adjust the mechanical properties of the hydrogel. An optimal alginate and cross-linker concentration were found that allowed both robust cell proliferation and hydrogel mechanical stability. Cell proliferation was inversely proportional to hydrogel stiffness, suggesting that stiffer hydrogels were preventing full cell spreading and migration within the hydrogel. Cells encapsulated in hydrogels of optimal stiffness responded immediately after the onset of ARF by upregulating calcium, and shortly after upregulating cyclooxygenase-2 and prostaglandin E2, and later forming mineralized tissue in culture. Interestingly, cells seeded on top of hydrogels did not demonstrate the same calcium flux as encapsulated cells, suggesting that encapsulating the cells provided an additional mechanical stimulus. COX-2 and PGE2 data from cells encapsulated in hydrogels with no ARF showed a similar response to encapsulation alone.

Conclusion

These studies support the idea of combining cell therapy with LIPUS-derived ARF to enhance mineralized tissue formation.

Lay Summary

Ultrasound has been a tool available to clinicians for a long time, and for a wide range of applications, including bone repair. Here, we show that it can stimulate stem cells that have been encapsulated in hydrogels to differentiate into bone cells through mechanical stimulation. This work would allow a clinician to implant stem cells into a large bone defect and stimulate them using ultrasound to differentiate into bone cells and help these large defects heal more quickly. This stimulation would be done transdermally and non-invasively, allowing the defect to heal with minimal intervention.

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Acknowledgements

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funding

Research reported in this publication was wholly supported by the National Science Foundation under award #1752915 and the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number R01AR073206.

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Authors and Affiliations

  1. Department of Biomedical Engineering, University of Connecticut, 260 Glenbrook Ave, Storrs, CT, 06269-3247, USA

    Fayekah Assanah, Hanna Anderson, Kevin Grassie, Lakshmi Nair & Yusuf Khan

  2. Connecticut Convergence Institute for Translation in Regenerative Engineering, UCONN Health, 263 Farmington Ave, CT, 06030, Farmington, USA

    Fayekah Assanah, Hanna Anderson, Kevin Grassie, Lakshmi Nair & Yusuf Khan

  3. Department of Orthopedic Surgery, UCONN Health, 263 Farmington Ave, Farmington, CT, 06030, USA

    Lakshmi Nair & Yusuf Khan

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  1. Fayekah Assanah
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Contributions

Conceptualization: Y. K., F. A. Experimental design: Y. K., F. A., H. A., K. G., L. N. Experimental work: F. A., H. A., K. G. Manuscript preparation, writing, and editing: Y. K., F. A., L. N. Final approval of manuscript: F. A., Y. K., K. G., H. A., L. N.

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Correspondence to Yusuf Khan.

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Supplementary Information

ESM 1

Supplementary Figure I: The COX-2 and the PGE2 quantification of 0.25 % and 0.5 % alginate hydrogels were exposed to a one-time ultrasound exposure of 300 mW/cm2 spatial intensity. (A) COX-2 gene expression was quantified through RT-PCR. Ultrasound treatment of BMSCs encapsulated in 0.5 % alginate hydrogel enhanced COX-2 expression over the control, although not significantly. (B) Normalized PGE2 expression per 100,000 cells quantified through ELISA. PGE2 expression was significantly upregulated in 0.5 % hydrogels when treated with ultrasound compared to the control groups. As the matrix stiffness increased from 0.25 % to the 0.5 % alginate hydrogel, both COX-2 and PGE2 levels were increased. Error is reported in bar graphs as the standard deviation of the mean. Statistical significance noted (*P < 0.05), (n = 3). (DOCX 440 kb)

ESM 2

Supplementary Figure II: (A) Rheology data showing the storage and loss modulus of 2 % alginate with varying concentrations of CaCl2. Both storage and loss modulus increase with increasing concentration of CaCl2 showing that increased ionic cross-linking within the alginate solution makes stiffer hydrogels. (B) Storage and loss modulus of varying alginate solution with 10 mM CaCl2. Both the storage and loss modulus increase with an increase in the concentration of alginate solution. (DOCX 274 kb)

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Assanah, F., Anderson, H., Grassie, K. et al. Ultrasound-Derived Mechanical Stimulation of Alginate Hydrogels for Bone Repair: an In Vitro Study. Regen. Eng. Transl. Med. (2023). https://doi.org/10.1007/s40883-023-00312-2

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