Cell density matters: Local cell density enhancement by sound to increase the therapeutic efficacy in regenerative medicine

Jun 29, 2022, 4:50 PM
10m
Room: S3 B

Room: S3 B

Speaker

Géraldine Guex, Anne (AO Research Institute Davos, Switzerland )

Description

Introduction
Within the scope of personalised healthcare in the field of regenerative medicine, patient-derived cells are key players. Their successful application is, however, often hampered by low cell numbers at the expense of donor-site morbidity and lengthy in vitro expansion. Novel biofabrication methods requiring lower initial cell numbers are therefore timely to address this unmet clinical challenge. In vitro, local cell density enhancement by use of sound induced morphogenesis (SIM) at low frequency of <100 Hz was shown to induce increased microvasculature formation at lower cell concentration than conventional methods.1 Based on this, we are developing cell-hydrogel biografts with local cell density enhancement and evaluate their performance after subcutaneous implantation at the back of nude mice. Our research is driven by the hypothesis that local cell density enhancement can improve the therapeutic efficacy in various clinical scenarios such as anastomosis within wounds or bone formation of non-union fractures.

Methodology
We followed a twofold approach, assessing on the one hand anastomosis of implanted human umbilical vein endothelial cells (expressing green fluorescent protein, GFP-HUVEC) and on the other hand ectopic bone formation of mesenchymal stem cells (MSC). To assess anastomosis, HUVEC and MSC were mixed at a 1:1 ratio and resuspended in PEG-based or Dextran-based hydrogels at a final concentration of 2×106 cells per mL. To assess ectopic bone formation, MSC were resuspended in PEG-based hydrogels at a final concentration of 2×106 or 5×106 cells per mL, with or without BMP-2. Cells were then placed on a custom-made acoustic bioprinter and assembled into distinct patterns at a frequency of 60 Hz. Four cell-hydrogel biografts of approximately 4 x 9 mm2 were implanted at the back of nude mice and harvested after 2 or 8 weeks, respectively. Explants were fixed and either imaged as whole constructs or embedded in paraffin for subsequent histological analysis.

Results
By adjusting formulations of PEG-based and Dextran-based hydrogels, time to gelation was increased from 4 minutes to 7 minutes, which proved essential for successful pattern formation by sound. During a 3-day in vitro culture, endothelial cells assembled into pre-vascular structures of tight cell-cell contacts. The animal experiments were conducted with zero mortality during the time of implantation and no other complications. Microscopic evaluation and visualisation of the GFP signal indicated that HUVEC were retained within the PEG-hydrogel after 2 weeks of implantation and formed a pre-vascular network. Further analysis will investigate anastomosis between the host vasculature and implanted HUVEC. Based on visual inspection, ectopic bone formation was more pronounced in samples and regions of higher cell density. In future experiments, the extent of bone formation will by quantified by micro-CT, followed by decalcification and histological evaluation.

Conclusions
Our results provide evidence that sound induced morphogenesis is a versatile method to produce cell-hydrogel biografts for subsequent pre-clinical evaluation. We demonstrated that local cell density enhancement by sound requires a lower initial cell concentration than conventional methods to achieve comparable microvasculature structures or local osteogenesis.

References
1 Petta et al., Biofabrication 2021, 13, 015004

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