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Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair
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Multiphoton Imaging of Maturation in Tissue Engineering.

Maximilian P Werner1,2, Vytautas Kučikas3, Kirsten Voß4

  • 1Department of Biohybrid & Medical Textiles (BioTex), Institute of Applied Medical Engineering (AME), Helmholtz Institute, RWTH Aachen University, Aachen, Germany.

Tissue Engineering. Part C, Methods
|December 20, 2023
PubMed
Summary

This study introduces a new 3D imaging method for assessing tissue-engineered (TE) cardiovascular implants without slicing. The technique quantifies tissue maturation, showing increased collagen and alpha-smooth muscle actin during development.

Keywords:
collagen synthesisprotein quantificationremodeling processsmooth muscle actintwo-photon microscopy

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Area of Science:

  • Biomedical Engineering
  • Regenerative Medicine
  • Cardiovascular Research

Background:

  • Tissue-engineered (TE) implants offer personalized cardiovascular disease treatment.
  • Current methods lack longitudinal, subcellular assessment of TE construct development.
  • Histological slicing is destructive and prevents longitudinal studies.

Purpose of the Study:

  • Establish a generalized labeling and 3D imaging protocol for TE constructs.
  • Enable quantified, longitudinal assessment of TE tissue maturation without slicing.
  • Analyze intracellular matrix (ICM) and extracellular matrix (ECM) development.

Main Methods:

  • Developed a whole-mount labeling and multiphoton laser scanning microscopy (MPLSM) protocol.
  • Imaged TE patches over 21 days of conditioning in 3D.
  • Quantified cell coverage, ICM, and ECM volume fractions using image analysis.

Main Results:

  • Achieved high-quality 3D imaging of intact TE constructs.
  • Demonstrated increased collagen and alpha-smooth muscle actin (α-SMA) volume fractions with maturation.
  • Observed developing, interconnected ICM and ECM networks.

Conclusions:

  • The developed MPLSM protocol allows non-destructive, quantitative assessment of TE construct maturation.
  • This method supports personalized TE therapy development for cardiovascular diseases.
  • Future work includes integrating MPLSM into bioreactors for dynamic monitoring.