@article{biehl_colmon_timofeeva_gracioso martins_dion_peters_freytes_2023, title={Scalable and High-Throughput In Vitro Vibratory Platform for Vocal Fold Tissue Engineering Applications}, volume={10}, ISSN={["2306-5354"]}, url={https://www.mdpi.com/2306-5354/10/5/602}, DOI={10.3390/bioengineering10050602}, abstractNote={The vocal folds (VFs) are constantly exposed to mechanical stimulation leading to changes in biomechanical properties, structure, and composition. The development of long-term strategies for VF treatment depends on the characterization of related cells, biomaterials, or engineered tissues in a controlled mechanical environment. Our aim was to design, develop, and characterize a scalable and high-throughput platform that mimics the mechanical microenvironment of the VFs in vitro. The platform consists of a 24-well plate fitted with a flexible membrane atop a waveguide equipped with piezoelectric speakers which allows for cells to be exposed to various phonatory stimuli. The displacements of the flexible membrane were characterized via Laser Doppler Vibrometry (LDV). Human VF fibroblasts and mesenchymal stem cells were seeded, exposed to various vibratory regimes, and the expression of pro-fibrotic and pro-inflammatory genes was analyzed. Compared to current bioreactor designs, the platform developed in this study can incorporate commercial assay formats ranging from 6- to 96-well plates which represents a significant improvement in scalability. This platform is modular and allows for tunable frequency regimes.}, number={5}, journal={BIOENGINEERING-BASEL}, author={Biehl, Andreea and Colmon, Ramair and Timofeeva, Anastasia and Gracioso Martins, Ana Maria and Dion, Gregory R. and Peters, Kara and Freytes, Donald O.}, year={2023}, month={May} }
 @article{gracioso martins_biehl_sze_freytes_2022, title={Bioreactors for Vocal Fold Tissue Engineering}, volume={28}, ISSN={["1937-3376"]}, DOI={10.1089/ten.teb.2020.0285}, abstractNote={It is estimated that almost one-third of the United States population will be affected by a vocal fold (VF) disorder during their lifespan. Promising therapies to treat VF injury and scarring are mostly centered on VF tissue engineering strategies such as the injection of engineered biomaterials and cell therapy. VF tissue engineering, however, is a challenging field as the biomechanical properties, structure, and composition of the VF tissue change upon exposure to mechanical stimulation. As a result, the development of long-term VF treatment strategies relies on the characterization of engineered tissues under a controlled mechanical environment. In this review, we highlight the importance of bioreactors as a powerful tool for VF tissue engineering with a focus on the current state of the art of bioreactors designed to mimic phonation in vitro. We discuss the influence of the phonatory environment on the development, function, injury, and healing of the VF tissue and its importance for the development of efficient therapeutic strategies. A concise and comprehensive overview of bioreactor designs, principles, operating parameters, and scalability are presented. An in-depth analysis of VF bioreactor data to date reveals that mechanical stimulation significantly influences cell viability and the expression of proinflammatory and profibrotic genes in vitro. Although the precision and accuracy of bioreactors contribute to generating reliable results, diverse gene expression profiles across the literature suggest that future efforts should focus on the standardization of bioreactor parameters to enable direct comparisons between studies. Impact statement We present a comprehensive review of bioreactors for vocal fold (VF) tissue engineering with a focus on the influence of the phonatory environment on the development, function, injury, and healing of the VFs and the importance of mimicking phonation on engineered VF tissues in vitro. Furthermore, we put forward a strong argument for the continued development of bioreactors in this area with an emphasis on the standardization of bioreactor designs, principles, operating parameters, and oscillatory regimes to enable comparisons between studies.}, number={1}, journal={TISSUE ENGINEERING PART B-REVIEWS}, author={Gracioso Martins, Ana M. and Biehl, Andreea and Sze, Daphne and Freytes, Donald O.}, year={2022}, month={Feb}, pages={182–205} }
 @article{biehl_martins_davis_sze_collins_mora-navarro_fisher_freytes_2022, title={Towards a standardized multi-tissue decellularization protocol for the derivation of extracellular matrix materials}, volume={12}, ISSN={["2047-4849"]}, DOI={10.1039/d2bm01012g}, abstractNote={The goal of tissue decellularization is to efficiently remove unwanted cellular components, such as DNA and cellular debris, while retaining the complex structural and molecular milieu within the extracellular matrix (ECM). Decellularization protocols to date are centered on customized tissue-specific and lab-specific protocols that involve consecutive manual steps which results in variable and protocol-specific ECM material. The differences that result from the inconsistent protocols between decellularized ECMs affect consistency across batches, limit comparisons between results obtained from different laboratories, and could limit the transferability of the material for consistent laboratory or clinical use. The present study is the first proof-of-concept towards the development of a standardized protocol that can be used to derive multiple ECM biomaterials (powders and hydrogels) via a previously established automated system. The automated decellularization method developed by our group was used due to its short decellularization time (4 hours) and its ability to reduce batch-to-batch variability. The ECM obtained using this first iteration of a unified protocol was able to produce ECM hydrogels from skin, lung, muscle, tendons, cartilage, and laryngeal tissues. All hydrogels formed in this study were cytocompatible and showed gelation and rheological properties consistent with previous ECM hydrogels. The ECMs also showed unique proteomic composition. The present study represents the first step towards developing standardized protocols that can be used on multiple tissues in a fast, scalable, and reproducible manner.}, journal={BIOMATERIALS SCIENCE}, author={Biehl, Andreea and Martins, Ana M. Gracioso M. and Davis, Zachary G. G. and Sze, Daphne and Collins, Leonard and Mora-Navarro, Camilo and Fisher, Matthew B. B. and Freytes, Donald O. O.}, year={2022}, month={Dec} }
 @article{martins_wilkins_ligler_daniele_freytes_2021, title={Microphysiological System for High-Throughput Computer Vision Measurement of Microtissue Contraction}, volume={6}, ISSN={["2379-3694"]}, DOI={10.1021/acssensors.0c02172}, abstractNote={The ability to measure microtissue contraction in vitro can provide important information when modeling cardiac, cardiovascular, respiratory, digestive, dermal, and skeletal tissues. However, measuring tissue contraction in vitro often requires the use of high number of cells per tissue construct along with time-consuming microscopy and image analysis. Here, we present an inexpensive, versatile, high-throughput platform to measure microtissue contraction in a 96-well plate configuration using one-step batch imaging. More specifically, optical fiber microprobes are embedded in microtissues, and contraction is measured as a function of the deflection of optical signals emitted from the end of the fibers. Signals can be measured from all the filled wells on the plate simultaneously using a digital camera. An algorithm uses pixel-based image analysis and computer vision techniques for the accurate multiwell quantification of positional changes in the optical microprobes caused by the contraction of the microtissues. Microtissue constructs containing 20,000-100,000 human ventricular cardiac fibroblasts (NHCF-V) in 6 mg/mL collagen type I showed contractile displacements ranging from 20-200 μm. This highly sensitive and versatile platform can be used for the high-throughput screening of microtissues in disease modeling, drug screening for therapeutics, physiology research, and safety pharmacology.}, number={3}, journal={ACS SENSORS}, author={Martins, Ana Maria Gracioso and Wilkins, Michael D. and Ligler, Frances S. and Daniele, Michael A. and Freytes, Donald O.}, year={2021}, month={Mar}, pages={985–994} }