A Flow Perfusion Bioreactor System for Vocal Fold Tissue Engineering Applications

Abstract

The human vocal folds (VFs) undergo complex biomechanical stimulation during phonation. The aim of the present study was to develop and validate a phono-mimetic VF flow perfusion bioreactor, which mimics the mechanical microenvironment of the human VFs in vitro. The bioreactor uses airflow-induced self-oscillations, which have been shown to produce mechanical loading and contact forces that are representative of human phonation. The bioreactor consisted of two synthetic VF replicas within a silicone body. A cell-scaffold mixture (CSM) consisting of human VF fibroblasts, hyaluronic acid, gelatin, and a polyethylene glycol cross-linker was injected into cavities within the replicas. Cell culture medium (CCM) was perfused through the scaffold by using a customized secondary flow loop. After the injection, the bioreactor was operated with no stimulation over a 3-day period to allow for cell adaptation. Phonation was subsequently induced by using a variable speed centrifugal blower for 2 h each day over a period of 4 days. A similar bioreactor without biomechanical stimulation was used as the nonphonatory control. The CSM was harvested from both VF replicas 7 days after the injection. The results confirmed that the phono-mimetic bioreactor supports cell viability and extracellular matrix proteins synthesis, as expected. Many scaffold materials were found to degrade because of challenges from phonation-induced biomechanical stimulation as well as due to biochemical reactions with the CCM. The bioreactor concept enables future investigations of the effects of different phonatory characteristics, that is, voice regimes, on the behavior of the human VF cells. It will also help study the long-term functional outcomes of the VF-specific biomaterials before animal and clinical studies.

A tissue-mimetic nano-fibrillar hybrid injectable hydrogel for potential soft tissue engineering applications

“…Alginate-based scaffolds, however, were shown to offer poorly regulated degradation due to the absence of enzymes in mammals to break down alginate 82 . The use of polyethylene glycol tetra acrylate as the cross-linking agent for a composite injectable hydrogel composed of thiolated-HA and thiolated-Ge was investigated to overcome the fast degradation rate and to enhance the mechanical and biochemical stability of the scaffolds 2 . Although no cytotoxicity was observed in vitro , and the encapsulated cells synthetized Col-I and Col-III after 7 days in culture, our recent in vivo study in rabbits showed serious inflammatory response.…”

“…The hybrid hydrogels had a half-life of more than 35 days and they featured a greater biochemical stability than that of the HA-based hydrogels previously used for STE with half-life of 2–3 weeks in cartilage, 3–5 days in vocal fold, less than one day in epidermis, and a few minutes in blood 2 , 85 . The hydrogels with higher GCS concentration (G1s) were more resistant to enzymatic degradation over the first 30 days than those with lower GCS concentration.…”

“…The degradation rate of the scaffolding materials should mirror the regeneration rate of the host tissue, especially for in vivo applications 2 , 65 , 86 . Also, a controlled biodegradation is appropriate for in vitro studies using tissue culture bioreactors, for which the hydrogel provides the scaffold for the cells to reside, interact with, and thus synthesize, deposit and remodel neo-tissue 2 .…”

“…Cell-biomaterial interactions were examined using cell viability, metabolic activity, proliferation, cell adhesion, cell migration and cell morphology tests. The proposed hydrogels (Col-I&III/GCS) were shown to support cell adhesion and implantation, and they provided longer half-life than previously investigated HA-based hydrogels 2 . Col-III/GCS hydrogels were observed to support various aspects of cell implantation.…”

“…Considerable efforts have been made over the past few decades to develop scaffolding materials which mimic the extracellular matrix (ECM) for Soft Tissue Engineering (STE), the process of synthesizing natural tissue for the repair or replacement of diseased or lost tissues 1 – 6 . These scaffolding materials are used in vivo , for in-situ tissue regeneration, or in vitro for the fabrication of tissue substitutes in tissue culture bioreactors 7 , 8 , or as controlled tissue-mimetic microenvironments to investigate the effects of biomechanical and biochemical stimuli on cell behavior 2 . The chemical composition and microstructure of the scaffolds considerably influence tissue regeneration and function restoration.…”