Fabrication and Characterization of Decellularized Periodontal Ligament Cell Sheet Constructs

Abstract

Decellularized tissue-engineered constructs have the potential to promote regeneration by providing a biomimetic extracellular matrix that directs tissue-specific regeneration when implanted in situ. Recently, the use of cell sheets has shown promising results in promoting periodontal regeneration. Here, we describe the fabrication of decellularized periodontal cell sheets with intact extracellular matrix structural and biological properties. Melt electro spun polycaprolactone (PCL) scaffolds are used as a carrier for the inherently fragile cell sheets, to provide support during the processes of decellularization. An optimized decellularization method is outlined using perfusion with a combination of NHOH and Triton X-100 together with a DNase treatment step for DNA removal. The maintenance of extracellular matrix structural and biological integrity is important, and here we describe the assessment of these properties using immunostaining for extracellular matrix proteins and ELISA for growth factor quantification.

Assessment of static and perfusion methods for decellularization of PCL membrane-supported periodontal ligament cell sheet constructs

“…The use of 3D biomimetic bioprinted constructs as well as polymer supported fibrous cell sheets constructs may be particularly suited to the regeneration of soft-hard tissue interfaces, such as the fibrocartilage phase (Gurkan et al, 2014) and the complex (bone-ligamentcementum) structure of periodontal attachment (A, Roberts, Yap, Gomez, & Neufeld, 2015;Ahn et al, 2015;Akizuki et al, 2005;Dan et al, 2014;Ma et al, 2012;Vaquette et al, 2012;Vaquette, Ivanovski, Hamlet, & Hutmacher, 2013). However, the utilization of decellularized periodontal ligament cell sheets has only recently been reported in the literature (Farag, Vaquette, Hutmacher, Bartold, & Ivanovski, 2017;Farag et al, 2014) and presents some unique challenges, with the inherent fragility of the sheets presenting issues with handling and delivery. These challenges are accentuated in the context of decellularization, which is a technique which requires considerable handling and manipulation.…”

Effect of In Vitro Culture Length on the Bone-Forming Capacity of Osteoblast-Derived Decellularized Extracellular Matrix Melt Electrowritten Scaffolds

“…While the precise mode of action by which decellularized ECM enhances tissue regeneration is not fully understood, it is hypothesized that the release of various biological cues such as growth factors, , cytokines, and extracellular vesicles plays a major role and highlights the importance of preserving ECM components. The decellularization protocol employed in the present study was previously optimized for the decellularization of periodontal ligament cell sheets while preserving the most important components of the ECM through short incubation with gentle chemical and enzymatic treatments. ,, Generally, the decellularization of native tissues requires the use of harsh treatments to allow efficient cell removal due to their densely organized ECM preventing fluid circulation. Consequently, this significantly damages the matrix and results in poor preservation of the biological cues embedded in the ECM. − Such decellularized constructs usually present poor recellularization potential and require further treatments such as electroporation or mechanical perforation to enhance cell infiltration. , The use of scaffolds with controlled porosity and subsequently decorated with ECM can overcome these limitations, allowing for better circulation of the decellularization solution and ultimately enabling homogeneous recellularization. ,, The strategy of using mild decellularization solution was efficiently applied to our constructs, resulting in 82.5% of DNA removal in the 4 week old constructs, and over 90% removal in the other groups.…”

“…The decellularization protocol employed in the present study was previously optimized for the decellularization of periodontal ligament cell sheets while preserving the most important components of the ECM through short incubation with gentle chemical and enzymatic treatments. 46,61,62 Generally, the decellularization of native tissues requires the use of harsh treatments to allow efficient cell removal due to their densely organized ECM preventing fluid circulation. Consequently, this significantly damages the matrix and results in poor preservation of the biological cues embedded in the ECM.…”

Biodegradable engineered fiber scaffolds fabricated by electrospinning for periodontal tissue regeneration

“…Decellularization technology is a cell-based therapy providing a biomimetic extracellular matrix without eliciting an adverse immunogenic response when implanted in vivo. 127,128 It can be combined with electrospinning to develop ligament constructs 129 for the utilization in periodontal field. Flores transplanted human periodontal ligament cell sheets into periodontal fenestration defects of immunodeficient rats and found an obvious formation of cementum layer and Sharpey's fibers.…”