Amnion-Derived Teno-Inductive Secretomes: A Novel Approach to Foster Tendon Differentiation and Regeneration in an Ovine Model.

Maria Rita Citeroni,Valentina Russo,Barbara Barboni,Maura Turriani,Annunziata Mauro,Michael Santer,Mohammad El Khatib,Giovanna Della Porta,Nicholas R Forsyth,Maria Camilla Ciardulli,Miriam Di Mattia,Nicola Maffulli

doi:10.3389/fbioe.2021.649288

Abstract

Regenerative medicine has greatly progressed, but tendon regeneration mechanisms and robust in vitro tendon differentiation protocols remain to be elucidated. Recently, tendon explant co-culture (CO) has been proposed as an in vitro model to recapitulate the microenvironment driving tendon development and regeneration. Here, we explored standardized protocols for production and storage of bioactive tendon-derived secretomes with an evaluation of their teno-inductive effects on ovine amniotic epithelial cells (AECs). Teno-inductive soluble factors were released in culture-conditioned media (CM) only in response to active communication between tendon explants and stem cells (CMCO). Unsuccessful tenogenic differentiation in AECs was noted when exposed to CM collected from tendon explants (CMFT) only, whereas CMCO upregulated SCXB, COL I and TNMD transcripts, in AECs, alongside stimulation of the development of mature 3D tendon-like structures enriched in TNMD and COL I extracellular matrix proteins. Furthermore, although the tenogenic effect on AECs was partially inhibited by freezing CMCO, this effect could be recovered by application of an in vivo-like physiological oxygen (2% O2) environment during AECs tenogenesis. Therefore, CMCO can be considered as a waste tissue product with the potential to be used for the development of regenerative bio-inspired devices to innovate tissue engineering application to tendon differentiation and healing.

Highlights

Tendon differentiation is a stepwise process characterized by the sequential expression of tissue specific markers, an in vivo occurrence through the interaction of inductive paracrine conditions and the recruitment of tissue progenitor stem cells (Brent, 2005; Nourissat et al, 2015; Citeroni et al, 2020)
Collagen type I (COL I) and Collagen type III (COL III) are the major component of tendon extracellular matrix (ECM) and their modulation is related to tendon homeostasis and healing (Maffulli et al, 2000; Sharma and Maffulli, 2005)
TNMD and THBS4 are both considered late tendon markers: TNMD is abundantly expressed in the mature tissue (Docheva et al, 2005), while THBS4 contributes to the regulation of extracellular matrix deposition and in the repair of myotendinous junction (MTJs) (Frolova et al, 2014; Subramanian and Schilling, 2014)

Summary

Introduction

Tendon differentiation is a stepwise process characterized by the sequential expression of tissue specific markers, an in vivo occurrence through the interaction of inductive paracrine conditions and the recruitment of tissue progenitor stem cells (Brent, 2005; Nourissat et al, 2015; Citeroni et al, 2020). Many of these genes are already expressed in mesenchyme-derived stem cell sources (Zarychta-Wisniewska et al, 2019), making threshold level determination in tenogensis difficult. The most prevalent tenogenic markers are Scleraxis (SCX), Tenomodulin (TNMD), Collagen type I (COL I), Collagen type III (COL III), and Thrombospondin 4 (THSB4) (Ciardulli et al, 2020b; Citeroni et al, 2020). Mice with loss of TNMD expression showed impaired tenocyte proliferation, reduced tenocyte density, and increased maximal and greater variation of fibril diameters (Docheva et al, 2005)

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