Amniotic epithelial cell transplantation induced alternative M2 macrophage activation supporting tendon regeneration

Amniotic epithelial cells (AECs) have a therapeutic potential in tissue repair because their multipotent characteristics and ability to modulate the immune response (1,2). AECs, directly and secreting paracrine factors, modulate tendon healing inducing a prompt regeneration in experimentally injured tendons (3). In the present research, the role exerted of AECs on macrophages (Mφ) polarization from pro-inflammatory M1Mφ to anti-inflammatory M2Mφ phenotype were studied and related to the early phases of tendon healing. Ovine PKH-26 labeled AECs were transplanted into experimentally induced calcaneal tendons defects (3). Control (CTR) and AEC-tendon explants were explanted at 7, 14, and 28 days for morphological/molecular analyses. Tendon healing was described using Hematoxylin and Eosin stain and immunohistochemical (IHC) analyses to detect the expression of collagen type I an extracellular matrix protein (COL1). IHC and RT-PCR analyses were carried out to identify Mφ (PanMφCD68) and the incidence of pro-inflammatory M1Mφ (CD86, IL12) and anti-inflammatory M2Mφ (CD206, YM1, IL10) sub-populations. AECs were always retrieved within the host tissue enhancing the early phase of tendon healing. In parallel, tendons displayed a different evolution in inflammatory events. PanMφCD68 expression indicated that inflammation progressively decreased in both tendons. However, while polarized Mφ and cytokines were similarly expressed in AECs and CTR tendons until day 7, the M2Mφ became prevalent in the presence of AECs at day 14 when the tissue started to recover its microarchitecture displaying COL1 parallel oriented fibers. Analogously, the mRNA content of M2Mφ anti-inflammatory cytokines YM1, IL10, CD206 increased, while M1Mφ pro-inflammatory markers IL12 and CD86 decreased. Mφ and cytokine expression were considerably reduced in regenerated AEC-tendons after 28 days, while they were still expressed in CTR tendons that maintained a disorganized tissue structure. In summary, these findings suggest that AECs modulate macrophage recruitment enhancing the M2Mφ regenerative phenotype and down regulating M1Mφ, pro-inflammatory phenotype contributing to tendon healing. The present results suggests novel insights into potential mechanisms underlying tendon regeneration, associated with extracellular matrix remodeling amelioration, induced by exogenous ovine amnios epithelial layer stem cells delivery.