The authors evaluated the relationship between vascular endothelial growth factor (VEGF) production, blood vessel extension, and steroidogenesis in small (<4 mm), medium (4-5 mm), and large (>5 mm) follicles isolated from gilts treated with eCG. VEGF and estradiol levels were measured in follicular fluid by an enzyme immunoassay and radioimmunoassay, respectively, and then each follicle wall was used to evaluate VEGF mRNA content and for the immunohistochemical analysis of blood vessels. VEGF production was low in small follicles (<3 ng/ml), high in large follicles (>10 ng/ml), and markedly differentiated in medium follicles; 44% exhibited values up to 15 ng/ml, whereas the levels never exceeded 3 ng/ml in the remaining aliquot. Medium follicles were then used as a model to investigate angiogenesis. Reverse transcription-polymerase chain reaction for VEGF mRNA demonstrated that granulosa cells represent the main component involved in the production of VEGF. The follicle wall, which presents two distinct concentric vessel networks, showed a vascular area (positive stained area/percent of field area) that was significantly wider in high VEGF follicles than in low VEGF follicles (2.54% +/- 0.58% vs. 1.29% +/- 0.58%, respectively). Medium follicles with high VEGF levels and extensive vascularization accumulated high estradiol levels (150-300 ng/ml), whereas follicles with low VEGF levels had basal estradiol levels that never exceeded 30 ng/ml. Early atretic medium-size follicles had undetectable levels of VEGF and estradiol paralleled by a marked reduction in blood vessel. The data presented propose an improved model for follicle dynamics in which the production of VEGF, stimulated by gonadotropin, creates the vascular conditions required for follicle growth and activity.

Amniotic epithelial cells (AECs) are ideal seed cells for tissue regeneration, but no research has yet been reported on their tendon regeneration potential. This study investigated the efficiency of AEC allotransplantation for tendon healing, as well as the mechanism involved. To this aim ovine AECs, characterized by specific surface and stemness markers (CD14-, CD49f, CD29, CD166, OCT4, SOX2, NANOG, TERT), were allotransplanted into experimentally induced tissue defects in sheep Achilles tendon. In situ tissue repair revealed that AEC-treated tendons had much better structural and mechanical recoveries than control ones during the early phase of healing. Immunohistochemical and biochemical analyses indicated that extracellular matrix remodeling was more rapid and that immature collagen fibers were completely replaced by mature ones in 28 days. Moreover, spatial-temporal analysis of cellularity, proliferation index, vascular area, and leukocyte infiltration revealed that AECs induced a specific centripetal healing process that first started in the tissue closer to the healthy portion of the tendons, where AECs rapidly migrated to then progress through the core of the lesion. This peculiar healing evolution could have been induced by the growth factor stimulatory influence (TGF-b1 and VEGF) and/or by the host progenitor cells recruitment, but also as the consequence of a direct tenogenic AEC differentiation resulting in the regeneration of new tendon matrix. These findings demonstrate that AECs can support tendon regeneration, and their effects may be used to develop future strategies to treat tendon disease characterized by a poor clinical outcome in veterinary medicine.

The in vitro expansion is detrimental to therapeutic applications of amniotic epithelial cells (AEC), an emerging source of fetal stem cells. This study provides molecular evidences of progesterone (P4) role in preventing epithelial-mesenchymal transition (EMT) in ovine AEC (oAEC). oAEC amplified under standard conditions spontaneously acquired mesenchymal properties through the up-regulation of EMT-transcription factors. P4 supplementation prevented phenotype shift by inhibiting the EMT-inducing mechanism such as the autocrine production of TGF-β and the activation of intracellular-related signaling. The effect of P4 still persisted for one passage after steroid removal from culture as well as steroid supplementation promptly reversed mesenchymal phenotype in oAEC which have experienced EMT during amplification. Furthermore, P4 promoted an acute up-regulation of pluripotent genes whereas enhanced basal and LPS-induced oAEC anti-inflammatory response with an increase in anti-inflammatory and a decrease in pro-inflammatory cytokines expression. Altogether, these results indicate that P4 supplementation is crucial to preserve epithelial phenotype and to enhance biological properties in expanded oAEC. Therefore, an innovative cultural approach is proposed in order to improve therapeutic potential of this promising source of epithelial stem cells.

Cell-based therapy holds great promise for tendon disorders, a widespread debilitating musculoskeletal condition. Even if the cell line remains to be defined, preliminary evidences have proven that amniotic-derived cells possess in vitro and in vivo a great tenogenic potential. This study investigated the efficacy of transplanted human amniotic epithelial cells (hAECs) by testing their early regenerative properties and mechanisms involved on a validated ovine Achilles tendon partial defect performed on 29 animals. The injured tendons treated with hAECs recovered rapidly, in 28 days, structural and biomechanical properties undertaking a programmed tissue regeneration, differently from the spontaneous healing tissues. hAECs remained viable within the host tendons establishing with the endogenous progenitor cells an active dialogue. Through the secretion of modulatory factors, hAECs inhibited the inflammatory cells infiltration, activated the M2 macrophage subpopulation early recruitment, and accelerated blood vessel as well as extracellular matrix remodelling. In parallel, some in situ differentiated hAECs displayed a tenocytelike phenotype. Both paracrine and direct hAECs stimulatory effects were confirmed analysing their genome profile before and after transplantation. The 49 human up-regulated transcripts recorded in transplanted hAECs belonged to tendon lineage differentiation (epithelial-mesenchymal transition, connective specific matrix components, and skeleton or muscle system development-related transcripts), as well as the in situ activation of paracrine signalling involved in inflammatory and immunomodulatory response. Altogether, these evidences support the hypothesis that hAECs are a practicable and efficient strategy for the acute treatment of tendinopathy, reinforcing the idea of a concrete use of amniotic epithelial cells towards the clinical practice.

BackgroundEvidence has been provided that a cell-based therapy combined with the use of bioactive materials may significantly improve bone regeneration prior to dental implant, although the identification of an ideal source of progenitor/stem cells remains to be determined.AimIn the present research, the bone regenerative property of an emerging source of progenitor cells, the amniotic epithelial cells (AEC), loaded on a calcium-phosphate synthetic bone substitute, made by direct rapid prototyping (rPT) technique, was evaluated in an animal study.Material And MethodsTwo blocks of synthetic bone substitute (∼0.14 cm3), alone or engineered with 1×106 ovine AEC (oAEC), were grafted bilaterally into maxillary sinuses of six adult sheep, an animal model chosen for its high translational value in dentistry. The sheep were then randomly divided into two groups and sacrificed at 45 and 90 days post implantation (p.i.). Tissue regeneration was evaluated in the sinus explants by micro-computer tomography (micro-CT), morphological, morphometric and biochemical analyses.Results And ConclusionsThe obtained data suggest that scaffold integration and bone deposition are positively influenced by allotransplantated oAEC. Sinus explants derived from sheep grafted with oAEC engineered scaffolds displayed a reduced fibrotic reaction, a limited inflammatory response and an accelerated process of angiogenesis. In addition, the presence of oAEC significantly stimulated osteogenesis either by enhancing bone deposition or making more extent the foci of bone nucleation. Besides the modulatory role played by oAEC in the crucial events successfully guiding tissue regeneration (angiogenesis, vascular endothelial growth factor expression and inflammation), data provided herein show that oAEC were also able to directly participate in the process of bone deposition, as suggested by the presence of oAEC entrapped within the newly deposited osteoid matrix and by their ability to switch-on the expression of a specific bone-related protein (osteocalcin, OCN) when transplanted into host tissues.