In Vitro Innovation of Tendon Tissue Engineering Strategies.

Tendinopathy is the term used to refer to tendon disorders. Spontaneous adult tendon healing results in scar tissue formation and fibrosis with suboptimal biomechanical properties, often resulting in poor and painful mobility. The biomechanical properties of the tissue are negatively affected. Adult tendons have a limited natural healing capacity, and often respond poorly to current treatments that frequently are focused on exercise, drug delivery, and surgical procedures. Therefore, it is of great importance to identify key molecular and cellular processes involved in the progression of tendinopathies to develop effective therapeutic strategies and drive the tissue toward regeneration. To treat tendon diseases and support tendon regeneration, cell-based therapy as well as tissue engineering approaches are considered options, though none can yet be considered conclusive in their reproduction of a safe and successful long-term solution for full microarchitecture and biomechanical tissue recovery. In vitro differentiation techniques are not yet fully validated. This review aims to compare different available tendon in vitro differentiation strategies to clarify the state of art regarding the differentiation process.

Fabrication and Plasma Surface Activation of Aligned Electrospun PLGA Fiber Fleeces with Improved Adhesion and Infiltration of Amniotic Epithelial Stem Cells Maintaining their Teno-inductive Potential.

Electrospun PLGA microfibers with adequate intrinsic physical features (fiber alignment and diameter) have been shown to boost teno-differentiation and may represent a promising solution for tendon tissue engineering. However, the hydrophobic properties of PLGA may be adjusted through specific treatments to improve cell biodisponibility. In this study, electrospun PLGA with highly aligned microfibers were cold atmospheric plasma (CAP)-treated by varying the treatment exposure time (30, 60, and 90 s) and the working distance (1.3 and 1.7 cm) and characterized by their physicochemical, mechanical and bioactive properties on ovine amniotic epithelial cells (oAECs). CAP improved the hydrophilic properties of the treated materials due to the incorporation of new oxygen polar functionalities on the microfibers' surface especially when increasing treatment exposure time and lowering working distance. The mechanical properties, though, were affected by the treatment exposure time where the optimum performance was obtained after 60 s. Furthermore, CAP treatment did not alter oAECs' biocompatibility and improved cell adhesion and infiltration onto the microfibers especially those treated from a distance of 1.3 cm. Moreover, teno-inductive potential of highly aligned PLGA electrospun microfibers was maintained. Indeed, cells cultured onto the untreated and CAP treated microfibers differentiated towards the tenogenic lineage expressing tenomodulin, a mature tendon marker, in their cytoplasm. In conclusion, CAP treatment on PLGA microfibers conducted at 1.3 cm working distance represent the optimum conditions to activate PLGA surface by improving their hydrophilicity and cell bio-responsiveness. Since for tendon tissue engineering purposes, both high cell adhesion and mechanical parameters are crucial, PLGA treated for 60 s at 1.3 cm was identified as the optimal construct.

Hypoxic Functional Regulation Pathways in the GI Tract: Focus on the HIF-1α and Microbiota's Crosstalk.

Hypoxia is an essential gastrointestinal (GI) tract phenomenon that influences both physiologic and pathologic states. Hypoxia-inducible factors (HIFs), the primary drivers of cell adaptation to low-oxygen environments, have been identified as critical regulators of gut homeostasis: directly, through the induction of different proteins linked to intestinal barrier stabilization (ie, adherent proteins, tight junctions, mucins, integrins, intestinal trefoil factor, and adenosine); and indirectly, through the regulation of several immune cell types and the modulation of autophagy and inflammatory processes. Furthermore, hypoxia and HIF-related sensing pathways influence the delicate relationship existing between bacteria and mammalian host cells. In turn, gut commensals establish and maintain the physiologic hypoxia of the GI tract and HIF-α expression. Based on this premise, the goals of this review are to (1) highlight hypoxic molecular pathways in the GI tract, both in physiologic and pathophysiologic settings, such as inflammatory bowel disease; and (2) discuss a potential strategy for ameliorating gut-related disorders, by targeting HIF signaling, which can alleviate inflammatory processes, restore autophagy correct mechanisms, and benefit the host-microbiota equilibrium.

In recent years, hypoxic conditions, with subsequent hypoxia-inducible factor activation, and the gut's microbiota composition have both received significant attention due to their correlation with gut homeostasis maintenance. However, their potential synergic action needs further investigation.

Hypoxia-Mimetic CoCl2 Agent Enhances Pro-Angiogenic Activities in Ovine Amniotic Epithelial Cells-Derived Conditioned Medium.

Amniotic epithelial stem cells (AECs) are largely studied for their pro-regenerative properties. However, it remains undetermined if low oxygen (O2) levels that AECs experience in vivo can be of value in maintaining their biological properties after isolation. To this aim, the present study has been designed to evaluate the effects of a hypoxia-mimetic agent, cobalt chloride (CoCl2), on AECs' stemness and angiogenic activities. First, a CoCl2 dose-effect was performed to select the concentration able to induce hypoxia, through HIF-1α stabilization, without promoting any cytotoxicity effect assessed through the analysis of cell vitality, proliferation, and apoptotic-related events. Then, the identified CoCl2 dose was evaluated on the expression and angiogenic properties of AECs' stemness markers (OCT-4, NANOG, SOX-2) by analysing VEGF expression, angiogenic chemokines' profiles, and AEC-derived conditioned media activity through an in vitro angiogenic xeno-assay. Results demonstrated that AECs are sensitive to the cytotoxicity effects of CoCl2. The unique concentration leading to HIF-1α stabilization and nuclear translocation was 10 µM, preserving cell viability and proliferation up to 48 h. CoCl2 exposure did not modulate stemness markers in AECs while progressively decreasing VEGF expression. On the contrary, CoCl2 treatment promoted a significant short-term release of angiogenic chemokines in culture media (CM). The enrichment in bio-active factors was confirmed by the ability of CoCl2-derived CM to induce HUVEC growth and the cells' organization in tubule-like structures. These findings demonstrate that an appropriate dose of CoCl2 can be adopted as a hypoxia-mimetic agent in AECs. The short-term, chemical-induced hypoxic condition can be targeted to enhance AECs' pro-angiogenic properties by providing a novel approach for stem cell-free therapy protocols.

Amniotic Epithelial Stem Cells Counteract Acidic Degradation By-Products of Electrospun PLGA Scaffold by Improving Their Immunomodulatory Profile In Vitro.

Electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds with highly aligned fibers (ha-PLGA) represent promising materials in the field of tendon tissue engineering (TE) due to their characteristics in mimicking fibrous extracellular matrix (ECM) of tendon native tissue. Among these properties, scaffold biodegradability must be controlled allowing its replacement by a neo-formed native tendon tissue in a controlled manner. In this study, ha-PLGA were subjected to hydrolytic degradation up to 20 weeks, under di-H2O and PBS conditions according to ISO 10993-13:2010. These were then characterized for their physical, morphological, and mechanical features. In vitro cytotoxicity tests were conducted on ovine amniotic epithelial stem cells (oAECs), up to 7 days, to assess the effect of non-buffered and buffered PLGA by-products at different concentrations on cell viability and their stimuli on oAECs' immunomodulatory properties. The ha-PLGA scaffolds degraded slowly as evidenced by a slight decrease in mass loss (14%) and average molecular weight (35%), with estimated degradation half-time of about 40 weeks under di-H2O. The ultrastructure morphology of the scaffolds showed no significant fiber degradation even after 20 weeks, but alteration of fiber alignment was already evident at week 1. Moreover, mechanical properties decreased throughout the degradation times under wet as well as dry PBS conditions. The influence of acid degradation media on oAECs was dose-dependent, with a considerable effect at 7 days' culture point. This effect was notably reduced by using buffered media. To a certain level, cells were able to compensate the generated inflammation-like microenvironment by upregulating IL-10 gene expression and favoring an anti-inflammatory rather than pro-inflammatory response. These in vitro results are essential to better understand the degradation behavior of ha-PLGA in vivo and the effect of their degradation by-products on affecting cell performance. Indeed, buffering the degradation milieu could represent a promising strategy to balance scaffold degradation. These findings give good hope with reference to the in vivo condition characterized by physiological buffering systems.

Insight into Hypoxia Stemness Control.

Recently, the research on stemness and multilineage differentiation mechanisms has greatly increased its value due to the potential therapeutic impact of stem cell-based approaches. Stem cells modulate their self-renewing and differentiation capacities in response to endogenous and/or extrinsic factors that can control stem cell fate. One key factor controlling stem cell phenotype is oxygen (O2). Several pieces of evidence demonstrated that the complexity of reproducing O2 physiological tensions and gradients in culture is responsible for defective stem cell behavior in vitro and after transplantation. This evidence is still worsened by considering that stem cells are conventionally incubated under non-physiological air O2 tension (21%). Therefore, the study of mechanisms and signaling activated at lower O2 tension, such as those existing under native microenvironments (referred to as hypoxia), represent an effective strategy to define if O2 is essential in preserving naïve stemness potential as well as in modulating their differentiation. Starting from this premise, the goal of the present review is to report the status of the art about the link existing between hypoxia and stemness providing insight into the factors/molecules involved, to design targeted strategies that, recapitulating naïve O2 signals, enable towards the therapeutic use of stem cell for tissue engineering and regenerative medicine.

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

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.

Electrospun PLGA Fiber Diameter and Alignment of Tendon Biomimetic Fleece Potentiate Tenogenic Differentiation and Immunomodulatory Function of Amniotic Epithelial Stem Cells.

Injured tendons are challenging in their regeneration; thus, tissue engineering represents a promising solution. This research tests the hypothesis that the response of amniotic epithelial stem cells (AECs) can be modulated by fiber diameter size of tendon biomimetic fleeces. Particularly, the effect of electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned microfibers possessing two different diameter sizes (1.27 and 2.5 µm: ha1- and ha2-PLGA, respectively) was tested on the ability of AECs to differentiate towards the tenogenic lineage by analyzing tendon related markers (Collagen type I: COL1 protein and mRNA Scleraxis: SCX, Tenomodulin: TNMD and COL1 gene expressions) and to modulate their immunomodulatory properties by investigating the pro- (IL-6 and IL-12) and anti- (IL-4 and IL-10) inflammatory cytokines. It was observed that fiber alignment and not fiber size influenced cell morphology determining the morphological change of AECs from cuboidal to fusiform tenocyte-like shape. Instead, fleece mechanical properties, cell proliferation, tenogenic differentiation, and immunomodulation were regulated by changing the ha-PLGA microfiber diameter size. Specifically, higher DNA quantity and better penetration within the fleece were found on ha2-PLGA, while ha1-PLGA fleeces with small fiber diameter size had better mechanical features and were more effective on AECs trans-differentiation towards the tenogenic lineage by significantly translating more efficiently SCX into the downstream effector TNMD. Moreover, the fiber diameter of 1.27 µm induced higher expression of pro-regenerative, anti-inflammatory interleukins mRNA expression (IL-4 and IL-10) with favorable IL-12/IL-10 ratio with respect to the fiber diameter of 2.5 µm. The obtained results demonstrate that fiber diameter is a key factor to be considered when designing tendon biomimetic fleece for tissue repair and provide new insights into the importance of controlling matrix parameters in enhancing cell differentiation and immunomodulation either for the cells functionalized within or for the transplanted host tissue.