The mesentery as the epicenter for intestinal regeneration.

The mesentery, a newly minted organ, plays various anatomical and physiological roles during animal development. In echinoderms, and particularly in members of the class Holothuroidea (sea cucumbers) the mesentery plays an additional unique role: it is crucial for the process of intestinal regeneration. In these organisms, a complete intestine can form from cells that originate in the mesentery. In this review, we focus on what is known about the changes that take place in the mesentery and what has been documented on the cellular and molecular mechanisms involved. We describe how the events that unfold in the mesentery result in the formation of a new intestine.

Insights into intestinal regeneration signaling mechanisms.

The cellular mechanisms underlying the amazing ability of sea cucumbers to regenerate their autotomized intestines have been widely described by us and others. However, the signaling pathways that control these mechanisms are unknown. Previous studies have shown that Wnt homologs are upregulated during early intestinal regenerative stages, suggesting that the Wnt/ß-catenin pathway is active during this process. Here, we used small molecules, putative disruptors of the Wnt pathway, to determine the potential role of the canonical Wnt pathway on intestine regeneration in the sea cucumber Holothuria glaberrima. We evaluated their effects in vivo by using histological analyses for cell dedifferentiation, cell proliferation and apoptosis. We found that iCRT14, an alleged Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a presumed Wnt pathway activator, increased its size. The possible cellular mechanisms by which signaling pathway disruptors affect the gut rudiment size were further studied in vitro, using cultures of tissue explants and additional pharmacological agents. Among the tested signaling activators, those that act through GSK-3 inhibition, LiCl, 1-Azakenpaullone, and CHIR99021 were found to increase muscle cell dedifferentiation, while the inhibitor iCRT14 blocked cell dedifferentiation. Differently, cell proliferation was reduced by all GSK-3 inhibitors, as well as by iCRT14 and C59, which interferes with Wnt ligand secretion. The in vivo temporal and spatial pattern of ß-catenin activity was determined using an antibody against phosphorylated ß-catenin and shown to correlate with cell proliferative activity. In vitro treatment using C59 decreased the number of cells immunostained for nuclear phosphorylated ß-catenin. Our results showed that the cell dedifferentiation observed during intestinal regeneration can be decoupled from the cell proliferation event and that these cellular processes can be modulated by particular signaling pathway inhibitors and activators. These results open the door for future studies where the cellular signaling pathways involved at each regeneration stage can be determined.

Bioactive Scaffolds as a Promising Alternative for Enhancing Critical-Size Bone Defect Regeneration in the Craniomaxillofacial Region.

Reconstruction of critical-size bone defects (CSDs) in the craniomaxillofacial (CMF) region remains challenging. Scaffold-based bone-engineered constructs have been proposed as an alternative to the classical treatments made with autografts and allografts. Scaffolds, a key component of engineered constructs, have been traditionally viewed as biologically passive temporary replacements of deficient bone lacking intrinsic cues to promote osteogenesis. Nowadays, scaffolds are functionalized, giving rise to bioactive scaffolds promoting bone regeneration more effectively than conventional counterparts. This review focuses on the three approaches most used to bioactivate scaffolds: (1) conferring microarchitectural designs or surface nanotopography; (2) loading bioactive molecules; and (3) seeding stem cells on scaffolds, providing relevant examples of in vivo (preclinical and clinical) studies where these methods are employed to enhance CSDs healing in the CMF region. From these, adding bioactive molecules (specifically bone morphogenetic proteins or BMPs) to scaffolds has been the most explored to bioactivate scaffolds. Nevertheless, the downsides of grafting BMP-loaded scaffolds in patients have limited its successful translation into clinics. Despite these drawbacks, scaffolds containing safer, cheaper, and more effective bioactive molecules, combined with stem cells and topographical cues, remain a promising alternative for clinical use to treat CSDs in the CMF complex replacing autografts and allografts.

Intestine Explants in Organ Culture: A Tool to Broaden the Regenerative Studies in Echinoderms.

The cellular events underlying intestine regrowth in the sea cucumber Holothuria glaberrima have been described by our group. Currently, the molecular and signaling mechanisms involved in this process are being explored. One of the limitations to our investigations has been the absence of suitable cell culture methodologies, required to advance the regeneration studies. An in vitro system, where regenerating intestine explants can be studied in organ culture, was established previously by our group. However, a detailed description of the histological properties of the cultured gut explants was lacking. Here, we used immunocytochemical techniques to study the potential effects of the culture conditions on the histological characteristics of explants, comparing them to the features observed during gut regeneration in our model in vivo. Additionally, the explant outgrowths were morphologically described by phase-contrast microscopy and SEM. Remarkably, intestine explants retain most of their original histoarchitecture for up to 10 days, with few changes as culture time increases. The most evident effects of the culture conditions on explants over culture time were the reduction in the proliferative rate, the loss of the polarity in the localization of proliferating cells, and the appearance of a subpopulation of putative spherulocytes. Finally, cells that migrated from the gut explants could form net-like monolayers, firmly attached to the culture substrate. Overall, regenerating explants in organ culture represent a powerful tool to perform short-term studies of processes associated with gut regeneration in H. glaberrima under controlled conditions.

In vitro evaluation of human osteoblast adhesion to a thermally oxidized gamma-TiAl intermetallic alloy of composition Ti-48Al-2Cr-2Nb (at.%).

Ti-48Al-2Cr-2Nb (at.%) (gamma-TiAl), a gamma titanium aluminide alloy originally designed for aerospace applications, appears to have excellent potential as implant material. Thermal treatment of gamma-TiAl renders this alloy extremely corrosion resistant in vitro, which could improve its biocompatibility. In this study, the surface oxides produced by thermal oxidation (at 500 degrees C, and at 800 degrees C for 1 h in air) on gamma-TiAl were characterized by X-ray photoelectron spectroscopy (XPS). hFOB 1.19 cell adhesion on thermally oxidized gamma-TiAl was examined in vitro by a hexosaminidase assay, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) after 1, 7 and 14 days. Ti-6Al-4V surfaces were used for comparison. Hexosaminidase assay data and CLSM analysis of focal contacts and cytoskeleton organization showed no differences in cell attachment on autoclaved and both heat-treated gamma-TiAl surfaces at the different time points. SEM images showed well organized multi-layers of differentiated cells adhered on thermally oxidized gamma-TiAl surfaces at day 14. Unexpectedly, thermally oxidized Ti-6Al-4V surfaces oxidized at 800 degrees C exhibited cytotoxic effects on hFOB 1.19 cells. Our results indicate that thermal oxidation of gamma-TiAl seems to be a promising method to generate highly corrosion resistant and biocompatible surfaces for implant applications.

Fabrication and Evaluation of Polycaprolactone Beads-on-String Membranes for Applications in Bone Tissue Regeneration.

Tissue engineering leads to the development of biomaterial scaffolds where its biocompatibility and bioactivity are often improved after performing physical or chemical surface modification treatments. Micropatterning, soft lithography, and biofabrication are also approaches that provide a biomimetic microenvironment but have proven very costly and time consuming. In this concern, an appropriate substrate with suitable sites for cell attachment represents a major factor in cell behavior and biological functions. For this reason, our strategy was to fabricate a standard fibrous biomaterial with reproducible surface topography, incorporating microbeads and nanofeatures, and show the positive outcomes of the new substrate reflected on cell functions of bone cells. The electrospun polycaprolactone (PCL) beads-on-string membranes were obtained by adjusting the spinning solution at different concentrations until continuous beads were formed. Cell adhesion and proliferation, on the PCL scaffold, were analyzed the subsequent 2 days after initial culture. Complementary studies of cytoskeleton spreading and differentiation were analyzed after 7 and 14 days of the initial incubation. The scanning electron microscopy (SEM) images showed evidence of the formation of beads-on-string nanofibers and suggested that as-formed microstructures worked as attachment sites for osteoblasts. We investigated cell proliferation using anti-BrdU fluorescence assay, and results show a similar proliferation rate of cells cultured between PCL scaffolds and control. Finally, Phalloidin TRITC and antisialoprotein antibody were used to analyze cell spreading and differentiation after 7 and 14 days, respectively. This work shows a low-cost fabrication method to produce a biodegradable scaffold with micro/nanostructured characteristics that favor cell adhesion, proliferation, maturation, and subsequent differentiation of osteoblasts. According to the results, the biocompatibility of PCL beads-on-string could be comparable to other complex biomaterials, and we conclude that our scaffold is optimal for applications in bone tissue regeneration.

Assessing the Suitability of Cellulose-Nanodiamond Composite As a Multifunctional Biointerface Material for Bone Tissue Regeneration.

Interfacial surface properties, both physical and chemical, are known to play a critical role in achieving long-term stability of cell-biomaterial interactions. Novel bone tissue engineering technologies, which provide a suitable interface between cells and biomaterials and mitigate aseptic osteolysis, are sought and can be developed via the incorporation of nanostructured materials. In this sense, engineered nanobased constructs provide an effective interface and suitable topography for direct interaction with cells, promoting faster osseointegration and anchoring. Therefore, herein we have investigated the surface functionalization, biocompatibility, and effect of cellulose-nanodiamond conjugates on osteoblast proliferation and differentiation. Cellulose nanocrystals (CNC) were aminated through a 3-aminopropyltriethyoxysilane (APTES) silylation, while nanodiamonds (ND) were treated with a strong acid oxidation reflux, as to produce carboxyl groups on the surface. Thereafter, the two products were covalently joined through an amide linkage, using a common bioconjugation reaction. Human fetal osteoblastic cells (hFOB) were seeded for 7 days to investigate the in vitro performance of the cellulose-nanodiamond conjugates. By employing immunocytochemistry, the bone matrix expression of osteocalcin (OC) and bone sialoprotein (BSP) was analyzed, demonstrating the viability and capacity of osteoblasts to proliferate and differentiate on the developed composite. These results suggest that cellulose-nanodiamond composites, which we call oxidized biocompatible interfacial nanocomposites (oBINC), have the potential to serve as a biointerface material for cell adhesion, proliferationand differentiation because of their osteoconductive properties and biocompatibility; furthermore, they show promising applications for bone tissue regeneration.

Primary cell cultures of regenerating holothurian tissues.

The ability to culture different cell types is essential for answering many questions in developmental and regenerative biology. Studies in marine organisms, in particular echinoderms, have been limited by the lack of well-described cellular culture systems. Here we describe a cell culture system, for normal or regenerating holothurian cells, that allows cell characterization by immunohistochemistry and scanning electron microscopy. These cell cultures can now be used to perform multiple types of experiments in order to explore the cellular, biochemical, and genomic aspects of echinoderm regenerative properties.