Tissue Bioengineering Research Articles

Salivary Glands: Function, Dysfunction, Regeneration, and Repair.

Salivary gland dysfunctions are common conditions variously related to aging, inflammatory players, and any other factor able to alter their normal physiology. These conditions may significantly impact oral and systemic health, affecting the overall quality of life. Over time, numerous therapeutic strategies have been explored to regenerate, repair, or replace injured salivary glands, focusing on those molecular and cellular mechanisms able to be safely translated into a clinical landscape. In this context, stem cells, tissue engineering, and the novel organoids technology, have gained exciting results, even if such approaches may require some optimization for their long-term maintenance. Despite extensive research, a composite stem cell population capable of regenerating functional glandular tissue remains elusive; nonetheless, to overcome these current limitations, recently, the transplantation of allogeneic stem cells has emerged as a reliable solution. This overview comprehensively examines the salivary glands in the light of modern biotechnologies, with the aim of better understanding the current state of the art in salivary gland regeneration and repair by using tissue engineering, biomimetic strategies, target therapies, and three-dimensional (3D) organoids technology. This work investigates the main salivary gland dysfunctions and their impact on oral and systemic health. It then discusses the most promising advanced strategies for oral tissue bioengineering, focusing on the potential of stem cells and organoids.

Advanced Hydrogels: Enhancing Tissue Bioengineering with RGD Peptides and Carbon Nanomaterials.

The advancement of tissue engineering (TE) is driven by the development of scaffolds that mimic the mechanical, spatial, and biological environment of the extracellular matrix (ECM), crucial for regulating cell behavior and tissue repair. Hydrogels, 3D networks of polymer chains, are particularly suited for TE due to their high biocompatibility, ability to mimic tissue water content, facilitate cell migration, sustain growth factor release, and offer controllable physical properties. However, hydrogels mimicking the ECM often face challenges related to cell adhesion due to the absence of specific receptors. This issue can be addressed by incorporating ECM components into the polymer matrix, such as the peptide sequence arginine-glycine-aspartic acid (RGD), known for its role in cell adhesion. Additionally, carbon nanomaterials (CNMs) offer unique physicochemical properties that can improve scaffold-cell interactions. Despite the potential benefits, there are limited reports on their combination. RGD-CNM hydrogels enable a more accurate emulation of the natural cellular environment, enhancing tissue engineering applications. This hybrid approach may promote robust cell adhesion along with exceptional mechanical and electrical properties. This review outlines the potential benefits of these hybrid scaffolds and their synergistic potential, aiming to inspire new research directions in this innovative field.

Advanced bioengineering strategies broaden the therapeutic landscape for corneal failure.

The cornea acts as the eye foremost protective layer and is essential for its focusing power. Corneal blindness may arise from physical trauma or conditions like dystrophies, keratitis, keratoconus, or ulceration. While conventional treatments involve medical therapies and donor allografts-sometimes supplemented with keratoprostheses-these options are not suitable for all corneal defects. Consequently, the development of bioartificial corneal tissue has emerged as a critical research area, aiming to address the global shortage of human cornea donors. Bioengineered corneas hold considerable promise as substitutes, with the potential to replace either specific layers or the entire thickness of damaged corneas. This review first delves into the structural anatomy of the human cornea, identifying key attributes necessary for successful corneal tissue bioengineering. It then examines various corneal pathologies, current treatments, and their limitations. Finally, the review outlines the primary approaches in corneal tissue engineering, exploring cell-free, cell-based, and scaffold-based options as three emerging strategies to address corneal failure.

Bone Regeneration with Dental Pulp Stem Cells in an Experimental Model.

The therapeutic approach to bone mass loss and bone's limited self-regeneration is a major focus of research, emphasizing new biomaterials and cell therapy. Tissue bioengineering emerges as a potential alternative to conventional treatments. In this study, an experimental model of a critical bone lesion in rats was used to investigate bone regeneration by treating the defect with biomaterials Evolution® and Gen-Os® (OsteoBiol®, Turín, Italy), with or without mesenchymal stromal cells from dental pulp (DP-MSCs). Forty-six adult male Wistar rats were subjected to a 5-mm critical bone defect in the right mandible, which does not regenerate without intervention. The rats were randomly assigned to a Simulated Group, Control Group, or two Study Groups (using Evolution®, Gen-Os®, and DP-MSCs). The specimens were euthanized at three or six months, and radiological, histological, and ELISA tests were conducted to assess bone regeneration. The radiological results showed that the DP-MSC group achieved uniform radiopacity and continuity in the bone edge, with near-complete structural defect restitution. Histologically, full bone regeneration was observed, with well-organized, vascularized lamellar bone and no lesion edges. These findings were supported by increases in endoglin, transforming growth factor-beta 1 (TGF-β1), protocollagen, parathormone, and calcitonin, indicating a conducive environment for bone regeneration. The use of DP-MSCs combined with biomaterials with appropriate three-dimensional matrices is a promising therapeutic option for further exploration.

Transient Notch Activation Converts Pluripotent Stem Cell-Derived Cardiomyocytes Towards a Purkinje Fiber Fate.

Cardiac Purkinje fibers form the most distal part of the ventricular conduction system. They coordinate contraction and play a key role in ventricular arrhythmias. While many cardiac cell types can be generated from human pluripotent stem cells, methods to generate Purkinje fiber cells remain limited, hampering our understanding of Purkinje fiber biology and conduction system defects. To identify signaling pathways involved in Purkinje fiber formation, we analyzed single cell data from murine embryonic hearts and compared Purkinje fiber cells to trabecular cardiomyocytes. This identified several genes, processes, and signaling pathways putatively involved in cardiac conduction, including Notch signaling. We next tested whether Notch activation could convert human pluripotent stem cell-derived cardiomyocytes to Purkinje fiber cells. Following Notch activation, cardiomyocytes adopted an elongated morphology and displayed altered electrophysiological properties including increases in conduction velocity, spike slope, and action potential duration, all characteristic features of Purkinje fiber cells. RNA-sequencing demonstrated that Notch-activated cardiomyocytes undergo a sequential transcriptome shift, which included upregulation of key Purkinje fiber marker genes involved in fast conduction such as SCN5A, HCN4 and ID2, and downregulation of genes involved in contractile maturation. Correspondingly, we demonstrate that Notch-induced cardiomyocytes have decreased contractile force in bioengineered tissues compared to control cardiomyocytes. We next modified existing in silico models of human pluripotent stem cell-derived cardiomyocytes using our transcriptomic data and modeled the effect of several anti-arrhythmogenic drugs on action potential and calcium transient waveforms. Our models predicted that Purkinje fiber cells respond more strongly to dofetilide and amiodarone, while cardiomyocytes are more sensitive to treatment with nifedipine. We validated these findings in vitro , demonstrating that our new cell-specific in vitro model can be utilized to better understand human Purkinje fiber physiology and its relevance to disease.

Native mechano-regulative matrix properties stabilize alternans dynamics and reduce spiral wave stabilization in cardiac tissue.

The stability of wave conduction in the heart is strongly related to the proper interplay between the electrophysiological activation and mechanical contraction of myocytes and extracellular matrix (ECM) properties. In this study, we statistically compare bioengineered cardiac tissues cultured on soft hydrogels ( kPa) and rigid glass substrates by focusing on the critical threshold of alternans, network-physiological tissue properties, and the formation of stable spiral waves that manifest after wave breakups. For the classification of wave dynamics, we use an improved signal oversampling technique and introduce simple probability maps to identify and visualize spatially concordant and discordant alternans as V- and X-shaped probability distributions. We found that cardiac tissues cultured on ECM-mimicking soft hydrogels show a lower variability of the calcium transient durations among cells in the tissue. This lowers the likelihood of forming stable spiral waves because of the larger dynamical range that tissues can be stably entrained with to form alternans and larger spatial spiral tip movement that increases the chance of self-termination on the tissue boundary. Conclusively, we show that a dysfunction in the excitation-contraction coupling dynamics facilitates life-threatening arrhythmic states such as spiral waves and, thus, highlights the importance of the network-physiological interplay between contractile myocytes and the ECM.

Impact of Uniaxial Static Strain on Myoblast Differentiation in Collagen-Coated PCL Microfilament Scaffolds: Role of Onset Time of Mechanical Stimulation.

Tissue engineering endeavors to create in vitro constructs that replicate the properties of native tissue, such as skeletal muscle. This study investigated the use of mechanical stimulation to promote myogenic differentiation and enhance the functionality of bioengineered tissues. Specifically, it aimed to facilitate the differentiation of myoblasts within a three-dimensional scaffold using a defined pattern of mechanical stimulation. C2C12 cells were cultured on a collagen-coated PCL microfilament scaffold and subjected to 24 h of uniaxial static strain using a biomechanical stimulation system. Two onset times of stimulation, 72 h and 120 h post-seeding, were evaluated. Cell proliferation, myogenic marker expression, and alterations in cell morphology and orientation were assessed. Results indicate that static strain on the scaffold promoted myoblast differentiation, evidenced by morphological and molecular changes. Notably, strain initiated at 72 h induced an early differentiation stage marked by MyoD expression, whereas stimulation beginning at 120 h led to a mid-stage differentiation characterized by the co-expression of MyoD and Myogenin, culminating in myotube formation. These results highlight the critical influence of myoblast maturity at the time of strain application on the differentiation outcome. This study provides insights that could guide the optimization of mechanical stimulation protocols in tissue engineering applications.

Stem cells, cell therapies, and bioengineering in lung biology and diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade, increasing evidence from preclinical models suggests that mesenchymal stromal cells, which are not normally resident in the lung, can be used to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathological remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

<i>In situ</i> and <i>in silico</i> modeling of the hematopoiesis-inducing effect of chelidonic acid

The current trend in regenerative medicine, in the context of an aging population, is the search for new ways and means to optimize tissue bioengineering. One of the convenient models for in situ studying bone marrow regeneration is the subcutaneous ectopic osteogenesis test on scaffolds that imitate the architecture of bone tissue. Chelidonic acid (CA), a small molecule, is capable of participating in various cellular processes and metabolic pathways, and it can activate the osteogenic differentiation of mesenchymal stem cells. However, the molecular mechanisms behind the regulatory effects of CA remain unknown. The aim of this study was to investigate the modulatory effect of CA on the in situ formation of hematopoietic foci, as well as to predict target genes and intracellular signalling pathways involved in the hematopoietic activity of CA. An aqueous solution of CA, isolated from an extract of the Saussurea controversa plant. Course (daily for 35 days) oral administration of CA. Ectopic osteogenesis testing in Balb/c mice. Morphometric analysis of histological sections after 45 days and in silico modelling of gene expression with statistical analysis. CA, when administered orally in a low dose (10 mg/kg), threefold increases the normalized area of bone marrow in the composition of bone tissue plates grown in situ in a test of ectopic subcutaneous osteogenesis in mice. This effect is associated essentially (a probability of CA activity Pa 0.5 and a probability of inactivity Pi 0.5) with enhanced expression of 358 hematopoiesis-related genes, as predicted by in silico analysis. The top list with the highest Pa value included 10 target genes, such as GATA1, CITED2, SFRP1, EP300, LGALS9, VNN1, IL10RB, RARA, CD83, and HMOX1. CA has a significant ability to enhance the reparative remodelling of hematopoietic tissue in situ. The next phase of research will be to test actual target genes and signalling pathways that mediate the regulatory effect of HC on hematopoiesis both in vitro and in vivo, as well as in clinical settings.

Turning Portunus pelagicus Shells into Biocompatible Scaffolds for Bone Regeneration.

Bone tissue engineering (BTE) provides an alternative for addressing bone defects by integrating cells, a scaffold, and bioactive growth factors to stimulate tissue regeneration and repair, resulting in effective bioengineered tissue. This study focuses on repurposing chitosan from blue swimming crab (Portunus pelagicus) shell waste as a composite scaffold combined with HAP and COL I to improve biocompatibility, porosity, swelling, and mechanical properties. The composite scaffold demonstrated nearly 60% porosity with diameters ranging from 100-200 μm with an interconnected network that structurally mimics the extracellular matrix. The swelling ratio of the scaffold was measured at 208.43 ± 14.05%, 248.93 ± 4.32%, 280.01 ± 1.26%, 305.44 ± 20.71%, and 310.03 ± 17.94% at 1, 3, 6, 12, and 24 h, respectively. Thus, the Portunus pelagicus scaffold showed significantly lower degradation ratios of 5.64 ± 1.89%, 14.34 ± 8.59%, 19.57 ± 14.23%, and 29.13 ± 9.87% for 1 to 4 weeks, respectively. The scaffold supports osteoblast attachment and proliferation for 7 days. Waste from Portunus pelagicus shells has emerged as a prospective source of chitosan with potential application in tissue engineering.

The Role of Fibroblasts in Skin Homeostasis and Repair.

Fibroblasts are typical mesenchymal cells widely distributed throughout the human body where they (1) synthesise and maintain the extracellular matrix, ensuring the structural role of soft connective tissues; (2) secrete cytokines and growth factors; (3) communicate with each other and with other cell types, acting as signalling source for stem cell niches; and (4) are involved in tissue remodelling, wound healing, fibrosis, and cancer. This review focuses on the developmental heterogeneity of dermal fibroblasts, on their ability to sense changes in biomechanical properties of the surrounding extracellular matrix, and on their role in aging, in skin repair, in pathologic conditions and in tumour development. Moreover, we describe the use of fibroblasts in different models (e.g., in vivo animal models and in vitro systems from 2D to 6D cultures) for tissue bioengineering and the informative potential of high-throughput assays for the study of fibroblasts under different disease contexts for personalized healthcare and regenerative medicine applications.

Hydrogel-chitosan and polylactic acid-polycaprolactone bioengineered scaffolds for reconstruction of mandibular defects: a preclinical in vivo study with assessment of translationally relevant aspects.

Background: Reconstruction of mandibular bone defects is a surgical challenge, and microvascular reconstruction is the current gold standard. The field of tissue bioengineering has been providing an increasing number of alternative strategies for bone reconstruction. Methods: In this preclinical study, the performance of two bioengineered scaffolds, a hydrogel made of polyethylene glycol-chitosan (HyCh) and a hybrid core-shell combination of poly (L-lactic acid)/poly ( -caprolactone) and HyCh (PLA-PCL-HyCh), seeded with different concentrations of human mesenchymal stromal cells (hMSCs), has been explored in non-critical size mandibular defects in a rabbit model. The bone regenerative properties of the bioengineered scaffolds were analyzed by in vivo radiological examinations and ex vivo radiological, histomorphological, and immunohistochemical analyses. Results: The relative density increase (RDI) was significantly more pronounced in defects where a scaffold was placed, particularly if seeded with hMSCs. The immunohistochemical profile showed significantly higher expression of both VEGF-A and osteopontin in defects reconstructed with scaffolds. Native microarchitectural characteristics were not demonstrated in any experimental group. Conclusion: Herein, we demonstrate that bone regeneration can be boosted by scaffold- and seeded scaffold-reconstruction, achieving, respectively, 50% and 70% restoration of presurgical bone density in 120days, compared to 40% restoration seen in spontaneous regeneration. Although optimization of the regenerative performance is needed, these results will help to establish a baseline reference for future experiments.

Model-predicted effect of radial flux distribution on oxygen and glucose pericellular concentration in constructs cultured in axisymmetric radial-flow packed-bed bioreactors

Radial flow packed-bed bioreactors (rPBBs) overcome the transport limitations of static and axial-flow perfusion bioreactors and enable development of clinical-scale bioengineered tissues. We developed criteria to design rPBBs with uniform medium radial flux distribution along bioreactor length ensuring uniform construct perfusion. We report a model-based analysis of the effect of non-uniform axial distribution of medium radial flux on pericellular concentration of oxygen and glucose. Albeit pseudo-homogeneous, the model predicts how medium flux, solutes transport and cellular consumption interact and determine the pericellular oxygen and glucose concentrations in the presence of pore transport resistance to design optimal axisymmetric rPBBs and enable control of pericellular environment. Thus, oxygen and glucose supply may match cell requirements as tissue matures. Flow and solute transport in bioreactor empty spaces and construct was described with Navier-Stokes and Darcy-Brinkman equations, and with convection–diffusion and convection–diffusion-reaction equations, respectively. Solute transport in construct accounted for Michaelian cellular consumption and bulk medium-to-cell surface oxygen transport resistance in terms of a transport-equivalent bed of Raschig rings. The effect of relevant dimensionless groups on pericellular and bulk solute concentrations was predicted under typical tissue engineering operation and evaluated against literature data for bone tissue engineering. Axial distribution of medium radial flux influenced the distribution of pericellular solutes concentration, more so at high cell metabolic activity. Increasing medium feed flow rates relieved non-uniform solute concentration distribution and decayed at cell surface for metabolic consumption, also starting from axially non-uniform radial flux distribution. Model predictions were obtained in runtimes compatible with on-line control strategies.

Design approaches for 3D cell culture and 3D bioprinting platforms.

The natural habitat of most cells consists of complex and disordered 3D microenvironments with spatiotemporally dynamic material properties. However, prevalent methods of in vitro culture study cells under poorly biomimetic 2D confinement or homogeneous conditions that often neglect critical topographical cues and mechanical stimuli. It has also become increasingly apparent that cells in a 3D conformation exhibit dramatically altered morphological and phenotypical states. In response, efforts toward designing biomaterial platforms for 3D cell culture have taken centerstage over the past few decades. Herein, we present a broad overview of biomaterials for 3D cell culture and 3D bioprinting, spanning both monolithic and granular systems. We first critically evaluate conventional monolithic hydrogel networks, with an emphasis on specific experimental requirements. Building on this, we document the recent emergence of microgel-based 3D growth media as a promising biomaterial platform enabling interrogation of cells within porous and granular scaffolds. We also explore how jammed microgel systems have been leveraged to spatially design and manipulate cellular structures using 3D bioprinting. The advent of these techniques heralds an unprecedented ability to experimentally model complex physiological niches, with important implications for tissue bioengineering and biomedical applications.

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research.

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

A Review on Carbon Nanotubes Family of Nanomaterials and Their Health Field.

The use of carbon nanotubes (CNTs), which are nanometric materials, in pathogen detection, protection of environments, food safety, and in the diagnosis and treatment of diseases, as efficient drug delivery systems, is relevant for the improvement and advancement of pharmacological profiles of many molecules employed in therapeutics and in tissue bioengineering. It has contributed to the advancement of science due to the development of new tools and devices in the field of medicine. CNTs have versatile mechanical, physical, and chemical properties, in addition to their great potential for association with other materials to contribute to applications in different fields of medicine. As, for example, photothermal therapy, due to the ability to convert infrared light into heat, in tissue engineering, due to the mechanical resistance, flexibility, elasticity, and low density, in addition to many other possible applications, and as biomarkers, where the electronic and optics properties enable the transduction of their signals. This review aims to describe the state of the art and the perspectives and challenges of applying CNTs in the medical field. A systematic search was carried out in the indexes Medline, Lilacs, SciELO, and Web of Science using the descriptors "carbon nanotubes", "tissue regeneration", "electrical interface (biosensors and chemical sensors)", "photosensitizers", "photothermal", "drug delivery", "biocompatibility" and "nanotechnology", and "Prodrug design" and appropriately grouped. The literature reviewed showed great applicability, but more studies are needed regarding the biocompatibility of CNTs. The data obtained point to the need for standardized studies on the applications and interactions of these nanostructures with biological systems.

IGF2BP3-mediated enhanced stability of MYLK represses MSC adipogenesis and alleviates obesity and insulin resistance in HFD mice

Mesenchymal stem cells (MSCs) hold immense potential as multipotent stem cells and serve as a primary source of adipocytes. The process of MSC adipogenesis plays a crucial role in maintaining systemic metabolic homeostasis and has garnered significant attention in tissue bioengineering. N6-methyladenosine (m6A), the most prevalent RNA modification, is known to regulate cell fate and disease. However, the precise involvement of m6A readers in MSC adipogenesis remains unclear. In this study, we investigated the impact of IGF2BP3, a prominent m6A reader, on MSC adipogenesis. Our findings revealed a decrease in IGF2BP3 expression during the natural adipogenic differentiation of MSCs. Furthermore, IGF2BP3 was found to repress MSC adipogenesis by augmenting the levels of MYLK, a calcium/calmodulin-dependent kinase. Mechanistically, IGF2BP3 interacted with MYLK mRNA in an m6A-dependent manner, extending its half-life and subsequently inhibiting the phosphorylation of the ERK1/2 pathway, thereby impeding the adipogenic differentiation of MSCs. Additionally, we successfully achieved the overexpression of IGF2BP3 through intraperitoneal injection of adeno-associated virus serotype Rec2, which specifically targeted adipose tissue. This intervention resulted in reduced body weight and improved insulin resistance in high-fat diet mice. Overall, our study provides novel insights into the role of IGF2BP3 in MSC adipogenesis, shedding light on adipocyte-related disorders and presenting potential targets for related biomedical applications.

Characterization of the mechanical properties of the mouse Achilles tendon enthesis by microindentation. Effects of unloading and subsequent reloading

The fibrocartilaginous tendon enthesis, i.e. the site where a tendon is attached to bone through a fibrocartilaginous tissue, is considered as a functionally graded interface. However, at local scale, a very limited number of studies have characterized micromechanical properties of this transitional tissue. The first goal of this work was to characterize the micromechanical properties of the mineralized part of the healthy Achilles tendon enthesis (ATE) through microindentation testing and to assess the degree of mineralization and of carbonation of mineral crystals by Raman spectroscopy. Since little is known about enthesis biological plasticity, our second objective was to examine the effects of unloading and reloading, using a mouse hindlimb-unloading model, on both the micromechanical properties and the mineral phase of the ATE. Elastic modulus, hardness, degree of mineralization, and degree of carbonation were assessed after 14 days of hindlimb suspension and again after a subsequent 6 days of reloading. The elastic modulus gradually increased along the mineralized part of the ATE from the tidemark to the subchondral bone, with the same trend being found for hardness. Whereas the degree of carbonation did not differ according to zone of measurement, the degree of mineralization increased by >70 % from tidemark to subchondral bone. Thus, the gradient in micromechanical properties is in part explained by a mineralization gradient. A 14-day unloading period did not appear to affect the gradient of micromechanical properties of the ATE, nor the degree of mineralization or carbonation. However, contrary to a short period of unloading, early return to normal mechanical load reduced the micromechanical properties gradient, regardless of carbonate-to-phosphate ratios, likely due to the more homogeneous degree of mineralization. These findings provide valuable data not only for tissue bioengineering, but also for musculoskeletal clinical studies and microgravity studies focusing on long-term space travel by astronauts.

Mesenchymal Stem Cells and Tissue Bioengineering Applications in Sheep as Ideal Model.

The most common technologies in tissue engineering include growth factor therapies; metal implants, such as titanium; 3D bioprinting; nanoimprinting for ceramic/polymer scaffolds; and cell therapies, such as mesenchymal stem cells (MSCs). Cell therapy is a promising alternative to organ grafts and transplants in the treatment of numerous musculoskeletal diseases. MSCs have increasingly been used in generative medicine due to their specialized self-renewal, immunomodulation, multiplication, and differentiation properties. To further expand the potential of these cells in tissue repair, significant efforts are currently dedicated to the production of biomaterials with desirable short- and long-term biophysical properties that can aid the differentiation and expansion of MSCs. Biomaterials support MSC differentiation by modulating their characteristics, such as composition, mechanical properties, porosity, and topography. This review aimed to describe recent MSC approaches, including those associated with biomaterials, from experimental, clinical, and preclinical studies with sheep models.

Transplantation of a bioengineered tissue patch promotes uterine repair in the sheep.

Innovative bioengineering strategies utilizing extracellular matrix (ECM) based scaffolds derived from decellularized tissue offer new prospects for restoring damaged uterine tissue. Despite successful fertility restoration in small animal models, the translation to larger and more clinically relevant models have not yet been assessed. Thus, our study investigated the feasibility to use a 6 cm2 graft constructed from decellularized sheep uterine tissue, mimicking a future application to repair a uterine defect in women. Some grafts were also recellularized with fetal sheep bone marrow-derived mesenchymal stem cells (SF-MSCs). The animals were followed for six weeks post-surgery during which blood samples were collected to assess the systemic immune cell activation by fluorescence-activated cell sorting (FACS) analysis. Tissue regeneration was assessed by histology, immunohistochemistry, and gene expression analyses. There was a large intra-group variance which prompted us to implement a novel scoring system to comprehensively evaluate the regenerative outcomes. Based on the regenerative score each graft received, we focused our analysis to map potential differences that may have played a role in the success or failure of tissue repair following the transplantation therapy. Notably, three out of 15 grafts exhibited major regeneration that resembled native uterine tissue, and an additional three grafts showed substantial regenerative outcomes. For the better regenerated grafts, it was observed that the systemic T-cell subgroups were significantly different compared with the failing grafts. Hence, our data suggest that the T-cell response play an important role for determining the uterus tissue regeneration outcomes. The remarkable regeneration seen in the best-performing grafts after just six weeks following transplantation provides compelling evidence that decellularized tissue for uterine bioengineering holds great promise for clinically relevant applications.