Regenerative Medicine Approaches Research Articles

Decellularized extracellular matrix from bovine ovarian tissue maintains the protein composition of the native matrisome

Recent approaches of regenerative reproductive medicine investigate the decellularized extracellular matrix to develop a transplantable engineered ovary (TEO). However, a full proteomic analysis is not usually performed after the decellularization process to evaluate the preservation of the extracellular matrix (ECM). In this study, the ECM of the bovine ovarian cortex was analyzed before and after decellularization using mass spectrometry and bioinformatics. A total of 155 matrisome proteins were identified in the native ECM of the bovine ovarian cortex, with 145 matrisome proteins detected in the decellularized ECM. After decellularization, only 10 matrisome proteins were lost, and notably, none belonged to the category of reproductive biological processes. Histology and histochemistry analyses were employed to assess the general morphology of both native and decellularized ECM, allowing for the identification of the most abundant ECM proteins. Moreover, our study highlighted collagen type VI alpha 3 and heparan sulfate proteoglycan 2 as the most abundant components in the bovine ovarian ECM, mirroring the composition observed in the human ovary. These findings enhance our understanding of the composition of both native and decellularized ECM, with the potential implications for the development of a TEO. SignificanceThe significance of the present study lies on the possibility of advancing towards developing a bioengineered ovary, which is the ultimate strategy to regain fertility in women. The results demonstrate that the decellularized extracellular matrix of the bovine ovary maintains the protein composition of the native matrisome, using a recently described decellularization protocol. The decellularized matrix may serve as scaffolding for seeding ovarian stromal cells and follicles to create a bioengineered ovary, and as closer its composition is to the native matrix the better. Also, comparing the bovine ovarian matrisome, which was described for the first time here, with the human ovarian matrisome, we could see a great similarity, suggesting that the bovine ovary decellularized matrix may serve as a model for developing a human bioengineered ovary.

Establishing a rabbit model with massive supraspinatus tendon defect for investigating scaffold-assisted tendon repair

BackgroundShoulder pain and disability from rotator cuff tears remain challenging clinical problem despite advancements in surgical techniques and materials. To advance our understanding of injury progression and develop effective therapeutics using tissue engineering and regenerative medicine approaches, it is crucial to develop and utilize animal models that closely resemble the anatomy and display the pathophysiology of the human rotator cuff. Among various animal models, the rabbit shoulder defect model is particularly favored due to its similarity to human rotator cuff pathology. However, a standardized protocol for creating a massive rotator cuff defect in the rabbits is not well defined. Therefore, the objective of our study was to establish a robust and reproducible model of a rotator cuff defect to evaluate the regenerative efficacy of scaffolds.ResultsIn our study, we successfully developed a rabbit model with a massive supraspinatus tendon defect that closely resembles the common rotator cuff injuries observed in humans. This defect involved a complete transection of the tendon, spanning 10 mm in length and encompassing its full thickness and width. To ensure stable scaffolding, we employed an innovative bridging suture technique that utilized a modified Mason-Allen suture as a structural support. Moreover, to assess the therapeutic effectiveness of the model, we utilized different scaffolds, including a bovine tendon extracellular matrix (ECM) scaffold and a commercial acellular dermal matrix (ADM) scaffold. Throughout the observation period, no scaffold damage was observed. Notably, comprehensive histological analysis demonstrated that the regenerative tissue in the tendon ECM scaffold group exhibited an organized and aligned fiber structure, indicating tendon-like tissue regeneration while the tissue in the ADM group showed comparatively less organization.ConclusionsThis study presents a comprehensive description of the implemented procedures for the development of a highly reproducible animal model that induces massive segmental defects in rotator cuff tendons. This protocol can be universally implemented with alternative scaffolds to investigate extensive tendon defects and evaluate the efficacy of regenerative treatments. The application of our animal model offers a standardized and reproducible platform, enabling researchers to systematically evaluate, compare, and optimize scaffold designs. This approach holds significant importance in advancing the development of tissue engineering strategies for effectively repairing extensive tendon defects.

Infected wound repair correlates with collagen I induction and NOX2 activation by cold atmospheric plasma

Cold atmospheric plasma (CAP) is a promising complement to tissue repair and regenerative medicine approaches. CAP has therapeutic potential in infected cutaneous wounds by mechanisms which remain enigmatic. Here, CAP is shown to activate phagocyte NADPH oxidase complex NOX2. CAP induced increased intracellular reactive oxygen species, alleviated by NOX2 inhibitors. Genetic and pharmacological inhibitions of NOX2 in macrophages and bioengineered skin infected with Staphylococcus aureus and treated with CAP reduced intracellular oxidants and increased bacterial survival. CAP triggered Rac activation and phosphorylation of p40phox and p47phox required for NOX2 assembly and activity. Furthermore, CAP induced collagen I expression by fibroblasts. Infection and healing kinetics showed that murine skin wounds infected with S. aureus and treated with CAP are characterized by decreased bacterial burden, increased length of neoepidermis and extracellular matrix formation. Collectively, our findings identify mechanisms triggered by CAP that subdue infection and result in enhanced repair following skin injury.

Regenerative Medicine Approaches for Skin Wound Healing: from Allografts to Engineered Skin Substitutes

Abstract Purpose of the Review Recent advancements in tissue engineering and regenerative medicine have paved the way for innovative solutions in skin regeneration, particularly for extensive burns and full-thickness wounds where traditional approaches are limited. The purpose of the review is to explore the integration of bioactive materials, stem cell therapies, and tissue-engineered skin substitutes and their role in revolutionizing wound healing and skin transplantation. Recent Findings Studies leveraging natural and synthetic biomaterials as scaffolds, alongside the regenerative capabilities of mesenchymal stem cells (MSCs) and other cellular therapies, underscore the potential to enhance tissue repair, minimize scarring, and improve overall clinical outcomes. The development of multifunctional biomaterials and the advent of cutting-edge techniques such as 3D bioprinting and nanomedicine further propel the field, offering personalized and effective solutions. As these technologies evolve, they hold promise for more efficient, patient-specific skin grafting, reducing the need for systemic immunosuppression and enhancing graft survival. Summary The critical advancements in biomaterials, stem cell therapies, and tissue engineering, outline a course toward more effective and personalized skin regeneration therapies.

Dipeptidyl peptidase-4 marks distinct subtypes of human adipose stromal/stem cells with different hepatocyte differentiation and immunoregulatory properties

BackgroundHuman adipose-derived stromal/stem cells (hASCs) play important roles in regenerative medicine and numerous inflammatory diseases. However, their cellular heterogeneity limits the effectiveness of treatment. Understanding the distinct subtypes of hASCs and their phenotypic implications will enable the selection of appropriate subpopulations for targeted approaches in regenerative medicine or inflammatory diseases.MethodshASC subtypes expressing dipeptidyl peptidase-4 (DPP4) were identified via fluorescence-activated cell sorting (FACS) analysis. DPP4 expression was knocked down in DPP4+ hASCs via DPP4 siRNA. The capacity for proliferation, hepatocyte differentiation, inflammatory factor secretion and T-cell functionality regulation of hASCs from DPP4−, DPP4+, and control siRNA-treated DPP4+ hASCs and DPP4 siRNA-treated DPP4+ hASCs were assessed.ResultsDPP4+ hASCs and control siRNA-treated DPP4+ hASCs presented a lower proliferative capacity but greater hepatocyte differentiation capacity than DPP4− hASCs and DPP4 siRNA-treated DPP4+ hASCs. Both DPP4+ hASCs and DPP4− hASCs secreted high levels of vascular endothelial growth factor-A (VEGF-A), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6), whereas the levels of other factors, including matrix metalloproteinase (MMP)-1, eotaxin-3, fractalkine (FKN, CX3CL1), growth-related oncogene-alpha (GRO-alpha, CXCL1), monokine induced by interferon-gamma (MIG), macrophage inflammatory protein (MIP)-1beta, and macrophage colony-stimulating factor (M-CSF), were significantly greater in the supernatants of DPP4+ hASCs than in those of DPP4− hASCs. Exposure to hASC subtypes and their conditioned media triggered changes in the secreted cytokine profiles of T cells from healthy donors. The percentage of functional T cells that secreted factors such as MIP-1beta and IL-8 increased when these cells were cocultured with DPP4+ hASCs. The percentage of polyfunctional CD8+ T cells that secreted multiple factors, such as IL-17A, tumour necrosis factor alpha (TNF-α) and TNF-β, decreased when these cells were cocultured with supernatants derived from DPP4+ hASCs.ConclusionsDPP4 may regulate proliferation, hepatocyte differentiation, inflammatory cytokine secretion and T-cell functionality of hASCs. These data provide a key foundation for understanding the important role of hASC subpopulations in the regulation of T cells, which may be helpful for future immune activation studies and allow them to be customized for clinical application.

Differential effects of macrophage subtype-specific cytokines on fibroblast proliferation and endothelial cell function in co-culture system.

Macrophages are involved in several critical activities associated with tissue repair and regeneration. Current approaches in regenerative medicine are focusing on leveraging the innate immune response to accelerate tissue regeneration and improve long-term healing outcomes. Of particular interest in this regard are the currently known, four main M2 macrophage subtypes: M2interleukin (IL)-4,IL-13, M2IC, M2IL-10, M2non-selective adenosine receptor agonists (NECA) (M2IL-4,IL-13 → M2NECA). In this study, rat bone marrow-derived macrophages (M0) were polarized to each of the four subtypes M2IL-4,IL-13 → M2NECA and cultured for 72 h in vitro. Luminex assay results highlighted increased production of tissue inhibitor of metalloproteinases-1 (TIMP-1) for M2IL-4,IL-13, higher amounts of transforming growth factor-beta 1 (TGF-β1) for M2IL-10, and elevated vascular endothelial growth factor A (VEGF-A) from M2NECA. Co-culture experiments performed with M2IL-10 macrophages and L929 fibroblasts highlighted the increased production of soluble collagen within the media as well as higher amounts of collagen in the extracellular matrix. Human umbilical vein endothelial cells (HUVECs) were co-cultured with M2NECA macrophages, which demonstrated an increase in intercellular adhesion molecule (ICAM) and platelet endothelial cell adhesion molecule (PECAM), as well as increased formation of endothelial tubes. The findings of this study emphasize a critical demand for further characterization and analyses of distinct M2 subtypes and careful selection of specific macrophage populations for regeneration of specific tissue types. The current, broad classification of "M2" may be sufficient in many general tissue engineering applications, but, as conditions are constantly in flux within the microenvironment in vivo, a higher degree of specificity and control over the initial M2 subtype could result in more consistent long-term outcomes where macrophages are utilized as part of an overall regenerative strategy.

Unraveling devitalization: its impact on immune response and ectopic bone remodeling from autologous and allogeneic callus mimics.

Endochondral bone regeneration is a promising approach in regenerative medicine. Callus mimics (CMs) are engineered and remodeled into bone tissue upon implantation. The long-term objective is to fabricate a sustainable off-the-shelf treatment option for patients. Devitalization was introduced to facilitate storage and using allogeneic (donor) cells would further propel the off-the-shelf approach. However, allogeneic CMs for bone regeneration pose a potential antigenicity concern. Here, we explored the impact of devitalization on antigenicity and osteoinductive bone formation when implanting syngeneic or allogeneic CM in a vital or devitalized state. For this, we implanted chondrogenically differentiated rat-derived mesenchymal stromal cells using an allogeneic immunocompetent ectopic rat model. Vital syngeneic CMs demonstrated the highest bone formation, and vital allogeneic CMs showed the lowest bone formation, while both devitalized CMs showed comparable intermediate levels of bone formation. Preceding bone formation, the level of tartrate-resistant acid phosphatase staining at 7 and 14 days was proportional to the level of eventual bone formation. No differences were observed for local innate immune responses at any time point before or after bone formation. In contrast, allogeneic CMs elicit a mild adaptive immune response, which still permits bone formation in an immunocompetent environment, albeit at a reduced rate compared to the autologous living counterpart. Overall, devitalization delays bone formation when autologous CMs are implanted, whereas it accelerates bone formation in allogeneic CMs, highlighting the potential of this approach for achieving off-the-shelf treatment.

Comparison of Bioengineered Scaffolds for the Induction of Osteochondrogenic Differentiation of Human Adipose-Derived Stem Cells.

Osteochondral lesions may be due to trauma or congenital conditions. In both cases, therapy is limited because of the difficulty of tissue repair. Tissue engineering is a promising approach that relies on designed scaffolds with variable mechanical attributes to favor cell attachment and differentiation. Human adipose-derived stem cells (hASCs) are a very promising cell source in regenerative medicine with osteochondrogenic potential. Based on the assumption that stiffness influences cell commitment, we investigated three different scaffolds: a semisynthetic animal-derived GelMA hydrogel, a combined scaffold made of rigid PEGDA coated with a thin GelMA layer and a decellularized plant-based scaffold. We investigated the role of different biomechanical stimulations in the scaffold-induced osteochondral differentiation of hASCs. We demonstrated that all scaffolds support cell viability and spontaneous osteochondral differentiation without any exogenous factors. In particular, we observed mainly osteogenic commitment in higher stiffness microenvironments, as in the plant-based one, whereas in a dense and softer matrix, such as in GelMA hydrogel or GelMA-coated-PEGDA scaffold, chondrogenesis prevailed. We can induce a specific cell commitment by combining hASCs and scaffolds with particular mechanical attributes. However, in vivo studies are needed to fully elucidate the regenerative process and to eventually suggest it as a potential approach for regenerative medicine.

Pharmacological Treatments for Erectile Dysfunction in Diabetic Patients: Efficacy, Safety, and Emerging Therapies

Men with diabetes mellitus, particularly those with type 2 diabetes (T2DM), are known to experience erectile dysfunction (ED), a condition whose prevalence has risen to 75% in recent years. The pathophysiology of ED in diabetic patients involves vascular impairment, neuropathy, hormonal dysregulation, and chronic hyperglycemia, all of which contribute to endothelial dysfunction, reduced nitric oxide availability, and nerve damage. Phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil, tadalafil, and vardenafil, are the first-line pharmacological treatments for ED, though their efficacy is reduced in diabetic patients due to the extent of vascular and neurological damage. Other treatments, including testosterone replacement therapy (TRT), intracavernosal injections, and vacuum erection devices (VEDs), provide alternatives for patients unresponsive to PDE5 inhibitors. Emerging therapies such as low-intensity extracorporeal shockwave therapy (LI-ESWT) and regenerative medicine approaches, including stem cell and platelet-rich plasma (PRP) injections, show potential in improving erectile function in diabetic patients. Glycemic control plays a critical role in both preventing ED and enhancing the efficacy of treatments by improving vascular function and reversing nerve damage. This review was conducted through an extensive analysis of current literature on pharmacological and emerging treatments for ED in diabetic patients. In conclusion, integrating traditional pharmacotherapy with emerging treatments and emphasizing glycemic control holds promise for optimizing ED management in diabetic patients. Keywords: Erectile Dysfunction (ED), Diabetes Mellitus (T2DM), Phosphodiesterase Type 5 (PDE5) Inhibitors, Testosterone Replacement Therapy (TRT), Glycemic Control

L-carnitine cause to increase cell proliferation of C-Kit+ hematopoietic progenitor cells via decreasing the PI3K and FOXO-1 protein expression

BackgroundStem cell-based therapy has emerged as an attractive approach for regenerative medicine. Poor survival and maintenance of the cells used in regenerative medicine are considered as serious barriers to enhance the efficacy of the cell therapy. Using some antioxidants has been reported to prevent the aging of stem cells, and finding effective factors to reduce the senescence of these cells has impressive potential in cell therapy. The PI3K pathway adversely regulates the transcription factors known as FOXO, which are thought to have an inhibitory influence on cell proliferation. By downregulating FOXO and other targets, PI3K signaling controls the growth of cells. For this reason, the aim of the present study is to investigate the effect of L-carnitine (LC) as antioxidant on the cell proliferation and the protein expression of PI3K and FOXO. MethodsFor understanding the in vitro effect of LC on the PI3K and FOXO-1 expression of C-kit+ hematopoietic progenitor cells, the bone marrow mononuclear cells were isolated, and C-kit+ cells was enriched by the magnetic-activated cell sorting (MACS). Next, the identification of enriched C-kit+ cells were done by flowcytometry and immunocytochemistry. Then, C-kit+ cells were treated with 0.2 mM LC, the cells were collected at the end of the treatment period (48 h), and the proteins were extracted. In the following, the protein expression of PI3K and FOXO-1 was measured by western blotting. In addition, flowcytometry was done to assess the Ki-67 expression as a key marker for cell proliferation investigation. Results0.2 mM LC cause to significantly decrease in the protein expression of PI3K and FOXO-1 (*P<0.05 and **P<0.01, respectively). Also, the expression of Ki-67 was significantly increased in the presence of 0.2 mM LC (***P<0.001). ConclusionBriefly, LC can be considered an effective factor in increasing the proliferation of C-kit+ cells via some signaling pathways.

3D printable gelatin/nisin biomaterial inks for antimicrobial tissue engineering applications.

Modern regenerative medicine approaches can rely on the fabrication of personalised medical devices and implants; however, many of these can fail due to infections, requiring antibiotics and revision surgeries. Given the rise in multidrug resistant bacteria, developing implants with antimicrobial activity without the use of traditional antibiotics is crucial for successful implant integration and improving patient outcomes. 3D printed gelatin-based implants have a broad range of applications in regenerative medicine due to their biocompatibility, ease of modification and degradability. In this paper, we report on the development of gelatin biomaterial inks loaded with the antimicrobial peptide, nisin, for extrusion-based 3D printing to produce scaffolds with controlled porosity, high shape fidelity, and structural stability. Rheological properties were comprehensively studied to develop inks that had shear thinning behaviour and viscoelastic properties to ensure optimal printability and extrudability, and enable precise deposition and structural integrity during 3D printing. The 3D printed scaffolds fabricated from the gelatin/nisin inks demonstrated excellent antimicrobial efficacy (complete kill) against Gram positive bacteria methicillin-resistant Staphylococcus aureus (MRSA). Overall, this ink's high printability and antimicrobial efficacy with the model antimicrobial peptide, nisin, offers the potential to develop customisable regenerative medicine implants that can effectively combat infection without contributing to the development of multidrug resistant bacteria.

Advances in Tissue Engineering and Its Future in Regenerative Medicine Compared to Traditional Reconstructive Techniques: A Comparative Analysis.

Tissue engineering represents a revolutionary approach in regenerative medicine, offering promising alternatives to traditional reconstructive techniques. This systematic review explores recent advances in tissue engineering, comparing their efficacy, postoperative outcomes, and patient satisfaction to conventional methods. A comprehensive literature search was conducted across PubMed, Cochrane Library, and Google Scholar, covering studies published from 2000 to 2024. Fourteen studies were selected for final analysis based on inclusion criteria focusing on outcomes such as scar quality, postoperative pain, and patient satisfaction. The review demonstrated that tissue engineering techniques consistently provided superior cosmetic outcomes with minimal scarring compared to traditional methods. Patients undergoing tissue-engineered procedures experienced mild-to-moderate postoperative pain with rapid resolution, whereas traditional techniques resulted in moderate to severe pain requiring extended management. Furthermore, patients treated with tissue engineering reported high satisfaction rates due to improved cosmetic and functional outcomes. Despite challenges such as ensuring adequate vascularization, controlling scaffold degradation, and overcoming regulatory and cost barriers, ongoing research and development are essential to fully realize the potential of these innovative therapies. Tissue engineering offers significant advantages over traditional reconstructive techniques and has the potential to profoundly improve patient care in regenerative medicine.

Mesenchymal Stem Cells and Secretome as a New Possible Approach to Treat Cartilage Damage: An In Vitro Study.

Introduction: Osteoarthritis is a degenerative condition of the cartilage, often common among the population and occurs frequently with aging. Many factors are decisive for the development of its pathogenesis such as age, obesity, trauma, mechanical load, and modification of synovial biology. The main features of osteoarthritis are chondrocytes and cartilage matrix loss, which lead to pain, loss of function of the whole joint, and disability, representing a relevant health problem. Recently, a new therapeutic approach based on cell therapy has been studying the regenerative ability of mesenchymal stem cells for osteoarthritic chondrocytes. Aim: This in vitro study clarifies the regenerative effects of multipotent adipose-derived stem cells and the pluripotent amniotic epithelial stem cells on arthrosis chondrocytes by performing co-culture experiments. Methods: We studied the regenerative potential of secretome (soluble factors and extracellular vesicles), mesenchymal stem cells, and the adipose stromal vascular fraction. The regenerative effects were evaluated by gene and protein expression analysis of articular cartilage-specific genes and proteins like col2a1, acan, and sox9. Results: Mesenchymal stem cells, secretome, and adipose stromal vascular fractions influenced the cartilage genes and protein expression. Conclusions: The results indicate that the treatment with mesenchymal stem cells could be the best biological approach for cartilage regenerative medicine.

Substrate stress relaxation regulates monolayer fluidity and leader cell formation for collectively migrating epithelia.

Groups of cells must coordinate their movements in order to sculpt organs during development and maintain tissues. The mechanical properties of the underlying substrate on which cells reside are known to influence key aspects of single and collective cell migration. Despite being a nearly universal feature of biological tissues, the role of viscoelasticity (i.e., fluid-like and solid-like behavior) in collective cell migration is unclear. Using tunable engineered biomaterials, we demonstrate that sheets of epithelial cells display enhanced migration on slower-relaxing (more elastic) substrates relative to faster-relaxing (more viscous) substrates. Building our understanding of tissue-substrate interactions and collective cell dynamics provides insights into approaches for tissue engineering and regenerative medicine, and therapeutic interventions to promote health and treat disease.

Photobiomodulation Dose-Response on Adipose-Derived Stem Cell Osteogenesis in 3D Cultures.

Osteoporosis and other degenerative bone diseases pose significant challenges to global healthcare systems due to their prevalence and impact on quality of life. Current treatments often alleviate symptoms without fully restoring damaged bone tissue, highlighting the need for innovative approaches like stem cell therapy. Adipose-derived mesenchymal stem cells (ADMSCs) are particularly promising due to their accessibility, abundant supply, and strong differentiation potential. However, ADMSCs tend to favor adipogenic pathways, necessitating the use of differentiation inducers (DIs), three-dimensional (3D) hydrogel environments, and photobiomodulation (PBM) to achieve targeted osteogenic differentiation. This study investigated the combined effects of osteogenic DIs, a fast-dextran hydrogel matrix, and PBM at specific wavelengths and fluences on the proliferation and differentiation of immortalized ADMSCs into osteoblasts. Near-infrared (NIR) and green (G) light, as well as their combination, were used with fluences of 3 J/cm2, 5 J/cm2, and 7 J/cm2. The results showed statistically significant increases in alkaline phosphatase levels, a marker of osteogenic differentiation, with G light at 7 J/cm2 demonstrating the most substantial impact on ADMSC differentiation. Calcium deposits, visualized by Alizarin red S staining, appeared as early as 24 h post-treatment in PBM groups, suggesting accelerated osteogenic differentiation. ATP luminescence assays indicated increased proliferation in all experimental groups, particularly with NIR and NIR-G light at 3 J/cm2 and 5 J/cm2. MTT viability and LDH membrane permeability assays confirmed enhanced cell viability and stable cell health, respectively. In conclusion, PBM significantly influences the differentiation and proliferation of hydrogel-embedded immortalized ADMSCs into osteoblast-like cells, with G light at 7 J/cm2 being particularly effective. These findings support the combined use of 3D hydrogel matrices and PBM as a promising approach in regenerative medicine, potentially leading to innovative treatments for degenerative bone diseases.

Identification of Effects of Parylene-C Coating on Electrospun Fibers

As various healthcare technologies such as regenerative medicine, precision medicine, and alternative approaches to animal testing develop, the interest in the use and application of nano- and microfibers is steadily increasing. In this study, the effect of parylene-C coating on electrospun fibers was investigated, and a pattern coating method was developed to expand the potential utilization of parylene-C-coated electrospun fibers. An SEM analysis demonstrated that parylene-C was successfully deposited on the electrospun fibers without any failure such as pinholes or air bubbles. Biocompatibility was investigated through cell tests, which indicated that the coated fibers were non-toxic and supported cell growth well. Tensile tests demonstrated a significant increase in the elastic modulus of the parylene-C-coated fibers, with it nearly quadrupling compared to the original PCL fibers, and the fracture strength almost doubled. At the same time, hydrophobicity was well maintained without any statistically significant changes. In particular, a non-adhesive magnet–metal masking was proposed in order to selectively coat the electrospun fibers with parylene-C with a specific pattern. Furthermore, it was presented that the magnet–metal mask-based coating electrospun nanofibers with parylene-C could be used in the fabrication of hybrid fibers composed of different diameters and materials.

Single-Cell Transcriptomic Profiling of Cholangiocyte Organoids Derived from Bile Ducts of Primary Sclerosing Cholangitis Patients

Background and AimsPrimary sclerosing cholangitis (PSC) is a chronic inflammatory liver disorder without effective medical treatment which is characterized by inflammation and fibrotic structures around the bile ducts. Biliary epithelial cells (cholangiocytes) are the target and potential disease drivers in PSC, yet little is known if cholangiocytes from PSC patients differ from non-PSC controls. To characterize cholangiocytes at early rather than end-stage disease, cholangiocyte organoids (COs) were derived from diseased bile ducts of PSC patients and compared to organoids generated from disease controls.MethodsCholangiocytes were obtained during endoscopic retrograde cholangiopancreatography (ERCP) brushing of diseased bile duct areas and expanded as organoids using previously established culture methods. Stable CO lines were analyzed for cell type identity, basic cholangiocyte function, and transcriptomic signature.ResultsWe demonstrate that cholangiocytes, derived from the damaged area within the bile ducts of PSC patients, can be expanded in culture without displaying functional or genetic disease-related features. We further show that COs from patients who later were diagnosed with dysplasia exhibit higher expression of the cancer-associated genes PGC, FXYD2, MIR4435-2HG, and HES1.ConclusionsOur results demonstrate that PSC organoids are largely similar to control organoids after culture and highlight the significance of COs as a tool for regenerative medicine approaches as well as their potential for discovering new potential biomarkers for diagnosing cholangiocarcinoma.

3D Bioprinitng technologies for the enhancement and application of functional lung organoid models

Respiratory diseases, ranging from minor infections to serious chronic diseases and malignancies, negatively affect the respiratory system and are influenced by various environmental factors such as air pollution, occupational hazards, and tobacco smoke, as well as lifestyle, genetic causes, and infectious agents. The prevalence and severity of respiratory diseases requires the development of advanced models to better understand their pathophysiology and develop effective treatments. In this context, 3D bioprinting technology is emerging as an innovative tool to create functional lung organoid models. The use of induced pluripotent stem cells (iPSCs) and extracellular matrix (ECM) in bioprinting enables the development of organoids that closely mimic human lung tissue. Bioprinting-based organoids can better replicate the dynamic environment of the human lung, facilitating more accurate disease modeling and drug testing. In this review, we highlight the potential of bioprinted lung organoids in understanding the mechanisms of chronic respiratory diseases, testing the efficacy and safety of new drugs, and exploring regenerative medicine approaches. The integration of advanced bioprinting and organoid technologies is a promising field in respiratory disease research and treatment, offering new hope for patients suffering from lung diseases.

Starvation and Inflammation Modulate Adipose Mesenchymal Stromal Cells' Molecular Signature.

Mesenchymal stromal cells (MSCs) and their released factors (secretome) are intriguing options for regenerative medicine approaches based on the management of inflammation and tissue restoration, as in joint disorders like osteoarthritis (OA). Production strategy may modulate cells and secretome fingerprints, and for the latter, the effect of serum removal by starvation used in clinical-grade protocols has been underestimated. In this work, the effect of starvation on the molecular profile of interleukin 1 beta (IL1β)-primed adipose-derived MSCs (ASCs) was tested by assessing the expression level of 84 genes related to secreted factors and 84 genes involved in defining stemness potential. After validation at the protein level, the effect of starvation modulation in the secretomes was tested in a model of OA chondrocytes. IL1β priming in vitro led to an increase in inflammatory mediators' release and reduced anti-inflammatory potential on chondrocytes, features reversed by subsequent starvation. Therefore, when applying serum removal-based clinical-grade protocols for ASCs' secretome production, the effects of starvation must be carefully considered and investigated.

Comparative insight into the regenerative mechanisms of the adult brain in zebrafish and mouse: highlighting the importance of the immune system and inflammation in successful regeneration.

Regeneration, the complex process of restoring damaged or absent cells, tissues, and organs, varies considerably between species. The zebrafish is a remarkable model organism for its impressive regenerative abilities, particularly in organs such as the heart, fin, retina, spinal cord, and brain. Unlike mammals, zebrafish can regenerate with limited or absent scarring, a phenomenon closely linked to the activation of stem cells and immune cells. This review examines the unique roles played by the immune response and inflammation in zebrafish and mouse during regeneration, highlighting the cellular and molecular mechanisms behind their divergent regenerative capacities. By focusing on zebrafish telencephalic regeneration and comparing it to that of the rodents, this review highlights the importance of a well-controlled, acute, and non-persistent immune response in zebrafish, which promotes an environment conducive to regeneration. The knowledge gained from understanding the mechanisms of zebrafish regeneration holds great promises for the treatment of human neurodegenerative diseases and brain damage (stroke and traumatic brain injuries), as well as for the advancement of regenerative medicine approaches.