Search Results (56)

Extracellular vesicles (EVs) have emerged as promising acellular tools for modulating immune responses for tissue engineering applications. This study explores the potential of human fibroblast-derived EVs delivered within a three-dimensional (3D) injectable scaffold composed of polycaprolactone (PCL) nanofibers and collagen (PNCOL) to reprogram macrophage behavior and support scaffold integrity under inflammatory conditions. EVs were successfully isolated from human fibroblasts using ultracentrifugation and characterized for purity, size distribution and surface markers (CD63 and CD9). Macrophage-laden PNCOL scaffolds were prepared under three conditions: macrophage-only (MP), fibroblast co-encapsulated (F-MP), and EV-encapsulated (EV-MP) groups. Structural integrity was assessed via scanning electron microscopy and Masson’s trichrome staining, while immunomodulatory effects were evaluated through metabolic assays, gene expression profiling, and immunohistochemistry for macrophage polarization markers (CD80, CD206). When co-encapsulated with pro-inflammatory (M1) macrophages in PNCOL scaffolds, fibroblast-derived EVs preserved scaffold structure and significantly enhanced macrophage metabolic activity compared to the control (MP) and other experimental group (F-MP). The gene expression and immunohistochemistry data demonstrated substantial upregulation of anti-inflammatory markers (TGF-β, CD163, and CCL18) and surface protein CD206, indicating a phenotypic shift toward M2-like macrophages for EV-encapsulated scaffolds relative to the other groups. The findings of this study demonstrate that fibroblast-derived EVs integrated into injectable PCL–collagen scaffolds offer a viable, cell-free approach to modulate inflammation, preserve scaffold structure, and support regenerative healing. This strategy holds significant promise for advancing immuno-instructive platforms in regenerative medicine, particularly in settings where conventional cell therapies face limitations in survival, cost, or safety. Full article

Chronic wounds represent a major therapeutic challenge, with limited effective treatment options currently available. Both cellular and acellular approaches are being explored to address this issue, with mesenchymal stromal cells (MSCs) emerging as a promising option. While these cells have been extensively studied, alternative stromal cell sources, such as fibroblasts (Fbs), may also possess comparable therapeutic potential. Thus, this review focuses on stromal cell-derived secretomes (conditioned medium) as a source of acellular therapy for chronic wounds and presents the available wound-healing models (in vitro, ex vivo, and in vivo) suitable for evaluating their therapeutic efficacy, prior to clinical application. By conducting an analysis of the existing studies, we present the impact of the cell culture conditions on the enhancement in the bioactivity of the MSC/Fb-derived conditioned medium, a research area that continues to evolve. Full article

Hypoxic preconditioning (HP) is a promising approach to enhance the therapeutic efficacy of mesenchymal stem cells (MSCs) by modulating their oxidative stress response, metabolic activity, and secretome composition. Conditioned media (CM) obtained from MSCs cultured under hypoxia contains bioactive molecules and extracellular vesicles (EVs) that support regenerative processes. However, the effects of varying oxygen levels on the redox status and physicochemical characteristics of MSC-derived CM remain incompletely understood. This study aimed to investigate how two physiologically relevant oxygen concentrations (1% and 5%) influence oxidative stress parameters and nanoparticle features in Wharton’s jelly-derived MSC (WJ-MSC)-conditioned media. Cells were cultured under 1% or 5% O₂ and subjected to serum starvation for 48 or 72 h. CM samples were analyzed for total oxidant status (TOS), total antioxidant status (TAS), and oxidative stress index (OSI). Nanoparticle size and zeta potential were evaluated using dynamic light scattering (DLS), and HIF-1α levels were quantified via ELISA. Results showed that CM from 1% O₂ cultures exhibited significantly higher oxidative stress, with elevated TOS and OSI values and reduced TAS levels, particularly after 72 h. Nanoparticle size was initially larger under 1% O₂ but decreased with time, whereas 5% O₂ supported more stable size profiles. Zeta potential measurements revealed more negative values under 5% O₂, indicating greater colloidal stability. HIF-1α expression markedly increased under 1% O₂, confirming hypoxia-induced cellular adaptation. In conclusion, this study demonstrates that graded hypoxia distinctly modulates oxidative stress and nanoparticle characteristics in MSC-derived CM. These findings provide a basis for optimizing hypoxic preconditioning protocols to improve the quality and therapeutic potential of acellular MSC-based therapies. Full article

Introduction: Ehlers–Danlos Syndromes (EDSs) are a heterogeneous group of monogenic connective tissue disorders (e.g., joint hypermobility and dislocation, skin hyperelasticity and fragility, chronic pain, delayed wound healing process,, etc.). The primary objective of this study was to present a specialized therapeutic wound management process for a burn-injured female patient diagnosed with EDS. Case Presentation: A 34-year-old female patient presented with extensive thermal burns (biofireplace explosion). The patient had a family history of diagnosed EDS. Additionally, the patient was in a poor mental condition and, since 2020, had been undergoing pharmacotherapy with antidepressant and anti-anxiety medication. This might be the first such clinical observation in the world, but a correlation has been observed between psychiatric medication use and EDS wound healing impairment. During the hospitalization process, the patient underwent a series of surgeries aimed at the fastest and most effective closure of wounds. The patient, after 182 days of hospitalization in our facility, was discharged home. Materials and Methods: During the patient’s hospital stay, the patient underwent multiple procedures involving debridement of necrotic tissues. Additionally, allogeneic acellular dermal matrix (ADM) grafting was performed on the wounds, and a procedure was conducted in which skin was grafted using the MEEK technique. The in vitro cultured skin cells, as the advanced therapy medicinal products (ATMPs), were used. During the patient’s stay in the hospital, images were taken using low-energy laser speckle contrast analysis (LASCA) to asses microperfusion or lack thereof. The measurements were taken at intervals of several days. Conclusions: The treatment of burn wounds in patients with EDS requires a long hospitalization period. It also may require a multi-stage approach utilizing innovative preparations (e.g., ADMs and ATMPs). The assessment of wound healing progress can be performed using advanced equipment, such as laser speckle contrast analysis (LASCA). Full article

This case report outlines the intricate clinical management of a 5-month-old infant with recurrent bacterial infections and a non-healing umbilical ulcer following the surgical excision of a urachal remnant. The infant’s medical history was significant for delayed umbilical cord detachment and multiple surgical site infections. The initial surgical approach included the excision of the residual urachus, wound debridement, and abdominal wall reinforcement using a collagen matrix combined with local flap closure. Despite an apparently uneventful postoperative course, the wound experienced dehiscence and failed to heal. As part of the diagnostic workup, genetic testing was conducted, revealing an autosomal dominant mutation in the RAC2 gene, which impairs neutrophil function. Given the urgent need for wound closure prior to hematopoietic stem cell transplantation (HSCT), further debridement and sessions of negative pressure therapy were performed, alongside attempted repair with acellular dermal regeneration matrices, which ultimately proved to be ineffective. Ultimately, HSCT was undertaken despite the infectious associated risks, resulting in spontaneous wound healing without requiring further surgical interventions. This case highlights the challenges of coordinating medical, surgical, and hematological treatments in such complex cases, necessitating effective communication and collaboration among multidisciplinary teams to optimize patient outcomes. Full article

The skin functions as the body’s primary defense barrier; when compromised, it can lead to dehydration, infection, shock, or potentially life-threatening conditions. Miniature pigs exhibit skin characteristics and healing processes highly analogous to humans. Mesenchymal stem cells contribute to skin injury repair through a paracrine mechanism involving exosomes. This research examines whether adipose-derived MSC exosomes effectively enhance healing following autologous skin grafting in miniature pigs. It also compares the roles and distinctions of ADSCs and ADSC-Exos in inflammatory responses and tissue regeneration. This study found significantly reduced levels of oxidative stress products and pro-inflammatory factors, while antioxidant factors, anti-inflammatory factors, and pro-regenerative factors were elevated, and anti-regenerative factor levels decreased. Moreover, the expression levels of key markers—namely, PI3K, Akt, and mTOR—in the regeneration-associated signaling pathway were increased. The alterations in these indicators indicate that ADSC-Exos can regulate inflammatory responses and promote regeneration. This study provides a novel theoretical foundation for the implementation of acellular therapy in clinical settings. Full article

A primary challenge following severe musculoskeletal trauma is incomplete muscle regeneration. Current therapies often fail to heal damaged muscle due to dysregulated healing programs and insufficient revascularization early in the repair process. There is a limited understanding of the temporal changes that occur during the early stages of muscle remodeling in response to engineered therapies. Previous work demonstrated that nanotopographically patterned scaffolds provide cytoskeletal guidance and direct endothelial angiogenic and anti-inflammatory phenotypes. The aim of this study was to evaluate how endothelial cell (EC) patterning guides temporal and histomorphological muscle remodeling after muscle injury. In the current study, mice were treated with EC-laden engineered constructs that exhibited either aligned or random patterning of collagen nanofibrils, following a volumetric muscle loss injury (VML). Remodeling was evaluated at 2, 7, and 21 days post injury. Over the 21-day study, all groups (Acellular Aligned, EC Aligned, EC Random) demonstrated similar significant increases in vascular density and myogenesis. Animals treated with acellular controls demonstrated a two-fold decrease in muscle cross-sectional area between days 2 and 21 post injury, consistent with VML-induced muscle atrophy; however, animals treated with patterned EC-laden constructs exhibited preservation of muscle mass. The implantation of an EC-laden construct led to a 50% increase in the number of animals exhibiting areas of fibrous remodeling adjacent to the construct, along with greater collagen deposition (p < 0.01) compared to acellular controls 21 days post injury. These findings suggest that nanotopographically patterned EC-laden constructs may guide early muscle-protective programs that support muscle mass retention through myo-vascular independent pathways. Full article

Osteoarthritis (OA) is a prominent cause of disability, and has severe social and economic ramifications across the globe. The main driver of OA’s pervasiveness is the fact that no current medical interventions exist to reverse or even attenuate the degeneration of cartilage within the articular joint. Crucial for cell-to-cell communication, extracellular vesicles (EVs) contribute to OA progression through the delivery of bioactive molecules in the inflammatory microenvironment. By repurposing this acellular means of signal transmission, therapeutic drugs may be administered to degenerated cartilage tissue in the hopes of encouraging regeneration. Positive outcomes are apparent in in vivo studies on this subject; however, for this therapy to prove itself in the clinical world, efforts towards standardizing the characterization, application, biological contents, and dosage are essential. Full article

Background: Radiation therapy is a crucial component of breast cancer treatment. However, it is well known to increase the risk of unsatisfactory cosmetic outcomes and higher complication rates. The aim of this study is to provide further insight into the use of acellular dermal matrices (ADMs) for the prevention of capsular contracture. Materials and Methods: This single-center, retrospective study analyzed irradiated patients who underwent post-mastectomy, ADM-assisted implant reconstructions. Of the 60 patients included, 26 underwent expander-to-implant substitution after radiotherapy (Group A), while 34 required implant replacement due to capsular contracture following radiotherapy (Group B). The primary objective was to evaluate the effectiveness of ADMs in reducing reconstructive failures, complications, and capsular contracture after breast irradiation. Results: We recorded a total of 15 complications and four implant losses. Reconstructive failures were attributed to implant exposure in two cases, full-thickness skin necrosis in one case, and severe Baker grade IV contracture in one case. Both Group A and Group B showed a significant decrease in postoperative Baker grades. US follow-up was used to demonstrate ADM integration with host tissues over time. Conclusions: Based on our findings, the use of ADM in selected cases appears to be a viable option for treating and preventing capsular contracture in irradiated breasts. This approach is associated with relatively low complication rates, a low rate of reconstructive failure, and satisfactory cosmetic outcomes and can be applied both in breast reconstructed with implants and with expanders. Full article

Spinal cord injury (SCI) treatment remains challenging globally, with limited breakthroughs. Tissue engineering offers promise, particularly using acellular spinal cord scaffolds. This study developed a 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)-crosslinked vascular endothelial growth factor (VEGF)-modified acellular spinal cord scaffold for sustained VEGF release. The results show sustained VEGF release over 20 days without altering the scaffold’s properties. Enhanced stability and mechanical properties were observed without increased cytotoxicity. In a rat SCI model, the system improved motor function, reduced glial scarring, and restored spinal cord morphology and histology, indicating potential for SCI therapy. Full article

About one-fourth of patients with pancreatic ductal adenocarcinoma (PDAC) are categorized as borderline resectable (BR) or locally advanced (LA). Chemotherapy and radiation therapy have not yielded the anticipated outcomes in curing patients with BR/LA PDAC. The surgical resection of these tumors presents challenges owing to the unpredictability of the resection margin, involvement of vasculature with the tumor, the likelihood of occult metastasis, a higher ratio of positive lymph nodes, and the relatively larger size of tumor nodules. Oncolytic virotherapy has shown promising activity in preclinical PDAC models. Unfortunately, the desmoplastic stroma within the PDAC tumor microenvironment establishes a barrier, hindering the infiltration of oncolytic viruses and various therapeutic drugs—such as antibodies, adoptive cell therapy agents, and chemotherapeutic agents—in reaching the tumor site. Recently, a growing emphasis has been placed on targeting major acellular components of tumor stroma, such as hyaluronic acid and collagen, to enhance drug penetration. Oncolytic viruses can be engineered to express proteolytic enzymes that cleave hyaluronic acid and collagen into smaller polypeptides, thereby softening the desmoplastic stroma, ultimately leading to increased viral distribution along with increased oncolysis and subsequent tumor size regression. This approach may offer new possibilities to improve the resectability of patients diagnosed with BR and LA PDAC. Full article

The vast regenerative potential of stem cells has laid the foundation for stem cell-based therapies. However, certain challenges limit the application of cell-based therapies. The therapeutic use of cell-free therapy can avoid limitations associated with cell-based therapies. Acellular stem cell-based therapies rely on the use of biological factors released by stem cells, including growth factors and extracellular vesicles such as exosomes. Due to their comparable regenerative potential, acellular therapies may provide a feasible and scalable alternative to stem cell-based therapies. Exosomes are small vesicles secreted by various types of cells, including stem cells. Exosomes contain parent cell-derived nucleic acids, proteins, lipids, and other bioactive molecules. They play an important role in intra-cellular communication and influence the biological characteristics of cells. Exosomes inherit the properties of their parent cells; therefore, stem cell-derived exosomes are of particular interest for applications of regenerative medicine. In comparison to stem cell-based therapy, exosome therapy offers several benefits, such as easy transport and storage, no risk of immunological rejection, and few ethical dilemmas. Unlike stem cells, exosomes can be lyophilized and stored off-the-shelf, making acellular therapies standardized and more accessible while reducing overall treatment costs. Exosome-based acellular treatments are therefore readily available for applications in patients at the time of care. The current review discusses the use of exosomes as an acellular therapy. The review explores the molecular mechanism of exosome biogenesis, various methods for exosome isolation, and characterization. In addition, the latest advancements in bioengineering techniques to enhance exosome potential for acellular therapies have been discussed. The challenges in the use of exosomes as well as their diverse applications for the diagnosis and treatment of diseases have been reviewed in detail. Full article

Bone/fracture healing is a complex process with different steps and four basic tissue layers being affected: cortical bone, periosteum, fascial tissue surrounding the fracture, and bone marrow. Stem cells and their derivatives, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, hematopoietic stem cells, skeletal stem cells, and multipotent stem cells, can function to artificially introduce highly regenerative cells into decrepit biological tissues and augment the healing process at the tissue level. Stem cells are molecularly and functionally indistinguishable from standard human tissues. The widespread appeal of stem cell therapy lies in its potential benefits as a therapeutic technology that, if harnessed, can be applied in clinical settings. This review aims to establish the molecular pathophysiology of bone healing and the current stem cell interventions that disrupt or augment the bone healing process and, finally, considers the future direction/therapeutic options related to stem cells and bone healing. Full article

Numerous challenges remain within conventional cell-based therapy despite the growing trend of stem cells used to treat various life-debilitating diseases. These limitations include batch-to-batch heterogeneity, induced alloreactivity, cell survival and integration, poor scalability, and high cost of treatment, thus hindering successful translation from lab to bedside. However, recent pioneering technology has enabled the isolation and enrichment of small extracellular vesicles (EVs), canonically known as exosomes. EVs are described as a membrane-enclosed cargo of functional biomolecules not limited to lipids, nucleic acid, and proteins. Interestingly, studies have correlated the biological role of MSC-EVs to the paracrine activity of MSCs. This key evidence has led to rigorous studies on MSC-EVs as an acellular alternative. Using EVs as a therapy was proposed as a model leading to improvements through increased safety; enhanced bioavailability due to size and permeability; reduced heterogeneity by selective and quantifiable properties; and prolonged shelf-life via long-term freezing or lyophilization. Yet, the identity and potency of EVs are still relatively unknown due to various methods of preparation and to qualify the final product. This is reflected by the absence of regulatory strategies overseeing manufacturing, quality control, clinical implementation, and product registration. In this review, the authors review the various production processes and the proteomic profile of MSC-EVs. Full article

In the dairy industry, bovine mastitis represents a major concern due to substantial production losses and costs related to therapies and early culling. The mechanisms of susceptibility and effective response to intra-mammary infections are still poorly understood. Therefore, we investigated innate immunity in acellular bovine skim milk through cytofluorimetric analyses of bacterial killing activity against both Gram-positive and Gram-negative pathogens. Freshly cultured E. coli and S. aureus strains were incubated with colostrum and milk samples at different lactation time points from two groups of cows, purportedly representing mastitis-resistant and mastitis-susceptible breeds; bacterial cells were analyzed for vitality by flow cytometry following incorporation of vital dyes. N-acetyl-β-D-glucosaminidase (NAGase) activity was also investigated in milk and colostrum samples. Our findings revealed that colostrum and milk bacterial killing activity was greater against S. aureus compared to E. coli., with this activity correlated with milk NAGase levels. Furthermore, both killing of S. aureus and NAGase activity were negatively correlated to the elapsed time of lactation. Interestingly, samples from the allegedly mastitis-resistant breed displayed higher bacterial killing and NAGase activities. Our study suggests that diverse control mechanisms are exerted against Gram-positive and Gram-negative pathogens in the mammary glands of cows, probably beyond those already described in the literature. Full article