The potential of the cell lysate, secretome, and extracellular vesicles (EVs) of mesenchymal stem cells (MSCs) to modulate the immune response and promote tissue regeneration has positioned them as a promising option for cell-free therapy. Currently, many clinical trials in stem cells-derived EVs and secretome are in progress various diseases and sometimes the results are failing. The major challenge on this roadmap is the lack of a standard extraction method for exosome, secretome, and lysate. The most optimal method for obtaining the secretome of MSCs for clinical utilization involves a comprehensive approach that includes non-destructive collection methods, time optimization, multiple collection rounds, optimization of culture conditions, and quality control measures. Further research and clinical studies are warranted to validate and refine these methods for safe and effective utilization of the MSC exosome, secretome, and lysate in various clinical applications. To address these challenges, it is imperative to establish a standardized and unified methodology to ensure reliable evaluation of these extractions in clinical trials. This review seeks to outline the pros and cons of methods for the preparation of MSCs-derived exosome, and secretome/lysate, and comparative analysis of their therapeutic potential.

Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease characterized by excessive deposition of extracellular matrix (ECM) and abnormal fibroblast proliferation, which is mainly caused by air pollution, smoking, aging, occupational exposure, environmental pollutants exposure, and microbial infections. Although antifibrotic agents such as pirfenidone and nintedanib, approved by the United States (US) Food and Drug Administration (FDA), can slow the decline in lung function and disease progression, their side effects and delivery inefficiency limit the overall prognosis of PF. Therefore, there is an urgent need to develop effective therapeutic targets and delivery approaches for PF in clinical settings. This review provides an overview of the pathogenic mechanisms, therapeutic drug targeting signaling pathways, and promising drug delivery strategies for treating PF.

Despite the promising potential of stromal cell therapy in treating myocardial infarction (MI), its effectiveness is limited by poor cell retention and engraftment in ischemic environments. This study introduces a novel strategy that combines the preconditioning of human adipose-derived stromal cells (hADSCs) using OLED-based photobiomodulation (OPBM) and culturing these cells into 3D spheroids. The preconditioned 3D spheroids (APCS group) exhibit significantly enhanced angiogenic, arterialized, and tissue remodeling capabilities compared with those of traditional 2D cultures and non-preconditioned spheroids. In vivo transplantation of these spheroids into the border zone of infarcted area significantly improve cardiac function and reduce adverse remodeling by enhancing anti-fibrosis and angiogenesis including arterialization. The combined strategy with OPBM preconditioning and 3D spheroid culture system can enhance therapeutic potential of hADSCs with multiple paracrine effects for cardiac repair. This novel approach provides next generation of cell therapeutics to overcome the limitation of adult stromal cell therapy in patients with post-MI heart failure.

Stem cell secretome offers a promising alternative to stem cell transplantation for treating myocardial infarction (MI). However, its clinical application faces two major challenges: how to enhance the levels of growth factors within the secretome to promote cardiac cell survival and vascularization, and how to efficiently deliver the secretome to the infarcted heart during the acute MI phase without risking rupture of the weakened myocardium. To address these challenges, we upregulated angiogenic growth factors in the secretome from adipose-derived stem cells (ADSC-secretome) by conditioning the cells under hypoxia and with insulin-like growth factor 1 (IGF-1). Our results show that exposure to 1 % O₂ condition significantly increased the expression of VEGF, bFGF, and PDGF-BB compared to 5 % O₂ condition. Co-treatment with IGF-1 further elevated the levels of these growth factors and, notably, reduced the secretion of pro-inflammatory cytokines such as TNFα, IL-1β, and IL-6 from the ADSCs. To rapidly and specifically deliver the secretome to the infarcted heart during acute MI, we encapsulated it within ischemia-targeting nanoparticles. These nanoparticles, designed for intravenous injection, preferentially accumulated in the infarcted region. The treatment significantly improved cardiac cell survival, tissue vascularization, and cardiac function. These findings suggest that ADSC secretome, enriched with angiogenic growth factors, holds strong potential for facilitating cardiac repair following MI.

Advanced bioengineering, popularly known as regenerative dentistry, has emerged and is steadily developing with the aim of replacement of lost or injured tissues in the mouth using stem cells and other biomaterials. Conventional therapies for reparative dentistry, for instance fillings or crowns, mainly entail the replenishment of affected tissues without much concern given to the regeneration of tissues. However, these methods do not enable the natural function and aesthetics of the teeth to be maintained in the long term. There are several regenerative strategies that offer the potential to address these limitations to the extent of biologically restoring the function of teeth and their components, like pulp, dentin, bone, and periodontal tissues. Hence, stem cells, especially dental tissue derived stem cells, such as dental pulp stem cells, periodontal ligament stem cells, or apical papilla stem cells, are quite promising in this regard. These stem cells have the potentiality of generating precise dental cell lineages and thus are vital for tissue healing and renewal. Further, hydrogels, growth factors, and synthetic scaffolds help in supporting the stem cells for growth, proliferation, and differentiation into functional tissues. This review aims at describing the process of stem cell-based tissue repair biomaterials in dental regeneration, and also looks into the practice and prospects of regenerative dentistry, analysing several case reports and clinical investigations that demonstrate the efficacy and limitations of the technique. Nonetheless, the tremendous potential for regenerative dentistry is a reality that is currently challenged by biological and technical constraints, such as scarcity of stem cell sources, inadequate vascularization, and the integration of the materials used in the procedure. As we move forward, the prospects for regenerative dentistry are in subsequent developments of stem cell technology, biomaterial optimization, and individualized treatment methods, which might become increasingly integrated in dental practices globally. However, there are regulatory, ethical and economic issues that may pose a hurdle in the further advancement of this discipline.

This comprehensive review aimed to provide insights into the Asherman syndrome's historical background, clinical manifestations, classifications, obstetrical challenges, and current treatment approaches. The syndrome is characterized by intrauterine adhesions and fibrotic changes within the uterine tract as well as symptoms including menstrual irregularities, pelvic pain and infertility. The primary causes of Asherman syndrome are often associated with iatrogenic complications and congenital uterine defects. The syndrome results in certain obstetrical challenges, including recurrent pregnancy loss, placenta abnormalities, preterm birth, and intrauterine growth retardation, emphasizing the need for effective management. Hysteroscopic adhesiolysis represents the current gold standard treatment, but challenges persist because of adhesion recurrence and obstetrical complications. In this sense, emerging therapies were explored, including paracrine-acting factors, tissue-engineered scaffolds, and cell-based therapies. Autologous CD133+ bone marrow-derived stem cell therapy shows promise, with clinical trials demonstrating improved endometrial conditions and positive obstetrical outcomes. The review concludes by highlighting the potential of single-cell RNA sequencing to unravel the molecular mechanisms behind Asherman syndrome. This advanced technology offers insights into the gene expression profiles of individual cells, fostering a deeper understanding of Asherman syndrome pathogenesis and the development of innovative therapeutic strategies.

Three-dimensional (3D) bio-derived materials are emerging as a promising approach to enhance wound healing therapies. These innovative materials can be tailored to meet the specific needs of various wound types and patients, facilitating the controlled release of therapeutic agents such as growth factors and antibiotics, which promote cell growth and tissue regeneration. Despite their potential, significant challenges remain in achieving optimal biocompatibility, ensuring structural integrity, and maintaining precise release mechanisms. Additionally, issues such as scalability, cost-effectiveness, and regulatory compliance pose substantial barriers to widespread use. However, recent advances in materials science and interdisciplinary research offer new opportunities to overcome these challenges. This review provides a comprehensive analysis of the current state of 3D bio-derived materials in biomedical applications, highlighting the types of materials available, their advantages and limitations, and the progress made in their design and development. It also outlines new directions for future research aimed at bridging the gap between scientific discoveries and their practical applications in injury healing strategies. The findings of this review underscore the significant potential of 3D bio-derived materials in revolutionizing wound healing and advancing personalized therapeutic approaches.

Myocardial infarction remains a leading cause of death worldwide due to the heart's limited regenerative capability and the current lack of viable therapeutic solutions. Therefore, there is an urgent need to develop effective treatment options to restore cardiac function after a heart attack. Stem cell-derived cardiac cells have been extensively utilised in cardiac tissue regeneration studies. However, the use of Matrigel as a substrate for the culture and maturation of these cells has been a major limitation for the translation of this research into clinical application. Hydrogels are emerging as a promising system to overcome this problem. They are biocompatible and can provide stem cells with a supportive scaffold that mimics the extracellular matrix, which is essential for repairing damaged tissue in the myocardium after an infarction. Thus, hydrogels provide an alternative and reproducible option in addressing myocardial infarction due to their unique potential therapeutic benefits. This review explores the different types of natural and synthetic polymers used to create hydrogels and their various delivery methods, the most common being via injection and cardiac patches and other applications such as bioprinting. Many challenges remain before hydrogels can be used in a clinical setting, but they hold great promise for the future of cardiac tissue regeneration.

Uterus bioengineering offers a potential treatment option for women with uterine factor infertility and for mitigating the risk of uterine rupture associated with women with defective uterine tissue. Decellularized uterine tissue scaffolds proved promising in further in vivo experiments in rodent and domestic species animal models. Variations in the extracellular matrix composition among different species and adaptations of the decellularization protocols make it difficult to compare the results between studies. Therefore, we assessed if our earlier developed sodium deoxycholate-based decellularization protocol for the sheep and the cow uterus could become a standardized cross-species protocol by assessing it on the non-human primate (baboon) uterus.

The baboon uterus was decellularized using sodium deoxycholate, and the remaining acellular scaffold was quantitatively assessed for DNA, protein, and specific extracellular matrix components. Furthermore, electron microscopy deepened morphology examination, while the chorioallantoic membrane assay examined the scaffolds' cytotoxicity, bioactivity, and angiogenic properties. The in vitro recellularization efficiency of the scaffolds using xenogeneic (rat) bone marrow-derived mesenchymal stem cells was also assessed. Finally, the immune potential of the scaffolds was evaluated by in vitro exposure to human peripheral blood mononuclear cells.

We obtained a decellularized baboon uterus with preserved extracellular matrix components by adding an 8-h sodium deoxycholate perfusion to our previously developed protocol for the sheep and cow models. This minor modification resulted in scaffolds with less than 1% of immunogenic host DNA content while preserving important uterine-specific collagen, elastin, and glycosaminoglycan structures. The chorioallantoic membrane assay and in vitro recellularization experiments confirmed that the scaffolds were bioactive and non-cytotoxic. As we have observed in other animal models, the enzymatic scaffold preconditioning with matrix metalloproteinases improved the recellularization efficiency further. Additionally, the preconditioning generated more immune-privileged scaffolds, as shown in a novel in vitro co-culture assay with human peripheral blood mononuclear cells.

For the first time, our data demonstrate the efficiency of our protocol for non-human primate uteri and its translational potential. This standardized protocol will facilitate cross-study comparisons and expedite clinical translation.

Peripheral nerve injuries are common complications in surgical and dental practices, often resulting in functional deficiencies and reduced quality of life. Current treatment choices, such as autografts, have limitations, including donor site morbidity and suboptimal outcomes. Adipose-derived stem cells (ADSCs) have shown assuring regenerative potential due to their accessibility, ease of harvesting and propagation, and multipotent properties. This review investigates the therapeutic potential of ADSCs in peripheral nerve regeneration, focusing on their use in bioengineered nerve conduits and supportive microenvironments. The analysis is constructed on published case reports, organized reviews, and clinical trials from Phase I to Phase III that investigate ADSCs in managing nerve injuries, emphasizing both peripheral and orofacial applications. The findings highlight the advantages of ADSCs in promoting nerve regeneration, including their secretion of angiogenic and neurotrophic factors, support for cellular persistence, and supplementing scaffold-based tissue repair. The regenerative capabilities of ADSCs in peripheral nerve injuries offer a novel approach to augmenting nerve repair and functional recovery. The accessibility of adipose tissue and the minimally invasive nature of ADSC harvesting further encourage its prospective application as an autologous cell source in regenerative medicine. Future research is needed to ascertain standardized protocols and optimize clinical outcomes, paving the way for ADSCs to become a mainstay in nerve regeneration.

Fractional carbon dioxide lasers and bone marrow mesenchymal stem cells (BMSCs) are commonly employed in the treatment of skin photoaging.

This study was developed to explore the effects of combination carbon dioxide laser treatment and BMSC injection on skin photoaging and the underlying molecular mechanisms.

In total, 24 mice with experimentally photoaged skin were separated into control, carbon dioxide fractional laser treatment, combination therapy, and BMSC injection groups. Samples of dorsal skin from these animals were subjected to hematoxylin and eosin staining or Masson's trichrome staining. In addition, immunohistochemical analyses and real-time polymerase chain reaction analyses were conducted to detect MMP-3 and MMP-9 expression.

After 1 week, both dermal thickness and collagen fiber density were significantly increased in the BMSC and combination treatment groups as compared to the control group (P<0.05), while both of these parameters were significantly increased in all treatment groups after 4 weeks relative to the control group (P<0.05), with the most pronounced effect in the combination therapy group (P<0.05). MMP-3 and MMP-9 mRNA and protein levels in the treatment groups were decreased relative to the control group after 4 weeks.

Combination BMSC and carbon dioxide laser therapy was more effective than either of these therapeutic approaches in isolation as a treatment for photoaged skin. The improvement of effect may be due to the decrease of MMP-3 and MMP-9 expression in combination therapy.

Stromal vascular fraction (SVF) is a complex mixture derived from adipose tissue, consisting of a variety of cells. Due to its potential for tissue repair, immunomodulation, and support of angiogenesis, SVF represents a promising frontier in regenerative medicine and offers potential therapy for a range of disease conditions. In this article, we delve into the mechanisms through which SVF exerts its effects and explore its potential applications in treating both male and female reproductive disorders, including erectile dysfunction, testicular injury, stress urinary incontinence and intrauterine adhesion.

Stem cells are unique, undifferentiated cells that have the ability to both replicate themselves and develop into specialized cell types. This dual capability makes them valuable in the development of regenerative medicine. Current development in stem cell research has widened their application in cell therapy, drug discovery, reproductive cloning in animals, and cell models for various diseases. Although there are substantial studies revealing the treatment of human degenerative diseases using stem cells, this is yet to be explored in livestock animals. Many diseases in livestock species such as mastitis, laminitis, neuromuscular disorders, autoimmune diseases, and some debilitating diseases are not covered completely by the existing drugs and treatment can be improved by using different types of stem cells like embryonic stem cells, adult stem cells, and induced pluripotent stem cells. This review mainly focuses on the use of stem cells for disease treatment in livestock animals. In addition to the diseases mentioned, the potential of stem cells can be helpful in wound healing, skin disease therapy, and treatment of some genetic disorders. This article explores the potential of stem cells from various sources in the therapy of livestock diseases and also their role in the conservation of endangered species as well as disease model preparation. Moreover, the future perspectives and challenges associated with the application of stem cells in livestock are discussed. Overall, the transformative impact of stem cell research on the livestock sector is comprehensively studied which will help researchers to design future research work on stem cells related to livestock diseases.

In this review, it is evaluated the progress in the application of stem cell therapy to ameliorate the symptoms of bipolar disorder, major depression, schizophrenia, and autism. These disorders are highly prevalent in clinical medicine and are responsible for high levels of psychosocial disability among patients. All of them share common biomedical features, such as complex and variable genetic substrates, significant susceptibility to environmental changes, and insufficient knowledge of their pathogenesis. In addition, the responsiveness of patients to pharmacological treatment is heterogeneous, and in some cases, no treatment is available. Therefore, the development of stem cell-based regenerative medicine and its possible combination with emerging therapeutic approaches that promote neural plasticity are expected to advance neuropsychiatry in the next few decades.

The tendon or ligament is attached to the bone by a triphasic but continuous area of heterogeneous tissue called the tendon-bone interface (TBI). The rapid and functional regeneration of TBI is challenging owing to its complex composition and difficulty in self-healing. The development of new technologies, such as decellularization, has shown promise in the regeneration of TBI. Several ex vivo and in vivo studies have shown that decellularized grafts and decellularized biomaterial scaffolds achieved better efficacy in enhancing TBI healing. However further information on the type of review that is available is needed.

In this review, we discuss the current application of decellularization biomaterials in promoting TBI healing and the possible mechanisms involved. With this work, we would like to reveal how tissues or biomaterials that have been decellularized can improve tendon-bone healing and to provide a theoretical basis for future related studies.

Decellularization is an emerging technology that utilizes various chemical, enzymatic and/or physical strategies to remove cellular components from tissues while retaining the structure and composition of the extracellular matrix (ECM). After decellularization, the cellular components of the tissue that cause an immune response are removed, while various biologically active biofactors are retained. This review further explores how tissues or biomaterials that have been decellularized improve TBI healing.

As the number of patients requiring organ transplants continues to rise exponentially, there is a dire need for therapeutics, with repair and regenerative properties, to assist in alleviating this medical crisis. Over the past decade, there has been a shift from conventional stem cell treatments towards the use of the secretome, the protein and factor secretions from cells. These components may possess novel druggable targets and hold the key to profoundly altering the field of regenerative medicine. Despite the progress in this field, clinical translation of secretome-containing products is limited by several challenges including but not limited to ensuring batch-to-batch consistency, the prevention of further heterogeneity, production of sufficient secretome quantities, product registration, good manufacturing practice protocols and the pharmacokinetic/pharmacodynamic profiles of all the components. Despite this, the secretome may hold the key to unlocking the regenerative blockage scientists have encountered for years. This review critically analyses the secretome derived from different cell sources and used in several tissues for tissue regeneration. Furthermore, it provides an overview of the current delivery strategies and the future perspectives for the secretome as a potential therapeutic. The success and possible shortcomings of the secretome are evaluated.

Psoriasis is a common chronic inflammatory disease in which keratinocytes proliferate abnormally due to excessive immune action. Psoriasis can be associated with various comorbidities and has a significant impact on health-related quality of life. Although many systemic treatments, including biologic agents, have been developed, topical treatment remains the main option for psoriasis management. Consequently, there is an urgent need to develop topical treatments with minimal side effects and high efficacy. Mesenchymal stem cells (MSCs) exhibit excellent immune regulation, anti-inflammatory activities, and therapeutic effects, and MSC-derived extracellular vesicles (EVs) can serve as crucial mediators of functional transfer from MSCs. Therefore, this study aimed to develop a safe and easy-to-use emulsion cream for treating psoriasis using MSC conditioned media (CM) containing EVs. We developed an enhanced Wharton's jelly MSC (WJ-MSC) culture method through a three-dimensional (3D) culture containing exogenous transforming growth factor-β3. Using the 3D culture system, we obtained CM from WJ-MSCs, which yielded a higher EV production compared to that of conventional WJ-MSC culture methods, and investigated the effect of EV-enriched 3D-WJ-MSC-CM cream on psoriasis-related inflammation. Administration of the EV-enriched 3D-WJ-MSC-CM cream significantly reduced erythema, thickness, and scaling of skin lesions, alleviated imiquimod-induced psoriasiform lesions in mice, and ameliorated histopathological changes in mouse skin. The upregulated mRNA expression of inflammatory cytokines, including IL-17a, IL-22, IL-23, and IL-36, decreased in the lesions. In conclusion, we present here a new topical treatment for psoriasis using an MSC EV-enriched cream.

Even with advancement of medical technologies, liver transplantation still faces several major challenges. Hence, other treatment modalities are urgently needed for patients with end-stage liver disease. Stem cells from human exfoliated deciduous teeth (SHED) was discovered to have highly proliferative and pluripotent properties; including differentiation into hepatocyte-like cells. This study aims to investigate the capability of intrasplenic transplanted SHED and SHED-Hep cells in inducing proliferation of stem cells and native hepatocytes in order to accelerate liver regeneration in liver fibrosis mice models.

Three carbon tetrachloride (CCl4)-injured male mice groups were used in this study. Two of those groups were transplanted with either SHED or SHED-Hep, while the other did not undergo transplantation. One age- and sex- matched healthy mice group was used as control. All specimens were immunohistochemically stained with anti-Ki-67 antibodies and anti-proliferating cell nuclear antigen (PCNA) antibodies before counter stained with hematoxylin-eosin.

Anti-Ki-67 antibodies staining: at both 8 and 12 weeks, proliferating activity was predominantly seen on both SHED- and SHED-Hep-transplanted CCl4-injured mice groups, while control and non-transplanted CCl4-injured mice group showed little to no sign of proliferation activity. Anti-PCNA staining: at both 8 and 12 weeks, significant proliferating activity was detected by PCNA staining, mainly on stem cells population area on SHED- and SHED-Hep-treated group.

In conclusion, this study has provided the evidence that transplantation of SHED or SHED-Hep on liver-injured mice induced proliferation of both transplanted stem cells and native liver cells in order to accelerate liver regeneration.

The establishment and maintenance of pregnancy depend on endometrial competence. Asherman syndrome (AS) and intrauterine adhesions (IUA), or endometrial atrophy (EA) and thin endometrium (TE), can either originate autonomously or arise as a result from conditions (i.e. endometritis or congenital hypoplasia), or medical interventions (e.g. surgeries, hormonal therapies, uterine curettage or radiotherapy). Affected patients may present an altered or inadequate endometrial lining that hinders embryo implantation and increases the risk of poor pregnancy outcomes and miscarriage. In humans, AS/IUA and EA/TE are mainly treated with surgeries or pharmacotherapy, however the reported efficacy of these therapeutic approaches remains unclear. Thus, novel regenerative techniques utilizing stem cells, growth factors, or tissue engineering have emerged to improve reproductive outcomes.

This review comprehensively summarizes the methodologies and outcomes of emerging biotechnologies (cellular, acellular, and bioengineering approaches) to treat human endometrial pathologies. Regenerative therapies derived from human tissues or blood which were studied in preclinical models (in vitro and in vivo) and clinical trials are discussed.

A systematic search of full-text articles available in PubMed and Embase was conducted to identify original peer-reviewed studies published in English between January 2000 and September 2023. The search terms included: human, uterus, endometrium, Asherman syndrome, intrauterine adhesions, endometrial atrophy, thin endometrium, endometritis, congenital hypoplasia, curettage, radiotherapy, regenerative therapy, bioengineering, stem cells, vesicles, platelet-rich plasma, biomaterials, microfluidic, bioprinting, organoids, hydrogel, scaffold, sheet, miRNA, sildenafil, nitroglycerine, aspirin, growth hormone, progesterone, and estrogen. Preclinical and clinical studies on cellular, acellular, and bioengineering strategies to repair or regenerate the human endometrium were included. Additional studies were identified through manual searches.

From a total of 4366 records identified, 164 studies (3.8%) were included for systematic review. Due to heterogeneity in the study design and measured outcome parameters in both preclinical and clinical studies, the findings were evaluated qualitatively and quantitatively without meta-analysis. Groups using stem cell-based treatments for endometrial pathologies commonly employed mesenchymal stem cells (MSCs) derived from the human bone marrow or umbilical cord. Alternatively, acellular therapies based on platelet-rich plasma (PRP) or extracellular vesicles are gaining popularity. These are accompanied by the emergence of bioengineering strategies based on extracellular matrix (ECM)-derived hydrogels or synthetic biosimilars that sustain local delivery of cells and growth factors, reporting promising results. Combined therapies that target multiple aspects of tissue repair and regeneration remain in preclinical testing but have shown translational value. This review highlights the myriad of therapeutic material sources, administration methods, and carriers that have been tested.

Therapies that promote endometrial proliferation, vascular development, and tissue repair may help restore endometrial function and, ultimately, fertility. Based on the existing evidence, cost, accessibility, and availability of the therapies, we propose the development of triple-hit regenerative strategies, potentially combining high-yield MSCs (e.g. from bone marrow or umbilical cord) with acellular treatments (PRP), possibly integrated in ECM hydrogels. Advances in biotechnologies together with insights from preclinical models will pave the way for developing personalized treatment regimens for patients with infertility-causing endometrial disorders such as AS/IUA, EA/TE, and endometritis.

https://osf.io/th8yf/.

Owing to the moist and curved interfaces of skin wounds, enhancing the adhesiveness while maintaining delivery efficacy of biomolecules has drawn significant attention in advanced wound dressings. Despite tremendous trials to load biomolecules with sound adhesiveness, the complicated fabricating processes and abnormal allergic responses that are attributed to chemical moiety-based adhesives remain as major problems. To this end, in this study a one-step fabrication process is developed to manufacture microstructures with both a therapeutic (cylindrical structure for embossed structure human adipose-derived stem cell sheet, ESS) and an adhesive part (octopi-inspired structure of adhesive, OIA), which ESOIA is called. OIA showed the highest adhesion strength in both dry (1.48 N cm-2) and wet pig skin conditions (0.81 N cm-2), maintaining the adhesive properties after repeated attach-detach trials. ESS from the therapeutic part of ESOIA also showed an enhanced angiogenic effect compared with the ones that are normally cultured in vitro. ESS also showed improved in vivo wound healing outcomes following enhanced cell engraftment compared to the cell injection group by means of intact cell-extracellular matrix interactions.

Obesity is a health problem characterized by large expansion of adipose tissue. During this expansion, genotoxic stressors can be accumulated and negatively affect the mesenchymal stem cells (MSCs) of adipose tissue. Due to the oxidative stress generated by these genotoxic stressors, senescence phenotype might be observed in adipose tissue MSCs. Senescent MSCs lose their proliferations and differentiation properties and secrete senescence-associated molecules to their niche thus triggering senescence for the rest of the tissue. Accumulation of senescent cells in adipose tissue results in decreased tissue regeneration and functional impairment not only in the close vicinity but also in the other tissues. Here we hypothesized that declined tissue regeneration might be associated with loss of stemness markers in MSCs population. We analyzed the expression of several stemness-associated genes of in vitro cultured MSCs originated from adipose tissue of high-fat diet and normal diet mice models. Since the heterogenous MSCs population covers a small percentage of the pluripotent stem cells, which have roles in proliferation and tissue regeneration, we measured the percentage of these cells via TRA-1-60 pluripotent state antigen. Additionally, by conducting a shotgun proteomic approach using LC-MS/MS, whole cell proteome of the adipose tissue MSCs of high-fat diet and normal diet mice were analyzed and identified proteins were evaluated via gene ontology and PPI network analysis. MSCs of obese mice showed senescent phenotype and altered cell cycle distribution due to a hostile environment with oxidative stress in adipose tissue where they reside. Additionally, the number of pluripotent markers expressing cells declined in the MSC population of the high-fat diet mice. Gene expression analysis evidenced the loss of stemness with a decrease in the expression of stemness-associated genes. Of the proteomic comparison of the normal and the high-fat diet group, MSCs revealed that stemness-associated molecules were decreased while inflammation and senescence-associated phenotypes emerged in obese mice MSCs. Our results showed us that the MSCs of adipose tissue may lose their stemness properties due to obesity-associated stress conditions.

Stem cell-based therapies offer promising avenues for treating inflammatory diseases owing to their immunomodulatory properties. However, challenges persist regarding their survival and efficacy in inflamed tissues. Our study introduces a novel approach by engineering adipose-derived stem cells (ADSCs) to enhance their viability in inflammatory environments and boost the secretion of paracrine factors for treating inflammatory bowel disease (IBD). An arginine-glycine-aspartate peptide-poly (ethylene glycol)-chlorin e6 conjugate (RPC) was synthesized and coupled with ADSCs, resulting in RPC-labeled ADSCs (ARPC). This conjugation strategy employed RGD-integrin interaction to shield stem cells and allowed visualization and tracking using chlorin e6. The engineered ARPC demonstrated enhanced viability and secretion of paracrine factors upon light irradiation, regulating the inflammatory microenvironment. RNA-sequencing analysis unveiled pathways favoring angiogenesis, DNA repair, and exosome secretion in ARPC(+) while downregulating inflammatory pathways. In in vivo models of acute and chronic IBD, ARPC(+) treatment led to reduced inflammation, preserved colon structure, and increased populations of regulatory T cells, highlighting its therapeutic potential. ARPC(+) selectively homed to inflammatory sites, demonstrating its targeted effect. Overall, ARPC(+) exhibits promise as an effective and safe therapeutic strategy for managing inflammatory diseases like IBD by modulating immune responses and creating an anti-inflammatory microenvironment.

Due to the limitations of the current skin wound treatments, it is highly valuable to have a wound healing formulation that mimics the extracellular matrix (ECM) and mechanical properties of natural skin tissue. Here, a novel biomimetic hydrogel formulation has been developed based on a mixture of Agarose-Collagen Type I (AC) combined with skin ECM-related components: Dermatan sulfate (DS), Hyaluronic acid (HA), and Elastin (EL) for its application in skin tissue engineering (TE). Different formulations were designed by combining AC hydrogels with DS, HA, and EL. Cell viability, hemocompatibility, physicochemical, mechanical, and wound healing properties were investigated. Finally, a bilayered hydrogel loaded with fibroblasts and mesenchymal stromal cells was developed using the Ag-Col I-DS-HA-EL (ACDHE) formulation. The ACDHE hydrogel displayed the best in vitro results and acceptable physicochemical properties. Also, it behaved mechanically close to human native skin and exhibited good cytocompatibility. Environmental scanning electron microscopy (ESEM) analysis revealed a porous microstructure that allows the maintenance of cell growth and ECM-like structure production. These findings demonstrate the potential of the ACDHE hydrogel formulation for applications such as an injectable hydrogel or a bioink to create cell-laden structures for skin TE.

Mesenchymal adipose stromal cells (ASCs) are considered the most promising and accessible material for translational medicine. ASCs can be used independently or within the structure of scaffold-based constructs, as these not only ensure mechanical support, but can also optimize conditions for cell activity, as specific features of the scaffold structure have an impact on the vital activity of the cells. This manuscript presents a study of the secretion and accumulation that occur in a conditioned medium during the cultivation of human ASCs within the structure of such a partial skin-equivalent that is in contact with it. It is demonstrated that the ASCs retain their functional activity during cultivation both within this partial skin-equivalent structure and, separately, on plastic substrates: they proliferate and secrete various proteins that can then accumulate in the conditioned media. Our comparative study of changes in the conditioned media during cultivation of ASCs on plastic and within the partial skin-equivalent structure reveals the different dynamics of the release and accumulation of such secretory factors in the media under a variety of conditions of cell functioning. It is also demonstrated that the optimal markers for assessment of the ASCs' secretory functions in the studied partial skin-equivalent structure are the trophic factors VEGF-A, HGF, MCP, SDF-1α, IL-6 and IL-8. The results will help with the development of an algorithm for preclinical studies of this skin-equivalent in vitro and may be useful in studying various other complex constructs that include ASCs.

People still hold the concept of using cell-based treatments to regenerate missing neurons in high esteem. CD117+ cells are considered favorable stem cells for regenerative medicine. The objective of this research was to examine the impact of Alginate-Gelatin (Alg-Gel) hydrogel on the process of neurogenic differentiation of CD117+ cells utilizing a cytokines secretion test conducted in a laboratory setting. To achieve this objective, bone marrow-CD117+ cells were isolated using the MACS technique and then transformed into neuron cells using a neurogenic differentiation medium. The characterization of enriched CD117+ cells has been done with flow cytometry as well as immunocytochemistry. Next, the cells underwent western blotting assay to evaluate the signaling pathways. Subsequently, the culture media was obtained from both groups in order to determine cytokine levels. The study revealed that the Alg-Gel hydrogel had a notable impact on enhancing the protein expression of neuron markers such as β-tubulin and Wnt/catenin signaling pathway components in CD117+ neurogenic differentiated cells. Furthermore, the cultured medium from the experimental group exhibited a notable abundance of IL-6 and IL-10 in comparison to the control group. The observed in vitro effects of Alg-Gel hydrogel on neurogenic differentiation of CD117+ cells are likely to be caused by the cytokines that are released.

Type 2 diabetes (T2D) is a progressive disease. Many drugs currently being used for the management of T2D have minimal effect on pancreatic beta cells regeneration. Cell-based therapies might provide potential benefits in this aspect.

A pilot study in five T2D patients with 12 months follow-up was performed to evaluate the effect of autologous bone marrow mononuclear stem cells (BM-MNCs) infusion into pancreatic arteries on the insulin requirement, beta-cell function, insulin resistance, and systemic inflammatory marker (CRP).

The primary endpoint, a 50 % reduction of total insulin doses from baseline, was not achieved in this study. However, a trend of increasing fasting C-peptide (p = 0.07) and C-peptide 60' (p = 0.07) and 90' (p = 0.07) after a mixed-meal tolerance test was observed 12 months post-infusion compared to baseline levels. A similar result was observed for the homeostatic model assessment of beta cell function (HOMA1-B), an index for beta cell function. No improvement was observed for insulin resistance measured by homeostasis model assessment of insulin resistance (HOMA1-IR) and systemic inflammatory parameter.

Intraarterial pancreatic autologous BM-MNCs infusion might potentially improve beta cell function in T2D patients, although further study is needed to confirm this finding.

Myocardial infarction (MI) stands out as a highly lethal disease that poses a significant threat to global health. Worldwide, heart failure resulting from MI remains a leading cause of human mortality. Mesenchymal stem cell (MSC) therapy has emerged as a promising therapeutic approach, leveraging its intrinsic healing properties. Nevertheless, pervasive issues, including a low cell retention rate, suboptimal survival rate, and incomplete differentiation of MSCs, present formidable challenges for further research. The introduction and advancement of biomaterials have offered a novel avenue for the exploration of MSC therapy in MI, marking considerable progress thus far. Notably, hydrogels, among the representative biomaterials, have garnered extensive attention within the biomedical field. This review delves into recent advancements, specifically focusing on the application of hydrogels to augment MSC therapy for cardiac tissue regeneration in MI.

This study aims to investigate the effects of a conditioned medium (CM) from human umbilical cord mesenchymal stem cells (HuMSCs) cultivated in gelatin sponge (GS-HuMSCs-CM) on hair growth in a mouse model.

CM was collected from the HuMSCs cultivated in a monolayer or in a gelatin sponge. Vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) levels in CMs were measured by enzyme-linked immunosorbent assays (ELISAs). A hair loss model by a C57 BL/6J mouse was prepared. The effects of GS-HuMSCs-CM and HuMSCs on hair regrowth in mice were investigated by intradermal injection in the depilated back skin with normal saline (NS) as the control. The time for hair regrowth and full covering in depilated areas was observed, and the hair growth was evaluated histologically and by grossly measuring hair length and diameter.

Compared with monolayer cultured cells, the three-dimensional (3D) culture of HuMSCs in gelatin sponge drastically increased VEGF, IGF-1, KGF, and HGF production. GS-HuMSCs-CM and HuMSCs injection both promoted hair regeneration in mice, while GS-HuMSCs-CM presented more enhanced effects in hair length, hair diameter, and growth rate. GS-HuMSCs-CM significantly promoted angiogenesis in injected skin areas, which might also contribute to faster hair regrowth.

GS-HuMSCs-CM exerted significant effects on inducing hair growth and promoted skin angiogenesis in C57BL/6J mice.

Inflammatory bowel disease (IBD) is characterized by a chronically dysregulated immune response in the gastrointestinal tract. Bone marrow multipotent mesenchymal stromal cells have an important immunomodulatory function and support regeneration of inflamed tissue by secretion of soluble factors as well as through direct local differentiation. CXCR4 is the receptor for CXCL12 (SDF-1, stromal-derived factor-1) and has been shown to be the main chemokine receptor, required for homing of MSCs. Increased expression of CXCL12 by inflamed intestinal tissue causes constitutive inflammation by attracting lymphocytes but can also be used to direct MSCs to sites of injury/inflammation. Trypsin is typically used to dissociate MSCs into single-cell suspensions but has also been shown to digest surface CXCR4. Here, we assessed the regenerative effects of CXCR4high and CXCR4low MSCs in an immune-deficient mouse model of DSS-induced colitis. We found that transplantation of MSCs resulted in clinical improvement and histological recovery of intestinal epithelium. In contrary to our expectations, the levels of CXCR4 on transplanted MSCs did not affect their regenerative supporting potential, indicating that paracrine effects of MSCs may be largely responsible for their regenerative/protective effects.

Human bone marrow-derived stem cells (hBMDSCs) are well characterized mediators of tissue repair and regeneration. An increasing body of evidence indicates that these cells exert their therapeutic effects largely through their paracrine actions rather than clonal expansion and differentiation. Here we studied the role of microRNAs (miRNAs) present in extracellular vesicles (EVs) from hBMDSCs in tissue regeneration and cell differentiation targeting endometrial stromal fibroblasts (eSF).

Extracellular vesicles (EVs) are isolated from hBMDSCs, characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) techniques. Extracted total RNA from EVs was subjected to RNA seq analysis. Transfection and decidualization studies were carried out in endometrial stromal fibroblasts (eSF). Gene expression was analyzed by qRTPCR. Unpaired t-test with Welch's correction was used for data analysis between two groups.

We identified several microRNAs (miRNAs) that were highly expressed, including miR-21-5p, miR-100-5p, miR-143-3p and let7. MiR-21 is associated with several signaling pathways involved in tissue regeneration, quiescence, cellular senescence, and fibrosis. Both miR-100-5p and miR-143-3p promoted cell proliferation. MiR-100-5p specifically promoted regenerative processes by upregulating TGF-ß3, VEGFA, MMP7, and HGF. MiR-100-5p blocked differentiation or decidualization as evidenced by morphologic changes and downregulation of decidualization mediators including HOXA10, IGFBP1, PRL, PR-B, and PR.

EVs delivered to tissues by hBMDSCs contain specific miRNAs that prevent terminal differentiation and drive repair and regeneration. Delivery of microRNAs is a novel treatment paradigm with the potential to replace BMDSCs in cell-free regenerative therapies.

Peripheral blood is an alternative source of stem/progenitor cells for regenerative medicine owing to its ease of retrieval and blood bank storage. Previous in vitro studies indicated that the conditioned medium derived from peripheral blood mononuclear cells (PBMCs) treated with the iron-quercetin complex (IronQ) contains potent angiogenesis and wound-healing properties. This study aims to unveil the intricate regulatory mechanisms governing the effects of IronQ on the transcriptome profiles of human PBMCs from healthy volunteers and those with diabetes mellitus (DM) using RNA sequencing analysis. Our findings revealed 3741 and 2204 differentially expressed genes (DEGs) when treating healthy and DM PBMCs with IronQ, respectively. Functional enrichment analyses underscored the biological processes shared by the DEGs in both conditions, including inflammatory responses, cell migration, cellular stress responses, and angiogenesis. A comprehensive exploration of these molecular alterations exposed a network of 20 hub genes essential in response to stimuli, cell migration, immune processes, and the mitogen-activated protein kinase (MAPK) pathway. The activation of these pathways enabled PBMCs to potentiate angiogenesis and tissue repair. Corroborating this, quantitative real-time polymerase chain reaction (qRT-PCR) and cell phenotyping confirmed the upregulation of candidate genes associated with anti-inflammatory, pro-angiogenesis, and tissue repair processes in IronQ-treated PBMCs. In summary, combining IronQ and PBMCs brings about substantial shifts in gene expression profiles and activates pathways that are crucial for tissue repair and immune response, which is promising for the enhancement of the therapeutic potential of PBMCs, especially in diabetic wound healing.

The field of regenerative medicine has recently witnessed groundbreaking advancements that hold immense promise for treating a wide range of diseases and injuries. At the forefront of this revolutionary progress are stem cells. Stem cells typically reside in specialized environments in vivo, known as microenvironments or niches, which play critical roles in regulating stem cell behavior and determining their fate. Therefore, understanding the complex microenvironments that surround stem cells is crucial for advancing treatment options in regenerative medicine and tissue engineering applications. Several research articles have made significant contributions to this field by exploring the interactions between stem cells and their surrounding niches, investigating the influence of biomechanical and biochemical cues, and developing innovative strategies for tissue regeneration. This review highlights the key findings and contributions of these studies, shedding light on the diverse applications that may arise from the understanding of stem cell microenvironments, thus harnessing the power of these microenvironments to transform the landscape of medicine and offer new avenues for regenerative therapies.

For many sportsmen, anterior cruciate ligament (ACL) tears are unfortunate but common injuries. Several growth factors, cytokine, chemokine, and protease inhibitors functions in stimulation of paracrine reactions in fibroblast, endothelial, and stem cells thereby promoting the tissue restorative processes. Augmented with dehydrated Human Amnion Chorion Membrane (dHACM) allograft reinforces the reconstructed ligament and aids in effective restoration.

In this case control study 15 patients undertaking ACL reconstruction with tripled peroneus augmented dHACM (G1) were prospectively monitored up for a period of 8 months along with 15 control patients (G2) without dHACM augmentation. Clinical and radiological outcomes were analysed and assessed about effect of augmenting the peroneus longus graft using dHACM. Clinical analysis included pre-operative two, four, six, and eight months post-operative Tegnor-Lysholm score, and radiological analysis included the 6th month postoperative MRI signal-to-noise ratio (SNR) measurements by mean signal-value at femoral insertion, midsubstance and tibial insertion of ACL graft.

Clinically, as a mean Lysholm score of all patients, they were revealed to be consecutively high in G1 than in Group 2 at four, six, and eight months. The signal-to-noise ratio from the MRI results showed majority having good healing in G1 group.

Based on 6-month MRI, an effective ligamentization (SNR<75) was noticed in 53.33% of patients in the dHACM allograft enhanced group on comparison with 33% in the controls. The overall results show that the augmentation of dHACM allograft to ACL reconstruction yields in good patient outcomes at post-operative follow-up.

Mesenchymal stem cells provide a valuable treatment option in orthopedic injuries in horses.

The aim of this study was to evaluate the hematological, biochemical, immunological and immunomodulatory parameters following intralesional treatment with tenogenic primed equine allogeneic peripheral blood-derived mesenchymal stem cells (tpMSCs) in client-owned horses with naturally occurring superficial digital flexor tendon (SDFT) and suspensory ligament (SL) injuries.

The immunogenicity and immunomodulatory capacities of tpMSCs were assessed in a modified mixed lymphocyte reaction, including peripheral blood mononuclear cells (PBMCs) of 14 horses with SDFT and SL injuries after treatment with tpMSCs. In a second study, 18 horses with SDFT and SL injuries received either an intralesional injection with tpMSCs (n = 9) or no treatment (n = 9).

The tpMSCs did not provoke a cellular immune response (p < 0.001) and were able to immunomodulate stimulated T lymphocytes (p < 0.001) in vitro. Therapeutic use of tpMSCs did not result in relevant hematologic or biochemical abnormalities.

Both studies had a small sample size. No statistical analyses were performed in the second study. Fibrinogen was only analyzed in a single horse prior to treatment.

Co-incubation of tpMSCs and PBMCs of horses that have been previously exposed to tpMSCs did not elicit a cellular immune response and tpMSCs were able to immunomodulate stimulated T lymphocytes. Intralesional treatment with tpMSCs did not provoke abnormal changes in hematological and biochemical parameters.

The desmoplastic bioarchitecture and microenvironment caused by fibroblasts have been confirmed to be closely related to the drug response behavior of pancreatic ductal adenocarcinoma (PDAC). Despite the extensive progress in developing PDAC models as in vitro drug screening platforms, developing efficient and controllable approaches for the construction of physiologically relevant models remains challenging. In the current study, multicellular spheroid models that emulate pancreatic cancer bioarchitecture and the desmoplastic microenvironment are bioengineered. An extrusion-based embedded dot bioprinting strategy was established to fabricate PDAC spheroids in a one-step process. Cell-laden hydrogel beads were directly deposited into a methacrylated gelatin (GelMA) suspension bath to generate spherical multicellular aggregates (SMAs), which further progressed into dense spheroids through in situ self assembly. By modulating the printing parameters, SMAs, even from multiple cell components, could be manipulated with tunable size and flexible location, achieving tunable spheroid patterns within the hydrogel bath with reproducible morphological features. To demonstrate the feasibility of this printing strategy, we fabricated desmoplastic PDAC spheroids by printing SMAs consisting of tumor cells and fibroblasts within the GelMA matrix bath. The produced hybrid spheroids were further exposed to different concentrations of the drug gemcitabine to verify their potential for use in cell therapy. Beyond providing a robust and facile bioprinting system that enables desmoplastic PDAC bioarchitecture bioengineering, this work introduces an approach for the scalable, flexible and rapid fabrication of cell spheroids or multi-cell-type spheroid patterns as platforms for advanced drug therapy or disease mechanism exploration.

Stem cell factors (SCFs) are pivotal factors existing in both soluble and membrane-bound forms, expressed by endothelial cells (ECs) and fibroblasts throughout the body. These factors enhance cell growth, viability, and migration in multipotent cell lineages. The preferential expression of SCF by arteriolar ECs indicates that arterioles create a unique microenvironment tailored to hematopoietic stem cells (HSCs). Insufficiency of SCF within bone marrow (BM)-derived adipose tissue results in decreased their overall cellularity, affecting HSCs and their immediate progenitors critical for generating diverse blood cells and maintaining the hematopoietic microenvironment. SCF deficiency disrupts BM function, impacting the production and differentiation of HSCs. Additionally, deleting SCF from adipocytes reduces lipogenesis, highlighting the crucial role of SCF/c-kit signaling in controlling lipid accumulation. This review elucidates the sources, roles, mechanisms, and molecular strategies of SCF in bone renewal, offering a comprehensive overview of recent advancements, challenges, and future directions for leveraging SCF as a key agent in regenerative medicine.

Erectile dysfunction (ED) is a common condition that negatively affects men's quality of life. It can have various causes, including psychological, vascular, and neurologic factors. Existing treatments for ED mainly focus on symptom relief rather than addressing the underlying cause. Stem cells (SCs) have shown potential as a therapeutic approach for ED due to their anti-inflammatory properties.

This systematic review aims to assess the current status of trials and determine the potential impact of SCs on male sexual health.

A comprehensive search strategy was employed to gather relevant articles from 6 electronic databases. The search included articles published until March 2023. The reference lists of articles were manually reviewed to identify additional studies of relevance. The eligibility criteria for inclusion in the analysis focused on clinical trials involving humans that evaluated the safety and efficacy of SC therapy for ED. Exclusion criteria encompassed case reports, case series, abstracts, reviews, and editorials, as well as studies involving animals or SC derivatives. Data extraction was performed via a standardized form with a focus on erectile outcomes.

A total of 2847 articles were initially identified; 18 were included in the final analysis. These studies involved 373 patients with ED and various underlying medical conditions. Multiple types of SC were utilized in the treatment of ED: mesenchymal SCs, placental matrix-derived mesenchymal SCs, mesenchymal SC-derived exosomes, adipose-derived SCs, bone marrow-derived mononuclear SCs, and umbilical cord blood SCs.

SC therapy shows promise as an innovative and safe treatment for organic ED. However, the lack of standardized techniques and controlled groups in many studies hampers the ability to evaluate and compare trials.

Introduction: Mesenchymal stromal cells (MSCs) have demonstrated therapeutic properties both in vitro and in vivo to treat various diseases, including anti-inflammatory, immunomodulatory and pro-angiogenic effects. These therapeutic effects are mediated by their secretome composed of soluble factors and extracellular vesicles (EVs). The composition of EVs reflects the molecular and functional characteristics of parental cells. MSC preconditioning can alter the composition of EVs, thereby influencing their therapeutic potential. Methods: MSCs were subjected to preconditioning with two cytokines, TNFα and IFNγ. Following 24 h of preconditioning, MSC-EVs secreted into the culture supernatant were isolated through tangential filtration. Particle concentration and size distribution were measured by nanoparticle tracking analysis, and the surface antigen expression of the EV-specific CD63 was quantified via Enzyme Linked ImmunoSorbent Assay. The angiogenic potential of MSCEVs obtained after preconditioning MSCs was assessed by the analysis of their protein composition and their influence on human umbilical vein endothelial cell (HUVECs) proliferation, migration, and tube-forming ability. Results: Preconditioning with TNFα and IFNγ did not influence the MSC-EV profile but did induce changes in their protein content. Indeed, the expression of pro-angiogenic proteins increased in EVs from preconditioned MSCs compared to EVs from no-preconditioned MSCs. EVs from preconditioned MSCs tend to stimulate HUVEC migration, proliferation and tubeforming ability. These observations imply the presence of a pro-angiogenic potential in EVs obtained after preconditioning of MSCs with TNFα and IFNγ. Discussion: In conclusion, it appears that the pro-angiogenic potential of EVs is enhanced through preconditioning of MSCs with TNFα and IFNγ. The use of these MSCs-EVs in therapy would circumvent the limitations of current cell-based therapies. Indeed, the therapeutic potential of MSC-EVs presents an attractive strategy for exploiting the clinical benefits of MSC therapy. For example, in the field of regenerative medicine, the exploitation of cell-free therapy using highly pro-angiogenic MSC-EVs is of great interest.

Understanding and addressing post-radical prostatectomy (RP) erectile dysfunction (ED) is of paramount importance for clinicians. Cavernous nerve (CN) injury rat model studies have provided consistently promising experimental data regarding regaining erectile function (EF) after nerve damage-induced ED. However, these findings have failed to translate efficiently into clinical practice, with post-RP ED therapeutic management remaining cumbersome and enigmatic. This disparity highlights the need for further standardization and optimization of the elaborate surgical preparation protocols and multifaceted reporting parameters involved in reliable CN injury rat model experimentation. Even so, despite its technical complexity, this animal model remains instrumental in exploring the functional implications of RP, i.e., surgical lesions of the neurovascular bundles (NVBs). Herein, besides cavernous nerve (CN) dissection, injury, and electrostimulation, multiple pressure measurements, i.e., mean arterial pressure (MAP) and intra-cavernosal pressure (ICP), must also be achieved. A transverse cervical incision allows for carotid artery cannulation and MAP measurements. Conversely, ICP measurements entail circumcising the penis, exposing the ischiocavernous muscle, and inserting a needle into the corporal body. Finally, using an abdominal incision, the prostate is revealed, and the major pelvic ganglia (MPG) and CNs are dissected bilaterally. Specific surgical techniques are used to induce CN injuries. Herein, we provide a narrative and illustrative overview regarding these complex experimental procedures and their particular requirements, reflecting on current evidence and future research perspectives.