Ana Catarina Sousa

Peripheral nerve injuries remain a major clinical problem, and cell-free therapies using stem cell-derived bioproducts have emerged as promising alternatives. This study evaluated the influence of neurogenic differentiation and passage number on the secretomic and exosomal profile of human dental pulp stem cells (hDPCSs). Conditioned media from undifferentiated and neurodifferentiated hDPSCs, and exosomes derived from undifferentiated hDPSCs at passages 4 and 7, were analyzed using multiplex immunoassays, RT-PCR, and scanning electron microscopy (SEM). Neurodifferentiated hDPSCs at early passages secreted higher levels of neurotrophic, angiogenic and immunomodulatory factors, including FGF-2, IL-6, IL-8, and PDGF-AA. Exosomes from early-passage undifferentiated cells showed a more abundant and relevant neuroregenerative mRNA cargo in comparison to the later passages. Both cell types and exosomes adhered to the Reaxon^® nerve guidance conduit, confirming the permissive nature of the materials regarding cells and cellular products, allowing adhesion and survival. Neurite outgrowth assays performed on neurodifferentiated hDPSCs confirmed functional neural behavior. In later passages, a decline in secretory and exosomal activity was noted. These results highlight the relevance of early-passage hDPSCs as a source of bioactive factors and support their application in cell-free approaches for peripheral nerve regeneration.

Peripheral nerve injuries remain a major clinical problem, and cell-free therapies using stem cell-derived bioproducts have emerged as promising alternatives. This study evaluated the influence of neurogenic differentiation and passage number on the secretomic and exosomal profile of human dental pulp stem cells. Conditioned media from undifferentiated and neurodifferentiated hDPSCs, and exosomes derived from undifferentiated hDPSCs at passages 4 and 7, were analyzed using multiplex immunoassays, RT-PCR, and scanning electron microscopy (SEM). Neurodifferentiated hDPSCs at early passages secreted higher levels of neurotrophic, angiogenic and immunomodulatory factors, including FGF-2, IL-6, IL-8, and PDGF-AA. Exosomes from early-passage undifferentiated cells showed a more abundant and relevant neuroregenerative mRNA cargo. Both cell types and exosomes adhered to the Reaxon^® nerve guidance conduit, confirming the permissive nature of the materials regarding cells and cellular products allowing adhesion and survival. Neurite outgrowth assays performed on neurodifferentiated hDPSCs confirmed functional neural behavior. At later passages, a decline in secretory and exosomal activity was noted. These results highlight the relevance of early-passage hDPSCs as a source of bioactive factors and support their application in cell-free approaches for peripheral nerve regeneration.

Stem cell-derived secretome and exosomes present a promising cell-free strategy for tissue repair and wound healing. This study aimed to isolate and characterize, for the first time, exosomes derived from rat hair follicle stem cells (rHFSCs) and to evaluate their wound-healing potential alongside rHFSC secretome. Exosomes were isolated via ultracentrifugation and characterized using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), biomarker profiling and protein quantification. Scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS) confirmed their spherical morphology, diameter and elemental composition. Protein quantification showed higher protein content in the secretome than in exosomes. RT-PCR and biomarker profiling highlighted the therapeutic relevance of the exosomal cargo compared to parent rHFSCs. Functional analysis of 30 wound-healing biomolecules validated their pro-regenerative potential. Cytocompatibility was confirmed via the PrestoBlue™ viability assay, while scratch assays demonstrated significant wound closure in the treated groups, both with and without mitomycin C. These findings highlight the potential of rHFSC-derived exosomes and secretome as innovative, cell-free therapeutic agents for cutaneous regeneration. This study advances our understanding of their role in wound healing and underscores their broader applicability in regenerative medicine.

Background: Stem cell-derived secretome and exosomes present a promising cell-free strategy for tissue repair and wound healing. This study aimed to isolate and characterize, for the first time, exosomes derived from rat hair follicle stem cells (rHFSCs) and to evaluate their wound-healing potential alongside rHFSC secretome. Methods and Results: Exosomes were isolated via ultracentrifugation and characterized using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), biomarker profiling and protein quantification. Scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS) confirmed their spherical morphology, diameter and elemental composition. Protein quantification showed higher protein content in the secretome than in exosomes. RT-PCR and biomarker profiling highlighted the therapeutic relevance of the exosomal cargo compared to parent rHFSCs. Functional analysis of 30 wound-healing biomolecules validated their pro-regenerative potential. Cytocompatibility was confirmed via the PrestoBlue™ viability assay, while scratch assays demonstrated significant wound closure in treated groups, both with and without mitomycin C. Conclusions: These findings highlight the potential of rHFSC-derived exosomes and secretome as innovative, cell-free therapeutic agents for cutaneous regeneration. This study advances our understanding of their role in wound healing and underscores their broader applicability in regenerative medicine.

Axonotmesis, a common peripheral nerve injury in humans and animals, leads to significant biomechanical and physiological consequences. The lack of a standardized crushing protocol for complex animal models limits research and therapeutic translations for humans and clinically relevant animal species. This study aimed to assess the impact of different crushing forces on the biomechanical behavior of the sheep common peroneal nerve and to establish a force for standardized in vivo protocols. Fourteen nerves of equal length were harvested and preserved and their initial diameter measured. They were subjected to crushing forces of 0 N, 80 N, and 180 N for one minute. Post crushing, the diameter, ultimate tensile strength, displacement at rupture, stress, strain, and stiffness were evaluated. Results showed that increasing crushing forces significantly affected nerve biomechanical parameters. Nerves crushed with 180N displayed lower tensile strength, displacement, and stiffness but higher stress and strain, indicating greater physical damage and structural degradation. These findings suggest that 180N induces substantial nerve fiber rupture and disruption of nerve trunk support elements, making it a candidate force for an axonotmesis protocol in the ovine model. Future in vivo studies should validate its effectiveness in creating complete crush injuries with functional and histological consequences, facilitating protocol standardization and translational research.

Background/Objectives: Chronic wounds pose a significant health concern, with their prevalence increasing due to various etiologies. The global aging population further contributes to this rise, placing a substantial burden on healthcare systems in developed countries. This work aimed to develop new therapeutic options in the form of creams and dressings based on honey, gellan gum, and hyaluronic acid for preventing and treating chronic wounds across all stages. Methods: To address this, after the formulations were developed, in vitro cytocompatibility was determined. To confirm biocompatibility, an ovine wound model was used: full-thickness excisional wounds were treated with three formulations, namely gellan gum and honey sponges (GG-HNY), gellan gum, honey and hyaluronic acid sponges (GG-HA-HNY) and a honey-based cream (cream FB002). Daily assessments, including visual evaluation and wound scoring, were conducted for 30 days. Following the study period, tissues were collected for histological analyses. Results: The macroscopic examination revealed that all therapeutic groups facilitated lesion closure. Lesion size reduction, granulation tissue disappearance, and scar tissue development were consistent across all groups, with the group receiving cream demonstrating an advanced stage of healing. All groups achieved substantial wound closure by day 30, with no significant differences. Histopathological analysis following ISO standards revealed that GG-HA-HNY had the lowest ISO score, indicating minimal reactivity and inflammation, which corroborated the cytocompatibility. Conclusions: Combining these insights with previous findings enhances our understanding of wound regeneration dynamics and contributes to refining therapeutic strategies for chronic wounds. The formulations were designed to balance therapeutic efficacy with cost-effectiveness, leveraging low-cost raw materials and straightforward production methods.

Peripheral nerve injuries remain a significant clinical challenge, particularly in severe neurotmesis injuries requiring complex therapeutic interventions to restore functionality. This study evaluated the pro-regenerative potential of combining neural guide conduits with secretome from olfactory mucosa mesenchymal stem cells, compared to gold-standard surgical techniques, in a validated ovine model of common peroneal nerve injury. Recovery was assessed over 24 weeks using functional, kinematic, ultrasonographic, and electrophysiological evaluations, complemented by post-mortem nerve stereology and muscle histomorphometry. All therapeutic approaches promoted nerve and muscle regeneration, resulting in notable functional and structural improvements. However, irregularities were observed, as neural guide conduits and secretome did not consistently outperform standard techniques. Additionally, recovery often fell short of normal values in the control group, highlighting the complexity of peripheral nerve regeneration in challenging surgical scenarios. These findings underscore the translational potential of biomaterials and cell secretome-based therapies while emphasizing the need for further research in complex animal models before its application in real clinical cases. Such studies are essential to refine therapeutic strategies, address observed irregularities and establish cell secretome as a viable tool in peripheral nerve regeneration and repair.

Axonotmesis, a common peripheral nerve injury in humans and animals, leads to significant biomechanical and physiological consequences. The lack of a standardized crushing protocol for complex animal models limits research and therapeutic translations for humans and clinically relevant animal species. This study aimed to assess the impact of different crushing forces on the biomechanical behavior of the sheep common peroneal nerve and to establish a force for standardized in vivo protocols. Fourteen nerves of equal length were harvested, preserved, and their initial diameter measured. They were subjected to crushing forces of 0N, 80N, and 180N for one minute. Post-crushing, diameter, ultimate tensile strength, displacement at rupture, stress, strain, and stiffness were evaluated. Results showed that increasing crushing forces significantly affected nerve biomechanical parameters. Nerves crushed with 180N displayed lower tensile strength, displacement, and stiffness, but higher stress and strain, indicating greater physical damage and structural degradation. These findings suggest that 180N induces substantial nerve fiber rupture and disruption of nerve trunk support elements, making it a candidate force for an axonotmesis protocol in the ovine model. Future in vivo studies should validate its effectiveness in creating complete crush injuries with functional and histological consequences, facilitating protocol standardization and translational research.

Chronic wounds pose a significant health concern and their prevalence, stemming from different etiologies, is on the rise due to the global aging population, posing a substantial burden on healthcare systems in developed countries. This work aimed to develop and formulate new therapeutic options in the form of creams and dressings based on honey, gellan gum and hyaluronic acid, for preventing and treating chronic wounds across all stages. To address this, after the formulations were developed, in vitro cytocompatibility was determined. To confirm biocompatibility, a ovine wound model was used: full-thickness excisional wounds were treated with 3 formulations, namely gellan gum and honey sponges (GG-HNY), gellan gum, honey and hyaluronic acid sponges (GG-HA-HNY) and a honey-based cream (cream FB002). Daily assessments, including visual evaluation, and wound scoring, were conducted for 30 days. Following the study period, tissues were collected for histological analyses. The macroscopic examination revealed that all therapeutic groups facilitated lesion closure. Lesion size reduction, granulation tissue disappearance, and scar tissue development were consistent across all groups, with the group receiving cream demonstrating an advanced stage of healing. All groups achieved substantial wound closure by day 30, with no significant differences among them. Histopathological analysis following ISO standards revealed that GG-HA-HNY had the lowest ISO score, indicating minimal reactivity and inflammation, corroborated the in vitro cytocompatibility. Combining these insights with previous findings enhances our understanding of wound regeneration dynamics and contributes to refining therapeutic strategies for chronic wounds. The resultant formulations aim to outperform existing medical devices, delivering superior performance at a lower overall unit cost.

The application of three-dimensional (3D) printing/bioprinting technologies and cell therapies has garnered significant attention due to their potential in the field of regenerative medicine. This paper aims to provide a comprehensive overview of 3D printing/bioprinting technology and cell therapies, highlighting their results in diverse medical applications, while also discussing the capabilities and limitations of their combined use. The synergistic combination of 3D printing and cellular therapies has been recognised as a promising and innovative approach, and it is expected that these technologies will progressively assume a crucial role in the treatment of various diseases and conditions in the foreseeable future. This review concludes with a forward-looking perspective on the future impact of these technologies, highlighting their potential to revolutionize regenerative medicine through enhanced tissue repair and organ replacement strategies.