DSpace Repository :: Browsing by Author "Schuh, Christina"

Browsing by Author "Schuh, Christina"

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Aloe vera peel-derived nanovesicles display anti-inflammatory properties and prevent myofibroblast differentiation
(2024) Ramírez, Orlando; Pomareda, Florencia; Olivares, Belén; Huang, Ya-Lin; Zavala, Gabriela; Carrasco, Javiera; Álvarez, Simón; Leiva, Camila; Hidalgo, Valeria; Romo, Pablo; Sánchez, Matías; Vargas, Ayleen; Martínez, Jessica; Aguayo, Sebastian; Schuh, Christina
Background: Aloe vera (AV) is a medicinal plant, most known for its beneficial effects on a variety of skin conditions. Its known active compounds include carbohydrates and flavonoids such as quercetin and kaempferol, among others. In the past decade, plant nanovesicles (NVs) have gained considerable interest as interkingdom communicators, presenting an opportunity for clinical standardization of natural products. In this study, we aimed to assess the potential of AVpNVs for the treatment of burn wounds. Methods: AVpNVs were isolated and characterized regarding vesicle yield (nanoparticle tracking analysis) and structure (transmission electron microscopy and atomic force microscopy), as well as their protein content with proteomics. We assessed key characteristics for treating burn wounds in vitro, such as the anti-inflammatory potential in LPS-stimulated macrophages and keratinocytes, and the effect of AVpNVs on myofibroblast differentiation and contraction. Key findings: AVpNVs presented a homogenous NV population, vesicular shape, and NV-associated protein markers. AVpNVs significantly decreased the secretion of pro-inflammatory cytokines TNFα, IL-1β, and IL-6. Furthermore, AVpNVs inhibited myofibroblast differentiation and significantly decreased their contractile potential in collagen matrices. Observed effects were linked to proteins identified in the isolates through proteomics analysis. Conclusion: AVpNVs displayed characteristics as an inflammatory modulator, while simultaneously diminishing myofibroblast differentiation and contraction. Novel strategies for burn wound treatment seek to decrease scarring on a cellular and molecular level in the early stages of wound healing, which makes AVpNVs a promising candidate for future plant-vesicle-based treatments.
An Optimized Collagen-Fibrin Blend Engineered Neural Tissue Promotes Peripheral Nerve Repair
(2018) Schuh, Christina; Day, Adam G.E.; Redl, Heinz; Phillips, James
Tissue engineering approaches in nerve regeneration often aim to improve results by bridging nerve defects with conduits that mimic key features of the nerve autograft. One such approach uses Schwann cell self-alignment and stabilization within collagen gels to generate engineered neural tissue (EngNT). In this study, we investigated whether a novel blend of fibrin and collagen could be used to form EngNT, as before EngNT design a beneficial effect of fibrin on Schwann cell proliferation was observed. A range of blend formulations was tested in terms of mechanical behavior (gel formation, stabilization, swelling, tensile strength, and stiffness), and lead formulations were assessed in vitro. A 90% collagen 10% fibrin blend was found to promote SCL4.1/F7 Schwann cell viability and supported the formation of aligned EngNT, which enhanced neurite outgrowth in vitro (NG108 cells) compared to formulations with higher and lower fibrin content. Initial in vivo tests in an 8 mm rat sciatic nerve model using rolled collagen-fibrin EngNT rods revealed a significantly enhanced axonal count in the midsection of the repair, as well as in the distal part of the nerve after 4 weeks. This optimized collagen-fibrin blend therefore provides a novel way to improve the capacity of EngNT to promote regeneration following peripheral nerve injury.
Antibacterial Effect of Honey-Derived Exosomes Containing Antimicrobial Peptides Against Oral Streptococci
(2021) Leiva-Sabadini, Camila; Alvarez, Simon; Barrera, Nelson P.; Schuh, Christina; Aguayo, Sebastian
Purpose: Recently, our group found exosome-like extracellular vesicles (EVs) in Apis mellifera honey displaying strong antibacterial effects; however, the underlying mechanism is still not understood. Thus, the aim of this investigation was to characterize the molecular and nanomechanical properties of A. mellifera honey-derived EVs in order to elucidate the mechanisms behind their antibacterial effect, as well as to determine differential antibiofilm properties against relevant oral streptococci. Methods: A. mellifera honey-derived EVs (HEc-EVs) isolated via ultracentrifugation were characterized with Western Blot and ELISA to determine the presence of specific exosomal markers and antibacterial cargo, and atomic force microscopy (AFM) was utilized to explore their ultrastructural and nanomechanical properties via non-destructive immobilization onto poly-L-lysine substrates. Furthermore, the effect of HEc-EVs on growth and biofilm inhibition of S. mutans was explored with microplate assays and compared to S. sanguinis. AFM was utilized to describe ultrastructural and nanomechanical alterations such as cell wall elasticity changes following HEc-EV exposure. Results: Molecular characterization of HEc-EVs identified for the first time important conserved exosome markers such as CD63 and syntenin, and the antibacterial molecules MRJP1, defensin-1 and jellein-3 were found as intravesicular cargo. Nanomechanical characterization revealed that honey-derived EVs were mostly <150nm, with elastic modulus values in the low MPa range, comparable to EVs from other biological sources. Furthermore, incubating oral streptococci with EVs confirmed their antibacterial and antibiofilm capacities, displaying an increased effect on S. mutans compared to S. sanguinis. AFM nanocharacterization showed topographical and nanomechanical alterations consistent with membrane damage on S. mutans. Conclusion: Honey is a promising new source of highly active EVs with exosomal origin, containing a number of antibacterial peptides as cargo molecules. Furthermore, the differential effect of HEC-EVs on S. mutans and S. sanguinis may serve as a novel biofilm-modulating strategy in dental caries.
Bacterial adhesion to collagens: implications for biofilm formation and disease progression in the oral cavity
(2022) Álvarez, Simón; Leiva, Camila; Schuh, Christina; Aguayo, Sebastián
Collagen is the most abundant structural protein in the body and the main component of the extracellular matrix of most tissues, including dentine and periodontal tissues. Despite the well-characterized role of collagen and specifically type-I collagen, as a ligand for host cells, its role as a substrate for bacterial adhesion and biofilm formation is less explored. Therefore, the purpose of this review is to discuss recent findings regarding the adhesion of oral bacteria to collagen surfaces and its role in the progression and severity of oral and systemic diseases. Initial oral colonizers such as streptococci have evolved collagen-binding proteins (cbp) that are important for the colonization of dentine and periodontal tissues. Also, periodontal pathogens such as Porphyromonas gingivalis and Tannerella forsythia utilise cbps for tissue sensing and subsequent invasion. The implications of bacteria-collagen coupling in the context of collagen biomaterials and regenerative dentistry approaches are also addressed. Furthermore, the importance of interdisciplinary techniques such as atomic force microscopy for the nanocharacterization of bacteria-collagen interactions is also considered. Overall, understanding the process of oral bacterial adhesion onto collagen is important for developing future therapeutic approaches against oral and systemic diseases, by modulating the early stages of biofilm formation.
Exosome-like vesicles in Apis mellifera bee pollen, honey and royal jelly contribute to their antibacterial and pro-regenerative activity
(2019) Schuh, Christina; Aguayo, Sebastian; Zavala, Gabriela; Khoury, Maroun
Microvesicles are key players in cellular communication. As glandular secretions present a rich source of active exosomes, we hypothesized that exosome-like vesicles are present in Apis mellifera hypopharyngeal gland secretomal products (honey, royal jelly and bee pollen), and participate in their known antibacterial and pro-regenerative effects. We developed an isolation protocol based on serial centrifugation and ultracentrifugation steps and demonstrated the presence of protein-containing exosome-like vesicles in all three bee-derived products. Assessing their antibacterial properties, we found that exosome-like vesicles had bacteriostatic, bactericidal and biofilm-inhibiting effects on Staphylococcus aureus. Furthermore, we demonstrated that mesenchymal stem cells (MSCs) internalize bee-derived exosome- like vesicles and that these vesicles influence the migration potential of the MSCs. In an in vitro wound-healing assay, honey and royal jelly exosome-like vesicles increased migration of human MSCs, demonstrating their inter-kingdom activity. In summary, we have discovered exosome-like vesicles as a new, active compound in bee pollen, honey and royal jelly.
Exosomes on the border of species and kingdom intercommunication
(2019-08) Schuh, Christina; Cuenca, Jimena; Alcayaga-Miranda, Francisca; Khoury, Maroun
Over the last decades exosomes have become increasingly popular in the field of medicine. While until recently they were believed to be involved in the removal of obsolete particles from the cell, it is now known that exosomes are key players in cellular communication, carrying source-specific molecules such as proteins, growth factors, miRNA/mRNA, among others. The discovery that exosomes are not bound to intraspecies interactions, but are also capable of interkingdom communication, has once again revolutionized the field of exosomes research. A rapidly growing body of literature is shedding light at novel sources and participation of exosomes in physiological or regenerative processes, infection and disease. For the purpose of this review we have categorized 6 sources of interest (animal products, body fluids, plants, bacteria, fungus and parasites) and linked their innate roles to the clinics and potential medical applications, such as cell-based therapy, diagnostics or drug delivery.
Extrusomas de células natural killer: Vesículas artificiales derivadas de células como una alternativa innovadora de encapsulamiento de fármacos para el cáncer de pulmón
(Universidad del Desarrollo. Facultad de Medicina, 2024) Carrasco Rojas, Javiera Valentina; Schuh, Christina
El cáncer de pulmón (CP) tiene la tasa de mortalidad más alta a nivel mundial. Su patogénesis es multifactorial y, aunque se recomiendan terapias dirigidas, muchos pacientes deben depender de tratamientos clásicos (por ejemplo, quimioterapia), los cuales presentan limitaciones (efectos adversos, resistencias al tratamiento, entre otros). Los extrusomas (EXT), o vesículas derivadas artificialmente de células (ACDVs), han surgido como una herramienta prometedora para superar las barreras de la traslación hacia la clínica que presentan actualmente los tratamientos acelulares basados en vesículas. Este estudio propone la generación de EXTs encapsulados con docetaxel (DTX) a partir de células Natural Killer (NK) humanas (DTX-EXTs) y evaluar su internalización en células de CP y su efecto citotóxico. También se realizaron análisis preliminares utilizando macrófagos polarizados a fenotipo M1 (EXTs-M1) para validar este método como una estrategia prometedora en la producción de ACDVs para la liberación de fármacos en el tratamiento del CP. Los EXTs se generaron por extrusión celular y se evaluaron su morfología, estabilidad y composición. Se midió su efecto citotóxico en células de cáncer de pulmón, y se investigaron los mecanismos de captación celular mediante inhibidores de diferentes vías de endocitosis. Los EXTs-NK y EXTs-M1 mostraron una morfología en forma de copa (tamaño medio <200 nm), los EXTs-NK tuvieron composición estable y su análisis Proteómico reveló proteínas presentes de manera diferencial asociadas con las características de las vesículas extracelulares (EV) y los marcadores de células NK, además de un patrón de enriquecimiento proteico distintivo. Se observó un efecto citotóxico significativo con los tratamientos EXT-DTX en comparación con los EXTs y EVs en ambas líneas celulares, con mayor eficacia en las células A549; la inducción de apoptosis corroboró estos hallazgos. Los estudios de internalización identificaron una localización perinuclear y la participación de la endocitosis mediada por clatrina como mecanismo principal en todos los grupos de vesículas. Similar fueron los hallazgos obtenidos para los EXTs-M1, en donde los DTX EXTs-M1 fueron más eficaces en la reducción de la viabilidad celular en las líneas de CP H1975 y H1299. Asimismo, se identificó preliminarmente, la endocitosis mediada por clatrina como principal mecanismo de internalización en células H1975. En conjunto, los resultados de este estudio sugieren que el uso de EXTs derivados de células inmunes, ofrece un enfoque prometedor para mejorar la entrega de quimioterapéuticos como el DTX, permitiendo reducir las dosis necesarias y, en consecuencia, minimizar los efectos adversos, lo que podría traducirse en una mejora en la calidad de vida de los pacientes con CP.
Modulation of the biophysical and biochemical properties of collagen by glycation for tissue engineering applications
(2022) Vaez, Mina; Asgari, Meisam; Hirvonenc, Liisa; Bakir, Gorkem; Khattignavong, Emilie; Ezzo, Maya; Aguayo, Sebastian; Schuh, Christina; Gough, Kathleen; Bozec, Laurent
The structural and functional properties of collagen are modulated by the presence of intramolecular and intermolecular crosslinks. Advanced Glycation End-products (AGEs) can produce intermolecular crosslinks by bonding the free amino groups of neighbouring proteins. In this research, the following hypothesis is explored: The accumulation of AGEs in collagen decreases its proteolytic degradation rates while increasing its stiffness. Fluorescence Lifetime Imaging (FLIM) and Fourier-transform infrared spectroscopy (FTIR) detect biochemical changes in collagen scaffolds during the glycation process. The accumulation of AGEs increases exponentially in the collagen scaffolds as a function of Methylglyoxal (MGO) concentration by performing autofluorescence measurement and competitive ELISA. Glycated scaffolds absorb water at a much higher rate confirming the direct affinity between AGEs and interstitial water within collagen fibrils. In addition, the topology of collagen fibrils as observed by Atomic Force Microscopy (AFM) is a lot more defined following glycation. The elastic modulus of collagen fibrils decreases as a function of glycation, whereas the elastic modulus of collagen scaffolds increases. Finally, the enzymatic degradation of collagen by bacterial collagenase shows a sigmoidal pattern with a much slower degradation rate in the glycated scaffolds. This study identifies unique variations in the properties of collagen following the accumulation of AGEs. STATEMENT OF SIGNIFICANCE: In humans, Advanced Glycation End-products (AGEs) are naturally produced as a result of aging process. There is an evident lack of knowledge in the basic science literature explaining the biomechanical impact of AGE-mediated crosslinks on the functional and structural properties of collagen at both the nanoscale (single fibrils) and mesoscale (bundles of fibrils). This research, demonstrates how it is possible to harness this natural phenomenon in vitro to enhance the properties of engineered collagen fibrils and scaffolds. This study identifies unique variations in the properties of collagen at nanoscale and mesoscale following accumulation of AGEs. In their approach, they investigate the unique properties conferred to collagen, namely enhanced water sorption, differential elastic modulus, and finally sigmoidal proteolytic degradation behavior.
Modulatory Effect of Glycated Collagen on Oral Streptococcal Nanoadhesion
(2021) Benso, B.; Naulin, P.A.; Barrera, N.P.; Bozec, L.; Aguayo, S.; Schuh, Christina
Biofilm-mediated oral diseases such as dental caries and periodontal disease remain highly prevalent in populations worldwide. Biofilm formation initiates with the attachment of primary colonizers onto surfaces, and in the context of caries, the adhesion of oral streptococcito dentinal collagen is crucial for biofilm progression. It is known that dentinal collagen suffers from glucose-associated crosslinking as a function of aging or disease; however, the effect of collagen crosslinking on the early adhesion and subsequent biofilm formation of relevant oral streptococci remains unknown. Therefore, the aim of this work was to determine the impact of collagen glycation on the initial adhesion of primary colonizers such as Streptococcus mutans UA159 and Streptococcus sanguinis SK 36, as well as its effect on the early stages of streptococcal biofilm formation in vitro. Type I collagen matrices were crosslinked with either glucose or methylglyoxal. Atomic force microscopy nanocharacterization revealed morphologic and mechanical changes within the collagen matrix as a function of crosslinking, such as a significantly increased elastic modulus in crosslinked fibrils. Increased nanoadhesion forces were observed for S. mutans on crosslinked collagen surfaces as compared with the control, and retraction curves obtained for both streptococcal strains demonstrated nanoscale unbinding behavior consistent with bacterial adhesin-substrate coupling. Overall, glucose-crosslinked substrates specifically promoted the initial adhesion, biofilm formation, and insoluble extracellular polysaccharide production of S. mutans, while methylglyoxal treatment reduced biofilm formation for both strains. Changes in the adhesion behavior and biofilm formation of oral streptococci as a function of collagen glycation could help explain the biofilm dysbiosis seen in older people and patients with diabetes. Further studies are necessary to determine the influence of collagen crosslinking on the balance between acidogenic and nonacidogenic streptococci to aid in the development of novel preventive and therapeutic treatment against dental caries in these patients.
Motor and sensory Schwann cell phenotype commitment is diminished by extracorporeal shockwave treatment in vitro
(2020) Hercher, David; Redl, Heinz; Schuh, Christina
The gold standard for peripheral nerve regeneration uses a sensory autograft to bridge a motor/sensory defect site. For motor nerves to regenerate, Schwann cells (SC) myelinate the newly grown axon. Sensory SCs have a reduced ability to produce myelin, partially explaining low success rates of autografts. This issue is masked in pre-clinical research by the excessive use of the rat sciatic nerve defect model, utilizing a mixed nerve with motor and sensory SCs. Aim of this study was to utilize extracorporeal shockwave treatment as a novel tool to influence SC phenotype. SCs were isolated from motor, sensory and mixed rat nerves and in vitro differences between them were assessed concerning initial cell number, proliferation rate, neurite outgrowth as well as ability to express myelin. We verified the inferior capacity of sensory SCs to promote neurite outgrowth and express myelin-associated proteins. Motor Schwann cells demonstrated low proliferation rates, but strongly reacted to pro-myelination stimuli. It is noteworthy for pre-clinical research that sciatic SCs are a strongly mixed culture, not representing one or the other. Extracorporeal shockwave treatment (ESWT), induced in motor SCs an increased proliferation profile, while sensory SCs gained the ability to promote neurite outgrowth and express myelin-associated markers. We demonstrate a strong phenotype commitment of sciatic, motor, and sensory SCs in vitro, proposing the experimental use of SCs from pure cultures to better mimic clinical situations. Furthermore we provide arguments for using ESWT on autografts to improve the regenerative capacity of sensory SCs
Nanomechanical and Molecular Characterization of Aging in Dentinal Collagen
(2022) Schuh, Christina; Leiva, Camila; Huang, Sui; Barrera, Nelson; Aguayo, Sebastian
Methylglyoxal (MGO) is an important molecule derived from glucose metabolism with the capacity of attaching to collagen and generating advanced glycation end products (AGEs), which accumulate in tissues over time and are associated with aging and diseases. However, the accumulation of MGO-derived AGEs in dentin and their effect on the nanomechanical properties of dentinal collagen remain unknown. Thus, the aim of the present study was to quantify MGO-based AGEs in the organic matrix of human dentin as a function of age and associate these changes with alterations in the nanomechanical and ultrastructural properties of dentinal collagen. For this, 12 healthy teeth from <26-y-old and >50-y-old patients were collected and prepared to obtain crown and root dentin discs. Following demineralization, MGO-derived AGEs were quantified with a competitive ELISA. In addition, atomic force microscopy nanoindentation was utilized to measure changes in elastic modulus in peritubular and intertubular collagen fibrils. Finally, principal component analysis was carried out to determine aging profiles for crown and root dentin. Results showed an increased presence of MGO AGEs in the organic matrix of dentin in the >50-y-old specimens as compared with the <26-y-old specimens in crown and root. Furthermore, an increase in peritubular and intertubular collagen elasticity was observed in the >50-y-old group associated with ultrastructural changes in the organic matrix as determined by atomic force microscopy analysis. Furthermore, principal component analysis loading plots suggested different "aging profiles" in crown and root dentin, which could have important therapeutic implications in restorative and adhesive dentistry approaches. Overall, these results demonstrate that the organic matrix of human dentin undergoes aging-related changes due to MGO-derived AGEs with important changes in the nanomechanical behavior of collagen that may affect diagnostic and restorative procedures in older people.
Nanoscale Dynamics of Streptococcal Adhesion to AGE-Modified Collagen
(2023) Leiva, C.; Tiozzo, P.; Hidalgo, L.; Rivas Jiménez, Lina María; Robles, A.; Fierro, A.; Barrera, N.; Bozec, L.; Schuh, Christina; Aguayo. S.
The adhesion of initial colonizers such as Streptococcus mutans to collagen is critical for dentinal and root caries progression. One of the most described pathological and aging-associated changes in collagen-including dentinal collagen-is the generation of advanced glycation end-products (AGEs) such as methylglyoxal (MGO)-derived AGEs. Despite previous reports suggesting that AGEs alter bacterial adhesion to collagen, the biophysics driving oral streptococcal attachment to MGO-modified collagen remains largely understudied. Thus, the aim of this work was to unravel the dynamics of the initial adhesion of S. mutans to type I collagen in the presence and absence of MGO-derived AGEs by employing bacterial cell force spectroscopy with atomic force microscopy (AFM). Type I collagen gels were treated with 10 mM MGO to induce AGE formation, which was characterized with microscopy and enzyme-linked immunosorbent assay. Subsequently, AFM cantilevers were functionalized with living S. mutans UA 159 or Streptococcus sanguinis SK 36 cells and probed against collagen surfaces to obtain force curves displaying bacterial attachment in real time, from which the adhesion force, number of events, Poisson analysis, and contour and rupture lengths for each individual detachment event were computed. Furthermore, in silico computer simulation docking studies between the relevant S. mutans UA 159 collagen-binding protein SpaP and collagen were computed, in the presence and absence of MGO. Overall, results showed that MGO modification increased both the number and adhesion force of single-unbinding events between S. mutans and collagen, without altering the contour or rupture lengths. Both experimental and in silico simulations suggest that this effect is due to increased specific and nonspecific forces and interactions between S. mutans UA 159 and MGO-modified collagen substrates. In summary, these results suggest that collagen alterations due to aging and glycation may play a role in early bacterial adherence to oral tissues, associated with conditions such as aging or chronic hyperglycemia, among others.
Potential novel strategies for the treatment of dental pulp-derived pain: pharmacological approaches and beyond
(2019) Schuh, Christina; Benso, Bruna; Aguayo, Sebastian
The diagnosis and management of pain is an everyday occurrence in dentistry, and its effective control is essential to ensure the wellbeing of patients. Most tooth-associated pain originates from the dental pulp, a highly vascularized and innervated tissue, which is encased within mineralized dentin. It plays a crucial role in the sensing of stimuli from the local environment, such as infections (i.e. dental caries) and traumatic injury, leading to a local inflammatory response and subsequently to an increase in intra-pulp pressure, activating nerve endings. However, thermal, chemical, and mechanical stimuli also have the ability to generate dental pulp pain, which presents mechanisms highly specific to this tissue and which have to be considered in pain management. Traditionally, the management of dental pulp pain has mostly been pharmacological, using non-steroidal anti-inflammatory drugs (NSAIDs) and opioids, or restorative (i.e. removal of dental caries), or a combination of both. Both research areas continuously present novel and creative approaches. This includes the modulation of thermo-sensitive transient receptor potential cation channels (TRP) by newly designed drugs in pharmacological research, as well as the use of novel biomaterials, stem cells, exosomes and physical stimulation to obtain pulp regeneration in regenerative medicine. Therefore, the aim of this review is to present an up-to-date account of causes underlying dental pain, novel treatments involving the control of pain and inflammation and the induction of pulp regeneration, as well as insights in pain in dentistry from the physiological, pharmacological, regenerative and clinical perspectives
Royal jelly extracellular vesicles promote wound healing by modulating underlying cellular responses
(2023) Álvarez, Simón; Contreras, Pamina; Aguayo, Sebastian; Ramírez, Orlando; Vallejos, Catalina; Ruiz, Jorge; Carrasco, Eva; Troncoso, Stefanie; Morales, Bernardo; Schuh, Christina
Apis mellifera royal jelly (RJ) is a well-known remedy in traditional medicine around the world and its versatile effects range from antibacterial to anti-inflammatory properties and pro-regenerative properties. As a glandular product, RJ has been shown to contain a substantial number of extracellular vesicles (EVs), and, in this study, we aimed to investigate the extent of involvement of RJEVs in wound healing-associated effects. Molecular analysis of RJEVs verified the presence of exosomal markers such as CD63 and syntenin, and cargo molecules MRJP1, defensin-1, and jellein-3. Furthermore, RJEVs were demonstrated to modulate mesenchymal stem cell (MSC) differentiation and secretome, as well as decrease LPS-induced inflammation in macrophages by blocking the mitogen-activated protein kinase (MAPK) pathway. In vivo studies confirmed antibacterial effects of RJEVs and demonstrated an acceleration of wound healing in a splinted mouse model. This study suggests that RJEVs play a crucial role in the known effects of RJ by modulating the inflammatory phase and cellular response in wound healing. Transfer of RJ into the clinics has been impeded by the high complexity of the raw material. Isolating EVs from the raw RJ decreases the complexity while allowing standardization and quality control, bringing a natural nano-therapy one step closer to the clinics.
Spatiotemporal differences in gene expression between motor and sensory autografts and their effect on femoral nerve regeneration in the rat
(2019) Hercher, David; Kerbl, Markus; Schuh, Christina; Heinzel, Johannes; Gal, László; Stainer, Michaela; Schmidhammer, Robert; Hausner, Thomas; Redl, Heinz; Nógrádi, Antal; Hacobian, Ara
To improve the outcome after autologous nerve grafting in the clinic, it is important to understand the limiting variables such as distinct phenotypes of motor and sensory Schwann cells. This study investigated the properties of phenotypically different autografts in a 6 mm femoral nerve defect model in the rat, where the respective femoral branches distally of the inguinal bifurcation served as homotopic, or heterotopic autografts. Axonal regeneration and target reinnervation was analyzed by gait analysis, electrophysiology, and wet muscle mass analysis. We evaluated regeneration-associated gene expression between 5 days and 10 weeks after repair, in the autografts as well as the proximal, and distal segments of the femoral nerve using qRT-PCR. Furthermore we investigated expression patterns of phenotypically pure ventral and dorsal roots. We identified highly significant differences in gene expression of a variety of regeneration-associated genes along the central – peripheral axis in healthy femoral nerves. Phenotypically mismatched grafting resulted in altered spatiotemporal expression of neurotrophic factor BDNF, GDNF receptor GFRα1, cell adhesion molecules Cadm3, Cadm4, L1CAM, and proliferation associated Ki67. Although significantly higher quadriceps muscle mass following homotopic nerve grafting was measured, we did not observe differences in gait analysis, and electrophysiological parameters between treatment paradigms. Our study provides evidence for phenotypic commitment of autologous nerve grafts after injury and gives a conclusive overview of temporal expression of several important regeneration-associated genes after repair with sensory or motor graft.
The Reparative Abilities of Menstrual Stem Cells Modulate the Wound Matrix Signals and Improve Cutaneous Regeneration
(2018) Cuenca, Jimena; Le-Gatt, Alice; Castillo, Valentina; Belletti, José; Díaz, Macarena; Kurte, Mónica; González, Paz; Alcayaga, Francisca; Schuh, Christina; Ezquer, Fernando; Ezquer, Marcelo; Khoury, Maroun
Considerable advances have been made toward understanding the cellular and molecular mechanism of wound healing, however, treatments for chronic wounds remain elusive. Emerging concepts utilizing mesenchymal stem cells (MSCs) from umbilical cord, adipose tissue and bone marrow have shown therapeutical advantages for wound healing. Based on this positive outcome, efforts to determine the optimal sources for MSCs are required in order to improve their migratory, angiogenic, immunomodulatory, and reparative abilities. An alternative source suitable for repetitive, non-invasive collection of MSCs is from the menstrual fluid (MenSCs), displaying a major practical advantage over other sources. This study aims to compare the biological functions and the transcriptomic pattern of MenSCs with umbilical cord MSCs in conditions resembling the wound microenvironment. Consequently, we correlate the specific gene expression signature from MenSCs with changes of the wound matrix signals in vivo. The direct comparison revealed a superior clonogenic and migratory potential of MenSCs as well as a beneficial effect of their secretome on human dermal fibroblast migration in vitro. Furthermore, MenSCs showed increased immunomodulatory properties, inhibiting T-cell proliferation in co-culture. We further, investigated the expression of selected genes involved in wound repair (growth factors, cytokines, chemokines, AMPs, MMPs) and found considerably higher expression levels in MenSCs (ANGPT1 1.5-fold; PDGFA 1.8-fold; PDGFB 791-fold; MMP3 21.6-fold; ELN 13.4-fold; and MMP10 9.2-fold). This difference became more pronounced under a pro-inflammatory stimulation, resembling wound bed conditions. Locally applied in a murine excisional wound splinting model, MenSCs showed a significantly improved wound closure after 14 days, as well as enhanced neovascularization, compared to the untreated group. Interestingly, analysis of excised wound tissue revealed a significantly higher expression of VEGF (1.42-fold) among other factors, translating an important conversion of the matrix signals in the wound site. Furthermore, histological analysis of the wound tissue from MenSCs-treated group displayed a more mature robust vascular network and a genuinely higher collagen content confirming the pro-angiogenic and reparative effect of MenSCs treatment. In conclusion, the superior clonogenicity, immunosuppressive and migration potential in combination with specific paracrine signature of MenSCs, resulted in an enhanced wound healing and cutaneous regeneration process.
Type I collagen hydrogels as a delivery matrix for royal jelly derived extracellular vesicles
(2020) Ramírez, Orlando J.; Alvarez, Simón; Contreras-Kallens, Pamina; Barrera, Nelson P.; Aguayo, Sebastian; Schuh, Christina
Throughout the last decade, extracellular vesicles (EVs) have become increasingly popular in several areas of regenerative medicine. Recently, Apis mellifera royal jelly EVs (RJ EVs) were shown to display favorable wound healing properties such as stimulation of mesenchymal stem cell migration and inhibition of staphylococcal biofilms. However, the sustained and effective local delivery of EVs in nonsystemic approaches – such as patches for chronic cutaneous wounds – remains an important challenge for the development of novel EV-based wound healing therapies. Therefore, the present study aimed to assess the suitability of type I collagen -a well-established biomaterial for wound healing – as a continuous delivery matrix. RJ EVs were integrated into collagen gels at different concentrations, where gels containing 2mg/ml collagen were found to display the most stable release kinetics. Functionality of released RJ EVs was confirmed by assessing fibroblast EV uptake and migration in a wound healing assay. We could demonstrate reliable EV uptake into fibroblasts with a sustained promigratory effect for up to 7 d. Integrating fibroblasts into the RJ EV-containing collagen gel increased the contractile capacity of these cells, confirming availability of RJ EVs to fibroblasts within the collagen gel. Furthermore, EVs released from collagen gels were found to inhibit Staphylococcus aureus ATCC 29213 biofilm formation. Overall, our results suggest that type I collagen could be utilized as a reliable, reproducible release system to deliver functional RJ EVs for wound healing therapies.
Ultrastructural characterisation of young and aged dental enamel by atomic force microscopy
(2022) Leiva, Camila; Schuh, Christina; Barrera, Nelson; Aguayo, Sebastián
Recent advances in atomic force microscopy (AFM) have allowed the characterisation of dental-associated biomaterials and biological surfaces with high resolution. In this context, the topography of dental enamel - the hardest mineralised tissue in the body - has been explored with AFM-based approaches at the microscale. With age, teeth are known to suffer changes that can impact their structural stability and function; however, changes in enamel structure because of ageing have not yet been explored with nanoscale resolution. Therefore, the aim of this exploratory work was to optimise an approach to characterise the ultrastructure of dental enamel and determine potential differences in topography, hydroxyapatite (HA) crystal size, and surface roughness at the nanoscale associated to ageing. For this, a total of six teeth were collected from human donors from which enamel specimens were prepared. By employing intermittent contact (AC mode) imaging, HA crystals were characterised in both transversal and longitudinal orientation (respect to surface plane) with high resolution in environmental conditions. The external enamel surface displayed the presence of a pellicle-like coating on its surface that was not observable on cleaned specimens. Acid-etching exposed crystals that were imaged and morphologically characterised in high resolution at the nanoscale in both the external and internal regions of enamel in older and younger specimens. Our results demonstrated important individual variations in HA crystal width and roughness parameters across the analysed specimens; however, an increase in surface roughness and decrease in HA width was observed for the pooled older external enamel group compared to younger specimens. Overall, high-resolution AFM was an effective approach for the qualitative and quantitative characterisation of human dental enamel ultrastructure. Future work should focus on exploring the ageing of dental enamel with increased sample sizes to compensate for individual differences as well as other potential confounding factors such as behavioural habits and mechanical forces.

Browsing by Author "Schuh, Christina"

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