Chitosan conduits enriched with fibrin-collagen hydrogel with or without adipose-derived mesenchymal stem cells for the repair of 15-mm-long sciatic nerve defect

Hollow conduits of natural or synthetic origins have shown acceptable regeneration results in short nerve gap repair; however, results are still not comparable with the current gold standard technique “autografts”. Hollow conduits do not provide a successful regeneration outcome when it comes to critical nerve gap repair. Enriching the lumen of conduits with different extracellular materials and cells could provide a better biomimicry of the natural nerve regenerating environment and is expected to ameliorate the conduit performance. In this study, we evaluated nerve regeneration in vivo using hollow chitosan conduits or conduits enriched with fibrin-collagen hydrogels alone or with the further addition of adipose-derived mesenchymal stem cells in a 15 mm rat sciatic nerve transection model. Unexpected changes in the hydrogel consistency and structural stability in vivo led to a failure of nerve regeneration after 15 weeks. Nevertheless, the molecular assessment in the early regeneration phase (7, 14, and 28 days) has shown an upregulation of useful regenerative genes in hydrogel enriched conduits compared with the hollow ones. Hydrogels composed of fibrin-collagen were able to upregulate the expression of soluble NRG1, a growth factor that plays an important role in Schwann cell transdifferentiation. The further enrichment with adipose-derived mesenchymal stem cells has led to the upregulation of other important genes such as ErbB2, VEGF-A, BDNF, c-Jun, and ATF3.Spanish "Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica, Ministerio de Economia y Competitividad (Instituto de Salud Carlos III) FIS PI14-1343 FIS PI17-0393 FIS PI20-0318Fondo Europeo de Desarrollo Regional ERDF-FEDER European UnionPlan Andaluz de Investigacion, Desarrollo e Innovacion (PAIDI2020), Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, Junta de Andalucia, Espana P18-RT-5059Programa Operativo FEDER Andalucia 2014-2020, Universidad de Granada, Junta de Andalucia, Espana A-CTS-498-UGR18European Commissio

Generation of a novel human dermal substitute functionalized with antibiotic‑loaded nanostructured lipid carriers (NLCs) with antimicrobial properties for tissue engineering

Background: Treatment of patients affected by severe burns is challenging, especially due to the high risk of Pseudomonas infection. In the present work, we have generated a novel model of bioartificial human dermis substitute by tissue engineering to treat infected wounds using fibrin-agarose biomaterials functionalized with nanostructured lipid carriers (NLCs) loaded with two anti-Pseudomonas antibiotics: sodium colistimethate (SCM) and amikacin (AMK). Results: Results show that the novel tissue-like substitutes have strong antibacterial effect on Pseudomonas cultures, directly proportional to the NLC concentration. Free DNA quantification, WST-1 and Caspase 7 immunohistochemical assays in the functionalized dermis substitute demonstrated that neither cell viability nor cell proliferation were affected by functionalization in most study groups. Furthermore, immunohistochemistry for PCNA and KI67 and histochemistry for collagen and proteoglycans revealed that cells proliferated and were metabolically active in the functionalized tissue with no differences with controls. When functionalized tissues were biomechanically characterized, we found that NLCs were able to improve some of the major biomechanical properties of these artificial tissues, although this strongly depended on the type and concentration of NLCs. Conclusions: These results suggest that functionalization of fibrin-agarose human dermal substitutes with antibioticloaded NLCs is able to improve the antibacterial and biomechanical properties of these substitutes with no detectable side effects. This opens the door to future clinical use of functionalized tissues.NanoGSkin project of EuroNanoMed-III (ERA-NET Cofund scheme of the Horizon 2020 Research and Innovation Framework Programme), EUInstituto de Salud Carlos III AC17/00013Centro para el Desarrollo Tecnológico Industrial -CDTI 00108589Spanish GovernmentJunta de Andalucía PE-0395-2019Fundacion Benefica Anticancer San Francisco Javier y Santa Candida, Granada, SpainDepartment of Economic Development and Infrastructure of the Basque Government budget, through the HAZITEK business R + D support program ZE-2017/00014European Union (EU) OTRI.35A-0

A novel 3D biofabrication strategy to improve cell proliferation and differentiation of human Wharton’s jelly mesenchymal stromal cells for cell therapy and tissue engineering

Supported by grant PSETC-19-001 (Fibrigar3D) by Plan Propio de Investigación y Transferencia 2019, Universidad de Granada, Spain. Supported by Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation, Plan Estatal de Investigación Científica y Técnica y de Innovación (I+D+i), grants FIS PI18/0331, FIS PI21/0980, FIS PI22/0059, and FIS PI20/0317 and co-funded by FEDER funds, European Union, Una manera de hacer Europa. Supported by grant PE-0395-2019 from Consejería de Salud y Familias, Junta de Andalucía, Spain and grant B-CTS-450-UGR20 (Programa Operativo FEDER Andalucía 2014–2020, University of Granada and Consejería de Transformación Económica, Industria, Conocimiento y Universidades). Cofinanced by the European Regional Development Fund (ERDF) through the “Una manera de hacer Europa” program.The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2023.1235161/full#supplementary-materialPurpose: Obtaining sufficient numbers of cells in a short time is a major goal of cell culturing in cell therapy and tissue engineering. However, current bidimensional (2D) culture methods are associated to several limitations, including low efficiency and the loss of key cell differentiation markers on cultured cells. Methods: In the present work, we have designed a novel biofabrication method based on a three-dimensional (3D) culture system (FIBRIAGAR-3D). Human Wharton’s jelly mesenchymal stromal cells (HWJSC) were cultured in 3D using 100%, 75%, 50%, and 25% concentrations of fibrin-agarose biomaterials (FA100, FA75, FA50 and FA25 group) and compared with control cells cultured using classical 2D systems (CTR-2D). Results: Our results showed a significant increase in the number of cells generated after 7 days of culture, with cells displaying numerous expansions towards the biomaterial, and a significant overexpression of the cell proliferation marker KI67 was found for the FA75 and FA100 groups. TUNEL and qRT-PCR analyses demonstrated that the use of FIBRIAGAR-3D was not associated with an induction of apoptosis by cultured cells. Instead, the 3D system retained the expression of typical phenotypic markers of HWJSC, including CD73, CD90, CD105, NANOG and OCT4, and biosynthesis markers such as types-I and IV collagens, with significant increase of some of these markers, especially in the FA100 group. Finally, our analysis of 8 cell signaling molecules revealed a significant decrease of GM-CSF, IFN-g, IL2, IL4, IL6, IL8, and TNFα, suggesting that the 3D culture system did not induce the expression of pro-inflammatory molecules. Conclusion: These results confirm the usefulness of FIBRIAGAR-3D culture systems to increase cell proliferation without altering cell phenotype of immunogenicity and opens the door to the possibility of using this novel biofabrication method in cell therapy and tissue engineering of the human cornea, oral mucosa, skin, urethra, among other structures.Universidad de Granada, SpainConsejería de Transformación Económica, Industria, Conocimiento y UniversidadesFEDER PE-0395-2019Secretaría de Estado de Investigación, Desarrollo e Innovación FIS PI18/0331, FIS PI20/0317, FIS PI21/0980, FIS PI22/0059 I+D+iInstituto de Salud Carlos III ISCIIIMinisterio de Ciencia e Innovación MICINNEuropean Regional Development Fund ERDFConsejería de Salud y Familias, Junta de Andalucía B-CTS-450-UGR20Spanish National Plan for Scientific and Technical Research and Innovatio

Inactivation of human plasma alters the structure and biomechanical properties of engineered tissues

Acknowledgments The authors are grateful to Echevarne Laboratories for the coagulation experiments and to Gloria Carmona and Rosario Sánchez Pernaute as well as all members of the Unidad de Producción y Reprogramación Celular (UPRC) for technical help and support. All figures were created through BioRender.com accessed on 1 September 2021.Funding This research was funded by the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i) from Ministerio de Ciencia, Innovación y Universidades (Instituto de Salud Carlos III), grants FIS PI17/0391, RTC-2017- 6658-1, PI20/0317 and ICI19/00024 (BIOCLEFT), co-financed by Fondo Europeo de Desarrollo Regional ERDF-FEDER, European Union and PE-0395-2019 from Consejería de Salud y Familias, Junta de Andalucía, Spain.Fibrin is widely used for tissue engineering applications. The use of blood derivatives, however, carries a high risk of transmission of infectious agents, necessitating the application of pathogen reduction technology (PRT). The impact of this process on the structural and biomechanical properties of the final products is unknown. We used normal plasma (PLc) and plasma inactivated by riboflavin and ultraviolet light exposure (PLi) to manufacture nanostructured cellularized fibrin-agarose hydrogels (NFAHs), and then compared their structural and biomechanical properties. We also measured functional protein C, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and coagulation factors [fibrinogen, Factor (F) V, FVIII, FX, FXI, FXIII] in plasma samples before and after inactivation. The use of PLi to manufacture cellularized NFAHs increased the interfibrillar spacing and modified their biomechanical properties as compared with cellularized NFAH manufactured with PLc. PLi was also associated with a significant reduction in functional protein C, FV, FX, and FXI, and an increase in the international normalized ratio (derived from the PT), APTT, and TT. Our findings demonstrate that the use of PRT for fibrin-agarose bioartificial tissue manufacturing does not adequately preserve the structural and biomechanical properties of the product. Further investigations into PRT-induced changes are warranted to determine the applications of NFAH manufactured with inactivated plasma as a medicinal product.Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i) from Ministerio de Ciencia, Innovación y Universidades (Instituto de Salud Carlos III), grants FIS PI17/0391, RTC-2017- 6658-1, PI20/0317 and ICI19/00024 (BIOCLEFT)Fondo Europeo de Desarrollo Regional ERDF-FEDEREuropean UnionPE-0395-2019 from Consejería de Salud y Familias, Junta de Andalucía, Spai

A novel 3D biofabrication strategy to improve cell proliferation and differentiation of human Wharton’s jelly mesenchymal stromal cells for cell therapy and tissue engineering

Purpose: Obtaining sufficient numbers of cells in a short time is a major goal of cell culturing in cell therapy and tissue engineering. However, current bidimensional (2D) culture methods are associated to several limitations, including low efficiency and the loss of key cell differentiation markers on cultured cells.Methods: In the present work, we have designed a novel biofabrication method based on a three-dimensional (3D) culture system (FIBRIAGAR-3D). Human Wharton’s jelly mesenchymal stromal cells (HWJSC) were cultured in 3D using 100%, 75%, 50%, and 25% concentrations of fibrin-agarose biomaterials (FA100, FA75, FA50 and FA25 group) and compared with control cells cultured using classical 2D systems (CTR-2D).Results: Our results showed a significant increase in the number of cells generated after 7 days of culture, with cells displaying numerous expansions towards the biomaterial, and a significant overexpression of the cell proliferation marker KI67 was found for the FA75 and FA100 groups. TUNEL and qRT-PCR analyses demonstrated that the use of FIBRIAGAR-3D was not associated with an induction of apoptosis by cultured cells. Instead, the 3D system retained the expression of typical phenotypic markers of HWJSC, including CD73, CD90, CD105, NANOG and OCT4, and biosynthesis markers such as types-I and IV collagens, with significant increase of some of these markers, especially in the FA100 group. Finally, our analysis of 8 cell signaling molecules revealed a significant decrease of GM-CSF, IFN-g, IL2, IL4, IL6, IL8, and TNFα, suggesting that the 3D culture system did not induce the expression of pro-inflammatory molecules.Conclusion: These results confirm the usefulness of FIBRIAGAR-3D culture systems to increase cell proliferation without altering cell phenotype of immunogenicity and opens the door to the possibility of using this novel biofabrication method in cell therapy and tissue engineering of the human cornea, oral mucosa, skin, urethra, among other structures

Comprehensive ex vivo and in vivo preclinical evaluation of novel chemo enzymatic decellularized peripheral nerve allografts

As a reliable alternative to autografts, decellularized peripheral nerve allografts (DPNAs) should mimic the complex microstructure of native nerves and be immunogenically compatible. Nevertheless, there is a current lack of decellularization methods able to remove peripheral nerve cells without significantly altering the nerve extracellular matrix (ECM). The aims of this study are firstly to characterize ex vivo, in a histological, biochemical, biomechanical and ultrastructural way, three novel chemical-enzymatic decellularization protocols (P1, P2 and P3) in rat sciatic nerves and compared with the Sondell classic decellularization method and then, to select the most promising DPNAs to be tested in vivo. All the DPNAs generated present an efficient removal of the cellular material and myelin, while preserving the laminin and collagen network of the ECM (except P3) and were free from any significant alterations in the biomechanical parameters and biocompatibility properties. Then, P1 and P2 were selected to evaluate their regenerative effectivity and were compared with Sondell and autograft techniques in an in vivo model of sciatic defect with a 10-mm gap, after 15 weeks of follow-up. All study groups showed a partial motor and sensory recovery that were in correlation with the histological, histomorphometrical and ultrastructural analyses of nerve regeneration, being P2 the protocol showing the most similar results to the autograft control group.Spanish "Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion TecnologicaSpanish Government FIS PI17-0393 FIS PI20-0318Fondo Europeo de Desarrollo RegionalERDF-FEDER European Union P18-RT-5059Plan Andaluz de Investigacion, Desarrollo eInnovacion (PAIDI 2020)Consejeria de Transformacion Economica, Industria, Conocimiento y UniversidadesJunta de Andalucia PI-0086-2020ERDF-FEDER, theEuropean Union CPP2021-009070Ministerio de Ciencia e Innovacion, Union Europea (NextGeneration EU)Agencia Estatal de Investigacion, Espan

Nanostructured fibrin-based hydrogel membranes for use as an augmentation strategy in achilles tendon surgical repair in rats

Hydrogels are polymeric biomaterials characterised by their promising biological and biomechanical properties, which make them potential alternatives for use in tendon repair. The aim of the present study was to generate in vitro, and determine the therapeutic efficacy in vivo, of novel nanostructured fibrin-based hydrogels to be used as an augmentation strategy for the surgical repair of rat Achilles tendon injuries. Fibrin, fibrin-agarose and fibrin-collagen nanostructured hydrogels (NFH, NFAH and NFCH, respectively) were generated and their biomechanical properties and cell-biomaterial interactions characterised ex vivo. Achilles tendon ruptures were created in 24 adult Wistar rats, which were next treated with direct repair (control group) or direct repair augmented with the generated biomaterials (6 rats/group). After 4 and 8 weeks, the animals were euthanised for macroscopical and histological analyses. Biomechanical characterisation showed optimal properties of the biomaterials for use in tendon repair. Moreover, biological analyses confirmed that tendon-derived fibroblasts were able to adhere to the surface of the generated biomaterials, with high levels of viability and functionality. In vivo studies demonstrated successful tendon repair in all groups. Lastly, histological analyses disclosed better tissue and extracellular matrix organisation and alignment with biomaterial-based augmentation strategies than direct repair, especially when NFAH and NFCH were used. The present study demonstrated that nanostructured fibrin-collagen hydrogels can be used to enhance the healing process in the surgical repair of tendon ruptures.The study was supported by the Spanish Society of Orthopaedics and Traumatology (SECOT), the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Ministerio de Economía y Competitividad (Instituto de Salud Carlos III), the European Regional Development Fund (ERDF-FEDER) Grant number FIS PI20-0318 and the Grant number P18-RT-5059 from the Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020), Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Andalusian Regional Government, Spain. The authors are grateful to Dr Ariane Ruyffelaert for her advice on the English version of the manuscript and to Karen Shashok for editing the revised manuscript. The authors are also grateful to Amalia de la Rosa Romero and Concepción López Rodríguez (Experimental Unit, University Hospital Virgen de las Nieves, Granada, Spain) and Fabiola Bermejo Casares (Department of Histology, University of Granada, Spain) for their technical assistance

Histological characterization of the human scapholunate ligament

The scapholunate interosseous ligament (SLIL) plays a fundamental role in stabilizing the wrist bones, and its disruption is a frequent cause of wrist arthrosis and disfunction. Traditionally, this structure is considered to be a variety of fibrocartilaginous tissue and consists of three regions: dorsal, membranous and palmar. Despite its functional relevance, the exact composition of the human SLIL is not well understood. In the present work, we have analyzed the human SLIL and control tissues from the human hand using an array of histological, histochemical and immunohistochemical methods to characterize each region of this structure. Results reveal that the SLIL is heterogeneous, and each region can be subdivided in two zones that are histologically different to the other zones. Analysis of collagen and elastic fibers, and several proteoglycans, glycoproteins and glycosaminoglycans confirmed that the different regions can be subdivided in two zones that have their own structure and composition. In general, all parts of the SLIL resemble the histological structure of the control articular cartilage, especially the first part of the membranous region (zone M1). Cells showing a chondrocyte-like phenotype as determined by S100 were more abundant in M1, whereas the zone containing more CD73-positive stem cells was D2. These results confirm the heterogeneity of the human SLIL and could contribute to explain why certain zones of this structure are more prone to structural damage and why other zones have specific regeneration potential

Ex Vivo Generation and Characterization of Human Hyaline and Elastic Cartilaginous Microtissues for Tissue Engineering Applications

This study was supported by grants FIS PI17/0393 and PI20/0318 from the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III); grants PI-0257-2017 and PE-0395- 2019 from Consejería de Salud y Familias, Junta de Andalucía, España; grant P18-RT-5059 from Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía, España; grant A-CTS-498-UGR18 from the University of Granada and Junta de Andalucía, España. It was co-funded by FEDER-ERDF funds.Authors are grateful to Fabiola Bermejo Casares for the technical histological assistance. Special thanks to Ariane Ruyffelaert for her critical review and proofreading service. This work forms part of the doctoral thesis conducted by David Sánchez Porras (Doctoral Program in Biomedicine, Doctoral School, University of Granada, Spain).Considering the high prevalence of cartilage-associated pathologies, low self-repair capacity and limitations of current repair techniques, tissue engineering (TE) strategies have emerged as a promising alternative in this field. Three-dimensional culture techniques have gained attention in recent years, showing their ability to provide the most biomimetic environment for the cells under culture conditions, enabling the cells to fabricate natural, 3D functional microtissues (MTs). In this sense, the aim of this study was to generate, characterize and compare scaffold-free human hyaline and elastic cartilage-derived MTs (HC-MTs and EC-MTs, respectively) under expansion (EM) and chondrogenic media (CM). MTs were generated by using agarose microchips and evaluated ex vivo for 28 days. The MTs generated were subjected to morphometric assessment and cell viability, metabolic activity and histological analyses. Results suggest that the use of CM improves the biomimicry of the MTs obtained in terms of morphology, viability and extracellular matrix (ECM) synthesis with respect to the use of EM. Moreover, the overall results indicate a faster and more sensitive response of the EC-derived cells to the use of CM as compared to HC chondrocytes. Finally, future preclinical in vivo studies are still needed to determine the potential clinical usefulness of these novel advanced therapy products.Instituto de Salud Carlos III Spanish Government FIS PI17/0393 PI20/0318Junta de Andalucia PI-0257-2017 PE-03952019Junta de Andalucia P18-RT-5059University of Granada A-CTS-498-UGR18Junta de Andalucia A-CTS-498-UGR18FEDER-ERDF fund

In vivo Evaluation of Nanostructured Fibrin-Agarose Hydrogels With Mesenchymal Stem Cells for Peripheral Nerve Repair

The regenerative capability of peripheral nerves is very limited, and several strategies have been proposed to increase nerve regeneration. In the present work, we have analyzed the in vivo usefulness of a novel nanostructured fibrin-agarose bio-artificial nerve substitute (Nano) used alone or in combination with NeuraGen® collagen type I conduits (Coll-Nano) in laboratory rats with a 10-mm sciatic nerve defect. Control animals were subjected to the gold-standard autograft technique (Auto). Results first demonstrated that the percentage of self-amputations was lower in Nano and Coll-Nano groups as compared to the Auto group. Neurotrophic ulcers were more abundant in the Auto group (60%, with 66.6% of them being >2-mm) than Nano and Coll-Nano groups (0%) at 4 weeks, although Nano showed more ulcers after 12 weeks. Foot length was significantly altered in Auto animals due to neurogenic retraction, but not in Nano and Coll-Nano groups after 12 weeks. At the functional level, all animals showed a partial sensory recovery as determined by the pinch test, especially in Nano and Auto groups, but did not reach the levels of native animals. Toe-spread test revealed a partial motor function recovery only in Nano animals at 4 weeks and Auto and Nano at 12 weeks. Electromyography showed clear denervation signs in all experimental groups, with few differences between Auto and Nano animals. After 12 weeks, an important denervation decrease and an increase of the reinnervation process was found in Auto and Nano groups, with no differences between these groups. Histological analyses demonstrated an active peripheral nerve regeneration process with newly formed peripheral nerve fascicles showing S-100, GAP-43 and myelin in all experimental groups. The peripheral nerve regeneration process was more abundant in Auto group, followed by Nano group, and both were better than Coll-Nano group. Muscle histology confirmed the electromyography results and showed some atrophy and fibrosis signs and an important weight and volume loss in all groups, especially in the Coll-Nano group (56.8% weight and 60.4% volume loss). All these results suggest that the novel Nano substitutes used in in vivo were able to contribute to bridge a 10-mm peripheral nerve defect in rats