Bioinspired silk-based biomaterials for tissue engineering
Building on our extensive experience with natural silk fibers, we are focused on developing innovative bioinspired fibers for biomedical applications. Our main goal is to produce silk fibers and 3D structures from them, with controlled physical-chemical, mechanical, and geometric properties, in order to create biomaterials for various applications.
In collaboration with national and international research groups, we focus on the production of high-performance artificial fibers derived from either natural proteins (regenerated fibers) or genetically engineered proteins (bioinspired fibers), thanks to spinning methods, including wet spinning and electrospinning, with a particular emphasis on the Straining Flow Spinning Process ® (SFS). The SFS technology is licensed to Silk Biomed, S.L.
Figure 1. (a) Schematic of the Straining Flow Spinning Process. The inset shows a detail of the capillary–nozzle system in which the flow of the dope (Qd) and of the focusing fluid (Qf) are indicated. VR1: Velocity of the take-up roller; VR2: Velocity of the post-spinning roller. (b) Schematic of the Electro-spinning method that uses electrohydrodynamic forces to draw charged polymer solutions into nanofibers (c) Example of the silk fibroin solution flowing through the nozzle and forming the fibers by SFP. Adapted from P. Lozano’s Thesis. (d) Example of a tube of silk fibroin fibers produced by SFP. (e) Examples of silk-fibroin hydrogels of 2% and 6% to be used as drug-delivery vehicles for brain ischemic episodes. (f) Example of silk-fibroin mats produced by electro-spinning.
In addition to fibers, silk proteins are utilized to produce other biomaterial formats, such as hydrogels, mats, and nanoparticles, to create scaffolds for tissue engineering applications. We have developed electrospun silk fibroin mats for peripheral nerve regeneration, implantable hydrogels to promote healing after brain injury, and silk nanoparticles for drug delivery systems. Ongoing research includes the creation of silk fibroin scaffolds for skin regeneration, some tested in animal models. For hydrogels, key goals include precisely controlling gelation time, biodegradation, geometry, encapsulated molecule or cell release, and mechanical properties—key factors for implantation in living organisms and the development of scaffolds for tissue engineering, especially in neuronal regeneration.
Biomechanics of soft tissues and soft biomaterials
We investigate the mechanical behavior of collagen-rich tissues, such as blood vessels, esophagus, tendons, and pericardium, to enhance the analysis and treatment of cardiovascular diseases and trauma. Recently, we refined our techniques to assess the dynamic mechanical properties of the porcine esophagus, simulating physiological conditions for tissue repair (Orozco-Vega et al., 2022).
Figure 2. Study of the mechanical properties of soft tissues and development of soft biomaterials. (a) Scanning electron microscope (SEM) of the group. (b) Precision mechanical-testing machine for biological samples.(c) Samples of a case of study: native porcine esophagus and decellularized esophagus. Adapted from Orozco-Vega et al. 2022. (d) SEM images of the native and decellularized esophagus tissues. (e) Sample of a soft collagen tissue to be uniaxially tested. (f) Representative examples of stress vs. stretch of esophagus samples.
Functionalization of materials to enhance biocompatibility
Functionalization of implants. Our research focuses on developing functional coatings for conventional biomaterials, such as titanium alloys, to enhance their biocompatibility. This is achieved through Activated Vapour Silanization® (AVS), an innovative variant of chemical vapor deposition developed in our laboratory. AVS-functionalized surfaces exhibit a high density of amine groups, enabling the covalent attachment of biologically relevant molecules, such as adhesion proteins.
Specifically, to address the challenge of implant osseointegration, we have decorated Ti-6Al-4V implants with immobilized oligopeptides (RGD, CS-1, IKVAV, PHSRN), ensuring long-term stability. Among them, RGD demonstrates the best performance in vitro, promoting adhesion, proliferation, and osteogenic differentiation of mesenchymal stem cells.
Functionalization for single-molecule and single-cell force spectroscopy studies. We specialize in the functionalization of materials for single-molecule and single-cell force spectroscopy. In particular, we develop advanced procedures to immobilize molecules on silicon and glass substrates, as well as on Atomic Force Microscopy (AFM) cantilevers, for Affinity Atomic Force Microscopy (A-AFM) studies. This allows us to measure molecular interactions between functionalized substrates and AFM tips, as demonstrated in studies on the interaction strength between Pru p 3 and its ligand. Additionally, we apply single-cell force spectroscopy to analyze cell adhesion to biofunctionalized surfaces, providing valuable insights into cellular interactions in Titanium implants.
Figure 3. Functionalization of biomaterials: Scheme of the procedure used to functionalize Ti-6Al-4V samples. Adapted from Rezvanian et al. (Applied Surface Science, 2016). (b) Scheme of the covalent binding of fibronectin to the functionalized titanium substrate, highlighting the main cell-binding motifs. Adapted from Rezvanian et al. (J. Funct. Biomater., 2023). (c) Osteoblastic differentiation assays performed on polystyrene, bare Ti-6Al-4V, and RGD-decorated R-THAB® Ti-6Al-4V. Adapted from Álvarez-Lopez et al. (Biomimetics, 2025). (d) Functionalized cantilevers for single-molecule force spectroscopy. (e) Measurement of interaction strength using force-displacement curves in AFM. Figures d-e were adapted from Corregidor et al. (Molecules 2023). (f) Functionalized cantilevers and substrates for single-cell force spectroscopy to assess cell adhesion strength to functionalized titanium implants. Figures g-i were adapted from Álvarez-López’s thesis (2024).
Mechanobiology
Cell and tissue mechanical properties are particularly important for cancer progression, cell differentiation, immune activation and treatments. Our research focuses on understanding the relation between the mechanical properties of cells, cell internal ordering and cell function, in order to identify robust biomarkers of cell physio(patho)logical states. During the last three years we have focused on new applications of the micropipette aspiration technique, microfluidic devices and on improving the use of Atomic Force Microscopy in this field.
Atomic force microscopy in the field of mechanobiology was used for several investigations, including the mechanical and structural characterisation of decellularized porcine cardiac matrices as a possible scaffold for the regeneration of infarcted cardiac tissue and the measurement of the mass of different cell types, such as macrophages or T and B lymphocytes.
Figure 4. Nanotec nanolife© atomic force microscope. (b) Application of AFM to characterize topography (inner images 1, 2 and 3) and mechanical properties (inner image 4) of decellularized porcine cardiac matrices.(c) Automatic analysis of micropipette aspiration images with machine-learning tools (Abarca-Ortega, Biophysical Journal, 2024). (d) Biophysical changes occurring in T-cells during aging (Adapted from González-Bermúdez et al., Immunology, 2022). (e) Scheme of constriction-based cytometry to measure cell mechanical properties. (f) Numerical model considering the cell to be a quasi-linear hyper-viscoelastic material. (Adapted from Abarca-Ortega, Journal of Mechanical Sciences, 2024).
Using micropipette aspiration, we have studied the relationships between deformability, cytoskeletal components and biological functionality of mammalian immune T cells during aging. We have also applied machine learning techniques to automate the imagine analysis of micropipette aspiration tests. Recently, we have implemented a constriction-based method to compute the mechanical properties of cells with high-throughout. Finally, we collaborate with European partners to characterize multiple biophysical parameters on the same cells.
Representative projects
GENOSBIO: Generación de biomateriales de titanio con rigidez superficial modulable, propiedades osteoinductivas y mitigación de la respuesta fibrótical. PID2023-152058OB-I00. PI: José Pérez Rigueiro. Funding Agency: Ministerio de Economía y Competitividad. Period: 2021-2023
THOR: Building vascular networks and Blood-Brain-Barriers through a Biomimetic manufacturing Technology for the fabrication of Human tissues and ORgans. THOR-101099719-HORIZON-EIC-2022-PATHFINDEROPEN-01-01. PI: José Pérez Rigueiro. Funding Agency: Horizon Europe. Period: 2023-2026
COVIDTECH -CM: PLATAFORMA CIENTÍFICO-TECNOLÓGICA PARA EL PRONÓSTICO, DIAGNÓSTICO Y SEGUIMIENTO DE LA ENFERMEDAD COVID19. PI: Gustavo Guinea Tortuero. Funding Agency: Comunidad de Madrid. Period: 2020-2022
MODBIOMAT: Modulación de la respuesta a biomateriales de titanio funcionalizados. PI: José Pérez Rigueiro. Funding Agency: Ministerio de Economía y Competitividad. Period: 2021-2024
Centro Tecnológico para el Estudio y Tratamiento Integrado de los Desórdenes Neurológicos NEUROCENTRO-CM B2017/BMD-3760. PI: Gustavo V. Guinea. Funding Agency: Dirección General de Investigación e Innovación, Consejería de Educación e Investigación, Comunidad de Madrid. Period: 2018-2022
NUEVAS TECNOLOGÍAS APLICADAS AL ESTUDIO DE NANOMÁQUINAS BIOLÓGICAS TEC4BIO. PI at UPM: Gustavo R. Plaza Baonza. Funding Agency: Comunidad de Madrid. Period: 2019-2022. Doctorado Industrial IND2018/BMD-9804. PI: Gustavo V. Guinea. Funding Agency: Dirección General de Investigación e Innovación, Consejería de Educación e Investigación, Comunidad de Madrid. Period: 2018-2022.
Contratos Ayudante Investigación Plan Empleo Juvenil PEJ-2020-AI-BMD-18487, “Producción de biomateriales y experimentos en el área de los cultivos celulares y los modelos animales para su validación pre-clínica”. PI: Gustavo V. Guinea. Funding Agency: Dirección General de Investigación e Innovación, Consejería de Educación e Investigación, Comunidad de Madrid. Period: 2021-2023
Contratos Ayudante Investigación Plan Empleo Juvenil PEJ-2021-AI/IND-21188, “Producción de biomateriales y experimentos en el área de los cultivos celulares y los modelos animales para su validación pre-clínica”. PI: Gustavo V. Guinea. Funding Agency: Dirección General de Investigación e Innovación, Consejería de Educación e Investigación, Comunidad de Madrid. Period: 2022-2024.
Contratos Programa Investigo IdISSC- 09-PIN1-00011.2/2022. PI: Gustavo V. Guinea. Funding Agency: Dirección General de Investigación e Innovación, Consejería de Educación e Investigación, Comunidad de Madrid. Period: 2022-2023.
Madrid Innovative Neurotech Alliance, MINA-CM P2022/BMD-7236. PI: Gustavo V. Guinea. Funding Agency: Dirección General de Investigación e Innovación, Consejería de Educación e Investigación, Comunidad de Madrid. Period: 2023-2026.
Doctorado Industrial “Modulación de la inflamación a través de la estimulación del nervio vago, combinada con la inyección intracerebral de células madre mesenquimales encapsuladas en fibroína de seda, para terapia de ictus isquémico” IND2023/BMD-28865. PI: Gustavo V. Guinea. Funding Agency: Dirección General de Investigación e Innovación, Consejería de Educación e Investigación, Comunidad de Madrid. Period: 2021-2023.
“Implantable Ecosystems of Genetically Modified Bacteria for the Personalized Treatment of Patients with Chronic Diseases”, ISOS-101130454-HORIZON-EIC-2023-PATHFINDEROPEN-01-01. PI: Gustavo V. Guinea. Funding Agency: Unión Europea. Period: 2023-2026.
Ayudas para la contratación de personal investigador predoctoral para el año 2022, PIPF-2022_SAL-GL-25129, “Superando los límites de la medicina regenerativa: implantes bio-híbridos de seda para la regeneración diferencial de neuronas sensitivas y motoras”. PI: Gustavo V. Guinea. Funding Agency: : Dirección General de Investigación e Innovación, Consejería de Educación e Investigación, Comunidad de Madrid. Period: 2024-2027
SameMultiPhys: Novel Biophysical Tools to Measure Multiple Parameters In The Same Cell. PI: Gustavo Plaza. Funding Agency: Horizon Europe. Period: 2023-2027.
Augmentación biológica de la reparación quirúrgica de las roturas crónicas del manguito rotador mediante fibroína de seda con doble funcionalización. PI: Yaiza Lópiz. Funding Agency: Fundación Mutua Madrileña. Period: 2023-2026.
Variación madurativa de actividad de Glicoproteína P, su influencia en la respuesta a tratamientos protectores/anticonvulsivantes, y su modificación por cannabidiol en ratas neonatas tras hipoxia-isquemia. PI: José Martínez-Orgado. Funding Agency: Ministerio de Ciencia e InnovaciónPeriod: 2024-2026.