Granero-Moltó, F. (Froilán)
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- Engineering a Humanised Niche to Support Human Haematopoiesis in Mice: Novel Opportunities in Modelling Cancer(2020) Calvo, I.A. (Isabel A.); Granero-Moltó, F. (Froilán); Flandes-Iparraguirre, M. (María); McGovern, J.A. (Jacqui A.); Landgraf, M. (Marietta); Juan-Pardo, E.M. (Elena M.) de; Nicol, A. (Andrew); Mazo, M. (Manuel); Shafiee, A. (Abbas); Paiva, B. (Bruno); Lahr, C.A. (Christoph A.); Sanchez-Herrero, A. (Alvaro); Prosper-Cardoso, F. (Felipe); Saez, B. (Borja); Bray, L.J. (Laura J.)Despite the bone marrow microenvironment being widely recognised as a key player in cancer research, the current animal models that represent a human haematopoietic system lack the contribution of the humanised marrow microenvironment. Here we describe a murine model that relies on the combination of an orthotopic humanised tissue-engineered bone construct (ohTEBC) with patient-specific bone marrow (BM) cells to create a humanised bone marrow (hBM) niche capable of supporting the engraftment of human haematopoietic cells. Results showed that this model supports the engraftment of human CD34+ cells from a healthy BM with human haematopoietic cells migrating into the mouse BM, human BM compartment, spleen and peripheral blood. We compared these results with the engraftment capacity of human CD34+ cells obtained from patients with multiple myeloma (MM). We demonstrated that CD34+ cells derived from a diseased BM had a reduced engraftment potential compared to healthy patients and that a higher cell dose is required to achieve engraftment of human haematopoietic cells in peripheral blood. Finally, we observed that hematopoietic cells obtained from the mobilised peripheral blood of patients yields a higher number of CD34+, overcoming this problem. In conclusion, this humanised mouse model has potential as a unique and patient-specific pre-clinical platform for the study of tumour–microenvironment interactions, including human bone and haematopoietic cells, and could, in the future, serve as a drug testing platform.
- Molecular and Cellular Mechanisms of Delayed Fracture Healing in Mmp10 (Stromelysin 2) Knockout Mice(Wiley, 2021) Calvo, I.A. (Isabel A.); Granero-Moltó, F. (Froilán); Paramo, J.A. (José Antonio); Ripalda-Cemboráin, P. (Purificación); Montiel-Terrón, V. (Verónica); Aldazabal, J. (Javier); Orbe, J. (Josune); Rodriguez, J.A. (José Antonio); Lopez, T. (Tania); Valdés-Fernández, J. (José); Muiños-López, E. (Emma); Romero-Torrecilla, J.A. (Juan Antonio); Prosper-Cardoso, F. (Felipe); Saez, B. (Borja)The remodeling of the extracellular matrix is a central function in endochondral ossification and bone homeostasis. During secondary fracture healing, vascular invasion and bone growth requires the removal of the cartilage intermediate and the coordinate action of the collagenase matrix metalloproteinase (MMP)-13, produced by hypertrophic chondrocytes, and the gelatinase MMP-9, produced by cells of hematopoietic lineage. Interfering with these MMP activities results in impaired fracture healing characterized by cartilage accumulation and delayed vascularization. MMP-10, Stromelysin 2, a matrix metalloproteinase with high homology to MMP-3 (Stromelysin 1), presents a wide range of putative substrates identified in vitro, but its targets and functions in vivo and especially during fracture healing and bone homeostasis are not well defined. Here, we investigated the role of MMP-10 through bone regeneration in C57BL/6 mice. During secondary fracture healing, MMP-10 is expressed by hematopoietic cells and its maximum expression peak is associated with cartilage resorption at 14 days post fracture (dpf). In accordance with this expression pattern, when Mmp10 is globally silenced, we observed an impaired fracture-healing phenotype at 14 dpf, characterized by delayed cartilage resorption and TRAP-positive cell accumulation. This phenotype can be rescued by a non-competitive transplant of wild-type bone marrow, indicating that MMP-10 functions are required only in cells of hematopoietic linage. In addition, we found that this phenotype is a consequence of reduced gelatinase activity and the lack of proMMP-9 processing in macrophages. Our data provide evidence of the in vivo function of MMP-10 during endochondral ossification and defines the macrophages as the lead cell population in cartilage removal and vascular invasion
- Novel hybrid biocomposites for tendon grafts: the addition of silk to polydioxanone and poly(lactide-co-caprolactone) enhances material properties, in vitro and in vivo biocompatibility(2023) Doyle, B. (Barry); Granero-Moltó, F. (Froilán); Heidari, B.S. (Behzad Shiroud); Rajkhowa, R. (Rangam); Zheng, M. (Minghao); Allardyce, B. (Benjamin); Ruan, R. (Rui); De-Juan-Pardo, E.M. (Elena M.); Chen, P. (Peilin); Muiños-López, E. (Emma); Davachi, S.M. (Seyed Mohammad); Dilley, R. (Rodney); Harrington, E. (Emma)Biopolymers play a critical role as scaffolds used in tendon and ligament (TL) regeneration. Although advanced biopolymer materials have been proposed with optimised mechanical properties, biocompatibility, degradation, and processability, it is still challenging to find the right balance between these properties. Here, we aim to develop novel hybrid biocomposites based on poly(p-dioxanone) (PDO), poly(lactide-co-caprolactone) (LCL) and silk to produce high-performance grafts suitable for TL tissue repair. Biocomposites containing 1-15% of silk were studied through a range of characterisation techniques. We then explored biocompatibility through in vitro and in vivo studies using a mouse model. We found that adding up to 5% silk increases the tensile properties, degradation rate and miscibility between PDO and LCL phases without agglomeration of silk inside the com-posites. Furthermore, addition of silk increases surface roughness and hydrophilicity. In vitro experiments show that the silk improved attachment of tendon-derived stem cells and proliferation over 72 h, while in vivo studies indicate that the silk can reduce the expression of pro-inflammatory cytokines after six weeks of implantation. Finally, we selected a promising biocomposite and created a prototype TL graft based on extruded fibres. We found that the tensile properties of both individual fibres and braided grafts could be suitable for anterior cruciate ligament (ACL) repair applications.
- An engineered periosteum for efficient delivery of rhBMP-2 and mesenchymal progenitor cells during bone regeneration(2023) Granero-Moltó, F. (Froilán); López-Martínez, T. (Tania); Riera-Alvarez, L. (Luis); Rodriguez-Florez, N. (Naiara); Elizalde, R. (Reyes); Ripalda-Cemboráin, P. (Purificación); Ruiz-de-Galarreta-Moriones, S.(Sergio); Juan-Pardo, E.M. (Elena M.) de; Childs, P. (Peter); Echanove-González De Anleo, M. (Miguel); Lamo-de-Espinosa-Vázquez-de-Sola, J.M. (José María); Valdés-Fernández, J. (José); Salmeron-Sanchez, M. (Manuel); Muiños-López, E. (Emma); Romero-Torrecilla, J.A. (Juan Antonio); Prosper-Cardoso, F. (Felipe); López-Barberena, A. (Asier); Abizanda-Sarasa, G. (Gloria); Jayawarna, V. (Vineetha)During bone regeneration, the periosteum acts as a carrier for key regenerative cues, delivering osteochondroprogenitor cells and crucial growth factors to the injured bone. We developed a biocompatible, 3D polycaprolactone (PCL) melt electro-written membrane to act as a mimetic periosteum. Poly (ethyl acrylate) coating of the PCL membrane allowed functionalization, mediated by fibronectin and low dose recombinant human BMP-2 (rhBMP-2) (10-25 mu g/ml), resulting in efficient, sustained osteoinduction in vitro. In vivo, rhBMP-2 functionalized mimetic periosteum demonstrated regenerative potential in the treatment of rat critical-size femoral defects with highly efficient healing and functional recovery (80%-93%). Mimetic periosteum has also proven to be efficient for cell delivery, as observed through the migration of transplanted periosteum-derived mesenchymal cells to the bone defect and their survival. Ultimately, mimetic periosteum demonstrated its ability to deliver key stem cells and morphogens to an injured site, exposing a therapeutic and translational potential in vivo when combined with unprecedentedly low rhBMP-2 doses.
- Intra-articular injection of two different doses of autologous bone marrow mesenchymal stem cells versus hyaluronic acid in the treatment of knee osteoarthritis: long-term follow up of a multicenter randomized controlled clinical trial (phase I/II)(BMC, 2018) Cañizo, C. (C.) del; Granero-Moltó, F. (Froilán); Bondia, J.M. (J. M.); Aquerreta, D. (Dámaso); López-Elío, S. (Silvia); Villarón, E. (Eva); Mora, G. (Gonzalo); Andreu, E.J. (Enrique José); Blanco, J.F. (J. F.); Valentí-Nin, J.R. (Juan Ramón); Prosper-Cardoso, F. (Felipe); Nuñez-Cordoba, J.M. (Jorge M.); Lamo-Espinosa, J.M. (J. M.); Sanchez-Guijo, F.M. (Fermín M.); Valentí-Azcárate, A. (Andrés)Background: Mesenchymal stromal cells (MSCs) are a promising option to treat knee osteoarthritis (OA). Their safety and usefulness have been reported in several short-term clinical trials but less information is available on the longterm efects of MSC in patients with osteoarthritis. We have evaluated patients included in our previous randomized clinical trial (CMM-ART, NCT02123368) to determine their long-term clinical efect. Materials: A phase I/II multicenter randomized clinical trial with active control was conducted between 2012 and 2014. Thirty patients diagnosed with knee OA were randomly assigned to Control group, intraarticularly administered hyaluronic acid alone, or to two treatment groups, hyaluronic acid together with 10×106 or 100×106 cultured autol‑ ogous bone marrow-derived MSCs (BM-MSCs), and followed up for 12 months. After a follow up of 4 years adverse efects and clinical evolution, assessed using VAS and WOMAC scorings are reported. Results: No adverse efects were reported after BM-MSCs administration or during the follow-up. BM-MSCs-adminis‑ tered patients improved according to VAS, median value (IQR) for Control, Low-dose and High-dose groups changed from 5 (3, 7), 7 (5, 8) and 6 (4, 8) to 7 (6, 7), 2 (2, 5) and 3 (3, 4), respectively at the end of follow up (Low-dose vs Control group, p=0.01; High-dose vs Control group, p=0.004). Patients receiving BM-MSCs also improved clinically accord‑ ing to WOMAC. Control group showed an increase median value of 4 points (−11;10) while Low-dose and Highdose groups exhibited values of −18 (−28;−9) and −10 (−21;−3) points, respectively (Low-dose vs Control group p=0.043). No clinical diferences between the BM-MSCs receiving groups were found. Conclusions: Single intraarticular injection of in vitro expanded autologous BM-MSCs is a safe and feasible proce‑ dure that results in long-term clinical and functional improvement of knee OA.
- CC-Chemokine receptor-2 expression in osteoblasts contributes to cartilage and bone damage during post-traumatic osteoarthritis(2023) Granero-Moltó, F. (Froilán); Longobardi, L. (Lara); Willcockson, H. (Helen); Hooper, L.B. (Lola B.); Ozkan, H. (Huseyin); Valdés-Fernández, J. (José); Mucahit, E. (Esra); Musawwir, L. (Layla); Arbeeva, L. (Liubov); Prosper-Cardoso, F. (Felipe)In osteoarthritis (OA), bone changes are radiological hallmarks and are considered important for disease progression. The C-C chemokine receptor-2 (CCR2) has been shown to play an important role in bone physiology. In this study, we investigated whether Ccr2 osteoblast-specific inactivation at different times during post-traumatic OA (PTOA) progression improves joint structures, bone parameters, and pain. We used a tamoxifen-inducible Ccr2 inactivation in Collagen1 & alpha;-expressing cells to obtain osteoblasts lacking Ccr2 (CCR2-Col1 & alpha;KO). We stimulated PTOA changes in CCR2-Col1 & alpha;KO and CCR2+/+ mice using the destabilization of the meniscus model (DMM), inducing recombination before or after DMM (early- vs. late-inactivation). Joint damage was evaluated at two, four, eight, and twelve weeks post-DMM using multiple scores: articular-cartilage structure (ACS), Safranin-O, histomorphometry, osteophyte size/maturity, subchondral bone thickness and synovial hyperplasia. Spontaneous and evoked pain were assessed for up to 20 weeks. We found that early osteoblast-Ccr2 inactivation delayed articular cartilage damage and matrix degeneration compared to CCR2+/+, as well as DMM-induced bone thickness. Osteophyte formation and maturation were only minimally affected. Late Collagen1 & alpha;-Ccr2 deletion led to less evident improvements. Osteoblast-Ccr2 deletion also improved static measures of pain, while evoked pain did not change. Our study demonstrates that Ccr2 expression in osteoblasts contributes to PTOA disease progression and pain by affecting both cartilage and bone tissues.
- Anisotropic cryostructured collagen scaffolds for efficient delivery of RhBMP–2 and enhanced bone regeneration(MDPI AG, 2019) Ewald, A. (Andrea); Andreu-Arzuaga, I. (Ion); Granero-Moltó, F. (Froilán); Groll, J. (Jürgen); Flandes-Iparraguirre, M. (María); Elizalde, R. (Reyes); Ripalda-Cemboráin, P. (Purificación); Stuckensen, K. (Kai); Lopez, T. (Tania); Pons-de-Villanueva, J. (Juan); Muiños-López, E. (Emma); Nickel, J. (Joachim); Iglesias, E. (Elena); Prosper-Cardoso, F. (Felipe); Lamo-Espinosa, J.M. (J. M.); Abizanda-Sarasa, G. (Gloria); Gbureck, U. (Uwe)In the treatment of bone non-unions, an alternative to bone autografts is the use of bone morphogenetic proteins (BMPs), e.g., BMP–2, BMP–7, with powerful osteoinductive and osteogenic properties. In clinical settings, these osteogenic factors are applied using absorbable collagen sponges for local controlled delivery. Major side effects of this strategy are derived from the supraphysiological doses of BMPs needed, which may induce ectopic bone formation, chronic inflammation, and excessive bone resorption. In order to increase the efficiency of the delivered BMPs, we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic) or trabecular bone (random distributed porosity, isotropic). We hypothesize that an anisotropic structure would enhance the osteoconductive properties of the scaffolds by increasing the regenerative performance of the provided rhBMP–2. In vitro, both scaffolds presented similar mechanical properties, rhBMP–2 retention and delivery capacity, as well as scaffold degradation time. In vivo, anisotropic scaffolds demonstrated better bone regeneration capabilities in a rat femoral critical-size defect model by increasing the defect bridging. In conclusion, anisotropic cryostructured collagen scaffolds improve bone regeneration by increasing the efficiency of rhBMP–2 mediated bone healing.
- Phase II multicenter randomized controlled clinical trial on the efficacy of intra-articular injection of autologous bone marrow mesenchymal stem cells with platelet rich plasma for the treatment of knee osteoarthritis(Springer Nature, 2020) Granero-Moltó, F. (Froilán); Dámaso-Aquerreta, J. (Jesús); Pompei-Fernández, O. (Orlando); Mora, G. (Gonzalo); Moreno, V. (V.); Andreu, E.J. (Enrique José); Blanco, J.F. (J. F.); Sánchez, M. (Mikel); Vitoria-Sola, M. (María); Cañizo, M.C. (María del Consuelo) del; Valentí-Nin, J.R. (Juan Ramón); Crespo-Cullel, Í. (Íñigo); Prosper-Cardoso, F. (Felipe); Delgado-San-Vicente, D. (Diego); Nuñez-Cordoba, J.M. (Jorge M.); Lamo-Espinosa, J.M. (J. M.); Sanchez-Guijo, F.M. (Fermín M.); Valentí-Azcárate, A. (Andrés)Background: Mesenchymal stromal cells are a safe and promising option to treat knee osteoarthritis as previously demonstrated in different clinical trials. However, their efficacy, optimal dose and addition of adjuvants must be determined. Here, we evaluated the clinical effects of a dose of 100 × 106 bone marrow mesenchymal stromal cells (BM-MSCs) in combination with Platelet Rich Plasma (PRGF®) as adjuvant in a randomized clinical trial. Methods: A phase II, multicenter, randomized clinical trial with active control was conducted. Sixty patients diagnosed with knee OA were randomly assigned to 3 weekly doses of PRGF® or intraarticular administration of 100 × 106 cultured autologous BM-MSCs plus PRGF®. Patients were followed up for 12 months, and pain and function were assessed using VAS and WOMAC and by measuring the knee range of motion range. X-ray and magnetic resonance imaging analyses were performed to analyze joint damage. Results: No adverse effects were reported after BM-MSC administration or during follow-up. According to VAS, the mean value (SD) for PRGF® and BM-MSC with PRGF® went from 5 (1.8) to 4.5 (2.2) (p = 0.389) and from 5.3 (1.9) to 3.5 (2.5) (p = 0.01), respectively at 12 months. In WOMAC, the mean (SD) baseline and 12-month overall WOMAC scores in patients treated with PRGF® was 31.9 (16.2) and 22.3 (15.8) respectively (p = 0.002) while that for patients treated with BM-MSC plus PRGF® was 33.4 (18.7) and 23.0 (16.6) (p = 0.053). Although statistical significances between groups have been not detected, only patients being treated with BM-MSC plus PRGF® could be considered as a OA treatment responders following OARSI criteria. X-ray and MRI (WORMS protocol) revealed no changes in knee joint space width or joint damage. Conclusions: Treatment with BM-MSC associated with PRGF® was shown to be a viable therapeutic option for osteoarthritis of the knee, with clinical improvement at the end of follow-up. Further phase III clinical trials would be necessary to confirm the efficacy. Trial registration Clinical Trials.gov identifier NCT02365142. Nº EudraCT: 2011-006036-23.
- Silane-modified hydroxyapatite nanoparticles incorporated into polydioxanone/poly(lactide-co-caprolactone) creates a novel toughened nanocomposite with improved material properties and in vivo inflammatory responses(2023) Doyle, B. (Barry); Granero-Moltó, F. (Froilán); Heidari, B.S. (Behzad Shiroud); Zheng, M. (Minghao); Juan-Pardo, E.M. (Elena M.) de; Ruan, R. (Rui); Chen, P. (Peilin); Muiños-López, E. (Emma); Vahabli, E. (Ebrahim); Davachi, S.M. (Seyed Mohammad)The interface tissue between bone and soft tissues, such as tendon and ligament (TL), is highly prone to injury. Although different biomaterials have been developed for TL regeneration, few address the challenges of the TL bone interface. Here, we aim to develop novel hybrid nanocomposites based on poly(p-dioxanone) (PDO), poly (lactide-co-caprolactone) (LCL), and hydroxyapatite (HA) nanoparticles suitable for TL-bone interface repair. Nanocomposites, containing 3-10% of both unmodified and chemically modified hydroxyapatite (mHA) with a silane coupling agent. We then explored biocompatibility through in vitro and in vivo studies using a subcutaneous mouse model. Through different characterisation tests, we found that mHA increases tensile properties, creates rougher surfaces, and reduces crystallinity and hydrophilicity. Morphological observations indicate that mHA nanoparticles are attracted by PDO rather than LCL phase, resulting in a higher degradation rate for mHA group. We found that adding the 5% of nanoparticles gives a balance between the properties. In vitro experiments show that osteoblasts' activities are more affected by increasing the nanoparticle content compared with fibroblasts. Animal studies indicate that both HA and mHA nanoparticles (10%) can reduce the expression of pro inflammatory cytokines after six weeks of implantation. In summary, this work highlights the potential of PDO/ LCL/HA nanocomposites as an excellent biomaterial for TL-bone interface tissue engineering applications.
- No sales del laboratorio sin aprenderlo todo(2024) Patiño-García, A. (Ana); Granero-Moltó, F. (Froilán); Hernandez-Sanchez, M. (María); Fernandez-Robredo, P. (Patricia); Recalde, S. (Sergio)La asignatura consta de una estancia en tres laboratorios donde los alumnos realizan prácticas en las técnicas básicas de laboratorio. De este modo obtienen los conocimientos necesarios, tanto teóricos como prácticos, de investigación para poder iniciar el Diploma de Investigación. Hasta el momento, hemos observado que no había en muchos casos una correlación directa de lo que han aprendido y demostrado en las prácticas con lo que han desarrollado en el examen teórico que se les planteaba. Por ello el objetivo principal ha sido sustituir el examen teórico por un escape room en donde han tenido que trabajar en equipo y aplicar todos los conocimientos que han ido trabajando y adquiriendo durante las estancias para conseguir salir literalmente del laboratorio (abriendo una serie de candados y obteniendo la llave del laboratorio) en un tiempo razonable. La valoración la han realizado los profesores con rúbricas individuales y por equipo.