Human platelet-rich plasma improves the nesting and differentiation of human chondrocytes cultured in stabilized porous chitosan scaffolds

[EN] The clinical management of large-size cartilage lesions is difficult due to the limited regenerative ability of the cartilage. Different biomaterials have been used to develop tissue engineering substitutes for cartilage repair, including chitosan alone or in combination with growth factors to improve its chondrogenic properties. The main objective of this investigation was to evaluate the benefits of combining activated platelet-rich plasma with a stabilized porous chitosan scaffold for cartilage regeneration. To achieve this purpose, stabilized porous chitosan scaffolds were prepared using freeze gelation and combined with activated platelet-rich plasma. Human primary articular chondrocytes were isolated and cultured in stabilized porous chitosan scaffolds with and without combination to activated platelet-rich plasma. Scanning electron microscopy was used for the morphological characterization of the resulting scaffolds. Cell counts were performed in hematoxylin and eosin-stained sections, and type I and II collagen expression was evaluated using immunohistochemistry. Significant increase in cell number in activated platelet-rich plasma/stabilized porous chitosan was found compared with stabilized porous chitosan scaffolds. Chondrocytes grown on stabilized porous chitosan expressed high levels of type I collagen but type II was not detectable, whereas cells grown on activated platelet rich plasma/stabilized porous chitosan scaffolds expressed high levels of type II collagen and type I was almost undetectable. In summary, activated platelet-rich plasma increases nesting and induces the differentiation of chondrocytes cultured on stabilized porous chitosan scaffolds.The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants MAT2016-76039-C4-2-R (M.S.-T. and C.C.) and MAT 2013-46467-C4-1-R (M.A.G.-G. and J.L.G.R.) from the Ministry of Economy and Competitiveness of the Spanish Government and by the program VLC-Bioclinic from the University of Valencia and INCLIVA (Spain). CIBER-BBN and CIBERER are funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund. M.A.G.-G. acknowledges a grant from the BES-2011-044740.Sancho-Tello Valls, M.; Martorell-Tejedor, S.; Mata, M.; Millán, L.; Gamiz Gonzalez, MA.; Gómez Ribelles, JL.; Carda-Batalla, C. (2017). Human platelet-rich plasma improves the nesting and differentiation of human chondrocytes cultured in stabilized porous chitosan scaffolds. Journal of Tissue Engineering. 8:1-6. https://doi.org/10.1177/2041731417697545S168Muzzarelli, R. A. A., Greco, F., Busilacchi, A., Sollazzo, V., & Gigante, A. (2012). Chitosan, hyaluronan and chondroitin sulfate in tissue engineering for cartilage regeneration: A review. Carbohydrate Polymers, 89(3), 723-739. doi:10.1016/j.carbpol.2012.04.057XIE, A., NIE, L., SHEN, G., CUI, Z., XU, P., GE, H., & TAN, Q. (2014). The application of autologous platelet-rich plasma gel in cartilage regeneration. Molecular Medicine Reports, 10(3), 1642-1648. doi:10.3892/mmr.2014.2358Rodríguez-Vázquez, M., Vega-Ruiz, B., Ramos-Zúñiga, R., Saldaña-Koppel, D. A., & Quiñones-Olvera, L. F. (2015). Chitosan and Its Potential Use as a Scaffold for Tissue Engineering in Regenerative Medicine. BioMed Research International, 2015, 1-15. doi:10.1155/2015/821279Kim, J., Lin, B., Kim, S., Choi, B., Evseenko, D., & Lee, M. (2015). TGF-β1 conjugated chitosan collagen hydrogels induce chondrogenic differentiation of human synovium-derived stem cells. Journal of Biological Engineering, 9(1). doi:10.1186/1754-1611-9-1Shimojo, A. A. M., Perez, A. G. M., Galdames, S. E. M., Brissac, I. C. de S., & Santana, M. H. A. (2015). Performance of PRP Associated with Porous Chitosan as a Composite Scaffold for Regenerative Medicine. The Scientific World Journal, 2015, 1-12. doi:10.1155/2015/396131SHEN, J., GAO, Q., ZHANG, Y., & HE, Y. (2014). Autologous platelet-rich plasma promotes proliferation and chondrogenic differentiation of adipose-derived stem cells. Molecular Medicine Reports, 11(2), 1298-1303. doi:10.3892/mmr.2014.2875Krüger, J. P., Ketzmar, A.-K., Endres, M., Pruss, A., Siclari, A., & Kaps, C. (2013). Human platelet-rich plasma induces chondrogenic differentiation of subchondral progenitor cells in polyglycolic acid-hyaluronan scaffolds. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 102(4), 681-692. doi:10.1002/jbm.b.33047Dhurat, R., & Sukesh, M. (2014). Principles and methods of preparation of platelet-rich plasma: A review and author′s perspective. Journal of Cutaneous and Aesthetic Surgery, 7(4), 189. doi:10.4103/0974-2077.150734Sancho-Tello, M., Forriol, F., Gastaldi, P., Ruiz-Saurí, A., Martín de Llano, J. J., Novella-Maestre, E., … Carda, C. (2015). Time Evolution ofin VivoArticular Cartilage Repair Induced by Bone Marrow Stimulation and Scaffold Implantation in Rabbits. The International Journal of Artificial Organs, 38(4), 210-223. doi:10.5301/ijao.5000404Shimojo, A. A. M., Perez, A. G. M., Galdames, S. E. M., Brissac, I. C. S., & Santana, M. H. A. (2016). Stabilization of porous chitosan improves the performance of its association with platelet-rich plasma as a composite scaffold. Materials Science and Engineering: C, 60, 538-546. doi:10.1016/j.msec.2015.11.080Oktay, E., Demiralp, B., Demiralp, B., Senel, S., Cevdet Akman, A., Eratalay, K., & Akıncıbay, H. (2010). Effects of Platelet-Rich Plasma and Chitosan Combination on Bone Regeneration in Experimental Rabbit Cranial Defects. Journal of Oral Implantology, 36(3), 175-184. doi:10.1563/aaid-joi-d-09-00023Kutlu, B., Tiğlı Aydın, R. S., Akman, A. C., Gümüşderelioglu, M., & Nohutcu, R. M. (2012). Platelet-rich plasma-loaded chitosan scaffolds: Preparation and growth factor release kinetics. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 101B(1), 28-35. doi:10.1002/jbm.b.32806Bi, L., Cheng, W., Fan, H., & Pei, G. (2010). Reconstruction of goat tibial defects using an injectable tricalcium phosphate/chitosan in combination with autologous platelet-rich plasma. Biomaterials, 31(12), 3201-3211. doi:10.1016/j.biomaterials.2010.01.03

A cell-free approach with a supporting biomaterial in the form of dispersed microspheres induces hyaline cartilage formation in a rabbit knee model

[EN] The objective of this study was to test a regenerative medicine strategy for the regeneration of articular cartilage. This approach combines microfracture of the subchondral bone with the implant at the site of the cartilage defect of a supporting biomaterial in the form of microspheres aimed at creating an adequate biomechanical environment for the differentiation of the mesenchymal stem cells that migrate from the bone marrow. The possible inflammatory response to these biomaterials was previously studied by means of the culture of RAW264.7 macrophages. The microspheres were implanted in a 3¿mm-diameter defect in the trochlea of the femoral condyle of New Zealand rabbits, covering them with a poly(l-lactic acid) (PLLA) membrane manufactured by electrospinning. Experimental groups included a group where exclusively PLLA microspheres were implanted, another group where a mixture of 50/50 microspheres of PLLA (hydrophobic and rigid) and others of chitosan (a hydrogel) were used, and a third group used as a control where no material was used and only the membrane was covering the defect. The histological characteristics of the regenerated tissue have been evaluated 3 months after the operation. We found that during the regeneration process the microspheres, and the membrane covering them, are displaced by the neoformed tissue in the regeneration space toward the subchondral bone region, leaving room for the formation of a tissue with the characteristics of hyaline cartilage.Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CICPBA), Universidad Nacional de La Plata, Grant/Award Number: 11/X643; Agencia Estatal de Investigación/Fondo Europeo de Desarrollo Regional de la Unión Europea, Grant/Award Number: MAT2016-76039-C4-1 2-R; Spanish Ministry of Economy and Competitiveness (MINECO)Zurriaga Carda, J.; Lastra, ML.; Antolinos-Turpin, CM.; Morales-Román, RM.; Sancho-Tello, M.; Perea-Ruiz, S.; Milián, L.... (2020). A cell-free approach with a supporting biomaterial in the form of dispersed microspheres induces hyaline cartilage formation in a rabbit knee model. 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(2016). Poly (L-Lactic Acid) Porous Scaffold-Supported Alginate Hydrogel with Improved Mechanical Properties and Biocompatibility. The International Journal of Artificial Organs, 39(8), 435-443. doi:10.5301/ijao.5000516Conoscenti, G., Schneider, T., Stoelzel, K., Carfì Pavia, F., Brucato, V., Goegele, C., … Schulze-Tanzil, G. (2017). PLLA scaffolds produced by thermally induced phase separation (TIPS) allow human chondrocyte growth and extracellular matrix formation dependent on pore size. Materials Science and Engineering: C, 80, 449-459. doi:10.1016/j.msec.2017.06.011Dashtdar, H., Murali, M. R., Abbas, A. A., Suhaeb, A. M., Selvaratnam, L., Tay, L. X., & Kamarul, T. (2013). PVA-chitosan composite hydrogel versus alginate beads as a potential mesenchymal stem cell carrier for the treatment of focal cartilage defects. Knee Surgery, Sports Traumatology, Arthroscopy, 23(5), 1368-1377. doi:10.1007/s00167-013-2723-5Denlinger, L. C., Fisette, P. L., Garis, K. 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H., & Hui, J. H. P. (2013). Evidence-Based Status of Microfracture Technique: A Systematic Review of Level I and II Studies. Arthroscopy: The Journal of Arthroscopic & Related Surgery, 29(9), 1579-1588. doi:10.1016/j.arthro.2013.05.027Hangody, L., Kish, G., Kárpáti, Z., Udvarhelyi, I., Szigeti, I., & Bély, M. (1998). Mosaicplasty for the Treatment of Articular Cartilage Defects: Application in Clinical Practice. Orthopedics, 21(7), 751-756. doi:10.3928/0147-7447-19980701-04Hoemann, C., Kandel, R., Roberts, S., Saris, D. B. F., Creemers, L., Mainil-Varlet, P., … Buschmann, M. D. (2011). International Cartilage Repair Society (ICRS) Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials. CARTILAGE, 2(2), 153-172. doi:10.1177/1947603510397535Kumar, M. N. V. R., Muzzarelli, R. A. A., Muzzarelli, C., Sashiwa, H., & Domb, A. J. (2004). Chitosan Chemistry and Pharmaceutical Perspectives. Chemical Reviews, 104(12), 6017-6084. doi:10.1021/cr030441bKuo, T.-F., Lin, M.-F., Lin, Y.-H., Lin, Y.-C., Su, R.-J., Lin, H.-W., & Chan, W. P. (2011). Implantation of platelet-rich fibrin and cartilage granules facilitates cartilage repair in the injured rabbit knee: preliminary report. Clinics, 66(10), 1835-1838. doi:10.1590/s1807-59322011001000026Landis, J. R., & Koch, G. G. (1977). The Measurement of Observer Agreement for Categorical Data. Biometrics, 33(1), 159. doi:10.2307/2529310Lao, L., Tan, H., Wang, Y., & Gao, C. (2008). Chitosan modified poly(l-lactide) microspheres as cell microcarriers for cartilage tissue engineering. Colloids and Surfaces B: Biointerfaces, 66(2), 218-225. doi:10.1016/j.colsurfb.2008.06.014Lastra, M. L., Molinuevo, M. S., Blaszczyk-Lezak, I., Mijangos, C., & Cortizo, M. S. (2017). Nanostructured fumarate copolymer-chitosan crosslinked scaffold: An in vitro osteochondrogenesis regeneration study. Journal of Biomedical Materials Research Part A, 106(2), 570-579. doi:10.1002/jbm.a.36260Lastra, M. L., Molinuevo, M. S., Cortizo, A. M., & Cortizo, M. S. (2016). Fumarate Copolymer-Chitosan Cross-Linked Scaffold Directed to Osteochondrogenic Tissue Engineering. Macromolecular Bioscience, 17(5). doi:10.1002/mabi.201600219Lebourg, M., Martínez-Díaz, S., García-Giralt, N., Torres-Claramunt, R., Ribelles, J. G., Vila-Canet, G., & Monllau, J. (2013). Cell-free cartilage engineering approach using hyaluronic acid–polycaprolactone scaffolds: A study in vivo. Journal of Biomaterials Applications, 28(9), 1304-1315. doi:10.1177/0885328213507298Luzardo-Alvarez, A., Blarer, N., Peter, K., Romero, J. F., Reymond, C., Corradin, G., & Gander, B. (2005). Biodegradable microspheres alone do not stimulate murine macrophages in vitro, but prolong antigen presentation by macrophages in vitro and stimulate a solid immune response in mice. Journal of Controlled Release, 109(1-3), 62-76. doi:10.1016/j.jconrel.2005.09.015Mainil-Varlet, P., Van Damme, B., Nesic, D., Knutsen, G., Kandel, R., & Roberts, S. (2010). A New Histology Scoring System for the Assessment of the Quality of Human Cartilage Repair: ICRS II. The American Journal of Sports Medicine, 38(5), 880-890. doi:10.1177/0363546509359068Martinez-Diaz, S., Garcia-Giralt, N., Lebourg, M., Gómez-Tejedor, J.-A., Vila, G., Caceres, E., … Monllau, J. C. (2010). In Vivo Evaluation of 3-Dimensional Polycaprolactone Scaffolds for Cartilage Repair in Rabbits. The American Journal of Sports Medicine, 38(3), 509-519. doi:10.1177/0363546509352448McCormick, F., Harris, J. D., Abrams, G. D., Frank, R., Gupta, A., Hussey, K., … Cole, B. (2014). Trends in the Surgical Treatment of Articular Cartilage Lesions in the United States: An Analysis of a Large Private-Payer Database Over a Period of 8 Years. 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Mutations, Genes, and Phenotypes Related to Movement Disorders and Ataxias

26 páginas, 4 figuras, 3 tablasOur clinical series comprises 124 patients with movement disorders (MDs) and/or ataxia with cerebellar atrophy (CA), many of them showing signs of neurodegeneration with brain iron accumulation (NBIA). Ten NBIA genes are accepted, although isolated cases compatible with abnormal brain iron deposits are known. The patients were evaluated using standardised clinical assessments of ataxia and MDs. First, NBIA genes were analysed by Sanger sequencing and 59 patients achieved a diagnosis, including the detection of the founder mutation PANK2 p.T528M in Romani people. Then, we used a custom panel MovDisord and/or exome sequencing; 29 cases were solved with a great genetic heterogeneity (34 different mutations in 23 genes). Three patients presented brain iron deposits with Fe-sensitive MRI sequences and mutations in FBXO7, GLB1, and KIF1A, suggesting an NBIA-like phenotype. Eleven patients showed very early-onset ataxia and CA with cortical hyperintensities caused by mutations in ITPR1, KIF1A, SPTBN2, PLA2G6, PMPCA, and PRDX3. The novel variants were investigated by structural modelling, luciferase analysis, transcript/minigenes studies, or immunofluorescence assays. Our findings expand the phenotypes and the genetics of MDs and ataxias with early-onset CA and cortical hyperintensities and highlight that the abnormal brain iron accumulation or early cerebellar gliosis may resembling an NBIA phenotype.This work was supported by the Instituto de Salud Carlos III (ISCIII)—Subdirección General de Evaluación y Fomento de la Investigación within the framework of the National R + D+I Plan co-funded with European Regional Development Funds (ERDF) [Grants PI18/00147 and PI21/00103 to CE]; the Fundació La Marató TV3 [Grants 20143130 and 20143131 to BPD and CE]; and by the Generalitat Valenciana [Grant PROMETEO/2018/135 to CE]. Part of the equipment employed in this work was funded by Generalitat Valenciana and co-financed with ERDF (OP ERDF of Comunitat Valenciana 2014–2020). PS had an FPU-PhD fellowship funded by the Spanish Ministry of Education, Culture and Sport [FPU15/00964]. IH has a PFIS-PhD fellowship [FI19/00072]. ASM has a contract funded by the Spanish Foundation Per Amor a l’Art (FPAA)Peer reviewe

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

In Vivo Articular Cartilage Regeneration Using Human Dental Pulp Stem Cells Cultured in an Alginate Scaffold: A Preliminary Study

Osteoarthritis is an inflammatory disease in which all joint-related elements, articular cartilage in particular, are affected. The poor regeneration capacity of this tissue together with the lack of pharmacological treatment has led to the development of regenerative medicine methodologies including microfracture and autologous chondrocyte implantation (ACI). The effectiveness of ACI has been shown in vitro and in vivo, but the use of other cell types, including bone marrow and adipose-derived mesenchymal stem cells, is necessary because of the poor proliferation rate of isolated articular chondrocytes. In this investigation, we assessed the chondrogenic ability of human dental pulp stem cells (hDPSCs) to regenerate cartilage in vitro and in vivo. hDPSCs and primary isolated rabbit chondrocytes were cultured in chondrogenic culture medium and found to express collagen II and aggrecan. Both cell types were cultured in 3% alginate hydrogels and implanted in a rabbit model of cartilage damage. Three months after surgery, significant cartilage regeneration was observed, particularly in the animals implanted with hDPSCs. Although the results presented here are preliminary, they suggest that hDPSCs may be useful for regeneration of articular cartilage

Low glutathione peroxidase in rdl mouse retina increases oxidative stress and proteases

Malondialdehyde, reduced glutathione, glutathione peroxidase, glutathione reductase and cysteine protease cathepsins at postnatal (PN) days 2, 7, 14, 21 and 28 in controls (wt) and the retinal degeneration 1 (rd1) mouse model for retinitis pigmentosa retinas were measured to determine oxidative stress. In PN28 wt and PN2 rd1 retinas, elevated malondialdehyde and low glutathione peroxidase activity indicate higher oxidative load, despite higher reduced glutathione in PN2 rd1 retinas. This is due to physiological exposure to light and retinal vascular/neural restructuring, respectively. Compared with wt retinas, relatively high malondialdehyde at PN2 and cathepsin levels at PN14, 21 and 28 in rd1 retinas indicate that cells of the residual inner retina also contribute to the oxidative stress and retinal degeneration

Single-cell multi-omics analysis of COVID-19 patients with pre-existing autoimmune diseases shows aberrant immune responses to infection

This publication is part of the Human Cell Atlas (https://www.humancellatlas.org/publications/). The authors gratefully acknowledge the Sanger Cellular Genetics Informatics team, particularly A. Predeus and S. Murray for their assistance with aligning the published raw data from uninfected MS patients, as well as S. van Dongen, M. Prete, and Q. Lin for their help with online data hosting. We acknowledge the members of the Vento-Tormo and Ballestar groups for useful discussions. A.B. received additional support from a Gates Cambridge Scholarship. This research was funded/supported by the R+D+i project of the Spanish Ministry of Science and Innovation (grant number PID2020-117212RB-I00/ MICIN/AEI/10.13039/501100011033), the Chan Zuckerberg Initiative (grant 2020-216799) and Wellcome Sanger core funding (WT206194). This publication has been supported by the Unstoppable campaign of the Josep Carreras Leukaemia Foundation. B.G., F.J.C.-N., and N.K.W. were funded by Wellcome (206328/Z/17/Z), the MRC (MR/S036113/1), and the Aging Biology Foundation.In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up