Maria Molinos

Layer-by-Layer Self-Assembly of Chitosan and Poly(γ-glutamic acid) into Polyelectrolyte Complexes

Chitosan (Ch) is a nontoxic and biocompatible polysaccharide extensively used in biomedical applications. Ch, as a polycation, can be combined with anionic polymers by layer-by-layer (LbL) self-assembly, giving rise to multilayered complexed architectures. These structures can be used in tissue engineering strategies, as drug delivery systems, or artificial matrices mimicking the extracellular microenvironment. In this work, Ch was combined with poly(γ-glutamic acid) (γ-PGA). γ-PGA is a polyanion, which was microbially produced, and is known for its low immunogenic reaction and low cytotoxicity. Multilayered ultrathin films were assembled by LbL, with a maximum of six layers. The interaction between both polymers was analyzed by: ellipsometry, quartz crystal microbalance with dissipation, Fourier transform infrared spectroscopy, atomic force microscopy, and zeta potential measurements. Ch/γ-PGA polyelectrolyte multilayers (PEMs) revealed no cytotoxicity according to ISO 10993-5. Overall, this study demonstrates that Ch can interact electrostatically with γ-PGA forming multilayered films. Furthermore, this study provides a comprehensive characterization of Ch/γ-PGA PEM structures, elucidating the contribution of each layer for the nanostructured films. These model surfaces can be useful substrates to study cell-biomaterial interactions in tissue regeneration.

Systemic delivery of bone marrow mesenchymal stem cells for in situ intervertebral disc regeneration

Cell therapies for intervertebral disc (IVD) regeneration presently rely on transplantation of IVD cells or stem cells directly to the lesion site. Still, the harsh IVD environment, with low irrigation and high mechanical stress, challenges cell administration and survival. In this study, we addressed systemic transplantation of allogeneic bone marrow mesenchymal stem cells (MSCs) intravenously into a rat IVD lesion model, exploring tissue regeneration via cell signaling to the lesion site. MSC transplantation was performed 24 hours after injury, in parallel with dermal fibroblasts as a control; 2 weeks after transplantation, animals were killed. Disc height index and histological grading score indicated less degeneration for the MSC‐transplanted group, with no significant changes in extracellular matrix composition. Remarkably, MSC transplantation resulted in local downregulation of the hypoxia responsive GLUT‐1 and in significantly less herniation, with higher amounts of Pax5+ B lymphocytes and no alterations in CD68+ macrophages within the hernia. The systemic immune response was analyzed in the blood, draining lymph nodes, and spleen by flow cytometry and in the plasma by cytokine array. Results suggest an immunoregulatory effect in the MSC‐transplanted animals compared with control groups, with an increase in MHC class II+ and CD4+ cells, and also upregulation of the cytokines IL‐2, IL‐4, IL‐6, and IL‐10, and downregulation of the cytokines IL‐13 and TNF‐α. Overall, our results indicate a beneficial effect of systemically transplanted MSCs on in situ IVD regeneration and highlight the complex interplay between stromal cells and cells of the immune system in achieving successful tissue regeneration. Stem Cells Translational Medicine 2017;6:1029–1039

Improvement of Bovine Nucleus Pulposus Cells Isolation Leads to Identification of Three Phenotypically Distinct Cell Subpopulations.

OBJECTIVE Strategies to promote intervertebral disc (IVD) regeneration have been hindered by the lack of knowledge of IVD fundamental cellular/molecular components. One of the key points to be addressed is the characterization of nucleus pulposus (NP) cell population(s). This study establishes an improved method for bovine NP (bNP) cell isolation, whose procedure is still not consensual among the literature, allowing a thorough characterization of cell (sub)populations that exist in the young NP. METHODS bNP was digested with distinct enzymes (collagenase-type-I, collagenase-type-II, and collagenase-type-XI) at different concentrations (0.5, 1.0, and 2.0 mg/mL), for 4 and 19 h. Cell yield, viability/apoptosis, and morphology were analyzed by flow cytometry and imaging flow cytometry. Identification of cell subpopulations within NP and its phenotype was investigated by assessing expression of CD29, CD44, CD45, CD34, CD146, and Brachyury. RESULTS It was found that bNP cells present a similar morphology independently of the digestive enzyme used. However, cell yield was greatly improved by Coll-XI (2 mg/mL) treatment for a short digestion period. Interestingly, three subpopulations, with different sizes and auto-fluorescence, were consistently identified by flow cytometry. And crucially, differential expression of cell markers was found among these subpopulations. CONCLUSION This study demonstrated that collagenase-type-XI is an efficient enzyme that is used for digesting bNP. And most importantly, three phenotypically distinct subpopulations of cells where identified within the bNP. Such knowledge is key for a better understanding of NP cell biology and its potential endogenous regenerative capacity.

Integrated Analysis of Biological Samples by Imaging Flow Cytometry.

Instituto de Engenharia Biomédica (INEB). University of Porto, Porto, Portugal b.IMAGE – Bioimaging Center for Biomaterials and Regenerative Therapies, INEB, Porto, Portugal Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal Instituto de Ciências Biomédicas Abel Salazar (ICBAS). University of Porto. Porto, Portugal Faculdade de Engenharia da Universidade do Porto (FEUP), Porto, Portugal Faculdade de Medicina da Universidade do Porto (FMUP), Porto, Portugal Centre for Cell Biology and Department of Biology, University of Aveiro, Aveiro, Portugal College of Life and Environmental Sciences, Biosciences, University of Exeter, Exeter, Devon, United Kindgom.

Matrisome Profiling During Intervertebral Disc Development And Ageing

Intervertebral disc (IVD) degeneration is often the cause of low back pain. Degeneration occurs with age and is accompanied by extracellular matrix (ECM) depletion, culminating in nucleus pulpous (NP) extrusion and IVD destruction. The changes that occur in the disc with age have been under investigation. However, a thorough study of ECM profiling is needed, to better understand IVD development and age-associated degeneration. As so, iTRAQ LC-MS/MS analysis of foetus, young and old bovine NPs, was performed to define the NP matrisome. The enrichment of Collagen XII and XIV in foetus, Fibronectin and Prolargin in elder NPs and Collagen XI in young ones was independently validated. This study provides the first matrisome database of healthy discs during development and ageing, which is key to determine the pathways and processes that maintain disc homeostasis. The factors identified may help to explain age-associated IVD degeneration or constitute putative effectors for disc regeneration.

Improvement of Bovine Nucleus Pulposus Cells Isolation Leads to Identification of Three Phenotypically Distinct Cell Subpopulations

Introduction Strategies to promote intervertebral disc (IVD) regeneration have been hindered by the lack of knowledge of IVD fundamental cellular/molecular components. One of the key points to be addressed is the characterization of nucleus pulposus (NP) cell population(s). This study establishes an improved method for bovine NP (bNP) cell isolation, whose procedure is still not consensual among the literature, allowing a thorough characterization of cell (sub)populations that exist in the young NP. Material and Methods bNP was digested with distinct enzymes (collagenase-type-I, collagenase-type-II, and collagenase- type-XI) at different concentrations (0.5, 1.0, and 2.0 mg/mL), for 4 and 19 hour. Cell yield, viability/apoptosis, and morphology were analyzed by flow cytometry and imaging flow cytometry. Identification of cell subpopulations within NP and its phenotype was investigated by assessing expression of CD29, CD44, CD45, CD34, CD146, and Brachyury. Results It was found that bNP cells present a similar morphology independently of the digestive enzyme used. However, cell yield was greatly improved by Coll-XI (2 mg/mL) treatment for a short digestion period. Interestingly, three subpopulations, with different sizes and auto-fluorescence, were consistently identified by flow cytometry. And crucially, differential expression of cell markers was found among these subpopulations. Conclusion This study demonstrated that collagenase-type-XI is an efficient enzyme that is used for digesting bNP. And most importantly, three phenotypically distinct subpopulations of cells where identified within the bNP. Such knowledge is key for a better understanding of NP cell biology and its potential endogenous regenerative capacity.

A Nanotechnology-Based Therapy to Target Inflammation in Degenerated Intervertebral Disc: First Results from an ex vivo Disc Organ Culture

Introduction Resolution of intervertebral disc (IVD) degeneration-associated inflammation is a pre-requisite for tissue regeneration. Here, a pro-inflammatory disc organ culture model from bovine origin was established,1 and a therapy based on the intra-discal delivery of an anti-inflammatory drug (diclofenac). Material and Methods Bovine caudal IVD punches were needle-punctured and additionally stimulated with lipopolysaccharide (10 µg/mL) or interleukin-1β (IL-1β, 10–100 ng/mL) for 48 hours. An intradiscal therapeutic approach was tested based on a non-steroidal anti-inflammatory drug, diclofenac (Df), alone or incorporated into nanoparticles, previously developed.2 Results IL-1β-treated IVD organ cultures showed a statistically significant up-regulation of pro-inflammatory markers (IL-6, IL-8, PGE2) and metalloproteases (MMP1, MMP3) expression while ECM proteins (Collagen II, Aggrecan) were significantly down-regulated. The injection of the anti-inflammatory drug was able to reduce the levels of pro-inflammatory cytokines and MMPs and surprisingly increased ECM protein levels. Conclusion These results point the intradiscal application of anti-inflammatory drugs as promising therapeutics for disc degeneration. Moreover, the organ culture model established could be used to address cellular/molecular mechanisms that regulate inflammation and IVD degeneration, and moreover to test novel therapeutic drugs, thus reducing the number of animals in in vivo experimentation. Acknowledgments This work was supported by European funds from FP7-EU-project GENODISC (HEALTH-F2–2008–201626), by German funds from the German Spine Foundation (Deutsche Wirbelsäulenstitung), by the Luso-German Integrated Actions Program (DAAD/CRUP, ref. A20/12), by FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE, by Portuguese funds through QREN (project NORTE-07–0124-FEDER-000005) co-financed by North Portugal Regional Operational (ON.2-O Novo Norte). Graciosa Q. Teixeira and Catarina L. Pereira also acknowledge FCT for their PhD grants (SFRH/BD/88429/2012 and SFRH/BD/85779/2012, respectively). References Teixeira GQ, et al. Tissue Eng 2015; (Part C). Accepted for publication Gonçalves RM, et al. J Mater Sci Mater Med 2015

On the Path to Autologous IVD Regeneration

Introduction Intervertebral disc (IVD) degeneration is often the cause of low back pain. With age, extracellular matrix (ECM) depletion occurs, leading to nucleus pulpous (NP) extrusion and IVD destruction. Concomitantly, there is a decline in viable cell populations, which in turn remain poorly characterized. As current clinical treatments have not provided adequate solutions, we propose an in-depth study in terms of NP cell subpopulation characterization and ECM changes with aging, in view of regeneration. Materials and Methods The first step was to characterize NP cell populations harvested from bovine IVDs, to better understand IVDs potential to endogenously regenerate. We started by optimizing cell isolation procedure, still not consensual among the literature. For that, distinct digestive enzymes (collagenase type-I, collagenase type-II, and collagenase type-XI) were used at three different concentrations (0.5, 1.0, and 2.0 mg/mL, respectively), for 4 and 19 hours. To understand the cellular effect of the digestion, we evaluated the cell yield (trypan blue exclusion); cell viability/apoptosis (AnnexinV/Propidium Iodide staining, Flow Cytometry), and cell morphology (actin (phalloidin-AlexaFluor488) and nuclei (DRAQ-5) staining, Imaging Flow Cytometry). The presence of specific cell subpopulations within NP and its phenotype was then investigated, by assessing the expression of CD29, CD44, CD45, CD146, CD34, Gly-A, and Brachyury. Due to microenvironments’ growing importance for regeneration, in parallel, we compared the NP ECM proteomic profile of bovine IVDs from fetus, young and old animals by quantitative iTRAQ LC-MS/MS. The candidates differentially expressed in the three age groups are being validated by Western blot. Results Cell yield and viability improved, in a concentration dependent manner, with increasing collagenase activity against collagen I and II—collagenase XI rendered the highest cell yield and greatest viability. In addition, cell yield was higher for shorter digestion periods (4 hours). Furthermore, NP cells did not reveal major morphological changes dependent on the enzymes used. Interestingly, three viable subpopulations, with different sizes and autofluorescence, were consistently identified by flow cytometry, immediately after isolation. Expression of cell markers differed among these subpopulations, proving the existence of an heterogeneous cell population within the NP. Concerning iTRAQ analysis, a few interesting candidates emerged that are expressed in different proportions in fetus, young and old animals. These quantitative differences are being validated. Conclusion This study shows that collagenase type XI is the most efficient enzyme to be used for isolation and that different cell populations can be identified within the bovine NP. It also demonstrates that distinct ECM proteins are expressed in the IVD at different ratios depending on bovine age groups. Acknowledgments This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade—COMPETE, by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia. The authors would also like to acknowledge the AO SPINE Research Network (AO SRN) for funding. Disclosure of Interest None declared

How are Bovine Nucleus Pulposus Cell Populations Affected by Cell Isolation Methods

Introduction Strategies to promote intervertebral disk (IVD) regeneration have been hindered by the lack of knowledge of IVD fundamental cellular/molecular components. To date, many studies have been published culturing bovine IVD cells as a model to study IVD. However, the cellular mechanisms behind IVD de(re)generation remain largely unexplored. One of the key points to address is the characterization of the nucleus pulposus (NP) cell population(s), for which the isolation method is not consensual among the literature. These cells can be isolated using different enzymes and tissue digestion methods, but the effect of these parameters on the phenotype of cell population(s) has not been properly explored. This work aims to evaluate how the cell isolation method affects NP cell population(s). Materials and Methods NP cells were harvested from bovine caudal discs using six different digestion protocols - mechanical or enzymatic digestion, either with collagenase I, collagenase II, or both collagenase types (I + II). For mechanical tissue disruption, a gentleMACSDissociator was used. The dissociation time ranged from 4 to 19 hours. The effect of the dissociation method on NP cells immediately upon isolation was assessed by measuring cell yield (by counting cells), by viability (by flow cytometry), and by evaluating the cells morphology (by Imaging Flow Cytometry). Total 24 hours after tissue digestion, nonadherent cells were separated from the adherent fraction and the metabolic activity of both fractions was determined over time using resazurin assay. Prospective identification of different cell populations within bovine NP was performed using flow cytometry. Results Using the isolation methods described, the cell yield ranged from 4.9×105 to 1.1 × 106 cells per gram of tissue, with a cell viability between 59 and 82%. Comparing the different methods, collagenase type II digestion led to a higher cell yield (1.1 × 106 cells/g) and viability (82%). Cells isolated by the different protocols could be further characterized in terms of their morphology by measuring diverse parameters, such as cell length, elongation, shape, diameter, number of cell nucleus lobes, and number of vacuoles. Interestingly, three subpopulations of viable cells, with different sizes and auto-fluorescence, could be consistently identified by flow cytometry analysis, immediately upon isolation, without significant differences between the isolation protocols. Furthermore, two cell populations, adherent and nonadherent, could be isolated and cell metabolic activity suggests that it is possible to maintain both adherent and suspension cell populations in culture. Currently, the expression of specific cell markers for these populations is being evaluated. Conclusion Bovine NP cells were isolated using different separation methods. Digestion protocols using collagenase type II rendered higher cell yields and greater viability. Three different subpopulations of bovine NP cells were identified upon cell isolation. In monolayer culture two different cell subpopulations were separated and maintained in culture-adherent and nonadherent NP cells. Overall, this study demonstrates that the isolation method is crucial for the subsequent study of bovine NP cells. The identification of different NP cell populations and their dependence on the isolation method is particularly important when envisioning the identification of novel biological evidences that contribute to highlight IVD degeneration and its potential regenerative capacity. Acknowledgments This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade - COMPETE, by portuguese funds through FCT - Fundação para a Ciência e a Tecnologia. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

An In Situ Anti-Inflammatory Drug Delivery System for Intervertebral Disks

Introduction Intervertebral disk (IVD) degeneration is one of the major causes of low back pain and the exact reason why (and if) it relates with pain symptoms remains to be discovered (Prithvi Raj, 2008). Current conservative treatments include exercise, medications, and physical therapy. Moreover, there is a growing consensus that surgery, towards removing and/or replacing the damaged tissue, may not be effective. This work focuses on the development of a local and minimally invasive anti-inflammatory drug delivery system for IVD. This drug delivery system is based on the incorporation of Chitosan (Ch)/g-Poly(glutamic acid) (PGA) nanocarriers with Diclofenac (Df), a widely used anti-inflammatory drug, in a hyaluronic acid (HA)-based hydrogel. The Df local delivery in IVD would overcome the disadvantages associated with its oral administration such as poor biological half-life, high doses, and side effects such as peptic ulceration. Moreover, the use of a hydrogel envisages the treatment of degenerated IVD, which could be replaced by a novel cell-free or cell-loaded biocompatible gel. Materials and Methods The Ch/PGA nanocarriers with Df were produced by coacervation method. Briefly, PGA and Ch electrostatic interaction at a controlled pH (pH 5) was explored as previously described (Antunes, 2012). The nanoparticles obtained were characterized in terms of size and polydispersion index by dynamic light scattering (DLS) and the Df entrapment was evaluated by UV/Vis absorbance. As a vehicle, a commercially available hydrogel based on HA and Gelatin (HyStem, Glycosan) was used. The gelation of the HA-based gel with the nanoparticles was evaluated at different temperatures and dilutions while the gel net was verified using CryoSEM. The quantity and distribution of the nanoparticles within the gel were characterized by fluorimetry and Confocal Microscopy (using FITC-Ch). The Df release from the nanoparticles within the gel was evaluated at physiological pH by UV/Vis absorbance. In the end, the biological effect of this anti-inflammatory strategy was assessed using Lipopolysaccharide (LPS)-induced macrophages isolated from peripheral blood samples. Cell viability, phenotypic markers, and the levels of diverse pro- and anti-inflammatory cytokines were evaluated (IL-6, TNF-alfa, IL-10, etc.). Results Df entrapment within Ch/PGA nanoparticles was optimized, reaching approximately 93% of drug entrapment with complexes with size of 253 ± 32 nm. These nano complexes remain stable during several days at pH 5.0 and were successfully incorporated into HA-based hydrogels, being distributed along the gel net as observed by CryoSEM and Confocal Microscopy, despite some aggregates. The gelation of the HA-based gel was optimized: the time of gelation range 25 minutes for gels with 10% of nanoparticles (% v/v), at 37 °C. The decrease of temperature (until 4°C) was shown to increase time of gelation (until 45 minutes). The Df delivery from the nanocomplexes entrapped in the HA-based hydrogel was determined at pH 7.4 (PBS) and compared with Df delivery from free nanoparticles. In this case, an immediate release of the drug occurs (∼ 60% after 1 hour incubation), accomplished by particles aggregation. Nevertheless, the Df release rate was hindered by incorporation of the Df-nanoparticles within the HA hydrogel: after 2 h only 26% of the anti-inflammatory drug was released reaching 100% after 24 h. The Df release in a degenerated disk environment (pH 6.5) is currently being evaluated. The cytotoxic effect of this delivery system is being evaluated in LPS-activated human macrophages. Df, in the range of concentrations used, did not cause any toxicity, while cell metabolic activity did not decrease below 80% in the presence of the nanoparticles. The Df seems to promote the expression of CD 86 and MHC II, in CD14+ macrophages, suggesting a polarization towards M1 or M2b phenotype and an increase in IL-10 production. The levels of prostaglandin (PGE2) are also being determined, since PGE2 is inhibited by anti-inflammatory drugs as Df. Conclusion The self-assembly of PGA, Ch, and Df resulted in stable nanosystems which can be used for the controlled release of the drug. Sustained Df release at physiological pH is provided by the incorporation of these NPs into an injectable gel. This work shows the feasibility of an in situ anti-inflammatory drug delivery system, controlled by pH changes, for IVD. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Prithvi Raj P. Intervertebral disc: Anatomy-Physiology-Pathophisiology-Treatment. Pain Practice 2008;8(1):18–44 Antunes JC, et al. Layer-by-layer self-assembly of Chitosan and Poly(γ-glutamic acid) into polyelectrolyte complexes. Biomacromolecules 2012 In press