Catarina Leite Pereira

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.

The effect of hyaluronan-based delivery of stromal cell-derived factor-1 on the recruitment of MSCs in degenerating intervertebral discs.

Intervertebral disc (IVD) degeneration is the leading cause of low back pain and disability in the active population. Transplantation of mesenchymal stem cells (MSCs) in a hydrogel carrier can induce regenerative effects in degenerated IVDs. Moreover, it was found that degenerative discs release chemoattractants effective in MSC recruitment. Based on these findings, we hypothesized that an injectable hydrogel that can enhance the number of migrated MSCs in the IVD and provide a suitable matrix for their survival and differentiation would be ideal. The purpose of this study was to evaluate the potential of a thermoreversible hyaluronan-poly(N-isopropylacrylamide) (HAP) hydrogel as chemoattractant delivery system to recruit human MSCs in degenerative IVDs. The results demonstrate that HAP hydrogels containing stromal cell derived factor-1 (SDF-1) significantly increased the number of MSCs migrating into nucleotomized discs compared with discs treated with only HAP or SDF-1 in solution. HAP hydrogels releasing SDF-1 enhanced both the number of recruited cells and their migration distance in the IVD tissue. Furthermore, this phenomenon was dependent on MSC donor age. In conclusion, HAP SDF-1 is effective for the recruitment of stem cells in the IVD, thus opening new possibilities for the development of regenerative therapies based on endogenous cell migration.

Anti-inflammatory Chitosan/Poly-γ-glutamic acid nanoparticles control inflammation while remodeling extracellular matrix in degenerated intervertebral disc.

UNLABELLED Intervertebral disc (IVD) degeneration is one of the most common causes of low back pain (LBP), the leading disorder in terms of years lived with disability. Inflammation can play a role in LPB, while impairs IVD regeneration. In spite of this, different inflammatory targets have been purposed in the context of IVD regeneration. Anti-inflammatory nanoparticles (NPs) of Chitosan and Poly-(γ-glutamic acid) with a non-steroidal anti-inflammatory drug, diclofenac (Df), were previously shown to counteract a pro-inflammatory response of human macrophages. Here, the effect of intradiscal injection of Df-NPs in degenerated IVD was evaluated. For that, Df-NPs were injected in a bovine IVD organ culture in pro-inflammatory/degenerative conditions, upon stimulation with needle-puncture and interleukin (IL)-1β. Df-NPs were internalized by IVD cells, down-regulating IL-6, IL-8, MMP1 and MMP3, and decreasing PGE2 production, compared with IL-1β-stimulated IVD punches. Interestingly, at the same time, Df-NPs promoted an up-regulation of extracellular matrix (ECM) proteins, namely collagen type II and aggrecan. Allover, this study suggests that IVD treatment with Df-NPs not only reduces inflammation, but also delays and/or decreases ECM degradation, opening perspectives to new intradiscal therapies for IVD degeneration, based on the modulation of inflammation. STATEMENT OF SIGNIFICANCE Degeneration of the IVD is an age-related progressive process considered to be the major cause of spine disorders. The pro-inflammatory environment and biomechanics of the degenerated IVD is a challenge for regenerative therapies. The novelty of this work is the intradiscal injection of an anti-inflammatory therapy based on Chitosan (Ch)/Poly-(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) with an anti-inflammatory drug (diclofenac, Df), previously developed by us. This drug delivery system was tested in a pro-inflammatory/degenerative intervertebral disc ex vivo model. The main findings support the success of an anti-inflammatory therapy for degenerated IVD that not only reduces inflammation but also promotes native IVD matrix production.

A Degenerative/Proinflammatory Intervertebral Disc Organ Culture: An Ex Vivo Model for Anti-inflammatory Drug and Cell Therapy

Resolution of intervertebral disc (IVD) degeneration-associated inflammation is a prerequisite for tissue regeneration and could possibly be achieved by strategies ranging from pharmacological to cell-based therapies. In this study, a proinflammatory disc organ culture model was established. Bovine caudal disc punches were needle punctured and additionally stimulated with lipopolysaccharide (10 μg/mL) or interleukin-1β (IL-1β, 10-100 ng/mL) for 48 h. Two intradiscal therapeutic approaches were tested: (i) a nonsteroidal anti-inflammatory drug, diclofenac (Df) and (ii) human mesenchymal stem/stromal cells (MSCs) embedded in an albumin/hyaluronan hydrogel. IL-1β-treated disc organ cultures showed a statistically significant upregulation of proinflammatory markers (IL-6, IL-8, prostaglandin E2 [PGE2]) and metalloproteases (MMP1, MMP3) expression, while extracellular matrix (ECM) proteins (collagen II, aggrecan) were significantly downregulated. The injection of the anti-inflammatory drug, Df, was able to reduce the levels of proinflammatory cytokines and MMPs and surprisingly increase ECM protein levels. These results point the intradiscal application of anti-inflammatory drugs as promising therapeutics for disc degeneration. In parallel, the immunomodulatory role of MSCs on this model was also evaluated. Although a slight downregulation of IL-6 and IL-8 expression could be found, the variability among the five donors tested was high, suggesting that the beneficial effect of these cells on disc degeneration needs to be further evaluated. The proinflammatory/degenerative IVD organ culture model established can be considered a suitable approach for testing novel therapeutic drugs, thus reducing the number of animals in in vivo experimentation. Moreover, this model can be used to address the cellular and molecular mechanisms that regulate inflammation in the IVD and their implications in tissue degeneration.

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

Poly(γ-glutamic acid) and poly(γ-glutamic acid)-based nanocomplexes enhance type II collagen production in intervertebral disc

Intervertebral disc (IVD) degeneration often leads to low back pain, which is one of the major causes of disability worldwide, affecting more than 80% of the population. Although available treatments for degenerated IVD decrease symptoms’ progression, they fail to address the underlying causes and to restore native IVD properties. Poly(γ-glutamic acid) (γ-PGA) has recently been shown to support the production of chondrogenic matrix by mesenchymal stem/stromal cells. γ-PGA/chitosan (Ch) nanocomplexes (NCs) have been proposed for several biomedical applications, showing advantages compared with either polymer alone. Hence, this study explores the potential of γ-PGA and γ-PGA/Ch NCs for IVD regeneration. Nucleotomised bovine IVDs were cultured ex vivo upon injection of γ-PGA (pH 7.4) and γ-PGA/Ch NCs (pH 5.0 and pH 7.4). Tissue metabolic activity and nucleus pulposus DNA content were significantly reduced when NCs were injected in acidic-buffered solution (pH 5.0). However, at pH 7.4, both γ-PGA and NCs promoted sulphated glycosaminoglycan production and significant type II collagen synthesis, as determined at the protein level. This study is a first proof of concept that γ-PGA and γ-PGA/Ch NCs promote recovery of IVD native matrix, opening new perspectives on the development of alternative therapeutic approaches for IVD degeneration.

Mesenchymal Stem/Stromal Cells seeded on cartilaginous endplates promote Intervertebral Disc Regeneration through Extracellular Matrix Remodeling.

Intervertebral disc (IVD) degeneration is characterized by significant biochemical and histomorphological alterations, such as loss of extracellular matrix (ECM) integrity, by abnormal synthesis of ECM main components, resultant from altered anabolic/catabolic cell activities and cell death. Mesenchymal Stem/Stromal Cell (MSC) migration towards degenerated IVD may represent a viable strategy to promote tissue repair/regeneration. Here, human MSCs (hMSCs) were seeded on top of cartilaginous endplates (CEP) of nucleotomized IVDs of bovine origin and cultured ex vivo up to 3 weeks. hMSCs migrated from CEP towards the lesion area and significantly increased expression of collagen type II and aggrecan in IVD, namely in the nucleus pulposus. Concomitantly, hMSCs stimulated the production of growth factors, promoters of ECM synthesis, such as fibroblast growth factor 6 (FGF-6) and 7 (FGF-7), platelet-derived growth factor receptor (PDGF-R), granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin-like growth factor 1 receptor (IGF-1sR). Overall, our results demonstrate that CEP can be an alternative route to MSC-based therapies for IVD regeneration through ECM remodeling, thus opening new perspectives on endogenous repair capacity through MSC recruitment.

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

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