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Chitosan (Ch) has recently been used in different studies as a vaccine adjuvant with an ability to modulate the tumor microenvironment (TME). This systematic review aims to elucidate the added value of using Ch-based therapies for immunotherapeutic strategies in cancer treatment, through the exploration of different Ch-based formulations, their capacity to modulate immune cells in vitroand in vivo, and their translational potential for clinical settings. A systematic review was conducted on PubMed, following both inclusion and exclusion steps. Original articles which focused on the immunomodulatory role of Ch-based formulations in the TME were included, as well as its usage as a delivery vehicle for other immunomodulatory molecules. This review illustrates the added value of Ch-based systems to reshape the TME, through the modulation of immune cells using different Ch formulations, namely solutions, films, gels, microneedles and nanoparticles. Generally, Ch-based formulations increase the recruitment and proliferation of cells associated with pro-inflammatory abilities and decrease cells which exert anti-inflammatory activities. These effects correlated with a decreased tumor weight, reduced metastases, reversion of the immunosuppressive TME and increased survival in vivo. Overall, Ch-based formulations present the potential for immunotherapy in cancer. Nevertheless, clinical translation remains challenging, since the majority of the studies use Ch in formulations with other components, implicating that some of the observed effects could result from the combination of the individual effects. More studies on the use of different Ch-based formulations, complementary to standardization and disclosure of the Ch properties used are required to improve the immunomodulatory effects of Ch-based formulations in cancer.

IFN-γ therapy has been approved by the Food and Drug Administration (FDA) for the treatment of chronic granulomatous disease and severe malignant osteopetrosis. Despite the promising IFN-γ-based therapeutic applications, its limited success in clinical trials is related with limitations inherent to its molecular properties and with the difficulties to deliver it locally or with adequate periodicity to achieve a therapeutic effect. We have previously shown that chitosan (Ch)/poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) are immunostimulatory, impairing colorectal cancer cell invasion. Ch is a biocompatible cationic polysaccharide extensively studied and already approved for biomedical applications while γ-PGA is a poly(amino acid), biodegradable and negatively charged. Here, we evaluated the potential of Ch/γ-PGA NPs as vehicles for IFN-γ and their ability to modulate immune cells’ phenotype. In this study, Ch/IFN-γ/γ-PGA nanoparticles (IFN-γ-NPs) prepared by a co-acervation method, presenting a size of approximately 180 nm and a low polydispersity index, were tested for their immunomodulatory activity. These IFN-γ-NPs induced an immunostimulatory profile on dendritic cells (DCs) with increased cell surface costimulatory molecules and secretion of pro-inflammatory cytokines, including IL-6, IL-12p40 and TNF-α. IFN-γ-NPs also modulated the IL-10-stimulated macrophage profile, increasing their ability to secrete the pro-inflammatory cytokines IL-6, IL-12p40 and TNF-α. Concomitantly, these phenotypic alterations enhanced T cell proliferation. In addition, the ability of DCs and macrophages to induce colorectal cancer cell invasion was hampered in the presence of IFN-γ-NPs. Although the major observations were mediated by Ch/γ-PGA NPs, the incorporation of IFN-γ into NPs potentiated the expression of CD40 and CD86, and the impairment of colorectal cancer cell invasion. This work bridges the previously reported immunostimulatory capacity of Ch/γ-PGA NPs with their potential as carriers for immunomodulatory molecules, like IFN-γ, opening new avenues for their use in clinical settings.

Intervertebral disc (IVD) degeneration is characterized by an unbalanced cell catabolic/anabolic activity and cell death, resulting in the degradation of extracellular matrix components and water loss. Repopulating the IVD with new cells may help in recovering tissue homeostasis and reverting the degenerative process. In this study the regenerative potential of a hyaluronan (HA)-based chemoattractant delivery system able to recruit mesenchymal stem cells (MSCs) seeded on the cartilaginous endplate (CEP) of IVD was explored. A HA delivery system containing stromal cell derived factor-1 (SDF-1) (5 ng/μL) (HAPSDF5) was injected in the cavity of nucleotomized bovine discs. Human MSCs (1 × 106) were seeded on the opposite CEP and allowed to migrate for up to 21 days. Migration of fluorescently labelled MSCs from CEP toward the IVD was enhanced by HAPSDF5. Likewise, an increase in collagen type II was detected at earlier time points, whereas no effect on proteoglycan content within the nucleotomized IVDs was found. MSCs produced an increased concentration of pro-catabolic factors, such as interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1). Overall, this study demonstrates that HAPSDF5 increased MSC recruitment, while the higher number of recruited cells partially contributed to accelerate matrix remodeling in nucleotomized IVDs.

Lumbar disc herniation (LDH) is highly associated with inflammation in the context of low back pain. Currently, inflammation is associated with adverse symptoms related to the stimulation of nerve fibers that may lead to pain. However, inflammation has also been indicated as the main factor responsible for LDH regression. This apparent controversy places inflammation as a good prognostic indicator of spontaneous regression of LDH. This review addresses the molecular and cellular mechanisms involved in LDH regression, including matrix remodeling and neovascularization, in the scope of the clinical decision on conservative versus surgical intervention. Based on the evidence, a special focus on the inflammatory response in the LDH context is given, particularly in the monocyte/macrophage role. The phenomenon of spontaneous regression of LDH, extensively reported in the literature, is therefore analyzed here under the perspective of the modulatory role of inflammation.

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. StemCellsTranslationalMedicine2017;6:1029–1039

Optimized ratio in the codelivery of therapeutics is of crucial importance to promote the synergism rather than the antagonistic effects. In this study, a self-healing spongy coating was described to facilitate the surface-mediated delivery of drug “cocktails” proportionally. The formation of spongy structures within the coating was achieved by acidic treatment and freeze-drying. Various drug combinations can be readily integrated through wicking method and subsequent micropore self-healing. The ratio of drug loading can be precisely regulated by the composition of loading solution and the embedded drugs were released in proportion according to the initial ratio of drug combination.

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-6and IL-8expression 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 vivoexperimentation. 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.

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.

Cartilage damage and/or aging effects can cause constant pain, which limits the patient's quality of life. Although different strategies have been proposed to enhance the limited regenerative capacity of cartilage tissue, the full production of native and functional cartilaginous extracellular matrix (ECM) has not yet been achieved. Poly(γ-glutamic acid) (γ-PGA), a naturally occurring polyamino acid, biodegradable into glutamate residues, has been explored for tissue regeneration. In this work, γ-PGA's ability to support the production of cartilaginous ECM by human bone marrow mesenchymal stem/stromal cells (MSCs) and nasal chondrocytes (NCs) was investigated. MSC and NC pellets were cultured in basal medium (BM), chondrogenic medium (CM), and CM-γ-PGA-supplemented medium (CM+γ-PGA) over a period of 21 days. Pellet size/shape was monitored with time. At 14 and 21 days of culture, the presence of sulfated glycosaminoglycans (sGAGs), type II collagen (Col II), Sox-9, aggrecan, type XI collagen (Col XI), type X collagen (Col X), calcium deposits, and type I collagen (Col I) was analyzed. After excluding γ-PGA's cytotoxicity, earlier cell condensation, higher sGAG content, Col II, Sox-9 (day 14), aggrecan, and Col X (day 14) production was observed in γ-PGA-supplemented MSC cultures, with no signs of mineralization or Col I. These effects were not evident with NCs. However, Sox-9 (at day 14) and Col X (at days 14 and 21) were increased, decreased, or absent, respectively. Overall, γ-PGA improved chondrogenic differentiation of MSCs, increasing ECM production earlier in culture. It is proposed that γ-PGA incorporation in novel biomaterials has a beneficial impact on future approaches for cartilage regeneration.

Promoting angiogenesis in a damaged tissue is a major challenge for tissue regeneration. Recent findings in tissue engineering suggest that fibroblasts (FBs) play an important role in orchestrating the angiogenic process. Fibroblasts maintain the structural integrity of connective tissue by continuously secreting growth factors and extracellular matrix precursors, which are essential for endothelial cell (EC) adhesion and spreading, thus playing a crucial role in angiogenesis. We hypothesized that FBs immobilized in alginate gels grafted with the RGD peptidic sequence could influence the recruitment of ECs to improve vascularization. In this work, the modulation of immobilized human FBs within the 3D synthetic extracellular matrix was assessed. Experiments using cocultures of ECs and FBs in indirect contact as well as angiogenic assays were performed to assess the influence of FBs immobilized in RGD–alginate in ECs' viability, stabilization, sprouting, and assembly into capillary-like structures. This study demonstrates the ability of FBs immobilized within RGD–alginate microspheres to modulate and support capillary-like structures' assembly. These findings indicate that the microenvironment created by these stromal cells in the scaffold modulates capillary morphogenesis, thus stimulating angiogenesis in situ and can potentially be used in regenerative medicine in clinical scenarios where vascularization is essential.

Macrophages are phagocytic cells with great importance in guiding multiple stages of inflammation and tissue repair. By producing a large number of biologically active molecules, they can affect the behavior of other cells and events, such as the foreign body response and angiogenesis. Since protein adsorption to biomaterials is crucial for the inflammatory process, we addressed the ability of the pro-inflammatory molecule fibrinogen (Fg) to modulate macrophage behavior toward tissue repair/regeneration. For this purpose, we used chitosan (Ch) as a substrate for Fg adsorption. Freshly isolated human monocytes were seeded on Ch substrates alone or previously adsorbed with Fg, and allowed to differentiate into macrophages for 10 days. Cell adhesion and morphology, formation of foreign body giant cells (FBGC), and secretion of a total of 80 cytokines and growth factors were evaluated. Both substrates showed similar numbers of adherent macrophages along differentiation as compared with RGD-coated surfaces, which were used as positive controls. Fg did not potentiate FBGC formation. In addition, actin cytoskeleton staining revealed the presence of punctuate F-actin with more elongated and interconnecting cells on Ch substrates. Antibody array screening and quantification of inflammation- and wound-healing-related factors indicated an overall reduction in Ch-based substrates versus RGD-coated surfaces. At late times, most inflammatory agents were down-regulated in the presence of Fg, in contrast to growth factor production, which was stimulated by Fg. Importantly, on Ch+Fg substrates, fully differentiated macrophages produced significant amounts of macrophage inflammatory protein-1delta (MIP-1δ), platelet-derived growth factor-BB, bone morphogenetic protein (BMP)-5, and BMP-7 compared with Ch alone. In addition, other important factors involved in bone homeostasis and wound healing, such as growth hormone, transforming growth factor-β3, and insulin-like growth factor-binding proteins, as well as several angiogenic mediators, including endocrine gland-derived vascular endothelial factor, fibroblast growth factor-7, and placental growth factor, were significantly promoted by Fg. This work provides a new perspective on the inflammatory response in the context of bone repair/regeneration mediated by a pro-inflammatory protein (Fg) adsorbed onto a biomaterial (Ch) that does not otherwise exhibit osteogenic properties.

Successful wound healing is a process that has three overlying phases: inflammatory, proliferative and remodeling. Chronic wounds are characterized by a perpetuated inflammation that inhibits the proliferative and remodeling phases and impairs the wound healing. Macrophages are key modulators of the wound healing process. Initially, they are responsible for the wound cleaning and for the phagocytosis of pathogens and afterwards they lead to the resolution of the inflammatory response and they express growth factors important for angiogenesis and cytokines and growth factors needed for cell proliferation and deposition of extracellular matrix. The phenotype of the macrophage changes gradually throughout the healing process from the initial M1 pro-inflammatory phenotype characteristic of the acute response to the M2 pro-regenerative phenotype that allows an accurate tissue repair. In chronic wounds, M1 pro-inflammatory macrophages persist and impair tissue repair. As such, immunomodulatory biomaterials arise as promising solutions to accelerate the wound healing process. In this review, we discuss the importance of macrophages and their polarization throughout the different phases of wound healing; macrophage dysfunction in chronic wounds and the use of immunomodulatory biomaterials to overcome the critical problem of chronic wounds—the continued inflammatory phase that impairs healing.

An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an ‘ideal’ inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell–MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies.

The vascularization of new tissue within a reasonable time is a crucial prerequisite for the success of different cell- and material-based strategies. Considering that angiogenesis is a multi-step process involving humoral and cellular regulatory components, only in vivo assays provide the adequate information about vessel formation and the recruitment of endothelial cells. The present study aimed to investigate if neonatal human dermal fibroblasts could influence in vivo neovascularization. Results obtained showed that fibroblasts were able to recruit endothelial cells to vascularize the implanted matrix, which was further colonized by murine functional blood vessels after one week. The vessels exhibited higher levels of hemoglobin, compared with the control matrix, implanted without fibroblasts, in which no vessel formation could be observed. No significant differences were detected in systemic inflammation. The presence of vessels originated from the host vasculature suggested that host vascular response was involved, which constitutes a fundamental aspect in the process of neovascularization. Fibroblasts implanted within matrigel increased the presence of endothelial cells with positive staining for CD31 and for CD34 and the production of collagen influencing the angiogenic process and promoting the formation of microvessels. New strategies in tissue engineering could be delineated with improved angiogenesis using neonatal fibroblasts.

Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification.

The use of biomaterials to replace lost bone has been a common practice for decades. More recently, the demands for bone repair and regeneration have pushed research into the use of cultured cells and growth factors in association with these materials. Here we report a novel approach to engineer new bone using a transient cartilage scaffold to induce endochondral ossification. Chondrocyte/chitosan scaffolds (both a transient cartilage scaffold—experimental—and a permanent cartilage scaffold—control) were prepared and implanted subcutaneously in nude mice. Bone formation was evaluated over a period of 5 months. Mineralization was assessed by Faxitron, micro computed tomography, backscatter electrons, and Fourier transform infrared spectroscopy analyses. Histological analysis provided further information on tissue changes in and around the implanted scaffolds. The deposition of ectopic bone was detected in the surface of the experimental implants as early as 1 month after implantation. After 3 months, bone trabeculae and bone marrow cavities were formed inside the scaffolds. The bone deposited was similar to the bone of the mice vertebra. Interestingly, no bone formation was observed in control implants. In conclusion, an engineered transient cartilage template carries all the signals necessary to induce endochondral bone formation in vivo.

Self-assembled monolayers can be tailored with specific ligands to a certain protein and at the same time prevent the non-specific adsorption of other proteins. Cibacron Blue F3G-A (CB-thiol) was successfully immobilized onto tetra(ethylene glycol)-terminated alkanethiol (CB-thiol). The affinity of human serum albumin (HSA) to immobilized Cibacron Blue F3G-A was studied using mixed thiolate self-assembled monolayers on gold with different n-alkyl chain lengths and functional terminal groups (CH3-; OH- and tetra(ethylene glycol)). Surfaces were characterized using X-ray photoelectron spectroscopy and water contact angle measurements. Albumin adsorption and exchangeability of the adsorbed albumin molecules with other albumin molecules in solution were evaluated using 125I-radiolabeled HSA. Competitive adsorption between albumin and fibrinogen to the different self-assembled monolayers (SAMs) was also investigated. Results showed that the incorporation of CB-thiol on the monolayers does not increase the HSA adsorption and reversibility on the SAMs. However, although specific adsorption of HSA to the immobilized Cibacron Blue F3G-A was not demonstrated, the presence of CB-thiol decreases the affinity of fibrinogen to the OH-terminated SAMs.

In this study we demonstrate, for the first time, that dexamethasone and BMP2 stimulated alkaline phosphatase ALP activity in MRC5 fibroblasts, a cell line derived from human fetal lung. Previously we reported that the watersoluble matrix WSM of nacre obtained from the inner shell layer of the oyster Pinctada maxima, promoted an increase in ALP activity that was dosedependent. In this work, we show that the effect of WSM is also timedependent. As a comparison, the effect of WSM was also tested in bone marrow stromal cells because marrow and other bone surfacederived osteoblast stem cells have the inherent direct potential for osteogenesis. WSM promotes cell proliferation and ALP activity when tested with bone marrow cells in concentrations between 135 and 540 μg proteinmL. The effect of WSM on ALP activity of bone marrow stromal cells is similar to that obtained by dexamethasone. These results imply that MRC5 fibroblasts respond to differentiating factors that promote osteoblastic phenotype in bonederived cell cultures. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 57: 306–312, 2001

Osteoporosis is a systemic disease that results in loss of bone density and increased fracture risk, particularly in the vertebrae and the hip. This condition and associated morbidity and mortality increase with population ageing. Long noncoding (lnc) RNAs are transcripts longer than 200 nucleotides that are not translated into proteins, but play important regulatory roles in transcriptional and post-transcriptional regulation. Their contribution to disease onset and development is increasingly recognized. Herein, we present an integrative revision on the studies that implicate lncRNAs in osteoporosis and that support their potential use as therapeutic tools. Firstly, current evidence on lncRNAs involvement in cellular and molecular mechanisms linked to osteoporosis and its major complication, fragility fractures, is reviewed. We analyze evidence of their roles in osteogenesis, osteoclastogenesis, and bone fracture healing events from human and animal model studies. Secondly, the potential of lncRNAs alterations at genetic and transcriptomic level are discussed as osteoporosis risk factors and as new circulating biomarkers for diagnosis. Finally, we conclude debating the possibilities, persisting difficulties, and future prospects of using lncRNAs in the treatment of osteoporosis.

Despite the importance of immune cell–biomaterial interactions for the regenerative outcome, few studies have investigated how distinct three-dimensional biomaterials modulate the immune cell-mediated mesenchymal stem/stromal cells (MSC) recruitment and function. Thus, this work compares the response of varied primary human immune cell populations triggered by different model scaffolds and describes its functional consequence on recruitment and motility of bone marrow MSC. It was found that polylactic acid (PLA) and chitosan scaffolds lead to an increase in the metabolic activity of macrophages but not of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells or monocytes. PBMC and NK cells increase their cell number in PLA scaffolds and express a secretion profile that does not promote MSC recruitment. Importantly, chitosan increases IL-8, MIP-1, MCP-1 and RANTES secretion by macrophages while PLA stimulates IL-6, IL-8 and MCP-1 production, all chemokines that can lead to MSC recruitment. This secretion profile of macrophages in contact with biomaterials correlates with the highest MSC invasion. Furthermore, macrophages enhance stem cell motility within chitosan scaffolds by 44% but not in PLA scaffolds. Thus, macrophages are the cells that in contact with engineered biomaterials become activated to secrete bioactive molecules that stimulate MSC recruitment.

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,1and 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.2Results 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

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.

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain, a problem with a heavy economic burden, which has been increasing in prevalence as populations age. Deeper knowledge of the complex spatial and temporal orchestration of cellular interactions and extracellular matrix remodelling is critical to improve current IVD therapies, which have so far proved unsatisfactory. Inflammation has been correlated with degenerative disc disease but its role in discogenic pain and hernia regression remains controversial. The inflammatory response may be involved in the onset of disease, but it is also crucial in maintaining tissue homeostasis. Furthermore, if properly balanced it may contribute to tissue repair/regeneration as has already been demonstrated in other tissues. In this review, we focus on how inflammation has been associated with IVD degeneration by describing observational and in vitrostudies as well as in vivoanimal models. Finally, we provide an overview of IVD regenerative therapies that target key inflammatory players.

In this study, 125I-radiolabelling was explored to follow the kinetics and isotherm of fibronectin (FN) adsorption to porous polymeric scaffolds, as well as to assess the elution and exchangeability of pre-adsorbed FN following incubation in serum-containing culture medium. Chitosan (CH) porous scaffolds with two different degrees of acetylation (DA 4% and 15%) were incubated in FN solutions with concentrations ranging from 5 to 50 µg/mL. The kinetic and isotherm of FN adsorption to CH were successfully followed using 125I-FN as a tracer molecule. While on DA 4% the levels of adsorbed FN increased linearly with FN solution concentration, on DA 15% a saturation plateau was attained, and FN adsorbed amounts were significantly lower. These findings were supported by immunofluorescent studies that revealed, for the same FN solution concentration, higher levels of exposed cell-binding domains on DA 4% as compared with DA 15%. Following incubation in serum containing medium, DA 4% also revealed higher ability to exchange pre-adsorbed FN by new FN molecules from serum than DA 15%. In accordance, when assessing the efficacy of passively adsorbed FN to promote endothelial cell (EC) adhesion to CH, ECs were found to adhere at higher levels to DA 4% as compared with DA 15%, 5 µg/mL of FN being already efficient in promoting cell adhesion and cytoskeletal organization on CH with DA 4%. Taken together the results show that protein radiolabelling can be used as an effective tool to study protein adsorption to porous polymeric scaffolds, both from single and complex protein solutions.