The role of the complement system in disc degeneration and Modic changes.

Disc degeneration and vertebral endplate bone marrow lesions called Modic changes are prevalent spinal pathologies found in chronic low back pain patients. Their pathomechanisms are complex and not fully understood. Recent studies have revealed that complement system proteins and interactors are dysregulated in disc degeneration and Modic changes. The complement system is part of the innate immune system and plays a critical role in tissue homeostasis. However, its dysregulation has also been associated with various pathological conditions such as rheumatoid arthritis and osteoarthritis. Here, we review the evidence for the involvement of the complement system in intervertebral disc degeneration and Modic changes. We found that only a handful of studies reported on complement factors in Modic changes and disc degeneration. Therefore, the level of evidence for the involvement of the complement system is currently low. Nevertheless, the complement system is tightly intertwined with processes known to occur during disc degeneration and Modic changes, such as increased cell death, autoantibody production, bacterial defense processes, neutrophil activation, and osteoclast formation, indicating a contribution of the complement system to these spinal pathologies. Based on these mechanisms, we propose a model how the complement system could contribute to the vicious cycle of tissue damage and chronic inflammation in disc degeneration and Modic changes. With this review, we aim to highlight a currently understudied but potentially important inflammatory pathomechanism of disc degeneration and Modic changes that may be a novel therapeutic target.

Oestrogen and Vibration Improve Intervertebral Disc Cell Viability and Decrease Catabolism in Bovine Organ Cultures.

Postmenopausal women are at an increased risk for intervertebral disc degeneration, possibly due to the decrease in oestrogen levels. Low-magnitude, high-frequency vibration (LMHFV) is applied as a therapeutic approach for postmenopausal osteoporosis; however, less is known regarding possible effects on the intervertebral disc (IVD) and whether these may be oestrogen-dependent. The present study investigated the effect of 17ß-oestradiol (E2) and LMHFV in an IVD organ culture model. Bovine IVDs (n = 6 IVDs/group) were treated with either (i) E2, (ii) LMHFV or (iii) the combination of E2 + LMHFV for 2 or 14 days. Minor changes in gene expression, cellularity and matrix metabolism were observed after E2 treatment, except for a significant increase in matrix metalloproteinase (MMP)-3 and interleukin (IL)-6 production. Interestingly, LMHFV alone induced cell loss and increased IL-6 production compared to the control. The combination of E2 + LMHFV induced a protective effect against cell loss and decreased IL-6 production compared to the LMHFV group. This indicates possible benefits of oestrogen therapy for the IVDs of postmenopausal women undergoing LMHFV exercises.

Terminal Complement Activation Is Induced by Factors Released from Endplate Tissue of Disc Degeneration Patients and Stimulates Expression of Catabolic Enzymes in Annulus Fibrosus Cells.

Terminal complement complex (TCC) deposition was identified in human degenerated discs. To clarify the role of terminal complement activation in disc degeneration (DD), we investigated respective activating mechanisms and cellular effects in annulus fibrosus (AF) cells. Isolated cells from human AF, nucleus pulposus (NP), and endplate (EP) were stimulated with human serum alone or with zymosan and treated with either the C3 inhibitor Cp40 or the C5 antibody eculizumab. Complement activation was determined via anaphylatoxin generation and TCC deposition detection. Thereby, induced catabolic effects were evaluated in cultured AF cells. Moreover, C5 cleavage under degenerative conditions in the presence of AF cells was assessed. Zymosan-induced anaphylatoxin generation and TCC deposition was significantly suppressed by both complement inhibitors. Zymosan induced gene expression of ADAMTS4, MMP1, and COX2. Whereas the C3 blockade attenuated the expression of ADAMTS4, the C5 blockade reduced the expression of ADAMTS4, MMP1, and COX2. Direct C5 cleavage was significantly enhanced by EP conditioned medium from DD patients and CTSD. These results indicate that terminal complement activation might be functionally involved in the progression of DD. Moreover, we found evidence that soluble factors secreted by degenerated EP tissue can mediate direct C5 cleavage, thereby contributing to complement activation in degenerated discs.

Neoepitope fragments as biomarkers for different phenotypes of intervertebral disc degeneration.

Background: During the intervertebral disc (IVD) degeneration process, initial degenerative events occur at the extracellular matrix level, with the appearance of neoepitope peptides formed by the cleavage of aggrecan and collagen. This study aims to elucidate the spatial and temporal alterations of aggrecan and collagen neoepitope level during IVD degeneration. Methods: Bovine caudal IVDs were cultured under four different conditions to mimic different degenerative situations. Samples cultured after 1- or 8-days were collected for analysis. Human IVD samples were obtained from patients diagnosed with lumbar disc herniation (LDH) or adolescent idiopathic scoliosis (AIS). After immunohistochemical (IHC) staining of Aggrecanase Cleaved C-terminus Aggrecan Neoepitope (NB100), MMP Cleaved C-terminus Aggrecan Neoepitope (MMPCC), Collagen Type 1α1 1/4 fragment (C1α1) and Collagenase Cleaved Type I and II Collagen Neoepitope (C1,2C), staining optical density (OD)/area in extracellular matrix (OECM) and pericellular zone (OPCZ) were analyzed. Conditioned media of the bovine IVD was collected to measure protein level of inflammatory cytokines and C1,2C. Results: For the bovine IVD sections, the aggrecan MMPCC neoepitope was accumulated in nucleus pulposus (NP) and cartilage endplate (EP) regions following mechanical overload in the one strike model after long-term culture; as for the TNF-α induced degeneration, the OECM and OPCZ of collagen C1,2C neoepitope was significantly increased in the outer AF region after long-term culture; moreover, the C1,2C was only detected in conditioned medium from TNF-α injection + Degenerative loading group after 8 days of culture. LDH patients showed higher MMPCC OECM in NP and higher C1,2C OECM in AF region compared with AIS patients. Conclusions: In summary, aggrecan and collagen neoepitope profiles showed degeneration induction trigger- and region-specific differences in the IVD organ culture models. Different IVD degeneration types are correlated with specific neoepitope expression profiles. These neoepitopes may be helpful as biomarkers of ECM degradation in early IVD degeneration and indicators of different degeneration phenotypes.

Interleukin-1ß and cathepsin D modulate formation of the terminal complement complex in cultured human disc tissue.

PURPOSE: Formation of terminal complement complex (TCC), a downstream complement system activation product inducing inflammatory processes and cell lysis, has been identified in degenerated discs. However, it remains unclear which molecular factors regulate complement activation during disc degeneration (DD). This study investigated a possible involvement of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) and the lysosomal protease cathepsin D (CTSD). METHODS: Disc biopsies were collected from patients suffering from DD (n = 43) and adolescent idiopathic scoliosis (AIS, n = 13). Standardized tissue punches and isolated cells from nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) were stimulated with 5% human serum (HS) alone or in combination with IL-1ß, CTSD or zymosan. TCC formation and modulation by the complement regulatory proteins CD46, CD55 and CD59 were analysed. RESULTS: In DD tissue cultures, IL-1ß stimulation decreased the percentage of TCC + cells in AF and EP (P < 0.05), whereas CTSD stimulation significantly increased TCC deposition in NP (P < 0.01) and zymosan in EP (P < 0.05). Overall, the expression of CD46, CD55 and CD59 significantly increased in all isolated cells during culture (P < 0.05). Moreover, cellular TCC deposition was HS concentration dependent but unaffected by IL-1ß, CTSD or zymosan. CONCLUSION: These results suggest a functional relevance of IL-1ß and CTSD in modulating TCC formation in DD, with differences between tissue regions. Although strong TCC deposition may represent a degeneration-associated event, IL-1ß may inhibit it. In contrast, TCC formation was shown to be triggered by CTSD, indicating a multifunctional involvement in disc pathophysiology.

Can UVA-light-activated riboflavin-induced collagen crosslinking be transferred from ophthalmology to spine surgery? A feasibility study on bovine intervertebral disc.

BACKGROUND: Collagen cross-links contribute to the mechanical resilience of the intervertebral disc (IVD). UVA-light-activated riboflavin-induced collagen crosslinking (UVA-CXL) is a well-established and effective ophthalmological intervention that increases the mechanical rigidity of the collagen-rich corneal matrix in Keratoconus. This study explores the feasibility, safety and efficacy of translating this intervention in reinforcing the IVD. METHODS: Annulus fibrosus (AF) cells were isolated from bovine IVDs and treated with different combinations of riboflavin (RF) concentrations (0.05-8 mM) and UVA light intensities (0.3-4 mW/cm2). Metabolic activity (resazurin assay), cell viability (TUNEL assay), and gene expression of apoptosis regulators C-FOS and PT5 were assessed immediately and 24 hours after treatment. Biomechanical effects of UVA-CXL on IVDs were measured by indentation analysis of changes in the instantaneous modulus and by peel-force delamination strength analysis of the AF prior and after treatment. RESULTS: Different intensities of UVA did not impair the metabolic activity of AF cells. However, RF affected metabolic activity (p < 0.001). PT53 expression was similar in all RF conditions tested while C-FOS expression decreased 24 hours after treatment. Twenty-four hours after treatment, no apoptotic cells were observed in any condition tested. Biomechanical characterizations showed a significant increase in the annular peel strength of the UVA-CXL group, when compared to controls of UVA and RF alone (p < 0.05). UVA-CXL treated IVDs showed up to 152% higher (p < 0.001) instantaneous modulus values compared to the untreated control. CONCLUSION: This is the first study on UVA-CXL treatment of IVD. It induced significantly increased delamination strength and instantaneous modulus indentation values in intact IVD samples in a structure-function relationship. RF concentrations and UVA intensities utilized in ophthalmological clinical protocols were well tolerated by the AF cells. Our findings suggest that UVA-CXL may be a promising tool to reinforce the IVD matrix.

Terminal complement complex formation is associated with intervertebral disc degeneration.

PURPOSE: The complement system is a crucial part of innate immunity. Recent work demonstrated an unexpected contribution to tissue homeostasis and degeneration. This study investigated for the first time, in human disc tissues, the deposition profile of the complement activation product terminal complement complex (TCC), an inflammatory trigger and inducer of cell lysis, and its inhibitor CD59, and their correlation with the degree of disc degeneration (DD). METHODS: Disc biopsies were collected from patients diagnosed with DD (n = 39, age 63 ± 12) and adolescent idiopathic scoliosis (AIS, n = 10, age 17 ± 4) and compared with discs from healthy Young (n = 11, age 7 ± 7) and Elder (n = 10, age 65 ± 15) donors. Immunohistochemical detection of TCC and CD59 in nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) was correlated with age, Pfirrmann grade and Modic changes. RESULTS: Higher percentage of TCC+ cells was detected in the NP and EP of DD compared to Elder (P < 0.05), and in the EP of Young versus Elder (P < 0.001). In DD, TCC deposition was positively correlated with Pfirrmann grade, but not with Modic changes, whereas for Young donors, a negative correlation was found with age, indicating TCC's involvement not only in DD, but also in early stages of skeletal development. Higher CD59 positivity was found in AIS and DD groups compared to Young (P < 0.05), and it was negatively correlated with the age of the patients. CONCLUSION: TCC deposition positively correlated with the degree of disc degeneration. A functional relevance of TCC may exist in DD, representing a potential target for new therapeutics.

Interleukin-1ß More Than Mechanical Loading Induces a Degenerative Phenotype in Human Annulus Fibrosus Cells, Partially Impaired by Anti-Proteolytic Activity of Mesenchymal Stem Cell Secretome.

Mesenchymal stem/stromal cell (MSC)-based therapies for low back pain and intervertebral disc (IVD) degeneration have been emerging, despite the poor knowledge of their full mechanism of action. As failure of the annulus fibrosus (AF) is often associated with IVD herniation and inflammation, the objective of the present study was to investigate the impact of the MSC secretome on human AF cells exposed to mechanical loading and a pro-inflammatory environment. Human AF cells isolated from IVD biopsies from patients with adolescent idiopathic scoliosis (AIS) or disc degeneration (DD) were exposed to physiological cyclic tensile strain (CTS) for 72 h in a custom-made device, with or without interleukin (IL)-1ß medium supplementation. AF cells stimulated with CTS + IL-1ß were then treated with secretome from IL-1ß-preconditioned MSCs for 48 h. AF cell metabolic activity, gene expression, protein secretion, matrix metalloproteinase (MMP) activity, and tissue inhibitor of MMPs (TIMP) concentration were evaluated. Expanded AF cells from AIS and DD patients revealed similar metabolic activity and gene expression profiles. CTS stimulation upregulated collagen type I (COL1A1) expression, while IL-1ß significantly stimulated IL-6, IL-8, MMP-1, and MMP-3 gene expression and prostaglandin E2 production by AF cells but downregulated COL1A1. The combination of CTS + IL-1ß had a similar outcome as IL-1ß alone, accompanied by a significant upregulation of elastin. The MSC secretome did not show any immunomodulatory effect on CTS + IL-1ß-stimulated AF cells but significantly decreased MMP-1, MMP-2, MMP-3, and MMP-9, while increasing the production of TIMP-1. The obtained results demonstrate a stronger impact of the inflammatory milieu on human AF cells than upper physiologic mechanical stress. In addition, a new MSC mechanism of action in degenerated IVD consisting of the modulation of AF MMP activity was also evidenced, contributing to the advancement of knowledge in AF tissue metabolism.

Infrared attenuated total reflection spectroscopic surface analysis of bovine-tail intervertebral discs after UV-light-activated riboflavin-induced collagen cross-linking.

The tensile strength of the intervertebral disc (IVD) is mainly maintained by collagen cross-links. Loss of collagen cross-linking combined with other age-related degenerative processes contributes to tissue weakening, biomechanical failure, disc herniation and pain. Exogenous collagen cross-linking has been identified as an effective therapeutic approach for restoring IVD tensile strength. The current state-of-the-art method to assess the extent of collagen cross-linking in tissues requires destructive procedures and high-performance liquid chromatography. In this study, we investigated the utility of infrared attenuated total reflection (IR-ATR) spectroscopy as a nondestructive analytical strategy to rapidly evaluate the extent of UV-light-activated riboflavin (B2)-induced collagen cross-linking in bovine IVD samples. Thirty-five fresh bovine-tail IVD samples were equally divided into five treatment groups: (a) untreated, (b) cell culture medium Dulbecco's Modified Eagle's Medium only, (c) B2 only, (d) UV-light only and (e) UV-light-B2. A total of 674 measurements have been acquired, and were analyzed via partial least squares discriminant analysis. This classification scheme unambiguously identified individual classes with a sensitivity >91% and specificity >92%. The obtained results demonstrate that IR-ATR spectroscopy reliably differentiates between different treatment categories, and promises an excellent tool for potential in vivo, nondestructive and real-time assessment of exogenous IVD cross-linking.

GEORG SCHMORL PRIZE OF THE GERMAN SPINE SOCIETY (DWG) 2018: combined inflammatory and mechanical stress weakens the annulus fibrosus: evidences from a loaded bovine AF organ culture.

PURPOSE: The pathomechanism of annulus fibrosus (AF) failure is still unknown. We hypothesise that mechanical overload and an inflammatory microenvironment contribute to AF structural weakening. Therefore, the objective of this study was to investigate the influence of these factors on the AF, particularly the translamellar bridging network (TLBN) which connects the AF lamellae. METHODS: A bovine AF organ culture (AF-OC) model of standardised AF rings was used to study the individual and combined effects of cyclic tensile strain (CTS) and IL-1ß (1 ng/mL) culture medium supplementation. AF-OCs were analysed for PGE2 production (ELISA) and deposition of IL-6, COX-2, fibrillin, and MMP3 in the tissue (immunohistochemistry, IHC). The mechanical strength of the TLBN was evaluated using a peel test to measure the strength required to separate an AF segment along a lamellar bound. RESULTS: The combination of CTS + IL-1ß led to a significant increase in PGE2 production compared to Control (p < 0.01). IHC evaluations showed that the CTS + IL-1ß group exhibited higher production of COX-2 and MMP3 within the TLBN regions compared to the adjacent lamellae and a significant increase in IL-6 ratio compared to Control (p < 0.05). A significant decrease in the annular peel strength was observed in the CTS + IL1ß group compared to Control (p < 0.05). CONCLUSION: Our findings suggest that CTS and IL-1ß act synergistically to increase pro-inflammatory and catabolic molecules within the AF, particularly the TLBN, leading to a weakening of the tissue. This standardised model enables the investigation of AF/TLBN structure-function relationship and is a platform to test AF-focused therapeutics. These slides can be retrieved under Electronic Supplementary Material.

Mesenchymal Stromal Cell Secretome: Influencing Therapeutic Potential by Cellular Pre-conditioning.

Mesenchymal stromal cells (MSCs) are self-renewing, culture-expandable adult stem cells that have been isolated from a variety of tissues, and possess multipotent differentiation capacity, immunomodulatory properties, and are relatively non-immunogenic. Due to this unique set of characteristics, these cells have attracted great interest in the field of regenerative medicine and have been shown to possess pronounced therapeutic potential in many different pathologies. MSCs' mode of action involves a strong paracrine component resulting from the high levels of bioactive molecules they secrete in response to the local microenvironment. For this reason, MSCs' secretome is currently being explored in several clinical contexts, either using MSC-conditioned media (CM) or purified MSC-derived extracellular vesicles (EVs) to modulate tissue response to a wide array of injuries. Rather than being a constant mixture of molecular factors, MSCs' secretome is known to be dependent on the diverse stimuli present in the microenvironment that MSCs encounter. As such, the composition of the MSCs' secretome can be modulated by preconditioning the MSCs during in vitro culture. This manuscript reviews the existent literature on how preconditioning of MSCs affects the therapeutic potential of their secretome, focusing on MSCs' immunomodulatory and regenerative features, thereby providing new insights for the therapeutic use of MSCs' secretome.

Role of the Complement System in the Response to Orthopedic Biomaterials.

Various synthetic biomaterials are used to replace lost or damaged bone tissue that, more or less successfully, osseointegrate into the bone environment. Almost all biomaterials used in orthopedic medicine activate the host-immune system to a certain degree. The complement system, which is a crucial arm of innate immunity, is rapidly activated by an implanted foreign material into the human body, and it is intensely studied regarding blood-contacting medical devices. In contrast, much less is known regarding the role of the complement system in response to implanted bone biomaterials. However, given the increasing knowledge of the complement regulation of bone homeostasis, regeneration, and inflammation, complement involvement in the immune response following biomaterial implantation into bone appears very likely. Moreover, bone cells can produce complement factors and are target cells of activated complement. Therefore, new bone formation or bone resorption around the implant area might be greatly influenced by the complement system. This review aims to summarize the current knowledge on biomaterial-mediated complement activation, with a focus on materials primarily used in orthopedic medicine. In addition, methods to modify the interactions between the complement system and bone biomaterials are discussed, which might favor osseointegration and improve the functionality of the device.

Immunomodulation of Human Mesenchymal Stem/Stromal Cells in Intervertebral Disc Degeneration: Insights From a Proinflammatory/Degenerative Ex Vivo Model.

STUDY DESIGN: Ex vivo experimental study. OBJECTIVE: To investigate the effect of proinflammatory/degenerative intervertebral disc (IVD) microenvironment on the regenerative and immunomodulatory behavior of mesenchymal stem/stromal cells (MSCs), using an ex vivo model from bovine origin. SUMMARY OF BACKGROUND DATA: Low back pain is a cause of disability worldwide, most frequently associated with IVD degeneration and inflammation, and characterized by increased levels of inflammatory mediators, often disregarded. MSC-based therapies to low back pain have been advocated, but the involvement of inflammation in IVD remodeling mechanism, promoted by MSCs has not yet been explored. METHODS: Bovine IVD organ cultures of nucleus pulposus punches were stimulated with needle puncture and culture medium supplementation with 10 ng/mL of interleukin (IL)-1ß, to induce a proinflammatory/degenerative environment, as previously established. Human bone marrow-derived MSCs were cultured on top of transwells, placed above nucleus pulposus punches, for up to 16 days. MSCs were analyzed by screening cell viability/apoptosis, metabolic activity, migration, and inflammatory cytokines production in response to the proinflammatory environment. IVD extracellular matrix (ECM) remodeling, gene expression profile of IVD cells, and inflammatory cytokine profile in the presence of MSCs in basal versus proinflammatory conditions were also evaluated. RESULTS: Proinflammatory/degenerative IVD conditions did not affect MSCs viability, but promoted cell migration, while increasing IL-6, IL-8, monocyte chemoattractant protein-1, and prostaglandin E2 and reducing transforming growth factor-ß1 production by MSCs. MSCs did not stimulate ECM production (namely type II collagen or aggrecan) in neither basal nor inflammatory conditions, instead MSCs downregulated bovine proinflammatory IL-6, IL-8, and TNF-α gene expression levels in IL-1ß-stimulated IVDs. CONCLUSION: The present study provides evidence for an immunomodulatory paracrine effect of MSCs in degenerated IVD without an apparent effect in ECM remodeling, and suggest an MSCs mechanism-of-action dependent on a cytokine feedback loop. LEVEL OF EVIDENCE: 5.

Pro-inflammatory chitosan/poly(γ-glutamic acid) nanoparticles modulate human antigen-presenting cells phenotype and revert their pro-invasive capacity.

Anticancer immune responses depend on efficient presentation of tumor antigens and co-stimulatory signals provided by antigen-presenting cells (APCs). However, it is described that immature dendritic cells (DCs) and macrophages at the tumor site may have an immunosuppressive profile, which limits the activity of effector T cells and supports tumor progression. Therapeutic targeting of these innate immune cells, either aiming at their elimination or re-polarization towards an immunostimulatory profile, has been pointed as an attractive approach to control tumor progression. In the present work, we assessed the potential of Chitosan (Ch)/Poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) to modulate macrophages and DCs inflammatory profile and to impair their ability to promote cancer cell invasion. Interestingly, Ch/γ-PGA NPs, prepared by co-acervation method, induced an immunostimulatory DCs phenotype, enhancing the expression of the co-stimulatory molecules CD86, CD40 and HLA-DR, and the secretion of the pro-inflammatory cytokines TNF-α, IL-12p40 and IL-6. Furthermore, Ch/γ-PGA NPs re-educated IL-10-stimulated macrophages towards a pro-inflammatory profile, decreasing the expression of CD163 and promoting the secretion of IL-12p40 and TNF-α. These alterations in the immune cells phenotype promoted CD4+ and CD8+ T cell activation/proliferation and partially inhibited APCs' ability to induce colorectal cancer cell invasion. Overall, our findings open new perspectives on the use of Ch/γ-PGA NPs as an immunomodulatory therapy for antigen-presenting cells reprogramming, providing a new tool for anticancer therapies. STATEMENT OF SIGNIFICANCE: The immune system is responsible to detect and destroy abnormal cells preventing the development of cancer. However, the immunosuppressive tumor microenvironment can compromise the immune response favoring tumor progression. Thus, immune system modulation towards an immunostimulatory profile can improve anticancer therapies. This research focus on the development of chitosan/poly(γ-glutamic acid) nanoparticles (NPs) to modulate human antigen-presenting cells (APCs) phenotype and to counteract their pro-invasive capacity. Interestingly, Ch/γ-PGA NPs had a prominent effect in inducing macrophages and dendritic cells immunostimulatory phenotype, thus favoring T cell proliferation and inhibiting colorectal cancer cell invasion. We propose that their combination with other immunomodulatory drugs or conventional anticancer therapies can improve patients' outcome.

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.

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)-1ß. Df-NPs were internalized by IVD cells, down-regulating IL-6, IL-8, MMP1 and MMP3, and decreasing PGE2 production, compared with IL-1ß-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-1ß (IL-1ß, 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-1ß-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.