Macrophage-based therapy for intervertebral disc herniation: preclinical proof-of-concept.

Intervertebral disc (IVD) degeneration and herniation is a leading cause of disability globally and a large unmet clinical need. No efficient non-surgical therapy is available, and there is an urgency for minimally invasive therapies capable of restoring tissue function. IVD spontaneous hernia regression following conservative treatment is a clinically relevant phenomenon that has been linked to an inflammatory response. This study establishes the central role of macrophages in IVD spontaneous hernia regression and provides the first preclinical demonstration of a macrophage-based therapy for IVD herniation. A rat model of IVD herniation was used to test complementary experimental setups: (1) macrophage systemic depletion via intravenous administration of clodronate liposomes (Group CLP2w: depletion between 0 and 2 weeks post-lesion; Group CLP6w: depletion between 2 and 6 weeks post-lesion), and (2) administration of bone marrow-derived macrophages into the herniated IVD, 2 weeks post-lesion (Group Mac6w). Herniated animals without treatment were used as controls. The herniated area was quantified by histology in consecutive proteoglycan/collagen IVD sections at 2 and 6 weeks post-lesion. Clodronate-mediated macrophage systemic depletion was confirmed by flow cytometry and resulted in increased hernia sizes. Bone marrow-derived macrophages were successfully administered into rat IVD hernias resulting in a 44% decrease in hernia size. No relevant systemic immune reaction was identified by flow cytometry, cytokine, or proteomic analysis. Furthermore, a possible mechanism for macrophage-induced hernia regression and tissue repair was unveiled through IL4, IL17a, IL18, LIX, and RANTES increase. This study represents the first preclinical proof-of-concept of macrophage-based immunotherapy for IVD herniation.

Long non-coding RNA H19 regulates matrisome signature and impacts cell behavior on MSC-engineered extracellular matrices.

BACKGROUND: The vast and promising class of long non-coding RNAs (lncRNAs) has been under investigation for distinct therapeutic applications. Nevertheless, their role as molecular drivers of bone regeneration remains poorly studied. The lncRNA H19 mediates osteogenic differentiation of Mesenchymal Stem/Stromal Cells (MSCs) through the control of intracellular pathways. However, the effect of H19 on the extracellular matrix (ECM) components is still largely unknown. This research study was designed to decode the H19-mediated ECM regulatory network, and to reveal how the decellularized siH19-engineered matrices influence MSC proliferation and fate. This is particularly relevant for diseases in which the ECM regulation and remodeling processes are disrupted, such as osteoporosis. METHODS: Mass spectrometry-based quantitative proteomics analysis was used to identify ECM components, after oligonucleotides delivery to osteoporosis-derived hMSCs. Moreover, qRT-PCR, immunofluorescence and proliferation, differentiation and apoptosis assays were performed. Engineered matrices were decellularized, characterized by atomic force microscopy and repopulated with hMSC and pre-adipocytes. Clinical bone samples were characterized by histomorphometry analysis. RESULTS: Our study provides an in-depth proteome-wide and matrisome-specific analysis of the ECM proteins controlled by the lncRNA H19. Using bone marrow-isolated MSC from patients with osteoporosis, we identified fibrillin-1 (FBN1), vitronectin (VTN) and collagen triple helix repeat containing 1 (CTHRC1), among others, as having different pattern levels following H19 silencing. Decellularized siH19-engineered matrices are less dense and have a decreased collagen content compared with control matrices. Repopulation with naïve MSCs promotes a shift towards the adipogenic lineage in detriment of the osteogenic lineage and inhibits proliferation. In pre-adipocytes, these siH19-matrices enhance lipid droplets formation. Mechanistically, H19 is targeted by miR-29c, whose expression is decreased in osteoporotic bone clinical samples. Accordingly, miR-29c impacts MSC proliferation and collagen production, but does not influence ALP staining or mineralization, revealing that H19 silencing and miR-29c mimics have complementary but not overlapping functions. CONCLUSION: Our data suggest H19 as a therapeutic target to engineer the bone ECM and to control cell behavior.

The impact of matrix age on intervertebral disc regeneration.

With the lack of effective treatments for low back pain, the use of extracellular matrix (ECM)-based biomaterials have emerged with undeniable promise for IVD regeneration. Decellularized scaffolds can recreate an ideal microenvironment inducing tissue remodeling and repair. In particular, fetal tissues have a superior regenerative capacity given their ECM composition. In line with this, we unraveled age-associated alterations of the nucleus pulposus (NP) matrisome. Thus, the aim of the present work was to evaluate the impact of ECM donor age on IVD de/regeneration. Accordingly, we optimized an SDS (0.1 %, 1 h)-based decellularization protocol that preserves ECM cues in bovine NPs from different ages. After repopulation with adult NP cells, younger matrices showed the highest repopulation efficiency. Most importantly, cells seeded on younger scaffolds produced healthy ECM proteins suggesting an increased capacity to restore a functional IVD microenvironment. In vivo, only fetal matrices decreased neovessel formation, showing an anti-angiogenic potential. Our findings demonstrate that ECM donor age has a strong influence on angiogenesis and ECM de novo synthesis, opening new avenues for novel therapeutic strategies for the IVD. Additionally, more appropriate 3D models to study age-associated IVD pathology were unveiled.

Circulating microRNAs Correlate with Multiple Myeloma and Skeletal Osteolytic Lesions.

Multiple myeloma (MM) is the second most frequent hematological disease and can cause skeletal osteolytic lesions. This study aims to evaluate the expression of circulating microRNAs (miRNAs) in MM patients and to correlate those levels with clinicopathological features, including bone lesions. A panel of miRNAs associated with MM onset and progression, or with bone remodeling, was analyzed in the plasma of 82 subjects (47 MM patients; 35 healthy controls). Results show that miR-16-5p, miR-20a-5p, and miR-21-5p are differently expressed between MM patients and healthy controls. Receiver operating characteristic analyses indicate that their combined expression has potential as a molecular marker (Area Under the Curve, AUC of 0.8249). Furthermore, significant correlations were found between the analyzed miRNAs and disease stage, treatment, ß2 microglobulin, serum albumin and creatinine levels, but not with calcium levels or genetic alterations. In this cohort, 65.96% of MM patients had bone lesions, the majority of which were in the vertebrae. Additionally, miR-29c-3p was decreased in patients with osteolytic lesions compared with patients without bone disease. Interestingly, circulating levels of miR-29b-3p correlated with cervical and thoracic vertebral lesions, while miR-195-5p correlated with thoracic lesions. Our findings suggest circulating miRNAs can be promising biomarkers for MM diagnosis and that their levels correlate with myeloma bone disease and osteolytic lesions.

Therapeutic Strategies for IVD Regeneration through Hyaluronan/SDF-1-Based Hydrogel and Intravenous Administration of MSCs.

Intervertebral disc (IVD) degeneration involves a complex cascade of events, including degradation of the native extracellular matrix, loss of water content, and decreased cell numbers. Cell recruitment strategies for the IVD have been increasingly explored, aiming to recruit either endogenous or transplanted cells. This study evaluates the IVD therapeutic potential of a chemoattractant delivery system (HAPSDF5) that combines a hyaluronan-based thermoreversible hydrogel (HAP) and the chemokine stromal cell derived factor-1 (SDF-1). HAPSDF5 was injected into the IVD and was combined with an intravenous injection of mesenchymal stem/stromal cells (MSCs) in a pre-clinical in vivo IVD lesion model. The local and systemic effects were evaluated two weeks after treatment. The hydrogel by itself (HAP) did not elicit any adverse effect, showing potential to be administrated by intradiscal injection. HAPSDF5 induced higher cell numbers, but no evidence of IVD regeneration was observed. MSCs systemic injection seemed to exert a role in IVD regeneration to some extent through a paracrine effect, but no synergies were observed when HAPSDF5 was combined with MSCs. Overall, this study shows that although the injection of chemoattractant hydrogels and MSC recruitment are feasible approaches for IVD, IVD regeneration using this strategy needs to be further explored before successful clinical translation.

Modulation of the In Vivo Inflammatory Response by Pro- Versus Anti-Inflammatory Intervertebral Disc Treatments.

Inflammation is central in intervertebral disc (IVD) degeneration/regeneration mechanisms, and its balance is crucial to maintain tissue homeostasis. This work investigates the modulation of local and systemic inflammatory response associated with IVD degeneration/herniation by administration of PRO- versus ANTI-inflammatory treatments. Chitosan/poly-γ-glutamic acid nanocomplexes, known as pro-inflammatory (PRO), and soluble diclofenac, a non-steroidal anti-inflammatory drug (ANTI), were intradiscally administered in a rat IVD injury model, 24 h after lesion. Two weeks after administration, a reduction of disc height accompanied by hernia formation was observed. In the PRO-inflammatory treated group, IL-1ß, IL-6 and COX-2 IVD gene expression were upregulated, and loss of nucleus pulposus (NP) structure and composition was observed. Systemically, lower T-cell frequency was observed in the lymph nodes (LN) and spleen (SP) of the PRO group, together with an increase in CD4+ T cells subset in the blood (BL) and LN. In contrast, the ANTI-group had higher proteoglycans/collagen ratio and collagen type 2 content in the NP, while an increase in the frequency of myeloid cells, M1 macrophages and activated macrophages (MHCII+) was observed at the systemic level. Overall, this study illustrates the dynamics of local and systemic inflammatory and immune cell responses associated with intradiscal therapies, which will contribute to designing more successful immunomodulatory treatments for IVD degeneration.

Osteogenic, anti-osteoclastogenic and immunomodulatory properties of a strontium-releasing hybrid scaffold for bone repair.

Strontium (Sr) is known to stimulate osteogenesis, while inhibiting osteoclastogenesis, thus encouraging research on its application as a therapeutic agent for bone repair/regeneration. It has been suggested that it may possess immunomodulatory properties, which might act synergistically in bone repair/regeneration processes. To further explore this hypothesis we have designed a Sr-hybrid system composed of an in situ forming Sr-crosslinked RGD-alginate hydrogel reinforced with Sr-doped hydroxyapatite (HAp) microspheres and studied its in vitro osteoinductive behaviour and in vivo inflammatory response. The Sr-hybrid scaffold acts as a dual Sr2+ delivery system, showing a cumulative Sr2+ release of ca. 0.3 mM after 15 days. In vitro studies using Sr2+concentrations within this range (0 to 3 mM Sr2+) confirmed its ability to induce osteogenic differentiation of mesenchymal stem/stromal cells (MSC), as well as to reduce osteoclastogenesis and osteoclasts (OC) functionality. In comparison with a similar Sr-free system, the Sr-hybrid system stimulated osteogenic differentiation of MSC, while inhibiting the formation of OC. Implantation in an in vivo model of inflammation, revealed an increase in F4/80+/CD206+ cells, highlighting its ability to modulate the inflammatory response as a pro-resolution mediator, through M2 macrophage polarization. Therefore, the Sr-hybrid system is potentially an appealing biomaterial for future clinical applications.

Profiling the circulating miRnome reveals a temporal regulation of the bone injury response.

Bone injury healing is an orchestrated process that starts with an inflammatory phase followed by repair and remodelling of the bone defect. The initial inflammation is characterized by local changes in immune cell populations and molecular mediators, including microRNAs (miRNAs). However, the systemic response to bone injury remains largely uncharacterized. Thus, this study aimed to profile the changes in the plasma miRnome after bone injury and determine its biological implications. Methods: A rat model of femoral bone defect was used, and animals were evaluated at days 3 and 14 after injury. Non-operated (NO) and sham operated animals were used as controls. Blood and spleen were collected and peripheral blood mononuclear cells (PBMC) and plasma were separated. Plasma miRnome was determined by RT-qPCR array and bioinformatics Ingenuity pathway analysis (IPA) was performed. Proliferation of bone marrow mesenchymal stem/stromal cells (MSC) was evaluated by Ki67 staining and high-throughput cell imaging. Candidate miRNAs were evaluated in splenocytes by RT-qPCR, and proteins found in the IPA analysis were analysed in splenocytes and PBMC by Western blot. Results: Bone injury resulted in timely controlled changes to the miRNA expression profile in plasma. At day 3 there was a major down-regulation of miRNA levels, which was partially recovered by day 14 post-injury. Interestingly, bone injury led to a significant up-regulation of let-7a, let-7d and miR-21 in plasma and splenocytes at day 14 relative to day 3 after bone injury, but not in sham operated animals. IPA predicted that most miRNAs temporally affected were involved in cellular development, proliferation and movement. MSC proliferation was analysed and found significantly increased in response to plasma of animals days 3 and 14 post-injury, but not from NO animals. Moreover, IPA predicted that miRNA processing proteins Ago2 and Dicer were specifically inhibited at day 3 post-injury, with Ago2 becoming activated at day 14. Protein levels of Ago2 and Dicer in splenocytes were increased at day 14 relative to day 3 post-bone injury and NO animals, while in PBMC, levels were reduced at day 3 (albeit Dicer was not significant) and remained low at day 14. Ephrin receptor B6 followed the same tendency as Ago2 and Dicer, while Smad2/3 was significantly decreased in splenocytes from day 14 relative to NO and day 3 post-bone injury animals. Conclusion: Results show a systemic miRNA response to bone injury that is regulated in time and is related to inflammation resolution and the start of bone repair/regeneration, unravelling candidate miRNAs to be used as biomarkers in the monitoring of healthy bone healing and as therapeutic targets for the development of improved bone regeneration therapies.

Injectable hybrid system for strontium local delivery promotes bone regeneration in a rat critical-sized defect model.

Strontium (Sr) has been described as having beneficial influence in bone strength and architecture. However, negative systemic effects have been reported on oral administration of Sr ranelate, leading to strict restrictions in clinical application. We hypothesized that local delivery of Sr improves osteogenesis without eliciting detrimental side effects. Therefore, the in vivo response to an injectable Sr-hybrid system composed of RGD-alginate hydrogel cross-linked in situ with Sr and reinforced with Sr-doped hydroxyapatite microspheres, was investigated. The system was injected in a critical-sized bone defect model and compared to a similar Sr-free material. Micro-CT results show a trend towards higher new bone formed in Sr-hybrid group and major histological differences were observed between groups. Higher cell invasion was detected at the center of the defect of Sr-hybrid group after 15 days with earlier bone formation. Higher material degradation with increase of collagen fibers and bone formation in the center of the defect after 60 days was observed as opposed to bone formation restricted to the periphery of the defect in the control. These histological findings support the evidence of an improved response with the Sr enriched material. Importantly, no alterations were observed in the Sr levels in systemic organs or serum.

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.