WO2023075623A1 - Macrophage phenoypes for use in the treatment of intravertebral discs - Google Patents

Abstract

The present invention refers to monocytes and macrophages for use in the treatment of intravertebral disc (IVD) herniation. Furthermore, the macrophage cell phenotype which is most effective for use in a phagocytosis immunotherapy is herein disclosed. Thus, the present invention provides compositions comprising such macrophage phenotypes and a method for differentiation, polarization, and purification of the most effective macrophage phenotype, as well as its preferred form of administration. Another aspect of the present invention refers to the compositions employed in the said polarization of the most effective macrophage phenotype. Yet another aspect of the present invention refers to the compositions employed in the purification of the most effective macrophage phenotype through negative selection. The uses, compositions and methods of the present invention can be advantageously employed in a cell-based therapy primarily for use in the treatment of intervertebral disc (IVD) herniation, as well as for other clinical conditions improved by the regression of regeneration-adverse reactive tissues, such as wound and surgery- related scar tissue, fibrosis and necrosis; the said clinical condition being selected from the list further consisting of myocardial infraction, endometriosis, pulmonary asthma, hepatic cirrhosis, spinal cord injury and cartilage injury.

Description

DESCRIPTION

MACROPHAGE PHENOTYPES FOR USE IN THE TREATMENT OF INTERVERTEBRAL

DISC

Technical field of the invention

The present invention relates to adoptive macrophage transfer via collection, ex vivo differentiation and polarization and subsequent cell administration, falling in the scope of cell-based immunotherapy .

State of the art

Intervertebral disc (IVD) herniation is highly associated to low back pain, is a highly disabling condition and a main cause of spinal surgery worldwide (GBD2017, 2018; Lorio et al. , 2020) . The intervertebral disc (IVD) is a unique tissue that lays between the vertebrae and warrants the structural stability of the spine. IVD herniation consists in the disruption of the annulus fibrosus (AF) , extrusion of the nucleus pulposus (NP) , release of inflammatory mediators and stimulation of spinal cord nerve fibers, leading to inflammation and pain. Symptoms originated by IVD herniation may disappear without any surgical treatment and in some of these patients this is accompanied by a reduction of the size of disc herniation on magnetic resonance imaging (MRI) . This phenomenon is known as spontaneous hernia regression. Clinical evidence shows that the regression may be partial or complete, with larger hernias being more prone to resorption (Chiu et al. , 2015) and that the level most commonly affected is L4-L5 (Macki et al. , 2014) . The first clinical approach to IVD herniation is nonsurgical, including physical therapy, nonsteroidal anti-inflammatory drugs, analgesics, and/or corticosteroid injections (Lorio et al. , 2020) , however this implies long periods of immobilization and great uncertainty in the outcome. Surgeries such as microdiscectomy provide faster pain relief but have complications associated such as neurological injury and high incidence of recurrence and revision surgery (Benzakour and Benzakour, 2019) . Decision on conservative versus surgical management of IVD herniation is mostly patient-clinician dependent, this is because the mechanisms behind spontaneous hernia regression are unclear and also because the prognosis of regression is uncertain. Understanding IVD hernia regression represents a major challenge in the spine. The main hypothesis behind spontaneous IVD hernia regression involves exposure of the herniated disc to the epidural vascular supply through the ruptured posterior longitudinal ligament, activation of immune cells that initiate a cascade of inflammation, angiogenesis and extracellular matrix (ECM) remodeling (reviewed in Cunha et al. , 2018) .

Macrophage infiltration has been consistently identified in herniated histological samples and especially in the sequestration subtype of IVD herniation, rather than subligamentous one, which is in accordance with the clinical evidence showing that sequestered hernias are more likely to regress (Ikeda et al. , 1996) . Using a rat IVD herniation model (Cunha et al. , 2017b) , the presence of CD68+ macrophages in the hernias was consistently observed, as well as of Pax5+ B-lymphocytes , and the percentage of macrophages is proportional to the hernia size (Cunha et al. , 2017a; Cunha et al. , 2017b) . Macrophages present in the hernia most likely result from blood monocyte recruitment (Kawakubo et al. , 2020) and what regulates the fate of these cells and to what extent they can assume the identity and function of resident macrophages is still unclear. Macrophages are known to promote homeostasis, not only as phagocytes, but also through trophic, regulatory, and repair functions. These highly plastic cells can display a range of phenotypes regulated by their microenvironment (including inflammatory cytokines and ECM composition) , which span from the classical pro-inflammatory Ml to the alternatively activated anti-inflammatory M2 phenotype (Locati et al. , 2020) . It is possible that the Ml phenotype may be more associated with increased IVD degeneration (Nakazawa et al. , 2018) but the detailed macrophage profile within the IVD remains undiscovered and a direct demonstration of macrophage role in IVD herniation is still missing.

Summary of the Invention

This study proposes compositions and methods to enable the macrophage potential for restoring intravertebral disc (IVD) homeostasis and hernia resolution, which may be relevant to the clinical practice. Ultimately, such therapy may improve human lifelong health and wellbeing and result in an outstanding innovation in a field central to lower back pain (LBP) .

Herein disclosed is an autologous cell-based therapy primarily for use in the treatment of intervertebral disc (IVD) herniation. This therapy consists of the specific monocyte and macrophage cell phenotypes which are most effective for use in IVD hernia phagocytosis and regression, as a cell immunotherapy. Thus, herein disclosed are monocytes and macrophages polarized to the M2 phenotype, more preferably to the M2a phenotype, for use in the treatment of IVD herniation, as well as other clinical conditions comprising regeneration-adverse reactive tissues, such as wound and surgery-related scar tissue, fibrosis and necrosis; the said clinical condition being selected from the list consisting of myocardial infraction, endometriosis, pulmonary asthma, hepatic cirrhosis, spinal cord injury and cartilage injury.

The present invention further provides a method for differentiation, polarization, and purification of the most effective macrophage phenotype, as well as a method of administration. Another aspect of the present invention refers to the compositions employed in the said polarization of the most effective macrophage phenotype .

Yet another aspect of the present invention refers to the compositions employed in the purification of the most effective macrophage phenotype through negative selection.

Detailed Description

The present invention relates to discloses the role of macrophages in IVD hernia regression in vivo using a well-established rat IVD herniation model and adoptive transfer of bone-marrow derived macrophages locally into the hernia (Figure 1) . In this embodiment, a lesion is performed in 3 consecutive coccygeal IVDs and unpolarized macrophages are differentiated from syngenic rat bone marrow precursors and adoptively transferred into the rat IVD hernia site. The animals are sacrificed at 6 weeks post-lesion (Mac6w) . Control groups without any treatment other than the lesion, are sacrificed at 2 (L2w) and 6 (L6w) weeks post-lesion. A detailed local and systemic analysis is performed to assess the functional potential of macrophage administration for in vivo hernia regression. Histological analysis consisting in delimitation of the hernia area throughout the IVD by proteoglycan/collagen staining reveals a statistically significant 44% decrease in the hernia sizes in the group in which macrophages were administered (Figure 2A) . There is also a decrease in the relative proportion of proteoglycans/collagen within the hernia in this group (Figure 2B) . Also, the number of macrophages present in the hernia and at 6 weeks post-lesion is lower in the macrophage group (Figure 2) .

The systemic effect of macrophage administration to the hernia is analysed in the blood and spleen. Flow cytometry analysis indicates a significant reduction (p<0.05) in the proportion of CD172+ myeloid cells in the blood, in the group administered with macrophages. No other significant alterations are found in the proportion of T cells or B cells (Figure 3A) . This decrease in macrophages in the blood might indicate a compensation mechanism in which decreased percentages of macrophages in the blood may be a consequence of the abnormally high number of macrophages in the IVD. Flow cytometry analysis of the spleen doesn't show any significant alterations in the proportion of immune cell populations associated to macrophage administration, but significant increases in myeloid and B cell proportions is detected at 6 weeks post-injury (Figure 3B) . There seems to be an enlargement of the spleen in the group administered with macrophages at 6 weeks post-injury, when compared with the control group 2 weeks post-injury, but this difference is not significant when compared with the control group 6 weeks post-injury (Figure 3C) . Immunohistochemistry analysis in the spleen shows a decrease in CD68+ macrophages in the Mac6w group, although not statistically significant. Furthermore, in proteomic analysis performed in the plasma by LC-MS/MS, the Principal Component Analysis does not separate the Mac6w and L6w groups, indicating that the majority of the proteins found in the plasma are unaltered by macrophage administration.

In another embodiment, human IVD cells with polarized macrophages are employed. Monocytes can be isolated from donors and monocytes polarized into MO, Ml, M2a, M2c and M2d macrophage phenotypes using specific combinations of cytokines in the culture media. The differently polarized macrophages co-cultured with human IVD surgical samples collected from patients undergoing microdiscectomy using transwell inserts allow to analyse the effect of each macrophage phenotype in herniated tissue. A phagocytosis assay conducted to determine the capability of each of the macrophage phenotype to resorb the herniated IVD tissue (Figure 4) indicates that, despite all macrophage phenotypes display similar number of phagocytosis bioparticles, higher area of phagocytic blobs is seen in macrophages of the M2 phenotype, more specifically, macrophages of the M2a phenotype. Thus, herein disclosed are macrophages of the M2a phenotype, as the functional cell phenotype which is most effective for use in a therapy for IVD hernia regression through a macrophagebased phagocytosis immunotherapy.

Based on this, it's possible to develop a method in which human primary monocytes are isolated from buffy coats, either from previously frozen peripheral blood mononuclear cells or from washed leukapheresis samples. In another embodiment, monocytes can be obtained from the bone marrow. Protocols know in the art for isolation of monocytes by negative selection are then employed, such as the RosetteSep monocyte enrichment cocktail (from StemCell Technologies) . Briefly, in one embodiment, buffy coats are centrifuged at room temperature for 20 min at 1200 g, without active acceleration or brake, for blood components separation. Peripheral blood mononuclear cell (PBMC) layer is collected and incubated with RosetteSep human monocyte enrichment isolation kit (from StemCell Technologies) for 20 min, under gentle mixing, according to the manufacturer' s instructions. The mixture is then diluted at a 1:1 ratio with 2% fetal bovine serum (BBS) in phosphate buffered saline (PBS) , gently layered over Histopaque-1077 (Sigma) and centrifuged as described above. The enriched monocyte layer is collected, and washed with PBS for platelet depletion, by centrifugation at 700 rpm for 17 min. Recovered monocytes are then seeded on 24-well plates in culture media (RPMI-1640 + Glutamax) supplemented with 10% heat inactivated Fetal Bovine Serum and 1% penicillin G-streptomycin (P/S) in the presence of 50ng/ml of macrophage colony-stimulating factor (M-CSF) and maintained in a humidified incubator, at 37 °C and with 5% CO₂. In another embodiment, isolated monocytes and macrophages can be polarized towards the M2a phenotype by supplementing the culture media comprising the cytokines selected from the list consisting of IL4 and IL-13.

In another embodiment, cultured polarized macrophages may be further purified by negative selection using antibodies selected from the list consisting of antibodies against human CD68, CD64, CD62L, CCR2, HLA-DR, CD80, CDllc, CD86, TLR2, TLR4, iNOS, pSTATl, CD163, TLR1, TLR8 and combinations thereof. Through conjugation with magnetic beads or another solid phase cells expressing any of these cell surface markers are captured, leaving the M2a monocytes untouched and free in solution.

Purified Macrophages are then put in contact with human IVD tissue or other tissues.

The purified macrophages can resuspended an appropriate vehicle comprising saline solution, polymer, hydrogel and a carrier biomaterial .

Each administration consists most preferably in IxlO⁶ cells in vehicle and is administered locally in the hernia site using a 31-G needle coupled to a microsyringe and using an adaptor to assure 2.5 mm depth administration to the hernia site.

This strategy is intended to degrade the IVD tissue using immune cells. Immune cells, particularly macrophages, have the risk of immune rejection and overall safety, which needs to be accounted for. For this reason, the herein disclosed use refers to use of monocyte- derived autologous macrophages isolated from the own patients' blood or bone marrow. In this way, potential immune rejection is minimized and indeed no local adverse effect is observed. Also, a minimally invasive percutaneous administration route is disclosed, which is the standard used in IVD cell therapy clinical trials. In regard to safety, a thorough analysis of possible systemic effects resulting from macrophage IVD hernia administration is conducted and the results are very positive in the sense that no side effects are observed in the blood, plasma or spleen (Figure 3) . This holds great promise for the safety and efficacy of such therapy. The advantage of the herein disclosed strategy over other cell therapies is that the natural physiological mechanisms of spontaneous hernia resorption are explored to potentiate hernia regression

The herein disclosed monocytes and macrophages, selected or polarized to the specific M2 phenotype, more preferably M2a, for use in the treatment of IVD herniation, may also be used in the treatment of other clinical conditions comprising regeneration-adverse reactive tissues, such as wound and surgery-related scar tissue, fibrosis and necrosis; the said clinical condition being selected from the list consisting of myocardial infraction, endometriosis, pulmonary asthma, hepatic cirrhosis, spinal cord injury and cartilage injury.

Brief Description of the Figures

Figure 1 : Experimental setup for macrophage local delivery for IVD hernia regression. One group of animals received macrophage administration at 2 weeks post-lesion and was sacrificed at 6 weeks post-lesion (Group Mac6w, n=6) . Control groups consist in lesioned animals sacrificed at 2w (L2w, n=6) and 6w (L6w, n=6) post-lesion, without macrophage administration.

In animal experiments of IVD lesion and herniation model, male Lewis (LEW/Crl) rats at 2-3 months of age were used for the IVD caudal herniation model, as previously described (Cunha et al. , 2017b) (Figure 1) . This inbred strain was used to reduce possible allogeneic cell rejection. Briefly, the animals were anaesthetized by isoflurane inhalation, placed in prone position and the tail skin was disinfected with ethanol. A percutaneous 21-G needle puncture was performed in the coccygeal IVDs Co5/6, Co6/7 and Co7 / 8 , using radiography for IVD identification. For the experiment, one group of animals received macrophage administration at 2 weeks post-lesion and sacrificed at 6 weeks post-lesion (Group Mac6w, n=6) . Control groups consist in lesioned animals sacrificed at 2w (L2w, n=6) and 6w (L6w, n=6) post lesion, without macrophage administration.

For macrophage isolation and administration into the rat IVD hernia, isolated monocytes are cultured ex vivo in a specific differentiation media, supplemented with rat recombinant colony stimulating factor (rrM-CSF) , for macrophage differentiation or for directing differentiation towards specific macrophage phenotypes. Unpolarized adherent and non-adherent cells are collected and resuspended an appropriate vehicle (saline solution, polymer, hydrogel, carrier biomaterial ) .

Each administration consists most preferably in IxlO⁶ cells in 10pl vehicle and is administered locally in the hernia site 2 weeks after IVD lesion, using a 31-G needle coupled to a microsyringe (Hamilton) and using an adaptor to assure 2.5 mm depth administration to the hernia site.

Figure 2: Hernia histopathological analysis 2- and 6-weeks postinjury and macrophage administration. A) Quantification of the hernia area (mm2) across the depth of all sections of an IVD with visible herniation, B) Proteoglycans/collagen content ratio in the hernia, C) Quantification of CD68+ cells within the hernia (positive cells are shown in brown, scale bar: 200pm) . Results are presented as box and whiskers plots. **p<0.001.

Each functional spinal unit (one IVD and two adjacent vertebrae) are collected and fixated in 10% neutral buffered formalin for 1 week at room temperature and decalcifying solution. Tissue is decalcified in EDTA-glycerol solution and processed for paraffin embedding. Sequential transversal 5-pm sections of the IVD are collected. Sections are deparaffinized in xylene, rehydrated through a graded series of ethanol and processed for Alcian blue/Picrosirius red staining and immunohistochemistry.

For Alcian blue/Picrosirius red staining, rehydrated sections are incubated in Weigert's Iron Hematoxylin for 10 min, washed in tap water and stained in Alcian blue solution pH 2.5 for 30 min. After rinsing in tap water, sections are incubated in Picrosirius red solution (0.1g Sirius red in 100 mL of saturated aqueous picric acid solution) for 1 hour, followed by wash in 0.01N HC1 for 2 minutes. Sections are dehydrated, mounted with Entellan (Merck) and analysed in a CX31 optical microscope equipped with a DP25 digital colour camera (Olympus) .

Hernia and NP area is determined by delimitating regions of interest (ROI) in each optical section, considering blue staining for proteoglycans and red staining for collagen. Hernia area is calculated as the mean of areas of each individual section throughout the IVD. Within hernia and NP ROIs, the % area of proteoglycans and collagen is determined by a custom ImageJ macro based on a color deconvolution technique used to separate the different color channels .

Immunohistochemistry (IHC) for detection of CD68+cells in the hernia is performed by the NovolinkTM Polymer Detection Kit (Leica Biosystems) , following the manufacturer's instructions. Antigen retrieval is performed through incubation in near-boiling point 10 mmol/L sodium citrate buffer, pH 6.0, for 1 minute. Sections are incubated with anti-CD68 antibody (clone EDI, 1: 100 dilution, BioRad Laboratories) , overnight at 4°C. The stained sections are imaged with light microscopy. CD68+ cells are quantified using ImageJ tools directly on the acquired images. From these, the %CD68 positivity is normalized to the total number of cells in the hernia.

Figure 3: Systemic immune response to macrophage administration to the herniated IVD . A) Blood immune cell populations profile by flow cytometry. B) Spleen immune cell populations profile by flow cytometry. C) Quantification of spleen length. D) Immunohistochemistry for CD68+ cells in the spleen. Scale bar: 200pm. Results are presented as box and whiskers plots. *p<0.05, **p<0.001, ***p<0.0001.

Animals are further dissected for collection of draining lymph nodes (iliac and inguinal) and spleen. Half of the draining lymph nodes was processed for histology and the other half was immediately processed for flow cytometry analysis. The spleen was photographed and half was processed for histology and half was immediately processed for flow cytometry analysis. Blood is centrifuged at 800g, for 20 min at room temperature and then plasma and buffy coats were separately collected. Plasma is further centrifuged twice at 2500g for 15min at 4°C to remove cell debris and kept at -80°C for proteomic analysis. Collected buffy coats were diluted with PBS, overlaid on Lymphoprep in a 1:1 ratio and centrifuged at 800g, for 30 min at room temperature, without brake, to isolate peripheral blood mononuclear cells (PBMC) . Lymph node cells are isolated by mechanical dissociation of lymph nodes on a 100-pm pore cell strainer. Spleen cells are collected by a similar process, injecting 100 U/mL Collagenase type I (Sigma) before mechanical dissociation. Red blood cells in spleen cell suspension are further lysed by incubation with NH₄C1 150 mM in Tris 10 mM solution for 8 min, at 37°C.

Flow cytometry analysis is performed for main immune cell populations, namely myeloid and lymphocytic (T, B and NK cells) lineages, with panels of f luorochrome-con ugated antibodies for cell surface lineage and activation markers. Staining is performed in 96- well plates in FACS buffer (0.5% BSA, 0.01% sodium azide, PBS) for 30 min on ice, after Fc receptors blocking. The following anti-rat antibodies are used: Mixl: CD161a(clone 10/78 ) -BB700, CD172 (clone OX-41) -BV421, CD3 (clone IF4) -APC, CD45R (clone HIS24) -PE, CDllb/c (clone OX-42 ) -PE-Cy7 , major histocompatibility complex class II (MHCII) (clone OX-6) -BV510, CD8a (clone OX-8) -FITC, CD4 (clone OX- 35) -APC-Cy7; Mix2 : CD172 (clone OX-41 ) -BV421 , CD86 (clone 24F) -PE, CDllb/c (clone OX-42 ) -PE-Cy7 , CD40 (clone HM40-3 ) -FITC, major histocompatibility complex class II (MHCII) (clone OX-6) -PerCP, CD163 (clone ED2 ) -DyLight®650. The CD163 is previously conjugated with DyLight®650 using the LYNX Rapid Plus Antibody Conjugation Kit, according to manufacturer's instructions. All antibodies can be obtained from commercial sourced, such as BD, except CD163 and conjugation kit, which are from BioRad. Cells are further incubated with the fixable viability dye eFluor®780 (Invitrogen) . Samples are acquired on a flow cytometer (FACSCanto II; BD) and data analyzed with FlowJo software version 8.7 (FlowJo, Ashland) .

Figure 4 : Phagocytosis assays performed on Human IVD tissue in Macrophage- IVD direct co-culture. The assay indicates that, despite all macrophage phenotypes display similar number of phagocytosis bioparticles, higher area of phagocytic blobs is seen in macrophages of the M2 phenotype, more specifically, macrophages of the M2a phenotype .

For Human IVD tissue collection, degenerated human lumbar IVD fragments, with grade III-IV (Pfirrmann scale) , are isolated from patients undergoing microdiscectomy, after informed consent and ethics committee approval. The surgeries are performed under general anesthesia in a sterile operating room and prophylactic antibiotic therapy with cefazolin 1 g IV was given. Patients are placed in a knee-chest position and a standard posterior approach to the appropriate interlaminar window is undertaken. After removal of the yellow ligament and medial retraction of the nerve root, the disc herniation is exposed. The posterior longitudinal ligament and the annulus fibrosus (AF) are incised and IVD fragments are excised, as deemed necessary by the surgeon. No more disc samples than those considered clinically appropriate are collected. In cases where the disc fragment is in contact with the epidural space or indistinguishable from the AF, the excised tissue is not included in these assays. Thus, AF-contained IVD fragments are collected. The collected fragments are punched with a 4mm puncher to uniformize the samples .

For Macrophage- IVD direct co-culture, after monocyte isolation, differentiation and polarization towards the MO, Ml, M2a, M2c and M2d phenotypes by supplementing the culture media with the following cytokines: IFNy (lOOpg/mL) , TNFa (lOOpg/mL) , IL-4 (lOOpg/mL) , IL-10 (lOOpg/mL) , IL-6 (lOOpg/mL) and LPS (Img/mL) , as follows: MO (no supplement) , Ml (LPS+IFNy) , M2a (IL4 and IL-13) , M2c (IL10 and TGF- p) , M2d (IL6 and TRLa) , the freshly collected IVD samples are introduced into the cell culture by placing the samples directly on top of the macrophages. The direct co-cultures are maintained for 3 days in RPMI-1640 media and incubated in a humidified incubator, at 37 °C and with 5% CO2.

For Phagocytosis assays, phagocytosis is assessed by using the pHrodo™ Deep Red BioParticles™ conjugates for Phagocytosis ( Invitrogen) , according to manufacturer's instructions. Briefly, the differently polarized macrophages are incubated with the pHrodo™ BioParticles™ Conjugates for 3h at 37°C, which increase intracellular fluorescence as the pH becomes more acidic. Cytochalasin D is used as a negative control phagocytosis inhibitor. Cells are imaged using a Leica DMI6000 FFW fluorescence microscope (Leica Microsystems) . Results are presented as median ± interquartile range (IQR) in box and whiskers plots. Statistical analysis is performed with nonparametric Kruskal-Wallis test, followed by Dunn's multiple comparison test, using GraphPad Prism 7. Statistical significance is set at p<0.05.

References :

Becher, B. , Tugues, S. , and Greter, M. (2016) . GM-CSF: From Growth Factor to Central Mediator of Tissue Inflammation. Immunity 45, 963- 973. Benzakour, T. , and Benzakour, A. (2019) . Disc herniation and disc disease: the present and the future of management. Int Orthop 43, 755-760.

Caldeira, J. , Santa, C. , Osorio, H. , Molinos, M. , Manadas, B., Goncalves, R. , and Barbosa, M. (2017) . Matrisome Profiling During Intervertebral Disc Development And Ageing. Sci Rep 7, 11629.

Chiu, C.C. , Chuang, T.Y. , Chang, K.H. , Wu, C.H. , Lin, P.W. , and Hsu, W.Y. (2015) . The probability of spontaneous regression of lumbar herniated disc: a systematic review. Clin Rehabil 29, 184-195. Cunha, C. , Almeida, C.R. , Almeida, M.I. , Silva, A.M. , Molinos, M. , Lamas, S. , Pereira, C.L. , Teixeira, G.Q. , Monteiro, A.T. , Santos, S.G. , et al. (2017a) . Systemic Delivery of Bone Marrow Mesenchymal Stem Cells for In Situ Intervertebral Disc Regeneration. Stem Cells Transl Med 6, 1029-1039.

Cunha, C. , Lamas, S. , Goncalves, R.M. , and Barbosa, M.A. (2017b) . Joint analysis of IVD herniation and degeneration by rat caudal needle puncture model. J Orthop Res 35, 258-268.

Danenberg, H.D. , Fishbein, I. , Gao, J. , Monkkonen, J. , Reich, R., Gati, I. , Moerman, E. , and Golomb, G. (2002) . Macrophage depletion by clodronate-containing liposomes reduces neointimal formation after balloon injury in rats and rabbits. Circulation 106, 599-605. GBD2017, D . a . I . I . a . P . C . (2018) . Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 392, 1789-1858.

Hughes, C.S. , Moggridge, S. , Muller, T. , Sorensen, P.H. , Morin, G.B. , and Krijgsveld, J. (2019) . Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc 14, 68-85.

Ikeda, T. , Nakamura, T. , Kikuchi, T. , Umeda, S. , Senda, H. , and Takagi, K. (1996) . Pathomechanism of spontaneous regression of the herniated lumbar disc: histologic and immunohistochemical study. J Spinal Disord 9, 136-140. Kawakubo, A. , Uchida, K. , Miyagi, M. , Nakawaki, M. , Satoh, M., Sekiguchi, H. , Yokozeki, Y . , Inoue, G. , and Takaso, M. (2020) . Investigation of resident and recruited macrophages following disc injury in mice. J Orthop Res 38, 1703-1709.

Li, Z. , Xu, X. , Feng, X. , and Murphy, P.M. (2016) . The Macrophagedepleting Agent Clodronate Promotes Durable Hematopoietic Chimerism and Donor-specific Skin Allograft Tolerance in Mice. Sci Rep 6, 22143.

Locati, M. , Curtale, G. , and Mantovani, A. (2020) . Diversity, Mechanisms, and Significance of Macrophage Plasticity. Annu Rev Pathol 15, 123-147.

Lorio, M. , Kim, C. , Araghi, A. , Inzana, J. , and Yue, J. J. (2020) . International Society for the Advancement of Spine Surgery Policy 2019-Surgical Treatment of Lumbar Disc Herniation with Radiculopathy. Int J Spine Surg 14, 1-17.

Macki, M. , Hernandez-Hermann, M. , Bydon, M. , Gokaslan, A. , McGovern, K. , and Bydon, A. (2014) . Spontaneous regression of sequestrated lumbar disc herniations: Literature review. Clinical Neurology and Neurosurgery 120, 136-141.

Meisel, H.J. , Siodla, V. , Ganey, T. , Minkus, Y . , Hutton, W.C. , and Alasevic, O.J. (2007) . Clinical experience in cell-based therapeutics: disc chondrocyte transplantation A treatment for degenerated or damaged intervertebral disc. Biomol Eng 24, 5-21.

Sunderkotter , C. , Nikolic, T. , Dillon, M.J. , Van Rooijen, N. , Stehling, M. , Drevets, D.A. , and Leenen, P.J. (2004) . Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. J Immunol 172, 4410-4417.

Weisser, S.B. , van Rooijen, N. , and Sly, L.M. (2012) . Depletion and reconstitution of macrophages in mice. J Vis Exp, 4105.

Porto, 26th October 2022

Claims

CLAIMS Monocyte-derived macrophages, more preferably macrophages with the M2 phenotype, even more preferably macrophages with the M2a phenotype, for use in the treatment of hernia of the intravertebral disc (IVD) as well as in other clinical conditions comprising regeneration-adverse reactive tissues such as wound and surgery-related scar tissue, fibrosis and necrosis, the said clinical conditions being selected from the list further consisting of myocardial infraction, endometriosis, pulmonary asthma, hepatic cirrhosis, spinal cord injury and cartilage injury. Composition for uses according to claim 1 characterized by, comprising monocyte-derived macrophages, more preferably macrophages with the M2 phenotype, even more preferably macrophages with the M2a phenotype in an appropriate vehicle selected from the list consisting of a saline solution, a polymer, a hydrogel or a carrier biomaterial. Composition according to claim 2 characterized by, comprising most preferably in IxlO⁶ cells in vehicle . Composition according to claims 2-3 characterized by, being administered by local injection using a 31-G needle coupled to a microsyringe and using a 2.5 mm depth adaptor. Method to obtain monocyte-derived macrophages for uses according to claim 1 characterized by, comprising the steps of: a) Collecting mononuclear cells from a sample obtained in vitro. b) Isolating the monocytes through negative selection. c) Culturing the recovered monocytes in a differentiation and polarization composition. d) Purification of the resulting macrophages by negative selection using a purification composition.

6. Method according to claim 5, in which the said sample obtained in vitro may comprise peripheral blood or bone marrow monocytes, fresh or frozen peripheral blood mononuclear cells or washed leukapheresis samples.

7. Method according to claim 5, in which the said differentiation and polarization composition is characterized by comprising macrophage colony-stimulating factor (M-CSF) and cytokines selected from the list consisting of IL-4 and IL-13.

8. Method according to claim 5, in which the said negative selection purification composition is characterized by comprising antibodies selected from the list consisting of antibodies against CD64, CD62L, CCR2, HLA-DR, CD80, CDllc, CD86, TLR2, TLR4, iNOS, pSTATl, CD163, TLR1, TLR8 and combinations thereof .

9. Composition for use in the treatment of hernia of the intravertebral disc (IVD) as well as in other clinical conditions comprising regeneration-adverse reactive tissues such as wound and surgery-related scar tissue, fibrosis and necrosis, the said clinical conditions being selected from the list further consisting of myocardial infraction, endometriosis, pulmonary asthma, hepatic cirrhosis, spinal cord injury and cartilage injury, characterized by comprising cytokines selected from the list consisting of IL-4 and IL-13, for polarization of resident macrophages to the M2 phenotype, most preferably the M2a phenotype.

Porto, 26th October 2022