Despite osteoarthritis (OA) representing a large burden for healthcare systems, there remains no effective intervention capable of regenerating the damaged cartilage in OA. Mesenchymal stromal cells (MSCs) are adult-derived, multipotent cells which are a candidate for musculoskeletal cell therapy. However, their precise mechanism of action remains poorly understood. The effects of an intra-articular injection of human bone-marrow derived MSCs into a knee osteochondral injury model were investigated in C57Bl/6 mice. The cell therapy was retrieved at different time points and single cell RNA sequencing was performed to elucidate the transcriptomic changes relevant to driving tissue repair. Mass cytometry was also used to study changes in the mouse immune cell populations during repair. Histological assessment reveals that MSC treatment is associated with improved tissue repair in C57Bl/6 mice. Single cell analysis of retrieved human MSCs showed spatial and temporal transcriptional heterogeneity between the repair tissue (in the epiphysis) and synovial tissue. A transcriptomic map has emerged of some of the distinct genes and pathways enriched in human MSCs isolated from different tissues following osteochondral injury. Several MSC subpopulations have been identified, including proliferative and reparative subpopulations at both 7 days and 28 days after injury. Supported by the mass cytometry results, the immunomodulatory role of MSCs was further emphasised, as MSC therapy was associated with the induction of increased numbers of regulatory T cells correlating with enhanced repair in the mouse knee. The transcriptomes of a retrieved MSC therapy were studied for the first time. An important barrier to the translation of MSC therapies is a lack of understanding of their heterogeneity, and the consequent lack of precision in its use. MSC subpopulations with different functional roles may be implicated in the different phases of tissue repair and this work offers further insights into repair process

The HIPGEN study funded under EU Horizon 2020 (Grant 7792939) has the aim to investigate the potential of the first regenerative cell therapy for the improvement of recovery after muscle injury in hip fracture patients. For this aim we intramuscularly injected placental derived mesenchymal stromal cells during hip fracture arthroplasty. Despite not having reached the primary endpoint, which was the Short Physical Performance Battery, we could observe an increase in abductor muscle strength and a faster return to balance looking at symmetry in insole measurements during follow up

Abstract. Objectives. Bone marrow aspirate concentrate (BMAC), together with fibrin glue (Tisseel, Baxter, UK) and Hyaluronic acid (HA) were used as a one-step cell therapy treating patients with ankle cartilage defects in our hospital. This therapy was proven to be safe, with patients demonstrating a significant improvement 12 months post-treatment. Enriched mesenchymal stem cells (MSCs) in BMAC are suggested inducers of cartilage regeneration, however, currently there is no point-of-care assessment for BMAC quality; especially regarding the proportion of MSCs within. This study aims to characterise the cellular component of CCR-generated BMAC using a point-of-care device, and to investigate if the total nucleated cell (TNC) count and patient age are predictive of MSC concentration. Methods. During surgery, 35ml of bone marrow aspirate (BMA) was collected from each patients’ iliac crest under anaesthesia, and BMAC was obtained via a commercial kit (Cartilage Regeneration kit, CCR, Innotec. ®. , UK). BMAC was then mixed with thrombin (B+T) for injection with HA and fibrinogen. In our study, donor-matched BMA, BMAC and B+T were obtained from consented patients (n=12, age 41 ± 16years) undergoing surgery with BMAC therapy. TNC, red blood cell (RBC) and platelet (PLT) counts were measured via a haematology analyser (ABX Micros ES 60, Horiba, UK), and the proportion of MSCs in BMA, BMAC and B+T were assessed via colony forming unit-fibroblast (CFU-F) assays. Significant differences data in matched donors were tested using Friedman test. All data were shown as mean ± SD. Results. Mean TNC counts in BMA and BMAC were not significantly different (14.0 ± 4.4 million/ml and 19.4 ± 32.9 million/ml, respectively, P>0.9999). However, TNC counts were significantly lower in B+T compared to BMAC (9.7 ± 24.5 million/ml and 19.4 ± 32.9 million/ml, respectively, P=0.0167). Similarly, PLT counts were decreased in B+T compared to BMAC (40.7 ± 30.7 million/ml and 417.5 ± 365.5 million/ml, respectively, P<0.0001), however, PLTs were significantly concentrated in BMAC compared to BMA (417.5 ± 365.5 million/ml and 114.8 ± 61.6 million/ml, respectively, P=0.0429). RBC counts were significantly decreased in BMAC and B+T compared to BMA (P=0.0322 and P<0.0001, respectively). Higher concentration of MSCs were observed in BMAC compared to BMA (0.006% ± 0.01% and 0.00007% ± 0.0001%, respectively, P=0.0176). Similar to TNCs and PLTs, the proportion of MSCs significantly decreased in B+T compared to BMAC (0.0004% ± 0.001% and 0.006% ± 0.01%, respectively, P=0.0023). Furthermore, patient age and TNC counts did not correlate with MSC concentration (Spearman's Rank test, P=0.3266 and P=0.4880, respectively). Conclusions. BMAC successfully concentrated PLTs, but BMAC preparations were highly variable. Mixing BMAC and thrombin however, as described in the CCR protocol, resulted in a dramatic reduction in TNCs, PLTs and MSCs. TNC counts and patient age could not be used to predict the MSC proportion in the BMAC based on current data. Future work aims to look at the biomolecule profile of BMAC plasma, and to correlate them to patient clinical outcomes. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Inflammation has been associated with early degradative changes in articular cartilage and immune responses are key factor influencing normal tissue regeneration and repair. With synovitis a prominent feature in osteoarthritis (OA) and associated with the progressive degradation of articular cartilage, immune factors need to be factored into efforts to achieve efficient cartilage repair/regeneration. Recent efforts have focused on the use of autologous or allogeneic mesenchymal stem/stromal cells (MSCs) to modulate the inflammatory environment in the injured or osteoarthritic joint. Intraarticular injection of MSCS has modulated cartilage degradation in a variety of pre-clinical OA models. Results from early clinical trials have also shown effects on pain and function-associated outcome measures. Other cell types may also have some capacity for use as a therapy for OA. For example, primary allogeneic chondrocytes also seem to have some immune-privilege in the synovial joint and are immunomodulatory in a rat model. Although MSCs isolated from bone marrow that are induced to undergo chondrogenic differentiation do not retain these properties, MSCs isolated from the synovium or chondroprogenitors generated from cartilage itself may represent the future of cell therapy for OA

Due to the presence of megakaryocytes, platelets and clotting factors, bone marrow aspirate (BMA) tends to coagulate. For the first time, starting from our previous studies on mesenchymal vertebral stem cells, it has been hypothesized that coagulated BMA represents a safe and effective autologous biological scaffold for bone regeneration in spinal surgery. The present research involved advanced preclinical in vitro models and the execution of a pilot clinical study.

Evaluation of cell morphology, growth kinetics, immunophenotyping, clonogenicity, trilineage-differentiation, growth-factors and HOX and TALE gene expression were analyzed on clotted- and un-clotted human V-BMA. In parallel, a pilot clinical study on ten patients with degenerative spine diseases submitted to instrumented posterior arthrodesis, is ongoing to assess the ability of clotted-V-BMA to improve spinal fusion at 6- and 12-months follow-up.

Results demonstrated that clotted-V-BMA have significantly higher growth-factor expression and mesenchymal stem cell (MSCs) viability, homogeneity, clonogenicity, and ability to differentiate towards the osteogenic phenotype than un-clotted-V-BMA. Clotted-V-BMA also highlighted significant reduced expression of PBX1 and of MEIS3 genes negatively involved in osteoblast maturation and differentiation. From December 2020, eight patients have already been enrolled with first promising results that will be finally evaluated in the next two months.

The application of V-BMA-clot as carrier of progenitors and cytokines and as natural scaffold with a structural texture represents a point-of-care orthobiologic product to improve spinal fusion. Clinical application seems to be efficacy, and we will confirm and strengthen these data with the final results of the pilot clinical study.

Long bone fractures in patients with diabetes mellitus (DM) are slow to heal, often resulting in delayed reunion or non-union. It is reasonable to postulate that the underlying cause of these DM-associated complications is a reduced population of bone marrow progenitor cells and/or their dysfunction. With the hypothesis that the administration of healthy, allogeneic adult bone marrow-derived mesenchymal stromal cells (MSCs) can enhance DM fracture healing, the aim of this endeavour was to assess the efficacy of MSC administration to support fracture repair using two doses. Here 250,000 or 500,000 human bone marrow-derived MSCs were locally introduced to femoral fractures in diabetic mice, and the quality of de novo bone assessed 8 weeks later. Preliminary bone bridging was evident in all animals; however, a large circumferential reparative callus was consistently retained indicating non-union. Micro-CT analysis elucidated consistent callus dimensions, bone mineral density, bone volume/total volume in all groups, but an increase in bone surface area/bone volume in cell-treated fractures. Moreover, greater amounts of mature bone were identified in fractures treated with a low dose of MSCs. Four-point bending evaluation of the mechanical integrity of the repairing fracture indicated a statistically significant improvement in flexure strength and flexure modulus in DM fractures treated with 250,000 MSCs as compared to controls. An improvement in total energy required for failure was observed in both groups that received MSCs. Therefore, the administration of non-DM bone marrow-derived MSCs supported the development of more mature bone in the reparative callus, resulting in greater mechanical integrity.

Background

Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. Within the REBORNE project, a multi-center orthopaedic clinical trial was focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union.

Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. The REBORNE project entailed a multi-center orthopaedic clinical trial focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial, to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union.

Peripheral blood was collected from patients at fixed time-endpoints, that is at 6,12 and 24 weeks post-surgery for implantation of expanded autologus MSC and bone-like particles. Bone-specific alkaline phosphatase (BAP), C-terminal-propeptide type I-procollagen (PICP), osteocalcin (OC), β-Cross-Laps Collagen (CTX), soluble receptor activator of NFkB (RANKL), osteoprotegerin (OPG) were measured by ELISA assays in blood samples of 22 patients at BM collection and at follow-up visits.

A significant relationship with age was found only at 6 months, with an inverse correlation for CTX, RANKL and OC, and positive for OPG. BTM levels were not related to gender. As an effect of local regenerative process, some BTM showed significant changes in comparison to the baseline value. In particular, the time course of BAP, PICP and RANKL was different in patients with a successful healing in comparison to patients with a negative outcome. The BTM profile apparently indicated a remarkable bone formation activity 12 weeks after surgery. However, the paucity of failed patients in our case series did not allow to prove statistically the role of BTM as predictors of the final outcome.

Blood markers related to bone cell function are useful to measure the efficacy of a expanded MSC-regenerative approach applied to long bone non-unions. Changes of the markers may provide a support to radiological assessment of bone healing.

Introduction

What is the most effective treatment of the early stages for osteonecrosis of the femoral head? Since the results of several treatment modalities such as multiple drilling, core decompression with or without bone graft, and vascularized fibular grafts have not been completely successful, we tried multiple drilling and stem cell transplantation to treat the early stages of osteonecrosis of the femoral head and to minimize patient morbidity. We report the clinical and radiological results of stem cell transplantation and core decompression.

Introduction

Cell-based therapies become more and more prominent for the treatment of intervertebral disc (IVD) injuries. Different strategies are under current development and address the restoration of either annulus fibrosus (AF) or nucleus pulposus (NP). Application of such Advanced Therapy Medicinal Products (ATMPs) is strictly regulated. One requirement is to show the identity of the cells, to make sure the cells are indeed AF or NP cells and retained their IVD cell character during manufacturing process before injection to the site of injury. Therefore, we recently identified novel marker genes that discriminate AF and NP cells on tissue level. However, expression of these AF and NP tissue markers has not been investigated in cultured cells, yet. The aim of this study was to proof the tissue marker”s specificity to discriminate cultured AF and NP cells. Furthermore, we evaluated the tissue markers robustness to different cell culture conditions.

Back pain is a leading cause of disability worldwide and it is primarily considered to be triggered by intervertebral disc (IVD) degeneration (IVDD). Current treatments may improve pain and mobility, but carry high costs and fail to address IVD repair or regeneration. As no effective therapeutic approach has been proposed to restore inflamed and degenerated IVDs, there is the urgent need to clarify the key pathomechanism of IVDD, the involvement of inflammation, particularly complement activation in matrix catabolism, and how to target them towards tissue repair/regeneration. Mesenchymal stem cell (MSC)-based therapies have become the focus of several regenerative IVD studies. Although patients in clinical trials reported less pain after cell therapy, the long-term success of cell engraftment is unclear due to the hostile IVD environment. The mechanism-of-action of MSCs is mostly dependent on the secreted soluble factors. Moreover, priming of MSC with interleukin (IL)-1β modulates the secretome content, improving its anti-inflammatory and regenerative effect on IVDD organ culture models. MSC-derived extracellular vesicles (EVs) have also been shown to modulate human IVD cells towards a healthy IVD phenotype in vitro. However, the mechanisms involved in the effect of secretome and EVs, particularly with regard to immunomodulation and matrix metabolism, are not fully understood. Our work investigates the effects of secretome and EVs secreted by IL-1β-primed MSCs to impair IVD matrix degradation and/or improve matrix formation in IVDD

Osteoarthritis (OA) is the most common type of arthritis and causes a significant deterioration in patients’ quality of life. The high prevalence of OA as well as the current lack of disease-modifying drugs led to a rise in regenerative medicine efforts. The hope is that this will provide a treatment modality with the ability to alter the course of OA via structural modifications of damaged articular cartilage (AC). Regenerative therapy in OA starts with the concept that administered cells may engraft to a lesion site and differentiate into chondrocytes. However, recent studies show that cells, particularly when injected in suspension, rapidly undergo apoptosis after exerting a transient paracrine effect. If the injected stem cells do not lead to structural improvements of a diseased joint, the high cost of cell therapy for OA cannot be justified, particularly when compared with other injection therapeutics such as corticosteroids and hyaluronic acid. Long-term survival of implanted cells that offer prolonged paracrine effects or possible engraftment is essential for a successful cell therapy that will offer durable structural improvements. In this talk, the history and current status of regenerative therapy in OA are summarized along with the conceptual strategy and future directionsfor a successful regenerative therapy that can provide structural modifications in OA

Osteoarthritis (OA) is the most common joint disease, affecting approximately 16% of the adult population worldwide. The chronic inflammation in the joint leads to the breakdown of cartilage, which leads to permanent pain and limitations in everyday life at an early stage of the disease. To date, there is no therapy that can interrupt the inflammatory state or reverse cartilage damage. The PROTO consortium (funded by the EU Horizon Europe program, Grant 101095635) aims to prevent the development of OA by correcting a pathological biomechanical pattern by a digital training intervention and to treat early stage OA with an innovative allogeneic cell therapy

Introduction and Objective. Osteoarthritis (OA) represents one of the leading cause of disability all over the world. Cell therapies, mainly based on mesenchymal stem cells (MSCs), have shown to modulate the pathogenesis of OA in basic, preclinical and clinical studies. Adipose tissue (AT) have emerged as a rich and promising source of MSCs called adipose derived stem cells (ASCs). Different systems are available for processing lipoaspirate to purify the samples from oily and haemorrhagic fractions, minimizing the risk of complications and maximizing the biological yield for subsequent grafting. However, few studies compared the efficacy of the different processing devices already used in clinical practice. This study aims to characterize the products obtained by the use of two different systems such as micro-fragmentation or nano-fragmentation comparing them with the starting material (AT) and the collagenase isolated ASCs. Materials and Methods. AT from 12 donors arrived without selection to the laboratories: 4 lipoaspirated (LA), 4 micro-fragmented (mF) and 4 nano-fragmented (nF). The samples were divided into three aliquots for paraffin embedding, RNA extraction and digestion with collagenase for ASCs isolation. Paraffin embedded tissue sections were stained with hematoxylin-eosin to analyze morphology. RNA was extracted, retro-transcribed and analyzed with real-time PCR to analyze the expression of pluripotency genes (SOX2, NANOG and POU5F1) and inflammatory genes (IL-1beta and iNOS). Data were analyzed using Graphpad Prism 8.0 and expressed as mean ± SD. One-way ANOVA followed by Tukey test was used to compare the different groups. Results. The LA comprised small lobules, with intact cell membranes and structurally integer adipocytes. mF samples showed the presence of integer adipocytes, small lobules and higher amount of cell clusters. nF samples showed the almost completely absence of adipocytes, a high amount of cells without lipid content and a high amount of stromal matrix. Real-time PCR results showed the lowest expression levels of pluripotency genes in LA samples that were assumed equal to 1.0 and used to calculate the expression levels of the other samples. mF showed expression levels of pluripotency genes similar to AT. nF showed expression levels of pluripotency genes higher than AT and mF, but without statistically significant differences. ASCs showed statistically significant higher expression levels of these genes compared to LA and mF (p ≤ 0.001). Likewise, the expression of inflammatory genes resulted to be lowest in LA samples (assumed equal to 1.0), higher in mF samples and in nF samples without statistical significance. As expected, the highest values were found in ASCs isolated cells compared to all the other samples (p ≤ 0.0001). Conclusions. These results confirmed that micro-fragmentation (mF) and nano-fragmentation (nF) permitted to separate a cell mixture enriched in ASCs from a lipoaspirate sample without activating the inflammatory pathways. Both processing methods gave a minimally manipulated product suitable for OA cell therapy application. Further studies are needed to elucidate possible different activities of the ASCs enriched AT-derivatives

Osteochondral injuries are a recognised factor in the development of osteoarthritis (OA). Mesenchymal stromal cells (MSCs) represent a promising biological therapeutic option as an OA-modifying treatment, and they also secrete factors that may have an anti-catabolic effect and/or encourage endogenous repair. We aim to study the effects of (i) intra-articular injection of human bone-marrow-derived MSCs and (ii) their secretome on recovery in a murine knee osteochondral injury model. The MSC secretome was generated by stimulating human bone-marrow-derived MSCs with tumour necrosis factor alpha (TNFα). Mice (n=48) were injected with i) MSC secretome, ii) MSCs or iii) cell culture medium (control). Pain was assessed by activity monitoring, and cartilage repair, subchondral bone volume and synovial inflammation were evaluated using histology and microCT. Both MSC- and MSC-secretome-injected mice showed significant pain reduction at day 7 when compared to control mice, but only the MSC-injected mice maintained a significant improvement over the controls at day 28. Cartilage repair was significantly improved in MSC-injected mice. No significant effects were observed with regards to synovial inflammation or subchondral bone volume. The MSC secretome demonstrates regenerative effects but this does not appear to be as sustained as a MSC cell therapy. Further studies are required to investigate if this can be overcome using different dosing regiments for injection of the MSC secretome. As we further understand the regenerative properties of the MSC secretome, we may be able to enhance the clinical translatability of these therapies. Direct intra-articular injection of MSCs for the treatment of OA also appears promising as a potential future strategy for OA management. Acknowledgements: MS is supported by a grant from the Wellcome Trust (PhD Programme for Clinicians)

Mesenchymal stromal cells (MSC) have been proposed as an emerging cell therapy for bone tissue engineering applications. However, the healing capacity of the bone tissue is often compromised by patient's age and comorbidities, such as osteoporosis. In this context, it is important to understand the impact of donor age on the therapeutic potential of MSC. Importantly, the impact on donor age is not restricted to cells themselves but also to their microenvironment that is known to affect cell function. The extracellular matrix (ECM) has an important role in stem cell microenvironment, being able to modulate cell proliferation, self-renewal and differentiation. Decellularized cell-derived ECM (dECM) has been explored for regenerative medicine applications due to its bioactivity and its resemblance to the in vivo microenvironment. Thus, dECM offers the opportunity not only to develop microenvironments with customizable properties for improvement of cellular functions but also as a platform to study cellular niches in health and disease. In this study, we investigated the capacity of the microenvironment to rescue the impaired proliferative and osteogenic potential of aged MSC. The goal of this work was to understand if the osteogenic capacity of MSC could be modulated by exposure to a dECM derived from cells obtained from young donors. When aged MSC were cultured on dECM derived from young MSC, their in vitro proliferative and osteogenic capacities were enhanced. Our results suggest that the microenvironment, specifically the ECM, plays a crucial role in the osteogenic differentiation capacity of MSC. dECM might be a valuable clinical strategy to overcome the age-related decline in the osteogenic potential of MSC by recapitulating a younger microenvironment, attenuating the effects of aging on the stem cell niche. Overall, this study opens new possibilities for developing clinical strategies for elderly patients with limited bone formation capacity who currently lack effective treatments. Acknowledgements: The authors thank FCT for funding through the project DentalBioMatrix (PTDC/BTM-MAT/3538/2020) and to the research institutions iBB (UIDB/04565/2020 and UIDP/04565/2020) and Associate Laboratory i4HB (LA/P/0140/2020)

Mincing cartilage with commercially available shavers is increasingly used for treating focal cartilage defects. This study aimed to compare the impact of mincing bovine articular cartilage using different shaver blades on chondrocyte viability. Bovine articular cartilage was harvested using a scalpel or three different shaver blades (2.5 mm, 3.5 mm, or 4.2 mm) from a commercially available shaver. The cartilage obtained with a scalpel was minced into fragments smaller than 1 mm. 3. All four conditions were cultivated in a culture medium for seven days. After Day 1 and Day 7, metabolic activity, RNA isolation, and gene expression of anabolic (COL2A1, ACAN) and catabolic genes (MMP1, MMP13), Live/Dead staining and visualization using confocal microscopy, and flow cytometric characterization of minced cartilage chondrocytes were measured. The study found that mincing cartilage with shavers significantly reduced metabolic activity after one and seven days compared to scalpel mincing (p<0.001). Gene expression of anabolic genes was reduced, while catabolic genes were increased after day 7 in all shaver conditions. The MMP13/COL2A1 ratio was also increased in all shaver conditions. Confocal microscopy revealed a thin line of dead cells at the lesion site with viable cells below for the scalpel mincing and a higher number of dead cells diffusely distributed in the shaver conditions. After seven days, there was a significant decrease in viable cells in the shaver conditions compared to scalpel mincing (p<0.05). Flow cytometric characterization revealed fewer intact cells and proportionally more dead cells in all shaver conditions compared to the scalpel mincing. Mincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing. This indicates that mincing cartilage with a shaver should be considered a matrix rather than a cell therapy. Further experimental and clinical studies are required to standardize the mincing process with a shaver. Acknowledgements: This study received unrestricted funding from KARL STORZ SE & Co. KG

Intervertebral disc degeneration (IDD) affects more than 80% of the population and is often linked to a reduction of the proteoglycan content within the nucleus pulposus (NP). The nutritional decline and accumulation of degraded matrix products promote the inflammatory process favoring the onset of disease. Several regenerative approaches based on cell therapy have been explored. Recently, paracrine factors and extracellular vesicles (EVs) such as exosomes have been described to play a fundamental role in the cross-talk between mesenchymal stem cells (MSCs) and NP in the microenvironment. EVs vehicule different molecules: proteins, nucleic acids and lipids involved in intercellular communication regulating the homeostasis of recipient cells. Therefore, MSCs-derived exosomes are an interesting emerging tool for cell-free therapies in IDD. The aim of this study was to evaluate the in vitro effects of MSCs derived exosomes on human NP cells (hNPCs). Exosomes were isolated through a multistep ultracentrifugation of bone marrow-MSCs (BM-MSCs) conditioned media (CM), obtained by culturing BM-MSCs without fetal bovine serum (FBS) for 48 hours. Exosomal morphology was characterized by transmission electron microscope (TEM). The exosomes were quantified by bicinchoninic acid assay (BCA) and cryopreserved at –80 °C. hNPCs derived from surgical speciments digested with type II collagenase. After culture expansion in vitro, hNPCs in alginate beads (three-dimensional culture system) were treated with growth medium (controls), exosomes, CM, interleukin-1 beta (IL-1b), IL-1b plus exosomes, IL-1b plus CM. After 24 hours, total RNA was extracted and reverse-transcribed. Gene expression levels of catabolic and anabolic genes were analyzed through real time-polymerase chain reaction (qPCR). TEM analysis confirmed the cup-shaped vescicles in our preparations. Gene expression levels resulted to be modulated by both exosomes and CM compared to controls. In addition, both treatments were capable to alter the inflammatory stimuli of IL-1b. Interestingly, exosomes were able to change anabolic and catabolic gene expression levels differently from CM. In our experimental conditions, both exosomes and CM from BM-MSCs could be an interesting alternative strategy in intervertebral disc regeneration, overcoming the costs and translational limits of cell therapy to the clinical practice

Intervertebral disc degeneration (IDD) affects more than 80% of the population all over the world. Current strategies for the treatment of IDD are based on conservative or surgical procedures with the aim of relieving pain. Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy in recent decades, but studies showed that the particularly hostile microenvironment in the intervertebral disc (IVD) can compromise cells survival rate. The use of exosomes, extracellular vesicles released by various cell types, possess considerable economic advantages including low immunogenicity and toxicity. Exosomes allow intercellular communication by conveying functional proteins, RNA, miRNA and lipids between cells. The purpose of this study is to assess the therapeutic effects of exosomes derived from Wharton Jelly mesenchymal stromal cells (WJ-MSC) on human nucleuspulposus cells (hNPC) in an in vitro 3D culture model. Exosomes (exos) were isolated by tangential flow filtration of WJ-MSC conditioned media and characterized by: quantification with BCA test; morphological observation with TEM, surface marker expression by WB and size evaluation by NTA. Confocal microscopy has been used to identify exosomes marked with PKH26 and monitor fusion and/or incorporation in hNPC. hNPC were isolated from waste surgical material from patients undergoing discectomy (n = 5), expanded, encapsulated in alginate beads and treated with: culture medium (control group); WJ-MSC exos (WJ-exos) at different concentrations (10 μg/ml, 50 μg/ml and 100 μg/ml). They were then analysed for: cell proliferation (Trypan Blu); viability (Live/Dead Assay); quantification of nitrites (Griess) and glycosaminoglycans, GAG (DMBB). The hNPC in alginate beads treated for 7 days were included in paraffin and histologically analysed to determine the presence of extracellular matrix (ECM) components. Finally, the expression levels of catabolic and anabolic genes were evaluated through real-time polymerase chain reaction (qPCR). All concentrations of WJ-exos under exam were capable to induce a significant increase in cell proliferation after 10 and 14 days of treatment (p < 0.01 and p < 0.001, respectively). Live/Dead assay showed a decrease in cell death at 50 μg/ml of WJ-exos (p < 0.05). While cellular oxidative stress indicator, nitrite production, was reduced in a dose-dependent way and statistically significant only with 100 μg/ml of WJ-exos (p < 0.05). WJ-exos at 10 and 100 μg/ml induced a significant increase in GAG content (p < 0.05; p < 0.01, respectively) confirmed by Alcian Blu staining. Exos derived from WJ-MSC modulated gene expression levels by increasing expression of ACAN and SOX-9 genes and reducing significantly of IL-6, MMP-1, MMP-13 and ADAMTS-5 levels (p < 0.05; p < 0.01) compared to the control group. Our results supported the potential use of exosomes from WJ-MSC for the treatment of IDD. Exosomes improved hNPC growth, attenuated ECM degradation and reduced oxidative stress and inflammation. This study offers a new scenario in IVD regeneration, promoting the potential use of extracellular vesicles as an alternative strategy to cell therapy

Low back pain is thought to relate to intervertebral disc (IVD) degeneration. Although the mechanisms have not been clearly identified, exhaustion of nucleus pulposus cells and their producing matrix is regarded as one cause. The matrix of the IVD is continuously replenished and remodeled by tissue-specialized cells and are crucial in supporting the IVD function. However, due to aging, trauma, and genetic and lifestyle factors, the cells can lose their potency and viability, thereby limiting their collective matrix production capacity. We have discovered the link between loss of angiopoietin-1 receptor (Tie2)-positive human NP progenitor cells (NPPC) and IVD degeneration. Tie2+ cells were characterized as undifferentiated cells with multipotency and possessing high self-renewal abilities. Thus we and others have proposed Tie2+ NPPC as a potent cell source for regenerative cell therapies against IVD degeneration. However, their utilization is hindered by low Tie2-expressing cell yields from NP tissue, in particular from commonly available older and degenerated tissue sources. Moreover, NPPC show a rapid Tie2 decrease due to cell differentiation as part of standard culture processes. As such, a need exists to optimize or develop new culture methods that enable the maintenance of Tie2-expressing NPPC. Trials to overcome these difficulties will be shared