Introduction

Low back pain (LBP) is a worldwide leading cause of disability. This preclinical study evaluated the safety of a combined advanced therapy medicinal product developed during the European iPSpine project (#825925) consisting of mesendoderm progenitor cells (MEPC), derived from human induced pluripotent stem cells, in combination with a synthetic poly(N-isopropylacrylamide) hydrogel (NPgel) in an ovine intervertebral disc degeneration (IDD) model.

Introduction

Osteoarthritis (OA) is a predominant chronic degenerative disease exerting a deep impact on quality of life and healthcare systems. Recent evidences suggest that pyroptosis, a programmed cell death characterized by inflammatory cytokine release, may play a significant role in modulating OA pain. The aim of the study is to investigate the potential role of extracellular vesicles derived from umbilical cord Wharton's jelly (WJ-MSC EVs) in the attenuation of the pyroptotic process on human chondrocytes (hOAC) pre-treated with synovial fluid in a 3D in vitro model.

Low back pain (LBP) is a worldwide leading cause of disability. Treatment of intervertebral disc (IVD) with stem cells has been used on degenerate discs (IDD), cause of around 40% of LBP cases. Despite pain reduction, clinical studies' follow-up have not shown a structural IVD improvement. A valid alternative may be the use of notocordal cells (NC) or their precursors. Mesendoderm progenitor cells (MEPC) have the ability to replicate and differentiate toward NC. In this preliminary study we evaluated in a preclinical IDD model the viability and NC differentiation of MEPC derived from induced pluripotent stem cells (iPSC).

MEPC derived from iPSC were developed during the iPSpine project (# 825925), thawed, plated for 24h on laminin and labeled with PKH26.

Two adult sheep were subjected to nucleotomy of five lumbar discs for the induction of IDD. After 5 weeks, 3 degenerated discs were treated with MEPC at 3 different doses (low, medium and high). One sheep was sacrificed after 7 days and one after 30 days. Clinical parameters were collected to evaluate the safety of treatment. Discs were analysed using histological techniques. Survival (PKH26), proliferation (PCNA), notocordal cell differentiation (Brachyury, Cytokeratin 8/18/19, Sox9, Foxa2) and endodermal differentiation (Sox17) were evaluated.

At 7 days from treatment, both sheep lost about 20% of body weight. Only in discs treated with the highest dose PKH26 stained cells were alive up to 30 days. These cells turn out to be: proliferating (PCNA); positive for Brachyury, cytokeratin 8/18/19 and Foxa2; positive for SOX17 in a small percentage.

This preliminary study shows that MEPC, derived from iPSC and injected into ovine discs degenerated by nucleotomy, are able to survive up to 30 days and differentiate within the disc predominantly towards the notocordal phenotype.

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.

Introduction and Objective

Low back pain (LBP) is a disorder strongly associated with intervertebral disc degeneration (IDD) with an important impact on the quality of life of affected people. To date, LBP treatment is based on conservative methods with the aim to reduce back pain without restoring the degenerative environment of the disc. The main cause of IDD is the drastic reduction of the proteoglycan content within the nucleus pulposus (NP), eventually leading to the loss of disc water content, micro-architecture, biochemical and mechanical properties. A promising approach for disc regeneration is represented by the transplantation of mesenchymal stromal cells (MSCs). The exact mechanism remains unknown. Growing evidence suggests that MSCs can influence cells and modulate cells’ behaviour by secreting a set of bioactive factors. MSCs secretome is composed of several molecules such as soluble protein, lipids, nucleic acids and extracellular vesicles (EVs) involved in inflammation, immunomodulation, cell survival and intercellular communication. The aim of this study was to evaluate the in vitro effects of MSCs secretome on human NP cells (hNPCs) in a 3D culture model with and without inflammatory stimulus.

In the last decade, skeletal muscle has been recognized as an endocrine organ able to release molecules that may act as paracrine or endocrine factors, namely myokines. Among these, irisin is secreted upon muscle contraction after physical exercise (PE) and has been demonstrated to yield anabolic effects on different cell types. Recently, irisin has been shown to improve cortical bone mass, geometry and strength, hence resembling the effect of PE. It has also been reported that irisin levels in the serum and synovial fluid of patients with knee osteoarthritis (OA) were negatively correlated with OA severity. Therefore, we hypothesized that irisin may improve cartilage metabolism and blunt the osteoarthritic process.

Human osteoarthritic chondrocytes (hOAC) were isolated from osteochondral specimens of patients undergoing total knee joint replacement. After in vitro expansion, hOAC were put in a three-dimensional culture system (alginate beads) and treated with either phosphate-buffered saline (control) or irisin (25 ng/mL). After 1 week, the amount of glycosaminoglycans (GAG) was evaluated using dimethylmethylene blue (DMMB) and PicoGreen assays. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect interleukin (IL)-1 and -6, matrix metalloproteinase (MMP)-1 and -13, inducible nitric oxide synthase (iNOS) and tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -3 gene expression levels.

hOAC treated with irisin showed a significant higher GAG content compared to the control group (p < 0.01). Moreover, irisin was able to reduce the expression of catabolic (MMP-1, -13, iNOS) and pro-inflammatory (IL-1, IL-6) markers, while incrementing the expression of TIMP-1 and -3 (p < 0.001).

Our results showed that irisin was able to stimulate GAG synthesis and diminish extracellular matrix catabolism in hOAC, demonstrating the existence of a cross-talk between cartilage and muscle possibly supporting the beneficial role of PE on cartilage homeostasis.

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.