## 5 Exosome Therapy for IVD Regeneration  
Emerging cell therapies have demonstrated promising results by supplementing damaged IVDs with stem cells harvested from different sources [97]. Despite this, there is a growing interest in cell-free therapy. Recent research has proposed that exosomes derived from different tissues such as bone marrow and adipose tissue could represent an emerging tool in regenerative medicine [98]. The use of MSC-derived exosomes may offer several advantages. In fact, they do not require tissue engraftment but present high biocompatibility, low immunogenicity and toxicity, less oncogenic potential and substantial economic benefits compared to cell therapy. Such aspects may promote the use of MSCs-derived exosomes for IVD regeneration as an attractive alternative to intradiscal cell therapy [99]. However, before the exploitation and application of exosomes, it is necessary to isolate them efficiently from biological fluids or supernatants from in vitro cell cultures [100; 101]. Ultracentrifugation is the most used technique and is often coupled with ultrafiltration to improve the purity of the sample, although this provides the risk of nanoparticle deformation [102; 103]. Other sophisticated approaches include tangential flow filtration, dimensional exclusion chromatography and polymeric precipitation [104; 105; 106]. Several methods are available for exosome characterization. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are often employed to verify their morphology. Nanoparticle tracking analyzers (NTA) measure the diameter size and specific EV markers (CD9, CD63, CD81, TSG101 and ALIX) can be identified through flow cytometry [107]. The role of exosomes has been evaluated with regards to ECM synthesis, metabolic dysfunction, anti-apoptotic processes, inflammation, and wound healing (Table 2) [108; 109]. \begin{table}
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Table 2: Summary of available evidence on the role of EVs in IDD. The beneficial effect of MSC-derived exosomes on NPCs in acidic conditions has been recently demonstrated. BM-MSC-exosomes increased proliferation and attenuated the apoptosis of NPCs and enhanced the expression of ECM components [115]. Moreover, exosomes are able to inhibit IL-1B-induced inflammation and apoptosis in AF cells and they promote cell proliferation [116]. On the other hand, [PERSON] et al. identified various similarities between CEP stem cells and MSCs, including their capacity to communicate with IVD cells by secreting extracellular vesicles [121]. Indeed, IVD repair may be promoted by CEP stem cell exosomes by inhibiting NPC apoptosis and encouraging the migration of CEP stem cells into the IVD and differentiate into NPCs via autocrine mechanisms [117; 118]. AF-derived exosomes have also showed a protective effect in IDD [120]. Aging and degenerative tissues usually accumulate in endoplasmic reticulum (ER) advanced glycation end-products (AGEs) related to inflammation, cell death, metabolic dysfunction, and stress [122; 123]. [PERSON] et al. found that the injection of MSCs-derived exosomes in an in vivo rat IDD model modulated ER stress by protecting NPCs against cell death and ameliorating IDD through an attenuation of AGE-induced ER stress mediated by AKT and ERK pathways [110]. Exosomes may also induce differentiation of stem cells towards different phenotypes, although the underlying mechanisms are still unknown. It was reported that the inhibition of Notch1 may promote exosome-induced differentiation of MSCs into NP-like cells in vitro [111]. [PERSON] et al. analyzed exosomes secreted by both NP cells and BM-MSCs in a co-culture system. They highlighted that NPC-derived exosomes induced BM-MSC differentiation towards a NP-like phenotype, whereas BM-MSC-derived exosomes promoted NPC proliferation and ECM production [112]. Recently, a study by [PERSON] et al. described the protective role of exosomes in a rabbit IDD model.