pharmaceuticals

Review The Case of Medication-Related Osteonecrosis of the Addressed from a Pathogenic Point of View. Innovative Therapeutic Strategies: Focus on the Most Recent Discoveries on Oral -Derived Exosomes

Amerigo Giudice 1 , Alessandro Antonelli 1 , Emanuela Chiarella 2 , Francesco Baudi 2 , Tullio Barni 2 and Anna Di Vito 2,*

1 Department of Health Sciences, University Magna Graecia of Catanzaro, 88100 Catanzaro, Italy; [email protected] (A.G.); [email protected] (A.A.) 2 Department of Experimental and Clinical Medicine, University Magna Graecia of Catanzaro, 88100 Catanzaro, Italy; [email protected] (E.C.); [email protected] (F.B.); [email protected] (T.B.) * Correspondence: [email protected]

 Received: 16 October 2020; Accepted: 23 November 2020; Published: 25 November 2020 

Abstract: -related osteonecrosis of the jaw (BRONJ) was firstly reported by Marx in 2003. Since 2014, the term medication-related osteonecrosis of the jaw (MRONJ) is recommended by the American Association of Oral and Maxillofacial Surgeons (AAOMS). Development of MRONJ has been associated to the assumption of bisphosphonates but many MRONJ-promoting factors have been identified. A strong involvement of immunity components has been suggested. Therapeutic intervention includes surgical and non-surgical treatments, as well as regenerative medicine procedures for the replacement of the lost tissues. The literature confirms that the combination of mesenchymal stem cells (MSCs), biomaterials and local biomolecules can support the regeneration/repair of different structures. In this review, we report the major open topics in the pathogenesis of MRONJ. Then, we introduce the oral tissues recognized as sources of MSCs, summing up in functional terms what is known about the exosomes release in physiological and pathological conditions.

Keywords: medication-related osteonecrosis of the jaw (MRONJ); oral mesenchymal stem cells; exosomes; immunity; inflammation

1. Introduction It’s widely accepted that cell-to-cell communications take place via both cell contacts and release of paracrine factors. Paracrine factors in particular could be released as soluble factors, protein complex and packaged mediators [1,2]. In the case of mesenchymal stem cells (MSCs), it becomes evident that tissue regeneration capability is mainly dependent on paracrine effects such as the secretion of trophic factors, cytokines and extracellular vesicles (EVs). EVs are phospholipid bilayer membrane-enclosed structures which can be secreted by the majority of cell types and then released in extracellular space and fluids. EVs are classified on the basis of both vesicle size and biogenesis in four categories: exosomes, released via exocytosis with the size of 30–150 nm; microvesicles, released via budding with the size of 100–1000 nm; retrovirus-like particles, 90–100 nm, which appear similar to retroviral particles and contain a portion of retroviral protein; apoptotic bodies, released during apoptotic events and generally larger than 1000 nm [3,4].

Pharmaceuticals 2020, 13, 423; doi:10.3390/ph13120423 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, 423 2 of 24

More recently, a different type of vesicles smaller than 50 nm has been characterized and termed exomeres [5,6]. EVs can be generated by a variety of pathways which generally accounted for the release of a heterogeneous populations of EVs, which differ for size as well as for composition and functions. Aside from these aspects, many attempts have been made in order to characterize the exact composition of EVs, however it’s clear that their composition results extremely variable and is cell- and environment-dependent. Indeed, proteomic and genomic complexities of EVs are still scarcely recognized. MSC-derived EVs composition appears even less characterized in pathological conditions. Medication-related osteonecrosis of the jaw (MRONJ) has been recognized as one of the most disabling associated to the assumption of bisphosphonates (BPs). In the last decades, researchers investigate on the pathogenesis of MRONJ and potential therapeutic approach. The most intriguing and promising tool results to be the application of undifferentiated cells able to regenerate and restore the destroyed tissues. Mesenchymal cells recovered by different sources such as marrow, dental , periodontal ligament have been applied in experimental therapeutic approaches both as cell suspension or cell sheet. MSCs have also been employed in combination with biocompatible scaffolds. The evidence that MSCs administration contributes to tissues restoring above all via the release of paracrine factors shift the attention to the exact nature and composition of such soluble factors [7]. In this review, we attempted to summarize the most recent discoveries about the composition of exosomes released by oral MSCs. We reported the major open topics in the pathogenesis of MRONJ, suggesting new experimental approaches in this field. Then, we introduced the different oral tissues recognized as source of MSCs, summing up in functional terms what is known about the exosomes release in physiological and pathological conditions.

2. Pathogenesis of Bisphosphonates-Related Osteonecrosis of the Jaw Osteonecrosis of the jaw is defined by at least three pathognomonic signs: (1) exposed bone in the maxillofacial region that does not heal within 8 weeks after the identification; (2) exposure of the patient to an antiresorptive agent; (3) no history of radiation therapy to the craniofacial region [8]. The first case of MRONJ was reported by Marx in 2003 [9]. Initially, osteonecrosis was reported only after treatment with BPs and then named -related osteonecrosis of the jaw (BRONJ); since 2014, the term medication-related osteonecrosis of the jaw (MRONJ) was recommended by the American Association of Oral and Maxillofacial Surgeons (AAOMS) [10]. Indeed, accumulating evidence suggested the involvement of other antiresorptive and antiangiogenic treatments among the causes of MRONJ, such as bevacizumab, sunitinib, aflibercept and [11–14]. More recently, the administration of other antiangiogenics such as dasatinib, erlotinib, imatinib, axitinib, sorafenib and cabozantinib has been associated with MRONJ [15]. A detailed review of the drugs related to the development of MRONJ has been recently published [15]. Today, the nitrogen-BPs (N-BPs) alendronate and zoledronate, and the human monoclonal antibody targeting the receptor activator of nuclear factor kappa-B ligand (RANKL), denosumab, have been suggested as drugs most commonly associated to the development of MRONJ. The antiresorptive effects of N-BPs and denosumab are different. Briefly, N-BPs are analogs of pyrophosphate able to bind to the hydroxyapatite crystals of bone. Upon incorporation within , they inhibit the enzyme farnesyl pyrophosphate (FPP) synthase and decrease prenylation of low molecular weight-proteins [16,17]. Moreover, the inhibition of FPP-synthase accounts for the accumulation of isopentenyl diphosphate (IPP), the production of a toxic metabolite (ApppI) and cell [18]. Conversely, the monoclonal antibody denosumab, approved by the U.S. Food and Drug Administration (FDA) in June 2010 for the treatment of in postmenopausal (PM) women with a high fracture risk [19,20], displays the capability to bind and inhibit RANKL reducing . Importantly, BPs accumulate in the skeleton for a long time, in contrast with denosumab whose half-life is approximately 26 days [21]. The most recent classification or staging system for MRONJ has been proposed by the AAOMS. Briefly, patients were assigned to different stages according Pharmaceuticals 2020, 13, 423 3 of 24 to the criteria reviewed by Ruggiero in 2014, and then classified as “at risk”, stage 0, stage 1, stage 2 characterized by the appearance of infection and stage 3. What is the trigger for the MRONJ? Although the development of MRONJ has been associated to the assumption of N-BPs, many MRONJ-promoting factors have been also identified, such as dental treatments and pre-existing oral infections. In the past, many experiments have been carried out in order to investigate the effects of N-BPs and non-N-BPs on the development of MRONJ. Most of the research focused on their pro- and/or anti-inflammatory action, showing both overlapped and opposed effects. To this purpose, typical experimental model of in vitro inflammation requires the administration of lipopolysaccharide (LPS), recognized as a major anaerobic component able to trigger inflammatory cascade in oral tissues. Indeed, oral cavity harbors many microenvironments constitutively exposed to which are directly or indirectly involved in periodontal diseases such as periodontitis and MRONJ [22]. In particular, a state of deficit healing process and reduced vascular supply of the is a favorable condition to establish an infective disease like or MRONJ, caused by commensal bacteria of the oral cavity [23]. Moreover, the loss of oral mucosal integrity and submucosal infections increases the infection risk by saprophytic bacteria of the oral cavity which in basal conditions have low virulence [24,25]. Several studies indicated Actinomyces spp. as one of the main players in MRONJ onset, however, other bacteria typical of the oral microbiota like Streptococcus spp. and aciduric bacteria play an important role in the bone colonization [26,27]. Furthermore, as evidenced in a clinical study by Zirk et al., the majority of the oral bacteria isolated in necrotic bone samples was quite different from bacteria isolated from the submucosal infected samples. A high Actinomyces spp. presence was detected in the submucosal infections, instead Viridansstreptococcae, Parvimonasmicra, Prevotella, and Veillonella species were identify both in bone and soft tissue samples. Usually an anaerobic bacteria dominance was noticed in the bone and submucosal infections, followed by facultative anaerobic gram-positive bacteria [28]. Regarding the cellular and molecular mechanisms involved in the development of MRONJ, the reduced vascularization observed in BPs-treated patients has been suggested as the key player for the of the jaw. However, as suggested by Dodson, infections-associated lesions often precede the development of necrosis of the jaw, so we can conclude that events occurring during the establishment of the infection result as the trigger for the exacerbation of MRONJ [29]. To date, the order by which infection, inflammation, and inhibition of angiogenesis appear during the pathogenesis of MRONJ remains to be elucidated.

3. The Effects of BPs on Cells Residing in the Bone BPs exert their anti-resorptive action targeting bone-resorbing . However, in the last two decades, many evidence challenges the long-held dogma that BPs act only in the skeleton, showing direct or indirect effects not only on , , fibroblast and epithelial cells [30–36], but also on cells and tumor-associated (TAMs). The molecular mechanisms involved in N-BPs interaction with cells other than osteoclasts have been investigated recently. As a matter of the fact, there are still no robust evidence suggesting that endothelial cells, osteoblast and immunity cells can internalize N-BPs. During bone remodeling that strongly occurs in alveolar bone, osteoclasts are the only cells that are able to release N-BPs via the production of HCl accounting for bone dissolving. In this acidic environment, N-BPs result lipophilic and then able to go across the cell membrane [37]. Nevertheless, Rogers et al. suggested that free BPs or BPs complexed to bone particles or matrix proteins are internalized by osteoclasts into membrane-bound vesicles by endocytosis [16]. It’s interesting to note that after the release of BPs into osteoclast cytosol, free BPs may accumulate in the cytosol or migrate by transcytosis in the extracellular space for their recycling [38]. In neutral environment, N-BPs must be taken into cells by specific transporters such as phosphate transporters. N-BPs enter in the osteoclasts via transporters belonging to the solute carriers (SLC) 20 and/or SLC34 family; non-N-BPs enter via transporters of SLC17 family [39]. There is evidence suggesting that Pharmaceuticals 2020, 13, 423 4 of 24 similar to osteoclasts, also TAMs take in BPs by endocytosis or by phagocytosis of BPs bound to microcalcification [38,40,41]. It’s generally accepted that a major event during the establishment of bacterial infection in MRONJ, is the suppression of the immunological reactions; the immunosuppressive state occurs as a result of BPs action on the different cell populations in the oral microenvironment, such as macrophages, γδ T-cells, endothelial cells [42].

3.1. BPs-Macrophages Cross-Talk Inflammation and necrosis have been identified as two hallmarks of N-BPs action [43–45]. Some evidence suggested that such effects depend on the N-BP concentrations: inflammation becomes evident at lower concentration while necrosis appears as a consequence of higher drug concentrations. Among the cell populations residing in alveolar socket, macrophages play a key role in the conservation of tissue by regulating both the development and the maintenance of inflammation. Since 1993, it became evident that some BPs are able to interfere with immunity response, via direct effects on macrophages, granulocytes and osteoclasts in vivo [46]. N-BPs inhibited monocytes/macrophages release of cytokines, osteoclast commitment, and dendritic differentiation [47–49]. More recently, it was described that N-BPs are able to regulated polarization. Macrophages have been classified into M1 and M2 subtypes: M1 macrophages are functionally pro-inflammatory, while M2 macrophages are anti-inflammatory. N-BPs are able to inhibit M2-polarization and stimulate the pro-inflammatory M1 phenotype both in vivo and in vitro [50,51]. Molecular mechanisms involved in such phenomena have been partially investigated. Macrophages may be stimulated by both damage-associated molecular pattern (DAMPs) and pathogen-associated molecular patterns (PAMPs) which trigger inflammatory responses through innate immune receptors, such as TLRs, and downstream pathways such as nuclear factor-κB (NF-κB). Zhu et al. showed that zoledronate in vivo administration accounted for Toll-Like Receptor 4 (TLR4) activation, NF-κB translocation in the nucleus and M1-polarization of macrophages in the alveolar socket after teeth extraction (Figure1)[ 51]. Moreover, it’s known that generally such stimuli promote the assembly of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome complex, the activation of caspase-1 and the production of mature form of interleukin (IL)-1β and IL-18 (Figure1)[52]. The role of IL-1 signaling in N-BPs-mediated inflammation has been described [53]. Both the two isoforms of IL-1, IL-1α and IL-1β are produced as a 31 kDa precursor which can be converted to proper 17 kDa mature form via enzymatic cleavage. The overexpression of caspase-1 and IL-1β reported in zoledronate treated cells seems to also involve epigenetic mechanisms. Indeed, zoledronate treatment increases the expression of two related JmjC-domain-containing proteins, lysine demethylase 6 A (Kdm6a) and Kdm6b, leading to demethylation of the caspase-1 and IL-1β promoters and their overexpression (Figure1)[ 54]. Interestingly, N-BPs-associated IL-1β overexpression is not always accompanied by its overproduction; such effect is due to the fact that caspase-1 expression might not be stimulated by N-BPs [53]. Similar mechanisms have been suggested to be involved in the N-BP-induced fever; indeed, fever occurs among patient receiving the first BP treatment, while does not occur in patients receiving second or repeated intravenous N-BPs. In the latter case, the inhibition of osteoclast, recognized as a major source of IL-1β, accounts for normal or reduced IL-1β level [53,55]. Together, these observations indicate a key role of IL-1β in the N-BPs-induced acute immune response. Pharmaceuticals 2020, 13, x FOR PEER REVIEW 4 of 25

It’s generally accepted that a major event during the establishment of bacterial infection in MRONJ, is the suppression of the immunological reactions; the immunosuppressive state occurs as a result of BPs action on the different cell populations in the oral microenvironment, such as macrophages, γδ T-cells, endothelial cells [42].

3.1. BPs-Macrophages Cross-Talk and necrosis have been identified as two hallmarks of N-BPs action [43–45]. Some evidence suggested that such effects depend on the N-BP concentrations: inflammation becomes evident at lower concentration while necrosis appears as a consequence of higher drug concentrations. Among the cell populations residing in alveolar socket, macrophages play a key role in the conservation of tissue homeostasis by regulating both the development and the maintenance of inflammation. Since 1993, it became evident that some BPs are able to interfere with immunity response, via direct effects on macrophages, granulocytes and osteoclasts in vivo [46]. N-BPs inhibited monocytes/macrophages release of cytokines, osteoclast commitment, and dendritic differentiation [47–49]. More recently, it was described that N-BPs are able to regulated macrophage polarization. Macrophages have been classified into M1 and M2 subtypes: M1 macrophages are functionally pro-inflammatory, while M2 macrophages are anti-inflammatory. N-BPs are able to inhibit M2-polarization and stimulate the pro-inflammatory M1 phenotype both in vivo and in vitro [50,51]. Molecular mechanisms involved in such phenomena have been partially investigated. Macrophages may be stimulated by both damage-associated molecular pattern (DAMPs) and pathogen-associated molecular patterns (PAMPs) which trigger inflammatory responses through innate immune receptors, such as TLRs, and downstream pathways such as nuclear factor-κB (NF-κB). Zhu et al. showed that zoledronate in vivo administration accounted for Toll-Like Receptor 4 (TLR4) activation, NF-κB translocation in the nucleus and M1-polarization of macrophages in the alveolar socket after teeth extraction (Figure 1) [51]. Moreover, it’s known that generally such stimuli promote the assembly of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3)

Pharmaceuticalsinflammasome2020 ,complex,13, 423 the activation of caspase-1 and the production of mature form5 of of 24 interleukin (IL)-1β and IL-18 (Figure 1) [52].

PharmaceuticalsPharmaceuticals 2020Pharmaceuticals, 13, x2020 FOR, 13 PEER, x 2020 FOR REVIEW, 13PEER, x FOR REVIEW PEER REVIEW 5 of 25 5 of 25 5 of 25 Figure 1. Schematic representation of ZOL-macrophage cross-talk mechanisms. ZOL-mediated activationFigure of1.Figure Schematic TLR4 1.Figure Schematic in representation macrophages1. Schematic representation ofrepresentation ZOL-macropha accounted of ZOL-macropha ofge ZOL-macropha for cross-talk thege overexpressioncross-talk mechanisms.ge cross-talk mechanisms. ZOL-mediated mechanisms. of ZOL-mediated mature ZOL-mediated form of IL-1 β leading activationactivation of TLR4activation ofin TLR4macrophages inof macrophagesTLR4 accountedin macrophages accounted for the accounted overexpression for the overexpression for the of overexpression mature of formmature of of IL-1form matureβ ofleading IL-1 formβ leadingof IL-1β leading to M1-to M1- polarization. polarization.to M1- polarization.to M1- Red Red polarization. arrows Red arrows indicatearrows Redindicate indicate thearrows action indicatethe of the action ZOL actionthe onof action ZOLTLR4 of on ofactivation ZOLTLR4ZOL on activation on andTLR4 TLR4 Kdm6 activation and proteins activationKdm6 and proteins Kdm6 and proteins Kdm6 proteins

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3.2. BPs-γδTheT role CellsThe of roleIL-1 Cross-TalkThe ofsignaling IL-1role signalingof inIL-1 N-BPs-mediated signaling in N-BPs-mediated in N-BPs-mediated inflammation inflammation hasinflammation been has described been has described been [53]. describedBoth [53]. the Both [53]. the Both the two isoformstwo isoforms oftwo IL-1, isoforms ofIL-1 IL-1,α and IL-1of IL-1,IL-1α andβ IL-1 are IL-1α produced andβ are IL-1 producedβ as are a 31produced kDaas a 31precursor kDa as a precursor31 whichkDa precursor can which be convertedcan which be converted can to be converted to to Theproper mechanisms proper17 kDa 17 propermature kDa 17 maturebyform kDa which via matureform enzymatic via N-BPs form enzymatic viacleainhibit enzymaticvage. clea Thevage. osteoclast overexpressionclea Thevage. overexpression The also overexpression of caspase-1 account of caspase-1 and of for caspase-1IL-1 and theβ IL-1 establishment andβ IL-1β of both reportedreported in zoledronatereported in zoledronate in treated zoledronate treatedcells seems treatedcells seemsto cells also to seemsinvolve also toinvolve epigeneticalso involveepigenetic mechanisms. epigenetic mechanisms. 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Similar Similarmechanisms Similarmechanisms have mechanisms been have suggested been have suggested been to be suggested involved to be involved toin bethe involved N-BP-inducedin the N-BP-induced in the N-BP-inducedfever; indeed,fever; indeed, fever; indeed, IPP extrusionfever occursfever is occursamongfever mediated amongoccurspatient amongpatientreceiving by ATP-binding patientreceiving the firstreceiving the BP first treatment, cassettethe BP first treatment, BPwhile transportertreatment, doeswhile not doeswhile occur not A1 does inoccur(ABCA1) patientsnot in occur patients in in patients cooperation with butyrophilin-3receivingreceiving second (BTN3A1)receiving secondor repeated secondor andrepeated intravenous or apolipoprotein repeated intravenous N-BPs. intravenous N-BPs.In the A-I N-BPs.latterIn (ApoA-I)the case, latterIn the the case, latter (Figureinhibition the case, inhibition 2ofthe)[ osteoclast, 60inhibition ,of61 osteoclast,]. of osteoclast, Arecognized lot ofrecognized papers as arecognized major as have a source major as reported aofsource major IL-1β of ,source accounts IL-1 thatβ of, accounts patientsIL-1 for βnormal, accounts for normaltreatedor reduced for normalor reduced with IL-1 orβ level N-BPsreduced IL-1 β[53,55]. level IL-1 showed [53,55].Together,β level Together,[53,55]. a significant Together, alteration of these observationsthese observationsthese indicate observations indicate a key role indicate a key of IL-1role a keyβ of in IL-1 roletheβ N-BPs-inducedof in IL-1 the βN-BPs-induced in the N-BPs-induced acute immune acute immune response.acute immune response. response. γδ T-cells population in peripheral blood despite a higher level of infiltrating γδ T-cells in the lesion area [623.2.– BPs-65].3.2.γδ In BPs-T Cells particular3.2.γδ Cross-Talk TBPs- Cellsγδ Cross-Talk T Cells note, Cross-Talk zoledronate was reported to activate T cell surveillance in both bone niche andThe blood, mechanismsThe together mechanismsThe by mechanisms which with by N-BPswhich antiangiogenic by inN-BPs whichhibit osteoclast inN-BPshibit osteoclastin and hibitalso accountosteoclast immunomodulatory also account for also the accountforestablishment the establishmentfor the mechanisms ofestablishment both of both of [both66]. The fact that inflammatoryinflammatory andinflammatory necrotic and necrotic events. and necrotic events.The most events.The relevant most The relevant involvesmost relevant involves the production involves the production the of IPPproduction ofduring IPP duringofthe IPP duringthe the singleinhibition or repeatedinhibition of FPPinhibition administration synthaseof FPP synthase of in FPP oste synthaseoclasts. in oste of oclasts.Indeed, in BPs oste mayoclasts.Indeed, IPP stimulates Indeed,IPP stimulates to VIPP diγV ffstimulatesδerent2 VT-cellsγVδ2 results to T-cellsV γproduceVδ2 to T-cellsis produce interferon remarkable to produce interferon interferon [67]. Regarding the molecular(INF)-γ mechanisms(INF)- and tumorγ (INF)-and necrosistumorγ and involved necrosis tumorfactor necrosis(TNF) factor in triggeringγδ(TNF) factorT-cells-activation, triggering (TNF) acute triggering inflammatory acute inflammatory acute N-BPs reactionsinflammatory reactions seem[56,57]. reactions [56,57]. toDendritic induce [56,57].Dendritic the Dendritic release of soluble cells, monocytescells, monocytescells, and monocytes macrophages-release and macrophages-release and macrophages-release of IPP seemsof IPP toseems of also IPP tomediate seems also mediateto the also activation mediate the activation of the γδ activation T-cells of γδ T-cells of γδ T-cells Sema4D[16,58,59]. which[16,58,59]. IPP further [16,58,59].extrusion IPP stimulatesextrusion IPPis mediatedextrusion is mediated macrophages-release byis ATP-mediated bybinding ATP- bybinding cassetteATP-binding cassette oftransporter TNF cassettetransporterα. A1 Further transporter (ABCA1) A1 studies(ABCA1) A1in (ABCA1) arein required in in order to clarifycooperation thiscooperation point. withcooperation butyrophilin-3 with butyrophilin-3 with (BTN3A1)butyrophilin-3 (BTN3A1) and ap(BTN3A1) olipoproteinand apolipoprotein and apA-Iolipoprotein (ApoA-I) A-I (ApoA-I) (Figure A-I (ApoA-I) (Figure2) [60,61]. (Figure2) [60,61]. 2) [60,61]. The mechanisms by which N-BPs account for MRONJ establishment also involve other cell types. Administration of N-BPs and in particular of zoledronate accounts for reduced recruitment of neutrophils and reduced activity of natural killer (NK) cells [68]. During bone remodeling, osteoclasts stimulate NKs cells which, in turn, produce INFγ inhibiting osteoclastogenesis.

Figure Figure2. Mechanisms 2.Figure Mechanisms 2.of MechanismsγδT-cells of γδ activation.T-cells of γδ activation.T-cells The activation.ZOL-mediat The ZOL-mediat Theed overproductionZOL-mediated overproductioned overproductionof IPP ofin IPP inof IPP in osteoclastsosteoclasts accountsosteoclasts accounts for γδ T-cellsaccounts for γδ activationT-cells for γδ activationT-cells and triggersactivation and triggersacut ande inflammatory triggersacute inflammatory acut ereaction. inflammatory reaction. The cells reaction.The cells The cells involvedinvolved in IPP productioninvolved in IPP production in also IPP includedproduction also included monocyte also included monocytes, macrophages monocytes, macrophages ands, macrophages dendritic and dendritic cells. and Black dendritic cells. arrows Black cells. arrows Black arrows indicate indicatethe direction indicatethe direction of thethe direction flow;of the green flow;of thearrow green flow; indicatesarrow green indicates arrowthe activation indicates the activation of the acute activation of inflammatory acute ofinflammatory acute inflammatory

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 5 of 25

Figure 1. Schematic representation of ZOL-macrophage cross-talk mechanisms. ZOL-mediated activation of TLR4 in macrophages accounted for the overexpression of mature form of IL-1β leading to M1- polarization. Red arrows indicate the action of ZOL on TLR4 activation and Kdm6 proteins

overexpression. ( ): gene transcription; ( ): increase; (H3K27Me3 ): reduced methylation; all the other arrows indicate the direction of the flow; IL-1β, interleukin-1β; Kdm6a, lysine demethylase 6 A; Kdm6b, lysine demethylase 6 B; NF-κB, nuclear factor-κB; NLRP3, NACHT, LRR, and PYD domains-containing protein 3; TLR4, Toll-Like Receptor 4. See text for explanation [51,53,54].

The role of IL-1 signaling in N-BPs-mediated inflammation has been described [53]. Both the two isoforms of IL-1, IL-1α and IL-1β are produced as a 31 kDa precursor which can be converted to proper 17 kDa mature form via enzymatic cleavage. The overexpression of caspase-1 and IL-1β reported in zoledronate treated cells seems to also involve epigenetic mechanisms. Indeed, zoledronate treatment increases the expression of two related JmjC-domain-containing proteins, lysine demethylase 6 A (Kdm6a) and Kdm6b, leading to demethylation of the caspase-1 and IL-1β promoters and their overexpression (Figure 1) [54]. Interestingly, N-BPs-associated IL-1β overexpression is not always accompanied by its overproduction; such effect is due to the fact that caspase-1 expression might not be stimulated by N-BPs [53]. Similar mechanisms have been suggested to be involved in the N-BP-induced fever; indeed, fever occurs among patient receiving the first BP treatment, while does not occur in patients receiving second or repeated intravenous N-BPs. In the latter case, the inhibition of osteoclast, recognized as a major source of IL-1β, accounts for normal or reduced IL-1β level [53,55]. Together, these observations indicate a key role of IL-1β in the N-BPs-induced acute immune response.

3.2. BPs-γδ T Cells Cross-Talk The mechanisms by which N-BPs inhibit osteoclast also account for the establishment of both inflammatory and necrotic events. The most relevant involves the production of IPP during the Pharmaceuticalsinhibition2020 of, 13FPP, 423 synthase in osteoclasts. Indeed, IPP stimulates VγVδ2 T-cells to produce interferon6 of 24 (INF)-γ and tumor necrosis factor (TNF) triggering acute inflammatory reactions [56,57]. Dendritic cells, monocytes and macrophages-release of IPP seems to also mediate the activation of γδ T-cells Osteoclast[16,58,59]. suppression IPP extrusion induced is bymediated N-BPs accountsby ATP- forbinding NK cellscassette deregulation transporter and A1 the (ABCA1) maintenance in of an immunosuppressivecooperation with butyrophilin-3 microenvironment, (BTN3A1) delaying and apolipoprotein wound healing A-I (ApoA-I) [69]. (Figure 2) [60,61].

γδ FigureFigure 2. Mechanisms 2. Mechanisms of T-cellsof γδT-cells activation. activation. The ZOL-mediated The ZOL-mediat overproductioned overproduction of IPP of in IPP osteoclasts in accounts for γδT-cells activation and triggers acute inflammatory reaction. The cells involved in IPP osteoclasts accounts for γδT-cells activation and triggers acutPharmaceuticalse inflammatory 2020, 13, x reaction.FOR PEER REVIEWThe cells 6 of 25 productioninvolved also in included IPP production monocytes, also macrophagesincluded monocyte and dendritics, macrophages cells. Blackand dendritic arrows indicate cells. Black the directionarrows of the flow;indicate green the arrow direction indicates of thethe activationflow; green of acutearrow inflammatory indicates the response. response.activation :of inhibitioninhibition acute inflammatory of of FPP FPP synthase. synthase. ABCA1: ATP-binding cassette transporter A1; ApoA-I: ABCA1: ATP-binding cassette transporter A1; ApoA-I: apolipoproteinapolipoprotein A-I; BTN3A1: A-I; BTN3A1: butyrophilin-3; butyrophilin-3; FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl

FPP: farnesylpyrophosphate; GGPP:geranylgeranyl diphosphate; HMG-CoA:diphosphate; Hydroxymethylglutaryl-CoA HMG-CoA: Hydroxymethylglutaryl-CoA Reductase; INF-γ: interferon-γ; IPP: isopentenyl diphosphate; TNF, tumor necrosis factor. See text for explanation [16,56–61]. Reductase; INF-γ: interferon-γ; IPP: isopentenyl diphosphate; TNF, tumor necrosis factor. See text for explanation [16,56–61]. A lot of papers have reported that patients treated with N-BPs showed a significant alteration of γδ T-cells population in peripheral blood despite a higher level of infiltrating γδ T-cells in the lesion 3.3. BPs-Vascular Endothelium Cross-Talk area [62–65]. In particular note, zoledronate was reported to activate T cell surveillance in both bone niche and blood, together with antiangiogenic and immunomodulatory mechanisms [66]. The fact N-BPs have been shown to inhibit angiogenesis both inthat vivo singleand orin repeated vitro, hindering administration the mobilityof BPs may lead to different results is remarkable [67]. of cells involved in the immune response [70]. It becomes evidentRegarding that the themolecular inhibitory mechanisms action involved towards in γδ T-cells-activation, N-BPs seem to induce the osteoclast interferes with normal angiogenesis, due to the rolerelease of osteoclasts of soluble Sema4D in thecorrect which further patterning stimulates of macrophages-release of TNFα. Further studies the vessels in the bone [71]. Moreover, N-BPs exert their roleare in required inhibiting in order angiogenesis to clarify this throughpoint. both The mechanisms by which N-BPs account for MRONJ establishment also involve other cell direct and indirect effects. First of all, N-BPs such as zoledronatetypes. inhibit Administration endothelial of N-BPs cells and in endothelial particular of zoledronate accounts for reduced recruitment of progenitor cells proliferation, migration and differentiation,neutrophils accounting and reduced for cells activity apoptosis of natural [68,72 killer]. (NK) cells [68]. During bone remodeling, On the other hand, Ohlrich et al. showed overexpression of vascular-endothelialosteoclasts stimulate NKs growth cells factorwhich, (VEGF)in turn, produce INFγ inhibiting osteoclastogenesis. in gingival fibroblast after exposure to zoledronate, suggestingOsteoclast the suppression induction induced of a by proangiogenic N-BPs accounts for NK cells deregulation and the maintenance of an immunosuppressive microenvironment, delaying wound healing [69]. environment [73]. Similar contrasting effects may be understood in light of the fact that N-BPs alter VEGF receptors maturation in endothelial cells, so that the increased3.3. BPs-Vascular expression Endothelium of VEGF Cross-Talk does not exert any effect [74]. In addition, zoledronate treatment may inhibit angiogenesisN-BPs have been by reducing shown to the inhi expressionbit angiogenesis both in vivo and in vitro, hindering the of osteopontin (an angiogenesis inducer) in both andmobility of cells [ 75involved]. Finally, in the N-BPs immune may response also [70]. It becomes evident that the inhibitory action interfere with endothelial differentiation of mesenchymal cellstowards [76]. osteoclast interferes with normal angiogenesis, due to the role of osteoclasts in the correct patterning of the vessels in the bone [71]. Moreover, N-BPs exert their role in inhibiting angiogenesis through both direct and indirect effects. First of all, N-BPs such as zoledronate inhibit endothelial 4. Current and Emerging Treatment Options cells and endothelial progenitor cells proliferation, migration and differentiation, accounting for During the years several authors have discussed MRONJcells treatment. apoptosis However, [68,72]. On while the theother objective hand, Ohlrich et al. showed overexpression of vascular-endothelial growth factor (VEGF) in gingival fibroblast after exposure to zoledronate, seems to be to minimize the risk of MRONJ onset, unfortunatelysuggesting this the is notinduction always of a possible. proangiogenic In 2014, environment [73]. Similar contrasting effects may be the American Association of Oral and Maxillofacial Surgeonsunderstood provided in light of some the fact guidelines that N-BPs alter in the VEGF receptors maturation in endothelial cells, so treatment of MRONJ. They argued that each MRONJ stagethat should the increased be treated expression with a specificof VEGF therapy;does not exert any effect [74]. In addition, zoledronate stage 0 (patients without any clinical evidence of necrotictreatment bone,but may with inhibit non-specific angiogenesis by symptoms reducing the expression of osteopontin (an angiogenesis inducer) in both maxilla and mandible [75]. Finally, N-BPs may also interfere with endothelial or clinical and radiographic findings) should be strictlydifferentiation monitored andof mesenchymal eventually cells treated [76]. with medication for chronic pain and infections; stage 1 (patients with exposed necrotic bone or fistulae without any evident infection) should benefit from medical4. Current management and Emerging with Treatment topic antimicrobial Options and 0.12% rinses, however immediate surgicalDuring treatment the years is not several suggested; authors have stage disc 2ussed MRONJ treatment. However, while the (patients with exposed necrotic bone or fistulae and clear signsobjective of infection) seems to be shouldto minimize be treatedthe risk of with MRONJ a onset, unfortunately this is not always possible. In 2014, the American Association of Oral and Maxillofacial Surgeons provided some guidelines in the treatment of MRONJ. They argued that each MRONJ stage should be treated with a specific therapy; stage 0 (patients without any clinical evidence of necrotic bone, but with non-specific symptoms or clinical and radiographic findings) should be strictly monitored and eventually treated with medication for chronic pain and infections; stage 1 (patients with exposed necrotic bone or fistulae without any evident infection) should benefit from medical management with topic antimicrobial and chlorhexidine 0.12% rinses, however immediate surgical treatment is not suggested; stage 2 (patients with exposed necrotic bone or fistulae and clear signs of infection) should be treated with a combination of oral antibiotics, antibacterial mouth rinses and debridement of necrotic areas to relieve soft tissues irritation and to control infection spread; stage 3 (patients with exposed necrotic bone or fistulae, clear signs of infection and huge necrotic bone areas, pathological fractures, extra-oral fistula, oral antral/oral nasal communication and process extended to the inferior mandibulae border or sinus floor) should be managed with antibiotics and pain control,

Pharmaceuticals 2020, 13, 423 7 of 24 combination of oral antibiotics, antibacterial mouth rinses and debridement of necrotic areas to relieve soft tissues irritation and to control infection spread; stage 3 (patients with exposed necrotic bone or fistulae, clear signs of infection and huge necrotic bone areas, pathological fractures, extra-oral fistula, oral antral/oral nasal communication and osteolysis process extended to the inferior mandibulae border or sinus floor) should be managed with antibiotics and pain control, antibacterial mouth rinses and surgical debridement/resection of all necrotic zones. However, there is no clarity about the best treatment to prevent or to manage MRONJ. Several authors affirmed as medical therapy with a wide spectrum antibiotic prophylaxis is a vital part to reduce and to manage the symptomatology of MRONJ [77]. However, for the advanced stage of MRONJ is mandatory a combined protocol of antibiotics administration and surgical approach [78]. Although in the past it has been highlighted how conservative surgical treatment can be useful to control bone necrosis progression in the early stages of MRONJ [79], recently it has been observed that radical surgical treatment of these stage is more effective in reducing the recurrence of MRONJ [80]. Furthermore, many innovative tools have been developed to perform a better management of surgical phases: the use of piezoelectric surgery, low-laser therapy and VELscope® device to distinguish the limits of necrotic bone [81–83]. Several authors assessed the efficacy of the application of platelet-rich fibrin, and transplantation of autologous bone mixed to MSCs to facilitate a faster tissue healing after surgery [84–87]. In the last decade, attention was focused on the possible effect of MSCs in the healing of wound caused by BPs [88]. In particular, the first case of MRONJ successfully treated with autologous stem cells transplantation has been reported by Cella et al. [89]. Classically, tissue engineering approach involves scaffolds, growth factors and MSCs, used either separately or in combination in order to restore functional tissues. In this context, it’s important to take in considerations the fact that oral MSCs are influenced by BPs administration, which accounts for the impairment of their capability in tissue repair and osteogenic differentiation [90,91].

5. An Overview of the Oral MSCs Ontogenesis The site specificity of MRONJ has been associated to intramembranous formation of maxilla and mandible, which was responsible for the different of jaw respect to long . Moreover, jaw displays highest bone turnover according to the majority of researches, and surgical interventions account for increase bone remodeling [92]. Jaw is particularly susceptible to bacterial infections compared to other bones, due to the anatomy of mucosal barrier which appears very thin and vulnerable. Finally, given the fact that jaw derived from neural crest cells (NCCs), it appeared that specific molecular pathways might drive both the differentiation of such structures and the pathogenesis of the jaw [93]. A rapid overview of jaw and teeth development may contribute to the understanding of the pathogenesis of the MRONJ. Briefly, during embryonic development, bone formation occurs by two distinct mechanisms: endochondral ossification and intramembranous ossification. Both mechanisms begin with the formation of mesenchymal condensation. During endochondral ossification, mesenchymal nodule forms a cartilage matrix and mesenchymal cells differentiate in chondroblasts; conversely, during intramembranous ossification mesenchymal nodule differentiates directly in , without chondrogenic phase. Craniofacial skeleton predominantly develops through intramembranous ossification involving NCCs-derived progenitor cells [94]. NCCs give rise not only to bone but also to cartilage and periodontium, via reciprocal interactions between the epithelium and the mesenchyme [95]. After their formation, at the interface between the surface ectoderm and the roof plate of the neural tube, NCCs undergo to epithelial-mesenchymal transition. Thereafter, they migrate to different sites. In particular, NCC responsible for maxilla and mandible formation migrate from midbrain and hindbrain (rhombomeres 1 and 2) through the first pharyngeal arch; teeth morphogenesis arises from the sixth week of development and go through four phases: thickening stage, bud stage, cap stage and bell stage. Thereafter, the development of , alveolar bone and periodontal ligament (periodontium) completes tooth morphogenesis before the eruption. Moreover, the Hertwig epithelial Pharmaceuticals 2020, 13, 423 8 of 24 root sheath (HERS) deteriorates and the residual cells form clusters termed as the epithelial rests of Malassez. In the erupted teeth, neural crest cells give rise to different cell types: dental pulp stem cells (DPSCs), dental follicle stem cells (DFSCs), stem cells from apical papilla (SCAPs), stem cells from exfoliated deciduous teeth (SHEDs) and periodontal ligament stem cells (PDLSCs). These cells maintain both self-renewal and differentiation properties [96]. Furthermore, several studies proved that many factors can influence oral MSCs behavior, such as the source, donor age and cell culture conditions [97]. Accordingly, we have previously described the influence of particular cell culture conditions and differentiation factors on both proliferative and osteogenic potential of PDLSCs [98].

6. Exosomes Although the endo-exocytosis intracellular pathways represent a bulky component of classic cellular biology, in the last decades the attention has been directed to intracellular vesicles and particularly to exosomes. The rapid evolution of knowledge in this field is proven by the challenging in the nomenclature of the different types of cellular vesicles [4]. Great interest derived by the discovery of this cellular activity that must not be considered part of a simple waste disposal, but somewhat represents a specific cellular function whose comprehension is still underway. So, the different functions of EVs could open to an evolving number of diagnostic and therapeutic applications. Initially, the different EVs was classified on the basis of biogenesis, charge or size into exosomes, microvesicles and apoptotic bodies. The exosomes, the more relevant EVs with regard to this review, so defined by Johnstone in 1987, appear in cryo-electron microscopy as round structures made up by a double layer membrane. Exosomes originate as intraluminal vesicles (ILVs) via inward budding of the limiting membrane of maturing endosomes, which are usually referred to as multivesicular endosomes (MVEs) or multivesicular bodies (MVBs) [99–101]. Depending on the cell of origin and specific physiological or pathological conditions, the exosomes can contain nucleic acids (DNA, RNA, most frequently miRNAs but also other species long noncoding RNA, circular RNA), lipids, proteins, and metabolites. Cells release exosomes in extracellular medium, where they exert several biological effects on neighboring cells or cells that are significantly distant from the place of production. Quantification and characterization of EVs and exosomes appear not easy, and the results obtained in the different experimental settings may vary, depending on the variety of separation and concentration methods, impacting also the functional studies. For these reasons, the International Society for Extracellular Vesicles (ISEV) provides some guidelines for the analyses of these vesicles useful to adopting the best practice [3]. Some databases are also available to the researchers, such as VESICLEPEDIA (http://www.microvesicles.org) or the ExoCarta [102,103]. For many years, EVs release has been considered the mechanism by which cells maintain cellular homeostasis, by the clearance of cellular undesirable and toxic material. Currently, a growing number of evidence of their involvement in many physiological and pathological situations pointed to EVs as mediators of intercellular communication. These pleiotropic functions involve all the principal biological pathways: gene expression regulation, proliferation, apoptosis, differentiation, development, immune response, signal transduction, migration and regulation. As a consequence, EVs deregulation has been implicated in several disorders from cancer to neurodegenerative diseases. This multifunctional property depends on the highly heterogeneity of the sources and the cargo content [104]. Moreover, it was suggested that cells secrete distinct populations of exosomes with unique size, protein and RNA composition, which display differential effects on the gene expression programs in recipient cells [105]. Although the endosomal theory resulted to be the most prevalent hypothesis on exosomes biogenesis, some evidence suggested a shared pathway from plasma membrane and endosomes [106,107]. The endocytic pathway is organized in key sorting stations in the endosomal system, which are represented by the early endosomes (EEs) and late endosomes (LEs). Transport from EEs to LEs is also accompanied by major proteins and lipids remodeling [108]. During their path, EEs can fuse each other or with vesicles containing acid hydrolases, thus forming intermediate structure, Pharmaceuticals 2020, 13, 423 9 of 24 called MVB and containing ILVs which eventually fuse with the LEs. The MVBs could be view as a crossroad containing one population of ILVs with different fates because the cargo can be packaged into lysosomes for degradation, but can also be routed toward other destinations. In particular, ILVs can be released extracellularly as exosomes. The cargo molecules could also be alternately recycled back to the plasma membrane or to the trans Golgi network to retrograde transport. Moreover, the vesicles trafficking inside these dynamic compartments is accompanied by membrane fusion in selected group of heterotypic and homotypic organelles by fusion and fission, so that the exchange and redistribution of the cargo are also possible [109]. In this context, it has been suggested that the specific targeting of the organelles could be useful in the characterization of EVs [108]. The molecular mechanisms regulating the complex EVs trafficking are still incompletely understood [108]. The biogenesis of MVBs is regulated from several processes, likely coexisting and redundant, otherwise interconnected leading to the production of a different subpopulation of ILVs. This has been comprehensively reviewed by [101] and [110]. As pointed out by Palmulli, four major checkpoints can be identified in the process leading to EVs production [101]. The first checkpoint in MVBs production regards the expression of the specific protein cargo; so, the availability of specific proteins in the cell of origin appears crucial for the production of EEs. Of course, the expression of proteins cargo is both cells- and environment-dependent. The internalization of the protein cargo take place via either clathrin-dependent or clathrin-independent endocytosis. The second checkpoint regards the fate of EEs, which can be recycled to the plasma membrane or directed to MVBs. This process is regulated by the postsynaptic density protein, disc-large, zonulin-1(PDZ) protein syntenin, which together with the endosomal sorting complex required for transport (ESCRT) accessory protein ALIX and thesyndecans account for exosomes release. At this point, MVBs also include proteins derived from the Golgi apparatus. In order to reach the plasma membrane for exosomes release, MVBs need to avoid the alternative fusion with lysosomes or autophagosomes. In particular, the ubiquitination status of the proteins plays a key role in the determination of proteins fate [110]. Most of these processes require the participation of ESCRT machinery. In particular, four biochemically distinct protein complexes (ESCRT-0, -I, -II, and -III) have been characterized to play a key role in the maturation of EEs in LEs together with accessory proteins [19]. In addition to ESCRT machinery-dependent pathway, other mechanisms seem to be involved in exosomal release. In particular, a syndecan/sintenin- and a lipid rafts-dependent processes could act in parallel or separately to recruit exosomal cargoes and generate ILVs [111]. The transport of ILVs on the cytoskeleton to the plasma membrane can be considered as the last major checkpoint in the release of exosomes. This process is mediated by molecular motors, such as dynein and kinesin, small GTPases and specific SNARE complexes [110].

6.1. Exosomes in Oral Mesenchymal Cells MSCs can be readily isolated from human oral tissues like dental pulp, apical papilla, periodontal ligament, gingiva, dental follicle, and others [96]. Among them, neural crest-derived MSCs, such as periodontal ligament and dental pulp, achieved greater attention due to specific and important properties associated to their easily harvesting and propagation, multipotential capabilities, and the absence of ethical concerns. Therefore, MSCs have been recognized as a therapeutic tool in the regenerative medicine field, according to their high ability to regenerate tissues from different origin [7]. In addition, MSCs transplantation results in low engraftment rate, so their therapeutic effects might be related to the secretion of soluble mediators acting in a paracrine fashion. In this scenario, the application of oral MSCs-derived exosomes might assume a crucial role as therapeutic approach in the future also for patients with MRONJ. The major literature available regarding the isolation and characterization of exosomes from different sources of oral MSCs is reported in Table1. In the following sections, authors describe the most recent discoveries on oral MSCs-derived exosomes. Pharmaceuticals 2020, 13, 423 10 of 24

6.2. Exosomes Derived from Human Dental Pulp Stem Cells (DPSCs) To date, a limited number of papers have described the isolation, composition and function of DPSCs exosomes [112–120]. Recently, a proangiogenic action for DPSCs-derived exosomes has been demonstrated. DPSCs-derived exosomes are efficiently taken up by HUVECs and are able to induce cell proliferation and tubule formation via the up-regulation of angiogenesis-related molecules and thePharmaceuticals involvement 2020 of, 13 p38, x FOR MAPK PEER REVIEW pathway [116]. Regarding the mechanism by which pro-angiogenic 10 of 25 action occurs, Gonzalez-King et al., in 2017 showed the recruitment of Jagged-1, the only Notch ligand ligand present in the exosomes derived from human dental pulp MSCs [114]. It’s known that Notch present in the exosomes derived from human dental pulp MSCs [114]. It’s known that Notch signaling signaling plays a key role in angiogenesis through its regulation of the balance between tip cells and plays a key role in angiogenesis through its regulation of the balance between tip cells and stalk stalk cell formation during sprouting process, in which Dll4 and Jagged1 ligands display opposing cellroles. formation They showed during that sprouting HIF-1α process,-overexpressing in which MSCs Dll4 secrete and Jagged1 higher ligandslevel of exosomes display opposing than MSCs, roles. α Theywith showed an higher that concentration HIF-1 -overexpressing of Jagged-1 MSCs able secreteto induce higher angiogenesis level of exosomes both in vivo than and MSCs, in withvitro an higher(Figure concentration 3A) [114]. of Jagged-1 able to induce angiogenesis both in vivo and in vitro (Figure3A) [ 114].

FigureFigure 3. 3. SchematicSchematic representation representation of the of paracrine the paracrine effects of e ffexosomesects of exosomesderived from derived DPSCs fromin angiogenesis, osteogenic differentiation and CD4+ cells alternative differentiation. Jagged1 Pharmaceuticals 2020, 13, x FOR PEER REVIEWDPSCs in angiogenesis, osteogenic differentiation 5 of 25 and CD4+ cells alternative differentiation. Jagged1overexpression overexpression in hypoxic in hypoxicDPSCs-derived DPSCs-derived exosomes antagonizes exosomes antagonizes Dll4 in Notch Dll4 binding, in Notch promoting binding, Figure 1. Schematic representation of ZOL-macrophage cross-talk mechanisms. ZOL-mediated promotingangiogenesis angiogenesis and increasing and increasing the number the of number tip cells of (A tip). cellsExosomes (A). Exosomes derived from derived DPSCs from induce DPSCs activation of TLR4 in macrophages accounted for the overexpressionPharmaceuticals of mature 2020, 13form, x FORof IL-1 PEERβ leading REVIEW 6 of 25 to M1- polarization. Red arrowsinduce indicateosteogenic the osteogenic action differentiation of ZOL di onff erentiationTLR4 activationof DPSCs ofand and DPSCs Kdm6 BMSCs proteins and ( BMSCsB) and induce (B) and Treg induce differentiation Treg differentiation of CD4+ cells of ( CD4C). + overexpression. ( ): gene transcription;cells (:C overexpression;). (: ): in overexpression;crease; (H3K27Me3 response. : inhibition ): : reduced inhibition inhibition of Th17 methylation; of differentiati FPP of Th17synthase. dion; ffABCA1: erentiation;all the ATP-bindingother arrows all the cassette indicate other transporter arrows the direction indicateA1; ApoA-I: all the other arrows indicate the direction of the flow; IL-1β, interleukin-1β; Kdm6a, lysine theof directionthe flow. BMSCs: of the flow. boneapolipoprotein marrow BMSCs: A-I;stem bone BTN3A1: cells; marrow CD4+: butyrophilin-3; stempositive cells; FPP:for CD4 farnesyl +expression;: pyrophosphate; positive DPSCs: for CD4GGPP: dental expression; geranylgeranyl pulp demethylase 6 A; Kdm6b, lysine demethylase 6 B; NF-κB, nucleardiphosphate; factor-κB; NLRP3, HMG-CoA: NACHT, LRR,Hydroxymethylglutaryl-CoA Reductase; INF-γ: interferon-γ; IPP: DPSCs:stem cells; dental IL-17: pulp interleukin- stem cells; IL-17:17; IL-10: interleukin-17; interleukin-10; IL-10: TGF- interleukin-10;β: transforming TGF- growthβ: transforming factor β; Th17: growth T and PYD domains-containing protein 3; TLR4, Toll-Like Receptorisopentenyl 4. See diphosphate; text for explanation TNF, tumor necrosis factor. See text for explanation [16,56–61]. [51,53,54]. factorhelperβ; Th17:17 cells; T helperTNF-α 17, tumor cells; necrosis TNF-α, tumorfactor necrosisα; Treg: regulatory factor α; Treg: T cells. regulatory See text T for cells. explanation See text for

The role of IL-1 signaling inexplanation N-BPs-mediated[113,114]. [113 inflammation,114]. A lot has of been papers described have reported[53]. Both that the patients treated with N-BPs showed a significant alteration of two isoforms of IL-1, IL-1α and IL-1β are produced as a 31γδ kDa T-cells precursor population which can in peripheralbe converted blood to despite a higher level of infiltrating γδ T-cells in the lesion proper 17 kDa mature form via DPSCs-derivedenzymaticDPSCs-derived cleavage. The exosomesarea exosomesoverexpression [62–65]. were In particularwere of tested caspase-1 tested note, for and their zoledronate IL-1forβ capability their was capability reported to induce to activateto lineage induce T cell specific surveillancelineage diff erentiationspecific in both bone reported in zoledronate treated cells seems to also involve epigenetic mechanisms. Indeed, ofdifferentiation naïve MSCs [113 of]. niche Itnaïve appeared and MSCs blood, that together[113]. DPSCs-derived Itwith appeared antiangiogenic exosomes that and DPSC immunomodulatory triggereds-derived odontogenic exosomes mechanisms di fftriggerederentiation [66]. The fact zoledronate treatment increases the expression of two thatrelated single JmjC-domain-containing or repeated administration proteins, of BPs may lead to different results is remarkable [67]. lysine demethylase 6 A (Kdm6a)ofodontogenic both and DPSCsKdm6b, leadingdifferentiation and to BMSCs.Regarding demethylation of Moreover,the both ofmolecular the DPSCs caspase-1 the mechanisms and and exosomes IL-1BMβSCs. involved derivedMoreover, in γδfrom T-cells-activation, the cellsexosomes cultured N-BPsderived in seem thefrom to presence induce cells the promoters and their overexpression (Figure 1) [54]. Interestingly, N-BPs-associated IL-1β ofcultured odontogenic in the di ffpresenceerentiationrelease of of soluble odontogenic media Sema4D were which differe more furtherntiation potent stimulates inmedia inducing macrophages-release were odontogenicmore potent of TNF diinαff . erentiation,Furtherinducing studies overexpression is not always accompanied by its overproduction; such effect is due to the fact that caspase-1 expression mightmost notodontogenic be stimulated probably by differentiation, N-BPs as consequence [53].are required most in oforder probably altered to clarify geneticas this co nsequencepoint. and protein of altered exosomal genetic cargoand protein (Figure exosomal3B) [ 113 ]. Similar mechanisms haveDPSCs-derivedcargo been suggested(Figure to exosomes3B) be involved[113]. TheDPSCs-derived alsoin themechanismsinduced N-BP-induced BMSCsby exosomes fever;which indeed, migration N-BPs also account induced and for proliferation BMSCsMRONJ establishmentmigration [120]. Similarand also proliferation involve results other were cell fever occurs among patient [120].receiving Similar the first BPresults treatment,types. were Administration while confirmed does not occurof N-BPsby in Hupatients and et in particularal., 2019. of zoledronateThey characterized accounts for reducedthe microRNA recruitment of receiving second or repeatedconfirmed intravenous by N-BPs. Hu et In al., theneutrophils 2019.latter case, They andthe characterized inhibitionreduced of activity osteoclast, the of microRNA natural killer expression (NK) cells profiles [68]. During of exosomes bone remodeling, derived expression profiles of exosomes derived from DPSCs under odontogenic conditions and observed a recognized as a major sourcefrom of IL-1 DPSCsβ, accounts under for normal odontogenicosteoclasts or reduced stimulate IL-1 conditionsβ level [53,55].NKs cellsTogether, and which, observed in turn, a set produce of diff erentiallyINFγ inhibiting expressed osteoclastogenesis. miRNA these observations indicateinvolved a keyset role of ofdifferentially in IL-1 theβ in regulation the N-BPs-induced expressedOsteoclast of Transforming acutesuppression miRNA immune involved response.induced Growth by in FactorN-BPs the regulation accountsβ1 (TGF for βof 1)NK Tran/SMADS cellssforming deregulation signaling Growth and pathway the Factor maintenance [β1191 ]. (TGFβ1)/SMADS signaling pathway [119]. It’s noteworthy the fact that dental tissue-derived 3.2. BPs-γδ T Cells Cross-TalkIt’s noteworthy the factof an that immunosuppressive dental tissue-derived microenviron exosomesment, delaying can potentially wound healing direct [69]. their action to all cell exosomes can potentially direct their action to all cell populations present in the microenvironment. The mechanisms by whichpopulations N-BPs inhibit present osteoclast in3.3. also the BPs-Vascular account microenvironment. for the Endothelium establishment Cross-Talk Asof both a matter of the fact, immunosuppressive properties inflammatory and necrotic events.As a Thematter most relevantof the involvesfact, immunosuppressive the production of IPP during proper the ties of MSCs are also mediated by exosomes inhibition of FPP synthase in oste[118].oclasts. Ji Indeed,et al. IPP showed stimulatesN-BPs VthatγVδ 2 have T-cellsDPSC-derived been to produce shown interferon toexosomes inhi bit angiogenesis displayed both stro innger vivo immunomodulatoryand in vitro, hindering the (INF)-γ and tumor necrosis factorcapability (TNF) triggering than bone acutemobility inflammatorymarrow of cellsMSCs reactions involved (BMMSC)-derive [56,57]. in the Dendritic immune d response exosomes [70]. when It becomes co-cultured evident that with the peripheralinhibitory action cells, monocytes and macrophages-release of IPP seems towardsto also mediate osteoclast the activation interferes of γδwith T-cells normal angiogenesis, due to the role of osteoclasts in the correct [16,58,59]. IPP extrusion is bloodmediated mononuclear by ATP-binding cells cassette [117]. transporter Indeed, A1 DPSCs-derived(ABCA1) in exosomes inhibited the differentiation of patterning of the vessels in the bone [71]. Moreover, N-BPs exert their role in inhibiting angiogenesis cooperation with butyrophilin-3 (BTN3A1) and apolipoprotein A-I (ApoA-I) (Figure 2) [60,61]. naïve CD4+ T cells intothrough T helper both direct 17 cells and indirect(Th17), effects. with theFirst consequent of all, N-BPs reductionsuch as zoledronate of IL-17 inhibitand TNF- endothelialα production. At the cellssame and time, endothelial Treg cell progenitor differentiation cells proliferatio appearedn, migration to be promoted,and differentiation, followed accounting by an for increased release of cellsthe anti-inflammatoryapoptosis [68,72]. factorsOn the IL-10 other and hand, TGF- Ohlrichβ (Figure et 3C).al. However,showed overexpression also in this of vascular-endothelial growth factor (VEGF) in gingival fibroblast after exposure to zoledronate, suggesting the induction of a proangiogenic environment [73]. Similar contrasting effects may be understood in light of the fact that N-BPs alter VEGF receptors maturation in endothelial cells, so that the increased expression of VEGF does not exert any effect [74]. In addition, zoledronate treatment may inhibit angiogenesis by reducing the expression of osteopontin (an angiogenesis inducer) in both maxilla and mandible [75]. Finally, N-BPs may also interfere with endothelial differentiation of mesenchymal cells [76].

4. Current and Emerging Treatment Options During the years several authors have discussed MRONJ treatment. However, while the objective seems to be to minimize the risk of MRONJ onset, unfortunately this is not always possible. Figure 2. Mechanisms of γδT-cells activation. The ZOL-mediatIn 2014, theed overproductionAmerican Association of IPP in of Oral and Maxillofacial Surgeons provided some guidelines in osteoclasts accounts for γδT-cells activation and triggersthe acut treatmente inflammatory of MRONJ. reaction. TheThey cells argued that each MRONJ stage should be treated with a specific involved in IPP production also included monocytes, macrophagestherapy; and stage dendritic 0 (patients cells. Black without arrows any clinical evidence of necrotic bone, but with non-specific indicate the direction of the flow; green arrow indicates the activation of acute inflammatory symptoms or clinical and radiographic findings) should be strictly monitored and eventually treated with medication for chronic pain and infections; stage 1 (patients with exposed necrotic bone or fistulae without any evident infection) should benefit from medical management with topic antimicrobial and chlorhexidine 0.12% rinses, however immediate surgical treatment is not suggested; stage 2 (patients with exposed necrotic bone or fistulae and clear signs of infection) should be treated with a combination of oral antibiotics, antibacterial mouth rinses and debridement of necrotic areas to relieve soft tissues irritation and to control infection spread; stage 3 (patients with exposed necrotic bone or fistulae, clear signs of infection and huge necrotic bone areas, pathological fractures, extra-oral fistula, oral antral/oral nasal communication and osteolysis process extended to the inferior mandibulae border or sinus floor) should be managed with antibiotics and pain control,

Pharmaceuticals 2020, 13, 423 11 of 24 of MSCs are also mediated by exosomes [118]. Ji et al. showed that DPSC-derived exosomes displayed stronger immunomodulatory capability than MSCs (BMMSC)-derived exosomes when co-cultured with peripheral blood mononuclear cells [117]. Indeed, DPSCs-derived exosomes inhibited the differentiation of naïve CD4+ T cells into T helper 17 cells (Th17), with the consequent reduction of IL-17 and TNF-α production. At the same time, Treg cell differentiation appeared to be promoted, followed by an increased release of the anti-inflammatory factors IL-10 and TGF-β (Figure3C). However, also in this case, the specific proteins, miRNAs, mRNAs or lipids involved in this process remain to be elucidated, by the application of both standards methodologies as well as new proteomic methods or bioinformatic platforms [121]. Recently, DPSC-derived exosomes have also been characterized for their capability to regulate migration and differentiation of Schwann cell, recognized as neural-crest-derived stem cells able to regenerate odontoblasts [122]. Exosomes derived from LPS-treated DPSCs accelerated Schwann cell proliferation and migration, via the strengthening of directional cell movement, as well as odontogenic differentiation, suggesting a role for Schwann cells in the regenerative processes [117,122]. DPSC-derived exosomes also displayed neuroprotective efficacy in an in vitro excitotoxicity model of neurodegeneration. In their comprehensive study, Venugopal et al. compared the neuroprotective effects using three strategies: hippocampal neuron-MSC co-culture, neuron-MSC condition medium treatment and neuron-MSC exosomes treatment in an in vitro model of kainic acid-induced excitotoxicity. They showed that neuroprotective action of DPSCs-derived exosomes was similar to that of co-culture strategy and conditioned medium treatment strategy [115].

6.3. Exosomes Derived from Human Periodontal Ligament Stem Cells (PDLSCs) An even lower number of papers have investigated the cargo of exosomes of PDLSCs. Rajan et al. first isolated exosomes from PDLSCs. Subsequent papers showed that PDLSCs-derived exosomes played a key role in events occurring during inflammation in periodontitis [123]. Exosomes derived from PDLSCs are involved in the altered balance of Th17/Treg reported in periodontal tissues of patients with periodontitis. Indeed, although PDLSCs in normal environment accounted for the maintenance of Th17/Treg balance, LPS-treatment of PDLSCs in vitro was responsible for a decreased expression of miR-155-5p in exosomes and a consequent up-regulation of SIRT-1 when transferred in CD4+ T cells culture. The increased expression of SIRT-1 accounted for the increase of Th17 differentiation and the downregulation of Treg phenotype (Figure4A) [ 124]. Moreover, exosomes derived from PDL fibroblasts exposed to LPS accounted for the inhibition of osteogenic activity and osteoprotegerin expression in osteoblasts (Figure4B). From these observations appear clear that PDLSCs-derived exosomes play a key role in bone remodeling that occurred during periodontal inflammation [125]. Pharmaceuticals 2020, 13, x FOR PEER REVIEW 12 of 25

Figure 4. Cont.

Figure 4. Schematic representation of the paracrine effects of exosomes derived from PDLSCs in CD4+ cells alternative differentiation, osteogenic differentiation, IL-1β production and angiogenesis. Exosomes derived from LPS-treated PDLSCs account for the upregulation of Th17 and the downregulation of Treg phenotype when transferred in CD4+ T cells culture (A). Exosomes derived from LPS-treated PDLSCs also accounted for the inhibition of osteogenic activity and osteoprotegerin expression in osteoblasts (B). PDLSCs under cyclic stretch release exosomes able to inhibit macrophage release of IL-1β (C). Inflamed PDLSCs release exosomes enriched in VEGFA,

leading to increased angiogenesis (D). : increase; decrease; : inhibition of the process; all the other arrows indicate the direction of the flow; +/-: plus or minus; CD4+: positive for CD4

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 12 of 25

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Figure 4. Schematic representation of the paracrine effects of exosomes derived from PDLSCs in CD4+ cellsFigure alternative 4. Schematic diff erentiation,representation osteogenic of the paracrine differentiation, effects of exosomes IL-1β production derived from and PDLSCs angiogenesis. in ExosomesCD4+ derived cells alternative from LPS-treateddifferentiation, PDLSCs osteogenic account differentiation, for the IL-1β upregulation production and of angiogenesis. Th17 and the downregulationExosomes ofderived Treg phenotypefrom LPS-treated when transferredPDLSCs account in CD4 for+ theT cells upregulation culture ( Aof). ExosomesTh17 and the derived PharmaceuticalsPharmaceuticals 2020, 13, x2020 FOR, 13 PEER, x FOR REVIEW PEER REVIEW 5 of 25 5 of 25 from LPS-treateddownregulation PDLSCs of Treg also phenotype accounted when for transferred the inhibition in CD4+ of osteogenic T cells culture activity (A). Exosomes and osteoprotegerin derived Figure 1.Figure Schematic 1. Schematic representationexpression representation fromof ZOL-macropha in osteoblastsLPS-treatedof ZOL-macrophage cross-talk (BPDLSCs).ge PDLSCscross-talk mechanisms. also mechanisms. under accounted ZOL-mediated cyclic ZOL-mediated stretchfor the release inhibition exosomes of osteogenic able to inhibit activity macrophage and activationactivation of TLR4 ofin TLR4macrophages in macrophages accountedosteoprotegerin accounted for the overexpression for theexpression overexpression of mature in Pharmaceuticalsosteoblasts of form mature of IL-1form (2020βB leadingof). ,IL-1PDLSCs 13, βx leadingFOR underPEER REVIEW cyclic stretch release exosomes able to 6 of 25 to M1- polarization.release Red arrows of IL-1indicateβ the(C ).action Inflamed of ZOL on PDLSCs TLR4 activation release and Kdm6 exosomes proteins enriched in VEGFA, leading to increased to M1- polarization. Red arrows indicateinhibit the action macrophage of ZOL on releaseTLR4 activation of IL-1 andβ (Kdm6C). Inflamed proteins PDLSCs release exosomes enriched in VEGFA, overexpression.overexpression. ( ): (geneangiogenesis transcription; ): gene transcription; ( (D ): ).in crease;(: ): increase;in (H3K27Me3crease; (H3K27Me3 ):decrease; reduced response. ): reducedmethylation; : methylation; inhibition inhibition of FPP of synthase. the process; ABCA1: all ATP-binding the other cassette arrows transporter A1; ApoA-I: all the allother the arrows other arrowsindicateindicate indicatethe directionleading thethe direction ofto increasedthe flow;of the of IL-1 angiogenesisflow; theβ, interleukin-1IL-1 flow;β, interleukin-1 (+/apolipoproteinDβ;). Kdm6a, : plusβ; : increase; Kdm6a,lysine orA-I; minus;lysine BTN3A1: decrease; CD4 butyrophilin-3;+ : : positiveinhibition FPP: forfarnesylof the CD4 process; pyrophosphate; expression; all GGPP: geranylgeranyl demethylasedemethylase 6 A; Kdm6b, 6 A; Kdm6b,lysine demethylase lysine demethylase 6 B; NF- κ6 B,B; nuclearNF-κB, factor-nuclearκ B;factor- NLRP3,κB;− NLRP3,NACHT, NACHT, LRR, LRR, β the other arrowsβ indicate the directiondiphosphate; of the HMG-CoA:flow; +/-: plus Hydroxymethylglutaryl-CoA or minus; CD4+: positive Reductase; for CD4 INF-γ: interferon-γ; IPP: and PYDand domains-containing PYD domains-containingIL-1 protein: interleukin-1 protein3; TLR4, 3; ToTLR4,ll-Like; LPS:To Receptorll-Like lipopolysaccharide; Receptor 4. See text4. Seefor textexplanation for OPG: explanation osteoprotegerin; PDLSCs: periodontal ligament isopentenyl diphosphate; TNF, tumor necrosis factor. See text for explanation [16,56–61]. [51,53,54].[51,53,54]. stem cells; SIRT-1: NAD-dependent deacetylase sirtuin-1; Th17: T helper 17 cells; Treg: regulatory T cells; VEGFA: Vascular endothelial growth factor A. See text for explanation [113,114]. See text for The roleThe of IL-1role signalingof IL-1 signaling in N-BPs-mediated in N-BPs-mediated inflammation inflammation has been has described Abeen lot described of [53].papers Both [53]. have the Both reported the that patients treated with N-BPs showed a significant alteration of two isoformstwo isoforms of IL-1, ofIL-1 IL-1,α and explanationIL-1 IL-1α andβ are IL-1 producedβ [are124 produced,126 as a– 31128 kDaas]. a precursor31 kDa precursorγδ which T-cells can which population be converted can be converted in to peripheral to blood despite a higher level of infiltrating γδ T-cells in the lesion proper proper17 kDa 17mature kDa matureform via form enzymatic via enzymatic cleavage. clea Thevage. overexpression The overexpressionarea of [62–65]. caspase-1 of caspase-1In andparticular IL-1 andβ IL-1note,β zoledronate was reported to activate T cell surveillance in both bone reportedreported in zoledronate in zoledronate treatedRecent treatedcells evidenceseems cells toseems also characterized toinvolve also involveepigeneticniche epigenetic the mechanisms. and anti-inflammatory blood,mechanisms. Indeed,together Indeed, with antiangiogenic role of PDLSCs-derived and immunomodulatory exosomes mechanisms in [66]. The fact zoledronatezoledronate treatment treatment increases increases the expression the expression of two ofrelated two relatedJmjC-domain-containing thatJmjC-domain-containing single or repeated proteins, proteins, administration of BPs may lead to different results is remarkable [67]. PDLSCs-macrophages cross-talk during both physiological and pathological conditions [127]. lysine demethylaselysine demethylase 6 A (Kdm6a) 6 A (Kdm6a) and Kdm6b, and Kdm6b, leading leadingto demethylation to demethylationRegarding of the caspase-1 of the the caspase-1 molecularand IL-1 andβ mechanismsIL-1β involved in γδ T-cells-activation, N-BPs seem to induce the promoterspromoters and their and overexpressiontheir overexpression (Figure (Figure1) [54]. 1) Interestingly,[54]. Interestingly, N-BPs-associated N-BPs-associated IL-1β IL-1β Exosomes released from PDLSCs underrelease cyclic of soluble stretch, Sema4D that which resembles further occlusal stimulates stimuli, macrophages-release were responsible of TNFα. Further studies overexpressionoverexpression is not always is not alwaysaccompanied accompanied by its over by production;its overproduction; such effect such is effect due to is thedue fact to thethat fact that caspase-1caspase-1 expression expression mightfor the not might be suppression stimulated not be stimulated by N-BPs of by IL-1 [53]. N-BPsβ production [53]. are required via the in inhibitionorder to clarify of thethis point. NF-κ B signaling pathway. Moreover, Similar Similarmechanisms mechanismsPDLSCs-derived have been have suggested been suggested exosomesto be involved to be involved werein the N-BP-induced alsoin the TheN-BP-induced able mechanisms tofever; inhibit indeed, fever; by indeed, NLRP3 which N-BPs inflammasome account for MRONJ signaling establishment in human also involve other cell fever occurs among patient receiving the first BP treatment, types.while doesAdministration not occur in of patients N-BPs and in particular of zoledronate accounts for reduced recruitment of fever occurs among patientmacrophages receiving the primed first BP treatment, with LPS while bydoes inhibiting not occur in patients the NF- κB signaling pathway (Figure4C) [ 127]. receivingreceiving second secondor repeated or repeated intravenous intravenous N-BPs. N-BPs.In the latterIn the case, latter theneutrophils case, inhibition the inhibition ofand osteoclast, reduced of osteoclast, activity of natural killer (NK) cells [68]. During bone remodeling, recognizedrecognized as a major as Also asource major ofinsource IL-1 thisβ of, accounts IL-1 case,β, accounts for PDLSCs-derived normal for normalor reduced or reduced IL-1osteoclasts exosomesβ level IL-1 [53,55].β levelstimulate play[53,55].Together, aTogether,NKs key cells role which, in the in maintenanceturn, produce ofINF periodontalγ inhibiting osteoclastogenesis. these observationsthese observations indicateimmune indicate a key role/ inflammatorya key of IL-1 roleβ of in IL-1 the βN-BPs-induced in homeostasis. the N-BPs-induced acuteOsteoclast immune acute immune response. suppression response. induced by N-BPs accounts for NK cells deregulation and the maintenance of an immunosuppressive microenvironment, delaying wound healing [69]. 3.2. BPs-3.2.γδ TBPs- Cellsγδ Cross-TalkT Cells Cross-Talk As pointed out by Wang et al., the fact that PDLSCs are the only ones among oral mesenchymal

The mechanismsThe mechanisms cellsby which showing by N-BPswhich inN-BPshibit cyclic osteoclastinhibit stretch-induced osteoclast also account also account for3.3. exosomethe BPs-Vascular establishmentfor the establishment secretion Endothelium of both of is both noteworthy Cross-Talk [127]. inflammatoryinflammatory and necrotic and necrotic events.Aberrant events.The most angiogenesis The relevant most relevant involves resulted involves the production as the pathognomonic production of IPP duringof IPP duringthe sign of the periodontitis. Interestingly, PDLSCs played inhibitioninhibition of FPP synthaseof FPP synthase in osteoclasts. in oste oclasts.Indeed, Indeed, IPP stimulates IPP stimulates VγVδ2 T-cellsVγVδ2N-BPs toT-cells produce tohave produce interferon been interferon shown to inhibit angiogenesis both in vivo and in vitro, hindering the (INF)-γ (INF)-and tumorγ and necrosistumora crucial necrosis factor role(TNF) factor in triggering (TNF) similar triggering acute event, inflammatory acute according inflammatory mobilityreactions to reactions the[56,57].of cells up-regulation [56,57].Dendritic involved Dendritic in the of immune VEGFA, response recognized [70]. It becomes as the mostevident potent that the inhibitory action cells, monocytescells, monocytes and macrophages-releaseagent and macrophages-release participating of IPP in seemsof modulation IPP to seems also mediateto also of mediate vasculartowardsthe activation the osteoclast endothelium.activation of γδ T-cells interferes of γδ T-cells According with normal to angiogenes Zhang etis, al., due inflammation to the role of osteoclasts was in the correct [16,58,59]. IPP extrusion is mediated by ATP-binding cassette transporter A1 (ABCA1) in [16,58,59]. IPP extrusion is mediated by ATP-binding cassette transporterpatterning A1 of (ABCA1)the vessels in in the bone [71]. Moreover, N-BPs exert their role in inhibiting angiogenesis cooperationcooperation with butyrophilin-3 withresponsible butyrophilin-3 (BTN3A1) for (BTN3A1) miR-17-5pand apolipoprotein and ap down-regulationolipoprotein A-I (ApoA-I) A-I (ApoA-I) (Figure in (Figure2) PDLSCs [60,61]. 2) [60,61]. which, in turn, accounted for increased expression of VEGFA in exosomes derived fromthrough inflamed bothPDLSCs. direct and Theindirect exosomes-mediated effects. First of all, N-BPs transfer such ofas VEGFAzoledronate to inhibit endothelial cells and endothelial progenitor cells proliferation, migration and differentiation, accounting for endothelial cells finally contributed tocells the increasedapoptosisvascularization [68,72]. On the of periodontalother hand, ligamentsOhlrich et in periodontitisal. showed overexpression of (Figure4D) [128]. vascular-endothelial growth factor (VEGF) in gingival fibroblast after exposure to zoledronate, suggesting the induction of a proangiogenic environment [73]. Similar contrasting effects may be understood in light of the fact that N-BPs alter VEGF receptors maturation in endothelial cells, so that the increased expression of VEGF does not exert any effect [74]. In addition, zoledronate treatment may inhibit angiogenesis by reducing the expression of osteopontin (an angiogenesis inducer) in both maxilla and mandible [75]. Finally, N-BPs may also interfere with endothelial differentiation of mesenchymal cells [76].

4. Current and Emerging Treatment Options During the years several authors have discussed MRONJ treatment. However, while the objective seems to be to minimize the risk of MRONJ onset, unfortunately this is not always possible. Figure 2.Figure Mechanisms 2. Mechanisms of γδT-cells of γδ activation.T-cells activation. The ZOL-mediat The ZOL-mediated overproductionIned 2014, overproduction the ofAmerican IPP ofin IPP Association in of Oral and Maxillofacial Surgeons provided some guidelines in osteoclastsosteoclasts accounts accounts for γδT-cells for γδ activationT-cells activation and triggers and triggersacute inflammatory acute theinflammatory treatment reaction. reaction. The of cells MRONJ. The cells They argued that each MRONJ stage should be treated with a specific involvedinvolved in IPP production in IPP production also included also included monocyte monocytes, macrophagess, macrophages and dendritictherapy; and dendritic cells. stageBlack cells. arrows 0 Black (patients arrows without any clinical evidence of necrotic bone, but with non-specific indicate indicatethe direction the direction of the flow;of the green flow; arrow green indicates arrow indicates the activation the activation of acute of inflammatory acute inflammatory symptoms or clinical and radiographic findings) should be strictly monitored and eventually treated with medication for chronic pain and infections; stage 1 (patients with exposed necrotic bone or fistulae without any evident infection) should benefit from medical management with topic antimicrobial and chlorhexidine 0.12% rinses, however immediate surgical treatment is not suggested; stage 2 (patients with exposed necrotic bone or fistulae and clear signs of infection) should be treated with a combination of oral antibiotics, antibacterial mouth rinses and debridement of necrotic areas to relieve soft tissues irritation and to control infection spread; stage 3 (patients with exposed necrotic bone or fistulae, clear signs of infection and huge necrotic bone areas, pathological fractures, extra-oral fistula, oral antral/oral nasal communication and osteolysis process extended to the inferior mandibulae border or sinus floor) should be managed with antibiotics and pain control,

Pharmaceuticals 2020, 13, 423 13 of 24

Immunomodulatory action of PDLSCs-derived exosomes was also supported by the in vivo research of [129]. They isolated exosomes/microvesicles from PDLSCs of patients showing relapsing-remitting multiple sclerosis, the most common form of multiple sclerosis with 85% of occurrence [130], and showed the capability of exosomes/microvesicles to inhibit NLRP3 inflammasome activation, exerting a protective role in experimental autoimmune encephalomyelitis mice model of multiple sclerosis. Similar effects were reported for conditioned media of PDLSCs of patients showing relapsing-remitting multiple sclerosis.

6.4. Exosomes Derived from Human Exfoliated Deciduous Teeth Stem Cells (SHEDs) Stem cells from exfoliated deciduous teeth are considered as advantageous stem cells source due to the fact that they represent a more immature populations of postnatal stem cells with high proliferation rate and osteoinductive capacity [131]. Exosomes have been successfully isolated by SHED and characterized for their capability of enhancing PDLSCs osteogenic differentiation via the Wnt/β-catenin and BMP/Smad signaling pathways [132]. Moreover, exosomes derived from osteogenic conditioned-SHED displayed higher osteogenic function in PDLSCs when compared to SHED without conditioning. Concerning the molecular mechanisms activated by SHED exosome, it was reported that Wnt3a and BMP2 up-regulation in exosomes accounted for the activation of Wnt/β-catenin and BMP/Smad signaling pathways in PDLSCs (FigurePharmaceuticals5)[132]. 2020, 13, x FOR PEER REVIEW 14 of 25

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Figure 1. Schematic representation of ZOL-macrophage cross-talk mechanisms. ZOL-mediated Figure 5. Schematic representation of the paracrine effects of exosomes derived from SHED. Figure 5. Schematic representationactivation of the of TLR4paracrine in macrophages effects accounted of exosomes for the overexpression derived offrom mature SHED.form of IL-1 β leading Exosomes derived from osteogenic-conditionedto M1- polarization. SHED Red arrows induce indicate PDLSCs the action osteogenic of ZOL on TLR4 diff activationerentiation and Kdm6 via proteins Exosomes derived from osteogenic-conditioned SHED induce PDLSCs osteogenic differentiation via the activation of Wnt/β-catenin and BMPoverexpression./Smad signaling( ): gene pathways.transcription; (: ): increase;increase; (H3K27Me3 all the other ): reduced arrows methylation; indicatethe activation the direction of Wnt/ of theβ-catenin flow; BMP2: andall theBMP/Smad Bone other morphogenetic arrows signaling indicate the proteinpa directionthways. 2; of PDLSCs: the : flow;increase; periodontalIL-1β, interleukin-1all the ligament otherβ; Kdm6a, lysine arrows indicate the direction of the flow;demethylase BMP2: 6 A; Bone Kdm6b, morphogeneti lysine demethylasec protein 6 B; NF-κ B,2; nuclear PDLSCs: factor- periodontalκB; NLRP3, NACHT, LRR, stem cells; SHED: stem cells fromand exfoliated PYD domains-containing deciduous protein teeth; 3; TLR4, SMAD: Toll-Like small Receptor mother 4. See text against for explanation decapentaplegic;ligament stem cells; Wnt3a: SHED: Wingless-relatedstem cells[51,53,54]. from exfolia integrationted deciduous site familyteeth; SMAD member: small 3A. mother See against text for decapentaplegic; Wnt3a: Wingless-related integration site family member 3A. See text for explanation [132]. The role of IL-1 signaling in N-BPs-mediated inflammation has been described [53]. Both the explanation [132]. two isoforms of IL-1, IL-1α and IL-1β are produced as a 31 kDa precursor which can be converted to Pivoraite˙ et al. first investigatedproper immunomodulatory 17 kDa mature form via and enzymatic anti-inflammatory cleavage. The overexpression function of of caspase-1 exosomes and IL-1β derivedPivorait from exfoliatedė et al. first deciduous investigatedreported teeth inimmunomodulain zoledronate an experimental treatedtory cells and animalseems anti-inflammatoryto also model involve of epigenetic paw inflammationfunction mechanisms. of Indeed, exosomes derived from exfoliatedzoledronate deciduous treatment teeth increases in thean expressionexperimental of two relatedanimal JmjC-domain-containing model of paw proteins, (obtained by subcutaneous injectionlysine of demethylase 1% λ-carrageenan) 6 A (Kdm6a) and [133 Kdm6b,]. Exosomes leading to demethylation administration of the caspase-1 exerted and IL-1β inflammation (obtained by subcutaneous injection of 1% λ-carrageenan) [133]. Exosomes strong anti-inflammatory effects, comparablepromoters and with their thoseoverexpression of . (Figure 1) [54]. One Interestingly, potential N-BPs-associated mechanism IL-1β administration exerted strong anti-inflammatoryoverexpression is not alwayseffects, accompanied comparable by its withoverproduction; those of such glucocorticoids. effect is due to the fact that One potential mechanism by whichcaspase-1 exosomes expression achieved might not besuch stimulated result by mayN-BPs involve[53]. the COX2, PLA2, Similar mechanisms have been suggested to be involved in the N-BP-induced fever; indeed, and iNOS signaling pathways, however,fever occurs to amongdate, thepatient supporting receiving the data first appearBP treatment, poor. while does not occur in patients Due to their embryological derivationreceiving second by cranialor repeated ne uralintravenous crest cells,N-BPs. whichIn the latter are precursorscase, the inhibition of both of osteoclast, neural and skeletal tissues, SHEDsrecognized is considered as a major source particularly of IL-1β, accounts suitable for normal for the or reducedinduction IL-1β levelof neural [53,55]. Together, these observations indicate a key role of IL-1β in the N-BPs-induced acute immune response. differentiation as well as for neuroprotective action. The first evidence for neuroprotective action of SHED-derived exosomes was prov3.2. BPs-idedγδ T Cellsby Cross-Talk[134], which tested the effects of exosomes and micro-vesicles derived from SHEDsThe on mechanisms human by dopaminergic which N-BPs inhibit neur osteoclastons alsoduring account oxidative for the establishment stress of both inflammatory and necrotic events. The most relevant involves the production of IPP during the induced by 6-OHDA. From the resultsinhibition itof FPPappeared synthase inthat oste exosomes,oclasts. Indeed, but IPP stimulatesnot micro-vesicles VγVδ2 T-cells to derived produce interferon from SHEDs, suppressed 6-OHDA–induced(INF)-γ and tumor necrosis ap optosisfactor (TNF) intriggering dopaminergic acute inflammatory neurons reactions [56,57].[134]. Dendritic SHED-derived exosomes have beencells, also monocytes tested and for macrophages-release potential neuroprotective of IPP seems to alsoaction mediate after the TBI.activation TBI ofis γδ T-cells [16,58,59]. IPP extrusion is mediated by ATP-binding cassette transporter A1 (ABCA1) in defined as damage to the braincooperation caused withby butyrophilin-3external mechanical (BTN3A1) and force; apolipoprotein at the A-I beginning, (ApoA-I) (Figure TBI 2) [60,61].is characterized by pro-inflammatory events involving the release of inflammatory factors by microglia. Then, there is a transition of microglia from M1 to M2 with anti-inflammatory action, followed by tissue reparation. In some cases, pro-inflammatory events are not inhibited, so that tissue repair did not occur. It has been showed that stem cell therapy, by using neural stem cells could ameliorate traumatic brain injury (TBI) sequel, also via a direct shift of microglia from M1 to M2. Authors showed that administration of SHED-derived exosomes could promote functional motor recovery in rats after TBI, via a direct shift of microglia polarization from M1 to M2 [135].

Figure 2. Mechanisms of γδT-cells activation. The ZOL-mediated overproduction of IPP in osteoclasts accounts for γδT-cells activation and triggers acute inflammatory reaction. The cells involved in IPP production also included monocytes, macrophages and dendritic cells. Black arrows indicate the direction of the flow; green arrow indicates the activation of acute inflammatory

Pharmaceuticals 2020, 13, 423 14 of 24 by which exosomes achieved such result may involve the COX2, PLA2, and iNOS signaling pathways, however, to date, the supporting data appear poor. Due to their embryological derivation by cranial neural crest cells, which are precursors of both neural and skeletal tissues, SHEDs is considered particularly suitable for the induction of neural differentiation as well as for neuroprotective action. The first evidence for neuroprotective action of SHED-derived exosomes was provided by [134], which tested the effects of exosomes and micro-vesicles derived from SHEDs on human dopaminergic neurons during oxidative stress induced by 6-OHDA. From the results it appeared that exosomes, but not micro-vesicles derived from SHEDs, suppressed 6-OHDA–induced apoptosis in dopaminergic neurons [134]. SHED-derived exosomes have been also tested for potential neuroprotective action after TBI. TBI is defined as damage to the brain caused by external mechanical force; at the beginning, TBI is characterized by pro-inflammatory events involving the release of inflammatory factors by microglia. Then, there is a transition of microglia from M1 to M2 with anti-inflammatory action, followed by tissue reparation. In some cases, pro-inflammatory events are not inhibited, so that tissue repair did not occur. It has been showed that stem cell therapy, by using neural stem cells could ameliorate traumatic brain injury (TBI) sequel, also via a direct shift of microglia from M1 to M2. Authors showed that administration of SHED-derived exosomes could promote functional motor recovery in rats after TBI, via a direct shift of microglia polarization from M1 to M2 [135].

6.5. Exosomes Derived from Gingival Mesenchymal Stem Cells (GMSC) More recently, gingival mesenchymal stem cells (GMSC)-derived exosomes have also been tested for their regenerative potentials. In particular, the study of Zhang et al. tested the regenerative potential of small intestinal submucosa–extracellular matrix (SISECM) constructs both with gingival MSCs or their derivative exosomes in taste bud regeneration during tongue reconstruction [136]. From their results it appeared that SIS-ECM construct, in association with GMSCs, exerted an overall better beneficial effect on the reconstruction of structure and taste bud regeneration with respect to exosome/SIS-ECM constructs. These observations suggest that some non exosomal factors secreted by GMSCs may also play a role in facilitating taste bud regeneration, so in this case exosomes appeared to exert a lower action respect to the cell of origin [136].

6.6. Exosomes Derived from Other Oral Sources Very recently, Zhang et al. tried to characterize exosomes in cells derived from Hertwig’s epithelial root sheath [137]. It’s clear that HERS is a transient structure assembled in the early period of the elongation of the root, so appeared difficult the use of the sample as experimental in vitro model. However, authors established an immortalized HERS cell line which resulted to be similar in morphology and characteristics to primary HERS cells, and could also induce odontogenic differentiation of dental papilla cells. HERS-derived exosomes are involved in this process. Similarly, rat maxillary sinus mucosa-derived cells and mandibular periosteum-derived cells have been successfully used to isolate exosomes [138]. In both cases, exosomes exposure was able to enhance the proliferation, migration, and osteogenic differentiation of rat BMMSCs in vitro. In addition, exosomes-scaffold materials construct accelerated bone formation in rat femoral defects, confirming the in vivo action. A detailed description of exosomes derived from stem cells from the apical papilla (SCAP) has been provided by [139]. They analyzed and compared PIWI-interacting RNAs (piRNAs) expression profiles in the exosomes of SCAP and the exosomes of BMMSCs, showing that they differ significantly. In particular, piRNAs are a new class of small ncRNAs forming RNA-induced silencing complexes by binding to PIWI-family subproteins [139]. According to the authors, the differentially expressed piRNAs could exert a specific role during the teeth morphogenesis and the formation of bone tissue, respectively. Functionally, exosomes derived from SCAP were able to promote BMMSC-based dentine-pulp complex regeneration when implanted subcutaneously into immunodeficient mice Pharmaceuticals 2020, 13, 423 15 of 24 together with tooth fragments, BMMSCs, and scaffold. Moreover, exosomes derived from SCAP promoted the specific dentinogenesis of BMMSCs [140]. Exosomes also displayed anti-apoptotic activity in odontoblasts. Indeed, exosomes derived from odontoblasts exposed to high concentration of LPS exerted an anti-apoptotic action on odontoblast exposed to low concentration of LPS, suggesting a protective role during inflammation caused by caries [141]. The involvement of oral MSCs-derived exosomes in malignant transformation has also been showed [142]. Oral is one of the most common malignancy types globally, and in the majority of cases evolve from oral premalignant lesions, such as and oral . The role of MSCs and above all cancer cells-MSCs cross-talk in malignant transformation has not been elucidated, however many evidence suggested a key role for oral MSCs. Similar to physiological conditions, also in this case indirect cell-to-cell interactions mediated by the release of EVs have been suggested [143]. Li et al. showed that exosomes derived from erythroplakia and oral leukoplakia patients exerted a key role in promoting proliferation, migration and invasion in vitro of two cancer cell line. i.e., the DOK cell line (oral hyperplasia cell line) and the SCC15 cell line (oral carcinoma cell line), an action mediated by the up-regulation of exosomal miR-8485 [142].

Table 1. Recent key studies with methodology for oral MSCs-derived exosomes isolation.

Cell Type Cell Source Isolation Method References DPSCs, PDLSCs, [112,114,116–118,124, human ultracentrifugation SHED, SCAP 125,128,132–134,141] SMCs (maxilla), PCs rat ultracentrifugation [138] Total exosome isolation reagent (Cat#4478359, DPSCs human [115,120] Invitrogen, Carlsbad, CA, USA) Total Exosome Isolation TM Hertwig’s epithelial root rat reagent (Life Technologies, [137] sheath-derived cells Carlsbad, CA, USA) ExoQuick-TC reagent (System DPSCs, PDLSCs, [113,123,126,129,135, human Biosciences, Mountain View, SHED, SCAP 139,140] CA, USA) Exo-spin exosome isolation DPSCs human reagent (Cell Guidance, [119] Cambridge, UK) PureExo® exosome isolation PDLSCs human kit (101Bio, Mountain View, [127] CA, USA) GMSC human N.A. [136] density gradient MSCs human [142] differential centrifugation DPSCs: dental pulp stem cells; PDLSCs: periodontal ligament stem cells; SHEDs: stem cells from exfoliated deciduous teeth; GMSC: gingival mesenchymal stem cells; SMC: sinus mucosa-derived cells; PCs: mandibular periosteum-derived cells; SCAP: stem cells from apical papilla; N.A.: not available.

6.7. Exosomes as Promising Tool for MRONJ Management Emerging role of exosomes as a tool for disease progression monitoring in humans and animals has been widely discussed in literature [144–146]. In the last decade, exosomes have been largely studied for their therapeutic potential, as well. In particular, biological properties of exosomes allow them to easily access to central nervous system, passing the blood brain barrier, so providing a vehicle for the delivery of drugs. The nanoscale size allows them to be rapidly uptake by cells, too. Exosomes can be engineered to target specific cell types or to carry specific cargo, via the stimulation or genetic alteration Pharmaceuticals 2020, 13, 423 16 of 24 of parental cells. Moreover, the advantages of using exosomes in craniofacial tissue engineering and regeneration have been recently reviewed [147–149]. Regarding osteonecrosis of the jaw, we find just one paper investigating the effects of EVs administration to prevent MRONJ both in vivo and in vitro. Watanabe et al. showed that MSCs-derived exosomes are able to prevent zoledronate-induced senescence in cells populating alveolar socket microenvironment, so reducing the alterations reported in the MRONJ [150]. It’s noteworthy the role of oral MSCs-derived exosomes in immunomodulation, which might have a crucial role in managing the MRONJ.

7. Conclusions Of course, the application of exosomes instead of MSCs may contribute to resolve risks of side effects associated to cell-based regenerative medicine, however many aspects attend to be resolved, among which the definition of standardized isolation and characterization protocol, the biological functions and molecular mechanisms of exosomes in MRONJ and their clinical translation.

Author Contributions: Conceptualization and writing of the original draft, A.D.V., T.B. and A.G.; Writing, review & editing, A.A., E.C., F.B.; Conceptualization, review & editing, A.D.V., A.G., T.B.; Images assembling, A.D.V. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: Publication of this article was supported by Dept. of Experimental and Clinical Medicine, University ”Magna Græcia” of Catanzaro, Italy. Conflicts of Interest: The authors declare no conflict of interest.

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