Hydrogen Sulfide in Bone Tissue Regeneration and Repair: State of the Art and New Perspectives

The importance of hydrogen sulfide (H2S) in the regulation of multiple physiological functions has been clearly recognized in the over 20 years since it was first identified as a novel gasotransmitter. In bone tissue H2S exerts a cytoprotective effect and promotes bone formation. Just recently, the scientific community has begun to appreciate its role as a therapeutic agent in bone pathologies. Pharmacological administration of H2S achieved encouraging results in preclinical studies in the treatment of systemic bone diseases, such as osteoporosis; however, a local delivery of H2S at sites of bone damage may provide additional opportunities of treatment. Here, we highlight how H2S stimulates multiple signaling pathways involved in various stages of the processes of bone repair. Moreover, we discuss how material science and chemistry have recently developed biomaterials and H2S-donors with improved features, laying the ground for the development of H2S-releasing devices for bone regenerative medicine. This review is intended to give a state-of-the-art description of the pro-regenerative properties of H2S, with a focus on bone tissue, and to discuss the potential of H2S-releasing scaffolds as a support for bone repair.


Introduction
Tissue development and repair has long been associated with sulfur metabolism: The first evidence dates back to the study by Williamson and Fromm (1955) which found an association between the activation of sulfur-containing amino acids metabolism and the wound healing process [1]. However, more than six decades have passed before the first studies linking biological functions of sulfurated amino acids to hydrogen sulfide (H 2 S) came to light. Notably, the identification (1996) of a physiological role of H 2 S in the brain tissue [2] opened the way to several studies unravelling its multiple biological functions. H 2 S is a gaseous molecule produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and, to a lower extent, 3-mercaptopyruvate sulfurtransferase (MPST) [3], within the transsulfuration pathway. A broad range of studies further clarified that H 2 S is not only a "secondary reaction product" of this pathway but is a critical mediator of physiological functions in many tissues.
Over the past years, several therapeutic applications of sulfur-containing compounds or H 2 S-releasing donors have emerged and a few clinical trials are ongoing [4]. However, while the studies on the systemic use of H 2 S-donors are at an advanced stage, the need for obtaining an organ-specific delivery of H 2 S to exploit the tissue-specific cytoprotective and regenerative properties of H 2 S has only recently emerged [5]. One field where H 2 S-releasing biomaterials hold a great potential interest is bone repair and regeneration.
Bone is a vascularized, dynamic tissue which provides the body with structural and movement support functions, organ protection, mineral reserve, and endocrine system function [6,7]. It is continuously remodeled by the coordination and balance of two key processes, bone formation and bone resorption [8,9], and is capable of repairing and reshaping without leaving scars [10]. However, bone lesions caused by trauma and pathological bone resorption (infectious diseases, biochemical disorders, congenital disorders, or abnormal skeletal development) have limited capacity to heal and require large amounts of autologous/allogenic bone or bone substitutes for reconstruction [11,12]. In recent years, research approaches have moved from a focus on inert biomaterials toward the complex design of biomaterials mimicking the structure of the native bone extracellular matrix and being able to provide a controlled release of biomimetic, pro-osteogenic factors [13].
Over the past few years, it has become increasingly clear that H 2 S regulates bone homeostasis and that systemic administration of H 2 S holds good therapeutic potential, as it prevented or reversed pathological bone loss [14][15][16]. However, whether H 2 S affects the processes of bone tissue regeneration and repair and whether it could be used as a biomimetic factor in regenerative medicine approaches has not been fully elucidated. This review is intended to first summarize the emerging evidence linking H 2 S and bone tissue repair and regeneration. Then, the state-of-the-art of the development of H 2 S-releasing scaffolds and biomaterials for promoting tissue regeneration will be described, with a focus on bone repair. Finally, implications and future challenges in the field of H 2 S-releasing scaffold for bone tissue engineering (BTE) will be discussed.

Bone Tissue Repair: H 2 S as a Suitable Biological Cue for Scaffold Functionalization
Bone remodeling is an orchestrated process constantly occurring to let the skeleton adapt to biomechanical loading, microfractures, environmental stress [17], and to control calcium and other ions levels in the circulation. Similarly, bone repair occurs in response to injury by activating complex and orchestrated regenerative processes to restore bone tissue integrity, structure, and homeostasis [18,19]. Despite the high innate regenerative capacity of bone, critical-size defects and non-unions fail to heal, thus making them a global health burden and a major challenge in regenerative medicine [12,[20][21][22]. Autograft is currently the gold standard therapy and achieve an 80% success rate [23]. However, the percentage of non-union remains significantly high and the procedure involves two surgical operations and donor site morbidity for patients, thus conditioning their quality of life. A wide variety of bone substitutes have been tested in clinics to improve bone repair, among which are allogenic bone and natural or synthetic osteoconductive matrices [24,25], obtaining promising results. In this context, BTE offers the opportunity to develop innovative alternative treatment options that will ideally eliminate the limitations of current treatments. BTE is based on various combinations of three principal components: biomaterials as scaffolds, regulatory signals such as growth factors (GF), and cells. Recently, the design of functionalized cell-free scaffolds has emerged as a useful tool to overcome current drawbacks linked to cell-based approaches (limited autologous cells, time/cost-intensive cell expansion procedures, relative low cell survival rate, and high risk of immune-rejection) [26]. Strategies of scaffold functionalization involve GF, chemokines, and peptides that are able to activate bone resident cells and promote bone regeneration [26]. These biological cues are usually incorporated into scaffolds via physical adsorption, chemical covalent coupling, and encapsulation [26], and their controlled and site specific release is crucial for the success of tissue engineering strategies. Over the past 50 years, several studies aimed at understanding the biological processes which takes place in in vivo models of bone fractures during bone repair [27], particularly in in the presence of bone fractures [28,29], and allowed the identification of several biological cues. Accordingly, bioinspired and biomimetic cell-free scaffolds were recently developed to mimic the natural self-healing events of bone healing by using biological signals able to modulate inflammation and induce cell recruitment, vascularization, and osteogenic differentiation [26]. Figure 1 summarizes the biological processes involved in bone fracture repair, highlighting which of them may be regulated by H 2 S (discussed more in detail in the next sections). Schematic representation of the process of bone fracture healing. Immediately after bone injury, the healing process is initiated by hematoma formation and inflammatory response accompanied by the release of pro-inflammatory cytokines. In direct bone healing (occurring at fractures that are perfectly aligned and with no vascular disruption) the inflammation activates a process of intramembranous ossification which directly produces a new lamellar bone structure without requiring any remodeling step. In indirect bone healing (occurring in fractures with large, hypoxic defects and interfragmentary strains), the intramembranous ossification response occurs subperiosteally directly adjacent to the ends of the fracture, generating a hard callus. Adjacent to the fracture line, endochondral ossification occurs: A cartilaginous callus form and several cells migrates and proliferate within it, building an intermediate cartilaginous template which subsequently undergoes calcification (primary bone formation); the cartilage and primary mineralized matrix are then resorbed and secondary bone formation occurs; ultimately bone remodeling by osteoclasts and osteoblasts take place and the callus is totally replaced by new bone indistinguishable from adjacent, uninjured tissue. The processes by which H2S may modulate during bone fracture healing are highlighted by a yellow arrow.

H2S Regulates Inflammation in Bone Tissue
Recent investigations in the field of osteoimmunology have demonstrated extensive cross-talk between immune and skeletal systems [30]. In particular, upon bone injury, hematoma formation is closely followed by acute inflammation and release of inflammatory cytokines which activate a cascade of processes to promote bone healing. A tightly regulated balance between pro-inflammatory macrophages (M1) and pro-healing, anti-inflammatory macrophages (M2) results in inflammatory and mesenchymal stromal cells (MSCs) recruitment, promotion of neovascularization, and induction of MSCs towards osteogenesis [30]. Notably, their absence has been shown to completely abolish callus formation [31]. Treg also promotes tissue healing by inducing M2 polarization, inhibiting neutrophil infiltration, activity, and inducing their apoptosis [32], supporting MSCs differentiation [33] and inhibiting osteoclasts (OCs) differentiation [32]. The ablation of δγ-T-cells promotes bone healing since mice deficient in this cellular type have a shorter time to fracture union and show improved biomechanical strength compared to control mice [34]. Conversely high presence of T-cells promotes OCs differentiation and inhibit MSCs [35] causing additional "by-stander" tissue damage [30]. Therefore, scaffold functionalization strategies are currently aimed at modulating proinflammatory and anti-inflammatory activity, thus preventing chronic inflammation and unbalanced inflammatory responses responsible for the formation of fibrous capsules around scaffolds, which make scaffolds bioinert [36].

Figure 1.
Schematic representation of the process of bone fracture healing. Immediately after bone injury, the healing process is initiated by hematoma formation and inflammatory response accompanied by the release of pro-inflammatory cytokines. In direct bone healing (occurring at fractures that are perfectly aligned and with no vascular disruption) the inflammation activates a process of intramembranous ossification which directly produces a new lamellar bone structure without requiring any remodeling step. In indirect bone healing (occurring in fractures with large, hypoxic defects and interfragmentary strains), the intramembranous ossification response occurs subperiosteally directly adjacent to the ends of the fracture, generating a hard callus. Adjacent to the fracture line, endochondral ossification occurs: A cartilaginous callus form and several cells migrates and proliferate within it, building an intermediate cartilaginous template which subsequently undergoes calcification (primary bone formation); the cartilage and primary mineralized matrix are then resorbed and secondary bone formation occurs; ultimately bone remodeling by osteoclasts and osteoblasts take place and the callus is totally replaced by new bone indistinguishable from adjacent, uninjured tissue. The processes by which H 2 S may modulate during bone fracture healing are highlighted by a yellow arrow.

H 2 S Regulates Inflammation in Bone Tissue
Recent investigations in the field of osteoimmunology have demonstrated extensive cross-talk between immune and skeletal systems [30]. In particular, upon bone injury, hematoma formation is closely followed by acute inflammation and release of inflammatory cytokines which activate a cascade of processes to promote bone healing. A tightly regulated balance between pro-inflammatory macrophages (M1) and pro-healing, anti-inflammatory macrophages (M2) results in inflammatory and mesenchymal stromal cells (MSCs) recruitment, promotion of neovascularization, and induction of MSCs towards osteogenesis [30]. Notably, their absence has been shown to completely abolish callus formation [31]. Treg also promotes tissue healing by inducing M2 polarization, inhibiting neutrophil infiltration, activity, and inducing their apoptosis [32], supporting MSCs differentiation [33] and inhibiting osteoclasts (OCs) differentiation [32]. The ablation of δγ-T-cells promotes bone healing since mice deficient in this cellular type have a shorter time to fracture union and show improved biomechanical strength compared to control mice [34]. Conversely high presence of T-cells promotes OCs differentiation and inhibit MSCs [35] causing additional "by-stander" tissue damage [30]. Therefore, scaffold functionalization strategies are currently aimed at modulating pro-inflammatory and anti-inflammatory activity, thus preventing chronic inflammation and unbalanced inflammatory responses responsible for the formation of fibrous capsules around scaffolds, which make scaffolds bioinert [36].
H 2 S plays an important role during inflammation in several tissues [37] and the increased H 2 S levels occurring during inflammation have been proposed as a stress-response mechanism to the injury caused by oxidative stress [38]. In OCs, inhibition of reactive oxygen species (ROS) signaling by H 2 S occurs through direct and indirect mechanisms by triggering NRF-2-dependent antioxidant response and thus inhibiting osteoclast differentiation ( Figure 2) [39]. Interestingly, H 2 S was shown to be a key mediator in the immediate anti-inflammatory and antioxidant function of N-acetyl-cysteine, a broadly used mucolytic medicine [40]. H 2 S was shown to reduce edema formation [41], promote neutrophil apoptosis, suppress the expression of some leukocyte and endothelial adhesion molecules [42], stimulate macrophages polarization to M2 [43][44][45], and activate the AnxA1 pro-resolutive pathway [46]. Concerning bone tissue, in the animal model of hyperhomocysteinemia (CBS −/− mice), the autosomal recessive disease involving CBS, higher levels of inflammation has been correlated to lower bone tissue [16]. Particularly, these mice had increased acetylation of NF-kB p65, resulting in increased levels of IL-6 and TNF-α in the circulation and inhibition of Runx-2 and Ocn gene expression [16]. Administration of NaHS suppressed histone acetylation dependent NF-kB p65 signaling activation and inflammation and rescued osteogenic genes expression [16]. Moreover, H 2 S exogenous administration (by using the H 2 S-releasing moiety of ATB-346 and ATB-352 molecules) has been used to inhibit inflammation and inflammatory bone loss in experimental periodontitis [47,48]. A therapeutic platform has been developed to deliver H 2 S and inhibit P-selectin expression in human platelets, which play an essential role in the initial recruitment of leukocytes to the site of injury during inflammation [49]. Moreover, H 2 S has been shown to mediate the inhibition of inflammatory responses during bacterial infections in an ex vivo model of cells infected with mycoplasma [50]. The molecular pathways imply inhibition and activation of nuclear translocation of NF-κB, reducing the transcription of pro-inflammatory genes [50]. Finally, H 2 S plays a role in mediating both innate and adaptive immunity, since it was also found to be essential for Treg cell differentiation and function and enhances T-cell proliferation, activation, and cell death [51,52]. Moreover, CBS −/− mice developed autoimmune disease [53].
In tissues other than bone, H 2 S has been shown to improve wound healing by attenuating inflammation and increasing angiogenesis. H 2 S has been hypothesized to play a role in severe burns, which are associated with processes that causes increased permeability and edema [54], and has been proven to improve diabetic wound healing [55,56].
Taken together these data indicates that future design of H 2 S-releasing scaffolds for BTE may be tuned to temporally modulate pro-inflammatory and anti-inflammatory activities, thus promoting the formation of regenerated bone tissue.

H 2 S Regulates Migration and Survival of Cells Involved in Bone Repair
The recruitment of autogenous endothelial progenitor cells (EPCs) and MSCs to the site of bone defect is critical to induce bone tissue repair [57]. More in general, EPCs play a crucial role in endothelial restoration after arterial injury [58], and the migratory potential of MSCs has been demonstrated to be one of the most important factors for neovascularization and regeneration [59,60]. H 2 S has been found to induce migratory properties in both cell types. In particular, NaHS, an H 2 S donor, increases the mobilization of EPCs after vascular injury, enhancing their adhesion and colony formation capacities [61] and improving re-endothelization in nude mice with carotid artery injury [62]. CSE-mediated H 2 S deficiency in high glucose-treated MSCs impairs their migratory capacity, which was further restored by NaHS treatments [63]. Furthermore, H 2 S has been found to mediate the migratory potential of other cells types. In particular, NaHS accelerates skin fibroblasts and human keratinocytes in migration assays [64], thus enhancing wound healing. H 2 S promotes macrophage migration to an infarcted area, by accelerating internalization of integrin β1 and activating the downstream Src-FAK/Pyk2-Rac pathway, thus promoting infarction healing [65].
Moreover, it is well known that H 2 S promotes proliferation and survival of MSCs and of other cell types during several stresses such as hypoxia, oxidative damage, and serum deprivation. Particularly, it inhibits cellular death by apoptosis [66,67]. Therefore, H 2 S may act similarly to other GF, which initiate the repair process by facilitating proliferation and differentiation of the stem cells that give rise to the fracture callus [68].
Whether H 2 S modulates cells migration, survival, and proliferation to the bone defect to induce bone tissue repair is still an open question.

H 2 S Promotes the Expression of Angiogenic Factors in Bone Tissue
Vascularization is a primary driving force behind both intramembranous and endochondral ossification. To achieve a highly functional new regenerated tissue, the tight connection of new vasculature formation and osteogenesis is of utmost importance. New vessels are fundamental both in the initial and in the final step of bone repair. In the first phase, new blood vessels are essential to provide nutrients and facilitate cells migration toward the damaged site and in the final phases of repair, vascular remodeling occurs and vessels regresses to the original state [69]. Importantly, failure of angiogenesis can lead to nonunion [70]. Deletion of vascular endothelial growth factor (VEGF) in osteoblasts (OBs) interrupts the coupling of osteogenesis and angiogenesis, and delays the intramembranous ossification-mediated repair during cortical bone defect healing [71]. To date, there is no information linking H 2 S promotion of neo-angiogenesis during bone regeneration and repair. However, several evidences may suggest a role. H 2 S has been shown to stimulate in vitro the three processes crucial for the neo-angiogenesis: proliferation, migration, and tube-like network formation by endothelial cells [72]. Evidence of neo-vascularization of Matrigel implants in vivo further confirmed the ability of H 2 S in promoting new blood vessel formation [73,74]. These features are at least partly mediated by H 2 S-dependent activation of two factors critical for bone vascularization: hypoxia-inducible factor-1 (HIF-1α) and VEGF. Indeed, a number of studies have established extensive crosstalk between H 2 S and VEGF. Exogenously administered H 2 S has been shown to upregulate VEGF expression in several tissues and cells [75], among which skeletal muscle cells and bone cells in vitro and in vivo [76,77]. H 2 S also significantly upregulated HIF-1alpha protein levels and increased HIF-1alpha binding activity under hypoxic conditions [78]. Finally, H 2 S improved vascular density and blood flow in ischemic skeletal muscles of CBS +/− mice or diabetic type-2FAL mice models with hindlimb femoral artery ligation [63,79].

H 2 S Stimulates Bone Formation
Over the past few years, it has become increasingly clear that H 2 S affects bone tissue regeneration by acting at several levels such as regulation of bone cells activity, reduction of oxidative stress, regulation of calcium intake by bone cells, and promotion of angiogenesis. CBS and CSE, the H 2 S-producing enzymes, are expressed both in MSCs [15,80] and in OBs [80][81][82]. In particular, CSE is the predominant source of H 2 S in OBs [81]. H 2 S plays a cytoprotective role in bone cells; it protects OBs against homocysteine-induced mitochondrial toxicity [83]; and OBs against hydrogen peroxide (H 2 O 2 )-induced cell death and apoptosis [84].
H 2 S was shown to play an important role in sustaining osteogenic differentiation of human MSCs (hMSCs) [14,15] and in modulating OCs differentiation [39,[85][86][87]. CBS and CSE levels increased during osteogenic differentiation and play an essential role in osteogenic differentiation in vitro [15,80]. Notably, H 2 S-deficiency due to CBS knock-down was linked to impaired osteogenesis and bone loss in mice [15]. Several pathways are implicated in H 2 S-dependent induction of osteogenesis, summarized in Figure 2: the WNT pathway [14,88]; BMP pathway [77]; and p38-MAPK and ERK signaling pathways [84,89]. Moreover, S-sulfhydration of cysteine residues in critical proteins involved in osteogenesis, such as Runx-2 [81] and Ca 2+ TRP channels [15], has been shown to be a mechanism of H 2 S-dependent induction of osteogenesis. Furthermore, overexpression of CSE in OBs increased Runx-2 S-sulfhydration and enhanced OBs biologic function (increased ALP activity, Alizarin red-positive calcification nodules, and osteogenic gene expression). Interestingly, mechanical loading in periodontal tissues up-regulated CSE expression and OCs formation inducing bone remodeling and accelerating orthodontic tooth movement (OTM) speed. Similarly, OTM and bone remodeling are stimulated by the up-regulation of osteoclastogenesis and osteoblastogenesis induced by H 2 S exogenous administration [90]. overexpression resulted in improved bone fracture healing: less inflammatory infiltration, greater collagen expression, fibrocartilage, and osteocyte deposition. Interestingly, the authors showed increased endochondral ossification at the tissue repair site, and a full bridge between fracture sites in rat femurs [81].
A summary of the main studies investigating the effects of H2S-releasing donors on bone cells are reported in Table 1.  [15]; at the transcriptional level, H2S donors induce the expression of osteogenic signaling through stimulation of multiple signaling pathway [14,77,88]. In the context of inflammation, H2S triggers an antioxidant signaling in OC which involves the increased nuclear translocations of NRF-2 and results in the inhibition of OC differentiation [39,87].  [15]; at the transcriptional level, H 2 S donors induce the expression of osteogenic signaling through stimulation of multiple signaling pathway [14,77,88]. In the context of inflammation, H 2 S triggers an antioxidant signaling in OC which involves the increased nuclear translocations of NRF-2 and results in the inhibition of OC differentiation [39,87].
An imbalance in H 2 S metabolism is associated with defective bone homeostasis. Evidence from several preclinical models showed that the depletion of H 2 S levels is implicated in bone loss; data were reported in ovariectomized mice [14,91], in H 2 S-deficient CBS +/− mice [15], in a model of H 2 S-deficiency linked to diet-induced hyperhomocysteinemia [16,92], and in methionine-restricted fed rats [93]. Interestingly, when the treatment with H 2 S donors in these models was investigated, finalized to normalize the plasma level of H 2 S, it was found that H 2 S was able to prevent or even reverse the bone loss. Moreover, asides from these conditions, H 2 S exogenous administration has been shown to have therapeutic potential against bone loss induced by modelled microgravity [94] and other conditions such as distraction osteogenesis [95,96]. Overall, these data demonstrate that H 2 S regulates osteogenesis and bone formation in both healthy and pathological conditions.
In the context of bone repair, new bone formation occurs by direct and indirect mechanisms, involving intramembranous and endochondral ossification, as extensively reviewed elsewhere [97][98][99][100][101]. During these processes MSCs proliferate and differentiate into the osteogenic or chondrogenic lineages and increase the production of blood vessels from pre-existing vessels.
In models of fracture healing, treatment with H 2 S by various means affected both direct and indirect mechanisms of bone healing. In a mouse model of distraction osteogenesis, H 2 S injection was observed to accelerate the formation of mature microstructures of trabeculae and mineralization substituting the fibrous-connective tissue and improve the mechanical properties of the callus healing within the distraction gaps [95]. Moreover, in a rat model of intramedullary fixed fractural bone, Zheng et al. investigated the delivery of CSE adenovirus through absorbable gelatin. CSE overexpression resulted in improved bone fracture healing: less inflammatory infiltration, greater collagen expression, fibrocartilage, and osteocyte deposition. Interestingly, the authors showed increased endochondral ossification at the tissue repair site, and a full bridge between fracture sites in rat femurs [81].
A summary of the main studies investigating the effects of H 2 S-releasing donors on bone cells are reported in Table 1.

H 2 S-Releasing Scaffolds for Regenerative Medicine
Besides the efficacy of systemic administration of H 2 S, several reasons provide a rationale for the development of H 2 S-releasing scaffolds for bone regeneration.
First, cell-free scaffolds have undergone intense development in the recent years due to the drawbacks linked to the employment of cells in regenerative medicine [26]. Many physical, chemical, or biological functionalizations have led to tremendous progresses in the ability to support the physiological healing process of the bone [26]. For example, it was demonstrated that topographically defined implants are able to recruit osteoprogenitor cells at the site of bone injury and promote a more efficient repair in vivo [102,103]. Moreover, chemical modifications of the surface can endow biomaterials with increased osteoinductive capacity; for example, doping dense biomaterials with inorganic ions such as magnesium (Mg), Cobalt (Co), silicon (Sr), and Strontium (Sr) was shown to promote osteogenic differentiation in vitro and in vivo [104][105][106][107].
Second, the field of H 2 S-releasing materials is readily expanding. Several reports over the past few years showed promising results from in vitro and in vivo models and demonstrated that the biological properties of H 2 S can be successfully recapitulated in H 2 S-releasing devices for biomedical applications. In a recent report, Wu et al. successfully doped phosporodithioate donors (JK-1), a kind of H 2 S donors triggered by hydrolysis and releasing H 2 S in a pH-dependent fashion, with polycaprolactone (PCL) and the resulting fibers showing a slower kinetic of H 2 S-release compared to the native molecules in solution. When applied in an animal model of full-thickness skin damage, the doped PCL-JK1 fiber showed overall improved wound healing times as compared to non-doped fibers [108], thus confirming that H 2 S released by the fibers retained the previously evidenced properties of promoting wound healing [64,109,110]. The same authors developed a biomimetic hyaluronic acid (HY) hydrogel doped with JK1, to overcome the low permeability to oxygen of PCL fibers. This scaffold accelerated the wound repair process by decreasing inflammation, driving macrophages polarization toward M2, and increasing angiogenesis, and the effect was higher compared to adding JK1 to the wound microenvironment without being embedded in HY [45].
Nano-structured patches obtained by electrospun fibers are a valuable tool to promote tissue healing and to achieve local release of bioactive compounds [111]. Cacciotti et al. recently developed H 2 S-releasing poly(lactic) acid fibrous membranes functionalized with H 2 S donors of natural origin (from garlic oil-soluble extracts and diallyl disulfide) [112]. These donors where entrapped within the fibers and the H 2 S release by the membranes was prolonged and gradual and induces MSCs proliferation and anti-microbial activity. Following a similar approach, Feng et al. added the H 2 S donors (NSHd1) to PCL fiber spun by electrospinning to produce controlled, thiol-triggered H 2 S release [113]. Interestingly, by tuning the diameter of the fibers the authors achieved a controlled rate of H 2 S release. The authors showed that H 2 S increased gene expression of collagen type I and type III in fibroblasts while protecting them from the oxidative stress produced by H 2 O 2 , suggesting that these microfibers may support wound dressing. The group of Mauretti et al. developed a photopolymerizable PEG-fibrinogen hydrogel incorporating albumin microbubbles functionalized with thiosulfate cyanide sulfurtransferase, one of the enzymes able to catalyze the H 2 S-production [114]. H 2 S produced by the scaffold increased cardiac progenitor cell proliferation, thus retaining the feature previously described by others [115]. The group of Liang et al. loaded a partially oxidized alginate (ALG-CHO) with the H 2 S donor 2-aminopyridine-5-thiocarboxamide and tetraaniline (a conductive oligomer), and adipose-derived stem cells (ADSCs) [116]. The hydrogel, which mimics the slow and continuous release of endogenous H 2 S, increased the ejection fraction value and reduced the infarction size in rats with myocardial infarction, thus evidencing that H 2 S released retained the H 2 S-dependent protective role on myocardial infarction and heart failure previously described [115] and offering a promising therapeutic strategy for treating infarction.
Overall, the strategies to add H 2 S to the biomaterials described above were based on physical adsorption, chemical covalent coupling, and encapsulation. Beside these strategies, chemical research is also growing to develop methods for obtaining H 2 S-releasing donors with improved physical and chemical features. Of interest, an H 2 S-releasing gel was developed by the group of JB Matson, by using an H 2 S-releasing peptide that self-assembles into a robust hydrogel in water, obtained by chemical synthesis of an aromatic peptide amphiphile and the H 2 S moiety, S-aroylthiooxime-SATO-functional group. This H 2 S-releasing gel, showed a slow kinetic of H 2 S release and absence of cytotoxicity [117]. In a recent report, this H 2 S-releasing hydrogel substantially reduced intimal hyperplasia in human veins, showing efficacy at a dose five times lower than NaHS, a fast release H 2 S donor [118].
Altogether, these data evidence how H 2 S can be used to functionalize biomaterials inducing cell differentiation and function, and thus can be considered as a regulatory signal in the "tissue engineering triad" (Figure 3). chemical synthesis of an aromatic peptide amphiphile and the H2S moiety, S-aroylthiooxime-SATO-functional group. This H2S-releasing gel, showed a slow kinetic of H2S release and absence of cytotoxicity [117]. In a recent report, this H2S-releasing hydrogel substantially reduced intimal hyperplasia in human veins, showing efficacy at a dose five times lower than NaHS, a fast release H2S donor [118]. Altogether, these data evidence how H2S can be used to functionalize biomaterials inducing cell differentiation and function, and thus can be considered as a regulatory signal in the "tissue engineering triad" (Figure 3). . Schematic representation of potential H2S role in the "tissue engineering triad". Three main factors are the key players of tissue engineering strategies and compose the so-called "tissue engineering triad": 1) a scaffold that provides structure and support cells migration, colonization, proliferation, and differentiation, and is the substrate of loading of regulatory signals such as growth factors; 2) a source of cells to promote the required tissue formation (endogenous cells, primary cells, or cell lines pre-cultured or loaded within the scaffold); and 3) regulatory signals (growth factors, biophysical stimuli, etc.) to induce cell differentiation and tissue formation. We postulate that H2S may be considered as a regulatory signal able to functionalize biomaterials and induce endogenous or loaded cells differentiation and promote bone tissue formation.
The only reported H2S-releasing biomaterial targeting bone regeneration was developed by our group [77]. Based on previous preclinical studies showing efficient stimulation of osteogenesis by H2S donors, we postulated that a local release of physiologically relevant H2S concentrations may trigger osteogenic differentiation of h-MSCs. An H2S-releasing silk fibroin (SF) sponge was developed by first applying a salt leaching approach to add appropriate porosity to the SF fibers and by then doping the H2S donor (GYY4137) to the sponges. The resulting SF-GYY scaffolds showed unaltered mechanical properties and no cytotoxicity compared to the native SF scaffolds. When tested in a model of 3D culture of h-MSCs within a perfusion bioreactor, H2S induced a significant increase in the differentiation to mature OBs, revealed by increased deposition of the mineral matrix (as shown in Figure 4) and increased expression of osteogenic genes after three weeks in culture. This study first provided a proof-of-principle that exposing h-MSCs to H2S released by a scaffold induced their differentiation toward OBs, and demonstrated that loading osteoconductive biomaterials with an H2S donor is a suitable strategy to promote OBs differentiation at sites of bone regeneration.
Overall, the evidences reported in the present review set H2S as an important emerging candidate for promoting regenerative medicine and particularly for BTE. . Schematic representation of potential H 2 S role in the "tissue engineering triad". Three main factors are the key players of tissue engineering strategies and compose the so-called "tissue engineering triad": 1) a scaffold that provides structure and support cells migration, colonization, proliferation, and differentiation, and is the substrate of loading of regulatory signals such as growth factors; 2) a source of cells to promote the required tissue formation (endogenous cells, primary cells, or cell lines pre-cultured or loaded within the scaffold); and 3) regulatory signals (growth factors, biophysical stimuli, etc.) to induce cell differentiation and tissue formation. We postulate that H 2 S may be considered as a regulatory signal able to functionalize biomaterials and induce endogenous or loaded cells differentiation and promote bone tissue formation.
The only reported H 2 S-releasing biomaterial targeting bone regeneration was developed by our group [77]. Based on previous preclinical studies showing efficient stimulation of osteogenesis by H 2 S donors, we postulated that a local release of physiologically relevant H 2 S concentrations may trigger osteogenic differentiation of h-MSCs. An H 2 S-releasing silk fibroin (SF) sponge was developed by first applying a salt leaching approach to add appropriate porosity to the SF fibers and by then doping the H 2 S donor (GYY4137) to the sponges. The resulting SF-GYY scaffolds showed unaltered mechanical properties and no cytotoxicity compared to the native SF scaffolds. When tested in a model of 3D culture of h-MSCs within a perfusion bioreactor, H 2 S induced a significant increase in the differentiation to mature OBs, revealed by increased deposition of the mineral matrix (as shown in Figure 4) and increased expression of osteogenic genes after three weeks in culture. This study first provided a proof-of-principle that exposing h-MSCs to H 2 S released by a scaffold induced their differentiation toward OBs, and demonstrated that loading osteoconductive biomaterials with an H 2 S donor is a suitable strategy to promote OBs differentiation at sites of bone regeneration.
Overall, the evidences reported in the present review set H 2 S as an important emerging candidate for promoting regenerative medicine and particularly for BTE.

Perspectives for H2S-Loaded Scaffolds for BTE
Although research on the biological activities of H2S was first triggered by investigations in the field of neurology and circulation, a substantial body of evidence, summarized in this review, has recently shown that H2S also plays an important role in bone physiology and can stimulate the process of bone repair at multiple levels. The field of H2S-releasing scaffolds for BTE holds the promise to open new opportunities to treat bone tissue damages. However, it is still on its infancy and a number of challenges are still ahead of us, which will require joint efforts from material scientists, chemists, and biologists.
First, the range of biomaterials used for the combination with H2S is limited. Interestingly, all the H2S-releasing scaffold before mentioned (PCL, polylactic, PEG, alginate, and peptide amphiphilic) are made by biomaterials previously tested for bone regeneration alone or composite [119][120][121][122][123], making them of interest also in the field of BTE. However, further combination with other biomaterials, especially those which better mimics the composition and the biomechanical properties of bone, is necessary to take a step forward in this field.
Second, the design of new formulations of H2S donors should address the need for improved stability (e.g., in aqueous solution during scaffold development) and a "controlled" release of H2S (for avoiding the burst-release phenomenon). Notably, several formulations of H2S donors has been developed to release H2S triggered by a change in pH [124,125] or amounts of thiols, reactive oxygen

Perspectives for H 2 S-Loaded Scaffolds for BTE
Although research on the biological activities of H 2 S was first triggered by investigations in the field of neurology and circulation, a substantial body of evidence, summarized in this review, has recently shown that H 2 S also plays an important role in bone physiology and can stimulate the process of bone repair at multiple levels. The field of H 2 S-releasing scaffolds for BTE holds the promise to open new opportunities to treat bone tissue damages. However, it is still on its infancy and a number of challenges are still ahead of us, which will require joint efforts from material scientists, chemists, and biologists.
First, the range of biomaterials used for the combination with H 2 S is limited. Interestingly, all the H 2 S-releasing scaffold before mentioned (PCL, polylactic, PEG, alginate, and peptide amphiphilic) are made by biomaterials previously tested for bone regeneration alone or composite [119][120][121][122][123], making them of interest also in the field of BTE. However, further combination with other biomaterials, especially those which better mimics the composition and the biomechanical properties of bone, is necessary to take a step forward in this field.
Second, the design of new formulations of H 2 S donors should address the need for improved stability (e.g., in aqueous solution during scaffold development) and a "controlled" release of H 2 S (for avoiding the burst-release phenomenon). Notably, several formulations of H 2 S donors has been developed to release H 2 S triggered by a change in pH [124,125] or amounts of thiols, reactive oxygen species, or enzymes [5,[126][127][128][129] within the microenvironment. These formulations partially addressed this need and may provide further opportunities to tune the H 2 S release in bone microenvironments according to different phases of bone healing to better exploit its biological properties. However, changes in the technologies for scaffolds development require a steady advancement in the development of H 2 S-donors. As an example, H 2 S donors are able to endure the high temperature required for gel printing while preventing premature H 2 S release would be of great interest given the rapid expansion of 3D printing technology for the development of critical-size bone defect customized products.
Third, new formulations of H 2 S donors should be tailored for applications in BTE. As an example, H 2 S donors able to bind with high affinity bone or bone substitutes, such as hydroxyapatite, would be useful for optimizing the interaction of the H 2 S donor within the scaffold matrices. The use of H 2 S-donors hybridized with bisphosphonates, molecules able to bind avidly bone, such as the recently developed H 2 S-releasing DM-22 [130], could represent an interesting approach to this challenge.
The knowledge generated by studies addressing these points will be instrumental for the development of H 2 S-releasing scaffolds tailored for bone regeneration and would greatly impact the advancement in the field of BTE. Furthermore, the direct correlation between bone healing and local H 2 S release by bone scaffolds will be fundamental to corroborate the rationale of H 2 S's role in mediating bone repair.

Concluding Remarks
This review provides a concise and up-to-date overview of the role of H 2 S in bone tissue repair and of the ongoing research and future challenges for the development of H 2 S-releasing scaffolds for regenerative purposes. The research of new formulations of H 2 S-releasing scaffolds for BTE may provide patients with skeletal injuries with novel opportunities of treatment.
Author Contributions: Manuscript drafted and revised by F.G., L.G. and B.G.
Funding: This study was supported by grant "Ricerca Finalizzata" from the Italian Ministry of Health (RF PE-2011-02348395, "Novel approach for bone regeneration and repair using sulfur donor-based therapy").

Conflicts of Interest:
The authors declare no conflict of interest.The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.