HS, with an average molecular weight of 30 kDa, is produced by majority of cells in the human body [14]. Even though HS biosynthesis is a rapid process (occurring within approximately a minute), the process is dynamic and complex that involves the conjunctive action of at least 22 enzymes, with some possessing varied isoforms that are used to produce cell and tissue specific forms. Majority of these enzymes possess a transmembrane region that enables them to be intercalated into the Golgi membrane [15].

The synthesized HS with intricate structure is predominantly cell-specific and not PG type-specific so that all HS chains synthesized by a given cell are similar [8, 16].The structural heterogeneity and variability such as the wide-ranging chain lengths and sulfate modifications delineate the HS chain as a family of related polymers rather than being considered as a single compound [17]. The sulfated structural domain motifs are mainly responsible for the regulatory properties of HS and serve as trigger zones that encourage the interactions to clusters of basic amino acids on many different types of proteins [9, 18]. Interestingly, one disaccharide unit of HS contains only one sulfate group, unlike heparin that contains three sulfate groups [19].

The endoglycosidase specific for HS, namely the heparanase (endo-β-glucuronidase), located on cell surfaces and ECM, is responsible for the degradation of the aforementioned glycans to smaller fragments [20, 21]. The degradation of HS begins in the extracellular space and is subsequently completed in the lysosomes [8].

Over the past decades, the functions of HS have been well researched. HS indirectly regulates the process of cell proliferation, differentiation, and angiogenesis, leukocytic migration and degranulation by functioning as endogenous receptors for numerous extracellular ligands, growth factors, and chemokines [22, 23]. The primary role of HS is to regulate the interactions between cells and ECM, whereby it stimulates the adhesion of cells to the ECM by binding to matrix macromolecules such as fibronectin or laminin [19, 24]. The ECM performs both structural and biochemical functions: acting as a scaffold for cell components and initiating signals that regulate cell behaviours [25]. HS in ECM plays a crucial role in the regulation of tissue homeostasis by mediating and integrating communication events via their ability to bind and interact with a wide variety of proteins and communication peptides such as growth factors, cytokines, and chemokines [11, 25]. HS also acts as a storehouse for these communication peptides, thereby playing a key role in the structural and spatial organization of the cellular micro-environment. Due to their larger size, HSs appear to protect the HS-bound proteins from proteolytic degradation by steric hindrance, thereby preventing the access for proteases [7].

When exploring advanced therapeutic strategies to treat various bone defects and deformities, it is important to have a basic understanding of the physiologic process of bone repair and regeneration. Most bone fractures are repaired through a process known as endochondral ossification, and the process is divided into four overlapped stages: the formation of an initial hematoma, followed by inflammation; the soft callus formation; the hard callus formation; and the remodeling of the newly formed bone [26], as shown in Fig. 2. This process involves a synchronized interaction of cells, growth factors, and extracellular matrix in a well-staged sequence that commences immediately after an injury.

The process of bone repair

When tissue was damaged by chemical, thermal, irradiation, external or internal mechanical aggressions, bacterial or viral infections, oxidative stress, or other factors, the tissue undergoes extensive cellular destruction, which triggered the release and activation of proteases, glycanases, and inflammatory communication peptides by inflammatory and neighboring resident cells. Glycanases destructed the ECM scaffold by degrading HSs, in turn giving access to proteases, which destroyed matrix proteins and stored growth factors. Meanwhile, the circulating cells released the communication peptides, provoking an emergency response that encouraged rapid repair of surrounding tissues [7]. During cell death, communication peptides trapped by HS became available to trigger cellular regeneration. Also, HS played a critical role in facilitating intercellular chemical communication by binding and regulating the functions of various heparin-binding growth factors (HBGFs), enzymes, and inhibitors, which made their participation pivotal in the initial phrases of tissue healing [22, 27].

Following any trauma to the bone tissues, the immediate response was the formation of haematoma by the blood vessels at the site of injury [28]. Within 24–72 h of an injury, HS flooded acute wound fluid and bound with heparin-binding epidermal growth factor (Hb-EGF), which acted as a mitogenic agent for fibroblasts, leiomyocytes, and epitheliocytes later [29]. H-kininogen (HK) and plasma prekallikrein/kallikrein (PK) were involved and interacted with the HSPGs in non-endothelial cells when blood vessels were damaged. The HS on HSPGs bound to HK/PK ligands, mediating their cellular entry,then facilitated the endocytosis of intact HK/PK within lipid raft domains/caveolae or endosomes. Subsequently, HK/PK ligands dissociated from HS, and HSPGs recycled back to the cell surface. These processes further influenced innate immunity, inflammation, angiogenesis, and coagulation [30]. Previous study revealed that HS chains can interact with antithrombin and tissue factor pathway inhibitor (TFPI), two proteins that naturally prevent blood clotting. HS chains enhanced antithrombin activity and their degradation promoted TFPI release from the endothelial cell surface [31, 32]. The quantity and length of HS chains on the surface of endothelial cells affected antithrombin’s anticoagulant effect, and degradation of them by heparanase, enzyme to degrade HS chains, can decrease antithrombin’s effectiveness. Elevated levels of HS chains were observed following reperfusion after coronary ischemia, indicating activation of coagulation and post-ischemic shedding of HS chains. Injection of antithrombin before inducing ischemia reduced the shedding of HS chains into the blood [33]. A previous study demonstrated heparanase enhanced haematoma formation through separation of TFPI from the vascular surface and the stimulation of tissue factor expression [34]. Histidine-rich glycoprotein (HRG) primarily attached to cell-surface HSPGs and high HRG level was one risk factor for thrombosis. N-terminal domain of HRG binds to cell-surface HS and exert its antiangiogenic effect. The enhanced tumor angiogenesis in HRG-deficient mice further supported above argument [35].

Additionally, the haematoma formation is an aggregation of products from the localized vasculature that consists of platelets, leukocytes, macrophages, fibrin, soluble growth factors, and cytokines, which serves as a matrix permitting the migration of inflammatory cells, endothelial cells and fibroblasts [36]. Compared to blood leaked into the surrounding tissues, local acute inflammatory response and hypoxia begin from initial 12 to 14 h and lasts for 7 days after an injury. Neutrophils first arrive at the site of injury followed by macrophages and lymphocytes. More than 40 chemokines are identified to activate the movement of leukocytes along the endothelium to sites of inflammation, and all are expected to bind to heparin or HS [37]. Mutant chemokines that are unable to bind with HS have been observed to fail in recruiting leukocytes neither in vitro or in vivo [38, 39]. Additionally, the mice with heparanase overexpression and shortening HS chains, display impaired neutrophil crawling to wild-type chemokines [40]. During tissue repair, HS is seen to enhance the recruitment of inflammatory cells, since endothelial surface HS decreases neutrophil rolling rapidity by facilitating their adhesion via L-selectin [41]. On the contrary, some studies reveal that HS may inhibit neutrophils attachment. Loss of endothelial glycocalyx, including HS, following LPS infusion resulted in increased neutrophils adhesion of and anti-ICAM-1 microspheres in wild mice while not in heparanase-free mice [42]. Additionally, HS can promote the binding of neutrophils to the endothelial surface by the Mac-1-CD44v3 interaction, which is essential for neutrophils extravasation [43].

Macrophages play a critical role by producing numerous cytokines and growth factors that trigger the healing process including transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), interleukins 1,6 and 10 (IL-1, IL-6 and IL-10), tumor necrosis growth factor-alpha (TNF-α) andbone morphogenetic proteins (BMPs) [28, 44]. HS fragments released from cell surface and ECM during inflammation can stimulate immune activation through Toll-like receptor 4 (TLR4) signaling pathway and activating antigen-presenting cells (APCs), which enables them to be recognized as a sensor for tissue injury [45]. This action of HS in turn triggers the secretion of proinflammatory cytokines by macrophages and significantly enhances the maturation of dendritic cells (DCs) [46]. The increased expression of MHC-II, CD40, ICAM-1, CD80, CD86, and decreased intake of antigen further validates the above results [47]. Leukocyte cell-surface HS can also present growth factors or inflammatory mediators produced by leukocytes to their target cells, as demonstrated in studies on fibroblast growth factor 2 (FGF2). Activated macrophages were found to enhance the proliferative response of HS-deficient BaF32 cells to low concentrations of FGF2. This effect was abolished by heparinase treatment, which indicates that macrophage HS facilitates the trans-presentation of FGF2 to target cells in a manner that promotes effective signalling [48]. However, HS sometimes hinders ligand–receptor interactions and acts as a barrier. Macrophages lacking HS sulfation after Ndst1 deletion exhibit enhanced response to interferon-β (IFN-β) stimulation and increased production of proinflammatory cytokines and chemokines [49]. This suggests that HS influence the IFN-β signaling by sequestering it away from its receptor, thereby maintaining macrophages in a state of quiescence.

HS indirectly regulates lymphocyte activity by modulating APC maturation [45]. Soluble HS enhances the proliferation of T cells when co-incubated with allogeneic DCs, but this effect is not observed when the DCs are derived [50]. Unlike above macrophage to enhance the expression of HS chains, T cells reduced HS expression and lost the ability to bind FGF2 during successive rounds of proliferation [51]. APRIL, a crucial factor for IgA class switching in response to mucosal antigens, relies on the presence of cell-surface HS of B cells for optimal receptor activation and downstream signalling [52]. IL-7, another B cells factor, also binds to heparin. Treatment of primary B cell precursors with heparitinase reduces IL-7/HS binding and impairs IL-7–driven proliferation [53]. The detailed interactions between HS and lymphocytes remain unclear and more significant exploration are needed in future.

Soft callus formation typically lasts for about 2–3 weeks following the haematoma formation and inflammatory reaction. In this stage, the pain and swelling decrease and intramembranous ossification is also observed away from bone fracture site. As mentioned above, endochondral ossification is the main repair method for bone fracture and the successful osteochondral regeneration involves the restitution of the blood vessels, cartilage and subchondral bone that serve as a matrix for the ensuing mineralization process. Previous research revealed that the precursor cells or osteochondroprogenitors, originating from the periosteum, migrate into the haematoma and differentiate into osteoblasts and chondrocytes, contributing to bone healing [54].The soft callus characterized with fibroblasts and chondroblasts stimulation is also known as fibrocartilaginous callus [55]. In above callus with the reconstructed vasculature, the recruited fibroblasts and chondroblasts release the cartilaginous matrix, which further provide support for future bone tissue formation. Though the mechanism by which HS influences the periosteum differentiation to osteoblasts and chondrocytes are still not well-defined, previous research provides some clues to reveal how HS affect the soft callus formation.

Angiogenesis is a significant part of the dynamic process of tissue healing and the new blood vessels act as a crucial medium in transporting oxygen and nutrients to the metabolically active callus, exchanging gasses, removing waste products, providing a pathway for inflammatory cells, cartilage and bone precursor cells, and allowing access to systemically circulating factors [56, 57]. Angiogenesis involves the interplay of various growth factors and cytokines, among which VEGF has been recognized as the master regulator of angiogenesis [58]. In mammal, the VEGF family comprises of five members, VEGFA, B, C, D and placenta growth factor (PLGF), which are approximately 40 kDa dimeric glycoproteins. HSPGs are identified as the co-receptors of VEGF when it’s defined as binding molecule lacking established VEGF-induced catalytic function. The binding site for VEGF on HSPGs is the HS chain [59]. For preferable safety and cost-effectiveness, recent strategies focus on delivering VEGF in an orderly and controlled manner that can ensure their sustained release locally, without any potential overdose-related complications [60, 61]. Numerous studies show that HSPGs critically regulate VEGF localization and signaling duration, impacting VEGFA164 modulation of sprouting blood vessel tip cell migration via gradients [62]. Biochemical analyses reveal that heparin/HS binds both growth factors and their receptors, extending the receptor complex’s half-life [63]. The VEGF family members can undergo alternative splicing, resulting in the creation of isoforms that exhibit varying biologic activities. The isoforms in humans are referred to as VEGFA121, VEGFA145, VEGFA165, VEGFA189, and VEGFA206, with VEGFA121 and VEGFA165 being the most prevalent [59]. When HSPGs on adjacent pericytes present VEGFA165 to VEGFR2, there is a significant increase in the strength and duration of the signal [64]. HSPG even stimulates new blood vessels formation with potencies comparable to VEGF165 [65]. In addition, the HS chains of glypican-1 (GPC1) are responsible for facilitating its interaction with VEGF, as demonstrated by the fact that the ability of GPC1 to bind to VEGF165 was eliminated when the HS chains were removed by heparanase treatment. The binding of VEGFA/VEGFR was enhanced when exogenous GPC1 was added, indicating that GPC1 regulates angiogenesis [66]. Actually, the binding domain was encoded by exon 7 of the VEGF gene, it was supported that VEGF121 cannot bind with HSPG for above amino acids deficiency [67]. The interaction of HSPGs and other VEGF family members and angiogenesis molecules should be explored and revealed in the future research.

Previous research has indicated that HS has an inhibitory effect on the development of cartilage, mainly achieved by regulating fibroblast growth factors (FGFs). The FGF family comprises 22 polypeptides (FGF1 to FGF22) that exhibit various biologic functions and send signals to all types of cells and tissues [68]. FGFs can be divided into two categories based on their functions and biologic effects: mitogenic FGFs promote cell proliferation, while metabolic FGFs regulate substrate and energy metabolism. HS in the ECM is identified to decrease the FGF travel distance, further inhibit its stimulation to other tissues by trapping and combining with the mitogenic FGF [69]. Metabolic FGFs members, FGF2, FGF4, FGF7, FGF18 and FGF20, are representative members combining with HS chains on HSPG [70, 71]. By contrast to mitogenic, the metabolic FGF members with HS affinity deficiency, including FGF19, FGF21 and FGF23, drive broad-spectrum functions in regulating the metabolic homeostasis of bile acid, lipids, glucose, energy, and minerals without direct proliferation-promoting activity [69]. For the chondrogenesis, current research mainly focuses on the perlecan/FGF2 signaling pathway. Perlecan, HSPG existing in the ECM, is found in all basement membranes, pericellular matrices of hypertrophic vertebral growth plate, and cartilaginous endplate chondrocytes [72, 73]. It has been proved that perlecan plays a crucial role in maintaining the balance of articular cartilage, rudiment, and growth plate cartilage growth, as well as promoting maturational changes such as mineralization [74]. Growth plate chondrocytes produce a perlecan form that has both HS and chondroitin sulfate side chains, with the HS chains facilitating cell signaling through FGF/FGFR [75]. Perlecan HS chains are also proved to bind and regulate the signaling pathways of FGF1, FGF2, and FGF18 [76]. And the regulatory process about chondrogenesis is mainly via FGF2, while FGF18 for endochondral ossification or osteogenesis. The interaction between HSPGs and FGF2 is facilitated by the binding of the negatively charged sulfate groups on the saccharide chain with the basic amino acid motifs, which plays a crucial role in modulating the biologic activity of FGF2 [77, 78]. When cartilage is damaged or overburdened, FGF2 serves as a crucial perlecan ligand in cartilage and functions as a mechanotransducer to the chondrocyte [79]. The combination of FGF2 and perlecan is crucial for determining the growth factor location. When perlecan degrade, the perlecan/FGF2 becomes unstable, and FGF2 loses its designated position in ECM, then interacts with another two HS proteoglycans families on the cell surface, syndecan and glypican [80]. It was found that heparanase, the enzyme for HS degradation, promotes chondrogenesis and is increased in abnormal cartilage, which contributes to the development of exostosis [81]. In addition, chondrocyte monolayer cultures treated with heparanase to decrease HS levels in the culture medium showed higher rates of chondrocyte proliferation and deposition of GAG compared to control group [82]. In future, more research is encouraged to explore the relationship of perlecan and other FGFs.

In addition, HSs/HSPGs secreted by the fibroblasts and endothelial cells are present in the granulation tissue and responsible for the regulation of tissue healing and angiogenesis through an orchestrated binding and modulation of various paracrine agents, such as VEGF, FGF, TGF-β, PDGF-β, SDF-1 [83]. Previous findings reveal that TGF-β3/HS complex promotes the GAG expression, the cartilage matrix proteins secretion, and the specific cartilage genes expression, as compared to treatment with TGF-β3 or HS [84]. HSPGs located on the surface of chondrocytes also show the affinity to combine with fibronectin 1(FN1), another crucial factor for the differentiation of cartilage and bone cells [85]. A novel BMP2 carrier system incorporated with an HS-bearing perlecan domain 1 (PlnD1), and the generated hyaluronic acid-based microscopic hydrogel particles has been developed. The system delivered BMP2 in a controlled manner and the results showed that it significantly stimulated the production of cartilage-specific ECM [86]. The enhancement of HS to subchondral bone regeneration and chondral tissue repair is also confirmed in rabbits with the femoral trochlea deficiency [87].

For the cell-surface HSPGs, syndecan and glypican, there is few publications revealing their regulation to chondrogenesis process. Syndecan is confirmed to regulate the expression of all HBGFs including FGFs and VEGF [88].

In conclusion, numerous studies highlight the contribution of VEGF and FGF families to soft callus formation, with HS and HSPGs playing a critical role. While VEGF and FGF bind to HS chains on HSPGs, it is difficult to attribute the effect solely to HS. The entire HSPG molecule contributes, with HS providing the binding site. There is no published research investigating the regulation of other types HSPGs to chondrogenesis. Therefore, more detailed information about syndecan and glypican as regulators deserves more attention in the following research.

The new formed soft callus provides a scaffold for hard callus formation as a bridge at the fracture gap, which lasts for 3–4 months until it completely mineralizes into calcified bone tissue. Once the soft callus starts calcified, it suggests that the beginning of hard callus formation [89]. Endogenous HS is identified as the inhibitor of chondrocytes differentiation and osteogenesis process. The researchers discover that chondrocytes and their matrix exhibit a high level of perlecan while osteoblasts in trabecular bone and periosteal wall show seldom expression of perlecan [90].

Besides playing a role in regulating above soft callus formation, HS/FGFs also modulates the process of osteogenesis. It has been revealed that FGF18 is necessary for the proper growth of long bones through endochondral ossification in the FGF18 knockout mice [74]. The cells surrounding the ossification center express perlecan and FGF18 at a high level. FGF18 stimulates the transition of cells at the center of the ossification center from cartilage formation to bone formation, resulting in mineralization of this region [74]. Heparanase, the enzyme to degrade HS, is discovered in the perichondrium, periosteum, and chondroosseous junct