The Essential Role of Proteoglycans and Glycosaminoglycans in Odontogenesis

Abstract

Tooth development and regeneration are regulated through a complex signaling network. Previous studies have focused on the exploration of intracellular signaling regulatory networks, but the regulatory roles of extracellular networks have only been revealed recently. Proteoglycans, which are essential components of the extracellular matrix (ECM) and pivotal signaling molecules, are extensively involved in the process of odontogenesis. Proteoglycans are composed of core proteins and covalently attached glycosaminoglycan chains (GAGs). The core proteins exhibit spatiotemporal expression patterns during odontogenesis and are pivotal for dental tissue formation and periodontium development. Knockout of core protein genes Biglycan, Decorin, Perlecan, and Fibromodulin has been shown to result in structural defects in enamel and dentin mineralization. They are also closely involved in the development and homeostasis of periodontium by regulating signaling transduction. As the functional component of proteoglycans, GAGs are negatively charged unbranched polysaccharides that consist of repeating disaccharides with various sulfation groups; they provide binding sites for cytokines and growth factors in regulating various cellular processes. In mice, GAG deficiency in dental epithelium leads to the reinitiation of tooth germ development and the formation of supernumerary incisors. Furthermore, GAGs are critical for the differentiation of dental stem cells. Inhibition of GAGs assembly hinders the differentiation of ameloblasts and odontoblasts. In summary, core proteins and GAGs are expressed distinctly and exert different functions at various stages of odontogenesis. Given their unique contributions in odontogenesis, this review summarizes the roles of proteoglycans and GAGs throughout the process of odontogenesis to provide a comprehensive understanding of tooth development.

Keywords

stem cells extracellular matrix signal transduction tooth development tooth germ periodontium

Introduction

Odontogenesis relies on a finely orchestrated signaling network (Thesleff 2003). Recently, the role of extracellular regulatory networks in tooth development has been revealed (Yu and Klein 2020). While diverse functions of signaling pathways have been demonstrated during different stages of tooth development, including initiation, morphogenesis, and mineralization, the pivotal link connecting and transmitting signaling from the extracellular matrix (ECM) remains to be clarified (Yu and Klein 2020). Proteoglycans are essential components of ECM proteins and key signaling molecules widely involved in odontogenesis.

Proteoglycans are a class of glycoproteins that widely exist on the cell membrane and in the ECM. They are composed of core proteins and covalently attached glycosaminoglycans (GAGs), which are linear polysaccharides with repeated disaccharide units (Fig. 1). Proteoglycans can differ based on the disaccharide units of the attached GAG chains—namely, chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS). Accordingly, proteoglycans can be categorized as heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), dermatan sulfate proteoglycans (DSPGs), and keratan sulfate proteoglycans (KSPGs) (Fig. 1) (Iozzo and Schaefer 2015).

Core proteins are traditionally considered the functional parts of proteoglycans. Expression of core proteins has been demonstrated at various stages of odontogenesis, and these proteins are key regulators of dental matrix formation. Knockout of the Bgn, Dcn, Hspg2 and Fmod genes results in defects in enamel or dentin formation and periodontium development (Haruyama et al. 2009; Ida-Yonemochi et al. 2011; Wang et al. 2014). Apart from core proteins, GAGs in proteoglycans play a pivotal role in regulating cellular processes as binding sites for growth factors. In our previous studies, we demonstrated that the expression of GAG biosynthetic enzymes exerts remarkable spatiotemporal specificity during early odontogenesis, suggesting their potential role in mediating early epithelial–mesenchymal interactions (Chen et al. 2023). Furthermore, recent evidence indicates that GAGs play an essential role in dental stem cell homeostasis via the regulation of signaling pathways. In mice, GAG deficiency causes overactivation of the fibroblast growth factor 10 (FGF10)/fibroblast growth factor receptor 2b (FGFR2b) signaling pathway, interrupts Sox2⁺ dental stem cell homeostasis, and ultimately leads to the formation of supernumerary incisors (Wu et al. 2020). Moreover, GAGs are critical for dental stem cell differentiation. GAG deficiency has been shown to inhibit the differentiation of ameloblasts and odontoblasts (Liu et al. 2017). Hence, the type, content, and sulfation of proteoglycans and GAGs create a suitable environment for odontogenesis, providing the necessary signals for dental stem cell homeostasis. This review focuses on the spatiotemporal expression of proteoglycans and GAGs during odontogenesis and on proteoglycan-mediated developmental processes to provide new insights into the functions of proteoglycans and GAGs in tooth development.

Structural and Biological Features of Proteoglycans and Glycosaminoglycans

The various functions of proteoglycans largely stem from the enormous structural diversity of GAGs. The heterogeneity of GAGs relies on complex biosynthetic pathways involving more than 40 enzymes with a role in linkage formation, chain elongation, and modifications, including sulfation, deacetylation, and isomerization (Noborn et al. 2021) (Fig. 1). Moreover, this biosynthetic process is tightly regulated in a spatially and temporally controlled manner, generating GAGs with distinct structures that ensure selective interactions with various ligands under different physiological and pathological conditions. Proteoglycans and GAGs are thus endowed with multiple functional domains and participate in the regulation of signaling cascades by facilitating the morphogen diffusion gradient, acting as coreceptors, and serving as reservoirs for growth factors, morphogens, and cytokines (Smock and Meijers 2018; Yu et al. 2018). GAGs are indispensable for maintaining dental epithelial stem cell homeostasis, as they shape the normal ligand gradient. They can confine the FGF10 gradient in the dental epithelium to regulate stable FGF10/FGFR2B signaling, which maintains dental epithelial stem cell homeostasis (Wu et al. 2020). Notably, the sulfation patterns of GAGs (6-O-sulfation, 2-O-sulfation, 3-O-sulfation, and N-sulfation) are crucial for growth factor binding and subsequent signaling (Chen et al. 2018; Qiu et al. 2018). Modification of 6-O-sulfation affects cell surface HSPGs by binding Wnt10a and subsequently activating Wnt signaling, mediating odontoblast differentiation and dentin formation (Hayano et al. 2012). As evidenced by the abnormalities in embryonic development and organogenesis in GAG-defective animals, during organogenesis, proteoglycans also modulate and maintain stable extracellular distribution of morphogens, including hedgehog, Wnt, and FGF ligands (Huang et al. 2018; Yin et al. 2018), indicating that GAGs are indispensable at several stages of vertebrate development. In addition, these signaling pathways form a critical regulatory network during tooth development (Yu and Klein 2020). However, the role of proteoglycans in mammalian tooth development remains poorly understood.

Proteoglycans in Odontogenesis: Spatiotemporal Expression and Functions

The expression of proteoglycans occurs throughout the process of tooth development and exhibits remarkable spatiotemporal specificity (Tables 1 and 2 and Fig. 2).

Table 1.

Expression of Proteoglycans and Glycosaminoglycan Chain Biosynthetic Enzymes in Mouse Incisor Development.

	Detection	Incisor
	Detection	Lamina	Bud	Cap	Bell	Postnatal
Syndecan 1 (Wu et al. 2020)	mRNA	Dental mesenchyme	Mesenchymal cells	SR and mesenchyme	CL, SR, and mesenchyme	NM
Syndecan 1 (Filatova et al. 2014)	Protein	NM	Mesenchymal cells	DF and mesenchymal cells	DF and mesenchymal cells	IEE, SI, SR, OEE, and mesenchyme
Syndecan 1 (Fujikawa et al. 2022)	mRNA	NM	NM	NM	Papillary layer	Papillary layer
Syndecan 2 (Wu et al. 2020)	Protein	Dental epithelium and mesenchyme	Dental mesenchyme	Dental mesenchyme and dental sac	Dental sac	NM
Syndecan 2 (Fujikawa et al. 2022)	mRNA	NM	NM	NM	Not detected	Not detected
Syndecan 3 (Wu et al. 2020)	Protein	Dental epithelium	Dental epithelium	SR and IEE	SR and oral lamina	NM
Syndecan 3 (Fujikawa et al. 2022)	mRNA	NM	NM	NM	Odontoblasts (weak)	Mature odontoblasts, differentiating odontoblasts (weak)
Syndecan 4 (Wu et al. 2020)	mRNA	Dental epithelium	Dental epithelium	Dental epithelium	SR and SI	NM
Syndecan 4 (Fujikawa et al. 2022)	mRNA	NM	NM	NM	Not detected	Mature odontoblasts (weak)
Syndecan 4 (Yan et al. 2014)	Protein	NM	NM	NM	NM	CL, SI, and mesenchymal cells
Biglycan (Randilini et al. 2020)	mRNA	NM	NM	NM	NM	Odontoblasts
Biglycan (Matsuura et al. 2001)	mRNA	NM	NM	NM	Secretory ameloblasts and dental papillary cells	NM
Decorin (Randilini et al. 2020)	mRNA	NM	NM	NM	NM	Differentiating odontoblasts
Decorin (Matsuura et al. 2001)	mRNA	NM	NM	NM	E19.0: secretory odontoblasts	NM
HA (Morita et al. 2016)	mRNA	NM	Tooth bud and mesenchyme	SR, DP, and DF	E16.0: SR, SI, DP E18.0: SR, SI, and dental pulp	Papillary layer, subodontoblastic layer, and DP
HAS1 (Morita et al. 2016)	mRNA	Not detected	Not detected	Not detected	Not detected	Not detected
HAS2 (Morita et al. 2016)	mRNA	NM	Mesenchyme and epithelial tooth bud	DP	DP	Not detected
HAS3 (Morita et al. 2016)	mRNA	NM	Epithelial tooth bud	IEE	Labial apical bud, SI, and IEE	Labial tooth bud and papillary layer near mature enamel

BM, basement membrane; CL, cervical loop; DF, dental follicle; DP, dental papilla; HA, hyaluronic acid; HAS1, hyaluronic acid synthetase 1; HAS2, hyaluronic acid synthetase 2; HAS3, hyaluronic acid synthetase 3; HERS, Hertwig’s epithelial root sheath; IEE, inner enamel epithelium; mRNA, messenger RNA; NM, not mentioned; OEE, outer enamel epithelium; SI, strata intermedium; SR, stellate reticulum.

Table 2.

Expression of Proteoglycans and Glycosaminoglycan Chain Biosynthetic Enzymes in Mouse Molar Development.

	Detection	Molar
	Detection	Lamina	Bud	Cap	Bell	Postnatal
Syndecan 1 (Wu et al. 2020)	mRNA	Dental mesenchyme	Dental epithelium and dental mesenchyme	Oral–dental epithelium junction and dental mesenchyme	IEE, CL, SI, and dental mesenchyme	NM
Syndecan 1 (Filatova et al. 2014)	Protein	NM	Mesenchyme	OEE, SR, DF, and DP	OEE, SR, DF, and DP	P3.0: SI, SR, and mesenchymal cells P10: SI, SR, outer dental epithelium, DF, and dental pulp
Syndecan 1 (Fujikawa et al. 2022)	mRNA	NM	NM	NM	Enamel organ and DP	P1.0: SI, SR, and the dental lamina P3.0: DF and DP P7.0: SI, SR, dental lamina, and cervical region of DP/DF near HERS
Syndecan 2 (Wu et al. 2020)	Protein	Weak in dental mesenchyme and epithelium	Dental mesenchyme	IEE and SI	CL	NM
Syndecan 2 (Fujikawa et al. 2022)	mRNA	NM	NM	NM	E16.0: Weak in enamel organ and DP E18.5: Not detected	Not detected
Syndecan 3 (Wu et al. 2020)	Protein	Dental epithelium	Dental epithelium	SR	IEE and CL	NM
Syndecan 3 (Fujikawa et al. 2022)	mRNA	NM	NM	NM	E16.0: Weak in enamel organ and DP E18.5: Weak in differentiating odontoblasts	P1.0: Strong in odontoblasts P3.0 and P7.0: Strong in mature odontoblasts, weak in differentiating odontoblasts
Syndecan 4 (Wu et al. 2020)	mRNA	Dental epithelium	Dental epithelium and mesenchyme	Dental epithelium and mesenchyme	SR and SI	NM
Syndecan 4 (Fujikawa et al. 2022)	mRNA	NM	NM	NM	Weak in enamel organ and DP	P1.0: Not detected P3.0 and P7.0: Weak in mature odontoblasts
Syndecan 4 (Yan et al. 2014)	Protein	NM	NM	NM	E18.0: Oral epithelium, SR, IEE, OEE, and mesenchymal cells	P2.0: Preameloblasts P4.0: Weak in newly differentiated ameloblasts and odontoblasts P7.0: Almost undetectable in ameloblasts and odontoblasts
Perlecan (Ida-Yonemochi et al. 2005)	mRNA	Strong in BM of the oral epithelium and weak in mesenchyme	Dental epithelial cells particularly at primary enamel knot and mild in mesenchymal cells	Enamel organ (particularly in secondary enamel knot), SR, and the mesenchyme	SR, ameloblast, and DP	P1.0: SR and immature dental pulp P6.0: Strong in odontoblasts P28.0: Odontoblasts
Perlecan (Ida-Yonemochi et al. 2005)	Protein	Strong in BM of the oral epithelium and weak in mesenchyme	BM and dental epithelial cells	SR and mesenchymal cells	SR and DP	P1.0: SR and immature dental pulp P6.0: SR and dental pulp P28.0: Predentin
Biglycan (Randilini et al. 2020)	mRNA	NM	Mesenchyme	Mesenchyme and DP	Odontoblasts	P1.0: DP, strong in mature odontoblasts but weak in differentiating odontoblasts P3.0: DP and odontoblastic layer P7.0: Weak in the odontoblastic layer
Biglycan (Randilini et al. 2020)	Protein	NM	NM	Mesenchyme and DP	Predentin, odontoblast, and osteoid	Dental pulp, predentin, and odontoblasts
Biglycan (Matsuura et al. 2001)	mRNA	NM	NM	NM	Tooth germ and presecretory ameloblasts	NM
Decorin (Randilini et al. 2020)	mRNA	NM	Mesenchyme	Mesenchyme	Differentiating odontoblasts and DP	P1.0: DP, strong in newly differentiating odontoblasts and reduced in mature odontoblasts P3.0: DP and newly differentiating odontoblasts P7.0: Newly differentiating odontoblasts at the cervical tip
Decorin (Randilini et al. 2020)	Protein	NM	NM	NM	Predentin, differentiating odontoblasts, and osteoid	Predentin
Decorin (Matsuura et al. 2001)	mRNA	NM	NM	NM	Secretory odontoblasts	NM
Fibromodulin (Randilini et al. 2020)	mRNA	NM	Not detected	Not detected	Not detected	Predentin
Fibromodulin (Randilini et al. 2020)	Protein	NM	NM	NM	Predentin, osteoid	Predentin
Lumican (Randilini et al. 2020)	mRNA	NM	Not detected	Not detected	Not detected	Predentin
Lumican (Randilini et al. 2020)	Protein	NM	NM	Osteoid	Predentin, osteoid	Predentin
Versican (Jiang et al. 2010)	mRNA	Not detected	Tooth bud and mesenchyme	E13.5: DP E15.0: DP, DF, and SR and weak in the IEE and OEE	E16.0: DP and enamel organ E18.5: DP	NM
Versican (Jiang et al. 2010)	Protein	Dental epithelium and BM	Tooth bud and mesenchyme	DP, DF, and SR	E16.0: DP, DF, and SR E18.5: SR, DP	NM
HA (Yang et al. 2016)	Protein	Dental epithelium	Tooth bud, dental mesenchyme	SR, DF, DP, IEE, and OEE	E16.5: SR, SI, DP, IEE, OEE, and mesenchyme E18.5: SR, SI, DP, preameloblast, and preodontoblast	NM
HA (Morita et al. 2016)	mRNA	Dental placode and mesenchyme	Dental placode and mesenchyme	SR, DP, and DF	SI and SR	P4 and P7: SR, SI, and HERS
HAS1 (Yang et al. 2016)	Protein	Dental epithelium	Tooth bud	SR, DP, IEE, and OEE	E16.5: SR, SI, DP, IEE, OEE, and mesenchyme E18.5: DP, CL	NM
HAS1 (Morita et al. 2016)	mRNA	Not detected	Not detected	Not detected	Not detected	Not detected
HAS2 (Yang et al. 2016)	Protein	Dental epithelium	Tooth bud	SR, DP, IEE, and OEE	E16.5: SR, DP, IEE, and OEE E18.5: CL and subodontoblastic layer of the coronal pulp	NM
HAS2 (Morita et al. 2016)	mRNA	Mesenchyme	Mesenchyme	E14.0: Mesenchyme, DP, and DF E15.0: Mesenchyme, DP	DP and mesenchyme	Not detected
HAS3 (Yang et al. 2016)	Protein	Dental epithelium	Tooth bud and the dental mesenchyme	SR, IEE (primary enamel knot), and DP	E16.5: SR, SI, DP, IEE, OEE, and mesenchyme E18.5: Subodontoblastic layer of the coronal pulp	NM
HAS3 (Morita et al. 2016)	mRNA	Dental placode	Enamel organ	IEE and SR	IEE, SR, and SI	P4.0 and P7.0: SI and HERS
Heparanase (Ida-Yonemochi et al. 2010)	Protein	Dental epithelial cells	NM	NM	E17.5: CL and dental lamina E19.0: SI	P1.0: SR P6.0: SR
HS (Ida-Yonemochi et al. 2010)	Protein	Dental epithelial cells, BM, and mesenchyme	NM	NM	E17.5: IEE and BM of the enamel organ E19.0: SI	P1.0: SI P6.0: SR and dental pulp
CS(CS-2B6) (Jiang et al. 2010)	Protein	Dental epithelium and BM	Tooth bud, BM, and mesenchyme	SR	E16.0: SR and DP E18.5: SR	NM
CS(CS-56) (Jiang et al. 2010)	Protein	Mesenchyme	Mesenchyme	DP, DF	E16.0: DP and DF E18.5: DP	NM
CS(CS-3B3) (Jiang et al. 2010)	Protein	Mesenchyme	Mesenchyme	E15.0: DP, DF E16.0: DP	DP	NM

BM, basement membrane; CL, cervical loop; CS, chondroitin sulfate; DF, dental follicle; DP, dental papilla; HA, hyaluronic acid; HAS1, hyaluronic acid synthetase 1; HAS2, hyaluronic acid synthetase 2; HAS3, hyaluronic acid synthetase 3; HERS, Hertwig’s epithelial root sheath; HS, heparan sulfate; IEE, inner enamel epithelium; mRNA, messenger RNA; NM, not mentioned; OEE, outer enamel epithelium; SI, strata intermedium; SR, stellate reticulum.

HSPGs in Odontogenesis

HSPGs can be divided into 3 groups based on their location: membrane HSPGs (syndecans, glypicans), secreted extracellular HSPGs (agrin, perlecan, and type XVIII collagen), and secretory vesicles (serglycin) (Iozzo and Schaefer 2015). Among HSPGs, syndecans and perlecan have been implicated in odontogenesis. The HS contained in syndecans, a main family of cell surface HSPGs, provides a docking site for molecules such as growth factors and morphogens (Afratis et al. 2017; Xie and Li 2019). In this family, syndecan 1 and 3 and syndecan 2 and 4 form 2 subfamilies. Syndecan 1 and 3 have additional CS chains added to the HS chains on both the membrane-distal and membrane-proximal ends of the ectodomain, while syndecan 2 and syndecan 4 contain GAG chains only on their membrane-distal ends (Afratis et al. 2017). Syndecan 1 and syndecan 3 are both related to matrix formation, but their distributions suggest different functions. Syndecan 1 distribution is correlated with preodontoblasts but not with differentiated odontoblasts, indicating that syndecan 1 might be highly related to the cytodifferentiation rather than the mineralization stage (Filatova et al. 2014). In contrast, syndecan 3 is more closely related to matrix formation, as it is mostly observed in mature odontoblasts (Fujikawa et al. 2022). These discrepancies may be attributed to structural differences, for example, the CS and HS content, which have been found to be positive regulators of osteogenesis and mineralization (Wang et al. 2017). Syndecan 2 appears to be more closely related to the initial stage of odontogenesis, as it is expressed only before the bud stage (Fujikawa et al. 2022). Moreover, its spatiotemporal expression pattern showed high concordance with the odontogenic shift from E11.5 to E13.5, suggesting a potential role in early epithelium–mesenchyme interaction (Chen et al. 2023). Unlike syndecan 2, syndecan 4 plays a critical role in amelogenesis, as suggested by its strong expression in preameloblasts. Furthermore, syndecan 4 is involved in cell proliferation and ameloblast differentiation via FGF10 signaling (Yan et al. 2014). Thus, syndecans may perform different functions during various stages of odontogenesis. However, their precise functions require further clarification through in vivo experiments.

Another HSPG member, perlecan, plays a distinct role in tooth morphogenesis. Perlecan has been strongly detected in primary and secondary enamel knots, where signaling transduction is fine-tuned to regulate tooth morphogenesis (Thesleff et al. 2001; Mogollón et al. 2021). This was further verified by the irregular teeth morphology, including abnormal crown, in keratin 5–perlecan Tg mice. Perlecan is a critical modulator of enamel formation. As an important component of the basement membrane, it facilitates the differentiation of polarized ameloblasts and the subsequent formation of Tomes processes. In addition, its normal function in the stellate reticulum is essential for nourishing the enamel organ. Furthermore, perlecan participates in the regulation of signaling molecules such as dentin sialophosphoprotein (DSPP), FGF-2, sonic hedgehog, and transforming growth factor β1 (TGF-β1) in a stage-specific manner to ensure normal development of the enamel organ (Ida-Yonemochi et al. 2011; Ida-Yonemochi et al. 2013). Because various signaling pathways appear to be finely orchestrated by perlecan during enamel organ development, its underlying regulatory roles require further exploration.

CS/DSPGs in Odontogenesis

CSPGs are the most studied proteoglycans involved in tooth development. Research on CSPGs has primarily focused on the later stages of odontogenesis. When the tooth germ develops into the bell stage and the enamel matrix is secreted and mineralized, the expression levels of CSPGs, including decorin and biglycan in predentine and enamel, significantly increase (Randilini et al. 2020), and they act as essential modulators in the secretion and mineralization of dentin and enamel. Decorin (DCN) and biglycan (BGN) are widely expressed in the late stages of tooth germ development and regulate the secretion and mineralization of the dental matrix (Goldberg et al. 2003). Both BGN and DCN exert positive effects on dentin mineralization, with Bgn^{– /–} and Dcn^–/– mice exhibiting significantly reduced dentin mineralization. However, dentin hypomineralization is more prominent in Dcn^–/– mice than Bgn ^–/– mice, possibly due to different compensatory mechanisms. Bgn^{– /–} odontoblasts and the subodontoblastic layer exhibit upregulated expression of dentinogenesis-related proteins, including DSPP, dentin matrix protein 1 (DMP-1), bone sialoprotein, and osteopontin, whereas Dcn^–/– mice exhibit decreased expression of these proteins (Goldberg et al. 2005). Interestingly, the interaction of BGN and DCN with the dentinogenesis-related protein DSPP is far more complex. Increased levels of BGN and DCN in the widened predentin and void spaces among the calcospherites in the dentin of Dspp^{– /–} mice indicate a potential regulatory network in dentin mineralization (Sreenath et al. 2003). To elucidate their regulation, Haruyama et al. (2009) constructed double-knockout mouse models and found that DCN and BGN had opposite roles in the interaction with DSPP. The deletion of BGN in Dspp^{– /–} mice resulted in an increased number of calcospherites, suggesting that the loss of BGN further inhibited the coalescence of calcospherites, thereby hindering predentine mineralization. In contrast, the knockout of Dcn in Dspp^{– /–} mice rescued the dentin hypomineralization phenotype, suggesting that a relatively normal level of DCN in Dspp^–/– mice negatively influenced the mineralization process at the dentin mineralization front (Sreenath et al. 2003; Haruyama et al. 2009). This discrepancy may be attributed to their distinct roles in odontoblasts as well as their interactions with different signaling molecules. These results identified a crucial role for BGN and DCN in orchestrating essential events during dentin mineralization, including potential regulation by DSPP. In contrast to their roles in dentin formation, BGN and DCN play contrasting regulatory roles in enamel formation. Knockout of DCN delays the formation of enamel and reduces the expression of enamelin, whereas the expression of amelogenin in Bgn ^{– /–} mouse tooth germ is significantly upregulated and accelerates enamel formation (Goldberg et al. 2005). These results have also indicated distinct roles in dentin and enamel formation, with different regulatory networks.

KSPGs in Odontogenesis

Compared to other proteoglycans, the expression and function of KSPGs are not well understood. Fibromodulin is widely expressed in the bone and dentin. It regulates collagen fibrillation in predentin and promotes dentin mineralization. Fibromodulin is involved in the early stages of enamel formation. Defects in enamel and dentin formation and mineralization were observed in newborn mice but became undetected in day 21 Fmod ^{– /–} mice; this may be attributed to compensatory mechanisms involving small integrin-binding ligand N-linked glycoproteins (SIBLINGs), a group that includes osteopontin, DMP-1, bone sialoprotein, matrix extracellular phosphoglycoprotein, dentin sialoprotein, and dentin phosphoprotein. The compensatory mechanisms appear to differ between molars and incisors, indicating that such regulation is also spatiotemporally specific (Goldberg et al. 2006; Goldberg et al. 2008; Goldberg et al. 2011). Another KSPG, osteoadherin, is found at the mineralization front of dentin and regulates mineralization in dental pulp cells in vitro (Nikdin et al. 2012). As preliminary results indicate a positive role for osteoadherin in mineralization, genetic knockout animals are needed to elucidate their exact roles and mechanisms.

Proteoglycans in Periodontal Tissue Development

In addition to dental tissue, the formation of periodontal tissue is an important part of tooth development. Proteoglycans are also involved in the development and homeostasis of periodontium. Previous reviews have discussed their distribution and regulatory roles under pathological conditions (Chen et al. 2021; Miguez et al. 2023). Periodontal tissue development is a complex process that involves the cementum, gingiva, periodontal ligament (PDL), and alveolar bone. Mesenchymal cells within the dental follicle are induced to differentiate into cementoblasts, fibroblasts, and osteoblasts, thereby giving rise to different tissue types. Several proteoglycans are highly expressed in fibroblasts and cementoblasts. Asporin, glypican-2, neurocan, osteomodulin, and versican are expressed at higher levels in fibroblasts, whereas aggrecan, fibromodulin, glypican-3, and serglycin are more abundant in cementoblasts. These findings indicate that the proteoglycans may play roles in constituting different stem cell niches and facilitating signaling transduction (Mun et al. 2022). However, to date, evidence owing to the heterogeneity of the dental follicle in vivo remains limited. Understanding this process in vivo will provide more clues, as lineage determination is an essential first step in the early development of periodontal tissue. Recently, this approach has been implemented using high-throughput techniques, including single-cell RNA sequencing and spatial transcriptome analysis.

Unraveling their distinct signatures in different cell lineages is the first step toward elucidating the roles of different proteoglycans; knockout animal models may provide additional information. The most abundant proteoglycans in the periodontal tissue belong to a unique family of small leucine-rich repeat proteoglycans (SLRPs), including fibromodulin, biglycan, decorin, lumican, asporin, and osteoadherin. SLRPs are characterized by their leucine-rich repeats, which impart the ability to bind collagen and regulate collagen fibril growth and fibrillogenesis (Miguez et al. 2023). Knockout animals with single or double SLRPs exhibit abnormalities in the PDL to different extents (Matheson et al. 2005) (Table 3). These findings indicate that SLRPs coordinate to regulate collagen fibril assembly and organization in the PDL. However, SLRPs might play different roles or participate in distinct regulation mechanisms, as these single knockout mice displayed unique collagen fibril morphology and organization. Furthermore, potential compensation also exists in the PDL, as double-knockout animals showed more severe phenotypes than single-knockout animals, as observed in Fmod^{– /–} Bgn^{– /–} and Fmod^{– /–} Lum^{– /–} mice (Matheson et al. 2005; Wang et al. 2014). Moreover, SLRP expression is spatiotemporally regulated to partially compensate for the depletion of Fmod and Bgn (Wang et al. 2014). Besides, SLRPs are also engaged in the regulation of signaling pathways, including TGF-β/bone morphogenetic proteins (BMP) signaling in the PDL. Notably, many proteoglycans function via signaling regulation under pathological conditions. However, our current understanding of their roles in signaling regulation in tooth development is limited, warranting further research.

Table 3.

Dental Phenotypes of Proteoglycan-Deficient and Proteoglycan-Overexpressed Animals.

Animal Models	Dental Phenotypes	Mechanisms	Reference
Keratin 5-perlecan Tg mice	1) Aberrant molar morphology (dull-ended crowns, greater attrition) 2) Deformed enamel organ (disordered ameloblast polarity, irregular Tomes process) and disturbed enamel formation	Elevated expression and localization of integrin β1, VEGF, bFGF, and TGF-β1 observed	(Ida-Yonemochi et al. 2011)
Bgn^{– /–} mice	1) Dentin hypomineralization and increased enamel formation in newborn mice 2) Abnormal collagen fibrils and fibril bundle morphology in the PDL	Abnormal upregulation of amelogenin resulted in increased enamel formation. Compensatory upregulation of SIBLINGs	(Goldberg et al. 2005; Matheson et al. 2005; Haruyama et al. 2009)
Dcn^{– /–} mice	1) Dentin hypomineralization (more severe than Bgn^{– /–} mice) and delayed enamel formation in newborn mice 2) Abnormal collagen fibrils and fibril bundle morphology in the PDL	Defective enamel formation probably resulted from the altered dentin formation. Downregulation of SIBLINGs might be attributed to dentin hypomineralization.	(Goldberg et al. 2005; Matheson et al. 2005; Haruyama et al. 2009)
Dcn^{– /–} Dspp^{– /–} double-knockout mice	Knockout of Dcn rescued the widened predentin in Dspp^{– /–} mice.	Not mentioned	(Haruyama et al. 2009)
Bgn^{– /–} Dspp^{– /–} double-knockout mice	Knockout of Bgn further inhibited calcospherite coalescence in Dspp^{– /–} mice.	Not mentioned	(Haruyama et al. 2009)
PALP-1^{– /–} mice	Abnormal structures of collagen fibrils in the PDL and decreased mechanical strength of the PDL	Not mentioned	(Kinoshita et al. 2023)
Lum^{– /–} mice	Abnormal collagen fibrils and fibril bundle morphology in the PDL	Not mentioned	(Matheson et al. 2005)
Lum^{– /–} Fmod^{– /–} double-knockout mice	Abnormal collagen fibrils and fibril bundle morphology in the PDL	Not mentioned	(Matheson et al. 2005)
Fmod^{– /–} mice	1) Dentin hypomineralization (enlarged diameter of collagen fibrils in predentin) in newborn and day 7 Fmod^{– /–} mice but no obvious structural defect detected at day 21 2) Delayed enamel formation and porous appearance from day 0 to day 21 3) Abnormal collagen fibrils and fibril bundle morphology in the PDL 4) Enhanced alveolar bone formation in newborn and day 7 Fmod^{– /–} mice, but no obvious structural defect detected at day 21	Molecule-specific and site-specific compensation involving SIBLINGs	(Goldberg et al. 2006; Goldberg et al. 2008; Goldberg et al. 2011; Ida-Yonemochi et al. 2011)
Fmod^{– /–} Bgn^{– /–} double-knockout mice	Abnormal collagen fibrils in PDL and altered remodeling of the alveolar bone	RANKL signaling and TGF-β/BMP signaling Compensatory upregulation of other SLRPs in an age- and tissue-specific manner observed	(Wang et al. 2014)
K14^Cre/+;Fam20B^fl/fl mice (epithelial conditional knockout)	Supernumerary tooth formation in a pattern reminiscent of replacement teeth	FGF10/FGFR2b signaling	(Wu et al. 2020)
K14^rtTA;tetO^Fgfr2b/Igh; K14^Cre/+;Fam20B^fl/fl mice (epithelial conditional knockout and FGFR2b inhibitor conditional overexpression)	Overexpression of the FGFR2b inhibitor partially rescued the supernumerary tooth formation in K14^Cre/+;Fam20B^fl/fl– mice.	FGF10/FGFR2b signaling	(Wu et al. 2020)
Sulf1^{– /–} Sulf2^{– /–} double-knockout mice	Thinner dentin matrix and shorter root	Wnt signaling	(Hayano et al. 2012)

bFGF, basic fibroblast growth factor; Bgn, biglycan; Dcn, decorin; Dspp, dentin sialophosphoprotein; Fam20B, family with sequence similarity 20; FGF10/FGFR2b, fibroblast growth factor10/fibroblast growth factor receptor 2b; Fmod, fibromodulin; K14, keratin 14; Lum, lumican; PDL, periodontal ligament; RANKL, receptor activator of nuclear factor-kappaB ligand; SIBLINGs, small integrin-binding ligand N-linked glycoproteins; SLRPs, small leucine-rich proteoglycans; Sulf1, sulfatase 1; Sulf2, sulfatase 2; TGF-β1, transforming growth factor β1; TGF-β/BMP, transforming growth factor β/bone morphogenic protein; VEGF, vascular endothelial growth factor.

As in other mineralized tissues, proteoglycans such as osteoadherin and fibromodulin are abundantly expressed in the alveolar bone (Petersson et al. 2003). Fibromodulin acts as a negative regulator of alveolar bone formation by affecting the number of mineralizing trabeculae (Goldberg et al. 2008). Moreover, fibromodulin appears to have dual roles in bone and dentin. Thus, the effects of Fmod deficiency persisted in the alveolar bone of day 21 Fmod ^{– /–} mice, whereas no significant phenotypes were observed in dentin. This finding might be attributed to the different fragmentation patterns of fibromodulins with different molecular weights (Goldberg et al. 2008) or to the potentially different compensatory mechanisms occurring in the dentin and alveolar bone. Fibromodulin may also play various tissue-specific roles. Fibromodulin and biglycan mediate homeostasis of the alveolar bone via TGF/BMP signaling, as demonstrated by the increased numbers of osteoclasts and the significantly higher expression of receptor-activator of nuclear factor-κB ligand (RANKL) in Fmod^–/– Bgn^–/– mice compared to wild-type mice (Wang et al. 2014). Taken together, these findings suggest that fibromodulin is a pivotal regulator in the development and homeostasis of the alveolar bone, exerting its biological functions in a highly tissue-specific manner.

Proteoglycans, such as versican, biglycan, and syndecans, are found in the cementum (Miguez et al. 2023). Versican is a crucial component of the ECM that forms highly hydrated complexes with hyaluronan and plays an important role in embryonic development (Nandadasa et al. 2021). Alternative splicing of versican generates 4 isoforms—namely, V0, V1, V2, and V3—with differences in the CS chain attachment (Ito et al. 1995). Versican is the only proteoglycan found in human dental phenotypes. Patients with VCAN mutations show a loss of cementum and abnormal root morphology. Whole-exome sequencing revealed an H2665L homozygous sequence variant predicted to affect full-length V0 isoforms, suggesting that the V0 isoform might be associated with cementum formation in periodontal tissues (Bigoni et al. 2018). However, the exact functions of versican isoforms need to be further clarified through additional in vitro and in vivo experiments. Understanding the differences in their functions and roles in cellular signaling will complement our current understanding of the structure–function relationships of GAGs.

Collectively, these results suggest that proteoglycans are spatiotemporally expressed and have different biological functions in tooth development (Fig. 3). However, their potential role in the earliest stages largely remains to be explored. Tooth development begins with the first epithelial signal in the dental lamina and mutual induction between the dental epithelium and mesenchyme. Understanding how proteoglycans act at this essential stage would provide clues for whole-tooth regeneration. Another challenge is to determine the specificity and redundancy of proteoglycans during odontogenesis. For example, BGN and DCN show an apparent functional overlap and compensation in the mineralization of bone (Wadhwa et al. 2007), whereas such compensation was only observed in dentin. Whether BGN and DCN have a functional overlap or compensatory effects during tooth mineralization remains unclear. Moreover, recent studies on single proteoglycan knockout animals have revealed no obvious dental phenotypes, and evidence of compensatory regulation by another proteoglycan has been little examined. Therefore, double-knockout animal models are needed to further elucidate the biological functions of proteoglycans and the potential compensation mechanisms between them.

Roles of Glycosaminoglycans in Odontogenesis

As mentioned above, functional discrepancies between different proteoglycans are highly relevant to GAGs. Thus, to a large extent, the types, contents, and sulfation patterns of GAGs determine the function of proteoglycans. There is substantial evidence illustrating the dynamic expression of different types of GAGs, suggesting that they are involved in different stages of odontogenesis, including cytodifferentiation, morphogenesis, and mineralization (Ida-Yonemochi et al. 2010; Jiang et al. 2010; Kero et al. 2018; Randilini et al. 2020). To date, analysis of GAGs has mostly focused on gradual mineralization from predentin to dentin, revealing changes in GAG types, chain lengths, and sulfation patterns. Mineralized dentin is characterized by higher CS content, longer chain length, and a relatively higher degree of 6-O-sulfation. Recent studies have provided more direct evidence of the essential roles of GAGs in regulating mineralization, such as by affecting fibrillogenesis and directly influencing collagen nucleation. The positive roles of GAGs in matrix formation and mineralization have been verified by the significant changes in structural and mechanical parameters as well as the decreased dentin remineralization observed after GAG removal (Milan et al. 2005; de Mattos Pimenta Vidal et al. 2017; Farina et al. 2019; Wojtas et al. 2020).

Compared to the established roles of GAGs in mineralization during late odontogenesis, their roles in early odontogenesis were only discovered recently. GAGs initiate tooth development by regulating the homeostasis of dental epithelial stem cells. GAGs facilitate the extracellular FGF10 gradient and ensure a stable level of FGF10/FGFR2b signaling to maintain the balance between the self-renewal and differentiation of dental epithelial stem cells. However, this balance is affected when GAG synthesis is disrupted in the dental epithelium, resulting in supernumerary incisors on the proximal lingual side of normal incisors. Furthermore, GAG deletion leads to abnormal activation of Sox2⁺ dental epithelial stem cells. Under normal conditions, Sox2⁺ stem cells are only localized in the labial cervical ring after the completion of tooth germ development as stem cell reserves. However, in GAG-deficient mice, a large amount of Sox2⁺ cells existed in the lingual side of the normal incisor, suggesting that the ablation of GAGs may interfere with the cell fate determination of Sox2⁺ stem cells and activate their potential for odontogenesis. Moreover, deficiency of GAGs in the dental epithelium caused unrestricted diffusion of FGF10, leading to overactivation of FGF10-FGFR2b. Thus, GAGs participate in the homeostasis of dental stem cells during early odontogenesis through mediating signaling transductions (Wu et al. 2020).

GAGs are also indispensable in the later stages of differentiation such as cytodifferentiation. GAGs affect cell folding and formation of the inner enamel epithelium, as well as the subsequent odontoblast and ameloblast differentiation, as shown by the significantly reduced expression of AMELX, DMP-1, and DSPP in GAG-deficient tooth germs (Liu et al. 2017; Jiang et al. 2019). Notably, sulfation levels have been shown to be pivotal determinants of cytodifferentiation via the regulation of signaling transduction. High sulfation levels have been observed in odontogenic cells, followed by their desulfation as they differentiated into odontoblasts, producing dentin matrix. Sulf1 and Sulf2 encode endosulfatases that remove sulfate groups from the HSPGs on the cell surface. Double knockout of Sulf1 and Sulf2 results in a thinner dentin matrix and decreases the expression of Dspp in mice, indicating the importance of HS sulfation in dentinogenesis. In Sulf2 knockdown odontoblasts, excessive sulfate groups trapped Wnt10a and hindered its interaction with its receptor, blocking Wnt signaling transduction and the subsequent downregulation of Dspp expression (Hayano et al. 2012). Hence, sulfated GAGs are significant factors in cytodifferentiation, and the postsynthetic modifications affecting the sulfation groups and levels are critical for signaling transduction.

Unlike sulfated GAGs, HA is a linear and unbranched GAG synthesized by 3 hyaluronan synthases with no sulfation modifications. HA is a major nonprotein component of the ECM that influences the biomechanical properties of tissues. In addition, HA can interact with cell surface receptors, such as CD44 and receptor for HA-mediated motility, and activate related signaling pathways to regulate cell proliferation, migration, and differentiation (Kobayashi et al. 2020). Interestingly, HA is associated with the naive state of dental cells. It is expressed in preameloblasts and preodontoblasts, but its expression disappears as the cells mature. HA plays a critical role in the control of cell proliferation, orientation, and migration during molar development. Inhibition of HA causes a significant increase in the proportion of proliferating cells in the dental organ, resulting in a larger first molar in ex vivo cell cultures. In addition, the disruption of cellular orientation, including elongation and reorientation of cell axes, hinders the budding of the second molar (Sánchez et al. 2020).

In summary, GAGs regulate the biological processes of tooth germ stem cells by maintaining a stable signaling transmission throughout odontogenesis. However, the specific roles of the different GAGs in odontogenesis have not been fully elucidated, and their regulatory mechanisms in dental stem cells require further exploration.

Conclusions and Perspectives

Proteoglycans play a significant role in odontogenesis and exert a profound influence on the fate of dental stem cells. However, the functions of proteoglycans in tooth growth and regeneration, as well as their underlying molecular mechanisms, remain unclear. GAGs are vital for proteoglycan functions. Thus, the type, relative content, chain length, and sulfation pattern of GAGs, including their sulfation level and sulfation groups, are all determining factors for proteoglycan functions. Therefore, it is crucial to better understand the role of GAGs in odontogenesis. The complex structures of GAGs and the variety of their modification sites add to the complexity. Moreover, the lack of specific animal models makes it difficult to elucidate their biological functions in vivo. In addition, there are functional overlaps and compensatory mechanisms between proteoglycans; therefore, the phenotypes associated with a single proteoglycan deficiency in in vivo and in vitro models might be weak and difficult to detect. Although the expression of proteoglycans during odontogenesis has been well described, it is critical to unravel their biological roles in physiological and pathological processes.

Importantly, tooth development is a gradual process that includes initiation, morphogenesis, and mineralization. Studies have demonstrated the regulatory roles of proteoglycans and GAGs in mineralization; recently, their involvement in early odontogenesis was noted. Further research is required to elucidate the roles of GAGs in the regulation of dental stem cell lineages and their underlying mechanisms. In addition, proteoglycans and GAGs exhibit high temporal and spatial specificities, and elucidation of the mechanism by which proteoglycans and GAGs coordinate the complex signaling network that guides normal odontogenesis remains a challenge. Further studies should harness glycomic methods, including using the GAG library, oligosaccharide sequencing, and microarrays, to comprehensively reveal the cooperation between GAGs and signaling molecules and understand their functions in odontogenesis.

Author Contributions

J. Chen, contributed to conception, design, drafted and critically revised the manuscript; T. Sun, B. Lin, contributed to design, critically revised the manuscript; B. Wu, J. Wu, contributed to conception and design, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by the National Natural Science Foundation of China (Grant No. 81900956, 81870755) and Applied Basic Research Fund of Guangdong Province (Grant No. 2020A1515110852).

ORCID iD

J. Chen

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