Proteoglycans, complex macromolecules that are prominent constituents of the ECM composed of a protein core to which are covalently attached variable length and composition glycosaminoglycan (GAG) chains (Schwartz, 2000). Because of their complex structure and chemistry, proteoglycans have been classified based on function, localization, and protein cores. To date, forty-three distinct proteoglycan-encoding genes have been identified, and are organized into four families based on their cellular and subcellular location, protein and genomic homologies, and unique protein modules shared by members of each specific family (Iozzo and Schaefer, 2015). Most proteoglycans interact with signaling molecules in multiple biological processes i.e., tissue development, wound healing and disease progression. These interactions are complex and often multivalent involving contributions by nonionic (hydrogen-bonding, Van der Waals and hydrophobic) forces, conformational changes, or clustering of binding complexes (Soares da Costa et al., 2017), or the ability of signaling factors to multimerize (Whalen et al., 2013). Among the families of sulfated proteoglycans, the heparan sulfate proteoglycans (HSPGs) are the best studied in terms of biointeractions with diverse ligands and various signaling molecules affecting cell behavior. A major ionic interaction is that between the carboxyl and sulfate groups in the GAG chains and positively charged amino acid (lysine and arginine) residues (Cardin-Weintraub sequence) in the N-terminal region of all hedgehog (HH) signaling molecules (Cardin and Weintraub, 1989) including sonic (SHH), indian (IHH) and desert (DHH). In addition to the HHs, proteoglycans with different modification patterns also function in fibroblast growth factor (FGF), wingless (WNT), transforming growth factor (TGFβ), chemokines and Slit/Robo signaling (Townley and Bulow, 2018).

In contrast to the HSPGs, the interactions of signaling molecules with chondroitin sulfate and dermatan sulfate proteoglycans (CSPG and DSPG), which are the focus of this review and are often the most abundant proteoglycans in tissues, are not as well understood. Chondroitin sulfate (CS) chains are found on multiple proteoglycans; the most common are the hyaluronan- and lectin-binding proteoglycans (hyalectans) which have structural similarity at both the protein and genomic levels. The hyalectan family consists of four members: aggrecan, versican, neurocan, and brevican, which all share a tri-domain structure: an N-terminal globular domain that binds hyaluronan, a central domain bearing the CS chains and a C-terminal region that binds lectins. The CS chains consist of repeating disaccharides of N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) decorated with different degrees and patterns of sulfation on the disaccharides: GalNAc may have sulfate on C4 (CS-A), C6 (CS-C) or both C4 and C6 (CS-E), all catalyzed by specific sulfotransferases. The GlcA unit may also be sulfated on the C2 position (CS-B or CS-D) depending on where the sulfate residue is on GalNAc. Dermatan sulfate (DS) derives from chondroitin sulfate by inversion of GlcA to iduronic acid (IdA), catalyzed by an epimerase enzyme.

Determining direct relationships between proteoglycan structure/function and the bi-directional signaling that regulates development remains challenging, mainly because few tools exist that allow alteration or removal of specific GAG motifs or sulfate substitutions. However, some progress has been made using in vitro and in vivo approaches. Although general principles of CSPG interaction with signaling molecules have been shown in several developing tissue systems, we use as example the formation of skeletal structures with a focus on the growth plate of long bones, a transient cartilage template that is, replaced by bone. A complex and highly orchestrated program regulates growth plate cartilage morphogenesis in which chondroblasts proliferate, differentiate to chondrocytes, alter their shape, proliferate in stacks along the longitudinal axis, terminally differentiate to hypertrophic chondrocytes, and elaborate a mineralized vascularized matrix, which is then replaced by osteocytes (Karsenty and Wagner, 2002; Kozhemyakina et al., 2015). Multiple signaling pathways control the growth plate morphogenesis process including: IHH, FGF, TGFβ, bone morphogenic protein (BMP), parathyroid hormone-related peptide (PTHrP), SMAD6 and SMURF, all of which play unique and sometimes interacting roles in growth plate morphogenesis (Wang et al., 2021) (Figure 1A).

Some well documented examples of the mechanisms by which signaling pathways are necessary for cartilage morphogenesis are illustrated in Figure 1A and include the IHH-PTHrP negative feedback loop which regulates the size of the proliferative zone and onset of hypertrophy (Vortkamp, 2001). PTHrP, secreted by the resting zone, preserves the reservoir of progenitor cells and promotes chondrocyte proliferation by interacting with IHH secreted by hypertrophic chondrocytes (Mizuhashi et al., 2018). Conversely, IHH antagonizes PTHrP signaling and promotes chondrocyte hypertrophy in the lower segment of the growth plate (Lee et al., 2019). Members of the TGFβ family promote chondrogenesis in undifferentiated mesenchyme cultures (Karsenty and Wagner, 2002), while long bone chondrocyte proliferation and hypertrophy is inhibited by TGFβ (Serra and Chang, 2003). Targeted deletion of the TGFβ2 gene product alters the overall size and shape of limb rudiments (Sanford et al., 1997), and naturally occurring mutations in the TGFβ2 gene cause Camurati-Engelmann Disease, characterized by thickening of the long bone collar (Campos-Xavier et al., 2001). Another major signaling family, the BMPs, positively regulate both chondrocyte proliferation and hypertrophy (Horiki et al., 2004), as shown in mice with mutations in the BMP receptor type 1B that develop brachyactyly (Baur et al., 2000), and in mice which over-express SMAD and SMURF (negative regulators of BMP signaling), leading to chondrocyte hypertrophy and dwarfism (Horiki et al., 2004). The patterning of bone and joints also requires the interaction of multiple signaling pathways, including BMP members, HH, WNT, and FGF families (Archer et al., 2003; Baldridge et al., 2010). Thus, as these examples illustrate, the processes of cartilage, bone, and joint development is dependent on multiple morphogens, growth factors, and cytokines. However, understanding the role of CSPGs, the major proteoglycans in the growth plate, in influencing the functions of these signaling molecules and pathways during skeletal development remains limited. In this review we provide evidence of the roles by which each major feature (core protein, GAG chains, sulfation) of proteoglycans contribute to regulation of chondrogenesis (Figure 2).

Several studies have described critical roles for CSPGs such as aggrecan, which is broadly expressed by chondrocytes, and versican, which is transiently expressed in undifferentiated mesenchyme, in chondrogenesis and joint morphogenesis (Schwartz and Domowicz, 2002; Snow et al., 2005; Shepard et al., 2007; Domowicz et al., 2009; Choocheep et al., 2010; Lauing et al., 2014). For most of these studies, mutant models of proteoglycan biosynthesis and metabolism have helped to unravel the role of proteoglycans in skeletal formation and maintenance. In fact, the strongest evidence that CSPGs are essential during differentiation of chondrocytes and maintenance of skeletal elements rests on the demonstration of abnormalities in CSPGs concomitant with aberrant growth patterns in human and animal models (Melvin and Schwartz, 1988; Schwartz and Domowicz, 1998). In particular, mutations in the aggrecan gene are the cause of several chondrodysplasias and inherited skeletal disorders in humans and animals (Schwartz and Domowicz, 2002; Schwartz and Domowicz, 2014). Mutations in the aggrecan core protein gene have been identified in human skeletal disorders including: spondyloepimetaphyseal dysplasia with premature and severe osteoarthritis and osteochondritis (Gleghorn et al., 2005) and the recessive skeletal dysplasia EMD aggrecan-type which results from a missence mutation affecting the C-type lectin domain of aggrecan (Tompson et al., 2009; Stattin et al., 2010). To date, eight human genetic diseases involving defects in aggrecan, now coined the aggrecanopathies (Gibson and Briggs, 2016) have been identified, but the impact of these aggrecan defects on signaling in humans has not yet been fully explored.

One of the earliest studied animal models was nanomelia (nm), a lethal chondrodystrophy of fowl (Landauer, 1965). The nanomelic chick cartilage can synthesize CS chains, but aggrecan core protein is absent due to a single nucleotide change that results in a premature stop codon in the aggrecan gene (Argraves et al., 1981; Li et al., 1993; Schwartz et al., 1993; Vertel et al., 1994). This severely truncated core protein is not glycosylated or transported through the secretory pathway leading to an altered cytoarchitecture (densely packed cellular growth plate devoid of matrix) and homeostasis (increased proliferation of hypertrophic chondrocytes and increased cell death in the proliferative zone) (Domowicz et al., 2000). Since all of these phenotypes are regulated by signaling pathways, these aggrecan mutants present ideal models for investigating the core protein interactions with growth plate regulators. Thus, further studies documented that loss of aggrecan results in defects in morphogen gradient distribution and gene expression profiles of the critical chondrocyte regulators (IHH, BMP, and FGF) (Domowicz et al., 2009; Schwartz and Domowicz, 2014).

A similar lethal mutation in the aggrecan genes of the cartilage-matrix deficiency (cmd) mouse is due to a 7-bp deletion in exon 5, resulting in a premature stop codon and no aggrecan product (Watanabe et al., 1994). A second mutation within the same locus and generating a similar phenotype, cmd ^bc , has been identified as the complete loss of exon 2 to 18, resulting in a significantly shortened mRNA and production of no aggrecan core protein (Krueger et al., 1999). In a landmark study, a novel transgenic mouse line (Tg COL2A1-ACAN) expressing a chick ACAN coding sequence driven by the mouse Col2A1 promoter has enabled the generation of cmdbc/cmdbc; Tg (COL2A1-ACAN) rescue embryos (Lauing et al., 2014). Robust re-expression of aggrecan in rescue embryos reversed the defects in different skeletal elements to varying degrees, most notably the reappearance of a hypertrophic zone and production of Col2a1 and Col10a1 in the limb growth plate. As well, transgene expression in rescue mice restored: i) Sox9 expression in resting and proliferative zones similar to wild type; ii) an increase in Ihh mRNA production in more chondrocytes in the pre-hypertrophic region; iii) relatively normal expression of Ptch1, the receptor of IHH within the bone marrow near the chondro-osteo junction, perichondrium and proliferative zones similar to wild type; iv) strong re-expression of Fgfr3, which encodes the receptor for negative regulators of chondrocyte proliferation such as FGF9 and FGF18 in the proliferative and early hypertrophic zones of the growth plate; all these features closely resemble those found in wild-type embryos. Taken together, the data obtained from RT-PCR, immunochemistry and mRNA in situ analyses confirm that the presence of aggrecan in the growth plate ECM is fundamental to maintaining normal expression and spatial localization of the essential signaling molecules that regulate chondrocyte organization, morphology, and maintenance during growth plate development (Lauing et al., 2014) (Figure 3).

These cited examples clearly present a requirement for CSPGs as demonstrated by mutations in the core protein, which lead to reduced or total absence of CSPGs. However, the complexity of CS/DSPGs and potential interplay with signaling molecules may also be due to interactions involving the long, linear GAG chains of repeating disaccharide units, as well as the contribution of sulfation components of proteoglycans. GAG chain initiation for CS, DS, HS, and heparin begin with addition of Xyl to a serine hydroxyl embedded in a specific core protein peptide sequence (Bourdon et al., 1987; Krueger et al., 1990), catalyzed by the chain-initiating enzyme xylosytransferase (Kearns et al., 1993; Schwartz, 1995). CS/DSPG GAG chain synthesis continues with addition of two galactose (Gal) residues and a GlcA residue, catalyzed by unique-glycosyltransferases (Schwartz and Rodén, 1974; Schwartz and Roden, 1975; Schwartz, 1976). In contrast to the common linkage region structures shared by most proteoglycans, their structural diversity and functional complexity derives from the long linear unbranched GAG chains comprised of unique repeating disaccharide units, which are then substituted with O- or N- linked sulfate groups. GAG chains engage in regulation of biological processes by interacting with various ligands; however, very little is known about these interactions since structural analysis of GAGs is difficult (Song et al., 2021).

Lastly, the impact of sulfation molecular diversity and patterning of CS/DS chains on signaling have also been investigated. Sulfation is particularly influential in GAG cross talk either indirectly by regulating protein folding via steric hindrance, exclusion or recruitment, or directly through electrostatic interactions that often are sequence specific. In the skeleton, sulfation plays two main roles: to generate osmotic swelling pressure which enables cartilage to withstand compressive loads and to foster direct cell-proteoglycan interactions with specific growth factors or signaling molecules. Interestingly, sulfation patterns change with maturation of cartilage (Bayliss et al., 1999) with an increasing ratio of CS-6 to CS-4 sulfated GAG chains and a diminished sensitivity to TGFβ (Hickery et al., 2003). Similarly, changes in sulfation patterns greatly influence skeletal development and maintenance by altering interactions with systemic soluble factors (IHH, PTH, FGFs, TGFβ and BMPs) (Klüppel et al., 2005), verifying that imbalance in GAG sulfation can modify the functioning of these signaling pathways. As mentioned, several sulfotransferases are involved in the 4- and 6- sulfation of GalNAC units and the GlcA unit. Examples of altered GAG sulfation involving sulfotransferases that cause abnormalities have been identified for both: mutations in chondroitin-6-sulfotransferase-1 (C6ST-1) which are associated with chondrodysplasia and progressive spinal involvement (Thiele et al., 2004), while mice deficient in chondroitin-4-sulfotransferase (C4ST-1) exhibit a more severe chrondrodysplasia. Detailed analysis of the mutant growth plate showed abnormal CS localization, chondrocyte differentiation and orientation, and strong up-regulation of TGFβ signaling with concomitant down-regulation of BMP signaling (Klüppel et al., 2002; Klüppel et al., 2005).

In addition to sulfotransferase-caused signaling defects, limiting the sulfate substrate for the sulfotransferases, phosphoadenosine phosphosulfate (PAPS), also leads to chondrodystrophies in mice (Orkin et al., 1976; Schwartz et al., 1978; Kurima et al., 1998) and humans (Faiyaz ul Haque et al., 1998). The brachymorphic (bm) mouse model (Sugahara and Schwartz, 1979; Sugahara and Schwartz, 1982a; Sugahara and Schwartz, 1982b; Sugahara and Schwartz, 1982c) has a mutation in the PAPSS2 gene which encodes PAPS synthetase 2 (PAPSS2), one of two isoforms in mammals that catalyze the synthesis of the universal sulfate donor (PAPS) (Kurima et al., 1998). At birth, mice are normal size but as development proceeds bm mice exhibit a 50% reduction in limb length, 25% reduction in axial skeleton and a normally organized growth plate but with a reduction in all zones (Schwartz et al., 1978). Aggrecan (Acan) and Col10a1 mRNA expression were comparable in wild type and bm mutants. In contrast, using a set of antibodies with specificity for all functional sulfate epitopes, immunohistochemistry revealed reduction in CS-4 and CS-6 epitopes and an increase in the CS-0 epitope in the bm growth plate EMC, compared to wild type. In contrast, N-sulfated HS showed comparable staining in wild type and bm growth plate. These data were verified by FACE and ³⁵S-sulfate incorporation experiments; only a reduction of sulfate incorporation into CSPGs of the predominantly CS-4 species and no change in HS-sulfate content in bm cartilage was observed; establishing the bm mouse as an excellent model for investigating the role of under-sulfated CS interactions with signaling molecules during cartilage development (Cortes et al., 2009).

Analysis of growth plate signaling showed that the PTHrP receptor (Pthr1) was expressed at high levels in the pre-hypertrophic zone in both bm and wild type. In contrast, Fgfr3 and Ihh (expressed in the pre-hypertrophic zone) and its receptor patched (Ptch1) expressed in the proliferative zone exhibited decreases in mRNA levels in bm cartilage by three methods, mRNA in situ, RT-PCR, and immunohistochemistry. In particular, IHH protein was not uniformly distributed between chondrocytes in bm samples, rather a restricted diffusion pattern characterized by protein aggregation was observed. The abnormal IHH distribution was verified by crossing bm mice with LacZ Ptch ± mice and determining the ratio of Gli activator (Gli1/Gli2) to Gli repressor (Gli3) to measure IHH pathway activation (Hilton et al., 2005). Since, a major function of IHH is to regulate chondrocyte proliferation, cell division was assessed. Significant decreases in BrdU-incorporation were observed, especially in the distal proliferative zone which correlates with the region of restricted IHH diffusion and decreased PTCH1 activation, verifying a decrease in cell division due to a disruption in IHH signaling in the under-sulfated bm growth plate (Cortes et al., 2009).

As with most previous studies on reciprocal interactions between signaling factors and CSPGs, the results are compelling, but not biochemically definitive. This ultimate goal was accomplished by three direct approaches. First, quantitative binding curves between IHH-alkaline phosphatase (AP) fusion protein and HS and CS GAG chains (with CS-4, CS-6, and unsulfated CS-0 motifs) showed a gradient of binding affinity (Kd) and binding capacity (Bmax) in order: HS, CS-4, CS-6, CS-0. Since CS-4 is the predominant species in postnatal cartilage and the binding affinity and capacity is higher for the CS-4 to CS-0 motif, a reduction in CS-4 is commensurate with abnormal IHH signaling. Secondly, to demonstrate that IHH interacts with CS specifically and does so through the IHH N-terminal Cardin-Weintraub motif, this motif was mutated which resulted in complete loss of binding to both HS and CS chains, suggesting that the interaction between IHH and CS is primarily mediated through this motif. Lastly, a direct interaction between CSPG and IHH-AP was demonstrated by quantitative immunoprecipitation with a specific aggrecan antibody, demonstrating that the major cartilage CSPG, aggrecan, directly interacts with IHH. Taken together, the biochemical and genetic evidence suggest a biological mechanism whereby undersulfated CSPGs result in restricted IHH diffusion through the ECM leading to a reduction in chondrocyte proliferation, which significantly impacts skeletal growth in the bm mutant (Cortes et al., 2009).

In addition to providing the most definitive evidence to date that molecular interactions occur between signaling factors and CSPGs, these landmark studies extend our understanding of the biological consequences of these interactions. First, the severe-to-mild spectrum of chondrodystrophies observed in CSPG-deficient models correlates directly with the location of the mutations in the CSPG synthetic pathway. Absence of aggrecan core protein (nanomelic chick and cmd mouse) leads to lethal phenotypes (Li et al., 1993; Krueger et al., 1999; Schwartz and Domowicz, 2002), whereas GAG chain addition (Pug) or sulfation (bm) models present with milder chondrodyplasia phenotypes (Cortes et al., 2009; Wilson et al., 2012; Lauing et al., 2014; Mis et al., 2014). Secondly, these studies provide a rational for the observations in complex ECMs consisting of more than one proteoglycan. In the cartilage matrix, CSPG is the predominant proteoglycan and contains a large number of CS chains per core protein (Krueger et al., 1990), therefore requiring more PAPS to sulfate the CS chains. In contrast, HSPGs contain fewer HS chains to be sulfated (Knox and Whitelock, 2006). However, HS sulfotransferases have higher affinity for PAPS and therefore result in preferential sulfation of HS chains even if PAPSS2 levels are reduced. Furthermore, the bm phenotype, in which only CSPG sulfation is reduced, is opposite of HS synthesis mutants. In particular, in the Ext1 gene trap mutant, reduction in HS results in an increased range of HH signaling (marked by increases in Ptch1 and Pthrp mRNA), increased chondrocyte proliferation and expansion of the proliferative zone (Koziel et al., 2004). In another study, additional HH binding sites were found using structural approaches (Whalen et al., 2013) that allowed multimerization of HSPGs close to the cell membrane needed for interaction with its receptor, while CSPGs, that are more broadly distributed in the ECM and have a lower affinity for HH, establish formation of the HH gradients, which may expand several cell-lengths from the site of production (Cortes et al., 2009) (Figure 1B). Thus, both CSPGs and HSPGs function as IHH modulators, and in concert, influence long-range HH signaling in the ECM growth plate. Third, it is important to highlight that temporal changes in GAG levels or composition are also critical to the mechanistic consequences in growth plate development. For example, reduction in GAGs during the formation of the growth plate (i.e., nm, cmd, pug) leads to accelerated maturation of chondrocytes, while reduction in GAGs in the mature growth plate (i.e., bm) leads to changes in morphogen distribution and altered rate of cell division. Lastly, two other mutant mouse models, a Golgi PAP phosphatase (Frederick et al., 2008) and nucleotidase Jaws/Bpnt2 (Sohaskey et al., 2008) both show only under-sulfated CSPGs and severe chondrodysphasias. All these findings suggest that sulfated CSPGs function in IHH signaling processes independent of HSPGs, and illustrate how bidirectional communication processes are especially important for regulating cell differentiation during normal tissue development (Gjorevski and Nelson, 2009; Clause and Barker, 2013).

As just summarized, many of the major developmental signaling pathways acting on growth plate cell populations function directly and indirectly through CSPGs, which also reciprocally influence the activity of these signaling pathways. As well, defects in CSPG-GAG metabolism has the potential to disrupt the function of essential regulators and is likely a major underlying mechanism for abnormal skeletogenesis progression (Alliston, 2010).

Because of the complexity of the ECM with multiple physical, biological, and chemical interactions involving temporal control of signaling molecule networks, it is challenging to recreate these environments experimentally. As well, growth factors and morphogens are intrinsically unstable, while the ECM is both an active participant and is needed for dimensionality, as the examples in this review have shown. Thus, innovative biomaterial design and tissue engineering strategies are required involving: i) autologous or xenographic cells from the tissue to be formed; ii) signaling molecules which provide instruction for expressing a desire phenotype; and iii) synthetic scaffolds that hold the cells together and shape the tissue formation (Bason et al., 2018). Basically, the goal is to recapitulate the embryonic development and patterning process. Because of the prevalence of skeletal injuries, especially in the pediatric population 15–30% of pediatric skeletal injuries involve the growth plate (Shen et al., 2020), and because injury often results in replacement of cartilage by bone which precludes additional skeletal length growth (Gigante and Martinez, 2019), the growth plate is a highly desirable target for repair. However, in order to successfully re-engineer cartilage tissue, the natural characteristics of the growth plate, i.e., gradients of cell states, composition of ECM, position and function of growth regulators and mechanical properties must be replicated; the growth plate still remains an active model for studying tissue engineering strategies (Wang et al., 2021). Examples include: i) use of various cell types, i.e., bone marrow mesenchymal stem cells (BMSCs) or chondrocytes; ii) and various growth factors (TGFβ, IGF1, and FGF2) (Chen et al., 2020; Wei et al., 2020); iii) as well as different scaffolds composed of natural or synthetic material (Abdollahiyan et al., 2020), all with varying results. Mesenchymal stem cells (MSCs) are widely used in engineering of cartilage due to their capability for self-renewal and their ability to secrete multiple growth factors (Gultekin et al., 2020). As well, there is an influx of MSCs to the injured growth plate site, suggesting that MSCs are naturally vital to the repair process (Zhou et al., 2004). Several studies have shown that MSCs can be derived from multiple sources (Uder et al., 2018), with different regenerative potentials (Maheshwer et al., 2021). Other studies have used autologous chondrocytes which prevent bone formation, build the desired columnar structure and avoid immune rejection (Jin et al., 2006; Tomaszewski et al., 2014; Boopalan et al., 2019). Advances also continue to be made in developing three dimensional cultures that successfully retain the chondrogenic potential (Chow et al., 2011), as well culturing chondrocytes on synthetic hydrogels prior to seeding has been shown to lead to synthesis of Sox-9, aggrecan and collagen, which accumulate over time (Chang et al., 2018). As mentioned, manipulating the microenvironment by addition of chondrogenic-factors (TGFβ1, FGF-2, IGF-1, etc.) stimulates chondrogenesis and synthesis of ECM components (Thielen et al., 2019) (see previous sections). A major improvement in the regeneration process is the introduction of 3D printing technology, which allows different parts of the scaffold to have distinct porosity and mechanical properties, thus more faithfully recapitulating a natural cartilage growth plate (Shaw et al., 2018). In sum, to maintain viable cells, preserve growth factor stability, develop biocompatible as well as degradable natural (or synthetic) scaffolds is an extremely active research area in the field of growth plate regeneration summarized in Wang et al. (2021). Furthermore, many of the concepts are also shared with the bone regeneration field where appropriate combinations of scaffolding and seeding cells with growth factors are also being developed to engineer missing pieces of bone lost due to genetic malformations, trauma, tumors or infections (Perez et al., 2018; Koons et al., 2020; Alonzo et al., 2021). Although results continue to move in promising directions, there are still many challenges and unsolved problems that need to be resolved to benefit clinical application, as might be expected for recapitulating such a complex system such as the skeletal growth plate.

Decades of studies have identified the hierarchical ECM-directed creation of the skeletal growth plate, which involves differentiation and growth of chondrocytes, positioning of cells, matrix and regulators into a highly integrated, bidirectional and temporally orchestrated process. As well, the reciprocity between ECM components, transcription factors, and signaling molecules that coordinate their expression has been revealed by the plethora of chondrodysplasias due to mutations in these regulatory molecules as well as proteoglycans and their biosynthetic enzymes that disrupt growth plate development and maturation summarized in Guasto and Cormier-Daire (2021). However, a more comprehensive molecular understanding is required to fully understand how chondrogenesis and growth plate expansion are regulated with such exquisite precision. As well, more detailed structural information to define the requisite structural interactions between CSPGs and pathway modulators are necessary to develop novel ECM/signaling paradigms, in order to reverse or ameliorate skeletal pathology.