## 2 The Role(s) of Non-Collagenous Bone Matrix Proteins in Bone  
### Basics of Non-Collagenous Proteins  
There is a plethora of NCPs in both skeletal and non-skeletal tissues, including bone, dentin, skin, tendon, cornea, and heart. Non-collagenous bone matrix proteins can be broadly classified into three main groups: glycoproteins, \(\gamma\)-carboxoglutamic acid (GLA)-containing proteins, and proteoglycans. In bone tissues, NCPs mostly reside at the interfibrillar and extrafibrillar compartments, i.e., between collagen fibrils or decorating them, as depicted by Figure 2a,b [53]. On a microstructural level, osteepontin (OPN), for instance, can be found within the cement lines and osteon reversal regions in the osteons of trabecular bone, whereas periostin preferentially localizes in the periosteum of bone [54; 55]. NCPs are believed to partake in a wide range of processes relevant for bone formation, growth and remodeling [56; 57]. Therefore, overexpression of, deficiencies in or missense mutations of one or more of these bone matrix proteins can provoke abnormal bone formation, presenting disease-like phenotypes. Deficiencies in certain NCPs, including biglycan and bone sialoprotein, also negatively impact the healing process of fractured bone [58; 59]. Figure 2: Non-collagenous proteins in bone. (**a**) A model depiction of matrix proteins OC–OPN complexes within the structural hierarchy of bone. Not to scale. Figure adapted from [60], with permission from the National Academy of Sciences of the USA. (**b**) Schematic showing that, ultrastructurally, NCPs are co-localized with apatite crystals, binding and bridging the collagen microfibrils. Figure reprinted from [61]. (**c**) Nanoscale localization of the matrix protein OC in human femur using high-resolution TEM: (**i**) OC proteins can be found between hydroxyapatite crystals with variousorientations, distorting the crystal lattice; (**ii**) Fast Fourier transform (FFT) showing characteristic reflections, confirming the presence of osteoclastcin and mineral octacalcium phosphate \"OCP\". Figures reprinted from [62]. (**d,e**) Possible structural roles of OPN and OC: (**d**) Intrafibrillar OPN network, and, similarly, OPN-containing NCP complexes, acting as a \"glue\" by forming sacrificial bonds that dissipate energy upon mechanical extension. Figures adapted from [63], with permission from the American Chemical Society. (**e**) OC-OPN protein complexes contribute to bone toughening under tensile stimulus via the formation of dilatational bands of \(\sim\)100 nm. Figure reprinted from [60], with permission from the National Academy of Sciences. OPN is a glycoprotein with intrinsically disordered regions. The flexibility conferred by this structural disorder and absence of a particular fold, together with its high binding affinity for Ca\({}^{2+}\) ions, means that OPN can intricately template and guide mineral deposition, crystallization, and growth [64]. A recent study by [PERSON] et al. also identified OPN as an essential protein for fibrillogenesis and collagen fibril organization, not just biomineralization, in bone using transmission electron microscopy (TEM) and nanobeam electron diffraction [36]. Osteocalcin (OC) possesses a strong affinity to hydroxyapatite minerals and has been proposed to guide the alignment of apatite minerals on collagen fibrils [65]. Using high-resolution TEM, [PERSON] et al. identified and located OC proteins, approximately 2 nm in diameter, in the form of connected strings attached to the tropocollagen molecules as well as in the form of individual protein globules within the mineral crystal structure (Figure 2c) [62]. Osteonectin (ON), also known as secreted protein acidic and rich in cysteine or SPARC, accounts for 23% of NCPs in bone and is highly expressed in immature bone [66]. This observation highlights its importance in bone formation and the osteonectin-mediated binding of type I collagen to apatite crystals [67].