Test – Development of tooth germ (answers)

1. Cell phenotype that is absolutely necessary for tooth formation:

A. epithelial B. mesenchymal

C. both epithelial and mesenchymal D. none of them

2. The first mineralized structure during tooth development:

A. predentin B. mantle-dentin C. intertubular dentin D. peritubular dentin

3. The borderline of enamel organ facing preodontoblasts:

A. inner enamel epithelium B. outer enamel epithelium C. papilla

D. folliculus

E. secretory ameloblasts

2. 1.2. Fibers and extracellular matrix of hard tissues –

Gabor Varga

The development of hard tissues starts with the synthesis of their organic extracellular matrix by specialized cells (osteoblasts, cementoblasts, odontoblasts and ameloblasts). This protein rich basic structure has an important role both in the initiation and the completion of mineralization. The main component of hard tissues is collagen. Although important in the building of the basic structure collagen does not itself influence the formation of the first crystallites. There is a significantly smaller amount of non-collagenous proteins (glycoproteins, phosphoproteins, proteoglycans, serum proteins) and these are the components that are responsible for the initiation, facilitation, modification and even for the inhibition of mineralization.

Figure 1.14. Figure 1. – Extracellular matrix of hard tissues

The protein content of the enamel secreted by epithelial cells differs considerably from that of the connective tissues that constitute most hard tissues. To read about enamel protein content in detail see the chapter on amelogenesis.

Figure 1.15. Figure 2. – Most important protein components of bone and dentin

The matrix of connective tissue consists mainly of collagen. This is not a single molecule but rather the name of a family of molecules. Type I collagen is the dominant type in the mineralized tissues. Its structure is built up from three left-coiled helices that are organized in a right-coiled triple helix.

Figure 1.16. Figure 3. – Collagen – three polypeptide chains forming a rope

All molecules classified as collagens have a triple helix region. The tissue distribution of different collagens is widespread.

Figure 1.17. Figure 4. – Types and distribution of collagen

Collagen is produced as a water-soluble precursor. During the conversion of procollagen to collagen, both the C- and N- termini of the molecule are cleaved in the extracellular space. Collagen consists of three independent polypeptide chains, called the α-chains, each comprising 1056 amino acids. Each α-chain has a left-handed helical structure and when they coil around each other they form a right-handed super helix that is similar to a rope. The amino acid composition of the chains has a characteristic periodicity; it contains repeated glycine-X-Y tripeptides, where X and Y are amino acids different from glycine. The residues in the X and Y positions are usually proline and lysine. There are typically 338 Gly-X-Y repeated triplets in the α-chains. The length of the triple helix (consisting of the three α-chains) is 297 nm. The glycine is the smallest amino acid and its predominance in the chains allows tight coiling. Proline and hydroxy-proline are limited in their ability to rotate in contrast to other amino acids therefore these amino acids enhance the stability of the triple helix structure.

Collagen molecules combine to form fibers with periodic cross-bonds.

Figure 1.18. Figure 5. – Structure of procollagen

The collagen chains are synthetized in the rough endoplasmic reticulum and then undergo posttranslational modifications, hydroxylation and glycosylation. The coiling into a triple helix is catalyzed by specific enzymes. The prosthetic groups of proline hydroxylase and lysine-hydroxylase both contain ferrous ions (Fe²⁺) and use oxygen. Ascorbic acid (vitamin C) is also essential for the function of both enzymes as a cofactor. This is the reason why vitamin C deficiency causes scurvy. An abnormal polypeptide sequence of collagen causes the hereditary disease osteogenesis imperfecta (also known as brittle bone disease).

The conversion of procollagen to collagen happens in the extracellular space and is facilitated by specific proteolytic enzymes. When the cross binding is completed the collagen becomes insoluble.

Figure 1.19. Figure 6. – Overview of collagen biosynthesis

Figure 1.20. Figure 7. – Hydroxylation during collagen biosynthesis

Figure 1.21. Figure 8. – Stages in collagen synthesis – rope formation

The stability of collagen is really important. Most of the proteolytic enzymes are unable to cleave it because of its cross-bonds and insolubility. Therefore, special metalloproteases (collagenases) are needed to degrade this molecule. These enzymes can cleave all the three chains at the same time.

Figure 1.22. Figure 9. – Enzymatic cleavage of collagen by mammalian collagenases

Non-collagenous proteins are known to interact with the surface charges of the hydroxyapatite crystals and thus influence the mineralization. There are many similarities but also significant differences between these proteins. Because of their multiple negative charges, these biomineralization proteins are intrinsically disordered proteins, even more so than transcription factors. The disordered structure and its flexibility can play a role in the rapid formation and strength of the interactions due to the fast conformational changes of these proteins.

Figure 1.23. Figure 10. – Interactions between hydroxyapatite crystals and ionic substances

Figure 1.24. Figure 11. – Disorder frequency of amino acid chains of proteins

participating in various biological functions

Most of the proteins present in connective tissues are glycoproteins. These contain one or more carbohydrate group typically sialic acid. Sialoproteins are very acidic and they represent 10% of the non-collagenous proteins of the bone. They contain 20% carbohydrate by weight. The main members of this group, sialoprotein I (osteopontin) and sialoprotein II also contain large amounts of phosphate.

Figure 1.25. Figure 12. – Sialic acid, a major constituent of sialoproteins

The modification by carboxylase in the γ position of glutamate in the peptide chains leads to the formation of γ-carboxyl-glutamate (Gla). This enzymatic reaction requires vitamin K and bicarbonate. Gla groups act as calcium-binding domains. Gla proteins include osteocalcin (OC), bone Gla protein (BGP) and matrix Gla protein (MGP). In vitamin K deficiency Gla proteins are not carboxylated and their incorporation may decrease significantly. However in this case bone structure does not change dramatically, there are only some alterations in the epiphysis. This finding led to recognition that these proteins actually inhibit or at least modulate the calcification. They also slow down the growth of hydroxyapatite crystals in vitro.

Figure 1.26. Figure 13. – Structure of proteoglycans

Proteoglycans. Proteoglycans are conjugates consisting of proteins and glycosaminoglycan prosthetic groups.

Glycosaminoglycans are built up from repeated units of two different sugars. One subunit is a hexosamine (D-galactosamine or glucosamine) that can be sulphated, and the other unit is glucuronic acid or galactose. They contribute to the jelly-like consistency of the matrix and can slow down mineralisation by their calcium binding property. Proteoglycans accumulate in the non-mineralized connective tissue and their amount decreases significantly during the mineralization process.

Figure 1.27. Figure 14. – Formation of γ-carboxyglutamyl residues

In phosphoproteins phosphate groups bind covalently to the peptide chain as phosphothreonine or phosphoserine. The affinity of phosphate ions for Ca²⁺ is very strong and the matrix-bound phosphate has a key role in the initiation of mineralization. In addition they serve as phosphate donors in the process.

Two well-known phosphoproteins in bone are osteopontin and osteonectin. The phosphoprotein of dentine, dentine sialophosphoprotein (DSPP), also known as phosphoforin, only contains phosphoserine.

Figure 1.28. Figure 15. – The most important amino acids in hard tissue

phosphoproteins

Plasma proteins can be extracted from both bone and dentine, where significant amounts of albumin and α2HS glycoprotein can be detected. However the relevance of this phenomenon is not known. The inert collagens do not have a direct role in the mineralization. The non-collageneous proteins mentioned above are primarily structural components that act as the initiators of mineralization or as enhancers or inhibitors of the process. At the same time, numerous peptides and proteins present in small amounts take part in the mineralisation process as transcription and differentiation factors, and as regulators of cell motility or enzymes.

Figure 1.29. Figure 16. – Some possible functions of proteins of hard tissue matrices affecting mineralization

2.1. Test – Fibers and extracellular matrix of hard tissues