Collagen is the main structural protein in vertebrates. It plays an essential role in providing a scaffold for cellular support and thereby affecting cell attachment, migration, proliferation, differentiation, and survival. As such, it also plays an important role in numerous approaches to the engineering of human tissues for medical applications related to tissue, bone, and skin repair and reconstruction. Currently, the collagen used in tissue engineering applications is derived from animal tissues, creating concerns related to the quality, purity, and predictability of its performance. It also carries the risk of transmission of infectious agents and precipitating immunological reactions. The recent development of recombinant sources of human collagen provides a reliable, predictable and chemically defined source of purified human collagens that is free of animal components. The triple-helical collagens made by recombinant technology have the same amino acid sequence as human tissue-derived collagen. Furthermore, by achieving the equivalent extent of proline hydroxylation via coexpression of genes encoding prolyl hydroxylase with the collagen genes, one can produce collagens with a similar degree of stability as naturally occurring material. The recombinant production process of collagen involves the generation of single triple-helical molecules that are then used to construct more complex three-dimensional structures. If one loosely defines tissue engineering as the use of a biocompatible scaffold combined with a biologically active agent (be it a gene or gene construct, growth factor or other biologically active agent) to induce tissue regeneration, then the production of recombinant human collagen enables the engineering of human tissue based on a human matrix or scaffold. Recombinant human collagens are an efficient scaffold for bone repair when combined with a recombinant bone morphogenetic protein in a porous, sponge-like format, and when presented as a membrane, sponge or gel can serve as a basis for the engineering of skin, cartilage and periodontal ligament, depending on the specific requirements of the chosen application.

Four human genes, two of them encoding the proa1 and proa2 chains of type I procollagen and two of them the two types of subunit of prolyl 4-hydroxylase (4-PH), were integrated into the genome of Pichia pastoris. The proa1 and proa2 chains expressed formed type I procollagen molecules with the correct 2 : 1 chain ratio, and the 4-PH subunits formed an active enzyme tetramer that fully hydroxylated the proa chains. Chains lacking their N but not C propeptides formed pCcollagen molecules with the 2 : 1 chain ratio and, surprisingly, the expression levels of pCcollagen were 1.5-3-fold relative to those of procollagen. Both types of molecule could be converted by pepsin treatment to collagen molecules that formed native-type fibrils in vitro. The expression levels obtained for the pCcollagen using only single copies of each of the four genes and a 2 l fermenter ranged up to 0.5 g/l, indicating that it should be possible to optimize this system for high-level production of recombinant human type I collagen for numerous medical applications.

Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of the 4-hydroxyproline residues that are essential for the generation of triple helical collagen molecules. The vertebrate C-P4Hs I, II, and III are [␣(I)] 2 ␤ 2 , [␣(II)] 2 ␤ 2 , and [␣(III)] 2 ␤ 2 tetramers with identical ␤ subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic ␣ subunit of C-P4H I to analyze its specific functions. The null mice died after E10.5, showing an overall developmental delay and a dilated endoplasmic reticulum in their cells. The capillary walls were frequently ruptured, but the capillary density remained unchanged. The C-P4H activity level in the null embryos and fibroblasts cultured from them was 20% of that in the wild type, being evidently due to the other two isoenzymes. Collagen IV immunofluorescence was almost absent in the basement membranes of the null embryos, and electron microscopy revealed disrupted basement membranes, while immunoelectron microscopy showed a lack of collagen IV in them. The amount of soluble collagen IV was increased in the null embryos and cultured null fibroblasts, indicating a lack of assembly of collagen IV molecules into insoluble structures, probably due to their underhydroxylation and hence abnormal conformation. In contrast, the null embryos had collagen I and III fibrils with a typical cross-striation pattern but slightly increased diameters, and the null fibroblasts secreted fibril-forming collagens, although less efficiently than wild-type cells. The primary cause of death of the null embryos was thus most likely an abnormal assembly of collagen IV.

Type II collagen is the main structural component of hyaline cartilages where it forms networks of thin fibrils that differ in morphology from the much thicker fibrils of type I collagen. We studied here in vitro the formation of fibrils of pepsin-treated recombinant human type II collagen produced in insect cells. Two kinds of type II collagen preparation were used: low hydroxylysine collagen having 2.0 hydroxylysine residues/1,000 amino acids, including 1.3 glycosylated hydroxylysines; and high hydroxylysine collagen having 19 hydroxylysines/1,000 amino acids, including 8.9 glycosylated hydroxylysines. A marked difference in fibril formation was found between these two kinds of collagen preparation, in that the maximal turbidity of the former was reached within 5 min under the standard assay conditions, whereas the absorbance of the latter increased until about 600 min. The critical concentration with the latter was about 10-fold, and the absorbance/microgram collagen incorporated into the fibrils was about onesixth. The morphology of the fibrils was also different, in that the high hydroxylysine collagen formed thin fibrils with essentially no interfibril interaction or aggregation, whereas the low hydroxylysine collagen formed thick fibrils on a background of thin ones. The data thus indicate that regulation of the extents of lysine hydroxylation and hydroxylysine glycosylation may play a major role in the regulation of collagen fibril formation and the morphology of the fibrils.The collagens are a family of extracellular matrix proteins that play a dominant role in maintaining the structural integrity of various tissues. A well coordinated deposition of the components of the extracellular matrix is essential to achieve and maintain their physiological function (for reviews, see Refs. 1-3).Type II collagen is the main structural component of hyaline cartilages and forms their fibrous scaffold, which interacts with various types of proteoglycan. It forms networks of thin fibrils that differ in morphology from the much thicker fibrils of type I collagen, the main fibril-forming collagen in most other tissues (1-3). Many factors such as the presence of various proteoglycans (4 -6) and interactions between collagen types (7-9) influence collagen fibril formation and the architecture of the resulting fibrils formed. Thus the differences in fibril architecture between tissues are not necessarily dependent on differences between the various collagen types themselves. However, fibril formation experiments with purified collagens in vitro have demonstrated that the differences in structure between type II and type I collagen molecules appear to be sufficient to explain many of the characteristic differences between these two types of fibril present in tissues (10). The kinetics for the assembly of type II collagen fibrils in such experiments differed markedly from those for the assembly of type I collagen, and the critical concentration for type II collagen at 37°C was about 50 times greater (10). In addition, the ty...

An efficient expression system for recombinant human collagens will have numerous scientific and medical applications. However, most recombinant systems are unsuitable for this purpose, as they do not have sufficient prolyl 4-hydroxylase activity. We have developed methods for producing the three major fibril-forming human collagens, types I, II and III, in the methylotrophic yeast Pichia pastoris. These methods are based on co-expression of procollagen polypeptide chains with the alpha- and beta-subunits of prolyl 4-hydroxylase. The triple-helical type-I, -II and-III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast cells and could be purified from the cell lysates by a procedure that included a pepsin treatment to convert the procollagens into collagens and to digest most of the non-collagenous proteins. All the purified recombinant collagens were identical in 4-hydroxyproline content with the corresponding non-recombinant human proteins, and all the recombinant collagens formed native-type fibrils. The expression levels using single-copy integrants and a 2 litre bioreactor ranged from 0.2 to 0.6 g/l depending on the collagen type.