Recombinant human type II collagen as a material for cartilage tissue engineering.

PURPOSE: Collagen type II is the major component of cartilage and would be an optimal scaffold material for reconstruction of injured cartilage tissue. In this study, the feasibility of recombinant human type II collagen gel as a 3-dimensional culture system for bovine chondrocytes was evaluated in vitro. METHODS: Bovine chondrocytes (4x106 cells) were seeded within collagen gels and cultivated for up to 4 weeks. The gels were investigated with confocal microscopy, histology, and biochemical assays. RESULTS: Confocal microscopy revealed that the cells maintained their viability during the entire cultivation period. The chondrocytes were evenly distributed inside the gels, and the number of cells and the amount of the extracellular matrix increased during cultivation. The chondrocytes maintained their round phenotype during the 4-week cultivation period. The glycosaminoglycan levels of the tissue increased during the experiment. The relative levels of aggrecan and type II collagen mRNA measured with realtime polymerase chain reaction (PCR) showed an increase at 1 week. CONCLUSION: Our results imply that recombinant human type II collagen is a promising biomaterial for cartilage tissue engineering, allowing homogeneous distribution in the gel and biosynthesis of extracellular matrix components.

The effect of cross-linking time on a porous freeze-dried collagen scaffold using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a cross-linker.

Collagen is a widely studied natural polymer for use as a scaffold material for various tissue engineering applications. Untreated collagen, however, has a fast biodegradation rate and low mechanical strength. Additionally, collagen cross-linking has been studied extensively and different cross-linking agents and methods have been explored. Although glutaraldehyde (GA) is the most convenient and traditional chemical agent currently used for this purpose, it nevertheless poses potential cytotoxicity. Therefore, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is widely being studied as well, due to its characteristic of being a zero-length cross-linker that does not remain in the collagen's structure. Nevertheless, cross-linking with EDC can be implemented in several ways. In this study, two methods of cross-linking with EDC, prior to and post freeze-drying, were examined for freezedried collagen scaffolds. Four different collagen concentrations between 0.3 and 2.0 w% were inspected and different cross-linking methods were examined by a differential scanning calorimeter (DSC) to determine the collagen's denaturation temperature (Td). Furthermore, the water uptake abilities of the scaffolds were tested in phosphate buffered saline (PBS) and the scaffolds' pore structure was examined with a scanning electron microscope (SEM). As assumed, the DSC measurements demonstrated that collagen Td values increased with increasing collagen concentration. With lower collagen concentrations, the cross-linking post freeze-drying enhanced the Td values, but with higher collagen concentrations, the Td values were increased only with cross-linking prior to freeze-drying. The water uptake measurement showed increased water uptake ability when cross-linking post freezedrying. The pore sizes of the different collagen scaffolds were between 50 and 120 mum.

Quantification of Pro alpha 1(I) collagen mRNA in skin biopsy specimens: levels of transcription in normal skin and in granuloma annulare.

The synthesis of type I collagen, the major component of the skin, is known to be modulated in aging and in various skin diseases and treatments. In vivo analysis of type I collagen expression, however, is difficult because of the low cell density of the dermis and the small amount of RNA obtainable from skin biopsy specimens. We present here a quantitative polymerase chain reaction method for the quantification of pro alpha 1(I) collagen mRNA in skin punch biopsy specimens. The targeted mRNA and a synthetic RNA as an internal standard were co-amplified together with the same primers. Collagen synthesis was found to decline after birth, so that the amount of pro alpha 1(I) collagen mRNA in the skin of 5- to 58-y-old donors was 17-37% of that in fetal skin. Slot-blot hybridization also indicated that the amount of pro alpha 1(I) collagen mRNA was much lower in adult skin than in fetal skin. In samples from lesional skin of two granuloma annulare patients, the number of pro alpha 1(I) mRNA molecules was increased 4- or 5-fold compared with values from nonlesional skin of the same patients. The method presented is a highly sensitive polymerase chain reaction application, requiring only very small amounts of total RNA.

Expression of mRNAs for lysyl oxidase and type III procollagen in cultured fibroblasts from patients with the Menkes and occipital horn syndromes as determined by quantitative polymerase chain reaction.

The Menkes syndrome and the occipital horn syndrome are two X-linked recessively inherited disorders characterized by abnormalities in copper metabolism. These abnormalities are associated with a reduction in the activity of lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme that initiates the crosslinking of collagens and elastin. We report here that the amount of lysyl oxidase mRNA, as studied by Northern blotting, and the number of lysyl oxidase mRNA molecules per picogram of RNA, as determined by a quantitative PCR method, were decreased in three cultured skin fibroblast lines from patients with the Menkes syndrome and two from patients with the occipital horn syndrome compared with four control cell lines. The decreased lysyl oxidase activity found in these disorders thus appears to be a least in part due to a pretranslational mechanism. No decrease was found in the number of the beta-actin mRNA molecules in the Menkes cell lines, but rather a slight increase, whereas a decrease was found in these molecules in the occipital horn cell lines. An additional abnormality found in the Menkes cell lines was a significant increase in the number of mRNA molecules for type III procollagen in two of the three cell lines investigated. The present and previous data indicate that the Menkes syndrome may involve several abnormalities in the expression of genes for connective tissue proteins.

Quantitative polymerase chain reaction of lysyl oxidase mRNA in malignantly transformed human cell lines demonstrates that their low lysyl oxidase activity is due to low quantities of its mRNA and low levels of transcription of the respective gene.

Lysyl oxidase (EC 1.4.3.13), an extracellular copper amino oxidase, initiates the cross-linking of collagens and elastin by catalyzing oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues. We developed here a polymerase chain reaction (PCR) method for the quantification of lysyl oxidase mRNA in which a synthetic RNA is used as an internal standard for coamplification with the targeted mRNA. The amount of lysyl oxidase mRNA when studied by Northern blot analysis and the number of lysyl oxidase mRNA molecules when determined by the quantitative PCR method were found to be markedly low in various malignantly transformed cell lines relative to control cell lines, quantitative PCR indicating values of about 2-10% of those in the controls. No difference was found in the number of beta-actin mRNA molecules between the transformed cells and the controls. Nuclear runoff experiments indicated that most if not all of the decrease in the number of lysyl oxidase mRNA molecules can be explained by diminished transcription of the respective gene.

Structure of the human lysyl oxidase gene.

Lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme, initiates the crosslinking of collagens and elastin by catalyzing oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues. We report here that the human lysyl oxidase gene is about 15 kb in size and consists of seven exons. Transcription is initiated at one major site and four minor sites, and the first exon consists of 273 bp of untranslated sequences (calculated to the major site) and 631 bp of translated sequences, which accounts for about half of all the translated sequences of the gene. The seventh exon, on the other hand, codes for only the last codon of amino acid 416 and for amino acid 417, which are followed by the translation termination codon and the 3' untranslated sequences. Exons 2-6 vary in size from 96 to 157 bp, and the introns from 331 bp to about 3.5 kb. The 5' flanking region contains a TATA-like sequence at -30 relative to the major transcription initiation site and a CCAAT motif at -109. The 5' flanking region and the downstream sequences present in the first exon and first intron contain altogether five possible binding sequences for Sp1, six for AP-2, one for AP-1, three for PEA3, three for MEP-1, and three CCCTCCC motifs, all of which may be involved in the regulation of the expression of the gene.

Major rearrangements in the alpha 5(IV) collagen gene in three patients with Alport syndrome.

The gene coding for the alpha 5 chian of type IV collagen (alpha 5(IV) collagen), which maps to Xq22, is a candidate gene for the X-linked dominant disease Alport syndrome (AS). Using three cDNA clones, covering the 3' end of the alpha 5(IV) collagen gene, 3 of 38 patients have been identified with mutations in this gene. Each of these patients shows a gross rearrangement of DNA: a deletion of at least 35 kb, an insertion/deletion event involving approximately 25 kb, and a duplication of at least 35 kb of DNA.

Molecular cloning of human lysyl oxidase and assignment of the gene to chromosome 5q23.3-31.2.

Lysyl oxidase (EC 1.4.3.13) initiates the crosslinking of collagens and elastin by catalyzing oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues. We report here on the isolation and characterization of cDNA clones for the enzyme from human placenta and rat aorta lambda gt11 cDNA libraries. A cDNA clone for human lysyl oxidase covers all the coding sequences, 230 nucleotides of the 5' and 299 nucleotides, of the 3' untranslated sequences, including a poly(A) tail of 23 nucleotides. This cDNA encodes a polypeptide of 417 amino acid residues, including a signal peptide of 21 amino acids. Sequencing of two rat lysyl oxidase cDNA clones indicated six differences between the present and the previously published sequence for the rat enzyme [Trackman et al. (1990) Biochemistry 29: 4863-4870], resulting in frameshifts in the translated sequence. The human lysyl oxidase sequence was found to be 78% identical to the revised rat sequence at the nucleotide level and 84% identical at the amino acid level, with the degree of identity unevenly distributed between various regions of the coded polypeptide. Northern blot analysis of human skin fibroblasts RNA indicated that the human lysyl oxidase cDNA hybridizes to at least four mRNA species; their sizes are about 5.5, 4.3, 2.4, and 2.0 kb. Analysis of a panel of 25 human x hamster cell hybrids by Southern blotting mapped the human lysyl oxidase gene to chromosome 5, and in situ hybridization mapped it to 5q23.3-31.2.(ABSTRACT TRUNCATED AT 250 WORDS)