Preparation and characterization of a novel humanized collagen III with repeated fragments of Gly300-Asp329.

Recombinant human collagens have attracted intensive interest in the past two decades, demonstrating considerable potential in medicine, tissue engineering, and cosmetics. Several humanized recombinant collagens have been produced, exhibiting similar characteristics as the native species. To get insight into the structural and bioactive properties of different parts of collagen, in this study, the segment of Gly300-Asp329 of type III collagen was first adopted and repeated 18 times to prepare a novel recombinant collagen (named rhCLA). RhCLA was successfully expressed in E. coli, and a convenient separation procedure was established through reasonably combining alkaline precipitation and acid precipitation, yielding crude rhCLA with a purity exceeding 90%. Additionally, a polishing purification step utilizing cation exchange chromatography was developed, achieving rhCLA purity surpassing 98% and an overall recovery of approximately 120 mg/L culture. Simultaneously, the contents of endotoxin, nucleic acids, and host proteins were reduced to extremely low levels. This fragmented type III collagen displayed a triple-helical structure and gel-forming capability at low temperatures. Distinct fibrous morphology was also observed through TEM analysis. In cell experiments, rhCLA exhibited excellent biocompatibility and cell adhesion properties. These results provide valuable insights for functional studies of type III collagen and a reference approach for the large-scale production of recombinant collagens.

Autologous Adipose-Derived Tissue Matrix Part I: Biologic Characteristics.

BACKGROUND: Autologous collagen is an ideal soft tissue filler and may serve as a matrix for stem cell implantation and growth. Procurement of autologous collagen has been limited, though, secondary to a sufficient source. Liposuction is a widely performed and could be a source of autologous collagen. OBJECTIVES: The amount of collagen and its composition in liposuctioned fat remains unknown. The purpose of this research was to characterize an adipose-derived tissue-based product created using ultrasonic cavitation and cryo-grinding. This study evaluated the cellular and protein composition of the final product. METHODS: Fat was obtained from individuals undergoing routine liposuction and was processed by a 2 step process to obtain only the connective tissue. The tissue was then evaluated by scanning electronic microscope, Western blot analysis, and flow cytometry. RESULTS: Liposuctioned fat was obtained from 10 individuals with an average of 298 mL per subject. After processing an average of 1 mL of collagen matrix was obtained from each 100 mL of fat. Significant viable cell markers were present in descending order for adipocytes > CD90+ > CD105+ > CD45+ > CD19+ > CD144+ > CD34+. Western blot analysis showed collagen type II, III, IV, and other proteins. Scanning electronic microscope study showed a regular pattern of cross-linked, helical collagen. Additionally, vital staing demonstrated that the cells were still viable after processing. CONCLUSIONS: Collagen and cells can be easily obtained from liposuctioned fat by ultrasonic separation without alteration of the overall cellular composition of the tissue. Implantation results in new collagen and cellular growth. Collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat and may provide long term results. LEVEL OF EVIDENCE: 5.

Recombinant human collagen for tissue engineered corneal substitutes.

We successfully fabricated transparent, robust hydrogels as corneal substitutes from concentrated recombinant human type I and type III collagen solutions crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). White light transmission through these gels is comparable or superior to that of human corneas. Hydrogels from both type I and type III collagens supported in vitro epithelium and nerve over-growth. While both these biocompatible hydrogels have adequate tensile strength and elasticity for surgical manipulation, type III collagen hydrogels tended to be mechanically superior. Twelve-month post-implantation results of type I recombinant collagen-based corneal substitutes into mini-pigs showed retention of optical clarity, along with regeneration of corneal cells, nerves and tear film. For clinical use, implants based on fully characterized, recombinant human collagen eliminate the risk of pathogen transfer or xenogeneic immuno-responses posed by animal collagens.

Characterization of whole fibril-forming collagen proteins of types I, III, and V from fetal calf skin by infrared matrix-assisted laser desorption ionization mass spectrometry.

Fibril-forming collagen proteins of the types I, III, and V were extracted from fetal calf skin, purified by differential salt precipitation, and analyzed by infrared matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IR-MALDI-TOF-MS). Glycerol was used as liquid IR-MALDI matrix. Noncovalently bound triple helices of the types I and V were detected from the NaCl precipitate. After heating at 43 degrees C for 10 min, resulting in the dissociation of the triple helix, the single alpha-chain subunits were detected. For type I, mass spectra acquired from molecular sieve chromatography fractions revealed the presence of further substructures of dimeric type and of supramolecular complexes up to the tetramer. Triple helices of type III, stabilized by covalent disulfide bonds, were detected from the total protein precipitate also after heat treatment. For type III, even hexamer and nonamer structures with molecular weights close to 600 and 900 kDa were recorded. For comparison, ultraviolet (UV-)MALDI-MS measurements with 2,5-dihydroxybenzoic acid as matrix were carried out with some of the samples. Here, only the single alpha-chains were detected with significantly reduced sensitivity.

Extraction and characterization of collagen with or without telopeptides from chicken skin.

Poultry by-products are not often processed into high-value products. Rather than being transformed into meal for animal feed, a large quantity of chicken skin could be used to produce collagen, which is valued for its unique functional properties. The purpose of this research project was to extract and characterize collagen from chicken skin. Skins were first ground and then were heated to 40 or 60 degrees C to extract the fat. After mechanical separation, the collagen contained in the resulting solid phase was extracted with pepsin or ethylene diamine. Types I and III collagen were then isolated and characterized by SDS PAGE, antigen labeling, determination of tyrosine residues, and transmission electron microscopy. The total collagen content of the skin was recovered from the solid phase following heat treatment at 40 degrees C. Extraction yields varied with the solubilization process: 38.9% of the collagen content in the solid phase was extracted with pepsin and 25.1% with ethylene diamine. Ratios of type I to type III collagen fractionated using NaCl were 74.4:19.8% with pepsin and 62.4:31.7% with ethylene diamine. Characterization tests further revealed the presence of telopeptides solely on ethylene diamine-solubilized collagen. Chicken skin thus appears to be a good alternative source of high-quality collagen.

Transgenic silkworms produce recombinant human type III procollagen in cocoons.

We describe the generation of transgenic silkworms that produce cocoons containing recombinant human collagen. A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroin light chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac vector. Silkworm eggs were injected with the vectors, producing worms displaying EGFP fluorescence in their silk glands. The cocoons emitted EGFP fluorescence, indicating that the promoter and fibroin L-chain cDNAs directed the synthesized products to be secreted into cocoons. The presence of fusion proteins in cocoons was demonstrated by immunoblotting, collagenase-sensitivity tests, and amino acid sequencing. The fusion proteins from cocoons were purified to a single electrophoretic band. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in bulk.

Identification of a pepsin-sensitive type III-like collagen in breast cancer MCF-7 cells.

Electrophoretic analysis of [3H]proline-labeled culture medium proteins of MCF-7 cells revealed the presence of disulfide-bonded, bacterial collagenase-sensitive component which comigrated with pro(alpha)1 chains of type III and type I collagens. However, it was pepsin- and trypsin-sensitive. Within 1 min of pepsin-digestion, a component with a size of alpha1 chain of type I or III collagen was produced which degraded after 5 min of digestion. Similarly, the pepsin-sensitive band was completely degraded by trypsin at 30 degrees C within 5 min. We examined CNBr peptides of the collagenous band and demonstrated that it was alpha1 chain of type III collagen. When MCF-7 cells were cultured in the presence of 2 nM estradiol, a marked increase in the level of collagen secreted into medium was found. The identified proteinase-sensitive type III-like collagen as major protein of extracellular matrix, would be expected to be more susceptible to degradation which might contribute to tumor progression.