Joyce A.M. Wootton

Local and remote matrix responses to chondrocyte-laden collagen scaffold implantation in extensive articular cartilage defects.

Chondrocyte-laden collagen scaffolds were evaluated in extensive cartilage defects in an equine model. Arthroscopic techniques were used to implant a chondrocyte-collagen culture product in 15-mm defects in the lateral trochlear ridge of the femoropatellar joint of 12 horses. Ungrafted control defects were formed in the opposite joint. Groups of six horses were terminated at 4 and 8 months after implantation and the repair sites, adjacent cartilage, and remote cartilage within each femoropatellar joint examined biochemically. Eight months following surgery the relative proportions of type II collagen in grafted and ungrafted defects, determined using the ratio of cyanogen bromide cleavage products alpha 1(II)CB10/alpha 2(I)CB3,5, were not significantly different (31.57 +/- 2.76% and 26.88 +/- 2.76%, respectively). Aggrecan content was significantly improved in grafted defects (85.61 +/- 6.51 and 74.91 +/- 10.31 micrograms/mg dry weight). Cartilage surrounding grafted defects also showed improved maintenance of cartilage glycosaminoglycan content. Thus, chondrocyte grafting in collagen scaffold vehicles improved the aggrecan content in extensive cartilage defects and surrounding normal cartilage. However, given the continued disparity between repair tissue and normal cartilage aggrecan content, and the low proportion of type II collagen in grafted defects, the utility of collagen scaffolds for chondrocyte grafting of large cartilage defects seems limited.

A comparative study of sterol biosynthesis in Annelida

1. 1. The metabolism acid has been studied in marine and terrestrial species from the Phylum Annelida with 14C substrates. 2. 2. Radioactive 3-β-hydroxy sterols were isolated from all polychaetes injected with 2-14C mevalonic acid; one of the labeled sterols found in Nereis diversicolor has been isolated and identified as cholesterol. 3. 3. In contrast, no 14C-3-β-hydroxy sterols were detected in the terrestrial species, Lumbricus terrestris, but a large amount of radioactivity was found in squalene; this species can synthesize squalene, but is unable to cyclize this intermediate to digitonin-precipitable sterols. 4. 4. Similar results were obtained both in the presence and absence of antibiotics.

[26] Detecting radiolabeled lipid-modified proteins in polyacrylamide gels

Publisher Summary This chapter discusses the methods for labeling cells and detecting tritium-labeled proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. The methods are specifically designed for use with fatty acid-modified proteins, most are equally applicable to other lipid modifications. Tritium-labeled precursors are generally best for metabolic labeling of cells owing to the higher specific activity (relative to 14 C derivatives) and lower level of metabolic interconversion. The length of the labeling period can be varied to minimize interconversion. Thus, the fate of exogenously added labeled fatty acids is checked by analysis of protein-bound label. This is usually done by high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC) analysis of fatty acids released by acid hydrolysis of methyl esters produced by alkaline methanolysis. If comparing labeling with the two fatty acids myristate and palmitate, a useful quick check method for labeling specificity is to process samples of total cell lysates on SDS-polyacrylamide gels followed by fluorography.

Canine COL1A2 Mutation Resulting in C-Terminal Truncation of Pro-α2(I) and Severe Osteogenesis Imperfecta

RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the probands RNA had a unique cleavage pattern in the region of COL1A2 encoding the C‐propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991‐3994 (“CTAG”) were replaced with “TGTCATTGG.” The first seven bases of the inserted sequence were identical to nucleotides 4002‐4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse‐transcription polymerase chain reaction (RT‐PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Increased density of pC‐α2(I) suggested comigration with the similarly sized pro‐α2(I) derived from the mutant allele. Furthermore, α‐chains were overhydroxylated and the ratio of α1(I):α2(I) was 3.2:1, consistent with the presence of α1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro‐α2(I) C‐propeptide and confirmed a diagnosis of OI.

Changes in the types of collagen synthesized during chondrogenesis of the mouse otic capsule

We have investigated the temporal relationship between the morphological differentiation of the mouse otic capsule and the pattern of collagen synthesis by mouse otocyst-mesenchyme complexes labeled in vitro. In 10.5- to 12-day embryos the mesenchyme surrounding the otocyst was loosely organized except for a few lateroventral condensations; explants from these embryos synthesized only small amounts of collagen. Collagen synthesis by whole explants increased by more than 50% between 12 and 13 days concomitant with metachromatic staining of the lateral periotic mesenchyme. Cartilage specific type II collagen was the predominant collagen synthesized by these explants as confirmed by SDS-PAGE, densitometry, CNBr cleavage, and V8 protease digestion. This biochemical expression of the cartilage phenotype preceded morphologic recognition of otic capsular cartilage by almost 2 days. Type II collagen synthesis continued to increase and predominate through Day 16 of gestation by which time the otic labyrinth was surrounded by mature cartilage. The minor cartilage collagen chains, 1 alpha, 2 alpha, and 3 alpha, first appeared on different days of gestation. The 1 alpha, and 3 alpha chains were synthesized by explants from 11-day embryos while the 2 alpha chain appeared during Day 13, just before overt differentiation of mature cartilage. These results suggested that the 1 alpha, 2 alpha, and 3 alpha chains may not form heterotrimers containing all three chains and that synthesis of the 2 alpha chain may be associated with stabilization of the cartilaginous matrix. Comparison of these data with the patterns of collagen production by mutant, diseased, or experimentally manipulated inner ear tissues may provide insights into the molecular basis of chondrogenic tissue interactions.