Connective Tissue VIII. Factors Effecting Collagen Synthesis by Sponge Biopsy Connective Tissue.

Abstract

The in vitro synthesis of collagen has been demonstrated in sponge biopsy connective tissue ( 1 ) , carrageenin granulomata ( 2 ) , rat uterus(3), rat cartilage(4) and rat bone(5) by either conversion of C14-lysine to C14-hydroxylysine or C14-proline to C14-hydroxyproline. Recently, homogenates from chick embryo have been reported capable of converting C14-proline to C14-hydroxyproline (6). Ascorbic acid was reported to be essential in the hydroxylation of proline to hydroxyproline in carrageenin granulomata from guinea pig ( 7,8). Designation of the hydrogen atoms of proline involved in the hydroxylation has been attempted by two groups of workers (8,9), each using both C14-and tritiated-proline; their results are not in agreemen t . Prockop et aZ.(lO), Scharpenseel et aZ.(1 1), and Fujimoto et aZ.( 12), using Ozs as a tracer, have reported that atmospheric oxygen rather than the oxygen of water is required for synthesis of collagen. The metabolism of collagen is still obscure. This study deals with a systematic survey of the op'timal conditions for collagen synthesis in sponge biopsy connective tissue.Methods. Sponge biopsy connective tissue, 2 weeks old, was obtained by the method ofBoucek and Noble( 13). Tissue slices ( 1 .OO g) were incubated in 5 ml Krebs-Henseleit medium( 14) with 1 ml saline containing 1 pc uniformly labelled L-pr01ine-C~~ (Schwarz BioResearch, Inc., specific activity 1.09 pc/ mg or 800 @c/mg) at 37OC, pH 7.35-7.45 under an atmosphere of 95% 02-5% COa, unless otherwise stated. After 6 hours of incubation, the slices were removed, blotted and homogenized in 0.85% sodium chloride containing 0.25% L-proline. The mixture was *This work was supported in part by U.S.P.H.S. grant.centrifuged at 15,000 x g for one-half hour and decanted. The residue was extracted with 0.1 N NaOH overnight and centrifuged at 105,OOO X g for 1 hour. This extraction was repeated once for 2 hours. To avoid the contamination of noncollagenous protein and C14-proline in the incubation medium, the NaCl solution and the base were discarded. Collagen was extracted from base-soluble residue by Fitch's method( 15). The purity of the collagen was indicated by demonstrating an hydroxyproline content of 13%. The collagen samples were hydrolyzed in 6N HCl at 110' for 16 hours in sealed tubes. Aliquots were taken for N2 determination by Kjeldahl digestion and Conway diffusion techniques (16). Radioactivity was counted in a low background (2 counts per minute) gas flow counter with micromil window. Our counting efficiency was 40%. Data were not corrected for counting efficiency. Specific activity was expressed as cpm/mg protein. Hydroxyproline and proline were separated on Whatman #4 paper in 2 solvent systems, 77% ethanol or water saturated phenol in a vapor phase of NHS (0.3% NH4OH) and HCN ( 5 % NaCN). Both solvents were proven to be satisfactory for separation of hydroxyproline from proline. Glutamic acid synthesis was negligible during 6 hours of incubation. The amino acids were eluted from the paper with water. Aliquots wer...

Aging in Connective Tissue: A Dynamic Process*

“…Thus far, our attempts to demonstrate the conversion of proline to hydroxyproline and of lysine to hydroxylysine in acellular systems have met with very little success. However, there are 2 in vitro tissue‐slice methods (18, 19, 22, 23) that can be adequately controlled for the study of various factors that may influence the formation and repair of connective tissue. One of us (KYK) has had considerable experience with connective tissue produced by sponges implanted under the skin of small laboratory animals.…”

“…Changes in the medium itself, both additions and subtractions, produced marked variations in the yields obtained. For example, the complete omission of sodium chloride from the medium reduced the collagen yield to 5 per cent of the control value and the hydroxyproline yield to approximately 1 per cent of the control value (23). The effect of other changes have been reported elsewhere (23).…”

“…For example, the complete omission of sodium chloride from the medium reduced the collagen yield to 5 per cent of the control value and the hydroxyproline yield to approximately 1 per cent of the control value (23). The effect of other changes have been reported elsewhere (23). Some preliminary work with both uterine slices and sponge‐implantation connective tissue indicates that collagen synthesis in vitro can be markedly influenced by hormonal changes in the donor animal.…”

The Lungs in Uremia

“…25 Severe pulmonary fibrosis is not usually a significant clinical problem during life. Acidosis has been shown to modify the composition of lung connective tissue by increasing the proportion of elastin, 44 perhaps by an action on fibroblasts, 45 but the relevance of this to fibrosis in renal failure is not known.…”

Wound Healing: A Review: II. Environmental Factors Affecting Wound Healing

“…Uitto and Prockop30 showed that temporary anoxia may result in the formation of under‐hydroxylated collagen fibers of low mechanical strength. Furthermore, Kao et al31 found that in vitro synthesis of collagen by connective tissue was accelerated with increasing ambient concentrations of oxygen. Another group demonstrated that maturation and cross‐linking of collagen in slices of skin of chick embryos increased at a linear rate when the oxygen concentration in incubators was raised from 20 to 95 volumes per cent 32.…”