Changes in the extracellular matrix of the autogenous patellar tendon graft after posterior cruciate ligament reconstruction: a biochemical study in sheep.

The left knee joints of female German sheep were operated, replacing the posterior cruciate ligament by the central third of the patellar tendon. After 2, 6, 16, 26 and 52 weeks the graft of the operated leg as well as the contralateral central third of the patellar tendon and the posterior cruciate ligament were dissected and used for biochemical analysis. The total glycosaminoglycan content in grafts increases within the first year up to 5 fold and is higher than that of the patellar tendon, but is lower than that of the cruciate ligament. The distribution pattern of glycosaminoglycans differ in the posterior cruciate ligament and the patellar tendon. In cruciate ligament the main components are chondroitin sulfate (76%) and hyaluronan (15%). In the patellar tendon a higher portion of dermatan sulfate (approx. 16%) was found, next to 52% chondroitin sulfate and 22% hyaluronan. During the examination time the grafts show changes in the concentration and the distribution pattern of glycosaminoglycans. The chondroitin sulfate content increases during the experimental period from 1.4 +/- 1.2 mumol/g dry weight (d.w.) to 8.7 +/- 2.9 mumol/g d.w. After 1 year the chondroitin sulfate content in the graft does not differ significantly from that of the cruciate ligament. In the grafts the concentration of chondroitin (non sulfated) increases after 6 weeks up to 1.3 +/- 0.6 mumol/g d.w. in comparison to the value after 2 weeks (0.2 +/- 0.1 mumol/g d.w.) and also in the other groups (16, 26 and 52 weeks) it remains significantly increased. After 1 year the dermatan sulfate content in the graft has increased up to the fifth-fold compared to the value after 2 weeks and is higher than in the patellar tendon and in the cruciate ligament. In graft the hyaluronan content (1.0 +/- 0.4 mumol/g d.w. does not differ significantly in the groups 2, 6, 16, 26 and 52 weeks after operation, but in all five groups it is lower than in the cruciate ligament (2.4 +/- 1.0 mumol/g d.w.). Dermatan and heparan sulfate are not or only little detected in all three tissues. The distribution pattern of glycosaminoglycans in graft shows chondroitin sulfate and dermatan sulfate being the major parts of glycosaminoglycans with a slight increase of these components in the different groups during the experimental period. Hyaluronan makes up to 24 +/- 5% of the whole content of glycosaminoglycans after 2 weeks and decrease to 8 +/- 1% after 52 weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

[Tissue proliferation by thrombocyte growth factor]. / Gewebsproliferation durch Thrombozytenwachstumsfaktor.

An essay for determining the growth stimulating activity of human platelets has been established. With the aid of this system a mitogenic factor from crude platelet extract was isolated. Besides a stimulation of DNA-synthesis an enhanced synthesis of collagen and glycosaminoglycans is induced by the platelet growth factor.

[Glycosaminoglycans in Dupuytren contracture]. / Glykosaminoglykane in der Dupuytren'schen Kontraktur.

Connective tissue from specimens of Dupuytren's disease showed higher contents and altered distribution patterns of glycosaminoglycans. Increasing chondroitin sulphate concentrations as well as increasing proportions of 6-sulphated galactosamines were found to be the characteristic changes.

[Biochemical aspects of chronic rheumatic inflammation]. / Biochemische Aspekte der chronisch-rheumatischen Entzündung)

Morphological phenomena in rheumatoid arthritis are closely correlated to the biochemical aberrations of connective tissue metabolism. Both, morphological and biochemical analysis of the altered tissue portions demonstrate evidence for uncontrolled proliferation resp. metabolism similar to human and experimental malignoma. The present state of biochemical knowledge in this field permits to describe the metabolic state of the afflicted tissue by means of enzyme activity and substrate pattern and to draw some conclusions in respect to the pathogenesis of inflammatory processes involved in chronic rheumatic diseases.

Metabolism and proliferation of cultured fibroblasts from specimens of human palmar fascia and Dupuytren's contracture. The pathobiochemistry of connective tissue proliferation, II.

Cell Cultures from 11 Dupuytren's contracture and 6 normal palmar fascia specimens were established. The rates of sulphated glycosaminoglycan, collagen and DNA synthesis by means of incorporation of labelled precursors ([35S]sulphate, [3H]proline, [3]thymidine) as well as the growth characteristics of the cell lines of both healthy and diseased were compared. The incorporation rates of [35S]sulphate and [3H]proline were found to be significantly higher in Dupuytren than in healthy palmar fascia-deriving cell lines. In contrast, no differences in cell growth or DNA synthesis could be demonstrated. The abnormal capacity to synthesize sulphated glycosaminoglycans and collagen is attributed to a permanent modulation of cell characteristics which can be propagated into cell culture.

A rare genetically determined variant of psuedocholinesterase in two German families with high plasma enzyme activity.

Activity of pseudocholinesterase (acylcholine-acyl-hydrolase) elevated up to four times has been detected in sera of members of two German families. The catalytic concentrations of the pseudocholinesterase of the afflicted members of both families (male and female) varied between 4800 U/l and 10 200 U/o (acetylthiocholine iodide substrate). The pseudocholinesterase of the propositi exhibits isoenzyme separation patterns in polyacrylamide electrophoresis as well as in electrofocussing which are different from those of pseudocholinesterase from normal persons. No differences could be seen as regards the Km of substrates or the inhibition by dibucaine, fluoride or succinyldiocholine.

The isolation and activity of growth-stimulating factors from human platelets.

Optimal conditions for determining the mitogenic activity of platelet growth factor in a homologous system (human palmar fascia fibroblasts/human platelet growth factor) were established. With the aid of this system it was possible to test the active fraction from crude platelet extract following affinity chromatography on heparin-Sepharose, chromatofocusing and molecular sieve filtration. This fraction has an isoelectric point of pH greater than 9 with a molecular mass of about 12000 daltons and reacts with beta-thromboglobulin antiserum. The addition of protease inhibitors proved essential for the isolation of the active growth factor, in order to prevent proteolytic degradation to a biologically inactive substance that is immunologically identical to beta-thromboglobulin. In addition to stimulation of DNA synthesis, the isolated growth factor induces enhanced synthesis of collagen and glycosaminoglycans in fibroblasts in vitro.

A comparative study of the activity of lysosomal and main metabolic pathway enzymes in tissue biopsies and cultured fibroblasts from Dupuytren's disease and palmar fascia. On the pathobiochemistry of connective tissue proliferation, I.

Activities of ten main metabolic pathway enzymes and seven lysosomal enzymes were determined in specimens from human normal palmar fascia and Dupuytren's contracture. The activities of the enzymes tested are 2-3 times higher in fresh specimens of Dupuytren's contracture. There are no differences in the activity distribution patterns of both these specimens, or in the absolute activities calculated in relation of the glyceraldehyde-3-phosphate dehydrogenase activities. With the exception of adenylate kinase and pyruvate kinase, the activities of main metabolic pathway enzymes based on the DNA content showed significantly lower activities in Dupuytren's contracture tissue than in palmar fascia. Lysosomal enzymes exhibit no significant differences of activity in the respective specimens. However, the lysosomal enzyme activities of cultured fibroblasts are lower than the corresponding activities from tissue specimens. The enzyme activities per DNA content in cultured fibroblasts are 10-50 times higher than in tissue specimens. The enzyme activities in cultured fibroblasts decrease with age or density of the cells in culture. The increased metabolic activity of the diseased tissues in Dupuytren's contracture is due to the higher cell content of the afflicted portions of the tissue, but individual enzymes show no qualitative changes in activity and there are no increases of enzyme activity per cell (DNA).

[A sensitive method for the analysis of the glycosaminoglycan distribution pattern, applied to 9 intervertebral discs of one human spine]. / Eine empfindliche Methode zur Bestimmung von Glykosaminoglykanverteilungsmustern, angewendet auf 9 Bandscheiben einer menschlichen Wirbelsäule.

A method has been established for analysing the glycosaminoglycan distribution pattern in small specimens from intervertebral discs and other connective tissue materials. The procedure is based on the consecutive digestion by the specific enzymes hyaluronate lyase, chondroitin sulfate lyase AC and ABC. The quantitative determination of the resulting disaccharides was achieved by high performance liquid chromatography with a detection limit of less than 0.1 nmmol. The undigested heparan- and keratansulfate were separated from each other by thin layer chromatography and quantified by the colorimetric determination of uronic acid and hexosamine, respectively. Correlation of distribution patterns obtained by this method with those obtained by ion exchange chromatography (Dowex 1 X 2) is not possible without the additional enzymatic characterization of the ion exchange chromatographic fractions. The method was applied to 9 discs of one human spine (anulus fibrosus and nucleus pulposus separately) with the following results: 1) there were no systematic changes to be seen in the distribution patterns with respect to the location of the discs in the spine, and 2) the galactosaminoglycan/keratansulfate ratio was higher in the anulus fibrosus than in the nucleus pulposus.

In-vitro culture of human chondrocytes from adult subjects.

An agarose gel matrix was utilized to grow chondrocytes from human donors of various ages in cell culture. The chondrocytes produced the pericellular matrix characteristic for such cells and synthesized collagen type II as well as glyco-saminoglycans. The latter exhibit the typical distribution pattern of the respective articular cartilage matrix. The electron-microscopic appearance of the cultured chondrocytes closely resembles that of chondrocytes in sections of the original cartilage.

The distribution of unsulphated and sulphated glycosaminoglycans in palmar fascia from patients with Dupuytren's disease and healthy subjects.

Eighty specimens from 20 patients with Dupuytren's disease and 7 biopsies of healthy palmar fascia were analysed for their glycosaminoglycan isomer patterns with a combined enzymatic/HPLC method. The diseased portions of palmar fascia tissue were characterized by elevated total glycosaminoglycans together with a relative increase in the sulphated fractions. The macroscopic stages of nodules, bands and unaffected tissue could be classified very well by multivariate statistical analysis on the basis of their glycosaminoglycan patterns. The biochemical analysis provided evidence of the pathological process even in those specimens that did not yet show any clinical symptoms of the disease.

High performance liquid chromatographic assay of disaccharides and oligosaccharides produced by the digestion of glycosaminoglycans with chondroitin sulphate lyases.

In high performance liquid chromatographic procedures hitherto described, SiO2, NH2 and RP columns have been used for the analysis of disaccharides produced by the digestion of glycosaminoglycans with the chondroitin sulphate lyases AC and ABC. The use of a potent anion exchanger offers the following advantages over these columns: superior separation characteristics for non-sulphated disaccharides, and improved column performance, coupled with more stable analytical conditions. Elution with dilute saline solutions permits separation of the two non-sulphated disaccharides from chondroitin and hyaluronate. The sequential application of chondroitinase AC and ABC permits the determination of hyaluronate, the chondroitin sulphate isomers and the dermatan sulphate isomers by high performance liquid chromatographic separation of the products of enzymatic hydrolysis. In a previously described method, hyaluronate lyase was used for the determination of hyaluronate. It has been found, however, that omission of the hyaluronate lyase step results in superior accuracy in the high performance liquid chromatographic separation of the non-sulphated disaccharides. The enzymatic analysis of human articular cartilage glycosaminoglycans has repeatedly yielded a fraction which is not digestable by chondroitinase AC, but is completely digestable by chondroitinase ABC. More extensive characterization has disclosed that this fraction differs structurally from chondroitin sulphate. Enzymatic characterization indicates that it should presumably be assigned to dermatan sulphate.