Kathryn G. Vogel

The effect of proteoglycans on the morphology of collagen fibrils formed in vitro.

The morphology of collagen fibrils at various times during formation in vitro was quantitatively examined by negative staining and by scanning electron microscopy. The presence of a small dermatan sulfate proteoglycan from bovine tendon (5 micrograms proteoglycan/100 micrograms collagen) resulted in collagen fibrils that were significantly thinner in width at all times by both methodologies. The rate of fibril diameter increase was also retarded by the small proteoglycans, suggesting that they inhibited the lateral aggregation of forming collagen fibrils. Large proteoglycans from cartilage did not produce this effect.

Structural Specialization in Tendons under Compression

Publisher Summary This chapter describes the dynamic characteristic of tendon––the ability of a tendon to modulate its structural and material properties to meet mechanical requirements distinct from the usual need for strength in tension. Tendon performs the mechanical role of transmitting the tensional force generated by contraction of its muscle of origin to the bone upon which it inserts. The histology, biochemistry, and cell biology of regions of the flexor digitorum profundus tendon (deep digital flexor tendon) either subjected to tension or to compression are described in the chapter. The hypothesis that tendon fibroblasts and the extracellular matrix they produce are modulated according to their mechanical requirements is explored. The development of cartilaginous tissue in the region of tendon subjected to compressive forces is genetically programmed. The stimulus to produce large Proteoglycan (PG) is generated by one or more soluble factors that are released under defined conditions and carried systemically to a population of target cells. Many fibroblasts in adult tissue have the capacity to generate a cartilage like tissue.

Characteristics of the In Vitro Interaction of a Small Proteoglycan (PG II) of Bovine Tendon with Type I Collagen

Binding of the small dermatan sulfate proteoglycan of bovine tendon (PG II type/decorin-like) to type I collagen was characterized in an in vitro fibril-forming assay, using native collagen prepared from bovine tendon by acid extraction and radiolabeled proteoglycans synthesized by bovine tendon fibroblasts in culture. Substantial binding to collagen was noted for both intact small proteoglycan and core protein from which the glycosaminoglycan chain was removed. However, binding to collagen was minimal for free glycosaminoglycan chains or large proteoglycans. Binding of the small proteoglycan was optimal at approximately physiological conditions of salt concentration and pH. Scatchard analysis showed a binding affinity constant of 3.3 x 10(7) M-1 with 0.054 proteoglycan binding sites/collagen molecule, when about 0.25-6 micrograms proteoglycan was combined with 100 micrograms collagen. Binding to preformed fibrils of native tendon collagen and to pepsin-treated bovine skin collagen was similar to binding to native tendon collagen. Binding occurred in non-ionic detergents at concentrations up to 1% and once bound, the proteoglycan was not released by washing with up to 2 M NaCl. When both PG I and PG II small proteoglycans were added to collagen, only PG II was bound. This difference is not readily explained by differences in disulfide bond position. These studies indicate a strong, specific interaction between type I collagen fibrils and the core protein of the small (PG II) proteoglycan of tendon.

BIOLOGY OF THE ROTATOR CUFF TENDON

Tendons are complex composite material composed primarily of water, collagen, proteolycans, and cells, designed to transmit tensile loads from muscle to bone. Although rotator cuff tendons differ in many ways from other tendons in the body, a knowledge of basic tendon structure and function is helpful in understanding rotator cuff tendon biology, injury, and repair. In addition to type I collagen, rotator cuff tendons contain small amounts of type III collagen, which play a role in healing and repair. In comparison with other tendons, the increased glycosaminoglycan and proteoglycan content seen in rotator cuff tendons may be adaptive, pathologic, or both. The etiology of rotator cuff pathology is probably related to trauma, aging, and degeneration. As our understanding of these processes increases, we will be able to develop and implement improved preventative and therapeutic interventions for rotator cuff pathology.

Aggrecan in bovine tendon

Large proteoglycans were purified by ion-exchange chromatography, gel filtration and CsCl gradient centrifugation from the compressed and tensional regions of adult bovine deep flexor tendon. Tryptic peptide maps of proteoglycan from the compressed region were very similar to maps of aggrecan from bovine articular cartilage, with evidence for the presence of all fifteen previously identified markers from the G1, G2 and G3 domains. The presence of aggrecan in these samples was confirmed by sequencing the G1 peptide YPIHTPR. The equivalent maps for large proteoglycan from tensional tendon were also consistent with the presence of aggrecan, and this was confirmed by sequencing three marker peptides from each of the G2 and G3 domains. However, G1 marker peptides were conspicuously absent from tensional samples. Northern blots for aggrecan mRNA showed high levels in cells from compressed tendon and articular cartilage. Extended exposure revealed a lower level of hybridization to RNA from tensional tendon as well. The results confirm that aggrecan, which is similar in core protein structure to articular cartilage aggrecan, is the predominant chondroitin sulfate-bearing large proteoglycan of compressed tendon. The results also indicate that aggrecan fragments lacking the G1 domain can account for the small amounts of chondroitin sulfate-bearing large proteoglycan in tensional regions of adult tendon.

Compression loading in vitro regulates proteoglycan synthesis by tendon fibrocartilage

The regulation of proteoglycan synthesis in a fibrocartilaginous tissue by mechanical loading was assessed in vitro. Discs of bovine tendon fibrocartilage were loaded daily with unconfined, cyclic, uniaxial compression (5 s/min, 20 min/day) and the synthesis of large and small proteoglycans was measured by incorporation of [35S]sulfate. All discs synthesized predominantly large proteoglycan when first placed in culture. After 2 weeks in culture nonloaded discs synthesized predominantly small proteoglycans whereas loaded discs continued to produce predominantly large proteoglycan. The turnover of 35S-labeled proteoglycan was not significantly altered by the compression regime. Increased synthesis of large proteoglycans was induced by a 4-day compression regime following 21 days of culture without compression. Inclusion of cytochalasin B during compression mimicked this induction. Autoradiography demonstrated that cell proliferation was minimal and confined to the disc edges whereas 35S-labeled proteoglycan synthesis occurred throughout the discs. These experiments demonstrate that mechanical compression can regulate synthesis of distinct proteoglycan types in fibrocartilage.

Functional morphology of the supraspinatus tendon

Grossly normal supraspinatus tendons were analyzed by stereomicroscope dissection and three-dimensional serial-section reconstruction. Four structurally independent subunits were identified: the tendon proper extended from the musculotendinous junction to approximately 2.0 cm medial to the greater tuberosity. It was composed of parallel collagen fascicles oriented along the tensional axis and separated by a prominent endotenon region. There was no interdigitation of fascicles, and an 18% incidence of fascicle convergence as the fascicles coursed from muscle toward greater tuberosity. The attachment fibrocartilage extended from the tendon proper to the greater tuberosity, consisted of a complex basket-weave of collagen fibers, and stained diffusely with alcian blue. The densely packed unidirectional collagen fibers of the rotator cable extended from the coracohumeral (CH) ligament posteriorly to the infraspinatus, coursing both superficial and deep to the tendon proper. The capsule was composed of thin collagen sheets each with uniform fiber alignment that differed slightly between sheets. These data describe a specialized tendon capable of internally compensating for changing joint angles through fascicles which are structurally independent and can slide past one another. The tendon attachment exhibits a structure adapted to tensional load dispersion and resistance to compression.

Ultrastructure and Proteoglycan Composition in the Developing Fibrocartilaginous Region of Bovine Tendon

Clear distinctions in morphology and proteoglycan composition have been described in regions of adult tendon that pass under bone and are subjected to compressive as well as tensional forces. In this study, developing bovine deep flexor tendon from early fetal stages through 6 months of age was examined biochemically and by light and electron microscopy. Longitudinal collagen fibers were seen in the tensional region of tendon throughout development; whereas a well established network arrangement of collagen fibers with wide interfibrillar spaces was seen in the compressed region by 7 months of fetal age. Collagen fibril diameters of both regions increased with age with the mean diameter in tensional tissue always greater than in compressed tissue. Glycosaminoglycan hexosamine content of the tensional region remained low throughout development (approximately 0.2% of dry tissue weight), but increased in the compressed region from 0.4% of dry weight at the 7-month fetal stage, to 1.0% dry weight at 6 months. Keratan sulfate was not detectable in tensional tendon at any age as measured by inhibition ELISA, but was found in increasing quantities in the pressure bearing region of tendon from young calves. Small proteoglycans predominated in both tensional and compressed regions throughout fetal and early neonatal development, and were of both PG I (biglycan) and PG II (decorin) types. Large proteoglycans represented only a small proportion of total proteoglycans in both regions of fetal tendon. By SDS/PAGE analysis, immunoreactivity, and molecular sieve chromatography, large proteoglycans of fetal compressed tendon were similar to large proteoglycans of adult tensional tendon in that they contained only chondroitin-6-sulfate, with little if any KS, and appeared to be slightly smaller than cartilage large proteoglycans.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hyaluronidase, trypsin, and EDTA on surface composition and topography during detachment of cells in culture☆

Cultured human embryo fibroblasts (HLM18) were labeled with [3H]glucosamine and Na35SO4, and then treated with testicular hyaluronidase, trypsin, or EDTA. Macromolecular material from the surface of these cells was characterized by DEAE-cellulose chromatography and cetylpyridinium chloride precipitation while the associated morphology of cell detachment was studied by phase contrast and scanning electron microscopy. Release of surface glycosaminoglycans by testicular hyaluronidase did not cause cell rounding or detachment. EDTA did not release cell-surface components, but caused cell contraction and detachment morphologically similar to that caused by trypsin. Large amounts of cell-surface glycoproteins and glycosaminoglycans were released by trypsin. From these observations it is concluded that hyaluronic acid is not a principal adhesive agent in the attachment of cells to a substrate. It is suggested that both EDTA and trypsin may have their primary effect upon the cytoskeleton.

Characterization and interactions of a fragment of the core protein of the small proteoglycan (PGII) from bovine tendon

Sequence analysis showed that Staphylococcus aureus V8 protease cleaved the core protein of the small dermatan sulfate proteoglycan of bovine tendon (PGII) on the carboxy side of a glutamic acid residue located 17 amino acids from the N-terminus of the intact molecule. The remaining 40 kDa core protein fragment inhibited collagen fibrillogenesis in an in vitro assay. V8 protease readily generated this fragment in tendon tissue, but it was not released from the tissue during treatment. These results indicate that neither the 17-amino acid N-terminal peptide nor the glycosaminoglycan chain attached to this peptide is required for maintaining the interaction of this proteoglycan with a collagen matrix.