Frederick H. Silver

Self-assembly of collagen fibers. Influence of fibrillar alignment and decorin on mechanical properties

Collagen is the primary structural element in extracellular matrices. In the form of fibers it acts to transmit forces, dissipate energy, and prevent premature mechanical failure in normal tissues. Deformation of collagen fibers involves molecular stretching and slippage, fibrillar slippage, and, ultimately, defibrillation. Our laboratory has developed a process for self-assembly of macroscopic collagen fibers that have structures and mechanical properties similar to rat tail tendon fibers. The purpose of this study is to determine the effects of subfibrillar orientation and decorin incorporation on the mechanical properties of collagen fibers. Self-assembled collagen fibers were stretched 0-50% before cross-linking and then characterized by microscopy and mechanical testing. Results of these studies indicate that fibrillar orientation, packing, and ultimate tensile strength can be increased by stretching. In addition, it is shown that decorin incorporation increases ultimate tensile strength of uncross-linked fibers. Based on the observed results it is hypothesized that decorin facilitates fibrillar slippage during deformation and thereby improves the tensile properties of collagen fibers.

Evaluation of Collagen Crosslinking Techniques

The properties of collagen films crosslinked by physical and chemical techniques were compared to the properties of films crosslinked with glutaraldehyde (GTA). Physical techniques studied include exposure to short wave (254 nm) u.v. irradiation and severe dehydration. Chemical techniques studied include immersion of collagen films in aqueous solutions of cyanamide or GTA. Collagen films exposed to combinations of aqueous solutions of cyanamide and severe dehydration had moduli of elasticity, swelling ratios and resistance to bacterial collagenase similar to films crosslinked with GTA. Theoretical calculations based on amino acid composition indicate that approximately seven times as many amino acid residues are capable of forming crosslinks using cyanamide or severe dehydration procedures as compared to GTA crosslinking. In addition, using severe dehydration or cyanamide forms crosslinks involving both amino and carboxyl residues which may allow these procedures to act synergistically. Based on our studies this two-step procedure effectively crosslinks collagen-based biomaterials while the only by-product of this reaction is water-soluble urea. Preliminary biocompatibility studies suggest that this crosslinking procedure may allow for pronounced tissue ingrowth.

Assembly of type I collagen: fusion of fibril subunits and the influence of fibril diameter on mechanical properties.

Structural stability of the extracellular matrix is primarily a consequence of fibrillar collagen and the extent of cross-linking. The relationship between collagen self-assembly, consequent fibrillar shape and mechanical properties remains unclear. Our laboratory developed a model system for the preparation of self-assembled type I collagen fibers with fibrillar substructure mimicking the hierarchical structures of tendon. The present study evaluates the effects of pH and temperature during self-assembly on fibrillar structure, and relates the structural effects of these treatments on the uniaxial tensile mechanical properties of self-assembled collagen fibers. Results of the analysis of fibril diameter distributions and mechanical properties of the fibers formed under the different incubation conditions indicate that fibril diameters grow via the lateral fusion of discrete approximately 4 nm subunits, and that fibril diameter correlates positively with the low strain modulus. Fibril diameter did not correlate with either the ultimate tensile strength or the high strain elastic modulus, which suggests that lateral aggregation and consequently fibril diameter influences mechanical properties during small strain mechanical deformation. We hypothesize that self-assembly is mediated by the formation of fibrillar subunits that laterally and linearly fuse resulting in fibrillar growth. Lateral fusion appears important in generating resistance to deformation at low strain, while linear fusion leading to longer fibrils appears important in the ultimate mechanical properties at high strain.

Mechanical properties of collagen fibres: a comparison of reconstituted and rat tail tendon fibres

This study involves comparison of the mechanical properties of reconstituted collagen fibres with those of collagen fibres obtained from rat tail tendons. Reconstituted collagen fibres were cross-linked in the presence of glutaraldehyde vapour for 2 and 4 d or using a combination of severe dehydration and carbodiimide treatment. Ultimate tensile strengths for reconstituted fibres cross-linked with glutaraldehyde ranged from 50 to 66 MPa while those cross-linked by severe dehydration and carbodiimide treatment had ultimate tensile strengths between 24 and 31 MPa. Rat tail tendon fibres had tensile strengths that ranged from 33 to 39 MPa. These results indicate that high-strength collagen fibres can be reconstituted in vitro and that these fibres may be useful in repair of dermal, dental, cardiovascular and orthopaedic defects.

Regeneration of achilles tendon with a collagen tendon prosthesis : results of a one-year implantation study

We previously reported on the short-term biocompatibility of a reconstituted type-I collagen prosthesis that had been tested in the Achilles tendons of rabbits. Preliminary results indicated that, by ten weeks after implantation, carbodiimide-cross-linked implants had been replaced by neotendon in a manner that was similar to that of autogenous tendon grafts that had been used as controls. Also by ten weeks after implantation, glutaraldehyde-cross-linked collagen implants were encapsulated and appeared to have caused an acute inflammatory response. In the present study, carbodiimide and glutaraldehyde-cross-linked collagen implants and autogenous grafts that served as controls were implanted for fifty-two weeks as a replacement for a three-centimeter section of the Achilles tendon of rabbits. The absence of a crimp in a cross-linked implant and the presence of a crimp in normal tendon and in tendon that formed after an implant had been resorbed made it possible to distinguish between a cross-linked implant and new host tendon that had replaced the implant after it was resorbed. New collagen that had replaced the implant and autogenous (control) tendon graft were compared with normal Achilles tendon with respect to the angle and length of the crimp. The autogenous grafts and the carbodiimide-cross-linked collagen implants had been completely resorbed and replaced by neotendon. The neotendon that was present fifty-two weeks after implantation was similar, but not identical, to normal tendon. In contrast, the glutaraldehyde-cross-linked implant was essentially inert, had not been resorbed, and was surrounded by a capsule of collagenous connective tissue. The neotendon in the capsule was also similar, but not identical, to normal tendon. There were more cells in the capsule than in the autogenous grafts or in the carbodiimide-cross-linked implants. The results of the present study indicate that rapid repair is achieved with a carbodiimide-cross-linked collagenous implant that has a structure and mechanical properties that are similar to those of an autogenous tendon graft and that biodegrades at a similar rate. Prolonged biodegradation of a glutaraldehyde-cross-linked collagenous implant results in formation of a capsule and only limited formation of neotendon.

Development of a reconstituted collagen tendon prosthesis. A preliminary implantation study.

A reconstituted collagen tendon prosthesis was developed and implanted in rabbit Achilles tendons. The prosthesis was prepared by extruding type-I collagen into fibers and crosslinking it either with glutaraldehyde or with dehydrothermal treatment followed by exposure to carbodiimide. A tendon prosthesis was assembled by coating a longitudinal array of the fibers with uncrosslinked collagen. In one leg of the rabbit, the Achilles tendon was replaced with the synthetic tendon; in the contralateral leg of the animal, the tendon was excised, devascularized, and anastomosed as an autogenous graft. The autogenous tendon grafts were seen to be infiltrated centrally by fibroblasts and capillaries ten weeks postoperatively and to have been partially replaced by repair tissue twenty weeks postoperatively. Three weeks after implantation, all collagen implants were noted to have been infiltrated with fibrous tissue. At ten weeks, reorganization of collagenous tissue was observed in and around the prostheses, and the carbodiimide-crosslinked implants had been resorbed and replaced by normal-appearing neotendon. The implants that had been treated with glutaraldehyde were resorbed more slowly and were surrounded by more inflammatory cells, compared with the prostheses that had been treated with carbodiimide. Neotendon in the glutaraldehyde-treated prostheses matured more slowly. When the implants were examined at intervals after the operation, their mechanical properties approached those of fresh tendon. The initial strength of the carbodiimide-treated implants was lower than that of the fresh autogenous grafts. Twenty weeks after implantation, the strength and modulus of the carbodiimide-treated implants approached those of fresh tendon.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of static axial strain on the tensile properties and failure mechanisms of self‐assembled collagen fibers

Collagen fibers form the structural units of connective tissue throughout the body, transmitting force, maintaining shape, and providing a scaffold for cells. Our laboratory has studied collagen self-assembly since the 1970s. In this study, collagen fibers were self-assembled from molecular collagen solutions and then stretched to enhance alignment. Fibers were tested in uniaxial tension to study the mechanical properties and failure mechanisms. Results reported suggest that axial orientation of collagen fibrils can be achieved by stretching uncrosslinked collagen fibers. Stretching by about 30% not only results in decreased diameter and increased tensile strength but also leads to unusual failure mechanisms that inhibit crack propagation across the fiber. It is proposed that stretching serves to generate oriented fibrillar substructure in self-assembled collagen fibers.

Mineral deposition in the extracellular matrices of vertebrate tissues: identification of possible apatite nucleation sites on type I collagen.

The possible means by which type I collagen may mediate mineralization in normal vertebrate bone, tendon, dentin and cementum as well as in pathological mineral formation are not fully understood. One consideration in this regard is that the structure of the protein is somehow important in binding calcium and phosphate ions in a stereochemical configuration conducive to nucleation of apatite crystals. In the present study, type I collagen, packed in a quarter-staggered arrangement in two dimensions and a quasi-hexagonal model of microfibrillar assembly in three dimensions, has been examined in terms of several of its charged amino acid residues. These included glutamic and aspartic acid, lysine, arginine, hydroxylysine and histidine, whose positions along the three α-chain axes of the collagen molecule were determined with respect to each other. It was found that the locations of these residues specified sites uniquely suited as potential apatite nucleation centers following binding of calcium and phosphate ions. From this analysis, it would appear that type I collagen provides a template of charged amino acid residues that dictates ion binding critical to subsequent nucleation events for mineral formation in vertebrate tissues.

Collagen fibrillogenesis in vitro: Comparison of types I, II, and III

The self-assembly of pepsin-extracted types I, II, and III collagen was studied to determine how differences in the triple-helical structure between collagen types influence in vitro collagen fibrillogenesis. Collagen types I, II, and III were extracted and purified from bovine sources, and were studied in solution by laser light scattering, pH titration, and determination of turbidity-time curves. The molecular weights were between 280,000 and 289,000, while the translational diffusion coefficients and particle scattering factors at 175.5 degrees were consistent with those expected for single collagen molecules. Titration of collagen types I, II, and III between pH 7.0 and 2.0 using HCl indicated that type I collagen had the most titratable carboxylic groups with type II and III having significantly fewer titratable groups. The self-assembly of these collagens was studied in vitro in phosphate-buffered saline. The time course and extent of fibril formation were studied turbidimetrically, and were found to be dependent on collagen type. Apparent rate constants were determined for both the lag and growth phases of fibril formation. The rates of both phases were greater for type III than for type I collagen, with the rates for type II collagen being intermediate. The extent of fibril formation was based on the turbidity per unit concentration (specific turbidity) extrapolated to zero concentration (intrinsic turbidity), which was found to be greater for type I than for type III collagen. Type II collagen had the smallest intrinsic turbidity. The specific and intrinsic turbidity values were consistent with the relative fibril diameters seen in dermis and cartilage by transmission electron microscopy. These observations indicate that helix-helix interactions are important in the regulation of the rate and extent of collagen fibrillogenesis and may be involved in the determination of fibril structure.

Structural and mechanical assessment of developing chick tendon

Abstract Changes in macroscopic stress-strain characteristics were used to assess alterations in tendon microstructure during chick tendon morphogenesis. Five periods of chick development were observed: post fertilization days 14 (stage 40), 16 (stage 42), 17 (stage 43), 21 (stage H0), 23 (stage H2). The shapes of the stress-strain curves, ultimate tensile strength, and strain to failure were used to follow changes in the biomechanical properties during development. The ultimate tensile strength increased from 2.05 MPa (1 MPa= 10 6 N/m 2 ) at stage 40 to 58.05 at 2 days post hatching. A statistically significant increase in measured tensile strength was observed between stage 42 and stage 43. Strain to failure was 13% at stage 40, 22% at stage 42, and 29% for stages 43, H0, and H2. Changes in total tendon collagen were assessed by measuring hydroxyproline content. Hydroxyproline/g of dry weight increased at each successive stage of morphogenesis and ranged from 39.17 mg/g dry weight at stage 40 to 83.41 mg/g dry weight at stage H2. Dry weight increased from 12.91% to 20.19% over the same period. The mean collagen fibril diameter ranged from approximately 45 nm to 62 nm. The fibril distribution was unimodal at all stages, but changed from a tight unimodal (stage 40, 42) to a broad unimodal distribution by stage H2.