ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 235, No. 1, November 15, pp. 178-185, 1984 Collagen Fibrillogenesis in Vitro: Comparison of Types I, II, and III DAVID E. BIRK’ AND FREDERICK H. SILVER Department of Pathology, University of Medic&e and Dentistry of New Jersey, Rutgers Medical School, Piscataway, New Jersey 08854 Received March 28, 1984, and in revised form July 18, 1984 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” 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. o 19% Academic press, inc. Collagen fibril structure and microar- chitecture is important in the determi- nation of tisssue structure and function. In tissues, collagen fibril structure is the consequence of a sequence of events in- volved in fibrillogenesis, which include interactions between the propeptides, the nonhelical telopeptides, and the helical regions. In this paper, we look at helix- helix interactions during fibrillogenesis of pepsin-extracted types I, II, and III col- lagen to determine whether these inter- actions may be partially responsible for differences in fibril diameters. 1 To whom correspondence should be addressed. In vivo, the collagen composition, dis- tribution of collagen types, and fibrillar organization is characteristic of different connective tissues. Type I collagen is found as dense bundles in tissues such as tendon and dermis. These bundles contain large- diameter collagen fibrils that provide me- chanical integrity and determine the strength of these tissues. Dermal type I collagen fibrils have diameters which range from 50 to 100 nm (1, 2). The diameters of type III fibrils in the skin have a narrower range than type I-con- taining fibrils, and a mean diameter of approximately 30 nm (2,3). Type III fibrils are found together with type I fibrils in 0003-9861/84 $3.00 Copyright b 19&4 by Academic Press, Inc All rights of reproduction in any farm reserved. 178