Effect of physical crosslinking methods on collagen-fiber durability in proteolytic solutions.

We previously demonstrated that ultraviolet (UV) or dehydrothermal (DHT) crosslinking partially denatured fibers extruded from an insoluble type I collagen dispersion. In this study denaturation effects were evaluated by measuring collagen-fiber sensitivity to trypsin. Shrinkage-temperature measurements and sensitivity to collagenase served as indices of crosslinking. UV or DHT crosslinking increased the collagen-fiber shrinkage temperature, resistance to degradation in collagenase, and durability under load in collagenase. However, in trypsin solutions, solubility was significantly increased for UV (approximately 11%) or DHT (approximately 15%) crosslinked fibers compared with uncrosslinked fibers (approximately 4%). Size-exclusion chromatography indicated that no intact collagen alpha-chains were present in the soluble fraction of fibers exposed to trypsin (MW < 1 kD). Interestingly, UV-crosslinked collagen fibers remained intact an order of magnitude longer (4840 +/- 739 min) than DHT-crosslinked (473 +/- 39 min) or uncrosslinked (108 +/- 53 min) fibers when placed under load in trypsin solutions. These data indicate that mechanical loading during incubation in a trypsin solution measures denaturation effects not detected by the trypsin-solubility assay. Our results suggest that DHT-crosslinked collagen fibers should not be used as load-bearing implants. UV-crosslinked fibers may retain more native structure and should exhibit greater resistance to nonspecific proteases in vivo.

Physical crosslinking of collagen fibers: comparison of ultraviolet irradiation and dehydrothermal treatment.

The strength, resorption rate, and biocompatibility of collagenous biomaterials are profoundly influenced by the method and extent of crosslinking. We compared the effects of two physical crosslinking methods, ultraviolet irradiation (UV) (254 nm) and dehydrothermal (DHT) treatment, on the mechanical properties and molecular integrity of collagen fibers extruded from an acidic dispersion of type I bovine dermal collagen. Collagen fibers exposed to UV irradiation for 15 min had ultimate tensile strength (54 MPa) and modulus (184 MPa) values greater than or equivalent to values for fibers crosslinked with DHT treatment for 3 or 5 days. UV irradiation is a rapid and easily controlled means of increasing the mechanical strength of collagen fibers. Characterization of collagen extracted from the crosslinked samples by dilute acetic acid and limited pepsin digestion indicate that both UV and DHT treatments cause fragmentation of at least a portion of the collagen molecules. Partial loss of the native collagen structure may influence attachment migration, and proliferation of cells on collagen fiberbased ligament analogs. These issues are currently being addressed in our laboratory.

Vascular graft sealants.

Although the use of vascular graft sealants is primarily intended to decrease the porosity of grafts upon implantation, sealants also may serve to passivate polymeric surfaces or act as a temporary scaffold for cell attachment and subsequent graft healing. The safety and efficacy of vascular grafts sealed with albumin, collagen, and gelatin have been established through many preclinical and clinical studies. Such products have been used in clinical practice since the mid-1980s. Any differences in the long-term human healing response to these sealants and the clinical relevance of such differences can be assessed only as the time of implantation increases and the data are evaluated.

Successful radioimmunotherapy for lung metastasis of human colonic cancer in nude mice.

The potential for radioimmunotherapy as an adjuvant treatment for early disseminated colonic cancer was investigated in an experimental lung metastasis model. Nude mice receiving intravenous injection with a suspension of human colonic cancer cells (GW-39) developed multiple (10-100) tumor nodules throughout the lungs, and more than 50% of the animals died of extensive tumor involvement within 5-10 weeks. Groups of eight or nine animals bearing 7-day-old tumor transplants were treated with a single intravenous injection of radioiodinated agents: either 0.15 or 0.30 mCi of whole IgG of the NP-4 murine monoclonal antibody (MAb) against carcinoembryonic antigen (CEA) or 0.15 or 0.30 mCi of whole IgG of Immu-31, an anti-alpha-fetoprotein (anti-AFP) MAb. Treatment of animals with 0.15 or 0.30 mCi of 131I-labeled NP-4 IgG 7 days after injection of tumor cells resulted in survival for 23 weeks after tumor implantation in four of eight and seven of nine animals, respectively. Microscopic examination revealed that over 90% of the lung tumor colonies had no evidence of surviving cells. Animals treated with 0.30 mCi of anti-AFP, an irrelevant MAb, survived 4 weeks longer than controls. Toxicity was evident in four of the 17 animals given 0.30 mCi of NP-4 IgG (specific) or anti-AFP IgG (irrelevant) MAb. These animals died within 1-3 weeks after radioantibody injection, suggesting that death was related to the radiation dose. None of the animals given 0.15 mCi of 131I-MAb died within this period.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a remote radioiodination system for radioimmunotherapy.

This report describes a remote radioiodination system which is inexpensive, easy to assemble, disposable, and capable of radioiodinating curie levels of activity safely. In addition to the safety afforded by this system, an immobilized oxidant and anion exchange resin are used to generate electrophilic iodine and remove free iodine, respectively. Reducing agents are not used and, therefore, when radioiodinating F(ab')2 fragments, degradation does not occur. In contrast, chloramine-T, sodium metabisulfite (CT/SMB) iodinations of F(ab')2 fragments resulted in products with up to 40% Fab' fragments. Radiolabeling yields (65.8% +/- 8.1%) and antibody immunoreactivity (68.8% +/- 8.0%) were not statistically different (p less than 0.001) from those obtained in remote CT/SMB iodinations. The system is currently being used to radioiodinate both IgG and F(ab')2 monoclonal antibodies with up to 450 mCi 131I for clinical radioimmunotherapy trials.

A simple remote system for the high-level radioiodination of monoclonal antibodies.

We have developed a system for the remote control radiolabeling of monoclonal antibodies (MoAb). The system is simple, inexpensive to assemble, and completely disposable. Reagents are pneumatically manipulated into vials containing Iodogen and an anion exchange resin, wherein iodination and separation of bound from free iodine occur, respectively, and sterilized (by filter) into a final vial. Radiolabeling yields of 38.9 +/- 5.5% were consistently produced with 99.3 +/- 0.2% of the isotope bound to the antibody. These results were not significantly different from those obtained in manual iodinations. However, the radiation dose to the hands of the laboratory worker in the remote mode was reduced at least ninefold. The system is presently being introduced for 300 to 400 mCi 131I iodinations of the anti-ovarian cancer MoAb OC 125 in a Phase I radioimmunotherapy trial.

Diffusivity of 125I-labelled macromolecules through collagen: mechanism of diffusion and effect of adsorption.

Diffusion of angiotensin II, albumin and aldolase was studied through collagen membranes with swelling ratios between 4 and 15. The diffusion coefficient was measured from the time-lag for the onset of steady-state flux through the membrane. Binding of macromolecules to collagen was evaluated from the results of sorption studies conducted as a function of macromolecular concentration. Results presented indicate that the diffusion of macromolecules through collagen membrane is slowed by electrostatic and hydrogen bonding between individual macromolecular chains and collagen. The extent of adsorption is increased as the molecular weight of the diffusant increases. Diffusion of water soluble macromolecules through collagen occurs rapidly, suggesting that diffusion occurs through water filled channels as opposed to between collagen molecules. The results of these studies are useful in understanding diffusion through connective tissues and in the design of drug delivery systems based on collagen.