(1) Field of the Invention
The present invention relates generally to crosslinked collagen scaffolds. More particularly, it relates to the method of crosslinking and producing collagen-based scaffolds with improved properties using light and photosensitizing reagent, and to the products produced by the method.
(2) Description of Related Art
Collagen is the most abundant protein in the extracellular matrix of human tissue and plays important roles in providing structural support as well as performing other functions in tissue growth and regeneration. Apart from collagen, other types of extracellular matrix components such as proteoglycans, elastin, etc. also play important roles in maintaining tissue structure and function. Producing scaffolds simulating natural tissue is an essential enabling technology in the tissue engineering industry.
Collagen is the best natural biomaterial for tissue engineering because of its close resemblance to nature, and its negligible immunogenecity and excellent biocompatibility. However, unprocessed collagen usually has insufficient mechanical properties for it to be useful in engineering tissues in particular the weight-bearing tissues such as tendons, ligaments, intervertebral discs, etc. Unprocessed collagen is also difficult to manipulate and put sutures through during the implantation process. Further, unprocessed collagen is highly water swellable and is vulnerable to enzymatic digestion and thermal denaturation.
Crosslinking has been used to improve the properties of collagen and therefore is crucial in the tissue engineering industry. Tissue engineering companies have disclosed various methods for crosslinking collagen constructs and scaffolds using chemical methods such as treatment with glutaraldehyde (Cavallaro, U.S. Pat. No. 5,718,012 for “Method of Strength Enhancement of Collagen Constructs”) and physical methods such as lyophilization (Kemp et al., U.S. Pat. No. 5,256,418 for “Collagen Constructs”) to enhance the strength and stability of the structures. Other physical crosslinking methods such as ultraviolet (UV) and gamma irradiation and dehydrothermal treatment have also been reported.
However, both chemical and physical crosslinking methods have encountered problems. Chemical crosslinking of collagen using a reagent such as glutaraldehyde, although efficient, compromises the biocompatibility of scaffolds because the toxic residual chemicals and degradation products induce cytotoxicity and calcification (Simmons D. M. et al., “Evaluation of Collagen Cross-Linking Techniques for the Stabilization of Tissue Matrices”, Biotechnol Appl Biochem. 17 (Pt 1):23-9 (1993)). Physical crosslinking methods are time-consuming (Weadock K. S. et al., “Crosslinking of Collagen Fibers: Comparison of Ultraviolet Irradiation and Dehydrothermal Treatment”, J Biomed Mater Res. 29(11):1373-9 (1995); and Billiar K. et al., “Effects of Carbodiimide Crosslinking Conditions on the Physical Properties of Laminated Intestinal Submucosa”, J. Biomed Mater Res. 56(1):101-8 (2001)), and compromise the stability of scaffolds because UV and gamma irradiation, and the harsh processing conditions in the dehydrothermal treatment method denature the protein (Weadock et al., supra; Billiar et al., supra). As a result, a long-felt need has existed for alternative methods of enhancing the physico-chemical properties of collagen coupled with features such as rapid and efficient processing, nil toxic substances, non-thermal processing and absence of denaturation of collagen.
Previous studies involving light-activated processes to stabilize xenografts (heterografts) such as pericardial tissues, and heart values for transplantation have been reported (Adams A. K. et al., “Crosslink Formation in Procine Valves Stabilized by Dye-Oxidated Photooxidation”, J. Biomed Mater Res, (57):582-587 (2001); Moore M. A. et al., “Stablization of pericardial tissue by dye-mediated photooxidation”, J. Biomed Mater Res, 28(5):611-618 (1994)). These studies were aimed at further stabilizing intact tissues having extracellular matrix protein networks and the intrinsic mechanical properties thereof. The xenograft repair approach differs greatly from that used in tissue engineering, in which tissue-like scaffold structures are built from basic units such as extracellular matrix components, cells and growth-stimulating bioactive factors.
Studies on the crosslinking of collagen proteins in solution using light-activated processes have been reported (Pitts J. D. et al., “New Photoactivators for Multiphoton Excited Three-Dimensional Submicron Cross-Linking of Proteins: Bovine Serum Albumin and Type I Collagen”, Photochem., Photobiol 76(2): 135-144). The average laser power used to create submicron structures ranged up to 1010 W/cm2. Although there was no evidence as to whether the collagen was denatured and whether the processed collagen structures have improved strength and stability, at such high power, collagen protein is very likely to coagulate and become denatured. Therefore, the crosslinking is based on thermal mechanism.
Mechanic, U.S. Pat. No. 5,147,514 for “Process for Crosslinking Collagenous Material and Resulting Products” describes a photooxidative process for crosslinking proteins using photocatalysts in the presence of oxygen. This patent discloses the oxygen dependence of the crosslinking process in that bubbling of air or oxygen, or stirring the reaction mixture vigorously was used to increase the concentration of oxygen. Further, a photocatalyst was used so that the compound did not change before and after the process. This is not the case for the photochemical crosslinking process of the present invention in that the photosensitive reagent participates in the crosslinking process. Moreover, the prior art crosslinking process does not strengthen or stiffen the crosslinked products and therefore cannot solve the major problem, namely, the inadequate mechanical properties of unprocessed collagen used as scaffolds for tissue engineering. Further, the patent does not reveal any intention to produce larger and thicker scaffolds. The thickness of collagen scaffolds to be crosslinked by irradiation is limited because of the depth of penetration of the light source.