Elsevier

Biomaterials

Volume 27, Issue 34, December 2006, Pages 5821-5827
Biomaterials

EDC cross-linking improves skin substitute strength and stability

https://doi.org/10.1016/j.biomaterials.2006.07.030Get rights and content

Abstract

Collagen-based scaffolds are extensively utilized as an analog for the extracellular matrix in cultured skin substitutes (CSS). To improve the mechanical properties and degradation rates of collagen scaffolds, chemical cross-linking is commonly employed. In this study, freeze-dried collagen-GAG sponges were crosslinked with increasing concentrations of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC; 0, 1, 5, 10, 50 mm). Cross-linking with EDC at concentrations >1 mm was shown to greatly decrease degradation by collagenase up to 21 days. Ultimate tensile strength (UTS) of acellular collagen sponges scaled positively with EDC concentration up to 10 mm. At 50 mm EDC, the UTS decreased dramatically likely due to the brittle nature of the highly crosslinked material. Co-culture of human fibroblasts (HF) and keratinocytes (HK) on these substrates reveals an apparent cytotoxicty of the EDC at high concentrations with reduced cell viability and poor cellular organization in CSS fabricated with scaffolds crosslinked with 10 or 50 mm EDC. From the data gathered in this study, intermediate concentrations of EDC, specifically 5 mm, increase collagen sponge stability and strength while providing an environment in which HF and HK can attach, proliferate and organize in a manner conducive to dermal and epidermal regeneration.

Introduction

Morbidity and mortality of patients with massive burns are closely related to the limited availability of donor sites [1]. Conventional treatments to promote recovery of these patients involve harvesting and grafting split-thickness skin grafts [2], [3], [4], [5]. Unfortunately, harvesting donor skin inflicts additional injury and in severely burned patients sufficient donor sites are not available. Thus, alternative means of skin replacement must be utilized. Viable [2], [6] allodermis, allodermis with autologous cultured keratinocytes [7], [8], [9], and acellular [10], [11] or fibroblast populated collagen sponges [12], [13] have been utilized to promote wound closure. For burns, however, it is commonly accepted that both the epidermis and dermis are required to achieve functional wound closure [14].

Bioengineered skin substitutes are able to generate greater surface area expansion from donor skin than conventional methods [15]. Collagen is commonly used as the scaffolding material for bioengineered skin [16], [17] due to its many advantageous properties including low antigenicity and high growth promotion. Unfortunately, poor mechanical properties and rapid degradation rates of collagen scaffolds can cause graft instability and difficult handling. In addition to suboptimal mechanical properties, native materials have inherent heterogeneity due to variability in source animals and processing conditions, which makes quality control of such scaffolds problematic.

The high rates of degradation and deficient mechanical properties of collagen often fail to meet the requirements of specific applications, consequently limiting the use of collagen-based scaffolds. Cross-linking collagen scaffolds via chemical methods has been widely utilized to slow degradation rates and optimize mechanical properties. Historically, glutaraldehyde (GA) has been the most widely utilized chemical cross-linking reagent [18]. However, GA cross-linked biomaterials have been shown to release toxic monomeric GA upon hydrolyzation of the material [19], [20]. GA crosslinked biomaterials have been reported to exhibit reduced cellular ingrowth in vitro and in vivo [19], [20]; thus alternate reagents have been employed. To overcome problems associated with reagent toxicity, carbodiimides have been used to cross-link collagen because they are members of the zero-length class of cross-linkers. Carbodiimides activate the carboxylic acid groups of glutamic or aspartic acid residues to react with amine groups of another chain, forming amide bonds [18], [21], [22], [23]. Cross-linking with carbodiimides is especially appealing for biological applications as the carbodiimide does not remain in the chemical bond but is released as a substituted urea molecule [24]. Collagen scaffolds crosslinked with 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC) have been shown to possess decreased degradation rates [21], [22], [23], [24], [25], increases denaturation temperature [26], improved the mechanical properties of collagen scaffolds [23], [27] and maintained porous structure of the matrix [28] while supporting the growth of human keratinocytes (HK) [29], smooth muscle cells [30], and fibroblasts [25], [31], [32], [33]. EDC cross-linked collagen has been investigated as a scaffold for dermal replacements [16], [28], [29], [33]. However, thus far no research has been reported on the effect of EDC on cultured skin substitutes (CSS) containing both dermal and epidermal components.

Freeze-dried, lyophilized collagen-GAG matrices have been successfully used, both clinically and experimentally, as scaffolds for CSS [34], [35], [36]. However, a subset of burn patients have cells which produce elevated levels of matrix metalloproteinases which can cause premature degradation of the collagen in CSS, leading to graft failure in vitro [37]. The goal of this study was to investigate the effect of increasing EDC cross-linking concentrations on the biostability, mechanical properties, and tissue morphogenesis of CSS to determine the optimal processing parameters to obtain stable, reproducible, and well organized skin substitutes.

Section snippets

Collagen scaffolds

Acellular collagen scaffolds were prepared via freeze-drying and lyophilization as previously described [15] from comminuted bovine hide collagen (Kensey Nash; Exton, PA) and chondroitin-6-sulfate (GAG) (Sigma; St. Louis, MO) except without chemical cross-linking with GA [38]. Briefly, bovine collagen powder was solubilized in 0.5 m acetic acid and co-precipitated with GAG to yield a final concentration of 0.6%wt/vol. The co-precipitate was cast into sheets, frozen, lyophilized and physically

Effect of cross-linking on physical and mechanical properties

Histological images reveal that the average pore area in the collagen scaffolds was not dramatically altered by cross-linking. The freeze drying process produced inherently heterogeneous sponges (Fig. 1) thus pore area varied depending on their location within the scaffold. The pore area analysis indicated that the mean pore area tended to increase with increasing EDC concentration (Fig. 2), and the control and 50 mm group were statistically different from each another.

In addition to preserving

Discussion

Physical characterization of control and cross-linked collagen sponges reveals that cross-linking produces no considerable alterations to scaffold morphology but distinct changes to scaffold stability, mechanical properties and cellular organization. Chemical cross-linking of collagen has been used for several years to improve scaffold stability [28], [31], [32], [33], [48] and the data collected here support these findings. Increases in EDC concentration associated with greater resistance to

Conclusion

The stability and strength of collagen scaffolds used for tissue engineering must satisfy their intended biomedical needs. Cross-linking of collagen sponges with EDC provides a means of easily tailoring a scaffolds biostability and mechanical properties. These techniques may be used to increase CSS quality and ease of handling in general and more specifically in cases where patient cells produce elevated levels of matrix metalloproteinases [37]. Based on these results, intermediate

Acknowledgments

HMP thanks the Shriners Hospitals for Children for a Post Docotoral Research Fellowship and Grant #8450 that supported these studies. The authors also thank Dr. David Witte within the Department of Pathology at the Cincinnati Children's Hospital Medical Center for the use of their scanning electron microscopy facilities.

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