Elsevier

Biomaterials

Volume 30, Issue 5, February 2009, Pages 703-710
Biomaterials

The stress relaxation characteristics of composite matrices etched to produce nanoscale surface features

https://doi.org/10.1016/j.biomaterials.2008.10.023Get rights and content

Abstract

Many synthetic and xenogenic natural matrices have been explored in tissue regeneration, however, they lack either mechanical strength or cell colonization characteristics found in natural tissue. Moreover natural matrices such as small intestinal submucosa (SIS) lack sample to sample homogeneity, leading to unpredictable clinical outcomes. This work explored a novel fabrication technique by blending together the useful characteristics of synthetic and natural polymers to form a composite structure by using a NaOH etching process that produces nanoscale surface features. The composite scaffold was formed by sandwiching a thin layer of PLGA between porous layers of gelatin–chitosan. The etching process increased the surface roughness of PLGA membrane, allowing easy spreading of the hydrophilic gelatin–chitosan solution on its hydrophobic surface and reducing the scaffold thickness by nearly 50% than otherwise. The viscoelastic properties of the scaffold, an area of mechanical analysis which remains largely unexplored in tissue regeneration was assessed. Stress relaxation experiments of the “ramp and hold” type performed at variable ranges of temperature (25 °C and 37 °C), loading rates (3.125% s−1 and 12.5% s−1) and relaxation times (60 s, 100 s and 200 s) found stress relaxation to be sensitive to temperature and the loading rate but less dependent on the relaxation time. Stress relaxation behavior of the composite matrix was compared with SIS structures at 25 °C (hydrated), 3.125% s−1 loading rate and 100 s relaxation time which showed that the synthetic matrix was found to be strain softening as compared to the strain hardening behavior exhibited by SIS. Popularly used quasi-linear viscoelastic (QLV) model to describe biomechanics of soft tissues was utilized. The QLV model predicted the loading behavior with an average error of 3%. The parameters of the QLV model predicted using nonlinear regression analysis appear to be in concurrence with soft tissues.

Introduction

Using biodegradable scaffolds that can support and guide the in-growth of cells have been a promising solution to regenerate tissue parts. Scaffolds generated from natural [1] and synthetic polymers or after removing the cellular components from xenogeneic tissues [2] have been used as scaffolds. Since naturally formed matrices such as small intestinal submucosa (SIS) are constrained by large-scale preparations of reliable, and reproducible products [3], forming synthetic matrices from biodegradable polymers has been an attractive alternative [4].

Synthetic polymers such as poly(glycolic acid), poly(lactic acid), PLGA and naturally derived polymers such as gelatin, chitosan and glycosaminoglycans have been explored in forming scaffolds [5]. However, the dearth of biomaterials that could form scaffolds eliciting controlled cellular responses with essential mechanical properties has necessitated search for novel biomaterials. Blending two polymers is an option to develop scaffolds with wide range of physicochemical properties and cellular interactions [5], [6], [7], [8], [9]. Recently, a composite matrix consisting of two porous compartments of chitosan reinforced with a thin membrane of PLGA was reported [10]. The composite was designed to mimic the biological properties, tensile properties and degradation characteristics of SIS. In this configuration, the mechanical and degradation properties can be tailored by selecting appropriate synthetic polymers. Further, biological properties can be tailored independently by altering natural polymer mixture.

Recent advances have shown that the physical properties of the scaffold such as pore size, and void fraction provide cues to guide cell colonization [11], apart from chemical properties such as cell-binding sites necessary for cell attachment. Surface features such as edges, grooves, and roughness also influence cell behavior [12], [13]. For example, PLGA membranes etched to produce nanoscale surface features using NaOH are reported to improve cell adherence and proliferation. Furthermore, cells from various origins can react very differently to changes in architectures, such as pore features and topographies [13], [14].

Cellular activity is also influenced by scaffold stiffness of the substrate [15], [16], [17]. Cells show reduced spreading and disassembly of actin even when soluble adhesive ligands are present in weak gels [18], [19]. However, it is long recognized that majority of the tissues [20] and extracellular matrix elements in the body behave as viscoelastic materials rather than pure elastic materials [21]. Viscoelastic materials store and dissipate energy within the complex molecular structure, producing hysteresis and allowing creep and stress relaxation to occur. Hence, a full description of the mechanical response of materials requires nonlinear viscoelastic behavior. Interestingly, very few studies have been performed to understand the viscoelastic nature of porous structures used in tissue regeneration [22].

The objective of this study was to assess the stress relaxation properties of the composite scaffolds formed using PLGA membrane with and without the etching process, and after incorporating porous structure of chitosan and gelatin. Stress relaxation behavior of the composite scaffold was also compared to the SIS. A quasi-linear viscoelastic model widely used in biomechanics was adapted. These results show significant differences between composite scaffold and SIS in addition to the etching process.

Section snippets

Sources for material

Chitosan (200–300 kDa molecular weight, Mw, 85% DO), Gelatin type-A (300 Bloom) are obtained from Sigma Aldrich Chemical (St. Louis, MO). 50:50 Poly(dl-lactictide-co-glycolide), ester terminated (nominal) with 90–120 kDa molecular weight was obtained from LACTEL absorbable polymers (Pelham, AL). SLS-1-7 × 10 (10 × 7-cm rectangles) were obtained from Cook® Biotech Inc. (W. Lafayette, IN). Apper Ethyl Alcohol, 200 proof, absolute, anhydrous and chloroform was obtained from Pharmaco.

Composite layered scaffold fabrication

The PLGA

Quasi-linear viscoelastic (QLV) model development

The QLV model, first proposed by Fung [24], is commonly used to characterize soft biological tissues [25]. It has the capability of modeling materials with time dependent viscoelastic behavior that undergo large deformations. We need to perform time dependent stress relaxation tests and fit the constants of the function that characterize stress relaxation as well as large deformation. The problem faced by the QLV model earlier was that it was applied to cases where the input ramp strain was

Effect of etching on roughness

To assess the effect of etching on the surface of the PLGA, membranes were analyzed via SEM. These results showed (Fig. 1A–C) significant difference between unetched and 10 min etched samples. After 15 min etching, clear indentations were visible relative to 10 min sample. However, 5 min of etching did not have significant influence on the surface (data not shown) roughness and appeared more like unetched sample. Hence, 5 min etching time was not pursued in subsequent analyses.

Surface

Discussion

This work explored a novel method of generating composite scaffolds with reduced thicknesses using the technique of etching which produces nanoscale surface features for tissue engineering applications. Composite matrices consisting of two porous compartments of chitosan–gelatin reinforced with a thin membrane of PLGA were formed. Porous compartment provides scaffolding for multilayered cell growth, while the membrane layer provides mechanical strength. In this configuration, the mechanical

Acknowledgements

The authors would like to thank Dr. Susheng Tan of the Microscopy Laboratory of OSU for assistance in AFM. Financial support was provided by the Oklahoma Center for Advancement of Science and Technology (HR05-075), National Institutes of Health (1R21DK074858-01A2) and Wentz Foundation for a scholarship that supported JP.

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