Ovalbumin-BasedPorous Scaffolds for Bone Tissue Regeneration

摘要

Cell differentiation on glutaraldehyde cross-linked ovalbumin scaffolds was the main focus of this research. Salt leaching and freeze drying were used to create a three-dimensional porous structure. Average pore size was 147.84±40.36 μm and 111.79±30.71 μm for surface and cross sectional area, respectively. Wet compressive strength and elastic modulus were 6.8±3.6 kPa. Average glass transition temperature was 320.1±1.4°C. Scaffolds were sterilized with ethylene oxide prior to seeding MC3T3-E1 cells. Cells were stained with DAPI and Texas red to determine morphology and proliferation. Average cell numbers increased between 4-hour- and 96-hour-cultured scaffolds. Alkaline phosphatase and osteocalcin levels were measured at 3, 7, 14, and 21 days. Differentiation studies showed an increase in osteocalcin at 21 days and alkaline phosphatase levels at 14 days, both indicating differentiation occurred. This work demonstrated the use of ovalbumin scaffolds for a bone tissue engineering application. Previous SectionNext Section 1. Introduction Autogenous bone is the most preferred bone grafting material. However, limitations and complications from using autografts include a limited quantity and chronic donor site pain [1, 2]. This has led to the need for an ideal bone graft substitute. An ideal substitute must have enhanced capabilities to reduce or eliminate the need for an autograft altogether [3]and are necessary to provide support, fill voids, and enhance biologic repair of defects. The need for tissue-engineered constructs is increasing and advances in the field have led to the use of scaffolds, cells, and factors to regenerate organs and tissues [3]. The integration of the biological, physical, and engineering sciences will create the new constructs that regenerate and restore the functional state of damaged tissues [4]. Using tissue engineered constructs such as biobased scaffolds as bone graft substitutes has emerged as an approach to regenerate bone. Tissue-engineered constructs, specifically biopolymers, can promote successful bone healing when originating from natural proteins found in the body. Ovalbumin (OA) is being used in this study because it is a biopolymer found in chicken egg whites, has a molecular mass of 45 kDa, and is comprised of 386 amino acids with 10% of the amino acid sequence conserved when compared to human serum albumin. OA is comprised mainly of α-helix and β-sheet, but when introduced to an alkaline environment (pH>7), it transforms to a predominantly β-sheet structure [5]. It can be used to create biocompatible scaffolds that aid in osteoblast adhesion and mineralization into 3D structures [6]. Ovalbumin is more readily available and cheaper ($40/kg, Sigma Aldrich) than using synthetic or other natural biopolymers [7]. Ovalbumin contains nineteen lysines per OA molecule which are necessary for chemical crosslinking with a common agent, glutaraldehyde (GA) [8]. Glutaraldehyde crosslinking is governed by reactions with ε-amino groups of lysines (Figure 1). The GA crosslinking process has been shown to alter cellular response due to its cytotoxicity [9] and may alter osteoblastic responses through modification of the scaffolds [10]. However, it has been previously reported that using GA as a crosslinker for other biopolymer scaffolds such as collagen, alginate, and keratin has not affected biocompatibility [11–13]. Therefore, small concentrations of GA will be used to prevent cytotoxicity, and OA scaffolds may also be created using this method. View larger version: