Reconstruction of a Hard Connective Tissue Utilizing a Pressed Silk Sheet and Type-I Collagen as the Scaffold for Fibroblasts

Abstract

A pressed silk sheet is a new biomaterial composed of a network of numerous cocoon filaments and having excellent mechanical strength and shape stability compared to a cotton-gauze sheet. To reconstruct a hard connective tissue using the silk sheet and type-I collagen as the scaffold for fibroblasts, three different three-dimensional floating culture systems were designed. "On sheet" system: fibroblasts were seeded on the silk sheet coated with collagen and the cell-attached sheet was cultured. "In gel" system: fibroblasts and the silk sheet were co-embedded in a collagen gel and the gel was cultured. "On vitrigel" system: fibroblasts were seeded on both sides of a collagen vitrigel involving the silk sheet and the vitrigel was cultured. The fibroblasts in all culture systems grew and formed disk-shaped connective tissue models involving the silk sheet by 14 days of culture. The "on sheet" and "on vitrigel" models retained a maximum elastic load of about 23 kgf and an ultimate tensile load of about 3.6 kgf, which were almost the same as for the individual silk sheet. However, the "in gel" system showed a low value for the tensile load. Cell damage following application of mechanical stress was lowest in the "on vitrigel" system. These data demonstrated the advantage of the "on vitrigel" system in reconstructing hard connective tissues. Such a novel culture method would contribute to a regenerative medicine for the failure of ligaments, tendons, and other connective tissues.

Predictive performance of the Vitrigel‐eye irritancy test method using 118 chemicals

“…In addition, it possesses excellent transparency and permeability of protein with high molecular weight and consequently the various studies utilizing it as a cell culture scaffold advances so well (2004, 2007a, 2007b, 2007c). We established a preparation method of a corneal epithelium model utilizing an air–liquid interface culture system that facilitates induction of layering rabbit corneal epithelial cells cultured on the CVM scaffold (Takezawa et al ., 2008).…”

Heat Shock-Induced Three-Dimensional-Like Proliferation of Normal Human Fibroblasts Mediated by Pressed Silk

“…On the other hand, silk fiber has recently been studied as a scaffold for tissue engineering because of its excellent biocompatibility and bioabsorbability and its low level of inflammatory potential [15–18]. A pressed silk sheet composed of cocoon filaments is a valuable scaffold that can facilitate 3D culture of fibroblasts [11]. More NHDFs adhered to the pressed silk than to HA, and a 3D-like proliferation pattern was formed more rapidly on pressed silk than on HA.…”

“…Pressed silk that consists of a network structure of numerous cocoon filaments each about 15 μm in diameter and with pore size axes shorter than 200 μm has recently been developed [11]. Cocoon filaments spread uniformly on a pressing machine were moistened and then pressed at a high temperature of 120 ºC to reagglutinate a protein of sericin covering a core protein of fibroin in order to yield a pressed silk sheet.…”

Scaffolds for tendon and ligament tissue engineering

“…However, gel-based systems lack mechanical properties required to replace or regenerate an injured tendon/ ligament, and are usually used only as a method of cell delivery. They have also been used, alone or in combination with cells, to repair partial tendon/ligament defects (Juncosa-Melvin et al 2006a;Hankemeier et al 2007), and combined with fibrous or spongy scaffolds to constitute hybrids scaffolds Juncosa-Melvin et al 2006b,c;Takezawa et al 2007). A plastic compression technique has also been used to rapidly process collagen gels into dense and strong matrices that could be better suitable for TE applications (Brown et al 2005).…”

“…Fibrous scaffolds are often combined with gel systems such as fibrin or collagen gel as a media for cell delivery Juncosa-Melvin et al 2006b,c;Takezawa et al 2007). However, the gel component is usually poorly integrated with the fibrous component, rendering the composite inherently unstable in mechanically dynamic environments like the knee joint.…”