Novel Freeze-Drying Methods to Produce a Range of Collagen–Glycosaminoglycan Scaffolds with Tailored Mean Pore Sizes

Abstract

• to copy, distribute, display, and perform the work.• to make derivative works. Under the following conditions:• Attribution -You must give the original author credit.• Non-Commercial -You may not use this work for commercial purposes.• The pore structure of three-dimensional scaffolds used in tissue engineering has been shown to significantly influence cellular activity. As the optimal pore size is dependant on the specifics of the tissue engineering application, the ability to alter the pore size over a wide range is essential for a particular scaffold to be suitable for multiple applications. With this in mind, the aim of this study was to develop methodologies to produce a range of collagen-glycosaminoglycan (CG) scaffolds with tailored mean pore sizes. The pore size of CG scaffolds is established during the freeze-drying fabrication process. In this study, freezing temperature was varied (À108C to À708C) and an annealing step was introduced to the process to determine their effects on pore size. Annealing is an additional step in the freeze-drying cycle that involves raising the temperature of the frozen suspension to increase the rate of ice crystal growth. The results show that the pore size of the scaffolds decreased as the freezing temperature was reduced. Additionally, the introduction of an annealing step during freeze-drying was found to result in a significant increase (40%) in pore size. Taken together, these results demonstrate that the methodologies developed in this study can be used to produce a range of CG scaffolds with mean pore sizes from 85 to 325 mm. This is a substantial improvement on the range of pore sizes that were possible to produce previously (96-150 mm). The methods developed in this study provide a basis for the investigation of the effects of pore size on both in vitro and in vivo performance and for the determination of the optimal pore structure for specific tissue engineering applications.

A biomimetic multi-layered collagen-based scaffold for osteochondral repair

“…In addition, a collagen-only single layer scaffold fabricated using the standard -40ºC freeze-drying recipe was used as a control in this study [34]. This collagen-only control was selected in order to allow the effect of adding components such as Col2, HyA and HA to be determined.…”

“…Digital images were captured at a magnification of 10x using a microscope (Eclipse 90i, Nikon, Japan) and a digital camera (DS Ri1, Nikon, Japan). Pore size analysis was carried out using a MATLAB (MathWorks Inc, MA, USA) based Pore Topology Analyser programme previously described [34]. The programme converts the digital images into binary form and calculates the average pore size based on the best fit elliptical lengths generated by the software.…”

“…The compressive modulus was defined as the slope of a linear fit to the stress-strain curve over 2-5% strain. [34,35].…”

Development of a gene-activated scaffold platform for tissue engineering applications using chitosan-pDNA nanoparticles on collagen-based scaffolds

“…Gas was removed from the slurries using a vacuum pump prior to freeze-drying (Advantage EL, Vis-Tir Co., Gardiner NY) to a final temperature of either -10°C (CHyA) or -40°C (Collagen and CHA) using a previously optimised freeze-drying profile [33]. The different freeze-drying temperatures used determine the resulting pore size with larger pores (~300µm) forming in scaffolds freeze-dried at -10°C compared to -40°C (~120µm) [40]. The scaffolds were crosslinked dehydrothermally (DHT) at 105 °C for 24 h at 0.05 bar in a vacuum oven (Vacucell 22; MMM, Germany) [41], followed by chemical cross-linking using a mixture of 6 mM N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 5.5 mM N-Hydroxysuccinimide (NHS).…”

Understanding the effect of mean pore size on cell activity in collagen-glycosaminoglycan scaffolds

“…[20][21][22] In a very recent study in our laboratory, which utilized improved technical capability of our freeze-drying system and introduced a novel annealing step during lyophilisation, we have been able to further expand the range of mean pore sizes produced in the CG scaffolds from 96-151 µm up to 85-325 µm. 23 We then investigated the effect of this new expanded range of scaffolds on initial cell attachment followed by migration and proliferation by monitoring cellular activity up to 7 days post-seeding (as opposed to 48 h in the earlier study 6 ) to see whether the pattern of specific surface area affecting initial cell adhesion as seen in the previous studies would continue as cells proliferated. 24 The results provide a possible insight into why there are conflicting reports in the literature on the optimal scaffold pore size for bone tissue engineering.…”