Novel freeze-drying methods to produce a range of collagen-glycosaminoglycan scaffolds with tailored mean pore sizes.

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 (−10 degrees C to −70 degrees C) 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 microm. This is a substantial improvement on the range of pore sizes that were possible to produce previously (96-150 microm). 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.

[1]  C B Sledge,et al.  Matrix collagen type and pore size influence behaviour of seeded canine chondrocytes. , 1997, Biomaterials.

[2]  P. Deluca,et al.  A study of the phase transitions in frozen antibiotic solutions by differential scanning calorimetry. , 1980, Journal of the Parenteral Drug Association.

[3]  P. Prendergast,et al.  Gene expression by marrow stromal cells in a porous collagen–glycosaminoglycan scaffold is affected by pore size and mechanical stimulation , 2008, Journal of materials science. Materials in medicine.

[4]  Geraldine Mitchell,et al.  The influence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo. , 2006, Biomaterials.

[5]  F. O'Brien,et al.  The effects of collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering. , 2009, Journal of the mechanical behavior of biomedical materials.

[6]  Fergal J O'Brien,et al.  Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds. , 2004, Biomaterials.

[7]  L G Griffith,et al.  Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. , 2001, Tissue engineering.

[8]  J. Schwartz,et al.  Effects of excipients on the crystallization of pharmaceutical compounds during lyophilization. , 1989, Journal of parenteral science and technology : a publication of the Parenteral Drug Association.

[9]  Patrick J Prendergast,et al.  The effect of pore size on permeability and cell attachment in collagen scaffolds for tissue engineering. , 2007, Technology and health care : official journal of the European Society for Engineering and Medicine.

[10]  Alessandro Sannino,et al.  Fabricating tubular scaffolds with a radial pore size gradient by a spinning technique. , 2006, Biomaterials.

[11]  Lorna J. Gibson,et al.  Cellular materials as porous scaffolds for tissue engineering , 2001 .

[12]  P. Prendergast,et al.  A collagen-glycosaminoglycan scaffold supports adult rat mesenchymal stem cell differentiation along osteogenic and chondrogenic routes. , 2006, Tissue engineering.

[13]  F. O'Brien,et al.  The effect of dehydrothermal treatment on the mechanical and structural properties of collagen-GAG scaffolds. , 2009, Journal of biomedical materials research. Part A.

[14]  G Rau,et al.  Control of pore structure and size in freeze-dried collagen sponges. , 2001, Journal of biomedical materials research.

[15]  Hatem Fessi,et al.  Freeze-drying of nanocapsules: impact of annealing on the drying process. , 2006, International journal of pharmaceutics.

[16]  M. Akyurt,et al.  Freezing phenomena in ice–water systems , 2002 .

[17]  H. Takita,et al.  Pore size of porous hydroxyapatite as the cell-substratum controls BMP-induced osteogenesis. , 1997, Journal of biochemistry.

[18]  박종철,et al.  Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking , 2002 .

[19]  T. V. van Kuppevelt,et al.  Construction of collagen scaffolds that mimic the three-dimensional architecture of specific tissues. , 2007, Tissue engineering.

[20]  Julien Andrieu,et al.  Freeze drying of pharmaceuticals in vials: Influence of freezing protocol and sample configuration on ice morphology and freeze-dried cake texture , 2007 .

[21]  J. Carpenter,et al.  Annealing to optimize the primary drying rate, reduce freezing-induced drying rate heterogeneity, and determine T(g)' in pharmaceutical lyophilization. , 2001, Journal of pharmaceutical sciences.

[22]  M. Spector,et al.  Collagen-GAG Substrate Enhances the Quality of Nerve Regeneration through Collagen Tubes up to Level of Autograft , 1998, Experimental Neurology.

[23]  Roger Zauel,et al.  3-D computational modeling of media flow through scaffolds in a perfusion bioreactor. , 2005, Journal of biomechanics.

[24]  L. Gibson,et al.  The effect of pore size on cell adhesion in collagen-GAG scaffolds. , 2005, Biomaterials.

[25]  James A. Searles.  Freezing and Annealing Phenomena in Lyophilization , 2004 .

[26]  A. Grodzinsky,et al.  The effects of cross-linking of collagen-glycosaminoglycan scaffolds on compressive stiffness, chondrocyte-mediated contraction, proliferation and biosynthesis. , 2001, Biomaterials.

[27]  Yu-Lin Lai,et al.  Osteoblastic response to collagen scaffolds varied in freezing temperature and glutaraldehyde crosslinking. , 2007, Journal of biomedical materials research. Part A.

[28]  Smadar Cohen,et al.  Tailoring the pore architecture in 3-D alginate scaffolds by controlling the freezing regime during fabrication. , 2002, Biomaterials.

[29]  Ioannis V. Yannas,et al.  Tissue and organ regeneration in adults , 2001 .