Gelatin microspheres crosslinked with genipin for local delivery of growth factors

A main challenge in tissue engineering and regenerative medicine is achieving local and efficient growth factor release to guide cell function. Gelatin is a denatured form of collagen that cells can bind to and degrade through enzymatic action. In this study, gelatin microspheres were used to release bone morphogenetic protein 2 (BMP2). Spherical microparticles with diameters in the range of 2–6 µm were created by an emulsification process and were stabilized by crosslinking with the small molecule genipin. The degree of crosslinking was varied by controlling the incubation time in genipin solution. Loading rate studies, using soy bean trypsin inhibitor as a model protein, showed rapid protein uptake over the first 24 h, followed by a levelling off and then a further increase after approximately 3 days, as the microspheres swelled. Growth factor release studies using microspheres crosslinked to 20%, 50% and 80% of saturation and then loaded with BMP2 showed that higher degrees of crosslinking resulted in higher loading efficiency and slower protein release. After 24 h, the concentration profiles produced by all microsphere formulations were steady and approximately equal. Microspheres incubated with adult human mesenchymal stem cells accumulated preferentially on the cell surface, and degraded over time in culture. BMP2‐loaded microspheres caused a three‐ to eight‐fold increase in expression of the bone sialoprotein gene after 14 days in culture, with more crosslinked beads producing a greater effect. These results demonstrate that genipin‐crosslinked gelatin microspheres can be used to deliver growth factors locally to cells in order to direct their function. Copyright © 2010 John Wiley & Sons, Ltd.

[1]  H. Sung,et al.  Effects of heparin immobilization on the surface characteristics of a biological tissue fixed with a naturally occurring crosslinking agent (genipin): an in vitro study. , 2001, Biomaterials.

[2]  S. Cryan,et al.  A comparative study of a range of polymeric microspheres as potential carriers for the inhalation of proteins. , 2008, International journal of pharmaceutics.

[3]  J. Jansen,et al.  In vitro growth factor release from injectable calcium phosphate cements containing gelatin microspheres. , 2009, Journal of biomedical materials research. Part A.

[4]  N. Adhirajan,et al.  Gelatin microspheres cross-linked with EDC as a drug delivery system for doxycyline: Development and characterization , 2007, Journal of microencapsulation.

[5]  R. Nerem,et al.  Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two- and three-dimensional culture. , 2003, Experimental cell research.

[6]  Yen Chang,et al.  Fixation of biological tissues with a naturally occurring crosslinking agent: fixation rate and effects of pH, temperature, and initial fixative concentration. , 2000, Journal of biomedical materials research.

[7]  H. Bohidar,et al.  Swelling and de-swelling kinetics of gelatin hydrogels in ethanol-water marginal solvent. , 2006, International journal of biological macromolecules.

[8]  Yen Chang,et al.  Gelatin microspheres encapsulated with a nonpeptide angiogenic agent, ginsenoside Rg1, for intramyocardial injection in a rat model with infarcted myocardium. , 2007, Journal of controlled release : official journal of the Controlled Release Society.

[9]  W. Bubnis,et al.  Chemical and Swelling Evaluations of Amino Group Crosslinking in Gelatin and Modified Gelatin Matrices , 1996, Pharmaceutical Research.

[10]  J. Ong,et al.  The effect of cross-linking of chitosan microspheres with genipin on protein release , 2007 .

[11]  J. Stegemann,et al.  The role of ERK signaling in protein hydrogel remodeling by vascular smooth muscle cells. , 2007, Biomaterials.

[12]  D. Rousseau,et al.  Kinetic and mechanistic considerations in the gelation of genipin-crosslinked gelatin. , 2006, International journal of biological macromolecules.

[13]  Antonios G Mikos,et al.  Biodegradable gelatin microparticles as delivery systems for the controlled release of bone morphogenetic protein-2. , 2008, Acta biomaterialia.

[14]  R L Reis,et al.  Materials in particulate form for tissue engineering. 1. Basic concepts , 2007, Journal of tissue engineering and regenerative medicine.

[15]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[16]  G. Plopper,et al.  Osteogenic differentiation of mesenchymal stem cells in defined protein beads. , 2008, Journal of biomedical materials research. Part B, Applied biomaterials.

[17]  J. Jansen,et al.  Introduction of gelatin microspheres into an injectable calcium phosphate cement. , 2008, Journal of biomedical materials research. Part A.

[18]  S. Waldman,et al.  Genipin Cross-Linked Fibrin Hydrogels for in vitro Human Articular Cartilage Tissue-Engineered Regeneration , 2009, Cells Tissues Organs.

[19]  P. Pudney,et al.  Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin , 2003 .

[20]  I. Chu,et al.  Type II collagen-chondroitin sulfate-hyaluronan scaffold cross-linked by genipin for cartilage tissue engineering. , 2009, Journal of bioscience and bioengineering.

[21]  Y. Ikada,et al.  Controlled release of growth factors based on biodegradation of gelatin hydrogel , 2001, Journal of biomaterials science. Polymer edition.

[22]  H. Sung,et al.  In vitro evaluation of the genotoxicity of a naturally occurring crosslinking agent (genipin) for biologic tissue fixation. , 2000, Journal of biomedical materials research.

[23]  R. Knuechel,et al.  The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds. , 2010, Biomaterials.

[24]  J. Tessmar,et al.  Matrices and scaffolds for protein delivery in tissue engineering. , 2007, Advanced drug delivery reviews.

[25]  H. Sung,et al.  Genipin-crosslinked gelatin microspheres as a drug carrier for intramuscular administration: in vitro and in vivo studies. , 2003, Journal of biomedical materials research. Part A.

[26]  Hsing-Wen Sung,et al.  Crosslinking structures of gelatin hydrogels crosslinked with genipin or a water‐soluble carbodiimide , 2004 .

[27]  H. Ly,et al.  Three catheter-based strategies for cardiac delivery of therapeutic gelatin microspheres , 2006, Gene Therapy.

[28]  Antonios G Mikos,et al.  Gelatin as a delivery vehicle for the controlled release of bioactive molecules. , 2005, Journal of controlled release : official journal of the Controlled Release Society.

[29]  R. Reis,et al.  Materials in particulate form for tissue engineering. 2. Applications in bone , 2007, Journal of tissue engineering and regenerative medicine.

[30]  Ikada,et al.  Protein release from gelatin matrices. , 1998, Advanced drug delivery reviews.

[31]  Alison P McGuigan,et al.  Modular tissue engineering: fabrication of a gelatin‐based construct , 2007, Journal of Tissue Engineering and Regenerative Medicine.

[32]  R. Reis,et al.  Chitosan microparticles as injectable scaffolds for tissue engineering , 2008, Journal of tissue engineering and regenerative medicine.

[33]  Y. Ikada,et al.  Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels. , 1998, Biomaterials.

[34]  Y. Ikada,et al.  Accelerated tissue regeneration through incorporation of basic fibroblast growth factor-impregnated gelatin microspheres into artificial dermis. , 2000, Biomaterials.

[35]  Antonios G. Mikos,et al.  In Vitro and In Vivo Release of Vascular Endothelial Growth Factor from Gelatin Microparticles and Biodegradable Composite Scaffolds , 2008, Pharmaceutical Research.

[36]  Chu Zhang,et al.  Evaluation of cross-linking methods for electrospun gelatin on cell growth and viability. , 2009, Biomacromolecules.