The design of biomaterials mimicking the physiological environment of the cells is important for biomedical material.  Extracellular matrix (ECM) provides the suitable biochemical and structural support to the cells in vivo and in vitro.  Basement membrane is the thin layer of ECM that divide the two different cell layers in developmental stages and whole body, such as embryo, skin, cornea, glands, muscle, and neuron.  Laminins-111 plays a critical in maintaining the many biological functions of basement membrane and is the major components of Matrigel, that is one of the widely used functional ECM hydrogels in vitro. We previously identified about 60 active sequences from laminin-111 through the peptide screening method from the entire sequence of laminin-111. Further, 29 biological active peptides from laminin-111 showed the biological activities when conjugated on chitosan membrane, and mixed-peptides conjugated chitosan membrane promoted laminin-111 like biological activities.  Here, we crosslinked chitosan by dicarboxilyc acids and conjugated laminin derived active peptides to the chitosan hydrgel. Ten dicarboxylic acids were tested to obtain the water soluble chitosan, and found that seven saturated liner dicarboxilyc acids could solve the chitosan to the water.  Then, chitosan was crosslinked by dicarboxilyc acids with directly addition of N-hydroxy succinimide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. As results, three chitosan/dicarboxilyc acid solutions showed the 3D hydrogel and chitosan/glutaric acid solution significantly generated transparent 3D hydrogel.  Next, we conjugated laminin active peptides to the chitosan/glutaric acid hydrogel and examined its biological activities.  The peptide-chitosan/glutaric acid hydrogel effectively promoted human dermal fibroblasts and mouse embryonic stem cell attachment that is stronger than cells on non-crosslinked peptide chitosan membrane.  When we culture the human salivary gland cells on the peptide-chitosan/glutaric acid hydrogel, some cells were differentiated and exhibited acinar like formation. The peptide-chitosan/glutaric acid hydrogel could use to analyze the cellular functions mediated by specific cell surface receptors and have a potential to use as a biomedical material.

Development of laminin active-peptide conjugated chitosan hydrogel crosslinked by dicarboxylic acids

254th National Meeting (2017)

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