Collagen Mimetic Peptide-PEG Hybrid Hydrogels:Preparation And Biomedical Applications

Collagen is one of the most widely used natural polymers in biomedicine, which plays an important role in tissue regeneration and drug delivery. Collagen-based biomaterials are characterized by inherent bio compatibility, biodegradability as well as bioactivity. However, they suffer from lot-to-lot variability and difficulties in controllable modification. In addition, they also raise concern about immunogenic response. Collagen mimetic peptides (CMPs), synthetic collagen models which were introduced to study the structure, self-assembly of collagen as well as collagen related diseases, are suggested to serve as alternative materials to address limitations of natural collagen.In this dissertation, we prepared collagen mimetic peptide-PEG hybrid hydrogels as alternatives of collagen gels and explore their applications in 3D culture, calcium phosphates biomineralization and local delivery of protein drugs. Specifically, CMPs possessing cysteine were conjugated to multi-arm (4/8) PEG-maleimide (PEG-MAL) by reaction between thiol and maleimide. Upon self-assembly of CMPs into triple helix, a polymeric three-dimensional network was formed where triple helix acted as physical crosslinks. This can be achieved at room temperature and is beneficial for biomacromolecules and cells encapsulated. Mechanical strength of the collagen mimetic peptide-PEG hybrid hydrogel was tunable as confirmed by rheology.By fixing the hydrogel in a Millicell insert, we establish a 3D culture system for bone marrow mesenchymal stem cells (BMSCs). LIVE/DEAD assay showed that BMSCs were viable in these hydrogels. Effects of 3D culture and growth factors (IGF-1, BMP3) on the expression of COL1A2, COL2A1, BMP2 and MMP13 of BMSCs were investigated. While no effects of the growth factors were observed, three-dimensional microenvironment of the collagen mimetic peptide-PEG hybrid hydrogels downregulated expression of COL1A2, which indicated that the 3D culture system might prevent hypertrophy of BMSCs during chondrogenic differentiation. Moreover, we showed that GEK motifs in collagen mimetic peptides could be crosslinked through the oxidation of lysine residues, a manner similar to that in biosynthesis of natural collagen.Bone is thought to form by hydroxyapatite (HA) crystallization in a collagenous hydrogel. Therefore, hydrogels are suggested to serve as ideal model systems to study biomineralization in vitro. We built a double-diffusion apparatus and investigated calcium phosphates mineralization in collagen mimetic peptide-PEG hybrid hydrogels. At physiological pH, dicalcium phosphate dihydrate (DCPD) and HA were observed on the first day. When the duration of mineralization was more than three days, only HA remained. This indicated HA in our study was formed by DCPD through phase transition. Moreover, we found the HA crystals displayed features characteristic of mesocrystals, which were regarded as novel functional materials. PH also plays a role in biomineralization. Calcium phosphates formed at pH6.0, pH7.0, pH7.4 and pH8.0 were DCPD, DCPD+HA, HA and HA, respectively. DCPD and HA were observed in patients suffering from kidney stone, the pH in urine of whom was frequently seen fluctuant. Therefore, our study may help to elucidate the pathological origin of kidney stone.Hydrogels are also attractive for their applications in drug delivery. We probed the versatility of collagen mimetic peptide-PEG hybrid hydrogels in local delivery of protein drugs. As confirmed by rheology, Cryo-SEM as well as release behavior of Dextran Blue, hydrogels prepared in PBS experienced heterogeneity, which led to poor stability and irregular erosion of resultant hydrogels. This can be addressed by preparing hydrogels in H2O, which slowed down the gelation process. In order to evaluate release behavior of entrapped protein, myoglobin was used as a model drug. Fitting release profiles by Ritger-Peppas equation revealed that release mechanism of myoglobin involved Fickian diffusion as well as surface erosion. In addition, we found that there might be unidentified interactions between BSA and the hydrogel network, which added more functionality for controlled release of protein drugs.In summary, we prepared collagen mimetic peptide-PEG hybrid hydrogels and showed they are good alternatives of collagen gels in 3D culture, biomineralization of HA/DCPD (physiologically as well as pathologically) and local delivery of protein drugs.