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Synthetic scaffolds that possess an intrinsic capability to protect and sequester sensitive growth factors is a primary requisite for developing successful tissue engineering strategies. Growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) is highly susceptible to premature degradation and to provide a meaningful clinical outcome require high doses that can cause serious side effects. We discovered a unique strategy to stabilize and sequester rhBMP-2 by enhancing its molecular interactions with hyaluronic acid (HA), an extracellular matrix (ECM) component. We found that by tuning the initial protonation state of carboxylic acid residues of HA in a covalently crosslinked hydrogel modulate BMP-2 release at physiological pH by minimizing the electrostatic repulsion and maximizing the Van der Waals interactions. At neutral pH, BMP-2 release is primarily governed by Fickian diffusion, whereas at acidic pH both diffusion and electrostatic interactions between HA and BMP-2 become important as confirmed by molecular dynamics simulations. Our results were also validated in an in vivo rat ectopic model with rhBMP-2 loaded hydrogels, which demonstrated superior bone formation with acidic hydrogel as compared to the neutral counterpart. We believe this study provides new insight on growth factor stabilization and highlights the therapeutic potential of engineered matrices for rhBMP-2 delivery and may help to curtail the adverse side effects associated with the high dose of the growth factor., De två första författarna delar förstaförfattarskapet.

Embryonic microenvironments can reverse the metastatic phenotype of aggressive tumors by inhibiting the Nodal signaling pathway. Here, we hypothesize that embryonic microenvironments can be transplanted for the purpose of oncotherapy. We report the development of an injectable bioactive hydrogel system containing the key antagonists of Nodal signaling-Cripto-1 receptor antibodies (2B11)-for the creation of embryonic microenvironments and the examination of their effect on tumor reversion treatment using a mouse model. Our in vitro results show that the hydrogel system can reduce the mitochondrial membrane potential of MDA-MB-231 and MCF-7, promote cell apoptosis, and reduce the invasive ability of cells. Our in vivo results illustrate that the hydrogel system can significantly inhibit tumor growth in both breast cancer and melanoma tumor-bearing mouse models, as well as transform the cell morphology of melanoma B16 cells to melanin-like cells. Furthermore, the results of the up-regulation of tumor suppressor genes and the down-regulation of oncogenes by high-throughput sequencing confirm that the developed system can also selectively turn on some tumor suppressor genes and turn off certain oncogenes so as to prompt the benign reversion of the tumor phenotype. Taken together, our results demonstrate the injectable biomaterial system is able to create an effective microenvironment for melanoma and breast tumor therapy.

This thesis presents deeper insights into bone applicable biomaterials’ design. Poor affinity of BMP-2 towards scaffolds required supra-physiological dose administration. Though molecules containing sulfate could sustain BMP-2 release, side effects occurred due to BMP-2 supra-dose, or these sulfate-containing biomolecules. Improved affinity between BMP-2 and scaffolds was first witnessed by using an acidic carrier (paper I). Hyaluronic acid (HA) hydrazone derived hydrogels having a pH of 4.5-loaded BMP-2 showed sustained release of bioactive BMP-2 in vitro and enhanced bone formation in vivo, while pH 7 HA hydrogels showed Fickian behavior and less bone formation in vivo. Computational evaluation revealed stronger electrostatic interactions between BMP-2, and HA were predominant at pH 4.5, whereas, weaker Van der Waals interactions played a key role at pH 7. During the pre-bone formation phase, endogenous cell responses to pH 4.5 and 7 with or without BMP-2 were investigated. HA hydrogels exhibited extraordinary biocompatibility and recruitment of neutrophils, monocytes, macrophages and stromal cells regardless of hydrogels’ pH and BMP-2 presence. The different inflammatory responses to HA hydrogels were observed (Appendix). Thiol derivatives can cleave the disulfide bond of BMP-2 to generate inactive monomeric BMP-2. In paper II, thiol-acrylate chemistry-based HA hydrogels (HA-SH) were compared to hydrazone-based HA hydrogels as BMP-2 carriers. Thiol modified HA disrupted BMP-2 integrity and bioactivity. HA-SH hydrogels with BMP-2 exhibited less bioactive BMP-2 release in vitro and induced less bone formation in vivo. Accumulated evidence has shown great osteogenic potential of lithium ions (Li). In paper III, we coordinated Li onto HA-PVA hydrazone hydrogels (Li-gel); Li-gel enhanced 3D cultured hMSCs osteogenic differentiation and induced higher bone formation in CAM defect model. Instead of BMP-2 protein, delivery of BMP-2-coding-plasmid can produce BMP-2 over

This thesis presents deeper insights into bone applicable biomaterials’ design. Poor affinity of BMP-2 towards scaffolds required supra-physiological dose administration. Though molecules containing sulfate could sustain BMP-2 release, side effects occurred due to BMP-2 supra-dose, or these sulfate-containing biomolecules. Improved affinity between BMP-2 and scaffolds was first witnessed by using an acidic carrier (paper I). Hyaluronic acid (HA) hydrazone derived hydrogels having a pH of 4.5-loaded BMP-2 showed sustained release of bioactive BMP-2 in vitro and enhanced bone formation in vivo, while pH 7 HA hydrogels showed Fickian behavior and less bone formation in vivo. Computational evaluation revealed stronger electrostatic interactions between BMP-2, and HA were predominant at pH 4.5, whereas, weaker Van der Waals interactions played a key role at pH 7. During the pre-bone formation phase, endogenous cell responses to pH 4.5 and 7 with or without BMP-2 were investigated. HA hydrogels exhibited extraordinary biocompatibility and recruitment of neutrophils, monocytes, macrophages and stromal cells regardless of hydrogels’ pH and BMP-2 presence. The different inflammatory responses to HA hydrogels were observed (Appendix). Thiol derivatives can cleave the disulfide bond of BMP-2 to generate inactive monomeric BMP-2. In paper II, thiol-acrylate chemistry-based HA hydrogels (HA-SH) were compared to hydrazone-based HA hydrogels as BMP-2 carriers. Thiol modified HA disrupted BMP-2 integrity and bioactivity. HA-SH hydrogels with BMP-2 exhibited less bioactive BMP-2 release in vitro and induced less bone formation in vivo. Accumulated evidence has shown great osteogenic potential of lithium ions (Li). In paper III, we coordinated Li onto HA-PVA hydrazone hydrogels (Li-gel); Li-gel enhanced 3D cultured hMSCs osteogenic differentiation and induced higher bone formation in CAM defect model. Instead of BMP-2 protein, delivery of BMP-2-coding-plasmid can produce BMP-2 over

This thesis presents deeper insights into bone applicable biomaterials’ design. Poor affinity of BMP-2 towards scaffolds required supra-physiological dose administration. Though molecules containing sulfate could sustain BMP-2 release, side effects occurred due to BMP-2 supra-dose, or these sulfate-containing biomolecules. Improved affinity between BMP-2 and scaffolds was first witnessed by using an acidic carrier (paper I). Hyaluronic acid (HA) hydrazone derived hydrogels having a pH of 4.5-loaded BMP-2 showed sustained release of bioactive BMP-2 in vitro and enhanced bone formation in vivo, while pH 7 HA hydrogels showed Fickian behavior and less bone formation in vivo. Computational evaluation revealed stronger electrostatic interactions between BMP-2, and HA were predominant at pH 4.5, whereas, weaker Van der Waals interactions played a key role at pH 7. During the pre-bone formation phase, endogenous cell responses to pH 4.5 and 7 with or without BMP-2 were investigated. HA hydrogels exhibited extraordinary biocompatibility and recruitment of neutrophils, monocytes, macrophages and stromal cells regardless of hydrogels’ pH and BMP-2 presence. The different inflammatory responses to HA hydrogels were observed (Appendix). Thiol derivatives can cleave the disulfide bond of BMP-2 to generate inactive monomeric BMP-2. In paper II, thiol-acrylate chemistry-based HA hydrogels (HA-SH) were compared to hydrazone-based HA hydrogels as BMP-2 carriers. Thiol modified HA disrupted BMP-2 integrity and bioactivity. HA-SH hydrogels with BMP-2 exhibited less bioactive BMP-2 release in vitro and induced less bone formation in vivo. Accumulated evidence has shown great osteogenic potential of lithium ions (Li). In paper III, we coordinated Li onto HA-PVA hydrazone hydrogels (Li-gel); Li-gel enhanced 3D cultured hMSCs osteogenic differentiation and induced higher bone formation in CAM defect model. Instead of BMP-2 protein, delivery of BMP-2-coding-plasmid can produce BMP-2 over

This thesis presents deeper insights into bone applicable biomaterials’ design. Poor affinity of BMP-2 towards scaffolds required supra-physiological dose administration. Though molecules containing sulfate could sustain BMP-2 release, side effects occurred due to BMP-2 supra-dose, or these sulfate-containing biomolecules. Improved affinity between BMP-2 and scaffolds was first witnessed by using an acidic carrier (paper I). Hyaluronic acid (HA) hydrazone derived hydrogels having a pH of 4.5-loaded BMP-2 showed sustained release of bioactive BMP-2 in vitro and enhanced bone formation in vivo, while pH 7 HA hydrogels showed Fickian behavior and less bone formation in vivo. Computational evaluation revealed stronger electrostatic interactions between BMP-2, and HA were predominant at pH 4.5, whereas, weaker Van der Waals interactions played a key role at pH 7. During the pre-bone formation phase, endogenous cell responses to pH 4.5 and 7 with or without BMP-2 were investigated. HA hydrogels exhibited extraordinary biocompatibility and recruitment of neutrophils, monocytes, macrophages and stromal cells regardless of hydrogels’ pH and BMP-2 presence. The different inflammatory responses to HA hydrogels were observed (Appendix). Thiol derivatives can cleave the disulfide bond of BMP-2 to generate inactive monomeric BMP-2. In paper II, thiol-acrylate chemistry-based HA hydrogels (HA-SH) were compared to hydrazone-based HA hydrogels as BMP-2 carriers. Thiol modified HA disrupted BMP-2 integrity and bioactivity. HA-SH hydrogels with BMP-2 exhibited less bioactive BMP-2 release in vitro and induced less bone formation in vivo. Accumulated evidence has shown great osteogenic potential of lithium ions (Li). In paper III, we coordinated Li onto HA-PVA hydrazone hydrogels (Li-gel); Li-gel enhanced 3D cultured hMSCs osteogenic differentiation and induced higher bone formation in CAM defect model. Instead of BMP-2 protein, delivery of BMP-2-coding-plasmid can produce BMP-2 over

This thesis presents deeper insights into bone applicable biomaterials’ design. Poor affinity of BMP-2 towards scaffolds required supra-physiological dose administration. Though molecules containing sulfate could sustain BMP-2 release, side effects occurred due to BMP-2 supra-dose, or these sulfate-containing biomolecules. Improved affinity between BMP-2 and scaffolds was first witnessed by using an acidic carrier (paper I). Hyaluronic acid (HA) hydrazone derived hydrogels having a pH of 4.5-loaded BMP-2 showed sustained release of bioactive BMP-2 in vitro and enhanced bone formation in vivo, while pH 7 HA hydrogels showed Fickian behavior and less bone formation in vivo. Computational evaluation revealed stronger electrostatic interactions between BMP-2, and HA were predominant at pH 4.5, whereas, weaker Van der Waals interactions played a key role at pH 7. During the pre-bone formation phase, endogenous cell responses to pH 4.5 and 7 with or without BMP-2 were investigated. HA hydrogels exhibited extraordinary biocompatibility and recruitment of neutrophils, monocytes, macrophages and stromal cells regardless of hydrogels’ pH and BMP-2 presence. The different inflammatory responses to HA hydrogels were observed (Appendix). Thiol derivatives can cleave the disulfide bond of BMP-2 to generate inactive monomeric BMP-2. In paper II, thiol-acrylate chemistry-based HA hydrogels (HA-SH) were compared to hydrazone-based HA hydrogels as BMP-2 carriers. Thiol modified HA disrupted BMP-2 integrity and bioactivity. HA-SH hydrogels with BMP-2 exhibited less bioactive BMP-2 release in vitro and induced less bone formation in vivo. Accumulated evidence has shown great osteogenic potential of lithium ions (Li). In paper III, we coordinated Li onto HA-PVA hydrazone hydrogels (Li-gel); Li-gel enhanced 3D cultured hMSCs osteogenic differentiation and induced higher bone formation in CAM defect model. Instead of BMP-2 protein, delivery of BMP-2-coding-plasmid can produce BMP-2 over

Degradation of plasmid DNA (pDNA) in the endosome compartment and its release to the cytosol are the major hurdles for efficient gene transfection. This is generally addressed by using transfection reagents that can overcome these limitations. In this article, the first report is presented which suggests that controlling the release of pDNA from endosome is the key for achieving efficient transfection. In this study, chondroitin sulfate (CS)-coated DNA-nanoplexes are developed using a modular approach where CS is coated post-pDNA/PEI nanoplex formation. To ensure good stability of the nanoplexes, imine/enamine chemistry is exploited by reacting aldehyde-modified chondroitin sulfate (CS-CHO) with free amines of pDNA/PEI complex. This supramolecular nanocarrier system displays efficient cellular uptake, and controlled endosomal pDNA release without eliciting any cytotoxicity. On the contrary, burst release of pDNA from endosome (using chloroqine) results in significant reduction in gene expression. Unlike pDNA/PEI-based transfection, the nanoparticle design presented here shows exceptional stability and gene transfection efficiency in different cell lines such as human colorectal cancer cells (HCT116), human embryonic kidney cells (HEK293), and mouse skin-derived mesenchymal stem cells (MSCs) using luciferase protein as a reporter gene. This new insight will be valuable in designing next generation of transfection reagents.

Site-specific administration of bone morphogenetic protein-2 (BMP-2) at the site of a bone fracture via scaffolds can minimise systemic side-effects and exhibit sustained biological effects. While this method requires sufficient scaffolds to preserve the BMP-2 structure and tuned release patterns, the incorporation of thiol-acrylate chemistry has shown great success in scaffold synthesis. However, thiolates attack the sulphur atoms of disulphide bonds, displacing the other sulphur atom and forming a new disulphide bond, hence at physiological conditions, thiol-modified scaffold components could potentially attack inter-chain disulphide bonds of BMP-2 by thiol-exchange reactions. This therefore led us to compare hyaluronic acid (HA) hydrogels synthesised via thiol-acrylate (HA-S) and hydrazone crosslinking chemistry (HA-H) formed BMP-2 carriers. The study revealed the integrity of BMP-2 dimer structures can be disrupted and reveals the osteogenic capacity of BMP-2 by HA derivatives (HA-SH). BMP-2 bioactivity released from HA-S hydrogels are decreased when compared to HA-H hydrogels. This was further confirmed via the rat ectopic bone model, showing that bone volume was significantly higher when induced by HA-H hydrogels with BMP-2 than compared to HA-S hydrogel with BMP-2. This study gives new insights into scaffolds synthesis, showing that biomolecule bioactivity needs to be considered when choosing a chemistry for scaffolds synthesis.

The poor affinity of rhBMP-2 to the scaffolds leads to high dose administration requirement resulted in massive side effects has been the hurdle for successful clinic translation for treating delayed unions or remained non-union at bone defect. Optimizing the scaffolds with the purpose of obtaining optimal BMP2 dose and release have been addressed as critical for BMP-2 administration, however, the results are contradictory concerning whether bone formation is more beneficial from burst or controlled release of BMP2. While this might be due to these studies incorporated other bioactive molecules onto scaffolds to immobilize BMP-2. In this study, we report the affinities of rhBMP-2 to the scaffolds can be improved by only tuning the pH of hyaluronic acid (HA) hydrazone crosslinking hydrogel without addition of other molecules. Neo bone induced by BMP-2 showed significantly higher volume with more impact structure and vascularization in pH 4.5 HA hydrogel compared to that in pH7 HA hydrogel. The mechanisms were demonstrated by In vitro BMP-2 release followed by diffusion quantitative calculation and computational simulation methods. Initial burst release of BMP-2 from pH 7 HA hydrogels with the fitting of Fickian behavior while sustained release from pH 4.5 HA hydrogel was observed. Computational stimulation revealed this is due to the protonation state of BMP2 at pH 4.5 resulted in stronger electrostatic interaction with negatively charged groups along the backbone of hyaluronic acid molecules compared to at pH 7. This study gives new direction to scaffolds designing for basic bioactive protein applications in future.