Shujiang Wang

Mild and Efficient Strategy for Site-Selective Aldehyde Modification of Glycosaminoglycans : Tailoring Hydrogels with Tunable Release of Growth Factor

Aldehydes have been used as an important bioorthogonal chemical reporter for conjugation of large polymers and bioactive substances. However, generating aldehyde functionality on carbohydrate-based biopolymers without changing its native chemical structure has always persisted as a challenging task. The common methods employed to achieve this require harsh reaction conditions, which often compromise the structural integrity and biological function of these sensitive molecules. Here we report a mild and simple method to graft aldehydes groups on glycosaminoglycans (GAGs) in a site-selective manner without compromising the structural integrity of the biopolymer. This regio-selective modification was achieved by conjugating the amino-glycerol moiety on the carboxylate residue of the polymer, which allowed selective cleavage of pendent diol groups without interfering with the C2-C3 diol groups of the native glucopyranose residue. Kinetic evaluation of this reaction demonstrated significant differences in second-order reaction rate for periodate oxidation (by four-orders of magnitude) between the two types of vicinal diols. We employed this chemistry to develop aldehyde modifications of sulfated and nonsulfated GAGs such as hyaluronic acid (HA), heparin (HP), and chondroitin sulfate (CS). We further utilized these aldehyde grafted GAGs to tailor extracellular matrix mimetic injectable hydrogels and evaluated its rheological properties. The composition of the hydrogels was also found to modulate release of therapeutic protein such as FGF-2, demonstrating controlled release (60%) for over 14 days. In short, our result clearly demonstrates a versatile strategy to graft aldehyde groups on sensitive biopolymers under mild conditions that could be applied for various bioconjugation and biomedical applications such as drug delivery and regenerative medicine.

Insights into the Mechanism and Catalysis of Oxime Coupling Chemistry at Physiological pH

The dynamic covalent-coupling reaction involving α-effect nucleophiles has revolutionized bioconjugation approaches, due to its ease and high efficiency. Key to its success is the discovery of aniline as a nucleophilic catalyst, which made this reaction feasible under physiological conditions. Aniline however, is not so effective for keto substrates. Here, we investigate the mechanism of aniline activation in the oxime reaction with aldehyde and keto substrates. We also present carboxylates as activating agents that can promote the oxime reaction with both aldehyde and keto substrates at physiological pH. This rate enhancement circumvents the influence of α-effect by forming H-bonds with the rate-limiting intermediate, which drives the reaction to completion. The combination of aniline and carboxylates had a synergistic effect, resulting in a ∼14-31-fold increase in reaction rate at pD 7.4 with keto substrates. The biocompatibility and efficiency of carboxylate as an activating agent is demonstrated by performing cell-surface oxime labeling at physiological pH using acetate, which showed promising results that were comparable with aniline.

Functionalised type-I collagen as a hydrogel building block for bio-orthogonal tissue engineering applications

In this study, we derivatized type I collagen without altering its triple helical conformation to allow for facile hydrogel formation via the Michael addition of thiols to methacrylates without the addition of other crosslinking agents. This method provides the flexibility needed for the fabrication of injectable hydrogels or pre-fabricated implantable scaffolds, using the same components by tuning the modulus from Pa to kPa. Enzymatic degradability of the hydrogels can also be easily fine-tuned by variation of the ratio and the type of the cross-linking component. The structural morphology reveals a lamellar structure mimicking native collagen fibrils. The versatility of this material is demonstrated by its use as a pre-fabricated substrate for culturing human corneal epithelial cells and as an injectable hydrogel for 3-D encapsulation of cardiac progenitor cells.

Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH

We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration-dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most biofriendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.

Smart design of stable hydrazone crosslinked extracellular matrix mimetic hydrogel for tissue engineering application

Adequate cellular in-growth into biomaterials is one of the fundamental requirements in regenerative medicine. Type-I-collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support. However, adequate cell in-growth and cell seeding has been suboptimal. Different densities of collagen scaffolds (0.3% to 0.8% (w/v)) with/without polymer knitting (poly-caprolactone (PCL)) were prepared. The structure of collagen scaffolds was characterized using scanning electronic microscopy (SEM) and HE staining. The mechanical strength of hybrid scaffolds was determined using tensile strength analysis. Cellular penetration and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and urothelium seeding. SEM and HE analysis showed the honeycomb structure and the hybrid scaffolds were adequately connected. The hybrid scaffolds were much stronger than collagen alone. The distribution of the beads and cells were highly dependent on the collagen density: at lower densities the beads and cells were more evenly distributed and penetrated deeper into the scaffold. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a 0.4% hybrid scaffold strengthened with knitting achieved the best cellular distribution.Human adult heart harbors a population of resident progenitor cells that can be isolated by Sca-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes and vascular cells in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, the survival rate and retention is really poor, less than 1% of injected cells being detectable in the hosttissue within few weeks. The present study aimed at investigating the possibility to produce scaffoldless, thick cardiac progenitor cell-derived cardiac patches by thermo-responsive technology. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces obtained by poly-N-isopropylacrylamide (PNIPAAm) surface immobilization. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and produced abundant extracellular matrix, while preserving their phenotype and plasticity. Cell phenotype and viability within the 3D construct were followed for 1 week, showing that no significant differentiation or apoptotic events occurred within the construct. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such approach to deliver stem cells.Spinal cord injury and repair is one of the important focus areas in tissue regeneration. Mechanical trauma caused due to factors such as contusion, compression or involuntary stretching induce post-traumatic secondary tissue damage in many Spinal Cord Injury (SCI) patients. Therefore, there is a need for scaffolds that provide a conducive threedimensionsal (3D) environment for injured cells to attach and grow. In this study we propose to synthesize 3D polymeric scaffolds in order to study the mechanical and adhesive properties & the nature of the interactions between hyaluronan-based (HY) biomaterials and cells and tissues both in vitroandin vivo. Here we have synthesized 3D HY-based hydrogels with robust mechanical and adhesive properties and demonstrate the use of this material for neuronal-related applications such as the treatment of SCI. Cell culture and survivability studies were done with NSC-34 cells. Live/Dead assay performed on the cells revealed significant differences in the staining of live cells and showed increased viability and proliferation. The number of live cells in the HY-based hydrogels with 0.1% collagen showed higher cell numbers compared with the other hydrogels. In this study we show that Injectable HYbased hydrogels with high elasticity, comparable to the mechanical properties of nervous tissue have been used in this study to study their biocompatibility and neuroprotective properties and they show better affinity for neuronal cells.Calcium phosphates (CaP) obtained by biomineralisation in Simulated Boby Fluid have been used for decades to assess the mineralisation capability of biomaterials. Recently, they have been envisioned as potential agents to promote bone formation. In this study, we have fabricated and coated with calcium phosphate melt electrospun scaffolds whereby macropores permit adequate cell migration and nutrient transfer. We have systematically investigated the effect of coating and osteoinduction onto the response of ovine osteoblasts and we observed that the coating up-regulated alkaline phosphatase activity regardless of the in vitro culture conditions. Micro Computed Tomography revealed that only scaffolds cultured in an osteoinductive cocktail were capable of depositing mineralised matrix, and that CaP coated scaffolds were more efficient at promoting mineralisation. Theses scaffolds were subcutaneously implanted in athymic rats and this demonstrated that the osteoinduction was a pre-requisite for bone formation in this ectopic model. It showed that although the bone formation was not significantly different after 8 weeks, the CaP coated scaffolds were superior at inducing bone formation as evidenced by higher levels of mineralisation at earlier time points. This work demonstrated that CaP coating is not sufficient to induce bone formation; however the combination of osteoinduction and CaP coating resulted in earlier bone formation in an ectopic model.Introduction: Bladder regeneration using minced bladder mucosa is an alternative to costly and time-consuming conventional in vitro culturing of urothelial cells. In this method, the uroepithelium ...