The ability to recapitulate the dynamic presentation of signals in a stem cell’s microenvironment remains a major hurdle in tissue engineering. Controlling cell growth and differentiation in 4 dimensions (i.e., time and 3D space) would allow for heterogeneous synthetic tissues to be produced that match the complexity of their native counterparts. Combining strategies in both light-programmable hydrogels and recombinant protein engineering, we control the cellular microenvironment using proteins site-specifically modified with a bioorthogonal handle and able to covalently bind to a photocaged reactive group in the hydrogel. Previously we’ve demonstrated the ability to photopattern gels with fluorescent proteins that excite at different wavelengths, and have shown that multiple proteins can be patterned independently within the same material with 4D control. These techniques have many potential applications, including improving joint replacement. Current joint replacement therapies often involve use of metals, plastics, and ceramics, and commonly require future revision surgeries. We propose that through our techniques, we can generate a patterned bone/cartilage interface to improve joint replacement, creating a longer-term option for joint replacement. Towards this, we have generated two photopatternable recombinant growth factors, BMP-2 (bone morphogenic protein 2) and TGF-β (transforming growth factor β), known to direct human mesenchymal stem cell (hMSC) osteogenesis and chondrogenesis. I will encapsulate hMSCs in a hydrogel and direct cell differentiation and growth in 4D using a combination of photopatterned BMP-2 and TGF-β proteins. This research will have significant impact in tissue engineering, as it will enable recreation of complex physiological structures, grown outside the body with the patient’s own cells, that can be used for personalized medicine. Our approach is unique in that it allows for unprecedented control over microscale tissue structures, ultimately matching the complexity of native tissue.

Proteins are naturally-occurring biopolymers that are useful in a wide range of therapeutic and imaging applications. Though natural proteins are comprised of just 20 monomeric amino acids, they can be post-translationally decorated to introduce expanded chemical functionality. Though several techniques have been developed, the majority do not provide site-specific modification nor stoichiometric conjugation necessary to preserve protein bioactivity. Additionally, proteins functionalized through these methods have proven difficult to purify after modification, thereby limiting potential clinical and industrial applications. Sortase-Tag Expressed Protein Ligation (STEPL) is a protein functionalization/purification method that solves many of these problems. This method allows functional probes to be site-specifically conjugated onto a protein, while simultaneously separating modified and unmodified proteins. Though this technique has been successfully demonstrated in the DeForest lab, a remaining challenge is to increase yield during protein expression. In our group, we exploit several microbiology techniques to optimize this process. Bacterial strain identity, growth conditions, reaction temperature, and protein solubility have all been shown to influence protein expression. Using Green Fluorescent Protein (GFP), we found that the STEPL yield increased when conducted for 4 hours at 37°C versus overnight at 18°C. This was determined through mass spectrometry, which showed hydrolyzed proteins after prolonged reaction. For some difficult to express proteins, including growth factors, the STEPL protocol resulted in a negligible protein yield. Through experimenting with a dialysis refolding protocol, functional growth factor Bone Morphogenic Protein-2 was produced at dramatically increased yields. Furthermore, experimenting with E. coli growth and STEPL reaction conditions has resulted in increased yield for many different functionalized proteins. Although progress has been made in producing functionalized protein with the STEPL method, yield remains below that obtained through other methods. Further experimentation to improve the robustness and yield of the STEPL protocol will increase the availability of functionalized proteins in biotherapeutics.

Hydrogels have proven to be excellent 3D cell culture platforms, serving as frameworks for synthetic tissue substitutes. Though given the complexity of the native extracellular environment, control over mechanical and biochemical cues within hydrogels is important in mimicking the extracellular matrix (ECM), the dynamic framework about which cells function. We seek to provide spatiotemporal control over cell fate within hydrogels formed through photo-mediated oxime ligation, a novel approach to photopolymerization seldom interfering with biological species present in cell encapsulation. By conjugating a photo-cleavable ortho-nitro benzyl (NPPOC) functionality to an aminooxy group, this otherwise spontaneous reaction may be spatiotemporally regulated with UV light. Excitation of the aminooxy-NPPOC complex upon UV light exposure cleaves NPPOC, enabling aminooxy to undergo ligation with an aldehyde. We have demonstrated the successful synthesis of 4-arm poly(ethylene glycol) (PEG) molecules separately functionalized with benzaldehyde and aminooxy-NPPOC end-groups. We have additionally demonstrated hydrogel formation following UV light exposure to 4-arm PEG solutions of varying compositions, verifying photo-mediated oxime ligation’s viability as a photopolymerization strategy. Following gelation, photo-mediated oxime ligation may be extended towards the incorporation of biochemical ligands via 3-dimensionally patterning NPPOC-conjugated proteins. Combining control over hydrogel geometry and stiffness with protein patterning, this dynamic hydrogel platform will expectedly aid in better understanding cell behavior through emulating the ECM’s heterogeneity. Consequently, an improved understanding of cell-matrix interactions may aid in advancing synthetic tissue substitutes.

Alcohol consumption among college students has long been a serious concern. Previous studies of college students have consistently found an association between heavy alcohol use and academic performance. This study examined the peak past month alcohol use as a function of sex, academic major, and academic performance among college students. 3003 participants completed a paid screening survey as part of a larger study evaluating the efficacy of a web-based intervention for heavy drinking and depressed college students. Measures included birth sex, academic major (STEM versus non-STEM; Science, Technology, Engineering, Mathematics), grade point average (GPA), and the peak number of drinks consumed on one occasion in the previous month. Hierarchical multiple regression analyses evaluated impact of sex, academic major, and GPA on peak alcohol consumption among college students. Significant main effects indicated that males, those with non-STEM majors, and those with lower GPAs drank more than other students. No two-way interactions were found. However, a significant three-way interaction indicated that males who are in STEM majors with high GPAs drank significantly less than those who have lower GPAs, while this relationship is not true for females, or for males who are in non-STEM majors. Implications and limitations of these findings are discussed. Future interventions on alcohol consumption among college students can be targeted to specific groups. Future studies using longitudinal designs to determine causality of the relationship between academic performance (attendance, activity involvement, etc., besides GPA) and alcohol consumption among college students are warranted.