Michael Floren

Carbon dioxide induced silk protein gelation for biomedical applications.

We present a novel method to fabricate silk fibroin hydrogels using high pressure carbon dioxide (CO(2)) as a volatile acid without the need for chemical cross-linking agents or surfactants. The simple and efficient recovery of CO(2) post processing results in a remarkably clean production method offering tremendous benefit toward materials processing for biomedical applications. Further, with this novel technique we reveal that silk protein gelation can be considerably expedited under high pressure CO(2) with the formation of extensive β-sheet structures and stable hydrogels at processing times less than 2 h. We report a significant influence of the high pressure CO(2) processing environment on silk hydrogel physical properties such as porosity, sample homogeneity, swelling behavior and compressive properties. Microstructural analysis revealed improved porosity and homogeneous composition among high pressure CO(2) specimens in comparison to the less porous and heterogeneous structures of the citric acid control gels. The swelling ratios of silk hydrogels prepared under high pressure CO(2) were significantly reduced compared to the citric acid control gels, which we attribute to enhanced physical cross-linking. Mechanical properties were found to increase significantly for the silk hydrogels prepared under high pressure CO(2), with a 2- and 3-fold increase in the compressive modulus of the 2 and 4 wt % silk hydrogels over the control gels, respectively. We adopted a semiempirical theoretical model to elucidate the mechanism of silk protein gelation demonstrated here. Mechanistically, the rate of silk protein gelation is believed to be a function of the kinetics of solution acidification from absorbed CO(2) and potentially accelerated by high pressure effects. The attractive features of the method described here include the acceleration of stable silk hydrogel formation, free of residual mineral acids or chemical cross-linkers, reducing processing complexity, and avoiding adverse biological responses, while providing direct manipulation of hydrogel physical properties for tailoring toward specific biomedical applications.

Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

UNLABELLED Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the context of implantable biomaterials. In this study, we utilized tunable hydrogels based on a simple high pressure CO2 method and silk fibroin (SF) the structural protein of Bombyx mori silk fibers. Modification of SF protein starting water solution concentration results in hydrogels of variable stiffness while retaining key structural parameters such as matrix pore size and β-sheet crystallinity. To further resolve the complex crosstalk of chemical signals with matrix properties, we chose to investigate the role of 3D hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Our data revealed the potential to upregulate matured vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Overall, our observations suggest that chemical and physical stimuli within the cellular microenvironment are tightly coupled systems involved in the fate decisions of hMSCs. The production of tunable scaffold materials that are biocompatible and further specialized to mimic tissue-specific niche environments will be of considerable value to future tissue engineering platforms. STATEMENT OF SIGNIFICANCE This article investigates the role of silk fibroin hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Specifically, we demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor.

Mussel-inspired polydopamine for bio-surface functionalization

Surface functionalization via molecular design has been a key approach to incorporate new functionalities into existing biomaterials for biomedical application. Mussel-inspired polydopamine (PDA) has aroused great interest as a new route to the functionalization of biomaterials, due to its simplicity and material independency in deposition, favorable interactions with cells, and strong reactivity for secondary functionalization. Herein, this review attempts to highlight the recent findings and progress of PDA in bio-surface functionalization for biomedical applications. The efforts made to elucidate the polymerization mechanism, PDA structure, and the preparation parameters have been discussed. Interactions between PDA coatings and the various cell types involved in different biomedical applications including general cell adhesion, bone regeneration, blood compatibility, and antimicrobial activity have also been highlighted. A brief discussion of post-functionalization of PDA and nanostructured PDA is also provided.

Processing Techniques and Applications of Silk Hydrogels in Bioengineering

Hydrogels are an attractive class of tunable material platforms that, combined with their structural and functional likeness to biological environments, have a diversity of applications in bioengineering. Several polymers, natural and synthetic, can be used, the material selection being based on the required functional characteristics of the prepared hydrogels. Silk fibroin (SF) is an attractive natural polymer for its excellent processability, biocompatibility, controlled degradation, mechanical properties and tunable formats and a good candidate for the fabrication of hydrogels. Tremendous effort has been made to control the structural and functional characteristic of silk hydrogels, integrating novel biological features with advanced processing techniques, to develop the next generation of functional SF hydrogels. Here, we review the several processing methods developed to prepare advanced SF hydrogel formats, emphasizing a bottom-up approach beginning with critical structural characteristics of silk proteins and their behavior under specific gelation environments. Additionally, the preparation of SF hydrogel blends and other advanced formats will also be discussed. We conclude with a brief description of the attractive utility of SF hydrogels in relevant bioengineering applications.

Porous poly(D,L-lactic acid) foams with tunable structure and mechanical anisotropy prepared by supercritical carbon dioxide †

The design and tunability of tissue scaffolds, such as pore size and geometry, is crucial to the success of an engineered tissue replacement. Moreover, the mechanical nature of a tissue scaffold should display properties similar to the tissue of interest; therefore, tunability of the foam mechanical properties is desirable. Polymeric foams prepared using supercritical carbon dioxide as a blowing agent has emerged in recent years as a promising technique to prepare porous scaffolds. While a number of groups have reported on the tailoring of scaffold morphologies by using gas foaming techniques, few have considered the effects of such processing conditions on the physical and mechanical anisotropy achieved. The aim of this study was to demonstrate the tunability of the structure and mechanical anisotropy of foams prepared using a variety of different gas foaming conditions. Porous poly(D,L lactic acid) foams were prepared by the systematic adjustment of processing conditions, namely pressure, temperature and venting time, resulting in an extensive range of scaffold morphologies. Characterization of sample anisotropy was achieved by mechanical evaluation of foam specimens both longitudinal and transverse to the foaming direction. The obtained mechanical properties demonstrated a strong dependence of the processing conditions on mechanical anisotropy and performance. Furthermore, results indicate that factors other than pore geometry may be necessary to define the mechanical behavior of the foam specimens. The favorable compressive moduli, coupled with large degrees of anisotropy, suggests these foams may have suitable application as scaffolds for bone tissue engineering.

A photoclickable peptide microarray platform for facile and rapid screening of 3-D tissue microenvironments

Microarrays are powerful experimental tools for high-throughput screening of cellular behavior in multivariate microenvironments. Here, we present a new, facile and rapid screening method for probing cellular behavior in 3D tissue microenvironments. This method utilizes a photoclickable peptide microarray platform developed using electrospun fibrous poly(ethylene glycol) hydrogels and microarray contact printing. We investigated the utility of this platform with five different peptide motifs and ten cell types including stem, terminally differentiated, cancer or immune cells that were from either primary origin or cell lines and from different species. We validated the capabilities of this platform to screen arrays consisting of multiple peptide motifs and concentrations for selectivity to cellular adhesion and morphology. Moreover, this platform is amenable to controlled spatial presentation of peptides. We show that by leveraging the differential attachment affinities for two cell types to two different peptides, this platform can also be used to investigate cell-cell interactions through miniature co-culture peptide arrays. Our fibrous peptide microarray platform enables high-throughput screening of 3D tissue microenvironments in a facile and rapid manner to investigate cell-matrix interactions and cell-cell signaling and to identify optimal tissue microenvironments for cell-based therapies.

High-Throughput Screening of Vascular Endothelium-Destructive or Protective Microenvironments: Cooperative Actions of Extracellular Matrix Composition, Stiffness, and Structure

Pathological modification of the subendothelial extracellular matrix (ECM) has closely been associated with endothelial activation and subsequent cardiovascular disease progression. To understand regulatory mechanisms of these matrix modifications, the majority of previous efforts have focused on the modulation of either chemical composition or matrix stiffness on 2D smooth surfaces without simultaneously probing their cooperative effects on endothelium function on in vivo like 3D fibrous matrices. To this end, a high-throughput, combinatorial microarray platform on 2D and 3D hydrogel settings to resemble the compositions, stiffness, and structure of healthy and diseased subendothelial ECM has been established, and further their respective and combined effects on endothelial attachment, proliferation, inflammation, and junctional integrity have been investigated. For the first time, the results demonstrate that 3D fibrous structure resembling native ECM is a critical endothelium-protective microenvironmental factor by maintaining the stable, quiescent endothelium with strong resistance to proinflammatory stimuli. It is also revealed that matrix stiffening, in concert with chemical compositions resembling diseased ECM, particularly collagen III, could aggravate activation of nuclear factor kappa B, disruption of endothelium integrity, and susceptibility to proinflammatory stimuli. This study elucidates cooperative effects of various microenvironmental factors on endothelial activation and sheds light on new in vitro model for cardiovascular diseases.

Biomimetic soft fibrous hydrogels for contractile and pharmacologically responsive smooth muscle

The ability to assess changes in smooth muscle contractility and pharmacological responsiveness in normal or pathological-relevant vascular tissue environments is critical to enable vascular drug discovery. However, major challenges remain in both capturing the complexity of in vivo vascular remodeling and evaluating cell contractility in complex, tissue-like environments. Herein, we developed a biomimetic fibrous hydrogel with tunable structure, stiffness, and composition to resemble the native vascular tissue environment. This hydrogel platform was further combined with the combinatory protein array technology as well as advanced approaches to measure cell mechanics and contractility, thus permitting evaluation of smooth muscle functions in a variety of tissue-like microenvironments. Our results demonstrated that biomimetic fibrous structure played a dominant role in smooth muscle function, while the presentation of adhesion proteins co-regulated it to various degrees. Specifically, fibre networks enabled cell infiltration and upregulated expression of actomyosin proteins in contrast to flat hydrogels. Remarkably, fibrous structure and physiologically relevant stiffness of hydrogels cooperatively enhanced smooth muscle contractility and pharmacological responses to vasoactive drugs at both the single cell and intact tissue levels. Together, this study is the first to demonstrate alterations of human vascular smooth muscle contractility and pharmacological responsiveness in biomimetic soft, fibrous environments with a cellular array platform. The integrated platform produced here could enable investigations for pathobiology and pharmacological interventions by developing a broad range of patho-physiologically relevant in vitro tissue models. STATEMENT OF SIGNIFICANCE Engineering functional smooth muscle in vitro holds the great potential for diseased tissue replacement and drug testing. A central challenge is recapitulating the smooth muscle contractility and pharmacological responses given its significant phenotypic plasticity in response to changes in environment. We present a biomimetic fibrous hydrogel with tunable structure, stiffness, and composition that enables the creation of functional smooth muscle tissues in the native-like vascular tissue microenvironment. Such fibrous hydrogel is further combined with the combinatory protein array technology to construct a cellular array for evaluation of smooth muscle phenotype, contraction, and cell mechanics. The integrated platform produced here could be promising for developing a broad range of normal or diseased in vitro tissue models.