Julie C. Liu

Characterization of Resilin-Based Materials for Tissue Engineering Applications

Modular proteins have emerged as powerful tools in tissue engineering because both the mechanical and biochemical properties can be precisely controlled through amino acid sequence. Resilin is an attractive candidate for use in modular proteins because it is well-known for having low stiffness, high fatigue lifetime, and high resilience. However, no studies have been conducted to assess resilins compressive properties, cytocompatibility with clinically relevant cells, or effect on cell spreading. We designed a modular protein containing repeating sequences of a motif derived from Anopheles gambiae and cell-binding domains derived from fibronectin. Rapid cross-linking with tris(hydroxymethyl)phosphine was observed. The hydrogels had a complex modulus of 22 ± 1 kPa and yield strain of 63%. The elastic modulus in compression, or unconfined compressive modulus, was 2.4 ± 0.2 MPa, which is on the same order as human cartilage. A LIVE/DEAD assay demonstrated that human mesenchymal stem cells cultured on the resilin-based protein had a viability of 95% after three days. A cell-spreading assay revealed that the cells interacted with the fibronectin-derived domain in a sequence-specific manner and resulted in a mean cell area ~1.4-fold larger than when cells were seeded on a sequence-scrambled negative control protein. These results demonstrate that our resilin-based biomaterial is a promising biomaterial for cartilage tissue engineering.

Incorporating the BMP-2 peptide in genetically-engineered biomaterials accelerates osteogenic differentiation

Protein-based biomaterials have emerged as powerful tools for tissue engineering applications. Recombinant DNA techniques can be used to precisely tune material properties at the molecular level, and multiple peptide modules can be incorporated into a single material. Here, we genetically engineered biomaterials that incorporate a peptide derived from bone morphogenetic protein-2 (BMP-2) and investigated whether the BMP-2 peptide, within the context of these materials, can promote stem cells to produce bone matrix and synergize with a cell-binding sequence (RGD). Our study is the first to demonstrate that when the BMP-2 peptide is incorporated within the backbone of protein-based biomaterials, it is active and accelerates osteogenic differentiation of mesenchymal stem cells. In particular, cells seeded on proteins containing the BMP-2 peptide had increased levels of alkaline phosphatase (AP) activity, calcium deposition, and expression of bone-related genes. However, the BMP-2 peptide did not synergize with the RGD cell-binding domain within the context of these protein-based materials. Overall, these results suggest that incorporation of the BMP-2 peptide within the context of modular proteins is a successful strategy for engineering biomaterials for applications in bone tissue engineering.

Synthesis and Characterization of Recombinant Abductin-Based Proteins

Recombinant proteins are promising tools for tissue engineering and drug delivery applications. Protein-based biomaterials have several advantages over natural and synthetic polymers, including precise control over amino acid composition and molecular weight, modular swapping of functional domains, and tunable mechanical and physical properties. In this work, we describe recombinant proteins based on abductin, an elastomeric protein that is found in the inner hinge of bivalves and functions as a coil spring to keep shells open. We illustrate, for the first time, the design, cloning, expression, and purification of a recombinant protein based on consensus abductin sequences derived from Argopecten irradians . The molecular weight of the protein was confirmed by mass spectrometry, and the protein was 94% pure. Circular dichroism studies showed that the dominant structures of abductin-based proteins were polyproline II helix structures in aqueous solution and type II β-turns in trifluoroethanol. Dynamic light scattering studies illustrated that the abductin-based proteins exhibit reversible upper critical solution temperature behavior and irreversible aggregation behavior at high temperatures. A LIVE/DEAD assay revealed that human umbilical vein endothelial cells had a viability of 98 ± 4% after being cultured for two days on the abductin-based protein. Initial cell spreading on the abductin-based protein was similar to that on bovine serum albumin. These studies thus demonstrate the potential of abductin-based proteins in tissue engineering and drug delivery applications due to the cytocompatibility and its response to temperature.

Cytocompatibility studies of a biomimetic copolymer with simplified structure and high‐strength adhesion

The development of adhesives suitable for biomedical applications has been challenging given that these materials must exhibit sufficient adhesion strengths and biocompatibility. Biomimetic materials inspired by mussel adhesive proteins appear to contain many of the necessary characteristics for biomedical adhesives. In particular, poly[(3,4-dihydroxystyrene)-co-styrene] has been shown to be a high strength adhesive material with bonding comparable to or even greater than several commercial glues. Herein, a thorough study on the cytocompatibility of this copolymer provides insights on the suitability of a mussel-mimicking adhesive for applications development. The cytotoxicity of poly[(3,4-dihydroxystyrene)-co-styrene] was evaluated through assessment of the viability, proliferation rate, and morphology of NIH/3T3 fibroblasts when cultured with copolymer extracts or directly in contact with the adhesive. After 1 and 3 days of culture, both the copolymer alone and copolymer cross-linked with periodate exhibited minimal effects on cell viability. Likewise, cells cultured on the copolymer displayed proliferation rates and morphologies similar to cells on the poly-L-lysine control. These results indicate that poly[(3,4-dihydroxystyrene)-co-styrene] is highly cytocompatible and therefore a promising material for use where biological contact is important.

Modular protein domains: an engineering approach toward functional biomaterials

Protein domains and peptide sequences are a powerful tool for conferring specific functions to engineered biomaterials. Protein sequences with a wide variety of functionalities, including structure, bioactivity, protein-protein interactions, and stimuli responsiveness, have been identified, and advances in molecular biology continue to pinpoint new sequences. Protein domains can be combined to make recombinant proteins with multiple functionalities. The high fidelity of the protein translation machinery results in exquisite control over the sequence of recombinant proteins and the resulting properties of protein-based materials. In this review, we discuss protein domains and peptide sequences in the context of functional protein-based materials, composite materials, and their biological applications.

Protein-engineered microenvironments can promote endothelial differentiation of human mesenchymal stem cells in the absence of exogenous growth factors

Peripheral artery disease often requires treatments with vascular grafts for vessel reconstruction. Endothelialization of the vascular grafts is important to achieve long-term patency because endothelial cells regulate thrombosis, inflammation, and growth of smooth muscle cells. One potential source of endothelial cells is human mesenchymal stem cells (hMSCs), which can be routinely differentiated towards the endothelial lineage using exogenous growth factors such as vascular endothelial growth factor (VEGF). However, there are few studies that investigate the effect of materials on endothelial differentiation in the absence of growth factors. This study demonstrates that exogenous growth factors are not needed to achieve endothelial differentiation of hMSCs and that protein-based microenvironments promote endothelial differentiation. Specifically, we genetically engineered proteins containing a VEGF-mimicking peptide and resilin repeats and demonstrated that cells grown on the protein-engineered microenvironments were viable, had normal metabolic activity, and displayed increased endothelial-specific markers and endothelial function compared to negative control cells. In particular, cells cultured on our proteins formed networks that were statistically equivalent to positive control cells. We confirmed that the mere presence of protein on surfaces was insufficient to promote endothelial differentiation of hMSCs, and the specific composition of the RZ-QK protein appeared to be necessary for promoting differentiation. Thus, our protein-based materials are promising tools for obtaining endothelial cells for use in vascular grafts.

Critical factors for the bulk adhesion of engineered elastomeric proteins

Many protein-based materials, such as soy and mussel adhesive proteins, have been the subject of scientific and commercial interest. Recently, a variety of protein adhesives have been isolated from diverse sources such as insects, frogs and squid ring teeth. Many of these adhesives have similar amino acid compositions to elastomeric proteins such as elastin. Although elastin is widely investigated for a structural biomaterial, little work has been done to assess its adhesive potential. In this study, recombinant elastin-like polypeptides were created to probe the factors affecting adhesion strength. Lap shear adhesion was used to examine the effects of both extrinsic factors (pH, concentration, cross-linker, humidity, cure time and cure temperature) and intrinsic factors (protein sequence, structure and molecular weight). Of the extrinsic factors tested, only humidity, cure time and cure temperature had a significant effect on adhesion strength. As water content was reduced, adhesion strength increased. Of the intrinsic factors tested, amino acid sequence did not significantly affect adhesion strength, but less protein structure and higher molecular weights increased adhesion strength directly. The strengths of proteins in this study (greater than 2 MPa) were comparable to or higher than those of two commercially available protein-based adhesives, hide glue and a fibrin sealant. These results may provide general rules for the design of adhesives from elastomeric proteins.

Supplementary material from "Critical factors for the bulk adhesion of engineered elastomeric proteins"

Many protein-based materials, such as soy and mussel adhesive proteins, have been the subject of scientific and commercial interest. Recently, a variety of protein adhesives have been isolated from diverse sources such as insects, frogs and squid ring teeth. Many of these adhesives have similar amino acid compositions to elastomeric proteins such as elastin. Although elastin is widely investigated for a structural biomaterial, little work has been done to assess its adhesive potential. In this study, recombinant elastin-like polypeptides were created to probe the factors affecting adhesion strength. Lap shear adhesion was used to examine the effects of both extrinsic factors (pH, concentration, cross-linker, humidity, cure time and cure temperature) and intrinsic factors (protein sequence, structure and molecular weight). Of the extrinsic factors tested, only humidity, cure time and cure temperature had a significant effect on adhesion strength. As water content was reduced, adhesion strength increased. Of the intrinsic factors tested, amino acid sequence did not significantly affect adhesion strength, but less protein structure and higher molecular weights increased adhesion strength directly. The strengths of proteins in this study (greater than 2 MPa) were comparable to or higher than those of two commercially available protein-based adhesives, hide glue and a fibrin sealant. These results may provide general rules for the design of adhesives from elastomeric proteins.