Synthetic charged proteins and counterionic surfactants were assembled into organofibers with extraordinary mechanical properties to promote bone-derived mesenchymal stem cell osteogenesis differentiation. Synthetic charged proteins and counterionic surfactants were assembled into organofibers with extraordinary mechanical properties to promote bone-derived mesenchymal stem cell osteogenesis differentiation. To date, there is a growing list of hydrogel and gel-derived fibers that have been widely used for tissue engineering, drug delivery systems, wound healing dressings, and wearable devices, boosting the advances in the development of hydrogel.1Zhang Y.S. Khademhosseini A. Advances in engineering hydrogels.Science. 2017; 356: eaaf3627Crossref PubMed Scopus (1126) Google Scholar Yet, despite the popularization of hydrogel, the outcomes of both hydrogel and gel-derived fibers are still unsatisfactory. For example, hydrogel and gel-derived fibers usually have low fracture toughness, storage modulus, and moisture retention, while hydrogel and gel-derived fibers, especially those used in cell biology, need to be equipped with high mechanical properties and moisture retention. Given the fragile mechanical properties of gel-based networks, there is no ideal biodegradable hydrogel with high strength, modulus, and toughness that can be used for tissue regeneration and transformation. Synthetic biology has been widely used to construct engineering bacteria to produce new kinds of proteins with specific functions by integrating computer simulation, genetic engineering, mechanical engineering, and information engineering in recent years.2Zhang J. Liu Y. Sun J. Gu R. Ma C. Liu K. Biological fibers based on naturally sourced proteins: mechanical investigation and applications.Materials Today Advances. 2020; 8: 100095Crossref Scopus (7) Google Scholar Through this strategy, properties of proteins, including the structure, molecular weight, hydrophilic and hydrophobic sites, and supramolecular bond bulk densities such as hydrogen bonds and positive and negative electrical properties can be effectively controlled. Moreover, the introduction of promoter, signal-peptide, and molecular chaperones could also be used to optimize the mechanical properties of expressed proteins.3Kolbe A. del Mercato L.L. Abbasi A.Z. Rivera Gil P. Gorzini S.J. Huibers W.H. Poolman B. Parak W.J. Herrmann A. De novo design of supercharged, unfolded protein polymers, and their assembly into supramolecular aggregates.Macromol. Rapid Commun. 2011; 32: 186-190Crossref PubMed Scopus (35) Google Scholar,4Veeregowda D.H. Kolbe A. van der Mei H.C. Busscher H.J. Herrmann A. Sharma P.K. Recombinant supercharged polypeptides restore and improve biolubrication.Adv. Mater. 2013; 25: 3426-3431Crossref PubMed Scopus (23) Google Scholar Excitingly, proteins with unique properties produced via synthetic biology and protein engineering are expected to manufacture hydrogel and protein organofibers with excellent mechanical properties. Anisotropic protein organofiber is a kind of protein-derived fiber whose internal structure or molecular alignment is arranged in a certain direction. The anisotropic structure endows these protein organofibers with excellent tensile strength and moisture retention. Recently, Dr. Liu and his colleagues synthesized highly charged elastin-like protein (ELP) organofibers with extraordinary mechanical properties via synthetic biology, protein engineering, and a supramolecular assembly strategy for stem cell differentiation.5Ma C. Li B. Shao B. Wu B. Chen D. Su J. Zhang H. Liu K. Anisotropic Protein Organofibers Encoded With Extraordinary Mechanical Behavior for Cellular Mechanobiology Applications.Angew. Chem. Int. Ed. Engl. 2020; 59: 21481-21487Crossref PubMed Scopus (20) Google Scholar These experimental results, for the first time, demonstrated the availability and feasibility of such anisotropic protein organofibers for cellular mechanobiology applications, showing great potential for tissue-regeneration translations. The structured organogel-derived protein fibers were designed by harnessing highly positively or negatively charged ELP and counterionic surfactants. Specifically, lysine (K) or glutamate acid (E) residues were precisely introduced into the fourth position of the characteristic pentapeptide unit (GVGXP)n. Then, the highly positively or negatively charged proteins were produced by engineered E. coli (Figure 1A) . Subsequently, the highly positively or negatively charged ELP proteins were assembled with the counterionic surfactants to form protein organofibers through various weak interactions including electrostatic interaction, cationic π effect, hydrophobic effect, etc. (Figure 1B). Compared with isotropic hydrogel, the obtained anisotropic protein-based organofibers exhibited extraordinary elasticity, which could be greatly stretched and self-heal in 5 s. Moreover, the overall mechanical properties of such anisotropic protein organofibers, including breaking strength, toughness, and elongation, were at least one order of magnitude higher than that of traditional isotropic hydrogels. Excitingly, the excellent mechanical properties of the (E-ELP)144-DEAB-THF fiber prepared in this work remained for as long as 6 months in ambient conditions. Encouraged by the ideal mechanical properties, researchers further investigated the applications of (K-ELP)144-SDBS-THF fibers for cellular mechanobiology and differentiation (Figure 1C). Specifically, arginine-glycine-aspartic acid (RGD) was introduced into the organofibers to facilitate cell adhesion, and then bone-derived mesenchymal stem cells (BMSCs) were seeded on the organofibers. After 1 week of co-culturing, the BMSCs started to adhere to the surface of the organofibers and grew along with the fiber direction. Runt-related transcription factor 2 (RUNX2), which is a transcription factor of osteoblasts and plays an important role in the formation of bone tissue, was used to characterize the osteogenic process. Interestingly, the fiber subgroup presented similar RUNX2 signals to that of typical osteogenic subgroup on plate, indicating that the osteogenesis occurred in the anisotropic protein organofibers. Typical osteogenic subgroups like bio-glasses and ceramics have been widely investigated for bone tissue engineering, but their non-degradability usually impairs their clinical application. Besides, a biomineralization assay was performed to validate the osteogenicity of BMSCs on the highly charged ELP organofibers. After silver staining, the brownish-black reduced silver was clearly observed on the organofibers, indicating that calcium phosphate, the biomarker of osteogenesis, was detected in the highly charged ELP organofibers. Thus, developing anisotropic protein organofibers with excellent mechanical properties as the degradable platform for stem cell differentiation may provide an important new insight in this field. Thus, synthetic biology and a protein engineering strategy provide the possibility of intelligently constructing well-defined proteins that are able to form various hydrogels with excellent properties for different applications. In this work, the anisotropic protein organofibers were developed from highly charged ELP, exhibiting superior strength, modulus, and toughness, as well as long-term stability, exhibiting great advantages of BMSCs differentiating via osteogenesis. Moreover, such anisotropic protein organofibers could be further explored as a universal platform for loading and releasing various kinds of therapeutics, such as transforming growth factor-β and osteogenesis inducible factor. However, before further clinical transformation, the cost of large-scale production of such anisotropic protein organofibers, their stability in physiological conditions, and the potential inflammatory response induced after implantation should be considered. In general, the hydrogel and gel-derived fibers synthesized via synthetic biology, protein engineering, and a supramolecular assembly strategy may expand the application of hydrogel in the field of cell biology, especially in the field of tissue regeneration and transformation.