A Bioengineering Approach for the Development of Fibroblast Growth Factor-7-Functionalized Sericin Biomaterial Applicable for the Cultivation of Keratinocytes.

Growth factors, including fibroblast growth factor-7 (FGF-7), are a group of proteins that stimulate various cellular processes and are often used with carriers to prevent the rapid loss of their activities. Sericin with great biocompatibility has been investigated as a proteinaceous carrier to enhance the stability of incorporated proteins. The difficulties in obtaining intact sericin from silkworm cocoons and the handling of growth factors with poor stability necessitate an efficient technique to incorporate the protein into a sericin-based biomaterial. Here, we report the generation of a transgenic silkworm line simultaneously expressing and incorporating FGF-7 into cocoon shells containing almost exclusively sericin. Growth-factor-functionalized sericin cocoon shells requiring simple lyophilization and pulverization processes were successfully used to induce the proliferation and migration of keratinocytes. Moreover, FGF-7 incorporated into sericin-cocoon powder exhibited remarkable stability, with more than 70% of bioactivity being retained after being stored as a suspension at 25 °C for 3 months. Transgenic sericin-cocoon powder was used to continuously supply biologically active FGF-7 to generate a three-dimensionally cultured keratinocyte model in vitro. The outcomes of this study propound a feasible approach to producing cytokine-functionalized sericin materials that are ready to use for cell cultivation.

Development of a cypovirus protein microcrystal-encapsulated Bacillus thuringiensis UV-tolerant and mosquitocidal δ-endotoxin.

The δ-endotoxin Cry4Aa from Bacillus thuringiensis israelensis (Bti) has insecticidal characteristics specific to insects of the order Diptera. Although Cry4Aa has shown potential as an effective proteinaceous pesticide against mosquitoes, it has an ultraviolet (UV)-intolerant property that limits its outdoor use. Our previous research showed that protein microcrystal polyhedra from Bombyx mori cypovirus can encapsulate diverse foreign proteins and maintain long-term protein activity under hostile environmental conditions, including UV irradiation. In this study, we report the development of polyhedra encapsulating the Cry4Aa insecticidal activity domain by using a modified baculovirus expression system. We confirmed the oral intake of recombinant polyhedra introduced into the experimental environment by the larvae of a mosquito, Aedes albopictus, and delivery of encapsulated proteins into the digestive tract. The polyhedra encapsulating partial Cry4Aa showed mosquito larvicidal activity during incubation of larvae with 50% lethal-dose value of 23.717×104 cubes for 10 Aedes albopictus larvae in 1 ml water. In addition, polyhedra showed a specific property to reduce the impact of UV-C irradiation on the activity of encapsulated partial Cry4Aa, thus demonstrating the effectiveness of encapsulating Bti δ-endotoxins inside polyhedra to increase the availability of proteinaceous pesticides for outdoor use for mosquito control.

Bioengineered Silkworm for Producing Cocoons with High Fibroin Content for Regenerated Fibroin Biomaterial-Based Applications.

Silk fibroin exhibits high biocompatibility and biodegradability, making it a versatile biomaterial for medical applications. However, contaminated silkworm-derived substances in remnant sericin from the filature and degumming process can result in undesired immune reactions and silk allergy, limiting the widespread use of fibroin. Here, we established transgenic silkworms with modified middle silk glands, in which sericin expression was repressed by the ectopic expression of cabbage butterfly-derived cytotoxin pierisin-1A, to produce cocoons composed solely of fibroin. Intact, nondegraded fibroin can be prepared from the transgenic cocoons without the need for sericin removal by the filature and degumming steps that cause fibroin degradation. A wide-angle X-ray diffraction analysis revealed low crystallinity in the transgenic cocoons. However, nondegraded fibroin obtained from transgenic cocoons enabled the formation of fibroin sponges with varying densities by using 1-5% (v/v) alcohol. The effective chondrogenic differentiation of ATDC5 cells was induced following their cultivation on substrates coated with intact fibroin. Our results showed that intact, allergen-free fibroin can be obtained from transgenic cocoons without the need for sericin removal, providing a method to produce fibroin-based materials with high biocompatibility for biomedical uses.

Effects of transgenic silk materials that incorporate FGF-7 protein microcrystals on the proliferation and differentiation of human keratinocytes.

The silk glands of silkworms produce large quantities of fibroin, which is a protein that can be physically processed and used as a biodegradable carrier for cell growth factors in tissue engineering applications. Meanwhile, protein microcrystals known as polyhedra, which are derived from cypovirus 1, have been used as a vehicle to protect and release encapsulated cell growth factors. We report the generation of transgenic silkworms that express recombinant fibroblast growth factor-7 (FGF-7) fused with the polyhedron-encapsulating signal in polyhedra produced in the middle (MSG) and posterior (PSG) silk glands. Immunofluorescence showed that polyhedra from silk glands are associated with FGF-7. The MSG and PSG from transgenic silkworms were processed into fine powdery materials, from which FGF-7 activity was released to stimulate the proliferation of human keratinocyte epidermal cells. Powders from PSGs exhibited higher FGF-7 activity than those from MSGs. Moreover, PSG powder showed a gradual release of FGF-7 activity over a long period and induced keratinocyte proliferation and differentiation in 3D culture to promote the formation of stratified epidermis expressing positive differentiation marker proteins. Our results indicate that powdery materials incorporating the FGF-7-polyhedra microcrystals from silk glands are valuable for developing cell/tissue engineering applications in vivo and in vitro.

Sustained Neurotrophin Release from Protein Nanoparticles Mediated by Matrix Metalloproteinases Induces the Alignment and Differentiation of Nerve Cells.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.