Pujiang Shi

Silk Fibroin-Based Complex Particles with Bioactive Encrustation for Bone Morphogenetic Protein 2 Delivery

Application of bone morphogenetic protein 2 (BMP-2) currently faces its challenges, and its efficacy of delivery has to be improved. The proper dosage of the powerful bioactive molecule is still under discussion and needs to be investigated further. In this work, pure silk fibroin particles and particles with calcium carbonate encrustation (complex particles) are designed, developed, and functionalized by BMP-2. These are used to deliver the bioactive molecule to mesenchymal stem cells (MSCs) to induce osteogenic differentiation. Results are compared with those of control groups of BMP-2 carriers under the same condition. Silk fibroin-based particles with size and component variations are prepared by self-assembly, desolvation, and soft template formation to improve BMP-2 loading efficiency. Results show that the particles significantly enhance osteogenic differentiation of MSCs, which is evident in the high ALP enzyme activity as well as the increased level of expression of osteogenic genes. Specifically, the combination of calcium compound and BMP-2 in the silk fibroin-calcium carbonate complex particles synergistically enhances osteogenesis. Release tests and mathematical modeling are applied to describe BMP-2 dissolution profiles, and the release mechanism is based on diffusion and polymer chain relaxation. In summary, the particles show high efficacies of BMP-2 delivery, and introduction of the complex particle can progressively enhance osteogenesis.

In vitro generation of a multilayered osteochondral construct with an osteochondral interface using rabbit bone marrow stromal cells and a silk peptide-based scaffold.

Tissue engineering of a biological osteochondral multilayered construct with a cartilage‐interface subchondral bone layer is a key challenge. This study presented a rabbit bone marrow stromal cell (BMSC)/silk fibroin scaffold‐based co‐culture approach to generate tissue‐engineered osteochondral grafts with an interface. BMSC‐seeded scaffolds were first cultured separately in osteogenic and chondrogenic stimulation media. The two differentiated pieces were then combined using an RADA self‐assembling peptide and subsequently co‐cultured. Gene expression, histological and biochemical analyses were used to evaluate the multilayered structure of the osteochondral graft. A complete osteochondral construct with a cartilage‐subchondral bone interface was regenerated and BMSCs were used as the only cell source for the osteochondral construct and interface regeneration. Furthermore, in the intermediate region of co‐cultured samples, hypertrophic chondrogenic gene markers type X collagen and MMP‐13 were found on both chondrogenic and osteogenic section edges after co‐culture. However, significant differences gene expression profile were found in distinct zones of the construct during co‐culture and the section in the intermediate region had significantly higher hypertrophic chondrocyte gene expression. Biochemical analyses and histology results further supported this observation. This study showed that specific stimulation from osteogenic and chondrogenic BMSCs affected each other in this co‐culture system and induced the formation of an osteochondral interface. Moreover, this system provided a possible approach for generating multilayered osteochondral constructs. Copyright

Enhancing analysis of cells and proteins by fluorescence imaging on silk-based biomaterials: modulating the autofluorescence of silk.

Silk is a versatile and established biomaterial for various tissue engineering purposes. However, it also exhibits strong autofluorescence signals-thereby hindering fluorescence imaging analysis of cells and proteins on silk-derived biomaterials. Sudan Black B (SB) is a lysochrome dye commonly used to stain lipids in histology. It has also been reported to be able to quench autofluorescence of tissues in histology and has been tested on artificial biomedical polymers in recent years. It was hypothesized that SB would exert similar quenching effects on silk, modulating the autofluorescence signals, and thereby enabling improved imaging analysis of cells and molecules of interests. The quenching effect of SB on the intrinsic fluorescence properties of silk and on commercial fluorescent dyes were first investigated in this study. SB was then incorporated into typical fluorescence-based staining protocols to study its effectiveness in improving fluorescence-based imaging of the cells and proteins residing with the silk-based biomaterials. Silk processed into various forms of biomaterials (e.g., films, sponges, fibers, and electrospun mats) was seeded with cells and cultured in vitro. At sacrificial time points, specimens were harvested, fixed, and prepared for fluorescence staining. SB, available commercially as a powder, was dissolved in 70% ethanol (0.3% [w/v]) to form staining solutions. SB treatment was introduced at the last step of typical immunofluorescence staining protocols for 15-120 min. For actin staining protocols by phalloidin toxin, SB staining solutions were added before and after permeabilization with Triton-X for 15-30 min. Results showed that ideal SB treatment duration is about 15 min. Apart from being able to suppress the autofluorescence of silk, this treatment duration was also not too long to adversely affect the fluorescent labeling probes used. The relative improvement brought about by SB treatment was most evident in the blue and green emission wavelengths compared with the red emission wavelength. This study has showed that the use of SB is a cost and time effective approach to enhance fluorescence-based imaging analyses of cell-seeded silk biomaterials, which otherwise would have been hindered by the unmodulated autofluorescence signals.

Ligament-to-bone Interface Tissue Regeneration Using a Functionalized Biphasic Silk Fibroin Scaffold

The emergence of the tissue engineering approach has shown to be a game changer in ligament reconstruction, making it possible to create multi-phasic scaffold structures for multi-specific tissue regeneration. To regenerate the bone-ligament-bone tissue, mimicking hard to soft tissue transition, we propose the use of a functionalized biphasic silk scaffold. Hydroxyapatite nanoparticles (nHA) and bone morphogenetic protein 2 (BMP2) were loaded in the ends of Bombyx mori silk fibroin (SF) scaffold system to enhance enthesis regeneration and bone tunnel healing, while the central onethird supported ligament regeneration. Two groups of biphasic scaffolds, distinguished by the different ends’ additive, were fabricated: nHA only (Ctrl) and n-HA/BMP2 (Exp). A series of bench work, small animal study and large animal preclinical trial was performed using MSC-seeded constructs for the reconstruction of excised ACL. The bioactivity of BMP2 was ascertained and shown to be eluting with an initial burst, followed by a lowered sustained release. Osteogenic genes were upregulated in both groups compared to pure SF. By 24 weeks in vivo, the ACL was regenerated and bone tunnel narrowing was observed with histological evidences indicating new bone and enthesis regeneration in Exp. Better graft to bone integration was observed in Exp compared to Ctrl. From this study, it was demonstrated that the BMP2 eluting biphasic silk scaffold is promising as an advanced tissue engineering treatment modality for complete bone-ligament-bone reconstruction.