Cryo-self-assembled silk fibroin sponge as a biodegradable platform for enzyme-responsive delivery of exosomes.

Although advances in protein assembly preparation have provided a new platform for drug delivery during tissue engineering, achieving long-term controlled exosome delivery remains a significant challenge. Diffusion-dominated exosome release using protein hydrogels results in burst release of exosomes. Here, a fibroin-based cryo-sponge was developed to provide controlled exosome release. Fibroin chains can self-assemble into silk I structures under ice-cold conditions when annealed above the glass transition temperature. Exosome release is enzyme-responsive, with rates primarily determined by enzymatic degradation of the scaffolds. In vivo experiments have demonstrated that exosomes remain in undigested sponge material for two months, superior to their retention in fibrin glue, a commonly used biomaterial in clinical practice. Fibroin cryo-sponges were implanted subcutaneously in nude mice. The exosome-containing sponge group exhibited better neovascularization and tissue ingrowth effects, demonstrating the efficacy of this exosome-encapsulating strategy by realizing sustained release and maintaining exosome bioactivity. These silk fibroin cryo-sponges containing exosomes provide a new platform for future studies of exosome therapy.

Comparison of three different acidic solutions in tendon decellularized extracellular matrix bio-ink fabrication for 3D cell printing.

Decellularized extracellular matrix (dECM) hydrogels are being increasingly investigated for use in bio-inks for three-dimensional cell printing given their good cytocompatibility and biomimetic properties. The osmotic pressure and stiffness of bio-ink are important factors affecting the biological functions of printed cells. However, little attention has been given to the osmotic pressure and stiffness of the dECM bio-inks. Here, we compared three types of commonly used acidic solutions in the bio-fabrication of a tendon derived dECM bio-ink for 3D cell printing (0.5 M acetic acid, 0.1 M hydrochloric acid and 0.02 M hydrochloric acid). We found that low pH value of 0.1 M hydrochloric acid could accelerate the digestion process for dECM powders. This could lead to a much softer dECM hydrogel with storage modulus less than 100 Pa. This soft dECM hydrogel facilitated the spreading and proliferation of stem cells encapsulated within it. It also showed better tendon-inducing ability compared with two others much stiffer dECM hydrogels. However, this over-digested dECM hydrogel was more unstable as it could shrink with the culture time going on. For 0.5 M acetic acid made dECM bio-ink, the hyperosmotic state of the bio-ink led to much lower cellular viability rates. Postprocess (Dilution or dialysis) to tailor the osmotic pressure of hydrogels could be a necessary step before mixed with cells. Thus, kindly choosing the type and concentration of acidic solution is necessary for dECM bio-ink preparation. And a balance should be made between the digestion period, strength of acidic solution, as well as the size and concentration of the dECM powders. STATEMENT OF SIGNIFICANCE: The dECM bio-ink has been widely used in 3D cell printing for tissue engineering and organ modelling. In this study, we found that different types of acid have different digestion and dissolution status for the dECM materials. A much softer tendon derived dECM hydrogel with lower stiffness could facilitate the cellular spreading, proliferation and tendon differentiation. We also demonstrated that the osmotic pressure should be taken care of in the preparation of dECM bio-ink with 0.5 M acetic acid. Thus, kindly choosing the type and concentration of acidic solution is necessary for dECM bio-ink preparation.

Stepwise Cross-Linking of Fibroin and Hyaluronic for 3D Printing Flexible Scaffolds with Tunable Mechanical Properties.

The development of 3D printing techniques has provided a promising platform to study tissue engineering and mechanobiology; however, the pursuit of printability limits the possibility of tailoring scaffolds' mechanical properties. The brittleness of those scaffolds also hinders potential clinical application. To overcome these drawbacks, a double-network ink composed of only natural biomaterials is developed. A shear-thinning hydrogel made of silk fibroin (SF) and methacrylated hyaluronic acid (MAHA) presents a high mechanical modulus with a low concentration of macromers. The physical cross-linking due to protein folding further increases the strength of the scaffolds. The proposed SF/MAHA scaffold exhibits a storage modulus 10 times greater than that of methacrylated gelatin scaffold, along with better flexibility and biodegradation. The synergistic effect between fibroin and hyaluronic allows us to tailor the mechanical strength of scaffolds without compromising their printability. The hierarchy porous structure of the SF/MAHA scaffolds offers a better spatial microenvironment for the migration and proliferation of cells compared to gelatin scaffolds. For the first time, this strategy achieves 3D printing of natural biomaterials with controlled mechanical characteristics by manipulating the cross-linking of peptide chains. The design of such ductile scaffolds with hydrolysis resistance provides a new platform for the mechanobiology research. It also shows promise in the tissue engineering of musculoskeletal system where structural strength is needed.

The tissue origin effect of extracellular vesicles on cartilage and bone regeneration.

Direct implantation of mesenchymal stem cells (MSCs) for cartilage and bone tissue engineering faces challenges, such as immune rejection and loss of cellular viability or functionality. As nanoscale natural particles, exosomes or small extracellular vesicles (EVs) of MSCs have potential to circumvent these problems. It is significant to investigate the impact of the tissue origin of MSCs on the therapeutic bioactivity of their corresponding EVs for cartilage and bone regeneration. Here, rat MSCs isolated from the adipose, bone marrow, and synovium are cultured to obtain their corresponding EVs (ADSC-EVs, BMSC-EVs, and SMSC-EVs, respectively). The ADSC-EVs stimulate the migration, proliferation, and chondrogenic and osteogenic differentiation of BMSCs in vitro as well as cartilage and bone regeneration in a mouse model more than the BMSC-EVs or SMSC-EVs. Proteomics analysis reveals that the tissue origin contributes to the distinct protein profiles among the three types of EVs, which induced cartilage and bone regenerative capacities by potential mechanisms of regulating signaling pathways including focal adhesion, ECM-receptor interaction, actin cytoskeleton, cAMP, and PI3K-Akt signaling pathways. Consequently, these findings provide insight that the adipose may be a superior candidate in EV-based nanomedicine for cartilage and bone regeneration. STATEMENT OF SIGNIFICANCE: Extracelluar vesicles (EVs) of mesenchymal stem cells (MSCs) have been considered as a promising approach in cartilage and bone tissue engineering. In this study, for the first time, we investigated the tissue origin effect of EVs on chondrogenesis and osteogenesis of MSCs in vitro and in vivo. The results demonstrated that EVs of adipose-derived MSCs showed the most efficiency. Meanwhile, protein proteomics revealed the potential mechanisms. We provide a novel evidence that the adipose is a superior reservoir in EV-based nanotechnologies and biomaterials for cartilage and bone regeneration.

Recent Progress in 3D Printing of Elastic and High-Strength Hydrogels for the Treatment of Osteochondral and Cartilage Diseases.

Despite considerable progress for the regenerative medicine, repair of full-thickness articular cartilage defects and osteochondral interface remains challenging. This low efficiency is largely due to the difficulties in recapitulating the stratified zonal architecture of articular cartilage and engineering complex gradients for bone-soft tissue interface. This has led to increased interest in three-dimensional (3D) printing technologies in the field of musculoskeletal tissue engineering. Printable and biocompatible hydrogels are attractive materials for 3D printing applications because they not only own high tunability and complexity, but also offer favorable biomimetic environments for live cells, such as porous structure, high water content, and bioactive molecule incorporation. However, conventional hydrogels are usually mechanically weak and brittle, which cannot reach the mechanical requirements for repair of articular cartilage defects and osteochondral interface. Therefore, the development of elastic and high-strength hydrogels for 3D printing in the repairment of cartilage defects and osteochondral interface is crucial. In this review, we summarized the recent progress in elastic and high-strength hydrogels for 3D printing and categorized them into six groups, namely ion bonds interactions, nanocomposites integrated in hydrogels, supramolecular guest-host interactions, hydrogen bonds interactions, dynamic covalent bonds interactions, and hydrophobic interactions. These 3D printed elastic and high-strength hydrogels may provide new insights for the treatment of osteochondral and cartilage diseases.

Digestion degree is a key factor to regulate the printability of pure tendon decellularized extracellular matrix bio-ink in extrusion-based 3D cell printing.

Improving the printability of pure, decellularized extracellular matrix (dECM) bio-ink without altering its physiological components has been a challenge in three-dimensional (3D) cell printing. To improve the printability of the bio-ink, we first investigated the digestion process of the powdered dECM material obtained from porcine tendons. We manifested the digestion process of tendon derived dECM powders, which includes dissolution, gelatinization and solubilization. After a short dissolution period (around 10 min), we observed a 'High viscosity slurry' status (3 h) of the dECM precursors, i.e. the gelatinization process, followed by the solubilization processes, i.e. a 'Medium viscosity slurry' period (12 h) and a 'Low viscosity slurry' period (72 h). The 'Medium viscosity slurry' status of the dECM bio-ink was inhomogeneous and could not be extruded out from the barrel after the pH value was neutralized to 7.4. Although the 'Low viscosity slurry' status of the dECM bio-ink has been reported to be extrudable, it has poor printability. This study explores the printability of the 'High viscosity slurry' status of the dECM bio-ink, which has not been addressed thus far. The results demonstrate that this less digested status of the dECM bio-ink yields higher shape fidelity and stacking accuracy than the traditional over-digested status of the dECM bio-ink; this indicates better printability of this less digested dECM bio-ink. We compared the performance of the two bio-inks using cell viability tests for 3D cell printing. Bone marrow mesenchymal stem cells derived from rats was printed using the 'High viscosity slurry' status of the dECM bio-ink, yielding high cellular viability lasting for 7 d after printing. Thus, the 'High viscosity slurry' status of tendon dECM bio-ink can be utilized to fabricate complicated 3D organoid structures; it also shows promise for applications such as regenerative medicine and biomimetic tissue engineering.

Biomechanically, structurally and functionally meticulously tailored polycaprolactone/silk fibroin scaffold for meniscus regeneration.

Meniscus deficiency, the most common and refractory disease in human knee joints, often progresses to osteoarthritis (OA) due to abnormal biomechanical distribution and articular cartilage abrasion. However, due to its anisotropic spatial architecture, complex biomechanical microenvironment, and limited vascularity, meniscus repair remains a challenge for clinicians and researchers worldwide. In this study, we developed a 3D printing-based biomimetic and composite tissue-engineered meniscus scaffold consisting of polycaprolactone (PCL)/silk fibroin (SF) with extraordinary biomechanical properties and biocompatibility. We hypothesized that the meticulously tailored composite scaffold could enhance meniscus regeneration and cartilage protection. Methods: The physical property of the scaffold was characterized by scanning electron microscopy (SEM) observation, degradation test, frictional force of interface assessment, biomechanical testing, and fourier transform infrared (FTIR) spectroscopy analysis. To verify the biocompatibility of the scaffold, the viability, morphology, proliferation, differentiation, and extracellular matrix (ECM) production of synovium-derived mesenchymal stem cell (SMSC) on the scaffolds were assessed by LIVE/DEAD staining, alamarBlue assay, ELISA analysis, and qRT-PCR. The recruitment ability of SMSC was tested by dual labeling with CD29 and CD90 by confocal microscope at 1 week after implantation. The functionalized hybrid scaffold was then implanted into the meniscus defects on rabbit knee joint for meniscus regeneration, comparing with the Blank group (no scaffold) and PS group. The regenerated meniscus tissue was evaluated by histological and immunohistochemistry staining, and biomechanical test. Macroscopic and histological scoring was performed to assess the outcome of meniscus regeneration and cartilage protection in vivo. Results: The combination of SF and PCL could greatly balance the biomechanical properties and degradation rate to match the native meniscus. SF sponge, characterized by fine elasticity and low interfacial shear force, enhanced energy absorption capacity of the meniscus and improved chondroprotection. The SMSC-specific affinity peptide (LTHPRWP; L7) was conjugated to the scaffold to further increase the recruitment and retention of endogenous SMSCs. This meticulously tailored scaffold displayed superior biomechanics, structure, and function, creating a favorable microenvironment for SMSC proliferation, differentiation, and extracellular matrix (ECM) production. After 24 weeks of implantation, the histological assessment, biochemical contents, and biomechanical properties demonstrated that the polycaprolactone/silk fibroin-L7 (PS-L7) group was close to the native meniscus group, showing significantly better cartilage protection than the PS group. Conclusion: This tissue engineering scaffold could greatly strengthen meniscus regeneration and chondroprotection. Compared with traditional cell-based therapies, the meniscus tissue engineering approach with advantages of one-step operation and reduced cost has a promising potential for future clinical and translational studies.

Investigation on the Structure and Mechanical Properties of Highly Tunable Elastomeric Silk Fibroin Hydrogels Cross-Linked by γ-Ray Radiation.

Silk fibroin (SF) is a natural polymer with low immunogenicity and good biocompatibility. However, most silk-based hydrogels formed through chemical or physical cross-linking are brittle, the preparation of which also inevitably introduces cytotoxic cross-linking agents. Herein, a simple strategy is presented for synthesizing SF hydrogels with tunable mechanical properties by combining γ-ray radiation with ethanol treatment. Neither toxic initiators nor chemical agents are utilized during the whole preparation procedure. For "soft" hydrogels, the compressive moduli are less than 29 kPa (SF-S hydrogels), while for "tough" hydrogels, the compressive moduli are between 1.21 and 2.41 MPa (SF-D hydrogels). Specifically, γ-ray radiation makes SF form uniform and stable chemical cross-linking sites within and between molecular chains, resulting in "soft" and highly elastic SF hydrogels. The physical cross-linking via ethanol treatment leads to the self-assembly of fibroin chains, transforming those soft hydrogels to tough hydrogels. These double cross-linked SF hydrogels (SF-D hydrogels) exhibit excellent mechanical strength. Effects of various cross-linking conditions on the secondary structure, pore structure, mechanical properties, gelation degree, swelling, and in vitro degradation properties are explored. A series of cell experiments demonstrate that the SF hydrogels with different mechanical strength can stimulate the expression of specific genes of rat bone marrow mesenchymal stem cells (BMSCs) in various differentiation directions. These results also show the application prospects in tissue engineering by customizing hydrogels for the mechanical strength of different tissues.

Advances in Porous Scaffold Design for Bone and Cartilage Tissue Engineering and Regeneration.

IMPACT STATEMENT: Challenges in musculoskeletal tissue regeneration affect millions of patients globally. Scaffolds for tissue engineering bone and cartilage provide promising solutions that increase healing and decrease need for complicated surgical procedures. Porous scaffolds have emerged as an attractive alternative to traditional scaffolds. However, the success of advanced materials, use of biological factors, and manufacturing techniques can vary depending on use case. This review provides perspective on porous scaffold manufacturing, characterization and application, and can be used to inform future scaffold design.

Laser-assisted Rapid Mineralization of Human Tooth Enamel.

The human body has difficulty repairing damaged dental enamel, an acellular hard tissue. Researchers have sought feasible biomimicry strategies to repair enamel defects; however, few have been successfully translated to clinical applications. In this study, we propose a new method for achieving rapid enamel mineralization under a near-physiological environment. Through treatment with a laser and chelating agents, 15 µm crystals could be grown compactly on an enamel substrate in less than 20 min. The compact crystal layer had similar structure as native enamel prisms and high elastic modulus. This layer also had the potential for further remineralization in saliva. The benefit of using laser can not only speed up the mineralization, but also control the crystal growth precisely where in need. A mechanism for how laser and chelating agents synergistically function is also proposed. This strategy offers a possibility for enamel-biomimicking repair in dental clinics.

Structurally and Functionally Optimized Silk-Fibroin-Gelatin Scaffold Using 3D Printing to Repair Cartilage Injury In Vitro and In Vivo.

Articular cartilage repair remains a great challenge for clinicians and researchers. Recently, there emerges a promising way to achieve one-step cartilage repair in situ by combining endogenic bone marrow stem cells (BMSCs) with suitable biomaterials using a tissue engineering technique. To meet the increasing demand for cartilage tissue engineering, a structurally and functionally optimized scaffold is designed, by integrating silk fibroin with gelatin in combination with BMSC-specific-affinity peptide using 3D printing (3DP) technology. The combination ratio of silk fibroin and gelatin greatly balances the mechanical properties and degradation rate to match the newly formed cartilage. This dually optimized scaffold has shown superior performance for cartilage repair in a knee joint because it not only retains adequate BMSCs, due to efficient recruiting ability, and acts as a physical barrier for blood clots, but also provides a mechanical protection before neocartilage formation and a suitable 3D microenvironment for BMSC proliferation, differentiation, and extracellular matrix production. It appears to be a promising biomaterial for knee cartilage repair and is worthy of further investigation in large animal studies and preclinical applications. Beyond knee cartilage, this dually optimized scaffold may also serve as an ideal biomaterial for the regeneration of other joint cartilages.

Synthesis of Europium-Doped Fluorapatite Nanorods and Their Biomedical Applications in Drug Delivery.

Europium (Eu)-doped fluorapatite (FA) nanorods have a biocompatibility similar to that of hydroxyapatite (HA) for use as cell imaging biomaterials due to their luminescent property. Here, we discuss the new application of europium-doped fluorapatite (Eu-FA) nanorods as an anticancer drug carrier. The Eu-FA nanorods were prepared by using a hydrothermal method. The morphology, crystal structure, fluorescence, and composition were investigated. The specific crystal structure enables the effective loading of drug molecules. Doxorubicin (DOX), which was used as a model anticancer drug, effectively loaded onto the surface of the nanorods. The DOX release was pH-dependent and occurred more rapidly at pH 5.5 than at pH 7.4. The intracellular penetration of the DOX-loaded Eu-FA nanorods (Eu-FA/DOX) can be imaged in situ due to the self-fluorescence property. Treatment of melanoma A375 cells with Eu-FA/DOX elicited a more effective apoptosis rate than direct DOX treatment. Overall, Eu-FA exhibits potential for tracking and treating tumors and may be potentially useful as a multifunctional carrier system to effectively load and sustainably deliver drugs.