Stiffness-dependent dynamic effect of inflammation on keratocyte phenotype and differentiation.

Although extensive studies have evaluated the regulation effect of microenvironment on cell phenotype and cell differentiation, further investigations in the field of the cornea are needed to gain sufficient knowledge for possible clinical translation. This study aims to evaluate the regulation effects of substrate stiffness and inflammation on keratocyte phenotype of corneal fibroblasts, as well as the differentiation from stem cells towards keratocytes. Soft and stiff substrates were prepared based on polydimethylsiloxane. HTK and stem cells were cultured on these substrates to evaluate the effects of stiffness. The possible synergistic effects between substrate stiffness and inflammatory factor IL-1ßwere examined by qPCR and immunofluorescence staining. In addition, macrophages were cultured on soft and stiff substrates to evaluate the effect of substrate stiffness on the synthesis of inflammatory factors. The conditioned medium of macrophages (Soft-CM and Stiff-CM) was collected to examine the effects on HTK and stem cells. It was found that inflammatory factor IL-1ßpromoted keratocyte phenotype and differentiation when cells were cultured on soft substrate (∼130 kPa), which were different from cells cultured on stiff substrate (∼2 × 103kPa) and TCP (∼106kPa). Besides, macrophages cultured on stiff substrates had significantly higher expression ofIL-1ßandTnf-αas compared to the cells cultured on soft substrates. And Stiff-CM decreased the expression of keratocyte phenotype markers as compared to Soft-CM. The results of our study indicate a stiffness-dependent dynamic effect of inflammation on keratocyte phenotype and differentiation, which is of significance not only in gaining a deeper knowledge of corneal pathology and repair, but also in being instructive for scaffold design in corneal tissue engineering and ultimate regeneration.

Advances in Regulatory Strategies of Differentiating Stem Cells towards Keratocytes.

Corneal injury is a commonly encountered clinical problem which led to vision loss and impairment that affects millions of people worldwide. Currently, the available treatment in clinical practice is corneal transplantation, which is limited by the accessibility of donors. Corneal tissue engineering appears to be a promising alternative for corneal repair. However, current experimental strategies of corneal tissue engineering are insufficient due to inadequate differentiation of stem cell into keratocytes and thus cannot be applied in clinical practice. In this review, we aim to clarify the role and effectiveness of both biochemical factors, physical regulation, and the combination of both to induce stem cells to differentiate into keratocytes. We will also propose novel perspectives of differentiation strategy that may help to improve the efficiency of corneal tissue engineering.

The application of human periodontal ligament stem cells and biomimetic silk scaffold for in situ tendon regeneration.

BACKGROUND: With the development of tissue engineering, enhanced tendon regeneration could be achieved by exploiting suitable cell types and biomaterials. The accessibility, robust cell amplification ability, superior tendon differentiation potential, and immunomodulatory effects of human periodontal ligament stem cells (hPDLSCs) indicate their potential as ideal seed cells for tendon tissue engineering. Nevertheless, there are currently no reports of using PDLSCs as seed cells. Previous studies have confirmed the potential of silk scaffold for tendon tissue engineering. However, the biomimetic silk scaffold with tendon extracellular matrix (ECM)-like structure has not been systematically studied for in situ tendon regeneration. Therefore, this study aims to evaluate the effects of hPDLSCs and biomimetic silk scaffold on in situ tendon regeneration. METHODS: Human PDLSCs were isolated from extracted wisdom teeth. The differentiation potential of hPDLSCs towards osteo-, chondro-, and adipo-lineage was examined by cultured in different inducing media. Aligned and random silk scaffolds were fabricated by the controlled directional freezing technique. Scaffolds were characterized including surface structure, water contact angle, swelling ratio, degradation speed and mechanical properties. The biocompatibility of silk scaffolds was evaluated by live/dead staining, SEM observation, cell proliferation determination and immunofluorescent staining of deposited collagen type I. Subsequently, hPDLSCs were seeded on the aligned silk scaffold and transplanted into the ruptured rat Achilles tendon. Scaffolds without cells served as control groups. After 4 weeks, histology evaluation was carried out and macrophage polarization was examined to check the repair effects and immunomodulatory effects. RESULTS: Human PDLSCs were successfully isolated, and their multi-differentiation potential was confirmed. Compared with random scaffold, aligned silk scaffold had more elongated and aligned pores and promoted the proliferation and ordered arrangement of hPDLSCs. After implantation into rat Achilles tendon defect, hPDLSCs seeded aligned silk scaffold enhanced tendon repair with more tendon-like tissue formation after 4 weeks, as compared to the scaffold-only groups. Higher expression of CD206 and lower expression of iNOS, IL-1ß and TNF-α were found in the hPDLSCs seeded aligned silk scaffold group, which revealed its modulation effect of macrophage polarization from M1 to M2 phenotype. CONCLUSIONS: In summary, this study demonstrates the efficacy of hPDLSCs as seed cells and aligned silk scaffold as a tendon-mimetic scaffold for enhanced tendon tissue engineering, which may have broad implications for future tendon tissue engineering and regenerative medicine researches.

Enzymatically crosslinked silk-nanosilicate reinforced hydrogel with dual-lineage bioactivity for osteochondral tissue engineering.

Osteochondral defects are characterized by damage to both articular cartilage and subchondral bone. Various tissue engineering strategies have been developed for osteochondral defect repair. However, strong mechanical properties and dual-lineage (osteogenesis and chondrogenesis) bioactivity still pose challenges for current biomaterial design. Silicate nanoclay has been reported to improve the mechanical properties and biofunctionality of polymer systems, but its effect on in vitro dual-lineage differentiation or in vivo osteochondral regeneration has not been extensively investigated before. Here, a novel enzymatically crosslinked silk fibroin (SF)-Laponite (LAP) nanocomposite hydrogel was fabricated and evaluated for osteochondral regeneration. The incorporation of a small amount of LAP (1% w/v) accelerated the gelation process of SF and greatly enhanced the mechanical properties and hydrophilicity of the hydrogel. In vitro investigations showed that the developed SF-LAP hydrogel was biocompatible and was able to induce osteogenic and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), validated by Alizarin red/Alcian blue staining, qPCR, and immunofluorescent staining. During an 8-week implantation into rabbit full-thickness osteochondral defects, the SF-LAP hydrogel promoted the simultaneous and enhanced regeneration of cartilage and subchondral bone. The repaired tissue in the chondral region was constituted mainly of hyaline cartilage with typical chondrocyte morphology and cartilaginous extracellular matrix (ECM). These findings suggested that the SF-LAP nanocomposite hydrogel developed in this study served as a promising biomaterial for osteochondral regeneration due to its mechanical reinforcement and dual-lineage bioactivity.

Cell-Free Biomimetic Scaffold with Cartilage Extracellular Matrix-Like Architectures for In Situ Inductive Regeneration of Osteochondral Defects.

The development of a biomimetic scaffold designed to provide a native extracellular matrix (ECM)-like microenvironment is a potential strategy for cartilage repair. The ECM in native articular cartilage is structurally composed of three different architectural zones, i.e., horizontally aligned, randomly arranged, and vertically aligned collagen fibers. However, the effects of scaffolds with these three different ECM-like architectures on in vivo cartilage regeneration are not clear. In this study, we aim to systematically investigate and compare their in situ inductive regenerative efficacy on cartilage defects. ECM-mimetic silk fibroin scaffolds with horizontally aligned, vertically aligned, and random pore architectures are fabricated using the controlled directional freezing technique. All of these scaffolds exhibit similar pore area, swelling ratio, and in vitro degradation behavior. Nevertheless, the aligned scaffolds have a higher pore aspect ratio and hydrophilicity, and increase the proliferation of bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. When implanted into rabbit osteochondral defects, the scaffold with vertically aligned pore architectures provides a more cell-favorable microenvironment conducive to endogenous BMSCs than other scaffolds and supports the simultaneous regeneration of cartilage and subchondral bone. These findings indicate that scaffolds with vertically aligned ECM-like architectures serve as an effective cell-free and growth factor-free scaffold for enhanced endogenous osteochondral regeneration.

Sustained Release of TPCA-1 from Silk Fibroin Hydrogels Preserves Keratocyte Phenotype and Promotes Corneal Regeneration by Inhibiting Interleukin-1ß Signaling.

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies that exists. Many studies focus on the pro-inflammatory and pro-angiogenic effects of interleukin-1ß (IL-1ß) on corneal wound healing. However, the effect of IL-1ß on keratocyte phenotype and corneal repair, as well as the underlying mechanisms, is not clear. This study reports, for the first time, that IL-1ß induces phenotype changes of keratocytes in vitro, by significantly down-regulating the gene and protein expression levels of keratocyte markers (Keratocan, Lumican, Aldh3a1 and CD34). Furthermore, it is found that the NF-κB pathway is involved in the IL-1ß-induced changes of keratocyte phenotype, and that the selective IKKß inhibitor TPCA-1, which inhibits NF-κB, can preserve keratocyte phenotype under IL-1ß simulated pathological conditions in vitro. By using a murine model of corneal injury, it is shown that sustained release of TPCA-1 from degradable silk fibroin hydrogels accelerates corneal wound healing, improves corneal transparency, enhances the expression of keratocyte markers, and supports the regeneration of well-organized epithelium and stroma. These findings provide insights not only into the pathophysiological mechanisms of corneal wound healing, but also into the potential development of new treatments for patients with corneal injuries.

An all-silk-derived functional nanosphere matrix for sequential biomolecule delivery and in situ osteochondral regeneration.

Endogenous repair of osteochondral defect is usually limited by the insufficient number of cells in the early stage and incomplete cell differentiation in the later stage. The development of drug delivery systems for sequential release of pro-migratory and pro-chondrogenic molecules to induce endogenous bone marrow-derived mesenchymal stem cells (BMSCs) recruitment and chondrogenic differentiation is highly desirable for in situ osteochondral regeneration. In this study, a novel, all-silk-derived sequential delivery system was fabricated by incorporating the tunable drug-loaded silk fibroin (SF) nanospheres into a SF porous matrix. The loading efficiency and release kinetics of biomolecules depended on the initial SF/polyvinyl alcohol (PVA) concentrations (0.2%, 1% and 5%) of the nanospheres, as well as the hydrophobicity of the loaded molecules, resulting in controllable and programmed delivery profiles. Our findings indicated that the 5% nanosphere-incorporated matrix showed a rapid release of E7 peptide during the first 120 h, whereas the 0.2% nanosphere-incorporated matrix provided a slow and sustained release of Kartogenin (KGN) longer than 30 days. During in vitro culture of BMSCs, this functional SF matrix incorporated with E7/KGN nanospheres showed good biocompatibility, as well as enhanced BMSCs migration and chondrogenic differentiation through the release of E7 and KGN. Furthermore, when implanted into rabbit osteochondral defect, the SF nanosphere matrix with sequential E7/KGN release promoted the regeneration of both cartilage and subchondral bone. This work not only provided a novel all-silk-derived drug delivery system for sequential release of molecules, but also a functional tissue-engineered scaffold for osteochondral regeneration.