OBJECTIVE: To assess the efficacy of GelMA hydrogel loaded with bone marrow stem cell-derived exosomes for repairing injured rat knee articular cartilage. METHODS: The supernatant of cultured bone marrow stem cells was subjected to ultracentrifugation separate and extract the exosomes, which were characterized by transmission electron microscopy, particle size analysis and Western blotting of the surface markers. The changes in rheology and electron microscopic features of GelMA hydrogel were examined after loading the exosomes. We assessed exosome release from the hydrogel was detected by BCA protein detection method, and labeled the exosomes with PKH26 red fluorescent dye to observe their phagocytosis by RAW264.7 cells. The effects of the exosomes alone, unloaded hydrogel, and exosome-loaded hydrogel on the polarization of RAW264.7 cells were detected by q-PCR and immunofluorescence assay. We further tested the effect of the exosome-loaded hydrogel on cartilage repair in a Transwell co-culture cell model of RAW264.7 cells and chondrocytes in a rat model of knee cartilage injury using q-PCR and immunofluorescence assay and HE and Masson staining. RESULTS: GelMA hydrogel loaded with exosomes significantly promoted M2-type polarization of RAW264.7 cells (P < 0.05). In the Transwell co-culture model, the exosome-loaded GelMA hydrogel significantly promoted the repair of injured chondrocytes by regulating RAW264.7 cell transformation from M1 to M2 (P < 0.05). HE and Masson staining showed that the exosome-loaded hydrogel obviously promoted cartilage repair in the rat models damage. CONCLUSION GelMA hydrogel loaded with bone marrow stem cell-derived exosomes can significantly promote the repair of cartilage damage in rats by improving the immune microenvironment.
Animals ; Bone Marrow Cells ; Cartilage ; Chondrocytes ; Exosomes ; Hydrogels/metabolism* ; Rats

In recent years, mesenchymal stem cell (MSCs)-derived exosomes have attracted much attention in the field of tissue regeneration. Mesenchymal stem cell-derived exosomes are signaling molecules for communication among cells. They are characterized by natural targeting and low immunogenicity, and are mostly absorbed by cells through the paracrine pathway of mesenchymal stem cells. Moreover, they participate in the regulation and promotion of cell or tissue regeneration. As a scaffold material in regenerative medicine, hydrogel has good biocompatibility and degradability. Combining the two compounds can not only improve the retention time of exosomes at the lesion site, but also improve the dose of exosomes reaching the lesion site by in situ injection, and the therapeutic effect in the lesion area is significant and continuous. This paper summarizes the research results of the interaction of exocrine and hydrogel composite materials to promote tissue repair and regeneration, in order to facilitate research in the field of tissue regeneration in the future.
Hydrogels/metabolism* ; Exosomes/metabolism* ; Wound Healing ; Regenerative Medicine ; Mesenchymal Stem Cells/metabolism*

Extracellular matrix (ECM) stiffness is closely related to the physiological and pathological states of breast tissue. The current study was aimed to investigate the effect of silk fibroin/collagen composite hydrogels with adjustable matrix stiffness on the growth and phenotype of normal breast epithelial cells. In this study, the enzymatic reaction of horseradish peroxidase (HRP) with hydrogen peroxide (H₂O₂) was used to change the degree of cross-linking of the silk fibroin solution. The rotational rheometer was used to characterize the composite hydrogel's biomechanical properties. Human normal mammary epithelial cell line MCF-10A were inoculated into composite hydrogels with various stiffness (19.10-4 932.36 Pa) to construct a three dimensional (3D) culture system of mammary epithelial cells. The CCK-8 assay was applied to detect the cell proliferation rate and active states in each group. Hematoxylin-Eosin (HE) staining and whole-mount magenta staining were used for histological evaluation of cell morphology and distribution. The results showed that with the increase of matrix stiffness, MCF-10A cells exhibited inhibited proliferation rate, decreased formation of acinus structures and increased branching structures. Meanwhile, with the increase of matrix stiffness, the polarity of MCF-10A cells was impeded. And the increase of matrix stiffness up-regulated the expression levels of mmp-2, mmp-3, and mmp-9 in MCF-10A cells. Among the genes related to epithelial-mesenchymal transition (EMT), the expression level of the epithelial marker gene E-cadherin was significantly down-regulated, while the interstitial cell marker gene Vimentin was up-regulated, and the expression levels of Snail, Wnt5b and Integrin β1 in the Wnt pathway were up-regulated. These results suggest that the silk fibroin/collagen composite hydrogels with adjustable matrix stiffness regulates the proliferation and the phenotype of MCF-10A cells. The effects of increased matrix stiffness may be closely related to the changes of the polar structures and function of MCF-10A cells, as well as the occurrence of ECM-remodeling and EMT.
Collagen/metabolism* ; Epithelial Cells/metabolism* ; Fibroins/pharmacology* ; Humans ; Hydrogels/metabolism* ; Hydrogen Peroxide ; Phenotype

Silk fibroin, a natural fibrin, is a suitable matrix biomaterial for wound repair due to its unique properties such as good biocompatibility, tunable biodegradation and mechanical properties, low host inflammatory response, low cost, ease of fabrication, etc. Silk fibroin can be used alone or in combination with other materials to construct various dressings including scaffolds, hydrogels, films, smart mats, and microneedles, which can meet the needs of different wound repair and regulate the wound repair process. Thus, the application research of silk fibroin in skin tissue engineering has increased dramatically. Compared with other natural materials, silk fibroin promotes tissue regeneration and wound repair by improving cell proliferation, migration, and differentiation behavior at different stages, showing unique advantages in different dimensions. Based on the development of silk fibroin wound repair materials in the recent years, this review focuses on the mechanism and application prospect of silk fibroin and its composite materials in wound repair.
Fibroins/metabolism* ; Biocompatible Materials/therapeutic use* ; Tissue Engineering ; Hydrogels ; Fibrin ; Tissue Scaffolds

OBJECTIVE: To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. METHODS: Firstly, hyaluronic acid was oxidized with NaIO ₄ and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young's modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: FTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young's modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point ( P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group ( P<0.05). CONCLUSION The MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.
Hydrogels ; Hyaluronic Acid/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Tissue Engineering/methods* ; Cell Differentiation ; Myoblasts/metabolism* ; Cell Proliferation

Hyaluronic acid (HA) is an important biomaterial as the extracellular matrix in human body. We produced HA by fermentation of Streptococcus iniae (Strep.). Production of HA by Strep. was evaluated and further improved by strain mutation by ultraviolet. One strain with higher HA yield and lower content of protein was obtained. Its HA yield increased from (82.3±3.3) mg/L to (120±10.6) mg/L, and protein decreased from (0.178±0.011) mg/L to (0.032±0.017) mg/L. The molecular weight (MW) of HA yield from Strep. is about 3.0×10⁵ Da. Using the method of freezing and thawing, HA aqueous solution was transferred into hydrogel. This HA hydrogel, casted on sterilized non-woven fabric, was applied to repair rabbit skin with full-thickness defect. The preliminary results of the animal tests displayed that HA hydrogel obviously reduced the inflammation around the wound and promoted the skin regeneration comparing with the control tests.
Animals ; Biocompatible Materials ; Fermentation ; Hyaluronic Acid ; biosynthesis ; Hydrogels ; Molecular Weight ; Rabbits ; Regeneration ; Skin ; drug effects ; growth & development ; Streptococcus iniae ; metabolism ; Wound Healing

Peptide RADA can undergo spontaneous assembly into well ordered nanofibers and hydrogels in its water solution. In this work, a variety of proteins, including IGF-1, aFGF and VEGF with different molecular weight and isoelectric points, were chosen and encapsulated within the RADA peptide hydrogel. UV-vis spectroscopy was used to determine the concentration of the released proteins in the solution. The release kinetics suggested that protein diffusion through nanofiber hydrogels depended primarily on the size of the protein and the density of the peptide nanofiber. Circular dichroism (CD) spectroscopy indicated that the encapsulation and release by RADA hydrogel did not affect the secondary structure of the proteins studied.
Circular Dichroism ; Delayed-Action Preparations ; Fibroblast Growth Factor 1 ; metabolism ; Hydrogels ; chemistry ; Insulin-Like Growth Factor I ; metabolism ; Nanofibers ; Peptides ; chemistry ; Protein Structure, Secondary ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts(ROB) in collagen hydrogel three-dimensional culture.

METHODSROB were obtained by enzyme digestion from the segregated neonatal SD rats skull and were embedded in 2 mg/mL rat tail collagen for three-dimensional culture. The growth state of ROB was observed by FDA/PI staining, HE staining and scanning electron microscopy. ROB were treated with icariin at the concentration of 1 × 10⁻⁴, 1 × 10⁻⁵, 1 × 10⁻⁶ and 1 × 10⁻⁷ mol/L respectively. The activity of alkaline phosphatase(ALP) was detected after 3, 6, 9 d of icariin treatment. Three-dimensional cultured ROB were treated with optimal concentration icariin for 12, 24, 36, 48 h and total RNA was extracted and the mRNA expressions of bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX-2) and Osterix were detected by real time RT-PCR. The protein expression of BMP-2, RUNX-2 and Osterix were examined by Western-blotting.

RESULTSROB were cultured in collagen hydrogel successfully. FDA/PI staining, HE staining, and scanning electron microscopy showed that ROB adhered with collagen tightly and distributed homogeneously. Icariin at final concentration of 1 × 10⁻⁵, 1 × 10⁻⁶ and 1×10⁻⁷ mol/L all enhanced the activity of ALP of collagen hydrogel three-dimensional cultured ROB, and 1 × 10⁻⁶ mol/L was the optimal concentration. Besides, icariin (1 × 10⁻⁶ mol/L) increased mRNA and protein expression of BMP-2、RUNX-2 and Osterix compared to control group.

CONCLUSIONIcariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly.

Alkaline Phosphatase ; metabolism ; Animals ; Bone Morphogenetic Protein 2 ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Collagen ; chemistry ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Drugs, Chinese Herbal ; Flavonoids ; pharmacology ; Hydrogels ; chemistry ; Osteoblasts ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley ; Skull ; cytology ; Transcription Factors ; metabolism