Engineering the viscoelasticity of gelatin methacryloyl(GelMA)hydrogels via small“dynamic bridges”to regulate BMSC behaviors for osteochondral regeneration

The dynamic extracellular matrix(ECM)constantly affects the behaviors of cells.To mimic the dynamics of ECM with controllable stiffness and energy dissipation,this study proposes a strategy in which a small molecule,3,4-dihydroxybenzaldehyde(DB),was used as fast"dynamic bridges"to construct viscoelastic gelatin methacryloyl(GelMA)-based hydrogels.The storage modulus and loss modulus of hydrogels were independently adjusted by the covalent crosslinking density and by the number of dynamic bonds.The hydrogels exhibited self-healing property,injectability,excellent adhesion and mechanical properties.Moreover,the in vitro results revealed that the viscous dissipation of hydrogels favored the spreading,proliferation,osteogenesis and chondrogenesis of bone marrow mesenchymal stem cells(BMSCs),but suppressed their adipogenesis.RNA-sequencing and immunofluorescence suggested that the viscous dissipation of hydrogels activated Yes-associated protein(YAP)by stabilizing integrinβ1,and further promoted nuclear translocation of smad2/3 andβ-catenin to enhance chondrogenesis and osteogenesis.As a result,the viscoelastic GelMA hydrogels with highest loss modulus showed best effect in cartilage and subchondral bone repair.Taken together,findings from this study reveal an effective strategy to fabricate viscoelastic hydrogels for modulating the interactions between cells and dynamic ECM to promote tissue regeneration.

Vanillin-based functionalization strategy to construct multifunctional microspheres for treating inflammation and regenerating intervertebral disc

Intervertebral disc degeneration(IVDD)is one of the main causes of low back pain.Although local delivery strategies using biomaterial carriers have shown potential for IVDD treatment,it remains challenging for intervention against multiple adverse contributors by a single delivery platform.In the present work,we propose a new functionalization strategy using vanillin,a natural molecule with anti-inflammatory and antioxidant properties,to develop multifunctional gelatin methacrylate(GelMA)microspheres for local delivery of transforming growth factorβ3(TGFβ3)toward IVDD treatment.In vitro,functionalized microspheres not only improved the release kinetics of TGFβ3 but also effectively inhibited inflammatory responses and promoted the secretion of extracellular matrix(ECM)in lipopolysaccharide-induced nucleus pulposus(NP)cells.In vivo,functionalized platform plays roles in alleviating inflammation and oxidative stress,preserving the water content of NP and disc height,and maintaining intact structure and biomechanical functions,thereby promoting the regeneration of IVD.High-throughput sequencing suggests that inhibition of the phosphatidylinositol 3-kinase(PI3K)-Akt signaling might be associated with their therapeutic effects.In summary,the vanillin-based functionalization strategy provides a novel and simple way for packaging multiple functions into a single delivery platform and holds promise for tissue regeneration beyond the IVD.

Mechanically conditioned cell sheets cultured on thermo-responsive surfaces promote bone regeneration

Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades.However,efficient harvest and handling of cell sheets remain challenging,including insufficient extracellular matrix content and poor mechanical strength.Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types.However,currently,there are no effective ways to apply mechanical loading to cell sheets.In this study,we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide)(PNIPAAm)to poly(dimethylsiloxane)(PDMS)surfaces.The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting.Subsequently,MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates.Upon maturation,the cell sheets were harvested by lowering the temperature.We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning.Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated.After implantation into the critical-sized calvarial defects of mice,the mechanically conditioned cell sheets significantly promoted new bone formation.Findings from this study reveal that thermo-responsive elastomer,together with mechanical conditioning,can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.