Time-dependent anabolic response of hMSC-derived cartilage grafts to hydrostatic pressure

It is generally accepted that the application of hydrostatic pressure (HP) is beneficial for MSC chondrogenesis. There is, however, evidence to suggest that the timing of application might determine its impact on cell fate and tissue development. Furthermore, understanding how the maturity of engineered cartilage affects its response to the application of HP can provide critical insight into determining when such a graft is ready for in vivo implantation into a mechanically loaded environment. In this study, we systematically examined chondrogenic maturation of hMSCs over 35 days in the presence of TGF-β3 in vitro. At specific timepoints, the response of hMSCs to the application of HP following the removal of TGF-β3 was assessed; this partially models conditions such grafts will experience in vivo upon implantation. In free swelling culture, the expression of chondrogenic (COL2A1 and ACAN) and hypertrophic (COL10A1) markers increased with time. At early timepoints, the expression of such markers continued to increase following TGF-β3 withdrawal; however, this was not observed after prolonged periods of chondrogenic priming (35 days). Interestingly, the application of HP was only beneficial after 35 days of chondrogenic priming, where it enhanced sGAG synthesis and improved key chondrogenic gene ratios. It was also found that HP can facilitate a metabolic shift towards oxidative phosphorylation, which can be viewed as a hallmark of successfully differentiating MSCs. These results point to the importance of mechanical loading as a key stimulus for maintaining a chondrogenic phenotype once MSCs are removed from chemically defined culture conditions. </p

Time-dependent anabolic response of hMSC-derived cartilage grafts to hydrostatic pressure

It is generally accepted that the application of hydrostatic pressure (HP) is beneficial for MSC chondrogenesis. There is, however, evidence to suggest that the timing of application might determine its impact on cell fate and tissue development. Furthermore, understanding how the maturity of engineered cartilage affects its response to the application of HP can provide critical insight into determining when such a graft is ready for in vivo implantation into a mechanically loaded environment. In this study, we systematically examined chondrogenic maturation of hMSCs over 35 days in the presence of TGF-β3 in vitro. At specific timepoints, the response of hMSCs to the application of HP following the removal of TGF-β3 was assessed; this partially models conditions such grafts will experience in vivo upon implantation. In free swelling culture, the expression of chondrogenic (COL2A1 and ACAN) and hypertrophic (COL10A1) markers increased with time. At early timepoints, the expression of such markers continued to increase following TGF-β3 withdrawal; however, this was not observed after prolonged periods of chondrogenic priming (35 days). Interestingly, the application of HP was only beneficial after 35 days of chondrogenic priming, where it enhanced sGAG synthesis and improved key chondrogenic gene ratios. It was also found that HP can facilitate a metabolic shift towards oxidative phosphorylation, which can be viewed as a hallmark of successfully differentiating MSCs. These results point to the importance of mechanical loading as a key stimulus for maintaining a chondrogenic phenotype once MSCs are removed from chemically defined culture conditions. </p

Hydrogels from TEMPO-Oxidized Nanofibrillated Cellulose Support <i>In Vitro</i> Cultivation of Encapsulated Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are the most prominent type of adult stem cells for clinical applications. Three-dimensional (3D) cultivation of MSCs in biomimetic hydrogels provides a more physiologically relevant cultivation microenvironment for in vitro testing and modeling, thus overcoming the limitations of traditional planar cultivation methods. Cellulose nanofibers are an excellent candidate biomaterial for synthesis of hydrogels for this application, due to their biocompatibility, tunable properties, availability, and low cost. Herein, we demonstrate the capacity of hydrogels prepared from 2,2,6,6-tetramethylpiperidine-1-oxyl -oxidized and subsequently individualized cellulose-nanofibrils to support physiologically relevant 3D in vitro cultivation of human MSCs at low solid contents (0.2–0.5 wt %). Our results show that MSCs can spread, proliferate, and migrate inside the cellulose hydrogels, while the metabolic activity and proliferative capacity of the cells as well as their morphological characteristics benefit more in the lower bulk cellulose concentration hydrogels

Integrating melt electrowriting and fused deposition modeling to fabricate hybrid scaffolds supportive of accelerated bone regeneration

Emerging additive manufacturing (AM) strategies can enable the engineering of hierarchal scaffold structures for guiding tissue regeneration. Here, the advantages of two AM approaches, melt electrowriting (MEW) and fused deposition modelling (FDM), are leveraged and integrated to fabricate hybrid scaffolds for large bone defect healing. MEW is used to fabricate a microfibrous core to guide bone healing, while FDM is used to fabricate a stiff outer shell for mechanical support, with constructs being coated with pro-osteogenic calcium phosphate (CaP) nano-needles. Compared to MEW scaffolds alone, hybrid scaffolds prevent soft tissue collapse into the defect region and support increased vascularization and higher levels of new bone formation 12 weeks post-implantation. In an additional group, hybrid scaffolds are also functionalized with BMP2 via binding to the CaP coating, which further accelerates healing and facilitates the complete bridging of defects after 12 weeks. Histological analyses demonstrate that such scaffolds support the formation of well-defined annular bone, with an open medullary cavity, smooth periosteal surface, and no evidence of abnormal ectopic bone formation. These results demonstrate the potential of integrating different AM approaches for the development of regenerative biomaterials, and in particular, demonstrate the enhanced bone healing outcomes possible with hybrid MEW-FDM constructs. </p

Integrating melt electrowriting and fused deposition modeling to fabricate hybrid scaffolds supportive of accelerated bone regeneration

Emerging additive manufacturing (AM) strategies can enable the engineering of hierarchal scaffold structures for guiding tissue regeneration. Here, the advantages of two AM approaches, melt electrowriting (MEW) and fused deposition modelling (FDM), are leveraged and integrated to fabricate hybrid scaffolds for large bone defect healing. MEW is used to fabricate a microfibrous core to guide bone healing, while FDM is used to fabricate a stiff outer shell for mechanical support, with constructs being coated with pro-osteogenic calcium phosphate (CaP) nano-needles. Compared to MEW scaffolds alone, hybrid scaffolds prevent soft tissue collapse into the defect region and support increased vascularization and higher levels of new bone formation 12 weeks post-implantation. In an additional group, hybrid scaffolds are also functionalized with BMP2 via binding to the CaP coating, which further accelerates healing and facilitates the complete bridging of defects after 12 weeks. Histological analyses demonstrate that such scaffolds support the formation of well-defined annular bone, with an open medullary cavity, smooth periosteal surface, and no evidence of abnormal ectopic bone formation. These results demonstrate the potential of integrating different AM approaches for the development of regenerative biomaterials, and in particular, demonstrate the enhanced bone healing outcomes possible with hybrid MEW-FDM constructs. </p

Grafting from a Hybrid DNA–Covalent Polymer by the Hybridization Chain Reaction

Nucleic acid–polymer conjugates are an attractive class of materials endowed with tunable and responsive character. Herein, we exploit the dynamic character of nucleic acids in the preparation of hybrid DNA–covalent polymers with extendable grafts by the hybridization chain reaction. Addition of DNA hairpins to an initiator DNA–dextran graft copolymer resulted in the growth of the DNA grafts as evidenced by various characterization techniques over several length scales. Additionally, aggregation of the initiator DNA–graft copolymer before the hybridization chain reaction was observed resulting in the formation of kinetically trapped aggregates several hundreds of nanometers in diameter that could be disrupted by a preheating step at 60 °C prior to extension at room temperature. Materials of increasing viscosity were rapidly formed when metastable DNA hairpins were added to the initiator DNA-dextran grafted copolymer with increasing concentration of the components in the mixture. This study shows the potential for hierarchical self-assembly of DNA-grafted polymers through the hybridization chain reaction and opens the door for biomedical applications where viscosity can be used as a readout