Spheroid trilineage differentiation model of primary mesenchymal stem/stromal cells under hypoxia and serum-free culture conditions

Due to their unique properties, human mesenchymal stem/stromal cells (MSCs) possess tremendous potential in regenerative medicine, particularly in cell-based therapies where the multipotency and immunomodulatory characteristics of MSCs can be leveraged to address a variety of disease states. Although MSC-based cell therapeutics have emerged as one of the most promising medical treatments, the clinical translation is hampered by the variability of MSC-based cellular products caused by tissue source-specific differences and the lack of physiological cell culture approaches that closely mimic the human cellular microenvironment. In this study, a model for trilineage differentiation of primary adipose-, bone marrow-, and umbilical cord-derived MSCs into adipocytes, chondrocytes and osteoblasts was established and characterized. Differentiation was performed in spheroid culture, using hypoxic conditions and serum-free and antibiotics-free medium. This platform was characterized for spheroid diameter and trilineage differentiation capacity reflecting functionality of differentiated cells, as indicated by lineage-specific extracellular matrix (ECM) accumulation and expression of distinct secreted markers. The presented model shows spheroid growth during the course of differentiation and successfully supports trilineage differentiation for MSCs from almost all tissue sources except for osteogenesis of umbilical cord-derived MSCs. These findings indicate that this platform provides a suitable and favorable environment for trilineage differentiation of MSCs from various tissue sources. Therefore, it poses a promising model to generate highly relevant biological data urgently required for clinical translation and therefore might be used in the future to generate in vitro microtissues, building blocks for tissue engineering or as disease models

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

Biofabrication of Poly(glycerol sebacate) Scaffolds Functionalized with a Decellularized Bone Extracellular Matrix for Bone Tissue Engineering

The microarchitecture of bone tissue engineering (BTE) scaffolds has been shown to have a direct effect on the osteogenesis of mesenchymal stem cells (MSCs) and bone tissue regeneration. Poly(glycerol sebacate) (PGS) is a promising polymer that can be tailored to have specific mechanical properties, as well as be used to create microenvironments that are relevant in the context of BTE applications. In this study, we utilized PGS elastomer for the fabrication of a biocompatible and bioactive scaffold for BTE, with tissue-specific cues and a suitable microstructure for the osteogenic lineage commitment of MSCs. In order to achieve this, the PGS was functionalized with a decellularized bone (deB) extracellular matrix (ECM) (14% and 28% by weight) to enhance its osteoinductive potential. Two different pore sizes were fabricated (small: 100–150 μm and large: 250–355 μm) to determine a preferred pore size for in vitro osteogenesis. The decellularized bone ECM functionalization of the PGS not only improved initial cell attachment and osteogenesis but also enhanced the mechanical strength of the scaffold by up to 165 kPa. Furthermore, the constructs were also successfully tailored with an enhanced degradation rate/pH change and wettability. The highest bone-inserted small-pore scaffold had a 12% endpoint weight loss, and the pH was measured at around 7.14. The in vitro osteogenic differentiation of the MSCs in the PGS-deB blends revealed a better lineage commitment of the small-pore-sized and 28% (w/w) bone-inserted scaffolds, as evidenced by calcium quantification, ALP expression, and alizarin red staining. This study demonstrates a suitable pore size and amount of decellularized bone ECM for osteoinduction via precisely tailored PGS elastomer BTE scaffolds

Grafting from a Hybrid DNA-covalent polymer by the hybridization chain reaction

\u3cp\u3eNucleic 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.\u3c/p\u3

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

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