Intervertebral disc human nucleus pulposus cells associated with back pain trigger neurite outgrowth in vitro and pain behaviors in rats.

Low back pain (LBP) is often associated with the degeneration of human intervertebral discs (IVDs). However, the pain-inducing mechanism in degenerating discs remains to be elucidated. Here, we identified a subtype of locally residing human nucleus pulposus cells (NPCs), generated by certain conditions in degenerating discs, that was associated with the onset of discogenic back pain. Single-cell transcriptomic analysis of human tissues showed a strong correlation between a specific cell subtype and the pain condition associated with the human degenerated disc, suggesting that they are pain-triggering. The application of IVD degeneration-associated exogenous stimuli to healthy NPCs in vitro recreated a pain-associated phenotype. These stimulated NPCs activated functional human iPSC-derived sensory neuron responses in an in vitro organ-chip model. Injection of stimulated NPCs into the healthy rat IVD induced local inflammatory responses and increased cold sensitivity and mechanical hypersensitivity. Our findings reveal a previously uncharacterized pain-inducing mechanism mediated by NPCs in degenerating IVDs. These findings could aid in the development of NPC-targeted therapeutic strategies for the clinically unmet need to attenuate discogenic LBP.

Modulus-dependent effects on neurogenic, myogenic, and chondrogenic differentiation of human mesenchymal stem cells in three-dimensional hydrogel cultures.

Human mesenchymal stromal cells (hMSCs) are of significant interest as a renewable source of therapeutically useful cells. In tissue engineering, hMSCs are implanted within a scaffold to provide enhanced capacity for tissue repair. The present study evaluates how mechanical properties of that scaffold can alter the phenotype and genotype of the cells, with the aim of augmenting hMSC differentiation along the myogenic, neurogenic or chondrogenic linages. The hMSCs were grown three-dimensionally (3D) in a hydrogel comprised of poly(ethylene glycol) (PEG)-conjugated to fibrinogen. The hydrogel's shear storage modulus (G'), which was controlled by increasing the amount of PEG-diacrylate cross-linker in the matrix, was varied in the range of 100-2000 Pascal (Pa). The differentiation into each lineage was initiated by a defined culture medium, and the hMSCs grown in the different modulus hydrogels were characterized using gene and protein expression. Materials having lower storage moduli (G' = 100 Pa) exhibited more hMSCs differentiating to neurogenic lineages. Myogenesis was favored in materials having intermediate modulus values (G' = 500 Pa), whereas chondrogenesis was favored in materials with a higher modulus (G' = 1000 Pa). Enhancing the differentiation pathway of hMSCs in 3D hydrogel scaffolds using simple modifications to mechanical properties represents an important achievement toward the effective application of these cells in tissue engineering.

Methacrylated fibrinogen hydrogels for 3D cell culture and delivery.

Methacrylation was performed on fibrinogen to design a new biomedical hydrogel for 3D cell culture or as a biodegradable delivery matrix for in vivo implantation. The methacrylation of denatured fibrinogen in solution was performed using methacrylic anhydride (MAA). The extent of fibrinogen methacrylation was quantified by proton NMR and controlled using stochiometric quantities of MAA during the reaction. The methacrylated fibrinogen (FibMA) hydrogels were formed by light-activated free-radical polymerization in the presence of macromolecular cross-linking polymers made from acrylated poly(ethylene glycol) (PEG). The biocompatibility and biodegradability of the FibMA hydrogels were characterized by in vitro assays and in vivo implantation experiments using quantitative magnetic resonance imaging (MRI) of the implant volume. The FibMA supported the growth and metabolic activity of human dermal fibroblasts in both 2D and 3D cultures. The methacrylation did not alter important biological attributes of the fibrinogen, including the ability to support cell adhesion and 3D cell culture, as well as to undergo proteolysis. Animal experiments confirmed the biodegradability of the FibMA for potential use as a scaffold in tissue engineering, as a bioink for 3D bioprinting, or as a biodegradable matrix for in vivo sustained delivery of bioactive factors. STATEMENT OF SIGNIFICANCE: This paper describes methacrylated fibrinogen (FibMA) and the formation of a biomedical hydrogel from FibMA for cell culture and other biomedical applications. Inspired from methacrylated gelatin (GelMA), the FibMA is made from blood-derived fibrinogen which is more suitable for clinical use. Sharing similar properties to other hydrogels made from methacrylated proteins, the FibMA has yet to be reported in the literature. In this manuscript, we provide the methodology to produce the FibMA hydrogels, we document the mechanical versatility of this new biomaterial, and we show the biocompatibility using 3D cell culture studies and in vivo implantations.

3D Bioprinting of Engineered Tissue Flaps with Hierarchical Vessel Networks (VesselNet) for Direct Host-To-Implant Perfusion.

Engineering hierarchical vasculatures is critical for creating implantable functional thick tissues. Current approaches focus on fabricating mesoscale vessels for implantation or hierarchical microvascular in vitro models, but a combined approach is yet to be achieved to create engineered tissue flaps. Here, millimetric vessel-like scaffolds and 3D bioprinted vascularized tissues interconnect, creating fully engineered hierarchical vascular constructs for implantation. Endothelial and support cells spontaneously form microvascular networks in bioprinted tissues using a human collagen bioink. Sacrificial molds are used to create polymeric vessel-like scaffolds and endothelial cells seeded in their lumen form native-like endothelia. Assembling endothelialized scaffolds within vascularizing hydrogels incites the bioprinted vasculature and endothelium to cooperatively create vessels, enabling tissue perfusion through the scaffold lumen. Using a cuffing microsurgery approach, the engineered tissue is directly anastomosed with a rat femoral artery, promoting a rich host vasculature within the implanted tissue. After two weeks in vivo, contrast microcomputer tomography imaging and lectin perfusion of explanted engineered tissues verify the host ingrowth vasculature's functionality. Furthermore, the hierarchical vessel network (VesselNet) supports in vitro functionality of cardiomyocytes. Finally, the proposed approach is expanded to mimic complex structures with native-like millimetric vessels. This work presents a novel strategy aiming to create fully-engineered patient-specific thick tissue flaps.

Alginate hydrogel beads embedded with drug-bearing polycaprolactone microspheres for sustained release of paclobutrazol.

In recent years there has been a growing demand for the development of agrochemical controlled release (CR) technologies. In the present study, we aimed to create a novel agricultural CR device using two polymeric systems that have been predominantly employed in biomedical applications: beads of alginate hydrogel embedded with drug-bearing Polycaprolactone (PCL) microspheres. The combined device utilizes the advantages of each polymer type for biodegradation and controlled release of Paclobutrazol (PBZ), a common growth retardant in plants. Surface morphology of the alginate beads was characterized by scanning electron microscopy (SEM) and water immersion tests were performed for stability and controlled release measurements. Bioassays were performed both in accelerated laboratory conditions and in field conditions. The results showed a capability to control the size of PBZ-loaded PCL microspheres through modification of homogenization speed and emulsifier concentration. Enlargement of PCL microsphere size had an adverse effect on release of PBZ from the alginate device. The growth of oatmeal plants as a model system was affected by the controlled release of PBZ. The preliminary field experiment observed growth retardation during two consecutive rainy seasons, with results indicating a substantial benefit of the sustained growth inhibition through the controlled release formulation. The final product has the potential to be used as a carrier for different substances in the agrochemical industry.

Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model.

Introduction: As many as 80% of the adult population experience back pain at some point in their lifetimes. Previous studies have indicated a link between back pain and intervertebral disc (IVD) degeneration. Despite decades of research, there is an urgent need for robust stem cell therapy targeting underlying causes rather than symptoms. It has been proposed that notochordal cells (NCs) appear to be the ideal cell type to regenerate the IVD: these cells disappear in humans as they mature, are replaced by nucleus pulposus (NP) cells, and their disappearance correlates with the initiation of degeneration of the disc. Human NCs are in short supply, thus here aimed for generation of notochordal-like cells from induced pluripotent cells (iPSCs). Methods: Human iPSCs were generated from normal dermal fibroblasts by transfecting plasmids encoding for six factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. Then the iPSCs were treated with GSK3i to induce differentiation towards Primitive Streak Mesoderm (PSM). The differentiation was confirmed by qRT-PCR and immunofluorescence. PSM cells were transfected with Brachyury (Br)-encoding plasmid and the cells were encapsulated in Tetronic-tetraacrylate-fibrinogen (TF) hydrogel that mimics the NP environment (G'=1kPa), cultured in hypoxic conditions (2% O2) and with specifically defined growth media. The cells were also tested in vivo in a large animal model. IVD degeneration was induced after an annular puncture in pigs, 4 weeks later the cells were injected and IVDs were analyzed at 12 weeks after the injury using MRI, gene expression analysis and histology. Results: After short-term exposure of iPSCs to GSK3i there was a significant change in cell morphology, Primitive Streak Mesoderm (PSM) markers (Brachyury, MIXL1, FOXF1) were upregulated and markers of pluripotency (Nanog, Oct4, Sox2) were downregulated, both compared to the control group. PSM cells nucleofected with Br (PSM-Br) cultured in TF hydrogels retained the NC phenotype consistently for up to 8 weeks, as seen in the gene expression analysis. PSM-Br cells were co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs) which, with time, expressed the NC markers in higher levels, however the levels of expression in BM-MSCs alone did not change. Higher expression of NC and NP marker genes in human BM-MSCs was found to be induced by iNC-condition media (iNC-CM) than porcine NC-CM. The annular puncture induced IVD degeneration as early as 2 weeks after the procedure. The injected iNCs were detected in the degenerated discs after 8 weeks in vivo. The iNC-treated discs were found protected from degeneration. This was evident in histological analysis and changes in the pH levels, indicative of degeneration state of the discs, observed using qCEST MRI. Immunofluorescence stains show that their phenotype was consistent with the in vitro study, namely they still expressed the notochordal markers Keratin 18, Keratin 19, Noto and Brachyury. Conclusion: In the present study, we report a stepwise differentiation method to generate notochordal cells from human iPSCs. These cells not only demonstrate a sustainable notochordal cell phenotype in vitro and in vivo, but also show the functionality of notochordal cells and have protective effect in case of induced disc degeneration and prevent the change in the pH level of the injected IVDs. The mechanism of this effect could be suggested via the paracrine effect on resident cells, as it was shown in the in vitro studies with MSCs.

Tailoring supramolecular guest-host hydrogel viscoelasticity with covalent fibrinogen double networks.

Supramolecular chemistry has enabled the design of tunable biomaterials that mimic the dynamic and viscoelastic characteristics of the extracellular matrix. However, the noncovalent nature of supramolecular bonds renders them inherently weak, limiting their applicability to many biomedical applications. To address this, we formulated double network (DN) hydrogels through a combination of supramolecular and covalent networks to tailor hydrogel viscoelastic properties. Specifically, DN hydrogels were formed through the combination of supramolecular guest-host (GH) hyaluronic acid (HA) networks with covalent networks from the photocrosslinking of acrylated poly(ethylene glycol) modified fibrinogen (PEG-fibrinogen) and PEG diacrylate. DN hydrogels exhibited higher compressive moduli, increased failure stresses, and increased toughness when compared to purely covalent networks. While GH concentration had little influence on the compressive moduli across DN hydrogels, an increase in the GH concentration resulted in more viscous behavior of DN hydrogels. High viability of encapsulated bovine mesenchymal stromal cells (MSCs) was observed across groups with enhanced spreading and proliferation in DN hydrogels with increased GH concentration. This combination of supramolecular and covalent chemistries enables the formation of dynamic hydrogels with tunable properties that can be customized towards repair of viscoelastic tissues.

Using bimodal MRI/fluorescence imaging to identify host angiogenic response to implants.

Therapies that promote angiogenesis have been successfully applied using various combinations of proangiogenic factors together with a biodegradable delivery vehicle. In this study we used bimodal noninvasive monitoring to show that the host response to a proangiogenic biomaterial can be drastically affected by the mode of implantation and the surface area-to-volume ratio of the implant material. Fluorescence/MRI probes were covalently conjugated to VEGF-bearing biodegradable PEG-fibrinogen hydrogel implants and used to document the in vivo degradation and liberation of bioactive constituents in an s.c. rat implantation model. The hydrogel biodegradation and angiogenic host response with three types of VEGF-bearing implant configurations were compared: preformed cylindrical plugs, preformed injectable microbeads, and hydrogel precursor, injected and polymerized in situ. Although all three were made with identical amounts of precursor constituents, the MRI data revealed that in situ polymerized hydrogels were fully degraded within 2 wk; microbead degradation was more moderate, and plugs degraded significantly more slowly than the other configurations. The presence of hydrogel degradation products containing the fluorescent label in the surrounding tissues revealed a distinct biphasic release profile for each type of implant configuration. The purported in vivo VEGF release profile from the microbeads resulted in highly vascularized s.c. tissue containing up to 16-fold more capillaries in comparison with controls. These findings demonstrate that the configuration of an implant can play an important role not only in the degradation and resorption properties of the materials, but also in consequent host angiogenic response.

Bis(1,3-di-tert-butylimidazolin-2-iminato) titanium complexes as effective catalysts for the monodisperse polymerization of propylene.

The use of bis(1,3-di-tert-butylimidazolin-2-iminato) titanium dichloride (1) and dimethyl (2) complexes in the polymerization of propylene is presented. The complexes were activated using different amounts of methylalumoxane (MAO), giving in each case a very active catalytic mixture and producing polymers with a narrow molecular weight distribution (polydispersity = 1.10). The use of the cocatalyst triphenylcarbenium (trityl) tetra(pentafluorophenyl)borate totally inhibits the reaction, producing the corresponding bis(1,3-di-tert-butylimidazolin-2-iminato) titanium(III) methyl complex, the trityl radical ((•)CPh(3)), the anionic MeB(C(6)F(5))(4)(-), B(C(6)F(5))(3), and the bis(1,3-di-tert-butylimidazolin-2-iminato) titanium(IV) dimethyl·B(C(6)F(5))(3) complex. The use of a combination of physical methods such as NMR, ESR-C(60), and MALDI-TOF analyses enabled us to propose a plausible mechanism for the polymerization of propylene, presenting that the polymerization is mainly carried out in a living fashion. In addition, we present a slow equilibrium toward a small amount of a dormant species responsible for 2,1-misinsertions and chain transfer processes.