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Nanofibrous spongy microspheres to deliver rabbit mesenchymal stem cells and anti-miR-199a to regenerate nucleus pulposus and prevent calcification.

ABSTRACT: Lower back pain is mainly caused by intervertebral disc degeneration, in which calcification is frequently involved. Here novel nanofibrous spongy microspheres (NF-SMS) are used to carry rabbit bone marrow mesenchymal stromal cells (MSCs) to regenerate nucleus pulposus tissues. NF-SMS are shown to significantly enhance the MSC seeding, proliferation and differentiation over control microcarriers. Furthermore, a hyperbranched polymer (HP) with negligible cytotoxicity and high microRNA (miRNAs) binding affinity is synthesized. The HP can complex with anti-miR-199a and self-assemble into "double shell" polyplexes which are able to achieve high transfection efficiency into MSCs. A double-emulsion technique is used to encapsulate these polyplexes in biodegradable nanospheres (NS) to enable sustained anti-miR-199 delivery. Our results demonstrate that MSC/HP-anti-miR-199a/NS/NF-SMS constructs can promote the nucleus pulposus (NP) phenotype and resist calcification in vitro and in a subcutaneous environment. Furthermore, injection of MSC/HP-anti-miR-199a/NS/NF-SMS can stay in place, produce functional extracellular matrix, maintain disc height and prevent intervertebral disc (IVD) calcification in a rabbit lumbar degeneration model.

SUBMITTER: Feng G

PROVIDER: S-EPMC7423691 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Dental pulp infection and necrosis are widespread diseases. Conventional endodontic treatments result in a devitalized and weakened tooth. In this work, we synthesized novel star-shaped polymer to self-assemble into unique nanofibrous spongy microspheres (NF-SMS), which were used to carry human dental pulp stem cells (hDPSCs) into the pulp cavity to regenerate living dental pulp tissues. It was found that NF-SMS significantly enhanced hDPSCs attachment, proliferation, odontogenic differentiation and angiogenesis, as compared to control cell carriers. Additionally, NF-SMS promoted vascular endothelial growth factor (VEGF) expression of hDPSCs in a 3D hypoxic culture. Hypoxia-primed hDPSCs/NF-SMS complexes were injected into the cleaned pulp cavities of rabbit molars for subcutaneous implantation in mice. After 4 weeks, the hypoxia group significantly enhanced angiogenesis inside the pulp chamber and promoted the formation of ondontoblast-like cells lining along the dentin-pulp interface, as compared to the control groups (hDPSCs alone group, NF-SMS alone group, and hDPSCs/NF-SMS group pre-cultured under normoxic conditions). Furthermore, in an in situ dental pulp repair model in rats, hypoxia-primed hDPSCs/NF-SMS were injected to fully fill the pulp cavity and regenerate pulp-like tissues with a rich vasculature and a histological structure similar to the native pulp.Statement of significanceVascularization is key to the regeneration of many vital tissues. However, it is challenging to create a suitable microenvironment for stem cells to regenerate vascularized tissue structure. This manuscript reports a novel star-shaped block copolymer that self-assembles into unique nanofibrous spongy microspheres, which as an injectable scaffold recapitulate the cell-cell and cell-matrix interactions in development. Using a clinically-relevant surgical procedure and a hypoxic treatment, the nanofibrous spongy microspheres were used to deliver stem cells and successfully regenerate dental pulp with a rich vasculature and a complex histologic structure similar to that of the native dental pulp. The novel microspheres can likely be used to regenerate many other vascularized tissues.

Project description:BackgroundThe development of mechanically active culture systems helps increase the understanding of the role of mechanical stress in intervertebral disc (IVD) degeneration. Motion segment cultures allow for preservation of the native IVD structure, and adjacent vertebral bodies facilitate the application and control of mechanical loads. The purpose of this study was to establish loading and organ culture methods for rabbit IVD motion segments to study the effect of static load on the whole disc organ.MethodsIVD motion segments were harvested from rabbit lumbar spines and cultured in no-loading 6-well plates (control conditions) or custom-made apparatuses under a constant, compressive load (3 kg, 0.5 MPa) for up to 14 days. Tissue integrity, matrix synthesis, and the matrix gene expression profile were assessed after 3, 7, and 14 days of culturing and compared with those of fresh tissues.ResultsThe results showed that ex vivo culturing of motion segments preserved tissue integrity under no-loading conditions for 14 days whereas the static load gradually destroyed the morphology after 3 days. Proteoglycan contents were decreased under both conditions, with a more obvious decrease under static load, and proteoglycan gene expression was also downregulated. However, under static load, immunohistochemical staining intensity and collagen Type II alpha 1 (COL2A1) gene expression were significantly enhanced (61.54 ± 5.91, P = 0.035) and upregulated (1.195 ± 0.040, P = 0.000), respectively, compared with those in the controls (P < 0.05). In contrast, under constant compression, these trends were reversed. Our initial results indicated that short-term static load stimulated the synthesis of collagen Type II alpha 1; however, sustained constant compression led to progressive degeneration and specifically to a decreased proteoglycan content.ConclusionsA loading and organ culture system for ex vivo rabbit IVD motion segments was developed. Using this system, we were able to study the effects of mechanical stimulation on the biology of IVDs, as well as the pathomechanics of IVD degeneration.

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Nanofibrous spongy microspheres to deliver rabbit mesenchymal stem cells and anti-miR-199a to regenerate nucleus pulposus and prevent calcification.

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