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Transforming Growth Factor-β3/Recombinant Human-like Collagen/Chitosan Freeze-Dried Sponge Primed With Human Periodontal Ligament Stem Cells Promotes Bone Regeneration in Calvarial Defect Rats.

ABSTRACT: Patients with a skull defect are at risk of developing cerebrospinal fluid leakage and ascending bacterial meningitis at >10% per year. However, treatment with stem cells has brought great hope to large-area cranial defects. Having found that transforming growth factor (TGF)-β3 can promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), we designed a hybrid TGF-β3/recombinant human-like collagen recombinant human collagen/chitosan (CS) freeze-dried sponge (TRFS) loading hPDLSCs (TRFS-h) to repair skull defects in rats. CFS with 2% CS was selected based on the swelling degree, water absorption, and moisture retention. The CS freeze-dried sponge (CFS) formed a porous three-dimensional structure, as observed by scanning electron microscopy. In addition, cytotoxicity experiments and calcein-AM/PI staining showed that TRFS had a good cellular compatibility and could be degraded completely at 90 days in the implantation site. Furthermore, bone healing was evaluated using micro-computed tomography in rat skull defect models. The bone volume and bone volume fraction were higher in TRFS loaded with hPDLSCs (TRFS-h) group than in the controls (p < 0.01, vs. CFS or TRFS alone). The immunohistochemical results indicated that the expression of Runx2, BMP-2, and collagen-1 (COL Ⅰ) in cells surrounding bone defects in the experimental group was higher than those in the other groups (p < 0.01, vs. CFS or TRFS alone). Taken together, hPDLSCs could proliferate and undergo osteogenic differentiation in TRFS (p < 0.05), and TRFS-h accelerated bone repair in calvarial defect rats. Our research revealed that hPDLSCs could function as seeded cells for skull injury, and their osteogenic differentiation could be accelerated by TGF-β3. This represents an effective therapeutic strategy for restoring traumatic defects of the skull.

SUBMITTER: Huang S

PROVIDER: S-EPMC8103166 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The structural and functional integrity of bone-periodontal ligament (PDL)-cementum complex stems from the load-bearing attachment sites (entheses) between soft (PDL) and hard (bone, cementum) tissues. These attachment sites are responsible for the maintenance of a bone-PDL-cementum complex biomechanical function. The objective was to investigate changes in spatiotemporal expression of key biomolecules in developing and functionally active entheses.Multilabeling technique was performed on hemimandibles of 3 wk and 3 mo-old scleraxis-GFP transgenic mice for CD146, CD31, NG2, osterix and bone sialoprotein. Regions of dominant stretch within the PDL were evaluated by identifying directionality of collagen fibrils, PDL fibroblasts and PDL cell cytoskeleton.CD146+ cells adjacent to CD31+ vasculature were identified at PDL-bone enthesis. NG2+ cells were located at coronal bone-PDL and apical cementum-PDL entheses in the 3-wk-old group, but at 3 mo, NG2 was positive at the entheses of the apical region and alveolar crest. NG2 and osterix were colocalized at the osteoid and cementoid regions of the PDL-bone and PDL-cementum entheses. Bone sialoprotein was prominent at the apical region of 3-wk-old mice. The directionality of collagen fibers, fibroblasts and their cytoskeleton overlapped, except in the apical region of 3 wk.Colocalization of biomolecules at zones of the PDL adjacent to attachment sites may be essential for the formation of precementum and osteoid interfaces at a load-bearing bone-PDL-tooth fibrous joint. Biophysical cues resulting from development and function can regulate recruitment and differentiation of stem cells potentially from a vascular origin toward osteo- and cemento-blastic lineages at the PDL-bone and PDL-cementum entheses. Investigating the coupled effect of biophysical and biochemical stimuli leading to cell differentiation at the functional attachment sites is critical for developing regeneration strategies to enable functional reconstruction of the periodontal complex.

Project description:BackgroundThe periodontal ligament is essential for homeostasis of periodontal tissue. A hypoxic milieu of the periodontal tissue is generated under periodontitis or during orthodontic treatment, which affects the periodontal and bone remodelling process. Here, we provide a comprehensive proteomic characterization of periodontal ligament cells under hypoxic conditions, aiming to reveal previously unappreciated biological changes and to help advance hypoxia-based therapeutic strategies for periodontal diseases.MethodsHuman periodontal ligament cells (hPDLCs) were characterized using immunohistochemistry (IHC) and flow cytometry (FACS). Successful hypoxia treatment of hPDLCs with 1% O2 was confirmed by qRT-PCR. Proliferation was evaluated using an MTT assay. The proteomic expression profile under hypoxia was studied with the isobaric tags for relative and absolute quantification (iTRAQ) approach followed by protein identification and bioinformatic analysis, and western blot verification was performed.ResultsThe hPDLCs were positive for vimentin, CD73 and CD105 and negative for keratin, CD34 and CD45. After hypoxia treatment, the mRNA expression of hypoxia-inducible factor 1a (HIF1a) was upregulated. The proliferation rate was elevated during the first 6 h but decreased from 6 h to 72 h. A total of 220 differentially expressed proteins were quantified in hPDLCs under hypoxia (1% O2, 24 h), including 153 upregulated and 67 downregulated proteins, five of which were verified by western blot analysis. The Gene Ontology enriched terms included the energy metabolic process, membrane-bound organelle and vesicle, and protein binding terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated several involved pathways, including glycolysis/gluconeogenesis, biosynthesis of amino acids, the HIF-1 signalling pathway, and focal adhesion. A protein-protein interaction (PPI) network demonstrated the dominant role of autophagy over apoptosis under hypoxia.ConclusionThe proteomic profile of hPDLCs under hypoxia was mainly related to energy metabolism, autophagy, and responses to stimuli such as adhesion and inflammation. Previously unrecognized proteins including solute carrier family proteins, heat shock proteins, ubiquitination-related enzymes, collagen and S100 family proteins are involved in adaptive response to hypoxia in hPDLCs and are thus of great research interest in future work.

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Transforming Growth Factor-β3/Recombinant Human-like Collagen/Chitosan Freeze-Dried Sponge Primed With Human Periodontal Ligament Stem Cells Promotes Bone Regeneration in Calvarial Defect Rats.

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