Journal: Biomaterials Research

Article Title: Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

doi: 10.34133/bmr.0320

Figure Lengend Snippet: Schematic illustration of the protein-based platform for the direct osteogenic reprogramming of human dermal fibroblasts (HDFs). (A) Production of recombinant Oct4-30Kc19 and Cbfβ-30Kc19 fusion proteins. Expression plasmids encoding reprogramming factors fused with the 30Kc19 moiety were introduced into an E. coli expression system to generate cell-permeable recombinant proteins. (B) Direct osteogenic reprogramming via intracellular protein transduction. The fusion proteins are intracellularly delivered through 30Kc19-mediated transport. Oct4-30Kc19 induces a state of cellular plasticity, while Cbfβ-30Kc19 promotes osteogenic lineage commitment, synergistically driving the conversion of HDFs into functional osteoblasts.

Article Snippet: Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) were cultured in Osteoblast Growth Medium (PromoCell) according to the manufacturer’s instructions.

Techniques: Recombinant, Expressing, Transduction, Functional Assay

Journal: Biomaterials Research

Article Title: Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

doi: 10.34133/bmr.0320

Figure Lengend Snippet: Direct reprogramming of HDFs into osteoblasts through ectopic overexpression of Oct4 and Cbfβ. (A) Schematic illustration of direct reprogramming process using the pCXLE episomal plasmid delivery system. The pCXLE-Oct4 and pCXLE-Cbfβ plasmids were delivered into HDFs via cationic polymer-based transfection, either individually or in combination. (B) Representative images of alkaline phosphatase (ALP) staining after 14 d of culture in osteogenic medium (OM). (C and D) Calcium deposition after 24 d of osteogenic induction, as detected by Alizarin Red S (ARS) and OsteoImage assays. (E and F) Immunofluorescence images showing expression of osteopontin (OPN) and osteocalcin (OCN), on day 24. Coexpression of Oct4 and Cbfβ induced robust expression of both osteogenic markers.

Article Snippet: Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) were cultured in Osteoblast Growth Medium (PromoCell) according to the manufacturer’s instructions.

Techniques: Over Expression, Plasmid Preparation, Polymer, Transfection, Staining, Immunofluorescence, Expressing

Journal: Biomaterials Research

Article Title: Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

doi: 10.34133/bmr.0320

Figure Lengend Snippet: Direct reprogramming of HDFs into osteoblasts using cell-permeable protein-based platform. (A) Schematic illustration of the reprogramming strategy using 30Kc19-fused recombinant proteins. HDFs were treated with a combination of Oct4-30Kc19 and Cbfβ-30Kc19 recombinant proteins 8 times over 8 d, followed by culture in OM. (B and C) ARS staining and subsequent quantification on day 24, showing robust calcium deposition in the group treated with both proteins. Data are presented as means ± SD ( n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. Unless otherwise indicated, comparisons were made to the nontreated control group (** P < 0.01; *** P < 0.001; ns, not significant). (D and E) Von Kossa staining and OsteoImage mineralization assay images, confirming mineralized matrix formation. Treatment with both Oct4-30Kc19 and Cbfβ-30Kc19 resulted in significantly enhanced mineralization compared with single-factor or untreated controls.

Article Snippet: Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) were cultured in Osteoblast Growth Medium (PromoCell) according to the manufacturer’s instructions.

Techniques: Recombinant, Staining, Control, Mineralization Assay

Journal: Biomaterials Research

Article Title: Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

doi: 10.34133/bmr.0320

Figure Lengend Snippet: Transcriptomic remodeling toward an osteoblast lineage by Oct4-30Kc19 and Cbfβ-30Kc19 fusion proteins. (A) Scatterplot showing differentially expressed genes (DEGs) between nontreated HDFs and protein-induced osteoblasts (piOBs) treated with Oct4-30Kc19 and Cbfβ-30Kc19 proteins (fold change ≥ 2). (B) Gene Ontology (GO) enrichment analysis of DEGs, highlighting overrepresentation of transcription-related biological processes. (C) Protein interaction network of enriched GO terms related to transcription regulation by RNA polymerase II. (D) Hierarchical clustering analysis of genes differentially expressed in both piOBs and primary human osteoblasts (hOBs), relative to HDFs. (E) Heatmap of DEGs associated with the ossification GO term, showing the expression of key osteogenic markers.

Article Snippet: Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) were cultured in Osteoblast Growth Medium (PromoCell) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Biomaterials Research

Article Title: Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

doi: 10.34133/bmr.0320

Figure Lengend Snippet: Efficient bone defect regeneration using a cell-permeable protein-based direct reprogramming platform. (A) Schematic illustration of the in vivo bone regeneration experiment. HDFs were pretreated 8 times with Oct4-30Kc19 and Cbfβ-30Kc19 proteins, seeded onto gelatin cryogels, and transplanted into 4-mm-sized calvarial defects in mice. Created with biorender.com . (B) Representative micro-CT 3D images showing bone regeneration 8 weeks post-transplantation. Green areas and arrows indicate newly regenerated bones, while yellow arrows denote bone defect regions. (C and D) Quantification of bone volume fraction (BV/TV) and trabecular separation in the regenerated bone tissue. Data are presented as means ± SD ( n = 4). Statistical significance was determined by Student’s t test (*** P < 0.001). (E) Hematoxylin and eosin (H&E) staining showing histological differences between defects implanted with untreated HDFs and those implanted with piOBs. (F) Masson’s trichrome (MTC) staining revealing collagen-rich new bone formation in the piOB-treated group. FT, fibrous tissues; NB, new bones. (G and H) Immunofluorescent staining for OPN and OCN, confirming the presence of mature osteoblast-derived matrix in piOB-transplanted defects.

Article Snippet: Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) were cultured in Osteoblast Growth Medium (PromoCell) according to the manufacturer’s instructions.

Techniques: In Vivo, Micro-CT, Transplantation Assay, Staining, Derivative Assay