<i>Ex Vivo</i> Regional Gene Therapy Compared to Recombinant BMP-2 for the Treatment of Critical-Size Bone Defects: An In Vivo Single-Cell RNA-Sequencing Study
<i>Ex vivo</i> regional gene therapy is a promising tissue-engineering strategy for bone regeneration: osteogenic mesenchymal stem cells (MSCs) can be genetically modified to express an osteoinductive stimulus (e.g., bone morphogenetic protein-2), seeded onto an osteoconductive scaffold,...
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2025-01-01
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| Series: | Bioengineering |
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| Online Access: | https://www.mdpi.com/2306-5354/12/1/29 |
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| author | Arijita Sarkar Matthew C. Gallo Jennifer A. Bell Cory K. Mayfield Jacob R. Ball Mina Ayad Elizabeth Lechtholz-Zey Stephanie W. Chang Osamu Sugiyama Denis Evseenko Jay R. Lieberman |
| author_facet | Arijita Sarkar Matthew C. Gallo Jennifer A. Bell Cory K. Mayfield Jacob R. Ball Mina Ayad Elizabeth Lechtholz-Zey Stephanie W. Chang Osamu Sugiyama Denis Evseenko Jay R. Lieberman |
| author_sort | Arijita Sarkar |
| collection | DOAJ |
| description | <i>Ex vivo</i> regional gene therapy is a promising tissue-engineering strategy for bone regeneration: osteogenic mesenchymal stem cells (MSCs) can be genetically modified to express an osteoinductive stimulus (e.g., bone morphogenetic protein-2), seeded onto an osteoconductive scaffold, and then implanted into a bone defect to exert a therapeutic effect. Compared to recombinant human BMP-2 (rhBMP-2), which is approved for clinical use, regional gene therapy may have unique benefits related to the addition of MSCs and the sustained release of BMP-2. However, the cellular and transcriptional mechanisms regulating the response to these two strategies for BMP-2 mediated bone regeneration are largely unknown. Here, for the first time, we performed single-cell RNA sequencing (10x Genomics) of hematoma tissue in six rats with critical-sized femoral defects that were treated with either regional gene therapy or rhBMP-2. Our unbiased bioinformatic analysis of 2393 filtered cells in each group revealed treatment-specific differences in their cellular composition, transcriptional profiles, and cellular communication patterns. Gene therapy treatment induced a more robust chondrogenic response, as well as a decrease in the proportion of fibroblasts and the expression of profibrotic pathways. Additionally, gene therapy was associated with an anti-inflammatory microenvironment; macrophages expressing canonical anti-inflammatory markers were more common in the gene therapy group. In contrast, pro-inflammatory markers were more highly expressed in the rhBMP-2 group. Collectively, the results of our study may offer insights into the unique pathways through which <i>ex vivo</i> regional gene therapy can augment bone regeneration compared to rhBMP-2. Furthermore, an improved understanding of the cellular pathways involved in segmental bone defect healing may allow for the further optimization of regional gene therapy or other bone repair strategies. |
| format | Article |
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| institution | Kabale University |
| issn | 2306-5354 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Bioengineering |
| spelling | doaj-art-f51325c6c5354e80b6aa93807dadfeb32025-01-24T13:23:00ZengMDPI AGBioengineering2306-53542025-01-011212910.3390/bioengineering12010029<i>Ex Vivo</i> Regional Gene Therapy Compared to Recombinant BMP-2 for the Treatment of Critical-Size Bone Defects: An In Vivo Single-Cell RNA-Sequencing StudyArijita Sarkar0Matthew C. Gallo1Jennifer A. Bell2Cory K. Mayfield3Jacob R. Ball4Mina Ayad5Elizabeth Lechtholz-Zey6Stephanie W. Chang7Osamu Sugiyama8Denis Evseenko9Jay R. Lieberman10Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USADepartment of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USADepartment of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USADepartment of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USADepartment of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USADepartment of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USADepartment of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USADepartment of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USADepartment of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USADepartment of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USADepartment of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USA<i>Ex vivo</i> regional gene therapy is a promising tissue-engineering strategy for bone regeneration: osteogenic mesenchymal stem cells (MSCs) can be genetically modified to express an osteoinductive stimulus (e.g., bone morphogenetic protein-2), seeded onto an osteoconductive scaffold, and then implanted into a bone defect to exert a therapeutic effect. Compared to recombinant human BMP-2 (rhBMP-2), which is approved for clinical use, regional gene therapy may have unique benefits related to the addition of MSCs and the sustained release of BMP-2. However, the cellular and transcriptional mechanisms regulating the response to these two strategies for BMP-2 mediated bone regeneration are largely unknown. Here, for the first time, we performed single-cell RNA sequencing (10x Genomics) of hematoma tissue in six rats with critical-sized femoral defects that were treated with either regional gene therapy or rhBMP-2. Our unbiased bioinformatic analysis of 2393 filtered cells in each group revealed treatment-specific differences in their cellular composition, transcriptional profiles, and cellular communication patterns. Gene therapy treatment induced a more robust chondrogenic response, as well as a decrease in the proportion of fibroblasts and the expression of profibrotic pathways. Additionally, gene therapy was associated with an anti-inflammatory microenvironment; macrophages expressing canonical anti-inflammatory markers were more common in the gene therapy group. In contrast, pro-inflammatory markers were more highly expressed in the rhBMP-2 group. Collectively, the results of our study may offer insights into the unique pathways through which <i>ex vivo</i> regional gene therapy can augment bone regeneration compared to rhBMP-2. Furthermore, an improved understanding of the cellular pathways involved in segmental bone defect healing may allow for the further optimization of regional gene therapy or other bone repair strategies.https://www.mdpi.com/2306-5354/12/1/29single-cell RNA sequencinggene therapyBMP-2mesenchymal stem cellsendochondral ossificationinflammation |
| spellingShingle | Arijita Sarkar Matthew C. Gallo Jennifer A. Bell Cory K. Mayfield Jacob R. Ball Mina Ayad Elizabeth Lechtholz-Zey Stephanie W. Chang Osamu Sugiyama Denis Evseenko Jay R. Lieberman <i>Ex Vivo</i> Regional Gene Therapy Compared to Recombinant BMP-2 for the Treatment of Critical-Size Bone Defects: An In Vivo Single-Cell RNA-Sequencing Study Bioengineering single-cell RNA sequencing gene therapy BMP-2 mesenchymal stem cells endochondral ossification inflammation |
| title | <i>Ex Vivo</i> Regional Gene Therapy Compared to Recombinant BMP-2 for the Treatment of Critical-Size Bone Defects: An In Vivo Single-Cell RNA-Sequencing Study |
| title_full | <i>Ex Vivo</i> Regional Gene Therapy Compared to Recombinant BMP-2 for the Treatment of Critical-Size Bone Defects: An In Vivo Single-Cell RNA-Sequencing Study |
| title_fullStr | <i>Ex Vivo</i> Regional Gene Therapy Compared to Recombinant BMP-2 for the Treatment of Critical-Size Bone Defects: An In Vivo Single-Cell RNA-Sequencing Study |
| title_full_unstemmed | <i>Ex Vivo</i> Regional Gene Therapy Compared to Recombinant BMP-2 for the Treatment of Critical-Size Bone Defects: An In Vivo Single-Cell RNA-Sequencing Study |
| title_short | <i>Ex Vivo</i> Regional Gene Therapy Compared to Recombinant BMP-2 for the Treatment of Critical-Size Bone Defects: An In Vivo Single-Cell RNA-Sequencing Study |
| title_sort | i ex vivo i regional gene therapy compared to recombinant bmp 2 for the treatment of critical size bone defects an in vivo single cell rna sequencing study |
| topic | single-cell RNA sequencing gene therapy BMP-2 mesenchymal stem cells endochondral ossification inflammation |
| url | https://www.mdpi.com/2306-5354/12/1/29 |
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