Saturday, October 17, 2015: 8:55 AM
Denver M. Lough, MD, PhD
,
Plastic and Reconstructive Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD
Christopher Madsen, MD
,
Plastic and Reconstructive Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD
Devin Miller, BA
,
Plastic and Reconstructive Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD
Edward W Swanson, MD
,
Plastic and Reconstructive Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD
Nikolai Sopko, MD, PhD
,
Plastic and Reconstructive Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD
Howard Wang, MD
,
Plastic and Reconstructive Surgery, Johns Hopkins School of Medicine, Baltimore, MD
Qiongyu Guo, PhD
,
Plastic and Reconstructive Surgery, The Johns Hopkins School of Medicine, Baltimore, MD
W. P. Andrew Lee, MD
,
Plastic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD
Gerald Brandacher, MD
,
Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD
Anand Kumar, MD
,
Plastic and Reconstructive Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD
Purpose: Large wounds resulting in the loss of significant soft tissue and bone elements remains a challenge for physicians and surgeons, even in today’s age of regenerative medicine and biomedical engineering. One reason for this continued challenge is that many of the “reconstructive and/or regenerative options” applied to wound beds act as a “filler” rather than as a functionally polarized tissue body. By simply applying a “soup” of acellular matrix or stem/progenitor cell entities, a wound is left with a milieu of disconnected signal pathways, complex lineage potential and ample extracellular matrices which, in the end, promote the migration, proliferation and differentiation of our body’s most reliable and dynamic cell—the fibroblast. It is here, that we employ a physiological combination of BMP-2 and type-I collagen substrate in order to both augment and direct the early fate decision of muscle derived stem cells in order to pursue the in vitro development of a translatable engineered scaffold for the neo-genesis of functionally polarized bone.
Methods: Utilizing a C57BL/6 murine model (n=60), we employed muscle derived stem cells (MDSc) to full-thickness craniectomy defects using BMP-2 enriched-collagen scaffold. At 8 weeks, defects were imaged using a mini-CT, laser scanning confocal microscopy and tissues collected for downstream assays including: focused osteo-induction gene and proteome arrays. Concurrently, in vitro studies utilizing baclovirus Premo Fucci® transduced MDSc (fluorescent correlation to cell cycle stage) were monitored using a FV10i-LIV® live cell confocal imaging system.
Results: While all groups depicted some form of healing within 8 weeks, defects treated with scaffolding enriched with hBMP-2 and isolated MDSc showed significantly higher rates of healing and reduced defect volume mm3 in 4 weeks. MDSc re-isolated from the healing wound construct showed significant up-regulation of osteo-induction pathway genes, while imaging and proteome assays validated relative expression and healing levels. In vitro studies indicated that MDSc more readily migrate, proliferate and differentiate when added to scaffolding and/or hBMP-2 vs. fibroblast controls. Subsequent, downstream gene and proteome arrays of in vitro defect modeling indicated significant MDSc lineage differentiation when compared to controls (p-value <0.05).
Conclusion: Our study provides a unique mechanism for the delivery of therapeutic BMP-2 enriched scaffolding, while employing the intrinsic capacity of the MDSc niche to induce intra-defect bone regeneration in less than half the time to repair a critical size cranial defect.
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