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Bone allografts combined with adipose-derived stem cells in an optimized cell/volume ratio showed enhanced osteogenesis and angiogenesis in a murine femur defect model

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Abstract

Critical sized defects, especially in long bones, pose one of the biggest problems in orthopedic surgery. By definition, these defects do not heal without further treatment. Different therapeutic options range from autologous bone grafts, for example, free vascularized bone grafts, to commercially available bone allografts. Disadvantages of these bone allografts are related to reduced osteogenesis, since they are solely composed of cell-free bone matrix. The purpose of this study was to investigate the cell seeding efficiency of human adipose-derived stem cells (hASCs) on human bone allografts in vitro and furthermore analyze these optimized seeded allografts in a critical sized defect model in vivo. Cancellous human bone allografts were colonized with human ASCs in vitro. Cell seeding efficiency was evaluated by Cell Counting Kit-8 assay. Thereafter, optimized hASC-seeded bone scaffolds were examined in a murine femur defect model, stabilized with the MouseExFix system. Subsequently, x-ray analysis and histology were performed. Examination of cell seeding efficiency revealed an optimum starting population of 84,600 cells per 100 mm3 scaffold. In addition, scaffolds seeded with hASCs showed increased osteogenesis compared with controls. Histological analysis revealed increased remodeling and elevated new bone formation within hASC-seeded scaffolds. Moreover, immunohistochemical stainings revealed increased proliferation, osteogenesis, and angiogenesis. In this study, we systemically optimized cell/volume ratio of two promising components of tissue engineering: hASCs and human bone allografts. These findings may serve as a basis for future translational studies.

Key messages

  • Bone tissue engineering.

  • Mesenchymal stem cells derived from human adipose tissue (hASCs).

  • Optimal cell/volume ratio of cell-seeded scaffolds.

  • Increased osteogenesis and angiogenesis in vivo.

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Abbreviations

hASC :

human adipose stem cell

DMEM :

Dulbecco’s Modified Eagle Medium

FCS :

fetal calf serum

DIZG :

Deutsches Institut für Zell- und Gewebeersatz

CCK-8 :

cell counting kit 8

PBS :

phosphate buffered saline

EDTA :

Ethylenediamine-tetraacetate

PCNA :

Proliferating-Cell-Nuclear-Antigen

RUNX2 :

Runt-related transcription factor

DAPI :

4,6-diamidino-2-phenylindole

Tunel :

TdT-mediated dUTP-biotin nick end labeling

hmito :

human mitochondrias

HIV :

human immunodeficiency virus

VEGF :

vascular endothelial growth factor

TCP:

tricalcium-phosphate

SVF :

stromal vascular fraction

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Author information

Author notes

  1. Johannes M. Wagner, Nicolas Conze and Guido Lewik contributed equally to this work.

Authors and Affiliations

  1. University Hospital BG Bergmannsheil Bochum, Bürkle-de-la-Camp Platz 1, 44789, Bochum, Germany

    Johannes M. Wagner, Nicolas Conze, Guido Lewik, Christoph Wallner, Stephanie Dittfeld, Henriette Jaurich, Mustafa Becerikli, Mehran Dadras, Kamran Harati, Marcus Lehnhardt & Björn Behr

  2. Deutsches Institut für Zell- und Gewebeersatz, Berlin, Germany

    Jan C. Brune

  3. BG Trauma Hospital Ludwigshafen, Ludwigshafen, Germany

    Sebastian Fischer

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  1. Johannes M. Wagner
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Correspondence to Björn Behr.

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All experiments were performed in adherence to the National Institute of Health guidelines for the use of experimental animals and after approval by the German legislation. The protocol was approved by the LANUV (NRW, Germany; Permit-Number: AZ 84-02.04.2013.A362).

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Wagner, J.M., Conze, N., Lewik, G. et al. Bone allografts combined with adipose-derived stem cells in an optimized cell/volume ratio showed enhanced osteogenesis and angiogenesis in a murine femur defect model. J Mol Med 97, 1439–1450 (2019). https://doi.org/10.1007/s00109-019-01822-9

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