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. 2023 Jun 27;12(13):1724.
doi: 10.3390/cells12131724.

Ex Vivo Preservation of Ovine Periosteum Using a Perfusion Bioreactor System

Hai Xin  1   2 , Sara Romanazzo  3 , Eva Tomaskovic-Crook  3   4   5 , Timothy C Mitchell  4 , Jui Chien Hung  4 , Steven G Wise  4 , Kai Cheng  1   6 , D S Abdullah Al Maruf  1   2 , Murray J Stokan  1 , Timothy G H Manzie  1 , Krishnan Parthasarathi  1 , Veronica K Y Cheung  2   7 , Ruta Gupta  2   7 , Mark Ly  2   8 , Carlo Pulitano  2   8 , Innes K Wise  9 , Jeremy M Crook  3   4   5 , Jonathan R Clark  1   2   6
Affiliations

Ex Vivo Preservation of Ovine Periosteum Using a Perfusion Bioreactor System

Hai Xin et al. Cells. .

Abstract

Periosteum is a highly vascularized membrane lining the surface of bones. It plays essential roles in bone repair following injury and reconstruction following invasive surgeries. To broaden the use of periosteum, including for augmenting in vitro bone engineering and/or in vivo bone repair, we have developed an ex vivo perfusion bioreactor system to maintain the cellular viability and metabolism of surgically resected periosteal flaps. Each specimen was placed in a 3D printed bioreactor connected to a peristaltic pump designed for the optimal flow rates of tissue perfusate. Nutrients and oxygen were perfused via the periosteal arteries to mimic physiological conditions. Biochemical assays and histological staining indicate component cell viability after perfusion for almost 4 weeks. Our work provides the proof-of-concept of ex vivo periosteum perfusion for long-term tissue preservation, paving the way for innovative bone engineering approaches that use autotransplanted periosteum to enhance in vivo bone repair.

Keywords: bone engineering; bone repair; perfusion; periosteum; transplantation; viability.

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Conflict of interest statement

The authors of this manuscript have no conflict of interest to disclose.

Figures

Figure 1
Figure 1
Periosteum procurement and ex vivo perfusion for tissue preservation. Vascularized periosteal flaps collected from sheep scapula (A,B). Primary components of the perfusion bioreactor system for periosteal maintenance (C,D). Perfusion system configuration for dry perfusion (E,F) and wet perfusion (G,H).
Figure 2
Figure 2
Live/dead cell analysis of biopsies collected at various time points from the 6 periosteal flaps during ex vivo perfusion. Images of periostea 1 and 2 are prepared by computational stitching (tile scanning) together with multiple images to show the large areas of the tissues. Quantitative assessments of periostea 3–6 indicate that the cell viability of periosteal tissues tended to increase with the increasing duration of perfusion and with successive trials. Live cells are indicated in green while dead cells are indicated in red.
Figure 2
Figure 2
Live/dead cell analysis of biopsies collected at various time points from the 6 periosteal flaps during ex vivo perfusion. Images of periostea 1 and 2 are prepared by computational stitching (tile scanning) together with multiple images to show the large areas of the tissues. Quantitative assessments of periostea 3–6 indicate that the cell viability of periosteal tissues tended to increase with the increasing duration of perfusion and with successive trials. Live cells are indicated in green while dead cells are indicated in red.
Figure 3
Figure 3
PrestoBlue-based cell viability and metabolic assay of biopsies collected at various time points from the 6 periosteal flaps during ex vivo perfusion.
Figure 4
Figure 4
H&E staining of periostea (×40). (A) Periosteum 3 maintained for 17 days within a wet perfusion system. (B) Periosteum 4 maintained for 18 days within a dry perfusion system. (C) Periosteum 5 maintained for 25 days within a wet perfusion system. (D) Periosteum 6 maintained for 25 days within a dry perfusion system. (E–G) Viable endothelial cells (blue arrow) on periostea 4, 5, and 6, respectively.
Figure 5
Figure 5
pH changes of the perfusion medium.
See this image and copyright information in PMC

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