Genetic modification of scAAV-equine-BMP-2 transduced bone-marrow-derived mesenchymal stem cells before and after cryopreservation: An "off-the-shelf" option for fracture repair

Abstract

Optimizing the environment of complex bone healing and improving treatment of catastrophic bone fractures and segmental bone defects remains an unmet clinical need both human and equine veterinary medical orthopaedics. The objective of this study was to determine whether scAAV-equine-BMP-2 transduced cells would induce osteogenesis in equine bone marrow derived mesenchymal stem cells (BMDMSCs) in vitro, and if these cells could be cryopreserved in an effort to osteogenically prime them as an "off-the-shelf" gene therapeutic approach for fracture repair. Our study found that transgene expression is altered by cell expansion, as would be expected by a transduction resulting in episomal transgene expression, and that osteoinductive levels could still be achieved 5 days after recovery, and protein expression would continue up to 14 days after transduction. This is the first evidence that cryopreservation of genetically modified BMDMSCs would not alter the osteoinductive potential or clinical use of allogeneic donor cells in cases of equine fracture repair. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1310-1317, 2019.

Keywords: bone biology; bone fracture; bone repair; progenitors and stem cells; therapeutics.

Figure 1.

BMP-2 protein expression (pg/mL): Error bars denote standard deviation from the mean. Letter changes denote significant differences between groups (p < 0.0001). ScAAV-equine-BMP-2 transduced cells produced significantly more BMP-2 than any other group. Transgene expression persisted without significant changes 14 days.

Figure 2.

(a) MSCs in culture treated with scAAV-equine-BMP-2, rhBMP-2, and osteogenic media exemplify osteogenic morphologic changes at day 7 following transduction. (b) Morphology Scores: Error bars denote standard deviation from the mean. Letter changes denote significant differences between groups. ScAAV-equine-BMP-2 transduced cells, rhBMP-2 treated cells, and osteogenic control cells appeared significantly more osteogenic than GFP transduced cells and negative controls (p < 0.0001). The morphological changes were evident by day 7.

Figure 3.

BMP-2 protein expression (pg/mL): Error bars denote standard deviation from the mean. Letter changes denote significant differences between groups (p < 0.0001). ScAAV-equine-BMP-2 transduced cells produced significantly more BMP-2 than any other group. Transgene expression persisted after cryopreservation.

Figure 4.

(a) MSCs in culture treated with scAAV-equine-BMP-2, rhBMP-2, and osteogenic media exemplify osteogenic morphologic changes at day 7 following transduction, cryopreservation, and recovery. (b) Morphology Scores: Error bars denote standard deviation from the mean. Letter changes denote significant differences between groups (p < 0.0001). Cryopreservation did not affect how scAAV-equine-BMP-2 transduced cells, rhBMP-2 treated cells, and osteogenic control cells appeared morphologically when compared to scAAV-GFP and negative controls (p < 0.0001). The morphological changes were evident by day 7 following transduction, cryopreservation, and recovery.

Figure 5.

Side by side comparison of day 4 and day 7 alkaline phosphatase expression (ng/mL). Expression patterns between cryopreserved and non-cryopreserved cells follow a similar trend when compared to BMP-2 protein expression (Figure 3).