Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Dec;4(1):22.
doi: 10.1186/s40634-017-0097-1. Epub 2017 Jun 21.

Impact of mechanical stimulation on the chondrogenic processes in human bone marrow aspirates modified to overexpress sox9 via rAAV vectors

Jagadeesh K Venkatesan  1 , Janina Frisch  1 , Ana Rey-Rico  1 , Gertrud Schmitt  1 , Henning Madry  1   2 , Magali Cucchiarini  3
Affiliations

Impact of mechanical stimulation on the chondrogenic processes in human bone marrow aspirates modified to overexpress sox9 via rAAV vectors

Jagadeesh K Venkatesan et al. J Exp Orthop. 2017 Dec.

Abstract

Background: Evaluation of gene-based approaches to target human bone marrow aspirates in conditions of mechanical stimulation that aim at reproducing the natural joint environment may allow to develop improved treatments for articular cartilage injuries. In the present study, we investigated the potential of rAAV-mediated sox9 gene transfer to enhance the chondrogenic differentiation processes in human bone marrow aspirates under established hydrodynamic conditions compared with the more commonly employed static culture conditions.

Methods: Fresh human bone marrow aspirates were transduced with rAAV-FLAG-hsox9 (40 μl) and maintained for up to 28 days in chondrogenic medium under mechanically-induced conditions in dynamic flow rotating bioreactors that permit tissue growth and matrix deposition relative to static culture conditions. The samples were then processed to examine the potential effects of sox9 overexpression on the cellular activities (matrix synthesis, proliferation) and on the chondrogenic differentiation potency compared with control treatments (absence of rAAV vector; reporter rAAV-lacZ, rAAV-RFP, and rAAV-luc gene transfer).

Results: Prolonged, significant sox9 overexpression via rAAV was achieved in the aspirates for at least 28 days when applying the rAAV-FLAG-hsox9 construct, leading to higher, prolonged levels of matrix biosynthesis and to enhanced chondrogenic activities relative to control treatments especially when maintaining the samples under mechanical stimulation. Administration of sox9 however did not impact the indices of proliferation in the aspirates. Remarkably, sox9 gene transfer also durably delayed hypertrophic and osteogenic differentiation in the samples regardless of the conditions of culture applied versus control treatments.

Conclusions: The current observations show the value of genetically modifying human bone marrow aspirates upon mechanical stimulation by rAAV sox9 as a promising strategy for future treatments to improve cartilage repair by implantation in lesions where the tissue is submitted to natural mechanical forces.

Keywords: Bone marrow aspirates; Cartilage repair; Chondrogenic differentiation; Mechanical stimulation; Recombinant adeno-associated virus.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Detection of transgene expression in rAAV-transduced human bone marrow aspirates in static versus dynamic culture conditions. Aspirates (150 μl) were transduced with rAAV-RFP, rAAV-lacZ, rAAV-luc, or rAAV-FLAG-hsox9 (40 μl each vector) or let untransduced for maintenance in dynamic or static culture conditions for up to 28 days (n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments) as described in the Methods. a Macroscopic views of the aspirates are presented for each cultivation systems (day 21, scale bars: 1.5 cm for the static cultures and 2 cm for the dynamic cultures; insets: day 28), b Detection of RFP expression by live fluorescence (day 21, scale bars: 50 μm; top insets: day 7; bottom insets: day 28), c detection of lacZ expression (day 21) by X-Gal staining (macroscopic views, scale bars: 1.5 cm for the static cultures and 2 cm for the dynamic cultures) and following histological processing of the samples (magnification x20, scale bars: 100 μm, all representative data), d detection of luc expression by analysis of luciferase activity with normalization to total cellular proteins, and e detection of SOX9 (day 28, magnification x40, scale bars: 40 μm; top insets: complete section, magnification x4; bottom insets: H&E staining, magnification x40; all representative data) as described in the Methods. aStatistically significant relative to rAAV-RFP application
Fig. 2
Fig. 2
Evalution of cartilage-specific components in rAAV-transduced human bone marrow aspirates in static versus dynamic culture conditions. Aspirates (150 μl) were prepared and transduced with rAAV-luc or rAAV-FLAG-hsox9 as described in Fig. 1 or let untransduced for maintenance in dynamic or static culture conditions for up to 28 days (n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments) as described in the Methods. The samples were processed (a) for safranin O staining and to detect the expression of type-II collagen after 7, 21, and 28 days as described in the Methods (magnification x10, scale bars: 200 μm; insets: complete section, magnification x4; all representative data) and (b) to evaluate the gene expression profiles (SOX9 and COL2A1, with GAPDH serving as a housekeeping gene and internal control) after 21 days by real-time RT-PCR amplification as described in the Methods. Ct values were obtained for each target and GAPDH as a control for normalization, and fold inductions (relative to untreated aggregates) were measured by using the 2-ΔΔCt method. Statistically significant relative to ano vector treatment, brAAV-luc application, and cstatic culture
Fig. 3
Fig. 3
Evaluation of hypertrophic and terminal differentiation processes in rAAV-transduced human bone marrow aspirates in static versus dynamic culture conditions. Aspirates (150 μl) were prepared and transduced with rAAV-luc or rAAV-FLAG-hsox9 as described in Fig. 1 and 2 or let untransduced for maintenance in dynamic or static culture conditions for up to 28 days (n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments) as described in the Methods. The samples were processed (a) for alizarin red staining and to detect the expression of type-X collagen after 7, 21, and 28 days as described in the Methods (magnification x10, scale bars: 200 μm, all representative data) and (b) to evaluate the gene expression profiles (COL10A1, ALP, MMP13, RUNX2, and β-catenin, with GAPDH serving as a housekeeping gene and internal control) after 21 days by real-time RT-PCR amplification as described in the Methods and in Fig. 2. Ct values were obtained for each target and GAPDH as a control for normalization, and fold inductions (relative to untreated aggregates) were measured by using the 2-ΔΔCt method. Statistically significant relative to ano vector treatment and brAAV-luc application
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Akiyama H, Lyons JP, Mori-Akiyama Y, Yang X, Zhang R, Zhang Z, Deng JM, Taketo MM, Nakamura T, Behringer RR, McCrea PD, de Crombrugghe B. Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev. 2004;18:1072–1087. doi: 10.1101/gad.1171104. - DOI - PMC - PubMed
    1. Babister JC, Tare RS, Green DW, Inglis S, Mann S, Oreffo RO. Genetic manipulation of human mesenchymal progenitors to promote chondrogenesis using “bead-in-bead” polysaccharide capsules. Biomaterials. 2008;29:58–65. doi: 10.1016/j.biomaterials.2007.09.006. - DOI - PubMed
    1. Beck SE, Jones LA, Chesnut K, Walsh SM, Reynolds TC, Carter BJ, Askin FB, Flotte TR, Guggino WB. Repeated delivery of adeno-associated virus vectors to the rabbit airway. J Virol. 1999;73:9446–9455. - PMC - PubMed
    1. Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B. Sox9 is required for cartilage formation. Nat Genet. 1999;22:85–89. doi: 10.1038/8792. - DOI - PubMed
    1. Buckwalter JA, Mankin HJ. Articular cartilage repair and transplantation. Arthritis Rheum. 1998;41:1331–1342. doi: 10.1002/1529-0131(199808)41:8<1331::AID-ART2>3.0.CO;2-J. - DOI - PubMed

LinkOut - more resources