TGF‐β gene transfer and overexpression viarAAV vectors stimulates chondrogenic events in human bone marrow aspirates

Abstract

Genetic modification of marrow concentrates may provide convenient approaches to enhance the chondrogenic differentiation processes and improve the repair capacities in sites of cartilage defects following administration in the lesions. Here, we provided clinically adapted recombinant adeno‐associated virus (rAAV) vectors to human bone marrow aspirates to promote the expression of the potent transforming growth factor beta (TGF‐β) as a means to regulate the biological and chondrogenic activities in the samples in vitro. Successful TGF‐β gene transfer and expression via rAAV was reached relative to control (lacZ) treatment (from 511.1 to 16.1 pg rhTGF‐β/mg total proteins after 21 days), allowing to durably enhance the levels of cell proliferation, matrix synthesis, and chondrogenic differentiation. Strikingly, in the conditions applied here, application of the candidate TGF‐β vector was also capable of reducing the hypertrophic and osteogenic differentiation processes in the aspirates, showing the potential benefits of using this particular vector to directly modify marrow concentrates to generate single‐step, effective approaches that aim at improving articular cartilage repair in vivo.

Genetic Modification of Human Peripheral Blood Aspirates Using Recombinant Adeno-Associated Viral Vectors for Articular Cartilage Repair with a Focus on Chondrogenic Transforming Growth Factor-β Gene Delivery

“…Recombinant vectors (rAAV) were packaged as conventional (not self‐complementary) vectors by using the 293 adenovirus‐transformed embryonic kidney cell line and helper functions provided by Adenovirus 5 and pAd8 helper plasmid as previously described . Vector preparations were purified, dialyzed, and titrated via real‐time polymerase chain reaction (PCR), resulting in 10 10 transgene copies/ml with approximately 1/500 functional recombinant viral particles , , .…”

“…Immediately after collection, the peripheral blood aspirates were divided into aliquots (100 μl/well in 96‐well plates) and transduced with 40‐μl vectors (8 × 10 5 functional recombinant viral particles, multiplicity of infection [MOI], 10 ± 3), followed by an addition of 50 μl of supplement‐free DMEM . The aspirates were incubated for 90 minutes at 37°C and 5% CO 2 , with subsequent addition of 60 μl of DMEM, 10% fetal bovine serum (growth medium), or chondrogenic medium to promote chondrogenesis (4.5 g/L DMEM high glucose, 100 U/ml penicillin, 100 μl/ml streptomycin, 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 μg/ml selenous acid, 5.35 μg/ml linoleic acid, 1.25 μg/ml bovine serum albumin, 1 mM sodium pyruvate, 37.5 μg/ml ascorbate 2‐phosphate, 10 −7 M dexamethasone, and 10 ng/ml TGF‐β3).…”

“…The aspirates were incubated for 90 minutes at 37°C and 5% CO 2 , with subsequent addition of 60 μl of DMEM, 10% fetal bovine serum (growth medium), or chondrogenic medium to promote chondrogenesis (4.5 g/L DMEM high glucose, 100 U/ml penicillin, 100 μl/ml streptomycin, 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 μg/ml selenous acid, 5.35 μg/ml linoleic acid, 1.25 μg/ml bovine serum albumin, 1 mM sodium pyruvate, 37.5 μg/ml ascorbate 2‐phosphate, 10 −7 M dexamethasone, and 10 ng/ml TGF‐β3). Incubation was pursued for up to 21 days, with careful medium change once per week .…”

“…Detection of lacZ expression was performed by X‐Gal staining (Roche Applied Science) and by immunohistochemistry using a specific primary antibody, a biotinylated secondary antibody, and diaminobenzidine (DAB) as the chromogen via the ABC method (Vector Laboratories, Alexis Deutschland GmbH, Grünberg, Germany, https://vectorlabs.com) , . The production of TGF‐β was monitored by ELISA and via immunohistochemistry.…”

“…Aspirates were collected after 21 days in a total volume of 100 μl of fresh, supplement‐free DMEM and digested with papain (final concentration, 75 μg/ml) at 60°C for ≥60 minutes . The DNA contents were measured by Hoechst 22358 assay; the proteoglycan contents were determined by binding to DMMB; and the type II, I, and X collagen contents determined by specific ELISAs . A protein assay (Pierce, ThermoFisher Scientific Life Sciences, Schwerte, Germany, http://www.thermofisher.com) was used to monitor total cellular proteins for normalization.…”