gms | German Medical Science

Objectives: Meniscal tears remain an unsolved problem in sports medicine and gene therapy may manage them with the clinically suited recombinant adeno-associated virus (rAAV) therapeutic gene vectors. The aim here was to monitor the effects of rAAV-mediated overexpression of FGF-2 and TGF-β via scaffold (alginate hydrogel)-guided vector delivery in primary human experimental meniscal lesions to improve the processes of meniscal healing.

Methods: rAAV vectors were routinely packaged, purified, and titrated, with rAAV-lacZ carrying the E. coli β-galactosidase (lacZ) reporter gene, rAAV-hFGF-2 a human FGF-2 sequence, and rAAV-hTGF-βa human TGF-β sequence, all controlled by the CMV-IE promoter/enhancer. rAAV (80 µl) were formulated in 1.5% alginate (AlgPH155) (80 µl) hydrogel (1:1, v/v) by direct mixing in a syringe. Alginate/rAAV hydrogel systems were implanted in human experimental meniscal lesions made with scalpel further maintained in DMEM, 10% bovine calf serum, 1% penicillin-streptomycin at 37°C under 5% CO2 for 21 days. Expression of FGF-2, TGF-β, type-I and -III collagen, IL-1 β, and TNF-α was detected by specific ELISAs (R&D Systems). The proteoglycan, DNA, and total protein contents were assessed by dimethylmethylene blue (DMMB), Hoechst 33258, and Pierce BCA Protein assays. Total RNA was extracted and reverse transcription carried out for cDNA amplification with Ct values acquired for each target genes (COL1A1, COL3A1, ɑ-SMA, COL2A1, COL10A1, IL-1 β, TNF-α) relative to GAPDH as a control for normalization using the 2-Δ ΔCt method. A t-test was employed where P≤0.05 was considered statistically significant.

Results: Overexpression of FGF-2 and TGF-β via rAAV was successfully achieved over time in FGF-2- and TGF-β-treated human meniscal explants, respectively, upon delivery with the alginate hydrogel (Figure 1 A, B [Fig. 1]). As a result, COL1A1 and COL3A1 expression increased in the FGF-2- and TGF-β-treated human meniscal lesions over time versus lacZ together with an increased expression of contractileα -SMA while no marked difference was noted between conditions when evaluating COL2A1 and COL10A1 expression (Figure 1C [Fig. 1]). Yet, administration of rAAV-hFGF-2 and rAAV-hTGF-β via alginate hydrogel decreased the expression of inflammatory mediators (IL-β, TNF-α) versus rAAV-lacZ. Furthermore, overexpression of FGF-2 and TGF-β via rAAV from the alginate hydrogel increased the proteoglycan contents in human meniscal tissue relative to lacZ treatment (Table 1 [Tab. 1]).

Conclusion: Delivery of rAAV-hFGF-2 and rAAV-hTGF-β guided by an alginate hydrogel is a valuable,non-invasive procedure for enhanced meniscal repair.