**1. Introduction**

Articular cartilage lesions represent serious clinical issues in orthopaedics as this specialized tissue does not fully heal on itself by lack of vascularization and of local chondroregenerative cells that may repopulate the defects [1,2]. Despite the availability of a number of clinical interventions (Pridie drilling, microfracture, cell transplantation), none can promote the generation of the original hyaline cartilage (proteoglycans, type-II collagen) in the lesions, with instead the appearance of a fibrocartilaginous repair tissue (type-I collagen) showing lesser mechanical properties and that may be prone to osteoarthritis [1–4]. Administration of chondroreparative mesenchymal stromal cells (MSCs) [5–7] in focal cartilage defects represents a valuable therapeutic alternative to activate the local healing processes [8,9], yet here again formation of the native hyaline cartilage is not observed [8,9], showing the necessity to develop improved treatments for adapted cartilage repair.

Scaffold-assisted gene transfer is an attractive therapeutic approach for cartilage repair as it has the potential to activate the intrinsic repair processes in sites of cartilage lesions by controlling the delivery of carriers coding for candidate genes [10–12], having been reported using nonviral [13–19] and lentiviral vectors [20–23]. While such gene vectors commonly support short-term transgene expression (nonviral vectors) or have the potential to activate oncogenes following genome integration (lentiviral vectors), vectors based on adeno-associated viruses (AAV) may be more adapted as they promote transgene expression over extended periods of time (some years) in a much safer manner due to the lack of viral protein coding sequences in the recombinant AAV (rAAV) backbone [10,11]. Thus far, biomaterial-assisted rAAV gene transfer for cartilage research has been described including polymeric micelles [24–27], hydrogels [28–32] and solid scaffolds [33], yet other materials may constitute valuable systems for rAAV delivery in experimental cartilage therapy. In this regard, carbon dots (CDs), a recently discovered class of carbon-dominated, biocompatible nanomaterials [34,35] used in drug delivery and theranostic approaches [35,36], may be good candidates to achieve this goal as they have been reported for their ability to intracellularly deliver nucleic acids and proteins in vitro [37] and in experimental models in vivo of cancer [38–40] and for regenerative medicine [41,42]. It remains to be seen whether CDs are capable of assisting rAAV vector transfer for cartilage repair as such vectors are more effective than nonviral vehicles to deliver genetic material in target cells [10,11].

The goal of this study was therefore to evaluate the potential of various CDs to associate with and release rAAV vectors as a means to target chondrogenically competent human MSCs (hMSCs), with a focus on transferring DNA sequences for the highly chondroreparative sex-determining region Y-type high mobility group box 9 (SOX9) transcription factor [43] and transforming growth factor beta (TGF-β) [5,6]. The data show that CDs are potent systems to efficiently vectorize and release rAAV, especially CD-2 nanoparticles, which allow hMSCs to be optimally targeted via rAAV gene transfer. Specific delivery of rAAV vectors carrying either the candidate SOX9 or TGF-β sequences assisted by CD-2 led to effective expression of the transgenes in these cells, enhancing cell proliferation and cartilage matrix deposition (glycosaminoglycans, type-II collagen) with reduced type-I and -X collagen production. These findings provide evidence on the ability of CD-assisted therapeutic rAAV gene delivery to target chondroreparative hMSCs in future non-invasive and safe applications to treat sites of cartilage injury.
