Three different heterologous substitutes for bone regeneration, manufactured with equine-derived cortical powder (CP), cancellous chips (CC) and demineralized bone matrix granules (DBM), were compared in in vitro and in vivo settings. We tested: a commercially available bone paste (Osteoplant-Activagen, consisting of aqueous collagenous carrier, CP, DBM; named A); a second-generation injectable paste (20 kDa polyethylene glycol/hydroxypropyl-methyl cellulose-based hydrogel, CP, DBM; B); a pre-formed bone filler (400 kDa polyethylene oxide/hydroxypropyl-methyl cellulose-based hydrogel, CP, CC, DBM; C). Vitamin C acted as a visco-modulator during C and B beta-rays sterilization, modifying graft injectability. For each filler, we examined dissolution in culture medium, gene expression of the substitute-exposed osteogenically-induced human bone marrow stromal cells (hBMSC), and performance in a rabbit bone defect model. A dissolved after 1 h, while fragmentation of B peaked after 8 h. C remained unaltered for 2 days, but affected the microenvironmental pH, slowing the proliferation of exposed cells. B-exposed hBMSC overexpressed bone sialoprotein, osteocalcin and RUNX2. For all fillers histological results evidenced bridged lesion margins, marrow replenishment and bone-remodeling. However, B-treated lesions displayed a metachromatic type II collagen-rich matrix with prehypertrophic-like cells, matching the in vitro expression of cartilage-specific markers, and suggesting a possible application of B/C double-layer monolithic osteochondral plugs for full-thickness articular defects.
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