2.7. In vitro cartilage regeneration of constructs

To create PRP + CPC constructs, 1 × 10⁶ CPCs at passage 3 were collected and suspended by 500 μL of PRPs, followed by treatment of a cocktail of 10% CaCl₂–40U/ml BT or not (Table 1). Constructs were cultured in DMEM supplemented with 10% vol/vol FBS, and incubated at 37 °C in an atmosphere of 5% CO₂. For chondrogenic differentiation, 1 × 10⁶ CPCs were centrifuged in polypropylene tubes at 300g for 10 min to form a pellet and maintained in chondrogenic induction medium consisting of DMEM, supplemented with 1% vol/vol insulin-transferrin-sodium selenite, 10⁻⁷ M dexamethasone, 1 mM sodium pyruvate, 50 μM ascorbate-2-phosphate, 50 μg/mL proline, and 20 ng/mL TGF-β3, which was regarded as a successful method to generate cartilage in vitro and can serve as positive control group according to previous studies [17,19,34]. On day 28, the constructs were fixed and sectioned, followed by evaluation of the cartilaginous matrix by HE, toluidine blue, and Safran-O staining. The expression of Col-II was detected by immunohistochemistry [35]. The images were captured using a microscope under brightfield mode and evaluated blindly by 5 graders according to the guidelines of the visual scoring system (Bern Score) based on published protocols for in vitro generated cartilaginous tissue [36]. For determining the biomechanical capacity of regenerated cartilage, the regenerated tissues from 6 groups were cut into square sections (2 mm³) along the long axis and subjected to biomechanical tests including tensile, compressive, and shear testing [19].

Experimental groups of the in vitro and in vivo studies.

PRP, platelet-rich plasma; CPC, chondrogenic progenitor cells; BT, bovine thrombin.