Table 1.
Preparation prior to in vitro co-culture experiment start Day 0.
Table 2.
Assays performed on 3,7, and 14 days.
Table 3.
Scoring criteria for chondrocyte morphology, lesion gross anatomy, adjacent articulate cartilage growth and subchondral bone repair.
Fig 1.
Histomorphometric measurements of lesion filling.
Five measurements were taken for lesion filling a) width of opening halfway between the lesion bottom and the projected original surface b) width, at the same depth as measurement a, of opening of physiological cartilage, c) depth of the opening at the center, d) depth at 100μm from edge of original lesion, and e) depth of original lesion, from the projected cartilage surface to the estimated original tidemark. Red dashed line represents the size of the original lesion (2.5 mm wide x ~ 1.5 mm deep).
Fig 2.
Co-culture chondrocyte cell counts and intracellular Col II production.
Cell counts (A) and Col II concentration (B) from chondrocytes cultured alone (white), with sECM (green), untransduced-sConstructs (blue) or BMP-2-sConstructs (red) on day 3, 7, and 14.
Fig 3.
Co-cultured chondrocyte monolayers and morphology scores.
Chondrocytes co-cultured with sECM or BMP-2-sConstructs. Selected photomicrographs, 20X, (A-F) and scores (G) from chondrocytes cultured alone (white), with sECM (green), untransduced-sConstructs (blue) or BMP-2-sConstructs (red) on day 3 and 7.
Fig 4.
Co-culture sConstruct MSC soluble biomarker concentrations.
HA (A), PG (B) and BMP-2 (C) from chondrocytes alone (white), sECM alone (green hatched), sECM chondrocyte co-culture (green), untransduced-sConstructs alone (blue hatched), untransduced-sConstructs chondrocyte co-cultures (blue) and BMP-2-sConstructs alone (red hatched), and BMP-2-sConstructs chondrocyte co-cultures (red) from media collected 1–3, 4–7, and 8–14 days.
Fig 5.
Co-culture sConstruct sMSC count, viability, and maturity.
Total cell count (A), % 7AAD (B), and % CD90 expression (C) with untransduced-sConstructs alone (blue hatched), untransduced-sConstructs in co-culture (blue), BMP-2-sConstructs alone (red hatched) and BMP-2-sConstructs in co-culture (red) after incubation for 3, 7, and 14 days.
Fig 6.
Lesion with sConstructs implants; Smooth fibrocartilage repair tissue, adjacent articular cartilage and subchondral bone repair.
Illustrative (median representative) photomicrographs (40x) of histochemical toluidine blue stained sections of rat knee lesions after 5 weeks of healing. Specimens selected for the photograph had the middle histology score for that group and thereby is a mid-representative of the differences. A) Lesions with BMP-2-sConstruct implants where the white arrow (⇨) points to smooth fibrocartilage repair tissue, the black arrow (➨) to chondrocyte/lacunae duplets, and the red dot, the histochemical staining for PG. B) Image as Fig 6A but from a control knee with suture only. C) Lesions with BMP-2-sConstruct implants where black arrow (➨) points to re-established subchondral bone and dotted line (- - - - -) is re-established tidemark, D) Similar image to Fig 6C from a control knee with suture only. Scale bar: 200 μm.
Fig 7.
Lesion with sConstruct implants; Growth scores and filling measurements.
Gross anatomy (A), articulate cartilage (B), and subchondral bone repair scores (C) and bottom (D), edge (E), sides inwards (F) and peripheral cartilage (G) measurement ratios. Lesion with no implant (white), with sECM (green), GFP-sConstructs (blue) or BMP-2-sConstructs (red).
Fig 8.
sConstructs recovered from implant site.
Histophotomicrographs (2X) of implants recovered from rat knee synovium. A) suture only with suture tracks (➨), B) sECM, C) GFP-sConstruct and D) BMP-2-sConstruct. Inserts are 20X magnification and C) was stained immunohistochemically for the presence of GFP. Scale bars represent 100 μm in the main picture and 10 μm for the inserts.