Fig 1.
KGN bead implantation in rat Achilles tendons in vivo.
A. KGN beads were prepared by squeezing drops of KGN+DMSO+alginate solution through a pipet into a calcium chloride solution. B. KGN beads air dried for 48 hrs. Diameter of the dried beads was 169 ± 32 μm. C. Rat Achilles tendon with the implanted bead. KGN implanted rats were allowed free cage activities until further analyses. Arrow (C) points to the KGN bead implanted site. Bar– 1 mm.
Table 1.
Rat RT-PCR primer sequences.
Fig 2.
Gross appearance of rat Achilles tendons after KGN bead implantation in vivo.
Five weeks after implantation, KGN bead implanted Achilles tendons showed thickened peritenon (A, triangle points to the skin), while the DMSO bead implanted control Achilles tendons had an apparent normal structure (B) similar to the normal rat Achilles tendon (C). Arrows indicate the site of KGN bead or DMSO bead implanted area. Bar– 1 mm.
Fig 3.
H&E staining of the KGN-bead implanted tendons.
Five weeks post-implantation of KGN beads (A), round chondrocyte-like cells (boxed) were observed in the KGN bead implanted tendons only. The boxed area in A is enlarged in C. Green arrows indicate chondrocyte-like cells. In contrast, DMSO-bead implantation did not affect tendon structure (B). The rectangular area in B is enlarged in D. Red arrows indicate normal tendon cells. Bar– 100 μm.
Fig 4.
Safranin O/Fast green (A, C, E) and Alcian blue/Fast nuclear red (B, D, F) staining of the KGN bead implanted sites in rat Achilles tendons in vivo.
Note that C is an enlargement of the rectangled region in A. Arrows point to the area outside the KGN-bead implantation site that appears normal. D is an enlargement of the yellow box in B. Triangles indicate the normal areas outside the KGN-bead implantation site. Positive staining was observed only in the KGN bead implanted sites (A, B, C, D) indicating the presence of glycosaminoglycan rich matrix representing chondrogenesis only at these sites. The DMSO bead implanted tissue sections stained minimally for these stains (E, F). Bar– 100 μm.
Fig 5.
Immunohistochemical staining of CD31 to visualize neovascularization in KGN bead implanted rat Achilles tendons.
CD31 positive staining (red) was observed (A, B) along the Alcian blue and fast red stained regions (C) in the KGN bead implanted Achilles tendon. Rest of the tendon was negative for CD31. Arrowheads indicate the chondrocyte-like cells and arrows point to the newly formed blood vessels. These vessels were positive for CD31. The DMSO bead implanted tendons were negative for CD31 staining (D, E), chondrocyte-like round cells and neovascularization (F). A, B–Red indicates CD31 staining; B, E–Blue indicates nuclear staining by Hoechst 33342. C, F–Alcian blue and fast red staining. Bar– 100 μm.
Fig 6.
Immunohistochemical staining of collagen type II (Col. II) in KGN bead implanted rat Achilles tendons.
The KGN bead implanted site in the Achilles tendon stained positive for Col. II (green in A, C–pink box; large boxes are enlargements of corresponding small boxes). Red arrows point to staining artifacts (green along the tendon edge), whereas the surrounding tissues and the DMSO bead implanted tissues (D, F) stained negative for Col. II. A, D–Green indicates positive Col. II staining; B, E–Blue indicates nuclei stained with Hoechst fluorochrome 33342; C, F–Overlay of Col. II and Hoechst staining. Bar– 150 μm.
Fig 7.
Release of KGN from KGN-alginate beads during their degradation in vitro at 37°C in PBS at various time points.
(A) Changes in the weight of KGN-DMSO-alginate beads during degradation. (B) KGN release kinetics during degradation of the KGN-DMSO-alginate beads.
Fig 8.
Safranin O staining of KGN treated rat Achilles TSCs in vitro (A-F).
TSCs treated with various KGN concentrations for 21 days were stained with Safranin O. Red color indicates the increase in the accumulation of glycosaminoglycan-rich matrix in cell cultures in a KGN concentration-dependent manner (KGN concentration: A– 0 nM; B– 1 nM; C– 10 nM; D– 100 nM; E– 1 μM; F– 10 μM). Gene expression analysis in KGN treated TSCs in vitro (G). Rat TSCs were cultured in growth medium containing various KGN concentrations for 72 hrs, followed by RNA isolation and qRT-PCR to determine chondrocyte specific gene expression. KGN up-regulated the expression of aggrecan, Col. II and Sox-9. Values represent mean ± SD. Asterisks indicate significant differences (P < 0.05) between the KGN treated and control groups (0 nM). Bar– 100 μm.