Figure 1.
rASCs culture and GFP labeling.
(A) rASCs at four passages, which appears to be a fibroblast-like uniform shape. (B) Fluorescence microscopic photograph of GFP labeled rASCs in Figure 1A. Most cells were GFP positive (Bar scales: 100 µm)..
Figure 2.
Proliferation and distribution of rASCs on a PLGA scaffold and secretion of the extracellular matrix (ECM).
(A) Gross view of the PLGA scaffold with a 7 mm diameter. (B) Scanning electron micrograph (SEM) images of PLGA scaffold without cells. (C) The view of the rASCs and PLGA scaffold at 7 days was observed by confocal microscopy to reveal the cell distribution on the scaffold (Bar scales: 20 µm). (D) Attachment of rASCs on the same PLGA scaffold and deposition of the ECM are shown by SEM (magnification×500). (E) The quantitative evaluation of the proliferation of rASCs seeded on the PLGA scaffold determined by DNA assay using Hoechst33258 dye. Abbreviations: rASCs, rabbit adipose stem cells; PLGA, polylactic-co-glycolie acid; SEM, scanning electronic microscope.
Figure 3.
Gross view of the grafts in each group at 12 weeks and 24 weeks post-surgery.
A, B, C: 0 weeks; D, E, F: 12 weeks; G, H, I: 24 weeks. A, D, G: Autologous rASCs+PLGA; B, E, H: PLGA alone; C, F, I: Only defect. At 12 weeks, the scaffold was mostly degraded and the corneas became nearly transparent in the group with implantation of the rASCs-PLGA construct while a partial scaffold still resides in the group with PLGA alone implantation. These two groups became transparent and showed no significant difference compared with the defect group and the normal cornea at 24 weeks. No inflammation or vascularization was found in all groups at 24 weeks after implantation.
Figure 4.
Histological analysis of tissue engineered grafts in each group at different time points.
A, D, G, J: autologous rASCs+PLGA group; B, E, H, K: PLGA group; C, F, I, L: Only defect group; A-F: 12 week; G-L: 24 week. HE stained histological sections showed that the corneal epithelial cells and endothelial cells were intact in all groups. Corneas in the group with implantation of autologous rASCs-PLGA had been repaired by newly formed tissue, forming more native structures at 24 weeks than at 12 weeks post transplantation. In PLGA alone and only defect group, collagen remodeling was not successful. Corneas were thinner, collagen arrangement was not in good order, and scars existed (A-C, G-I: Magnification ×100; D-F, J-L: Magnification ×200)..
Figure 5.
Distribution and diameter of collagen fibrils in different groups observed by transmission electron micrography.
A, B: the rASCs+PLGA group autologous; C: PLGA group; D: Only defect group; A: 12 weeks post-implantation; B-D: 24 weeks post-implantation. (E) Distribution and diameter of collagen fibrils in normal cornea. Electron microscopic examination showed larger interfibrillar interval existed in the neoformative stromal matrix (arrow in Figure 5A) at 12 weeks, while normal lamellae containing regularly spaced fibrils formed at 24 weeks. Fibril diameter analysis showed neoformative stroma regained the identical diameter distribution of collagen fibrils to those of normal rabbit corneas in auto-rASCs group at 24 weeks (F) (Magnification ×12000)..
Figure 6.
Survival of GFP-positive rASCs after 24 weeks of implantation and their differentiation to functional keratocytes.
(A-H) corneas implanted with autologous rASCs-PLGA constructs; (A, E) GFP expression, (green color); (B, F) nuclei staining by hoechst33258 at the same section, (blue color); (C) expression of keratocyte specific proteoglycan keratocan at same section, (red color); (G) expression of ALDH1A1 at same section, (red color); (D, H) GFP expression, nuclei staining and keratocan, ALDH1A1 immunostaining are superimposed with Adobe Photoshop software, showing keratocyte proteoglycan production by implanted GFP-positive rASCs (Bar scales: 50 µm)..
Figure 7.
Rabbit keratocan and ALDH1A1 expression in each group at 24 weeks post-implantation.
A, B, C: keratocan; D, E, F: ALDH1A1. (A, D) cornea implanted with autologous rASCs-PLGA constructs; (B, E) cornea implanted with PLGA alone; (C, F) cornea with defect only. (A-C) expression of keratocan; (D-F) expression of ALDH1A1. Both keratocan and ALDH1A1were positioned in the implanted layer of corneas implanted with the rASCs-PLGA complex, while the result was negative in corresponding layers in groups implanted with PLGA alone and defects only (Magnification×200)..
Figure 8.
Messenger RNA levels of keratocan and ALDH1A1 were determined by real-time reverse transcriptase - polymerase chain reaction analysis.
* p<0.05, **p<0.01.