Fig 2. Histology showing darkfield images along with ALP, TRAP and hematoxylin stained sections of defects implanted with (a-d) Chi-AHP at 4 weeks; (e-h) Chi-AHP+BMP at 4 weeks; (i-l) Chi-AHP at 8 weeks; and (m-p) Chi-AHP+BMP at 8 weeks.
Darkfield image: Accumulated mineral is shown in white color. Osteoblasts are EYFP positive (Topoz). Red. AC label indicates active mineralization in the last 24h; ALP staining: Red color indicates ALP positive osteoblats. Cell nuclei are stained white with DAPI; TRAP staining: Yellow color indicates presence of osteoclasts. Scale bar = 200 μm.
No mineralized tissue formation was observed in the Chi-AHP implanted defect site at 4 or 8 weeks of implantation. Chi-AHP+BMP showed an early woven bone-like mineralized tissue (indicated by #) at 4 weeks. At 8 weeks, a lamellar new bone (indicated by #) was observed in Chi-AHP+BMP implanted defect site along with marrow-like areas (indicated by ^) and blood vessels (indicated by O). Both Chi-AHP and Chi-AHP+BMP implanted defects showed the presence of non-degraded residual thermogel (indicated by *) and a periosteum-like layer composed of fibroblastic cells (indicated by arrow), at 4 and 8 weeks.
Fig 3. Histology showing darkfield images along with ALP, TRAP and hematoxylin stained sections of the critical sized defects implanted with Chi-AHP+C and Chi-AHP+C+BMP, at 4 weeks. (a) Darkfield image showing section of the whole calvaria; (b-f) High magnification images of Chi-AHP+C implanted defect site; (g-k) High magnification images of Chi-AHP+C+BMP implanted defect site
Darkfield image: Accumulated mineral is shown in white colour. Host derived osteoblasts are EYFP positive (Topaz) whereas donor derived osteoblasts are ECFP positive (Cyan). Red AC label indicates active mineralization in the last 24h; ALP staining: Red colour indicates ALP positive osteoblasts. Cell nuclei are stained white with DAPI; TRAP staining: Yellow colour indicates presence of osteoclasts. Scale bar: 500 µm. (n=5 at 4 week and n=6 at 8 weeks).
Chi-AHP+C showed an early woven bone-like mineralized tissue (indicated by #) at 4 weeks which was associated with ECFP positive donor cells with diffuse AC label, weak ALP activity and no appreciable TRAP activity. Chi-AHP+C+BMP group showed two types of mineralized areas (indicated by # and ^). The mineralized areas indicated by # showed ECFP positive donor cells with diffuse AC label, weak ALP and minimal TRAP activity. The area indicated by ^ showed EYFP positive, host derived osteoblasts with a sharp mineralization line (AC label), co-localized with ALP activity and good TRAP activity. Both Chi-AHP+C and Chi-AHP+C+BMP implanted defects showed the presence of non-degraded residual thermogel (indicated by *) and a periosteum-like layer composed of fibroblastic cells (indicated by arrow) at 4 and 8 weeks.
Fig 4. Histology showing darkfield images along with ALP, TRAP and hematoxylin stained sections of the critical sized defects implanted with Chi-AHP+C and Chi-AHP+C+BMP, at 8 weeks. (a) Darkfield image showing section of the whole calvaria; (b-f) High magnification images of Chi-AHP+C implanted defect site; (g-k) High magnification images of Chi-AHP+C+BMP implanted defect site
Darkfield image: Accumulated mineral is shown in white colour. Host derived osteoblasts are EYFP positive (Topaz) whereas donor derived osteoblasts are ECFP positive (Cyan). Red AC label indicates active mineralization in the last 24h; ALP staining: Red colour indicates ALP positive osteoblasts. Cell nuclei are stained white with DAPI; TRAP staining: Yellow colour indicates presence of osteoclasts. Scale bar: 500 µm. (n=5 at 4 week and n=6 at 8 weeks).
Chi-AHP+C showed an early woven bone-like mineralized tissue (indicated by #) predominantly associated with ECFP positive donor cells with diffuse AC label and co-localized ALP, along with a few EYFP positive host cells. Some TRAP activity was also observed and was mostly confined to the areas associated with the host EYFP cells. Chi-AHP+C+BMP group showed two types of mineralized areas (indicated by # and ^). The mineralized areas indicated by # showed ECFP positive donor cells with diffuse AC label, weak ALP and minimal TRAP activity. The area indicated by ^ showed EYFP positive, host derived osteoblasts with a sharp mineralization line (AC label), co-localized ALP activity and good TRAP activity. Both Chi-AHP+C and Chi-AHP+C+BMP implanted defects showed the presence of non-degraded residual thermogel (indicated by *) and a periosteum-like layer composed of fibroblastic cells (indicated by arrow) at 4 and 8 weeks.
Fig. 5. Merged overlay of darkfield, TRAP and ALP stained defect area implanted with Chi-AHP+C group at 8 weeks.
The defect area shows donor derived ECFP positive cells with diffuse AC and strong ALP activity (red color) but minimal associated TRAP activity indicating that these are immature early osteoblast cells making a woven bone like mineralized tissue even at 8 weeks. EYFP positive host cells detected in the center of the defect, however, show a sharp mineralization line (AC label), strong ALP activity and TRAP activity indicating the presence of actively mineralizing and remodeling bone matrix. Scale bar= 150 µm