3.

Constructs with BMSC sheet-scaffolds (mPCL-TCP) were implanted into nude rat, harvested after 28, 56 and 84 days. (A) Gross appearance of BMSC sheet-scaffolds constructs with osteogenic induction (Right) and non-induction (control) (Left) after 4 weeks. (B) X-ray detected bone like tissue formation in 28 day (C) 56 day (D) 84 day implants. (E) X-ray image of constructs without cell seeding (Control). (F) Micro-CT demonstrated the overall highly mineralized tissue similar to cortical (golden) and cancellous (red) bone in implanted constructs after 28 day. (G) Micro-CT images disclosed the hard tissue formation within constructs. Mineralized tissue with similar density with cancellous bone was detected (darker areas) while the lighter colour represented cortical bone. (H) Fluorescence was detected on the formed bone tissue after 28 days; the fluorescence came from the cFDA labeled BMSC. (I) Micro-CT quantification of implant tissue compositions depicted substantial bone formation in the induction group, accounting for 40% total volume for the 28, 56 and 84 day implantation, while the control group formed only connective tissue. (J) H&E staining shows lamellar bone-like tissue formed in both outer part and interior of constructs after 28 days. (K) High magnification image shows well organized lamellar bone like tissue (Bo) with distinct osteocytes located within bone tissue. (L) The typical osteoblasts (OB, black arrow) located on the surface of neo mineralized tissue with marrow cavities and blood vessels in 56 days implants. (M) Safranin-O staining demonstrated that hypertrophic chondrocytes (white arrow) were observed in 56 day implants in very low numbers. High magnification shows the chondrocytes (white arrow) with weak safranin-O staining surrounded by the osteocyte (black arrow). (N) OCN staining. Strong signals (arrow head) were detected on neo mineralized tissue while very weak to no-signals were detected on chondrocyte like cells (O). (P) Collagen type I staining. Extensive staining was detected on the neo mineralized tissue in constructs. (Q) Collagen type II staining. Limited signals (arrow head) were detected on chondrocyte like cells while no staining for the mineralized tissues. BV: blood vessel + red blood cells; Ma: marrow; OB: osteoblast; Bo: bone; OC: osteocyte; CY: chondrocyte. Scale bar: (J) 200μm; (H-Q) 50_m. Image reproduced from reference 11 with permission from Elsevier.