Fig. 1. (A) A schematic diagram detailing the fabrication of the mineralized alginate-chitosan beads and analysis. Mineralized polysaccharide beads were cultured in static or dynamic conditions. Transplanted beads were cultured in static conditions for one day before implantation. In this study, we looked at cell osteoinduction in 3-types of alginate microenvironment differing in ECM components. We compared the effectiveness of BM and BMP-2 in alginate beads to differentiate MSCs into osteogenic cells and osteoid tissue. (B) Standardized bead shape, size, shell structure and internal architecture of alginate beads; (i) Single alginate bead measuring 1 mm in diameter; (ii) light microscope image in polarized light to highlight the semi-crystalline structure of the calcium phosphate/ chitosan shell surrounding the alginate core (white arrow). (iii) Histological thin section of a single bead to demonstrate the internal architecture and external morphology; (iv) a single H & E stained sectioned bead to show the distribution of a fully loaded capsule for cell culture; (C) a diagrammatic representation of the four different types of augmented bead environments used in the study; (D) H&E staining of capsules containing hMSCs at different time points in the four individual bead microenvironments (BMP-2 added, BM proteins added, guest, added and normal alginate) to show the changes in cell distribution, density and proliferation. hMSCs inside BMP-2 beads, guest BMP-2 beads, and BM beads increased cell numbers compared to alginate beads. Also, in control group, hMSCs inside alginate beads distributed well separated and did not form clusters compared experiment groups. (E) hMSC numbers increased significantly in the guest BMP-2 beads and the BM beads compared to the MSCs beads and the BMP-2 beads. (* = Guest BMP-2 bead) (Scale bar = 200 μm).
Fig. 2. (A) Immunolabeling results to identify osteogenic related proteins contrasting between the hMSCs beads, BMP-2 beads, guest BMP-2 beads and BM beads. After two weeks culture, collagen I expression was stronger in the BMP-2 added group and BM added group compared to control group and guest BMP-2 group. The CD 90 expression was absent in all groups after two weeks culture. Thus, all encapsulating hMSCs lost stemness after two weeks culture. MSCs expressed Osterix, the marker for pre-osteoblasts, in alginate, and at higher levels in the BMP-2 and BM beads. Runx-2 staining was positive in the BMP-2 added, guest BMP-2 added and the BM beads, and absent in the control group. The osteocalcin cell expression in the BM beads compared with alginate beads, BMP-2 beads and nested BMP-2 beads; (scale bar = 400 μm); (B) percentages of positively versus negatively stained cells for Runx-2, osteocalcin, and CD90 to show the maturation phase of the hMSCs and their conversion into osteogenic lineage cells. (* = Guest BMP-2 bead). (C) Masson’s trichrome staining shows the in vivo generated structures from the four different kinds of alginate bead transplants within a mouse cranial bone defect. We find a difference between the MSC control beads inside the defect, and the added BMP-2 and BM alginate beads, which both show a narrowing of the pockets and contain osteoid tissue. More significantly, we see that the beads harboring a BMP-2 guest bead generated an extensive series of osteoid pockets across the defect region (C) accompanied by an even tighter narrowing of the regenerated tissue inside the defect. The IHC staining for OCN and OPN is negative among the control MSC beads. In contrast to the MSC beads, BMP-2 and BM laden beads were OPN and OCN positive. The spaces represent the alginate. Among the defects containing nested alginate beads, the staining for OCN and OPN was larger, more widespread and associated with the osteoid pockets (C; * = Guest BMP-2 bead; b