Isolation and Implantation of Bone Marrow–Derived Mesenchymal Stem Cells with Fibrin Micro Beads to Repair a Critical-Size Bone Defect in Mice

Abstract

Fibrin microbeads (FMBs) made using thermal treatment of fibrin drops in oil can efficiently isolate mesenchymal stem cells (MSCs) from bone marrow (BM) and other similar sources and culture them continuously in suspension culture. The pure mesenchymal profile of MSCs isolated using FMBs and their differentiation potency to different mesenchymal lineages were previously described in detail. In the current study, MSCs were isolated from the BM of (GFP+) C57/bl mice using FMBs. Addition of pro-osteogenic medium with 10 mM of ss-glycerolphosphate, 50 microg/mL of ascorbic acid, and 10(-8) M of dexamethasone for 1 month resulted in ossified bone-like solid cellular structures, as seen using fluorescence and scanning electron microscopy (SEM). Such spontaneously formed structures were implanted in full-depth approximately 5-mm-diameter drilled defects in the skulls of wild-type c57/bl mice. Two months later, the excised upper parts of the skulls with the defects were viewed using fluorescence microscopy for green fluorescence protein of the cells in the defect and using SEM. They were also scanned using micro-computed tomography to visualize the formation of new hard tissue. Then the samples were processed and sectioned for hematoxylin and eosin staining and immunohistochemistry. Implanted FMBs loaded with (GFP+) MSCs formed partially mature, dense bone-like tissue using a residual moderate inflammatory process containing remnants of FMBs and neo-angiogenesis. The filled defect with bone-like tissue had a Ca/P ratio similar to that of native bone. Limited merging of the implant with the skull indicated that the induced bone regeneration derived from the MSCs that were delivered with the implant. No repair was seen in the control animals without implants or where the defect was filled with FMBs only. Repair scoring (on a 0-5 scale) was found to be 3.38+/-0.35 in the experimental arm, relative to 0 in the controls (p < 0.001).

A review of fibrin and fibrin composites for bone tissue engineering

“…After 2 months, results showed that defects filled with bone-like tissue had a Ca/P ratio similar to that of the native bone while no repair was seen in the control animals without implants or when the defect was filled with FMBs alone. 102 In addition to efficiently repairing bone, the use of FMBs successfully eliminated stages of culturing the MSCs on tissue culture plastics, trypsinizing the cells that can damage their cellular membrane receptors, performing cell passages, and finally loading the cells onto the scaffolds. 102 Furthermore, FMBs may be used as a matrix onto which cells can be maintained for a long time at room temperature.…”

“…102 In addition to efficiently repairing bone, the use of FMBs successfully eliminated stages of culturing the MSCs on tissue culture plastics, trypsinizing the cells that can damage their cellular membrane receptors, performing cell passages, and finally loading the cells onto the scaffolds. 102 Furthermore, FMBs may be used as a matrix onto which cells can be maintained for a long time at room temperature. The matrix preserves their proliferation and bone differentiation potential, which in turn facilitates cellular transformation between different research and/or clinical centers.…”

Gender-Related Differences in a Process of the Age-Dependent Alterations of the Elements in Monkey Sino-Atrial Node

“…The phenomena would be mainly caused by the alterations of Ca, P, Fe, S, Zn and Mg. The gender difference was significant in the relation between Ca and P. The role of Ca/P has already been shown in bone [29,30], auditory ossicles [31], and teeth [32,33]. Furthermore, the Ca/P ratio plays a role for vascular calcification of aorta and coronary arteries [12,34].…”

The Use of Micro- and Nanospheres as Functional Components for Bone Tissue Regeneration

“…55,56 Moreover, these microspheres can also serve as a construct to support in vitro MSCs expansion and osteogenic differentiation, which consequently formed a calcified matrix embedding osteoblasts, 57 and further to induce ectopic bone formation 58 as well as reconstruction of critical-size bone defects in vivo. 59 However, the rapid degradation rate and poor mechanical stability of fibrin have been stated as the main limitation for bone tissue engineering. 54 To overcome this problem, composite microspheres were developed to stabilize the fibrin matrix.…”