Amniotic Growth Factor induced bone formation in a mouse ex-vivo model
Bamashmous, Abdullah Othman
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BACKGROUND: Cells, growth factors and scaffold are the 3 fundamental factors currently proposed necessary for tissue regeneration. The use of these components has to be orchestrated precisely for ideal functional tissue formation. Growth factors enhance cellular activities that may lead to angiogenesis, cell proliferation and extracellular matrix biosynthesis. Due to the complexity of biochemical reactions a single growth factor may have limited effect. In order to explore a mixed profile of growth factors, a new biomaterial containing multiple growth factors derived from human Amniotic Membrane was chosen to compare with a known single growth factor (rhPDGF-BB). AIM: To compare the potential for enhanced bone formation by a morselized amniotic membrane suspension (AmnioSpark) with a known single cytokine PDGF-BB (GEM21,Lynch) under ex-vivo calvaria culture conditions. MATERIALS AND METHODS: 45 Calvaria from 7-9 day neonatal CD-1 mice were surgically harvested under sterile conditions. The calvaria were split through the mid sagittal suture to create 90 test specimens. A 2mm diameter critical size defect was created by biopsy punch thru the center of each calvarial specimen. This defect was bridged with a non-crosslinked type I collagen membrane of the same diameter to act as a scaffold. To compare AmnioSpark (AGF) potential for tissue regeneration against a known single cytokine PDGF-BB, the calvarial specimen were divided into six experimental groups: 1) Defect only, 2) Defect + scaffold, 3) Defect + scaffold + a single dose of (rhPDGF-BB ) a known bone stimulant, 4) Defect + Scaffold + 4 doses (day 0,3,5,7) of ( rhPDGF-BB), 5) Defect + scaffold + a single dose of (AGF) and 6) Defect + scaffold + 4 doses (day 0,3,5,7) of (AGF). Each test group had (N=5). A unique static tissue culture method was used with DMEM medium supplemented with ascorbic acid (150 ug/ml) and bovine serum albumin (5 mg/ml) without fetal calf serum to enhance bone formation for up to 7 weeks. Culture medium was changed every 2 days after day 3 and the harvested media was used for the following analyses: A) Alkaline phosphatase (ALP) as an osteoblastic activity indicator, and B) Tartrate Resistant Acid phosphatase (TRAP) as an osteoclastic bone remodeling activity indicator1. Macro photography and Scanning electron microscope (SEM) image analysis at different magnifications was performed to evaluate surface conditions. Histological analysis was performed with light microscope images on standard 4 um sections using H&E, Tri chrome, Picrosirius red and a fluorescence stain for RUNX2 as an osteoblast marker. RESULTS: With a single dose of test material ALP activity in the AGF group was significantly higher at 5 and 7 days. In addition ALP activity was significantly higher compared to all groups for up to 3 weeks post-application in the multiple dose AGF group (P<0.05). In contrast there was a dramatic decline in ALP in all other groups within the first week. TRAP activity was not detectable in any group. SEM images showed that osteoblast like cells accumulated and new tissue formation occurred over the surface of the scaffold obliterating the defect/membrane interface at 21 days with the AGF stimulus while in the PDGF-BB group the scaffold was still distinguishable from surrounding bone with no new tissue formation or cell migration . Histologic images confirmed an organized distribution of cells along the surface of the scaffold and new bone formation around the periphery of the defect in the AGF group (FIG42), while no bone formation or cell migration occurred in PDGF-BB group (FIG 35-38). Further diagnostic stains confirmed the presence of active osteoblasts (RUNX2)and the production of collagens I and II ( Masson Tri Chrome and PSR). CONCLUSION: Our results indicate that growth factors from amniotic extract (AGF) have the potential to enhance calvarial bone regeneration under an ex-vivo culture condition. These findings suggest that AGF could be a candidate for use as a new type of therapeutic material for regenerative medicine.