الفهرس 
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Abstract
Aim: The aim of this study was to evaluate the bone regenerative potential of hGMSCs’ secretome
in critical-sized rabbits’ tibial bone defects by measuring mineral apposition rate, histomorphometric
analysis to the area of the newly formed bone and gene expression detection for osteocalcin using
Real-Time Quantitative Reverse Transcription PCR (qRT-PCR).
Methodology: 51 male rabbits were randomly divided; nine rabbits for the primary outcome
(mineral apposition rate), 42 rabbits for the secondary outcomes (histomorphometric analysis of
decalcified bone sections and gene expression detection for osteocalcin). Each rabbit received one
critical-sized bone defect in each tibia. Only the nine rabbits that were used for the primary outcome
were subcutaneously injected with 25 mg/kg oxytetracycline at 14 days and 20 mg/kg calcein green
at 28 days. The defects were equally randomized into three groups. group 1: the defects were left
empty untreated. group 2: the defects were filled with collagen sponge scaffold. group 3: the defects
were filled with collagen sponge scaffold loaded with hGMSCs’ secretome. At week 3, 21 rabbits
(with 42 total number of defects- 14 for each group) were euthanized for the secondary outcomes. At
week 6, the remaining rabbits for the secondary outcomes in addition to the nine rabbits of the primary.
outcome were euthanized. The bone specimens were dissected. For primary outcome undecalcified
sections were prepared and examined by fluorescence microscope. For secondary outcome A,
decalcified sections were prepared followed by histological and histomorphometrical examination.
For secondary outcome B, the tissues that were filling the defects were used for gene expression
analysis.
Results: The bone defects of Gp3 showed significant improved osseous healing as compared to the
other two groups in both 3 and 6 weeks. At 6 weeks, the histological examination of the bone defects
of Gp 1 &2 revealed open defects with less bone and more bone marrow spaces in-between, while
Gp 3 showed closed defects with bone. The mineral apposition rate was found significantly higher in
Gp 3 than the other two groups (P < 0.05). Moreover, the morphometric analysis performed between
groups revealed a significant increase in the bone area percentage in Gp 3 in comparison to the other
two groups. In addition, a statistically significant increase in the expression of osteocalcin gene was
detected in Gp 3 at the two times interval when compared to the other two groups (P < 0.05).
Conclusions: hGMSC’s secretome was effective in regeneration of critical-sized bone defects in
rabbits’ tibial model which provide a promising therapy for clinical applications.
Keywords: gingival mesenchymal stem cells, secretome, bone defects, bone healing.