Colloss and Colloss-E are sterile acellular lyophilizates extracted from bovine and equine bone matrix, respectively. Animal and clinical studies have shown that these xenogenic bone matrix extracts (BMEs) are effective as bone graft substitutes. In this report, we investigated the effect of Colloss and Colloss-E on human adult in vitro-expanded bone marrow-derived mesenchymal stem cells (BMMSCs). Specifically, we assessed whether these xenogenic BMEs induced osteoblastic differentiation of cultured BMMSC. We show that BMMSCs treated with either Colloss or Colloss-E exhibited characteristic osteoblastic morphological changes accompanied by the expression of osteoblast-specific markers, such as alkaline phosphatase activity, osteopontin secretion and calcium deposits, explicitly demonstrating that these bone matrix extracts induce osteoblastic differentiation of BMMSCs in vitro. Hence, xenogenic BMEs induce bone-specific differentiation of BMMSCs, presumably through providing stem cells with structural and soluble mediators that mimic the in vivo microenvironment. These results may explain the in vivo mode of action of these medical devices, and potentially provide a novel tissue engineering-based treatment of bone defect, using autologous BMMSCs pretreated with BMEs.
Inosine 5'-monophosphate dehydrogenase (IMPDH), which catalyzes a key step in the de novo biosynthesis of guanine nucleotide, is mediated by two highly conserved isoforms, IMPDH1 and IMPDH2. In this study, IMPDH2 genetic polymorphism was investigated in 96 individuals of Caucasian origin. Four single-nucleotide polymorphisms were identified, comprising one previously described single base-pair substitution in the close vicinity of the consensus donor splice site of intron 7 (IVS7+10T>C), and three novel polymorphisms, one silent substitution in exon 9 (c.915C>G), one single base-pair insertion (g.6971_6972insT) within the 3'-untranslated region of the gene, and one substitution located in the promoter region (c.-95T>G) in a transcription factor binding site CRE(A) (cyclic adenosine monophosphate [cAMP] response element). Considering the nature and location of this latter polymorphism, its functional relevance was examined by transfecting HEK293 and Jurkat cell lines with constructs of the related region of IMPDH2/luciferase reporter gene. The c.-95T>G mutation leads to a significant decrease of luciferase activity (HEK293: 55% decrease, p < 0.05; Jurkat: 65% decrease, p < 0.05) compared with the wild-type promoter sequence and, therefore, is likely to determine interindividual differences in IMPDH2 transcriptional regulation. These results might contribute to a better understanding of the variability in clinical outcome and dose adjustments of certain immunosuppressors that are metabolized through the IMPDH pathway or that are IMPDH inhibitors.
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