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Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVIII | Pages 4 - 4
1 Sep 2012
Chen C Uludag H Wang Z Jiang H
Full Access

Purpose

The data regarding the effects of noggin on bone morphogenetic protein (BMP)-induced osteogenesis of mesenchymal stem cells (MSCs) are controversial. Most studies performed in rodent cells/models indicated that noggin was a negative regulator of BMP-2-induced osteogenesis; however, one study conducted with human MSCs in culture showed that the addition of noggin induced osteogenesis in vitro. To clear the controversy, we designed this study to evaluate the effects of knocking down noggin gene expression on BMP-2-induced osteogenesis of human bone marrow-derived primary MSCs in vitro.

Method

MSCs were isolated from human tibial bone marrow by density gradient centrifugation. Two noggin small interfering RNAs (siRNAs) were used in this study to knockdown noggin gene expression. There were four study groups: MSCs with no transfection of siRNA (named as NT group), MSCs transfected with non-targeting negative control siRNA (named as control group), MSCs transfected with noggin siRNA1 (named as NOGsi1 group), and MSCs transfected with noggin siRNA2 (named as NOGsi2 group). After transfection, MSCs were induced to undergo osteogenic differentiation by incubating in basal medium containing 0.1 μg/ml BMP-2 for 35 days. The expression levels of osteoblastic marker genes were measured by real-time quantitative PCR on day 14. Also assessed was alkaline phosphatase (ALP) activity by a colorimetric kinetic assay and Fast Blue B staining on day 14. Calcium deposition was determined by the calcium assay on day 35.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 552 - 552
1 Nov 2011
Chen C Zhi L Pang X Uludag H Jiang H
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Purpose: The current clinical treatment protocol for bone healing applies super-physiological dose of rhBMP7. Unfortunately, it may result in adverse side effects. Some studies have demonstrated a dose-dependent osteogenic differentiation using rodent bone marrow derived stem cells (BMSCs). However, the dose effect of BMP7 on osteogenic differentiation of normal human BMSCs is largely unknown. In the present study, we investigated in vitro osteogenic differentiation of hBMSCs with a gradient concentration of rhBMP7. The interaction between rhBMP7 and osteogenic differentiation medium (ODM) was also examined.

Method: The primary BMSCs from human bone marrow were cultured and maintained in MSC growth medium (MGM). Six study groups were designed: MGM only, MGM with rhBMP7 of 0.1ug/ml, ODM only, and ODM with 3 concentration of rhBMP7 of 0.01μg/ml, 0.1μg/ml, and 1.0μg/ml, respectively. Alkaline phosphatase level (ALP) at day 17 and cumulative calcium deposit at both day 17 and day 35 were examined. mRNA expression level of osteogenic markers including osteocalcin (OC), osteopontin (OPN) and ALP were quantified using real-time RT-PCR at day 17.

Results: ALP activity at day17 did not increase in MGM with or without 0.1μg/ml of rhBMP7, ODM alone and ODM with 0.01μg/ml of rhBMP7. ALP activity was much higher with 0.1μg/ml of rhBMP7 plus ODM (0.22±0.02IU) than that in MGM with 0.1μg/ml of rhBMP7 (0.01±0.01IU, P< 0.05).

Conclusion: Our study demonstrated that rhBMP7 induced osteogenic differentiation of hBMSCs in a dose-dependent manner in the presence of ODM and the minimal dose for inducing in vitro osteoblastic differentiation was 0.1ug/ml of rhBMP7 under synergistic effect of ODM. The results of this study provide some insights into further investigation of synergy of rhBMP7 with other molecules. The types and amounts of simple molecules could significantly reduce therapeutic dose of rhBMP7 and achieve equivalent or better outcomes in clinical application warrant further investigation.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 552 - 552
1 Nov 2011
Chen C Uludag H Rezansoff A Jiang H
Full Access

Purpose: The osteogenic effects of BMPs on mesenchymal stem cells (MSCs) are less profound in human as compared to rodent. The mechanism for this phenomenon is unclear. This study evaluated the effects of macrophages on proliferation and BMP-2 induced osteogenic differentiation of human MSCs.

Method: MSCs were isolated from human bone marrow. Human monocytes THP-1 (human acute monocytic leukemia cell line) were induced into macrophages by phorbol myristate acetate. The conditioned media (CM) from monocytes and macrophages were collected separately. After treated with CM from monocytes or macrophages for 5 and 7 days, the proliferation rate of human MSCs was determined by WST-8 assay. A group without CM served as control. Pretreated human MSCs were then induced towards osteogenic differentiation by osteoinductive medium supplemented with 0.1ug/ml BMP-2. Expression levels of osteogenic markers were determined by real-time quantitative PCR. Alkaline phosphatase (ALP) activity and mineral deposition were assessed by p-NPP colorimetric kinetic assay and calcium assay, respectively.

Results: The number of MSCs was significantly decreased in the group with macrophage CM at both 5 and 7 days (both p< 0.001) as compared with control group, but not in the group with monocytes CM. Expression levels of ALP and bone sialoprotein 2 in the macrophage CM group were significantly lower than those in the control group (p=0.003 and p< 0.001, respectively). ALP activity was also significantly lower in the group with macrophage CM than control group (p< 0.001). Although the expression levels of osteocalcin and RUNX2 as well as calcium deposition in the macrophage CM group were reduced, they did not reach statistical significance.

Conclusion: Macrophages suppressed the proliferation of MSCs and inhibited BMP-2 induced osteogenic differentiation of human MSCs. In addition to known BMP antagonists, macrophages might be another important factor in suppressing the osteogenic effect of BMP-2 on human MSCs.