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Engineering functional BMP-2 expressing teratoma-derived fibroblasts for enhancing osteogenesis.

ABSTRACT: Bone morphogenetic protein 2 (BMP-2) is considered an effective growth factor for bone formation, and is used for making osteo-inductive scaffolds, but the related clinical investigations have shown low success rates. In this study, we genetically manipulated teratoma-derived fibroblast (TDF) cells by simultaneous introduction of BMP-2 and herpes simplex virus-thymidine kinase (HSV-tk) encoding genes. Self-production of BMP-2 in TDF cells strongly enhanced the alkaline phosphatase (ALP) activity, calcium content, and elevated the mRNA expression of osteogenic marker genes during in vitro osteogenesis. The bone formation volume was also remarkably enhanced in calvarial and femoral critical-size defect models. Ganciclovir (GCV) treatment induced apoptosis in TDF cells co-expressing HSV-tk and BMP-2, implying that HSV-tk suicide gene can modulate the side-effects of stem cell therapy, e.g., development of uncontrollable teratoma and tumor formation. Altogether, our findings revealed a safe and highly efficient technique with potential therapeutic applications for bone regeneration.

SUBMITTER: Go YY

PROVIDER: S-EPMC6167319 | biostudies-other | 2018 Oct

REPOSITORIES: biostudies-other

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Engineering functional BMP-2 expressing teratoma-derived fibroblasts for enhancing osteogenesis.

Go Yoon Young YY Mun Ji Yeon JY Chae Sung-Won SW Kim Shin Hye SH Song Hoseok H Song Jae-Jun JJ

Scientific reports 20181001 1

Bone morphogenetic protein 2 (BMP-2) is considered an effective growth factor for bone formation, and is used for making osteo-inductive scaffolds, but the related clinical investigations have shown low success rates. In this study, we genetically manipulated teratoma-derived fibroblast (TDF) cells by simultaneous introduction of BMP-2 and herpes simplex virus-thymidine kinase (HSV-tk) encoding genes. Self-production of BMP-2 in TDF cells strongly enhanced the alkaline phosphatase (ALP) activity ...[more]

PMID: 30275449

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Project description:Wnt-induced secreted protein 1 (WISP-1/CCN4) is a member of the CCN family that is highly expressed in skeletal tissue and in osteoprogenitor cells induced to differentiate in vitro. To determine the function of WISP-1 during osteogeneis, osteogenic bone marrow stromal cells (BMSCs) were transduced with WISP-1 adenovirus (adWISP-1) in the presence or absence of bone morphogenetic protein 2 (BMP-2) adenovirus (adBMP-2). WISP-1 overexpression enhanced the ability of BMP-2 to direct BMSCs toward osteogenic differentiation and appeared to work by stimulating Smad-1/5/8 phosphorylation and activation. The ability of WISP-1 to enhance BMP-2 activity also was shown in vivo using an ectopic osteogenesis assay with BMSCs transduced with WISP-1, BMP-2, or both. When BMSCs were infected with lentivirus containing human WISP1 shRNA, they formed less bone in vivo and were less responsive to BMP-2, confirming that WISP-1 and BMP-2 have a functional interaction. Immunoprecipitation (IP) and Western blot analysis showed that WISP-1 bound directly to BMP-2 and showed that WISP-1 increased BMP-2 binding to hBMSCs in a dose-dependent fashion. To understand how WISP-1 enhanced BMP-2 signaling, the influence of WISP-1 on integrin expression was analyzed. WISP-1 induced the mRNA and protein levels of α(5)-integrin and, further, was found to bind to it. Antibody-blocking experiments showed that the BMP-2 binding to BMSCs that was enhanced by WISP-1 was completely neutralized by treatment with anti-integrin α(5)β(1) antibody. Pilot studies and the use of transgenic mice that overexpressed human WISP-1 in preosteoblasts had increased bone mineral density (BMD), trabecular thickness, and bone volume (BV/TV) over wild-type controls, supporting observations using human osteoprogenitors that WISP-1 has a positive influence on osteogenesis in vivo. In conclusion, these studies show, for the first time, that WISP-1 has a positive influence on bone cell differentiation and function and may work by enhancing the effects of BMP-2 to increase osteogenesis through a mechanism potentially involving binding to integrin α(5)β(1).

Project description:ObjectivesThe inhibitory action of the superficial gingival connective tissues may limit the regenerative potential of alveolar bone in periodontal therapy or dental implant applications. The aims of this study were to investigate the hypothesis that gingival fibroblasts (GF) can inhibit bone morphogenetic protein (BMP)-induced osteoblastic differentiation, to determine their expression of BMP inhibitors, and finally to determine whether reduction of these inhibitors can relieve suppression of osteoblastic differentiation.MethodsGingival fibroblasts were co-cultured either directly or indirectly with calvarial osteoblasts to assess alkaline phosphatase inhibitory activity, a marker of osteoblastic differentiation. To test total BMP-inhibitory activity of rat GF, conditioned media (GFCM) were collected from cultures. ROS 17/2.8 osteoblastic cells were stimulated with BMP2, together with GFCM. Inhibitor expression was tested using RT-qPCR, Western blotting and in situ hybridization. Removal of inhibitors was carried out using immunoprecipitation beads.ResultsCo-culture experiments showed GF-secreted factors that inhibit BMP-stimulated ALP activity. 10 ng/ml BMP2 increased alkaline phosphatase expression in ROS cells by 41%. GFCM blocked BMP activity which was equivalent to the activity of 100 ng/ml Noggin, a well-described BMP inhibitor. Cultured gingival fibroblasts constitutively expressed BMP antagonist genes from the same subfamily, Grem1, Grem2 and Nbl1 and the Wnt inhibitor Sfrp1. Gremlin1 (6.7 × reference gene expression) had highest levels of basal expression. ISH analysis showed Gremlin1 expression was restricted to the inner half of the gingival lamina propria and the PDL. Removal of Gremlin1 protein from GFCM eliminated the inhibitory effect of GFCM on ALP activity in ROS cells. Subsequent addition of recombinant Gremlin1 restored the inhibitory activity.ConclusionsFactors secreted by gingival fibroblasts inhibit BMP-induced bone formation and a range of BMP inhibitors are constitutively expressed in gingival connective tissues. These inhibitors, particularly Gremlin1, may limit coronal alveolar bone regenerative potential during oral and periodontal surgery.

Project description:BackgroundMany growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo.ResultsUtilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™).ConclusionsA recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.

Project description:A growing body of work suggests that canine mesenchymal stromal cells (cMSCs) require additional agonists such as bone morphogenic protein-2 (BMP-2) for consistent in vitro osteogenic differentiation. BMP-2 is costly and may challenge the translational relevance of the canine model. Dexamethasone enhances osteogenic differentiation of human MSCs (hMSCs) and is widely utilized in osteogenic protocols. The aim of this study was to determine the effect of BMP-2 and dexamethasone on early- and late-stage osteogenesis of autologous and induced pluripotent stem cell (iPS)-derived cMSCs. Two preparations of marrow-derived cMSCs were selected to represent exceptionally or marginally osteogenic autologous cMSCs. iPS-derived cMSCs were generated from canine fibroblasts. All preparations were evaluated using alkaline phosphatase (ALP) activity, Alizarin Red staining of osteogenic monolayers, and quantitative polymerase chain reaction. Data were reported as mean ± standard deviation and compared using one- or two-way analysis of variance and Tukey or Sidak post hoc tests. Significance was established at P < 0.05. In early-stage assays, dexamethasone decreased ALP activity for all cMSCs in the presence of BMP-2. In late-stage assays, inclusion of dexamethasone and BMP-2 at Day 1 of culture produced robust monolayer mineralization for autologous cMSCs. Delivering 100 nM dexamethasone at Day 1 improved mineralization and reduced the BMP-2 concentrations required to achieve mineralization of the marginal cMSCs. For iPS-cMSCs, dexamethasone was inhibitory to both ALP activity and monolayer mineralization. There was increased expression of osteocalcin and osterix with BMP-2 in autologous cMSCs but a more modest expression occurred in iPS cMSCs. While autologous and iPS-derived cMSCs respond similarly in early-stage osteogenic assays, they exhibit unique responses to dexamethasone and BMP-2 in late-stage mineralization assays. This study demonstrates that dexamethasone and BMP-2 can be titrated in a time- and concentration-dependent manner to enhance osteogenesis of autologous cMSC preparations. These results will prove useful for investigators performing translational studies with cMSCs while providing insight into iPS-derived cMSC osteogenesis.

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Engineering functional BMP-2 expressing teratoma-derived fibroblasts for enhancing osteogenesis.

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Engineering functional BMP-2 expressing teratoma-derived fibroblasts for enhancing osteogenesis.

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