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Mapping calcium phosphate activated gene networks as a strategy for targeted osteoinduction of human progenitors.

ABSTRACT: Although calcium phosphate-containing biomaterials are promising scaffolds for bone regenerative strategies, the osteoinductive capacity of such materials is poorly understood. In this study, we investigated whether endogenous mechanisms of in vivo calcium phosphate-driven, ectopic bone formation could be identified and used to induce enhanced differentiation in vitro of the same progenitor population. To accomplish this, human periosteum derived cells (hPDCs) were seeded on hydroxyapatite/collagen scaffolds (calcium phosphate rich matrix or CPRM), or on decalcified scaffolds (calcium phosphate depleted matrix or CPDM), followed by subcutaneous implantation in nude mice to trigger ectopic bone formation. In this system, osteoblast differentiation occurred in CPRM scaffolds, but not in CPDM scaffolds. Gene expression was assessed by human full-genome microarray at 20 h after seeding, and 2, 8 and 18 days after implantation. In both matrices, implantation of the cell constructs triggered a similar gene expression cascade, however, gene expression dynamics progressed faster in CPRM scaffolds than in CPDM scaffolds. The difference in gene expression dynamics was associated with differential activation of hub genes and molecular signaling pathways related to calcium signaling (CREB), inflammation (TNF?, NFkB, and IL6) and bone development (TGF?, ?-catenin, BMP, EGF, and ERK signaling). Starting from this set of pathways, a growth factor cocktail was developed that robustly enhanced osteogenesis in vitro and in vivo. Taken together, our data demonstrate that through the identification and subsequent stimulation of genes, proteins and signaling pathways associated with calcium phosphate mediated osteoinduction, a focused approach to develop targeted differentiation protocols in adult progenitor cells can be achieved.

SUBMITTER: Eyckmans J

PROVIDER: S-EPMC4037407 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Mapping calcium phosphate activated gene networks as a strategy for targeted osteoinduction of human progenitors.

Eyckmans Jeroen J Roberts Scott J SJ Bolander Johanna J Schrooten Jan J Chen Christopher S CS Luyten Frank P FP

Biomaterials 20130326 19

Although calcium phosphate-containing biomaterials are promising scaffolds for bone regenerative strategies, the osteoinductive capacity of such materials is poorly understood. In this study, we investigated whether endogenous mechanisms of in vivo calcium phosphate-driven, ectopic bone formation could be identified and used to induce enhanced differentiation in vitro of the same progenitor population. To accomplish this, human periosteum derived cells (hPDCs) were seeded on hydroxyapatite/colla ...[more]

PMID: 23537666

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Project description:Objective: The objective of this study is to compare the bone induction of five kinds of calcium phosphate (Ca-P) biomaterials implanted in mice and explore the vascularization and particle-size-related osteoinductive mechanism. Methods: The following five kinds of Ca-P biomaterials including hydroxyapatite (HA) and/or tricalcium phosphate (TCP) were implanted in the muscle of 30 BALB/c mice (n = 6): 20 nm HA (20HA), 60 nm HA (60HA), 12 µm HA (12HA), 100 nm TCP (100TCP) and 12 µm HA + 100 nm TCP (HATCP). Then, all animals were put on a treadmill to run 30 min at a 6 m/h speed each day. Five and ten weeks later, three mice of each group were killed, and the samples were harvested to assess the osteoinductive effects by hematoxylin eosin (HE), Masson's trichrome and safranine-fast green stainings, and the immunohistochemistry of the angiogenesis and osteogenesis markers CD31 and type I collagen (ColI). Results: The numbers of blood vessels were 139 ±&nbsp;29,&nbsp;118&nbsp;±&nbsp;25,&nbsp;78&nbsp;±&nbsp;15,&nbsp;65&nbsp;±&nbsp;14 in groups HATCP, 100TCP, 60HA and 20HA, respectively, which were significantly higher than that of group 12HA (12 ± 5) in week 5 (p < 0.05). The area percentages of new bone tissue were (7.33 ± 1.26)% and (8.49 ±&nbsp;1.38)% in groups 100TCP and HATCP, respectively, which were significantly higher than those in groups 20HA (3.27 ±&nbsp;0.38)% and 60HA (3.43 ±&nbsp;0.27)% (p < 0.05); however, no bone tissue was found in group 12HA 10 weeks after transplantation. The expression of CD31 was positive in new blood vessels, and the expression of ColI was positive in new bone tissue. Conclusions: Nanoscale Ca-P biomaterials could induce osteogenesis in mice muscle, and the osteoinductive effects of TCP were about 124% higher than those of 20HA and 114% higher than those of 60HA. The particle size of the biomaterials affected angiogenesis and osteogenesis. There was a positive correlation between the number of blood vessels and the area percentage of new bone tissue; therefore, osteoinduction is closely related to vascularization. Our results provide an experimental basis for the synthesis of calcium-phosphorus matrix composites and for further exploration of the osteoinductive mechanism.

Project description:BackgroundTreatment of hemophiliacs with inhibitors remains challenging, and new treatments are in urgent need. Coagulation factor X plays a critical role in the downstream of blood coagulation cascade, which could serve as a bypassing agent for hemophilia therapy. Base on platelet-targeted gene therapy for hemophilia by our and other groups, we hypothesized that activated factor X (FXa) targeted stored in platelets might be effective in treating hemophilia A (HA) and B (HB) with or without inhibitors.MethodsTo achieve the storage of FXa in platelets, we constructed a FXa precursor and used the integrin αIIb promoter to control the targeted expression of FXa precursor in platelets. The expression cassette (2bFXa) was carried by lentivirus and introduced into mouse hematopoietic stem and progenitor cells (HSPCs), which were then transplanted into HA and HB mice. FXa expression and storage in platelets was examined in vitro and in vivo. We evaluated the therapeutic efficacy of platelet-stored FXa by tail bleeding assays and the thrombelastography. In addition, thrombotic risk was assessed in the recipient mice and the lipopolysaccharide induced inflammation mice.ResultsBy transplanting 2bFXa lentivirus-transduced HSPCs into HA and HB mice, FXa was observed stably stored in platelet α-granules, the stored FXa is releasable and functional upon platelet activation. The platelet-stored FXa can significantly ameliorate bleeding phenotype in HA and HB mice as well as the mice with inhibitors. Meanwhile, no FXa leakage in plasma and no signs of increased risk of hypercoagulability were found in transplantation recipients and lipopolysaccharide induced septicemia recipients.ConclusionsOur proof-of-principle data indicated that target expression of the FXa precursor to platelets can generate a storage pool of FXa in platelet α-granules, the platelet-stored FXa is effective in treating HA and HB with inhibitors, suggesting that this could be a novel choice for hemophilia patients with inhibitors.

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Mapping calcium phosphate activated gene networks as a strategy for targeted osteoinduction of human progenitors.

Publications

Mapping calcium phosphate activated gene networks as a strategy for targeted osteoinduction of human progenitors.

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