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Wnt protein delivery to skeletal stem cells for bone tissue regeneration

Wnt protein delivery to skeletal stem cells for bone tissue regeneration
Wnt protein delivery to skeletal stem cells for bone tissue regeneration
There is a pressing need to develop anabolic treatments that enhance bone regeneration. Bone fractures are a major socioeconomic problem, which is likely to increase as our population ages. A promising approach to address this problem may be the delivery of molecules targeted to stem cells responsible for the regeneration of bone. Wnt signalling is involved in regulating skeletal stem cells (SSCs), and is known to be an important regulator of fracture healing. The aim of this study was to test the hypothesis that Wnt protein can augment the osteogenic response of SSCs, and may be delivered in an active form at the fracture site using liposome nanoparticles.

Wnt signalling levels in bone marrow populations rich in SSCs were determined by qPCR. These populations were also transiently exposed to 100 ng/ml of Wnt3A, and their frequency, viability and proliferation were studied by flow cytometry. After 14 days of adherent culture, their colony forming unit fibroblastic and osteoblastic (CFUF/O) potentials were tested. Osteogenic differentiation was tested in cells exposed to transient and also to sustained Wnt stimulation. Liposomes were then investigated as a means of delivering active Wnt proteins. After optimising their lipid formulation and determining the association of Wnt with the nanoparticles, protein activity, nanoparticle uptake in vitro and biodistribution in vivo were characterised.

SSC-rich populations had elevated levels of Wnt signalling and were responsive to Wnt stimulation, which, when applied transiently, expanded the subset of osteoprogenitors and increased their osteodifferentiation. However, sustained stimulation inhibited this process. 100 nm liposomes enhanced Wnt activity and were readily taken up by cultured stromal cell populations, as well as by SSC-rich populations within fresh bone marrow isolates. At 24 and 48 hours following systemic injection, liposomes localised at the bone fracture site.

Wnt3A exposure primed SSCs and progenitors within fresh bone marrow isolates to an osteogenic fate, but sustained stimulation dramatically inhibited the osteogenic differentiation. This indicated that the timing of Wnt exposure is crucial, underlining the need for spatiotemporal delivery of this protein. The potential of liposomes to localise at the bone fracture site merits further study into the nanoparticle delivery of Wnt and its effects on bone regeneration.
University of Southampton
Janeczek, Agnieszka Aleksandra
56258fdc-f798-44ac-8bb4-886935283ffc
Janeczek, Agnieszka Aleksandra
56258fdc-f798-44ac-8bb4-886935283ffc
Evans, Nicholas
0982bf3e-b3f5-4682-9238-f362d6471992
Oreffo, Richard
ff9fff72-6855-4d0f-bfb2-311d0e8f3778
Tare, Rahul
587c9db4-e409-4e7c-a02a-677547ab724a

Janeczek, Agnieszka Aleksandra (2015) Wnt protein delivery to skeletal stem cells for bone tissue regeneration. University of Southampton, Faculty of Medicine, Doctoral Thesis, 301pp.

Record type: Thesis (Doctoral)

Abstract

There is a pressing need to develop anabolic treatments that enhance bone regeneration. Bone fractures are a major socioeconomic problem, which is likely to increase as our population ages. A promising approach to address this problem may be the delivery of molecules targeted to stem cells responsible for the regeneration of bone. Wnt signalling is involved in regulating skeletal stem cells (SSCs), and is known to be an important regulator of fracture healing. The aim of this study was to test the hypothesis that Wnt protein can augment the osteogenic response of SSCs, and may be delivered in an active form at the fracture site using liposome nanoparticles.

Wnt signalling levels in bone marrow populations rich in SSCs were determined by qPCR. These populations were also transiently exposed to 100 ng/ml of Wnt3A, and their frequency, viability and proliferation were studied by flow cytometry. After 14 days of adherent culture, their colony forming unit fibroblastic and osteoblastic (CFUF/O) potentials were tested. Osteogenic differentiation was tested in cells exposed to transient and also to sustained Wnt stimulation. Liposomes were then investigated as a means of delivering active Wnt proteins. After optimising their lipid formulation and determining the association of Wnt with the nanoparticles, protein activity, nanoparticle uptake in vitro and biodistribution in vivo were characterised.

SSC-rich populations had elevated levels of Wnt signalling and were responsive to Wnt stimulation, which, when applied transiently, expanded the subset of osteoprogenitors and increased their osteodifferentiation. However, sustained stimulation inhibited this process. 100 nm liposomes enhanced Wnt activity and were readily taken up by cultured stromal cell populations, as well as by SSC-rich populations within fresh bone marrow isolates. At 24 and 48 hours following systemic injection, liposomes localised at the bone fracture site.

Wnt3A exposure primed SSCs and progenitors within fresh bone marrow isolates to an osteogenic fate, but sustained stimulation dramatically inhibited the osteogenic differentiation. This indicated that the timing of Wnt exposure is crucial, underlining the need for spatiotemporal delivery of this protein. The potential of liposomes to localise at the bone fracture site merits further study into the nanoparticle delivery of Wnt and its effects on bone regeneration.

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Published date: September 2015
Organisations: University of Southampton, Human Development & Health

Identifiers

Local EPrints ID: 405272
URI: http://eprints.soton.ac.uk/id/eprint/405272
PURE UUID: a07152e6-b4cf-4046-9a47-218ea080772d
ORCID for Richard Oreffo: ORCID iD orcid.org/0000-0001-5995-6726
ORCID for Rahul Tare: ORCID iD orcid.org/0000-0001-8274-8837

Catalogue record

Date deposited: 18 Feb 2017 00:24
Last modified: 14 Mar 2019 01:48

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Contributors

Author: Agnieszka Aleksandra Janeczek
Thesis advisor: Nicholas Evans
Thesis advisor: Richard Oreffo ORCID iD
Thesis advisor: Rahul Tare ORCID iD

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