Wnt protein delivery to skeletal stem cells for bone tissue regeneration

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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Identifiers

ORCID for Richard Oreffo: orcid.org/0000-0001-5995-6726

ORCID for Rahul Tare: orcid.org/0000-0001-8274-8837

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