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Wnt signaling and metaphyseal bone healing
Linköping University, Department of Clinical and Experimental Medicine, Orthopaedics and Sports Medicine. Linköping University, Faculty of Health Sciences.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis relates to some new aspects on the regulation of bone healing. In the last few years, Wnt-signaling has been shown to play a central role in bone biology. As well as being involved in bone maintenance and repair, Wnt-signaling has been presented as one of the key pathways through which bone responds to mechanical load. Two secreted extracellular inhibitors of Wnt-signaling, sclerostin and dickkopf-1 are potent negative regulators of bone formation.

Using a rat fracture model we investigated how metaphyseal bone healing is influenced by changes in Wnt-signaling.

Antibodies were used to suppress levels of sclerostin and dickkopf-1, and thereby increase Wnt-signaling. Primarily, we investigated if those antibody treatments lead to improved bone healing. Also, we investigated if the response was coupled to the loading conditions of the bone.

Our findings suggest that suppression of either sclerostin or dickkopf-1 leads to increased bone formation and improved bone healing. Apart from just having an effect on healing, the treatment also improved bone formation in other parts of the skeleton. Depending on the loading conditions, the effects were different. Dickkopf-1 appeared to have a stronger effect on bone volume density in unloaded bone, implying a role mainly in mechano-transduction, while sclerostin had similar effect in both loaded and unloaded bone. To confirm these findings, we studied how the expression of several Wnt-related genes changed due to trauma and unloading in metaphyseal bone. We found that trauma led to upregulation of most of the genes with the largest effect seen in the unloaded bone. In untraumatized bone, there was mainly an effect on the sclerostin gene.

In conclusion, antibodies against sclerostin and dickkopf-1 appear to be able to improve metaphyseal bone healing. There appear to be some differences in how the effect of the two antibodies manifests itself, especially if the loading conditions of the bone are altered. These findings suggest a potential for clinical use to shorten fracture healing time.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2011. , 34 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1253
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-71287ISBN: 978-91-7393-103-8 (print)OAI: oai:DiVA.org:liu-71287DiVA: diva2:447010
Public defence
2011-10-14, Nils-Holger salen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2011-10-10 Created: 2011-10-10 Last updated: 2011-10-12Bibliographically approved
List of papers
1. Sclerostin Antibody Treatment Enhances Metaphyseal Bone Healing in Rats
Open this publication in new window or tab >>Sclerostin Antibody Treatment Enhances Metaphyseal Bone Healing in Rats
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2010 (English)In: JOURNAL OF BONE AND MINERAL RESEARCH, ISSN 0884-0431, Vol. 25, no 11, 2412-2418 p.Article in journal (Refereed) Published
Abstract [en]

Sclerostin is the product of the SOST gene Loss of-function mutations in the SOST gene result in a high bone-mass phenotype demonstrating that sclerostin is a negative regulator of bone mass Primarily expressed by osteocytes in bone sclerostin is reported to bind the LRP5/6 receptor thereby antagonizing canonical Wnt signaling and negatively regulating bone formation We therefore investigated whether systemic administration of a sclerostin neutralizing antibody would increase the regeneration of traumatized metaphyseal bone in rats Young male rats had a screw inserted in the proximal tibia and were divided into six groups given 25 mg/kg of sclerostin antibody or control twice a week subcutaneously for 2 or 4 weeks In four groups, the screws were tested for pull out strength At the time of euthanasia a similar screw also was inserted in the contralateral tibia and pull-out tested immediately Sclerostin antibody significantly increased the pull out force by almost 50% compared with controls after 2 and 4 weeks Also the screws inserted at the time of euthanasia showed increased pull out force Micro-computed tomography (mu CT) of the remaining two groups showed that the antibody led to a 30% increase in bone volume fraction in a region surrounding the screw There also was a general increase in trabecular thickness in cancellous bone Thus as measured by the amount of bone and its mechanical resistance the sclerostin antibody increased bone formation during metaphyseal repair but also in untraumatized bone

Place, publisher, year, edition, pages
American Society for Bone and Mineral Research, 2010
Keyword
bone formation;implants;bone repair;sclerostin;antibody
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-62733 (URN)10.1002/jbmr.135 (DOI)000284133500016 ()20499342 (PubMedID)
Available from: 2010-12-03 Created: 2010-12-03 Last updated: 2011-10-10
2. The effects of Dickkopf-1 antibody on metaphyseal bone and implant fixation under different loading conditions
Open this publication in new window or tab >>The effects of Dickkopf-1 antibody on metaphyseal bone and implant fixation under different loading conditions
2011 (English)In: BONE, ISSN 8756-3282, Vol. 48, no 5, 988-996 p.Article in journal (Refereed) Published
Abstract [en]

The secreted protein Dickkopf-1 (Dkk1) is an antagonist of canonical Wnt signaling, expressed during fracture healing. It is unclear how it is involved in the mechanical control of bone maintenance. We investigated the response to administration of a Dkk1 neutralizing antibody (Dkk1-ab) in metaphyseal bone under different loading conditions, with or without trauma. In this three part experiment, 120 rats had a screw or bone chamber inserted either unilaterally or bilaterally in the proximal tibia. Mechanical (pull-out) testing, mu CT and histology were used for evaluation. The animals were injected with either 10 mg/kg Dkk1-ab or saline every 14 days for 14, 28, or 42 days. Antibody treatment increased bone formation around the screws and improved their fixation. After 28 days, the pull-out force was increased by over 100%. In cancellous bone, the bone volume fraction was increased by 50%. In some animals, one hind limb was paralyzed with Botulinum toxin A (Botox) to create a mechanically unloaded environment. This did not increase the response to antibody treatment with regard to screw fixation, but in cancellous bone, the bone volume fraction increased by 233%. Thus, the response in unloaded, untraumatized bone was proportionally larger, suggesting that Dkk1 may be up-regulated in unloaded bone. There was also an increase in thickness of the metaphyseal cortex. In bone chambers, the antibody treatment increased the bone volume fraction. The results suggest that antibodies blocking Dkk1 might be used to stimulate bone formation especially during implant fixation, fracture repair, or bone disuse. It also seems that Dkk1 is up-regulated both after metaphyseal trauma and after unloading, and that Dkk1 is involved in mechano-transduction.

Place, publisher, year, edition, pages
Elsevier Science B.V., Amsterdam., 2011
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-68352 (URN)10.1016/j.bone.2011.02.008 (DOI)000289879900005 ()
Note
Original Publication: Fredrik Agholme, Hanna Isaksson, Stuart Kuhstoss and Per Aspenberg, The effects of Dickkopf-1 antibody on metaphyseal bone and implant fixation under different loading conditions, 2011, BONE, (48), 5, 988-996. http://dx.doi.org/10.1016/j.bone.2011.02.008 Copyright: Elsevier Science B.V., Amsterdam. http://www.elsevier.com/ Available from: 2011-05-20 Created: 2011-05-20 Last updated: 2011-10-10
3. Anti-sclerostin antibody and mechanical loading appear to influence metaphyseal bone independently in rats
Open this publication in new window or tab >>Anti-sclerostin antibody and mechanical loading appear to influence metaphyseal bone independently in rats
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2011 (English)In: Acta Orthopaedica, ISSN 1745-3674, Vol. 82, no 5, 628-632 p.Article in journal (Refereed) Published
Abstract [en]

Background and purpose: Sclerostin is produced by osteocytes and is an inhibitor of bone formation. Thus, inhibition of sclerostin by a monoclonal antibody increases bone formation and improves fracture repair. Sclerostin expression is upregulated in unloaded bone and is downregulated by loading. We wanted to determine whether an anti-sclerostin antibody would stimulate metaphyseal healing in unloaded bone in a rat model.

Methods: 10-week-old male rats (n = 48) were divided into 4 groups, with 12 in each. In 24 rats, the right hind limb was unloaded by paralyzing the calf and thigh muscles with an injection of botulinum toxin A (Botox). 3 days later, all the animals had a steel screw inserted into the right proximal tibia. Starting 3 days after screw insertion, either anti-sclerostin antibody (Scl-Ab) or saline was given twice weekly. The other 24 rats did not receive Botox injections and they were treated with Scl-Ab or saline to serve as normal-loaded controls. Screw pull-out force was measured 4 weeks after insertion, as an indicator of the regenerative response of bone to trauma.

Results: Unloading reduced the pull-out force. Scl-Ab treatment increased the pull-out force, with or without unloading. The response to the antibody was similar in both groups, and no statistically significant relationship was found between unloading and antibody treatment. The cancellous bone at a distance from the screw showed changes in bone volume fraction that followed the same pattern as the pull-out force.

Interpretation: Scl-Ab increases bone formation and screwfixation to a similar degree in loaded and unloaded bone.

Place, publisher, year, edition, pages
Taylor and Francis, 2011
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-71284 (URN)10.3109/17453674.2011.625539 (DOI)
Available from: 2011-10-10 Created: 2011-10-10 Last updated: 2014-10-17Bibliographically approved
4. Wnt gene expression during metaphyseal bone healing under different load conditions
Open this publication in new window or tab >>Wnt gene expression during metaphyseal bone healing under different load conditions
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Bone Wnt signalling has been presented as one of the key pathways through which bone responds to mechanical load. This pathway is also active during the healing process after bone trauma. Bone healing can be improved by pharmacological modulation of Wnt signalling. We investigated how the expression of several Wntrelated genes changed due to trauma and unloading in metaphyseal bone.

20 male rats had one hind limb unloaded by intramuscular Botox injections. In half of the animals a hole was drilled bilaterally in the proximal tibia. After 7 days, a cylindrical biopsy was taken from the bone surrounding the hole and at a corresponding site in animals without trauma. The biopsies were analyzed for the mRNA expression of Wnt1, Wnt3a, Wnt4, Wnt5a, Wnt5b, Sost, Dkk1, Dkk2, Sfrp1, Sfrp4, Lrp5, Lrp6, Wisp1, Wif1 and Wnt10b.

Trauma led to upregulation of most of the studied genes. This effect was most evident in unloaded bone, where 8 genes were upregulated, among them Wnt receptors, ligands and inhibitors. Unloading increased the expression of Sost in untraumatized bone, but did not significantly influence the other genes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-71285 (URN)
Available from: 2011-10-10 Created: 2011-10-10 Last updated: 2011-10-10Bibliographically approved

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