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Dual non-invasive visualization of recombinant human bone morphogenetic protein-2 release and associated bone regeneration
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics. (Sune Larsson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
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2014 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Bone morphogenetic proteins (BMP’s) are vital for bone and cartilage formation. BMP-2 is acknowledged as a key family member in osteoblast differentiation. In this study we investigated the possibility for longitudinal and non-invasive monitoring of implanted [125I]BMP-2 release and its correlation to ossification at the site of implantation. We used a unilateral rodent model with a critically sized femoral defect on the left side that was stabilized with an external fixator while the contralateral intact right femur was used as control. Male Sprague Dawley rats were randomized into two groups. The defect was filled with a hyaluronan hydrogel with 25% hydroxyapatite (n=2) alone or with the same compound combined a mixture of 30 µg of [125I]BMP-2 at a concentration of 150 µg/mL. Bone formation was followed by microCT (µCT) post surgery and thereafter at six occasions over 12 weeks. The release of [125I]BMP-2 was assessed with single photon emission computed tomography (SPECT), and the bone healing process was followed with [18F]sodium fluoride (NaF) using positron emission tomography (PET)at day 3 and at weeks 2, 4, and 6.

In the defects filled with hydrogel andBMP-2 a rapid burst release of [125I]BMP-2 was observed between day 3 and 2 weeks. A progressive increase in uptake levels of [18F]sodium fluoride followed, which reached a maximum 4 weeks post surgery. Bone formation was seen by µCT for all 12 weeks. In the hydrogel alone group, bone regeneration was observed by PET and CT at week 2 and 3 but later on no bone formation was observed in the defect. We propose that the functional, noninvasive imaging method described allow dual visualization of the release of BMP-2 and the following in vivo response, and should hereby be considered a potential novel tool for studies on bone regeneration. 

Place, publisher, year, edition, pages
Keyword [en]
bone tissue engineering; hydrogel; computed tomography; positron emission tomography; single-photon emission computed tomography; bone morphogenetic protein 2
National Category
Biological Sciences Other Medical Sciences
Research subject
URN: urn:nbn:se:uu:diva-234698OAI: oai:DiVA.org:uu-234698DiVA: diva2:758000
EU, FP7, Seventh Framework Programme
Available from: 2014-10-24 Created: 2014-10-23 Last updated: 2015-09-24
In thesis
1. Bone Regeneration with Cell-free Injectable Scaffolds
Open this publication in new window or tab >>Bone Regeneration with Cell-free Injectable Scaffolds
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bone is a remarkable multifunctional tissue with the ability to regenerate and remodel without generating any scar tissue. However, bone loss due to injury or diseases can be a great challenge and affect the patient significantly. Transplanting bone graft from one site in the patient to the site of fracture or bone void, i.e. autologous bone grafting is commonly used throughout the world. The transplanted bone not only fills voids, but is also bone inductive, housing the particular cells that are needed for bone regeneration. Nevertheless, a regenerative complement to autograft is of great interest and importance because the benefits from an off-the-shelf product with as good of healing capacity as autograft will circumvent most of the drawbacks with autograft. With a regenerative-medicine approach, the use of biomaterials loaded with bioactive molecules can avoid donor site morbidity and the problem of limited volume of material. Two such regenerative products that utilize bone morphogenetic protein 7 and 2 have been used for more than a decade in the clinic. However, some severe side effects have been reported, such as severe swelling due to inflammation and ectopic bone formation. Additionally, the products require open surgery, use of supra physiological doses of the BMPs due to poor localization and retention of the growth factors. The purpose of this thesis was to harness the strong inductive capability of the BMP-2 by optimizing the carrier of this bioactive protein, thereby minimizing the side effects that are associated with the clinical products and facilitating safe and localized bone regeneration at the desired site. We focused on an injectable hyaluronan-based carrier. The strategy was to use the body’s own regenerative pathway to stimulate and enhance bone healing in a manner similar to the natural bone-healing process. The hyaluronan-based carrier has a similar composition to the natural extracellular matrix and is degraded by resident hyaluronidase enzymes. Earlier studies have shown a more controlled release and improved mechanical properties when adding a weight of 25 percent of hydroxyapatite, a calcium phosphate that constitutes the inorganic part of the bone matrix. In Paper I, the aim was to improve the carrier by adding other forms of calcium phosphate. The results indicated that the bone formation was enhanced when using nano-sized hydroxyapatite. We wished to further develop the carrier system but were lacking an animal model with high output and easy access. We also wanted to provide paired data and were committed to the 3 Rs of refinement, reduction and replacement. To meet these challenges, we developed and refined an animal model, and this is described in Paper II. In Paper III, we characterized and optimized the handling properties of the carrier. In Paper IV, we discovered the importance of crushing the material, thus enhancing permeability and enlarging the surface area. In Paper V, we sought to further optimize biomaterial properties of the hydrogel through covalently bonding of bisphosphonates to the hyaluronan hydrogel. The results demonstrated exceptional retention of the growth factor BMP-2. In Paper VI, the in vivo response related to the release of the growth factor was examined by combining a SPECT/PET/µCT imaging method to visualize both the retention of the drug, and the in-vivo response in terms of mineralization.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 64 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1050
bone tissue engineering, hydrogel, computed tomography, positron emission tomography, large femoral bone defect, rat model, hydrogel, in vivo, osteogenesis, bone regeneration, 3R, single-photon emission computed tomography, bone morphogenetic protein 2, calcium phosphates, injectable, bisphosphonate
National Category
Biological Sciences Polymer Chemistry Other Medical Sciences Other Medical Sciences not elsewhere specified
Research subject
Medical Science; Biology; Chemistry with specialization in Polymer Chemistry; Orthopaedics
urn:nbn:se:uu:diva-234846 (URN)978-91-554-9093-5 (ISBN)
Public defence
2014-12-12, Enghofs salen, ingång 50 Akademiska sjukhuset, Uppsala, 09:15 (Swedish)
EU, FP7, Seventh Framework Programme, EUFP7-NMP.20102.3-1; Grant 262948
Available from: 2014-11-21 Created: 2014-10-24 Last updated: 2015-02-03

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