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Bone Regeneration with Cell-free Injectable Scaffolds
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics. (Sune Larsson)
2017 (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. Autologous bone grafting is commonly used throughout the world. Autograft both fills the void and is bone inductive, housing the particular cells that are needed for bone regeneration. However, a regenerative complement to autograft is of great interest as the use of biomaterials loaded with bioactive molecules can avoid donor site morbidity and the problem of a limited volume of material. Two such regenerative products that utilise bone morphogenetic protein (BMP)-7 and -2 have been used for more than a decade clinically. Unfortunately, several side effects have been reported, such as severe swelling due to inflammation and ectopic bone formation. Additionally, the products require open surgery and use of supra physiological doses of the BMPs due to poor localisation and retention of the growth factor. The purpose of this thesis was to harness the strong inductive capacity of the BMP-2 by optimising the carrier of this bioactive protein, thereby minimising the side effects that are associated with the clinical products and facilitating safe and localised bone regeneration. We focused on an injectable hyaluronan-based carrier developed through polymer chemistry at the University of Uppsala. 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 enzymes. Earlier studies have shown improved properties when adding 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 bone formation was enhanced when using nano-sized hydroxyapatite. In Paper II, we discovered the importance of crushing the material, thus enhancing permeability and enlarging the surface area. We wished to further develop the carrier system, but were lacking an animal model with relatively high throughput, facilitated access, paired data, and we were also committed to the 3Rs of refinement, reduction, and replacement. To meet these challenges, we developed and refined an animal model, and this is described in Paper III. In Paper IV, we sought to further optimise the biomaterial properties of the hydrogel through covalent bonding of bisphosphonates to the hyaluronan hydrogel. This resulted in exceptional retention of the growth factor BMP-2. In Paper V, SPECT/PET/µCT was combined as a tri-modal imaging method to allow visualisation of the biomaterial’s in situ action, in terms of drug retention, osteoblast activity and mineralisation. Finally, in Paper VI the correlation between existing in vitro results with in vivo outcomes was observed for an array of biomaterials. The study identified a surprisingly poor correlation between in vitro and in vivo assessment of biomaterials for osteogenesis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 67
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1287
Keywords [en]
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
Biomaterials Science Orthopaedics
Identifiers
URN: urn:nbn:se:uu:diva-310312ISBN: 978-91-554-9786-6 (print)OAI: oai:DiVA.org:uu-310312DiVA, id: diva2:1062801
Public defence
2017-02-24, Enghoffsalen, Akademiska sjukhuset, ingång 50, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework Programme, EUFP7-NMP.20102.3-1; Grant 262948Available from: 2017-02-02 Created: 2016-12-13 Last updated: 2018-01-13
List of papers
1. Calcium phosphates compounds in conjunction with hydrogel as carrier for BMP-2: A study on ectopic bone formation in rats
Open this publication in new window or tab >>Calcium phosphates compounds in conjunction with hydrogel as carrier for BMP-2: A study on ectopic bone formation in rats
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2011 (English)In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 7, no 8, p. 3042-3049Article in journal (Refereed) Published
Abstract [en]

Current treatment of fractures often involves the use of bone graft or bone morphogenetic proteins (BMP) to induce fracture healing, especially in patients with a compromised healing capacity. BMP has to be delivered in conjunction with a carrier. Unfortunately, there are drawbacks and limitations with current carriers, including their bovine origin which carries the risk of an immunological response. The physical properties also limit the use to open surgical procedures, as it cannot be injected. New carriers with improved properties are therefore needed. The aim of this study was to assess the ectopic bone forming capability of various calcium phosphate compounds when used in conjunction with a hydrogel as the carrier for BMP-2. Five different ceramic additives were tested, including beta-tricalcium phosphate and four types of hydroxyapatite (HAP) (nanoHAP, HAP, clods of HAP >100 mu m, and the biomimetic HAP Ostim35 (R)). The compounds were injected into the thigh muscle of rats, where it formed a gel in situ. After 4 weeks bone formation was evaluated by peripheral quantitative computed tomography and histology. The major finding was that the 20 nm nanoHAP yielded a higher bone density than the other additives (P = 0.0008, ANOVA with Tukey's multiple comparison test). We hypothesize that the higher bone density induced by nanoHAP might be due to nanocrystals of calcium phosphate acting as direct building blocks for biomineralization.

Keywords
Calcium phosphates, Hydrogel, Injectable, Nanosized, In vivo
National Category
Medical and Health Sciences Polymer Chemistry Engineering and Technology
Research subject
Chemistry with specialization in Polymer Chemistry; Engineering Science with specialization in Materials Science
Identifiers
urn:nbn:se:uu:diva-157018 (URN)10.1016/j.actbio.2011.04.021 (DOI)000293259500004 ()
Available from: 2011-08-16 Created: 2011-08-15 Last updated: 2018-06-26Bibliographically approved
2. Morphological differences in BMP-2-induced ectopic bone between solid and crushed hyaluronan hydrogel templates
Open this publication in new window or tab >>Morphological differences in BMP-2-induced ectopic bone between solid and crushed hyaluronan hydrogel templates
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2013 (English)In: Journal of materials science. Materials in medicine, ISSN 0957-4530, E-ISSN 1573-4838, Vol. 24, no 5, p. 1201-1209Article in journal (Refereed) Published
Abstract [en]

The possibility to affect bone formation by using crushed versus solid hydrogels as carriers for bone morphogenetic protein 2 (BMP-2) was studied. Hydrogels, based on chemical crosslinking between hyaluronic acid and poly(vinyl alcohol) derivatives, were loaded with BMP-2 and hydroxyapatite. Crushed and solid forms of the gels were analyzed both in vitro via a release study using I-125 radioactive labeling of BMP-2, and in vivo in a subcutaneous ectopic bone model in rats. Dramatically different morphologies were observed for the ectopic bone formed in vivo in the two types of gels, even though virtually identical release profiles were observed in vitro. Solid hydrogels induced formation of a dense bone shell around non-degraded hydrogel, while crushed hydrogels demonstrated a uniform bone formation throughout the entire sample. These results suggest that by crushing the hydrogel, the construct's three-dimensional network becomes disrupted. This could expose unreacted functional groups, making the fragment's surfaces reactive and enable limited chemical fusion between the crushed hydrogel fragments, leading to similar in vitro release profiles. However, in vivo these interactions could be broken by enzymatic activity, creating a macroporous structure that allows easier cell infiltration, thus, facilitating bone formation.

National Category
Engineering and Technology Natural Sciences Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-201244 (URN)10.1007/s10856-013-4877-6 (DOI)000318510200008 ()
Available from: 2013-06-10 Created: 2013-06-10 Last updated: 2017-12-06Bibliographically approved
3. A uni-cortical femoral defect model in the rat: evaluation using injectable hyaluronan hydrogel as a carrier for bone morphogenetic protein-2
Open this publication in new window or tab >>A uni-cortical femoral defect model in the rat: evaluation using injectable hyaluronan hydrogel as a carrier for bone morphogenetic protein-2
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2015 (English)In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, E-ISSN 1932-7005, Vol. 9, no 7, p. 799-807Article in journal (Refereed) Published
Abstract [en]

The development of biomaterial for bone regeneration requires animal models that are reliable and designed to mimic clinically relevant situations. We have previously investigated hydrogels comprised of modified hyaluronic acid and polyvinyl alcohol in models of ectopic bone formation. This hydrogel induces bone regeneration when loaded with bone morphogenetic proteins (BMPs). To allow further optimization of hydrogels, we developed a new, femoral, non-critical-sized cortical defect model. In the rat femur, we drilled standardized, elongated unilateral cortical defects that did not require stabilization and that could be created bilaterally to allow paired comparisons of biomaterials. After optimizing the defect size, subsequent stress fractures occurred in only 8% and the defect healed partially over the 40 day study period. In a time-course experiment, we treated bone defects with the previously studied hyaluronan hydrogel loaded with 10 µg hydroxyapatite and 6 µg BMP-2. The shape of the defect allowed controlled containment of the material within the defect. The defect in the right leg was left untreated, while the left defect was filled with 40 µl of the BMP hydrogel. As determined by pQCT analysis, the treated defects had a higher bone mineral content, bone area and bone density than control defects. The relative difference was greatest between the groups at 10 and 20 days and diminished as the defect healed in the untreated legs. We conclude that this animal model allows facile and rapid screening of biomaterials for bone regeneration in cortical femoral defects without requiring external fixation.

National Category
Other Natural Sciences Other Medical Sciences not elsewhere specified
Identifiers
urn:nbn:se:uu:diva-189868 (URN)10.1002/term.1655 (DOI)000357881900006 ()23225778 (PubMedID)
Available from: 2013-01-04 Created: 2013-01-04 Last updated: 2017-12-06Bibliographically approved
4. Bisphosphonate-Linked Hyaluronic Acid Hydrogel Sequesters and Enzymatically Releases Active Bone Morphogenetic Protein-2 for Induction of Osteogenic Differentiation
Open this publication in new window or tab >>Bisphosphonate-Linked Hyaluronic Acid Hydrogel Sequesters and Enzymatically Releases Active Bone Morphogenetic Protein-2 for Induction of Osteogenic Differentiation
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2013 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 14, no 9, p. 3055-3063Article in journal (Refereed) Published
Abstract [en]

Regeneration of bone by delivery of bone morphogenetic proteins (BMPs) from implantable scaffolds is a promising alternative to the existing autologous bone grafting procedures. Hydrogels are used extensively in biomaterials as delivery systems for different growth factors. However, a controlled release of the growth factors is necessary to induce bone formation, which can be accomplished by various chemical functionalities. Herein we demonstrate that functionalization of a hyaluronan (HA) hydrogel with covalently linked bisphosphonate (BP) ligands provides efficient sequestering of BMP-2 in the resulting HA-BP hydrogel. The HA-BP hydrogel was investigated in comparison with its analogue lacking BP groups (HA hydrogel). While HA hydrogel released 100% of BMP-2 over two weeks, less than 10% of BMP-2 was released from the HA-BP hydrogel for the same time. We demonstrate that the sequestered growth factor can still be released by enzymatic degradation of the HA-BP hydrogel. Most importantly, entrapment of BMP-2 in HA-BP hydrogel preserves the growth factor bioactivity, which was confirmed by induction of osteogenic differentiation of mesenchymal stem cells (MSCs) after the cells incubation with the enzymatic digest of the hydrogel. At the same time, the hydrogels degradation products were not toxic to MSCs and osteoblasts. Furthermore, BP-functionalization of HA hydrogels promotes adhesion of the cells to the surface of HA hydrogel. Altogether, the present findings indicate that covalent grafting of HA hydrogel with BP groups can alter the clinical effects of BMPs in bone tissue regeneration.

National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-219176 (URN)10.1021/bm400639e (DOI)000330095500012 ()
Available from: 2014-02-24 Created: 2014-02-24 Last updated: 2017-12-05Bibliographically approved
5. Non-invasive tri-modal visualisation of recombinant human bone morphogenetic protein-2 retention and associated bone regeneration: A proof of concept.
Open this publication in new window or tab >>Non-invasive tri-modal visualisation of recombinant human bone morphogenetic protein-2 retention and associated bone regeneration: A proof of concept.
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(English)Manuscript (preprint) (Other academic)
Keywords
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
Biology
Identifiers
urn:nbn:se:uu:diva-234698 (URN)
Funder
EU, FP7, Seventh Framework Programme
Available from: 2014-10-24 Created: 2014-10-23 Last updated: 2017-01-10
6. A surprisingly poor correlation between in vitro and in vivo testing of biomaterials for bone regeneration: Results of a multicentre analysis
Open this publication in new window or tab >>A surprisingly poor correlation between in vitro and in vivo testing of biomaterials for bone regeneration: Results of a multicentre analysis
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2016 (English)In: European Cells and Materials, ISSN 1473-2262, E-ISSN 1473-2262, Vol. 31, p. 312-322Article in journal (Refereed) Published
Abstract [en]

New regenerative materials and approaches need to be assessed through reliable and comparable methods for rapid translation to the clinic. There is a considerable need for proven in vitro assays that are able to reduce the burden on animal testing, by allowing assessment of biomaterial utility predictive of the results currently obtained through in vivo studies. The purpose of this multicentre review was to investigate the correlation between existing in vitro results with in vivo outcomes observed for a range of biomaterials. Members from the European consortium BioDesign, comprising 8 universities in a European multicentre study, provided data from 36 in vivo studies and 47 in vitro assays testing 93 different biomaterials. The outcomes of the in vitro and in vivo experiments were scored according to commonly recognised measures of success relevant to each experiment. The correlation of in vitro with in vivo scores for each assay alone and in combination was assessed. A surprisingly poor correlation between in vitro and in vivo assessments of biomaterials was revealed indicating a clear need for further development of relevant in vitro assays. There was no significant overall correlation between in vitro and in vivo outcome. The mean in vitro scores revealed a trend of covariance to in vivo score with 58 %. The inadequacies of the current in vitro assessments highlighted here further stress the need for the development of novel approaches to in vitro biomaterial testing and validated pre-clinical pipelines.

Keywords
in vivo, in vitro, correlation, biomaterials, multicentre study
National Category
Biomaterials Science
Identifiers
urn:nbn:se:uu:diva-306778 (URN)10.22203/eCM.v031a20 (DOI)000384895100020 ()27215739 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 262948
Available from: 2016-11-16 Created: 2016-11-03 Last updated: 2017-11-29Bibliographically approved

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