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Co-culture model using human osteoblasts and osteoclasts on bone discs for in situ monitoring of surface remodeling
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab. (Jöns Hilborn/Biomaterials)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
(English)In: Biomatter, ISSN 2159-2535Article in journal (Other academic) Submitted
Abstract [en]

Osteoporosis is marked by accelerated bone resorption than bone formation and is currently treated with suboptimal drugs associated with severe off-target effects. More robust in vitro models are needed to investigate the precise pharmacokinetic effects of new drug formulations on bone cells in coculture conditions. This would promote targeted drug development and could reduce the number of animals needed in pre-clinical trials. However, existing coculture models do not address the effect of soluble factors released from cells in coculture. To address this challenge, we developed a two-sided co-culture model comprising human osteoclasts and osteoblasts on opposite sides of a thin decellurized bone chip. Essential cellular functions such as resorption by osteoclasts and mineralization by osteoblasts were not disrupted in the two-sided co-culture, even though the bone chip physically separated the two cell types. In this model, we freshly quantified resorption pits and mineralization on opposite sides of the same material through microscopy assisted image analysis and histological staining, respectively. Mineralization by osteoblasts was assessed with alizarin red and showed downregulation by 25% in the presence of osteoclasts (relative to osteoblasts alone) on the bone chip. The drug Pamidronate reduced the osteoclast population by 10% without affecting the number of osteoblasts. Thus, this co-culture model significantly simplifies allows in-situ monitoring of the effect of soluble bone signaling factors and anti-osteoporotic drugs.

Place, publisher, year, edition, pages
Taylor & Francis Group.
National Category
Chemical Sciences
Research subject
Chemistry with specialization in Polymer Chemistry; Chemistry with specialization in Materials Chemistry; Biology with specialization in Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-259080OAI: oai:DiVA.org:uu-259080DiVA: diva2:843136
Projects
MultiTERM
Funder
EU, FP7, Seventh Framework Programme, 238551Swedish Research Council, 2014-6099
Available from: 2015-07-27 Created: 2015-07-27 Last updated: 2015-09-01Bibliographically approved
In thesis
1. Evaluation of Functionalized Biopolymers as a Step Toward Targeted Therapy of Osteoporosis
Open this publication in new window or tab >>Evaluation of Functionalized Biopolymers as a Step Toward Targeted Therapy of Osteoporosis
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work presented in this thesis focuses on the development of strategies and smart bioactive materials for the treatment of osteoporosis. High and low molecular weight soluble hyaluronic acid-bisphosphonate (HA-BP) derivatives were investigated for their ability to inhibit osteoclasts. Low molecular weight HA-BP (L-HA-BP) was most effective in inhibiting active resorption of both murine and human osteoclasts (without affecting osteoblasts) compared to free bisphosphonate (BP). Precursor monocytes were unaffected, suggesting the specificity of HA-BP towards osteoclasts. This new class of functionalized hyaluronic acid could lead to rapid development of tailor-made pro-drugs for targeted treatment of osteoporosis.

Polyphosphoesters (PEP) have been widely studied for their pro-osteoblast effects, primarily due to their involvement in cellular energy production pathway leading to the formation of inorganic phosphates that contribute to mineralized bone. Given that the effect of PEP on human osteoclasts is little studied, this work on poly(ethylene sodium phosphate) (PEP.Na) explores the potential to use PEP.Na as an inhibitor of osteoclast activity for the first time. PEP.Na exposure led to a dose-dependent toxicity of osteoclasts with reduction in their capacity to form resorption pits over 24h.

Currently, there is a dearth of in vitro cell-culture systems that can study osteoclast-related resorption and osteoblast-related mineralization in a single co-culture system, and to simultaneously quantify the effects of soluble factors on these processes. Described here, is the development of a novel and simple two-sided co-culture system that can overcome these limitations with reliable and quantifiable readouts. In comparison with traditional one-sided co-culture systems, the two-sided co-culture was able to generate similar readouts for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) markers. There is also the advantage of distinctly separate and quantifiable readouts for mineralization and resorption, which has been demonstrated using Pamidronate.

Finally, HA-BP was synthesized with pre-determined amounts of BP groups. The BP groups attached to HA allowed the tunable incorporation of BMP-2 in hydrogels. The charge-based affinity of BMP-2 and BP allowed stable incorporation of specific amounts of BMP-2, which could be tuned by the ratio of BP groups. 125I-labelled BMP-2 was loaded into hydrogels and their release was studied. Radioactive measurements revealed the tunable sequestration and controlled release of protein over time. This result was corroborated by ALP measurements of cells exposed to released BMP-2. ALP production was found to be almost 5-fold higher in HA-BP hydrogels loaded with BMP-2 which suggested that the sequestered BMP-2 is not only available to cells but also remains highly potent, even in entrapped form, The release of BMP-2 is dependent upon the rate of diffusion, swelling in hydrogels and degradation pattern of the gels and may assist in the long-term and rapid regeneration of osteoblasts in vitro.

Place, publisher, year, edition, pages
Uppsala: Uppsala universitet, 2015. 78 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1267
Keyword
Osteoporosis, Bone regeneration, Biomaterials, Hyaluronic acid, Bisphosphonates, Osteoclasts, Osteoblasts, Growth factors
National Category
Polymer Chemistry
Research subject
Chemistry with specialization in Polymer Chemistry; Chemistry with specialization in Materials Chemistry
Identifiers
urn:nbn:se:uu:diva-259386 (URN)978-91-554-9287-8 (ISBN)
Public defence
2015-09-24, Häggsalen, Ångström Laboratory, Lägerhyddsvägen 1, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2015-08-28 Created: 2015-08-01 Last updated: 2015-10-01

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Kootala, SujitZhang, YuHilborn, Jöns

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