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Surface modified biphasic calcium phosphate-particles enhance expression of bone markers in osteoblast-like cells
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Oral Surgery UHL.
Department of Clinical Dentistry, Center for Clinical Dental Research, Faculty of Medicine and Dentistry, University of Bergen, Bergen 5009, Norway.
Department of Oral& Maxillofacial Surgery, Gävle, County Hospital, Sweden/Department of Oral & Maxillofacial Surgery, Umeå University, Umeå Sweden/Center for Research and Development, Uppsala University/Gävleborg County Council, Sweden.
KOMET RHOBEST GmbH, Exlgasse 20a, 6020 Innsbruck, Austria.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Objectives: The aim of the present in vitro study was to evaluate the response of human osteoblast-like cells (HOB) to nano-crystalline-diamond-particle-modified (nDP-modified) and un-modified (control) deproteinized bovine bone (DBB) and biphasic calcium phosphate (BCP) scaffolds.

Materials and methods: nDP-modification of DBB and BCP- particles was carried out through different steps of preparation including grinding and  ultrasonic technique. In each experiment, 100 mg materials from each of the 4 groups (nDP-modified DBB, nDP-modified BCP, un-modified DBB and un-modified BCP) were plated into a 48 well cell culture plate and 200.000 cells/well were seeded onto the materials. Scanning electron microscopy (SEM) was carried out after 24 hours and 3 days. Real time-polymerase chain reaction (PCR) was carried out after 3 days, 1 week and 2 weeks of incubation. The following markers were analyzed; alkaline phosphatase (ALP), osteocalcin (OC), bone morphogenetic protein type 2 (BMP-2) and integrin alpha 10 (ITGA 10). The general tendency of DBB and BCP was compared.

Results: Cellular responses were evaluated in terms of attachment and differentiation. SEM after 24 hours and 3 days of incubation disclosed similar cell attachment and spreading for nDP-modified and non-modified DBB and BCP particles. Real-time PCR revealed significant up-regulation of mRNA expression of ALP and OC by HOB grown on nDP-modified DBB and BCP-particles after 1 and 2 weeks compared to non-modified  particles. A significant down-regulation of BMP-2 on nDP-modified DBB and a significant up-regulation of BMP-2 on nDP-modified BCP were disclosed for HOB in relation to un-modified particles. Cell adhesion marker ITGA 10 showed significant down-regulated in the mRNA level for both nDP-modified groups after 2 weeks of incubation (nDP-BCP (p<.01) and nDP-DBB (p<.05) compared to the non-modified materials.

Conclusion: nDP-modified BCP-particles seem to enhance the expression of osteogenic markers in HOB in vitro. The results indicate that nDP-modified BCP enhance the osteoblast phenotype and suggest that these scaffolds might be appropriate cell carriers, superior to nonmodified BCP-particles. The osteogenic markers of OC and ALP were increased for nDPmodified DBB particles but BMP-2 was down regulated in relation to non-modified DBB particles.

Keyword [en]
Tissue engineering, scaffolds, gene expression, human osteoblast-like cells, biphasic calcium phosphate, deproteinized bovine bone
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-72937OAI: oai:DiVA.org:liu-72937DiVA: diva2:463799
Available from: 2011-12-12 Created: 2011-12-12 Last updated: 2011-12-12Bibliographically approved
In thesis
1. On healing of titanium implants in biphasic calcium phosphate
Open this publication in new window or tab >>On healing of titanium implants in biphasic calcium phosphate
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Previously, autogenous bone was considered the gold standard for grafting procedures in implant surgery. Today, the use of bone graft substitutes is an alternative for sinus lift procedures. Nevertheless, only a few bone substitutes are well documented and can be recommended as an alternative to autogenous bone grafts. Both deproteinized bovine bone (DBB) and tricalcium phosphate (TCP) are materials that are frequently used and well documented. During the last years a novel biphasic calcium phosphate (BCP) has been introduced to the market. Until now only a few studies have been published.

Aims: Studies I, II, III and IV. The overall aim of the present thesis was to evaluate a new biphasic calcium phosphate for bone augmentation of the maxillary sinus. Deproteinized bovine bone was used as a control.Study V. The aim of this in vitro study was to evaluate the response of human osteoblast-like cells (HOB) to nano-crystalline-diamond-particle-modified (nDP-modified) and un-modified (control) deproteinized bovine bone (DBB) and biphasic calcium phosphate (BCP) scaffolds.

Materials and Methods: Studies I, II, III and IV. The studies were based on 11 patients (six women, five men) with a mean age of 67 years (range; 50 to 79 years). All patients showed severe maxillary resorption with less than 5mm of residual alveolar bone in the floor of the maxillary sinus, which excluded conventional implant treatment. Twenty-two maxillary sinuses were augmented with BCP on one side and with DBB acting as a control at the contralateral side. Simultaneously with the augmentation procedure 22 microimplants were placed inside the augmented materials. After 8 months of graft healing the microimplants and a surrounding bone core were retrieved for histomorphometrical analysis (Paper I) and for energy dispersive spectroscopy (Paper II). After retrieval of the microimplants, 62 conventional implants were placed and left to heal for 8 weeks before rehabilitation with fixed prosthetic constructions. The conventional implants were evaluated clinically at baseline, after 1- and 3 years of loading (Papers III and IV). After 3 years of graft healing 18 biopsies were harvested from 9 patients for histomorphometrical analysis (Paper IV).

Computerized tomography (CT) of the maxillary sinuses was performed after 3 years of graft healing to allow examination of the recipient sites.

Study V. Nano-crystalline-diamond particle-modification of DBB and BCP particles was carried out through different steps of preparation including grinding and ultrasonic techniques. Scanning electron microscopy (SEM) was carried out after 24 hours and 3 days. Real time-polymerase chain reaction (PCR) was carried out after 3 days, 1 week and 2 weeks of incubation. The following osteoblast differentiation markers were analyzed: alkaline phosphatase (ALP), osteocalcin (OC), bone morphogenetic protein type 2 (BMP-2) and integrin alpha 10 (ITGA 10).

Results: In paper I, the results revealed a similar degree of bone formation and bone-to-implant contact around sandblasted and acid-etched microimplants placed in sinuses augmented with BCP or DBB. No obvious signs of resorption of the BCP and DBB particles were noticed. There was a significantly higher amount of DBB particles in contact with newly formed bone compared to BCP (p=.007).

In paper II, the median Ca/P ratios (atomic %), determined from >200 discreet sites within residual graft particles and adjacent bone were analysed. The difference between the median values for BCP and DBB and for BCP-augmented bone compared with DBB-augmented bone were statistical significant (p=.028 in each case). The reduction in Ca/P ratio for BCP over the healing period is consistent with the dissolution of β-TCP and reprecipitation on the surface of calcium-deficient hydroxyapatite.

In paper III, the results revealed that no implant placed in residual bone was lost, one implant placed in BCP was lost after 3 months of functional loading due to infection, and one implant placed in DBB was lost only a few weeks after insertion due to lack of initial instability. The overall implant survival rate was 96.8%. Success rates for implants placed in BCP and DBB were 91.7% and 95.7% respectively. No significant differences in marginal bone loss were found around implants placed in BCP, DBB or residual bone respectively.

In paper IV, it was shown that after 36 months (range; 36 to 37 months) of functional loading the overall implant survival rate was 96.8%. Success rates for implants placed in BCP, DBB and residual bone were 91.7%, 95.7% and 86.7% respectively. No significant difference was found between implants placed in BCP, DBB and residual bone. The corresponding histological evaluation after 3 years of graft healing showed BCP particles under different levels of dissolution. Dissolution was mostly observed on the edges of the BCP particles but in some cases the entire particle was dissolving. In contrast, DBB particles showed no signs of resorption. The percentage of graft particles in contact with newly formed bone was not significantly different after 3 years of healing for BCP and DBB.

In paper V, cellular responses were evaluated in terms of attachment and differentiation. SEM after 24 hours and 3 days of incubation disclosed similar cell attachment and spreading for nDP-modified and non-modified DBB and BCP particles. Real-time PCR revealed significant up-regulation of mRNA expression of ALP and OC and by HOB grown on nDP-modified DBB and BCP-particles after 1 and 2 weeks compared to non-modified particles. A significant down-regulation of BMP-2 on nDP-modified DBB and a significant up-regulation of BMP-2 on DP-modified BCP was disclosed for HOB in relation to un-modified particles. Cell adhesion marker ITGA 10 showed significant down-regulation in the mRNA level for both nDP-modified groups after 2 weeks of incubation (mDP-BCP (p<.01) and nDP-DBB (p<.05) compared to the non-modified materials.

Conclusion: It is concluded that BCP can be used for maxillary floor augmentation and later placement of dental implants producing equal results to those for DBB. Nevertheless, the initial HA/β-TCP ratio in BCP might not be optimized for cell adhesion, which can affect the early healing phase. Furthermore, the results indicate that BCP is not optimized for gene expression in its present form and that nDP-modified BCP enhances the osteoblast phenotype suggesting that these scaffolds are appropriate cell carriers, superior to non-modified BCP particles. Surface modification of bone substitutes is a new interesting field in bone tissue engineering (BTE). Nevertheless, it´s still unclear if the modification will have any clinical impact.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. 63 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1274
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-72938 (URN)978-91-7393-026-0 (ISBN)
Public defence
2012-01-27, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2011-12-12 Created: 2011-12-12 Last updated: 2011-12-16Bibliographically approved

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