Global gene expression profile of osteoblast-like cells grown on polyester copolymer scaffolds.

One of the principal goals in tissue engineering is to produce scaffold materials that will guide cells to differentiate and regenerate functional replacement tissue at the site of injury. Poly(l-lactide-co-1,5-dioxepan-2-one) [Poly(LLA-co-DXO)], a potential scaffolding material for bone tissue engineering, has high hydrophilicity. Previous in vitro studies using human osteoblast-like cells (HOBs) demonstrated greater cytocompatibility and enhanced osteogenic differentiation when HOBs were seeded onto Poly(LLA-co-DXO) compared to Poly(l-lactide) [P(LLA)] scaffolds. The aim of the study was to identify the gene expression profiles of HOBs obtained from alveolar bone and grown on Poly(LLA-co-DXO) biodegradable polymer scaffolds compared to P(LLA) one. Illumina HumanWG-6 v3.0 Expression BeadChips were used for the gene expression analysis. Several genes were found as differentially expressed at 24 h and at 21 days. Expression of genes related to cell adhesion, cytoskeleton, antiapoptosis, proliferation, and bone mineralization was influenced by adding the monomer 1,5-dioxepan-2-one to the L-lactide. Genes related to three biological pathways involving Integrin, Notch, and Ras were found to be upregulated. For selected genes, results were confirmed by quantitative reverse transcriptase-polymerase chain reaction. Further, calcium content analysis revealed a significant enhancement of calcium deposition on both tested scaffolds. This observation was confirmed by Von Kossa and Alizarin Red S staining. Findings of this study are relevant to a better understanding of the molecular mechanisms underlying the behavior of HOBs in bone regenerative procedure.

Comparison of short-run cell seeding methods for poly(L-lactide-co-1,5-dioxepan-2-one) scaffold intended for bone tissue engineering.

Constructs intended for bone tissue engineering are influenced by the initial cell seeding procedure. The seeding method should be rapid, convenient, improve cell spatial distribution, and have no negative effects on cellular viability and differentiation. This study aimed to compare the effect of short-run seeding methods (centrifuge and vortex) with a static method on the scaffolds prepared from poly(L-lactide-co-1,5-dioxepan-2-one) by solvent-casting particulate-leaching (SCPL) technique. Human osteoblast-like cells (HOB) were seeded by the three methods described above. The seeding efficiency was determined by attached cell numbers. Cellular proliferation was analyzed by WST-1 and dsDNA assay. Cell distribution was examined by scanning electron (SEM) and fluorescence microscopy. Expression of alkaline phosphatase (ALP), collagen type I (Col I), osteocalcin (OC) and proliferating cell nuclear antigen (PCNA) were determined by real time RT-PCR. Results indicated that centrifuge and vortex increased seeding efficiency and had no negative effects on cellular viability. The data obtained by the fluorescence microscope confirmed the SEM results that the vortex method improved cell distribution through the scaffolds more than the other two methods (p<0.05). The RT-PCR results showed no significant differences on the expression of mRNA between the three methods of the above markers. The vortex method was found to be a simple and feasible seeding method for the poly(L-lactide-co-1,5-dioxepan-2-one) scaffolds.

Effect of endothelial cells on bone regeneration using poly(L-lactide-co-1,5-dioxepan-2-one) scaffolds.

Our recent in vitro study demonstrated that endothelial cells (ECs) might influence the differentiation of bone marrow stromal cells (BMSCs). Therefore, the aim of this study was to describe this effect in vivo, using a rat calvarial bone defect model. BMSCs were isolated from femurs of two-donor Lewis rats and expanded in α-minimum essential medium containing 10% fetal bovine serum. One fifth of BMSCs were induced and differentiated into ECs in an Endothelial Cell Growth Medium-2 and then characterized by a flow cytometry. The remaining BMSCs were cultured in freshly prepared osteogenic stimulatory medium, containing dexamethasone, ascorbic acid and ß-glycerophosphate. Either BMSCs alone (BMSC-group) or co-cultured ECs/BMSCs (CO-group) were seeded into poly(L-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO)] scaffolds, cultured in spinner flasks, and then implanted into symmetrical calvarial defects prepared in recipient rats. The animals were sacrificed after 2 months. The formation of new bone was evaluated by radiography and histology and by the expression of osteogenic markers using reverse transcriptase-polymerized chain reaction (RT-PCR). To investigate vessel formation, histological staining was performed with EC's markers. The radiographical and histological results showed more rapid bone formation in the CO- than in the BMSC-group. However, the expression of EC's marker was similar on both groups by histological analysis after 2 months postoperatively. Furthermore, the CO-group exhibited greater expression of osteogenic markers as demonstrated by RT-PCR. The results are consistent with the previous in vitro findings that poly(LLA-co-DXO) scaffold might be suitable candidate for bone tissue engineering. In vivo, bone regeneration was enhanced by a construct of the polymer scaffold loaded with co-cultured cells.

Growth and differentiation of bone marrow stromal cells on biodegradable polymer scaffolds: an in vitro study.

A fundamental component of bone tissue engineering is an appropriate scaffold as a carrier for osteogenic cells. The aim of the study was to evaluate the response of human bone marrow stromal cells (BMSC) to scaffolds made of three biodegradable polymers: poly(L-lactide-co-ε-caprolactone) (poly(LLA-co-CL)), poly(L-lactide-co-1,5dioxepan-2-one) (poly(LLA-co-DXO)), and poly(L-lactide) (poly(LLA)). Cellular response was evaluated in terms of attachment, proliferation, and differentiation. SEM disclosed earlier cell attachment and better spreading on poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds than on poly(LLA) after 1 h. At 24 h and 14 days postseeding, BMSCs had spread well, forming multiple cellular layers on the scaffolds. Cell proliferation was higher on poly(LLA-co-CL) and on poly(LLA-co-DXO) than on poly(LLA) after 1 and 7 days. Cell growth cycles of BMSC were longer on the scaffolds than on coverslips. After 7 and 14 days cultivation on scaffolds, the expression of osteogenic markers such as ALP, Col I, OPN, and Runx2 were stimulated by BMSC, which indicating that poly(LLA-co-DXO), poly(LLA-co-CL), and poly(LLA) could support the osteogenic differentiation of BMSC in vitro. Poly(LLA-co-CL) and poly(LLA-co-DXO) promoted better attachment and growth of BMSC than poly(LLA). BMSC also retained their osteogenic differentiation potential, indicating biological activity of BMSC on the scaffolds. The promising results of this in vitro study indicate that these copolymers warrant further evaluation for potential application in bone tissue engineering.

Polyester copolymer scaffolds enhance expression of bone markers in osteoblast-like cells.

In tissue engineering, the resorbable aliphatic polyester poly(L-lactide) (PLLA) is used as scaffolds in bone regeneration. Copolymers of poly(L-lactide)-co-(epsilon-caprolactone) [poly(LLA-co-CL)] and poly(L-lactide)-co-(1,5-dioxepan-2-one) [poly(LLA-co-DXO)], with superior mechanical properties to PLLA, have been developed to be used as scaffolds, but the influence on the osteogenic potential is unclear. This in vitro study of test scaffolds of poly(LLA-co-CL) and poly(LLA-co-DXO) using PLLA scaffolds as a control demonstrates the attachment and proliferation of human osteoblast-like cells (HOB) as measured by SEM and a methylthiazol tetrazolium (MTT) colorimetric assay, and the progression of HOB osteogenesis for up to 3 weeks; expressed as synthesis of the osteoblast differentiation markers: collagen type 1 (Col 1), alkaline phosphatase, bone sialoprotein, osteocalcin (OC), osteopontin and runt related gene 2 (Runx2). Surface analysis disclosed excellent surface attachment, spread and penetration of the cells into the pores of the test scaffolds compared to the PLLA. MTT results indicated that test scaffolds enhanced the proliferation of HOBs. Cells grown on the test scaffolds demonstrated higher synthesis of Col 1 and OC and also increased bone markers mRNA expression. Compared to scaffolds of PLLA, the poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds enhanced attachment, proliferation, and expression of osteogenic markers by HOBs in vitro. Therefore, these scaffolds might be appropriate carriers for bone engineering.

Endothelial cells influence the osteogenic potential of bone marrow stromal cells.

BACKGROUND: Improved understanding of the interactions between bone cells and endothelial cells involved in osteogenesis should aid the development of new strategies for bone tissue engineering. The aim of the present study was to determine whether direct communication between bone marrow stromal cells (MSC) and human umbilical vein endothelial cells (EC) could influence the osteogenic potential of MSC in osteogenic factor-free medium. METHODS: After adding EC to MSC in a direct-contact system, cell viability and morphology were investigated with the WST assay and immunostaining. The effects on osteogenic differentiation of adding EC to MSC was systematically tested by the using Superarray assay and results were confirmed with real-time PCR. RESULTS: Five days after the addition of EC to MSC in a ratio of 1:5 (EC/MSC) significant increases in cell proliferation and cellular bridges between the two cell types were detected, as well as increased mRNA expression of alkaline phosphatase (ALP). This effect was greater than that seen with addition of osteogenic factors such as dexamethasone, ascorbic acid and beta-glycerophosphate to the culture medium. The expression of transcription factor Runx2 was enhanced in MSC incubated with osteogenic stimulatory medium, but was not influenced by induction with EC. The expression of Collagen type I was not influenced by EC but the cells grown in the osteogenic factor-free medium exhibited higher expression than those cultured with osteogenic stimulatory medium. CONCLUSION: These results show that co-culturing of EC and MSC for 5 days influences osteogenic differentiation of MSC, an effect that might be independent of Runx2, and enhances the production of ALP by MSC.

Influence of modifying and veneering the surface of ceramic abutments on cellular attachment and proliferation.

OBJECTIVES: This in vitro study was aimed to investigate the attachment, spreading and proliferation of human gingival fibroblasts to milled and polished non-veneered ceramic surfaces in alumina and zirconia and to ceramic surfaces veneered by two different types of porcelain baseliners. MATERIALS AND METHODS: Fibroblasts were cultured on discs of pressed alumina or zirconia, on discs which had been milled, on discs comprising alumina or zirconia which had been polished, on discs of alumina veneered with NobelRondo baseliner Al, on discs of zirconia veneered with Cercon-S baseliner, and on alumina or zirconia discs veneered with the above baseliners and then polished. The surfaces were analyzed using an optical interferometer and scanning electron microscopy (SEM). Cell profile areas were measured using SEM and an image analyzer. Cell attachment was determined after 3 and 24 h as a ratio of the cell profiles and the total micrograph area and was expressed as percent of attachment. MTT analyses were undertaken to determine cellular attachment after 3 h of incubation and cellular proliferation after 7 days. RESULTS: The polished zirconia specimens had the smoothest surface in terms of average height deviation (S(a)=0.03 microm): the roughest were the zirconia specimens with milled surfaces (S(a)=0.36 microm). The application of the baseliners resulted in surfaces smoother than those of the non-veneered discs. The milled surfaces of both alumina and zirconia had significantly higher percentages of cell attachment and proliferation than the other surfaces whereas the milled surfaces in zirconia demonstrated better cellular attachment after 3 and 24 h of culture than the one in alumina. Fibroblasts attached and grew effectively on the surfaces veneered with NobelRondo throughout the experiments, whereas the zirconia surfaces veneered with Cercon-S had the lowest percentage of cell attachment and proliferation. CONCLUSIONS: Although the roughness of all surfaces investigated was <0.4 mum, the study disclosed significant differences in cellular attachment and proliferation associated with the various surface modifications.

Early loading of mandibular full-arch bridges screw retained after 1 week to four to five Monotype implants: 3-year results from a prospective multicentre study.

OBJECTIVES: This prospective multicentre study provides clinical experience up to 3 years to support a simplified treatment for mandibular edentulism within 1 week by using one-stage implant surgery and a screw-retained full-arch bridge. METHODS: Two hundred and fifty ITI Monotype implants were installed in 62 patients out of 66 patients; 60 patients got four implants each and two got five implants. After 1 week, a final bridge was in function. Radiographs were taken as baseline for vertical bone loss up to 3 years post-loading for the whole cluster and specific effects of gender, centre, age, bone class, implant length over time were compiled. Clinical (mPI, SBI) and subjective parameters such as general oral hygiene and patient satisfaction were recorded and repeated at specified intervals up to 3 years. RESULTS: Four patients were excluded at surgery and are not involved in the follow-ups. At 1 year, 61 patients (244 implants) were evaluable and all bridges were in function. After 3 years, 49 patients (194 implants) came to control. Eight patients died during the follow-up period. Three patients lost one implant each. The cumulative implant survival rate was 98.55% and the success rate for the prosthesis was 100%. As calculated from measurable radiographs, the mean bone level at baseline was 1.63+/-0.78 and at 1 and 3 years 2.50+/-0.60 and 2.56+/-0.74, respectively. Using the mixed model analysis and Friedman test, the time in situ, centre and bone class had significant effect on the bone resorption and to some small extent even, the implant length. Gender and age were unaffected. Oral hygiene and patient satisfaction of the treatment were improved. CONCLUSIONS: The results indicate that one-part self-tapping sandblasted, large-grit, acid-etched (SLA) implants are suitable for loading within 1 week. In the whole period, the mean bone crestal resorption was <1 mm, which is in agreement with other similar studies.

Early loading of mandibular full-arch bridges screw retained after 1 week to four to five Monotype® implants: 3-year results from a prospective multicentre study.

OBJECTIVES: This prospective multicentre study provides clinical experience up to 3 years to support a simplified treatment for mandibular edentulism within 1 week by using one-stage implant surgery and a screw-retained full-arch bridge. METHODS: Two hundred and fifty ITI Monotype® implants were installed in 62 patients out of 66 patients; 60 patients got four implants each and two got five implants. After 1 week, a final bridge was in function. Radiographs were taken as baseline for vertical bone loss up to 3 years post-loading for the whole cluster and specific effects of gender, centre, age, bone class, implant length over time were compiled. Clinical (mPI, SBI) and subjective parameters such as general oral hygiene and patient satisfaction were recorded and repeated at specified intervals up to 3 years. RESULTS: Four patients were excluded at surgery and are not involved in the follow-ups. At 1 year, 61 patients (244 implants) were evaluable and all bridges were in function. After 3 years, 49 patients (194 implants) came to control. Eight patients died during the follow-up period. Three patients lost one implant each. The cumulative implant survival rate was 98.55% and the success rate for the prosthesis was 100%. As calculated from measurable radiographs, the mean bone level at baseline was 1.63±0.78 and at 1 and 3 years 2.50±0.60 and 2.56±0.74, respectively. Using the mixed model analysis and Friedman test, the time in situ, centre and bone class had significant effect on the bone resorption and to some small extent even, the implant length. Gender and age were unaffected. Oral hygiene and patient satisfaction of the treatment were improved. CONCLUSIONS: The results indicate that one-part self-tapping sandblasted, large-grit, acid-etched (SLA) implants are suitable for loading within 1 week. In the whole period, the mean bone crestal resorption was <1 mm, which is in agreement with other similar studies.

Using optical tweezers for measuring the interaction forces between human bone cells and implant surfaces: System design and force calibration.

Optical tweezers were used to study the interaction and attachment of human bone cells to various types of medical implant materials. Ideally, the implant should facilitate cell attachment and promote migration of the progenitor cells in order to decrease the healing time. It is therefore of interest, in a controlled manner, to be able to monitor the cell adhesion process. Results from such studies would help foresee the clinical outcome of integrating medical implants. The interactions between two primary cell culture models, human gingival fibroblasts and bone forming human osteoblast cells, and three different implant materials, glass, titanium, and hydroxyapatite, were studied. A novel type of optical tweezers, which has a newly designed quadrant detector and a powerful 3 W laser was constructed and force calibrated using two different methods: one method in which the stiffness of the optical trap was obtained by monitoring the phase lag between the trap and the moved object when imposing a forced oscillation on the trapped object and another method in which the maximum trapping force was derived from the critical velocity at which the object escapes the trap. Polystyrene beads as well as cells were utilized for the calibrations. This is the first time that cells have been used directly for these types of force calibrations and, hence, direct measurements of forces exerted on cells can be performed, thus avoiding the difficulties often encountered when translating the results obtained from cell measurements to the calibrations obtained with reference materials. This more straightforward approach represents an advantage in comparison to established methods.

The influence of surface topography of ceramic abutments on the attachment and proliferation of human oral fibroblasts.

As different implant abutments are introduced to obtain a sufficient soft tissue barrier, the aim of this study was to determine the effect of three different surface modifications of densely sintered high-purity aluminium oxide on morphology, attachment and proliferation of human gingival fibroblasts. Fibroblasts were cultured on pressed aluminium oxide, milled, and then sintered to full density (1), on pressed, densely sintered (2), and on pressed, densely sintered and then polished surfaces (3). The different surfaces were analyzed using a confocal laser scanner, an atomic force microscope and a scanning electron microscope. The cell profile areas were measured using a semiautomatic interactive image analyzer and the figures were expressed as percent of attachment. The polished specimens had the smoothest surfaces and the roughest were the milled surfaces in terms of height deviation. No difference was found in the spacing between the peaks on the polished surfaces compared to the milled surfaces. Fibroblasts on the milled ceramic appeared to follow the direction of the fine irregularities on the surface. The analyses showed the polished surfaces had significantly higher percentages of initial cell attachment than the other surfaces (P < 0.05). After 3 days of cell culture, significantly more cells were attached to the milled and sintered surfaces than to the polished one, possibly indicating higher proliferation capacity on those types of surfaces.

A retrospective study of Mirage ceramic inlays over up to 9 years.

PURPOSE: The aim of this retrospective longitudinal study was to document the outcomes of resin-cemented ceramic inlays in patients treated in a private dental practice over up to 9 years. MATERIALS AND METHODS: Fifty-two patients received in all 109 Mirage ceramic inlays, 59 in molars and 50 in premolars. Follow-up data were collected on 51 patients and 107 inlays. The mean time in situ was 6.3 years. Technical failures or dislodgments of the inlays were recorded, as well as clinical and radiographic signs of periodontal and periapical diseases or caries lesions. RESULTS: During the follow-up period, 17 inlays had to be removed, 14 because of major fractures and 3 in teeth requiring endodontic treatment. The fractures were confined to 8 of the 51 patients available for follow-up and were significantly more common in molars than in premolars. Replacement of inlays was significantly more frequent in men than in women. During the study period, 20 further minor clinical interventions (eg, recementation of intact inlays, repair of minor fractures or caries defects) had been undertaken on the surviving inlays. Thus, major and minor complications together necessitated a total of 37 clinical interventions. The survival rate, based on the remaining 90 inlays still in situ, was 84%. CONCLUSION: In this material, the outcome of ceramic inlay therapy was influenced by both major and minor complications (35%). Eight of the 51 patients accounted for all the major fractures, implying that patient selection is an important determinant of the clinical outcome.

Production of transforming growth factor beta1 and prostaglandin E2 by osteoblast-like cells cultured on titanium surfaces blasted with TiO2 particles.

The surface roughness of an implant to which osteoblasts attach may influence endogenous expression of growth factor and cytokines at the implant-tissue interface, modulating the healing process and affecting the quality of bone formation. The present study, using cells derived from human mandibular bone, investigated the effect of varying roughness of titanium surfaces on production of transforming growth factor beta1 (TGF-beta1) and prostaglandin E2 (PGE2). The titanium surfaces were turned (control) and then roughened by blasting with 63-90 micro m, 106-180 micro m or 180-300 micro m TiO2 particles. The cells were cultured onto the surfaces till confluence was achieved. Fresh media were added in the presence or absence of 1,25-dihydroxyvitamin D3[1,25-(OH)2D3], the stimulator of osteogenic differentiation, and aliquots of conditioned cell media were used for assay 24 h later. Cellular morphology was determined by scanning electron microscopy. Cellular production of TGF-beta1 and PGE2 on each surface was assessed by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), respectively, using commercially available kits. All blasted surfaces showed significantly higher production of TGF-beta1 than the turned surfaces (P < 0.05). In response to stimulation by 1,25-(OH)2D3 cellular production of TGF-beta1, was also significantly greater (P < 0.05) on the blasted surfaces than on the turned one. The total amount of PGE2 in the conditioned media was higher than on the turned surfaces in the presence or absence of 1,25-(OH)2D3. There were no significant differences among the three blasted surfaces with respect to production of the local factors. However, we could not show a synergistic effect of surface roughness and vitamin D on the production of both TGF-beta1 and PGE2 using primary cell culture model.

Implant-supported welded titanium frameworks in the edentulous maxilla: a 5-year prospective multicenter study.

PURPOSE: This study evaluated the 5-year clinical and radiographic performance of fixed implant-supported maxillary prostheses with either welded titanium or conventional cast-gold alloy frameworks. MATERIALS AND METHODS: Fifty-eight consecutive patients were provided with 349 osseointegrated Brånemark system implants in the edentulous maxilla at six different implant centers. Twenty-eight of the patients received, at random, prostheses with laser-welded titanium frameworks, and the remaining 30 patients had prostheses with conventional cast-gold alloy frameworks. Clinical and radiographic data were collected for 5 years after prosthesis placement. RESULTS: The titanium and cast-gold framework groups exhibited similar cumulative survival and success rates (CSR). The 5-year implant CSR from time of placement was 91.4% and 94.0%, respectively, and from prosthesis delivery the rate was 94.9% and 95.6%, respectively. The corresponding 5-year prosthesis CSRs were 96.4% and 93.3%. One patient from each group lost all the implants and turned to complete dentures within the first year of function. Another patient with a cast-gold framework had the prosthesis replaced after 4 years, basically because of problems with the veneering material. No fractures of implant components were observed during the follow-up period. Bone loss was on average 0.59 mm (SD 0.97 mm) during 5 years, with no statistical difference between the two groups. CONCLUSION: Welded titanium frameworks presented a similar favorable clinical performance as conventional cast-gold alloy frameworks in fixed implant-supported prostheses in the edentulous maxilla after 5 years in function. Implant failures were concentrated in only a few patients in each study group.

Electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy analysis of titanium surfaces cultured with osteoblast-like cells derived from human mandibular bone.

Variations in the oxide films on titanium surfaces blasted with TiO(2) particles of various sizes were analyzed after cultures with cells derived from human mandibular bone. Turned titanium surfaces and surfaces blasted with 63-90-, 106-180-, and 180-300-microm TiO(2) particles were cultured with osteoblast-like cells. The surfaces were characterized before and after the cell culture with electrochemical impedance spectroscopy (EIS). The surface chemical composition of selected samples was analyzed with X-ray photoelectron spectroscopy (XPS). EIS revealed that with respect to the turned surfaces, the effective surface area was about 5, 6, and 4 times larger on the surfaces blasted with 63-90-, 106-180-, and 180-300-microm particles, respectively. After 28 days of the cell culture, the corrosion resistance on all sample types was unaffected. The impedance characteristics suggest a considerable effect of ion incorporation and precipitation during culturing. XPS revealed that before the cell culture, a typical surface layer consisted of TiO(2). After the culture, the surface oxide film contained both phosphorus and calcium, along with large amounts of oxidized carbon (carbonate) and nitrogen. There were lower concentrations of carbon and nitrogen on the blasted surfaces. We concluded that the effective surface area was several times higher on blasted surfaces than on turned surfaces. Cells derived from human mandibular bone affected ion incorporation into the implant surface.