Sølve Hellem

Endothelial cells influence the osteogenic potential of bone marrow stromal cells

BackgroundImproved 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.MethodsAfter adding EC to MSC in a direct-contact system, cell viability and morphology were investigated with the WST assay and immnostaining. 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.ResultsFive 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 β-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.ConclusionThese 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.

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 β-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 ECs markers. The radiographical and histological results showed more rapid bone formation in the CO- than in the BMSC-group. However, the expression of ECs 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.

Reproducibility of transcutaneous oximetry and laser Doppler flowmetry in facial skin and gingival tissue

Laser Doppler flowmetry (LDF) and transcutaneous oximetry (TcPO(2)) are non-invasive techniques, widely used in the clinical setting, for assessing microvascular blood flow and tissue oxygen tension, e.g. recording vascular changes after radiotherapy and hyperbaric oxygen therapy. With standardized procedures and improved reproducibility, these methods might also be applicable in longitudinal studies. The aim of this study was to evaluate the reproducibility of facial skin and gingival LDF and facial skin TcPO(2). The subjects comprised ten healthy volunteers, 5 men, aged 31-68 years. Gingival perfusion was recorded with the LDF probe fixed to a custom made, tooth-supported acrylic splint. Skin perfusion was recorded on the cheek. TcPO(2) was recorded on the forehead and cheek and in the second intercostal space. The reproducibility of LDF measurements taken after vasodilation by heat provocation was greater than for basal flow in both facial skin and mandibular gingiva. Pronounced intraday variations were observed. Interweek reproducibility assessed by intraclass correlation coefficient ranged from 0.74 to 0.96 for LDF and from 0.44 to 0.75 for TcPO(2). The results confirm acceptable reproducibility of LDF and TcPO(2) in longitudinal studies in a vascular laboratory where subjects serve as their own controls. The use of thermoprobes is recommended. Repeat measurements should be taken at the same time of day.

Effect of hyperbaric oxygen treatment on oxygen tension and vascular capacity in irradiated skin and mucosa

The aim of this study was to evaluate the effect of hyperbaric oxygen therapy (HBOT) on vascular function and tissue oxygenation in irradiated facial skin and gingival mucosa. Twenty-two patients, aged 51-90 years, were randomly allocated to a treatment or control group. All had a history of radiotherapy (50-70 Gy) to the orofacial region 2-20 years previously. Skin and mucosal perfusion were recorded with laser Doppler flowmetry (LDF). Tissue oxygenation was recorded by transcutaneous oximetry (TcPO(2)). Measurements were taken before HBOT and 3 and 6 months after a mean of 28 HBOT sessions (partial pressure of oxygen of 240 kPa for 90 min). For control subjects, measurements were taken on two occasions 6 months apart. After HBOT, blood flow in mucosa and skin after heat provocation increased significantly (P < 0.05). TcPO(2) increased significantly in the irradiated cheek (P < 0.05), but not at reference points outside the field of radiation. There were no differences between the 3- and 6-month follow-ups. In the control group, no significant changes in LDF or TcPO(2) were observed. It is concluded that oxygenation and vascular capacity in irradiated facial skin and gingival mucosa are increased by HBOT. The effects persist for at least 6 months.

A practical approach to interpretation of MRI of the temporomandibular joint

Background: Temporomandibular disorders (TMDs) such as pain, joint sounds, and impaired movement are common, and magnetic resonance imaging (MRI) is now the method of choice for diagnostic assessment. Purpose: To describe MR criteria chosen and the amount of temporomandibular joint (TMJ) pathology registered when examining MR images from patients referred to a university hospital for imaging of their TMJs. Material and Methods: The TMJs of 152 consecutive patients, 102 women and 40 men, referred for MRI during an 18 month period were imaged with a 1.5 T imaging system. Twelve asymptomatic students, seven women and five men, gave informed consent and acted as a control group. Results: Moderate to extensive disk displacement was registered in 53% of the patients’ TMJs, and 38% of the disks were deformed. Degenerative changes registered were flattening of the condyle heads in 50% of the TMJs and erosion of their cortical surfaces in 30%. Osteophytes were present in 31% of the condyles and bone marrow edema in 30%. Marked to extensive effusion in synovial compartments was registered in 39% of the studied TMJs. In the control group, none of the TMJs showed anterior disk displacement, deformed disks or degenerative changes, but 8 of the 24 joints showed marked effusion. A tendency for a higher amount of disk displacement and deformation was seen among young age groups and more degenerative changes in older age groups, but differences among groups were not significant when tested with chi-square analysis. Conclusion: Defined MR criteria that allow for comparative assessment are presented. According to these criteria, a large proportion of the patients referred for MR examination showed morphologic changes indicating TMJ pathology.

Hyperbaric oxygen stimulates vascularization and bone formation in rat calvarial defects

Hyperbaric oxygen (HBO) therapy is used to treat or prevent tissue necrosis in patients undergoing irradiation. Many such patients require reconstructive surgery, but little is known of the effects of HBO on bone vascularization and regeneration. In this study, copolymer poly(l-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) scaffolds were implanted into critical-sized calvarial defects in Wistar rats. The animals were randomly allotted to hyperbaric or normobaric oxygen groups. The treatment group received five sessions weekly for 90 min at increased atmospheric pressure, for up to 4 weeks. Samples were retrieved at weeks 2 and 8, i.e. after a total of 10 and 20 sessions, respectively. The samples were analyzed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and histology at week 2, and radiographically and histologically at week 8. At week 2, defects treated with HBO exhibited greater numbers of cells positive for the endothelial marker CD31, up-regulated gene expression of osteogenic markers, and down-regulated expression of pro-inflammatory cytokines. At week 8, radiographic examination revealed that calvarial defects subjected to HBO exhibited a higher percentage of radiopacities than normobaric controls, and histological examination disclosed enhanced bone healing. These results confirmed that HBO treatment was effective in stimulating vascularization and bone formation in rat calvarial defects.

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 hyperbaric oxygen treatment on irradiated oral mucosa: microvessel density

The aim of this study was to evaluate the effect of hyperbaric oxygen therapy (HBOT) on microvascular tissue and cell proliferation in the oral mucosa. Twenty patients, aged 51-78 years, were allocated randomly to a treatment or a control group. All had a history of radiotherapy (50-70 Gy) to the orofacial region 2-6 years previously. Tissue samples were taken from the irradiated buccal oral mucosa before HBOT and at 6 months after treatment. In the control group, tissue samples were taken on two occasions, 6 months apart. The samples were subjected to immunohistochemistry staining: double staining with CD31 and D2-40 for microvessels, or Ki-67 for the analysis of cell proliferation. Blood vessel density and area were significantly increased after HBOT (P=0.002-0.041). D2-40-positive lymphatic vessels were significantly increased in number and area in the sub-epithelial area (P=0.002 and P=0.019, respectively). No significant differences were observed in the control group. There were no significant differences in Ki-67-expressing epithelial cells between the two groups. It is concluded that the density and area of blood and lymphatic vessels in the irradiated mucosa are increased by HBOT 6 months after therapy. Epithelial cell proliferation is not affected by HBOT.

Variations of air-borne bacteria in a department of oral surgery.

The variation in the number of airborne bacteria before and after the reconstruction of an oral surgery department was determined in the policlinic and the operating theatre by means of sedimentation plates with bloodagar. The sedimentation plates were kept in analogous locations in both parts of the study and exposed for determined periods during the day. Along with the reconstruction, the hygienic standard was improved by introduction of several changes, such as obligatory use of oral/nasal mask, separated treatment units in the policlinic, decreased movement during surgical procedures, a better isolation of the operating theatre and overpressure ventilation with filtrated air. The results showed a generally significant variation (p<0.01) in the bacterial level correlated with the rate of activity in both parts of the study. Further results showed a very clear decrease in the number of bacteria for both localities. The percentage reduction was found to be 65 per cent in the operating theatre and 50 per ...