Bone engineering with adipose tissue derived stromal cells.

INTRODUCTION: Tissue engineering offers the means for replacing or repairing diseased or damaged organs within the patient's body. This current study deals with the general differentiation potential of human adipose tissue derived stromal cells (ATSCs) into mesenchymal tissues and particularly into bone. MATERIAL AND METHODS: ATSCs were isolated and cultivated until the 3rd passage. Three different ways of mesenchymal differentiation (adipogenic, osteogenic, myogenic) were induced and analysed histologically (v. Kossa, Sudan III) and immunhistologically (alpha-actin, osteocalcin). Nine different cell cultures were analysed in terms of calcium deposition (donor's age dependend) in the cell matrix and release of alkaline phosphatase (AP) during the differentiation period. Three-dimensional osteogenic constructs, based on collagenous micro-particles were performed and analysed histologically. RESULTS: Effective differentiation of ATSCs into osteogenic, adipogenic and myogenic cells could be demonstrated in histological and immunohistological analyses. There was an increase of calcium deposition in the cell matrix of osteogenically differentiated ATSCs that did not show any correlation with the age of the donor. Two out of three three-dimensional constructs showed clear zones of mineralization (v. Kossa) in the area of osteocalcin-producing cells. CONCLUSION: Adipose tissue derived stromal cells are a promising source for tissue engineering with a clear potential to differentiate into adipogenic, osteogenic and myogenic cells. The osteogenic differentiation potential did not show any correlation to the age of the donors (calcium deposition, AP-activity). Three-dimensional osteogenic constructs could be established.

Implant stability and histomorphometry: a correlation study in human cadavers using stepped cylinder implants.

The aim of the present study was to determine the correlation between the primary stability of dental implants placed in edentulous maxillae and mandibles, the bone mineral density and different histomorphometric parameters. After assessing the bone mineral density of the implant sites by computed tomography, 48 stepped cylinder screw implants were installed in four unfixed human maxillae and mandibles of recently deceased people who had bequeathed their bodies to the Anatomic Institute I of the University of Erlangen-Nuremberg for medical-scientific research. Peak insertion torque, Periotest values and resonance frequency analysis were assessed. Subsequently, histologic specimens were prepared, and bone-to-implant contact, the trabecular bone pattern factor (TBPf), the density of trabecular bone (BV/TV) and the height of the cortical passage of the implants were determined. The correlation between the different parameters was calculated statistically. The mean resonance frequency analysis values (maxilla 6130.4+/-363.2 Hz, mandible 6424.5+/-236.2 Hz) did not correlate with the Periotest measurements (maxilla 13.1+/-7.2, mandible -7.9+/-2.1) and peak insertion torque values (maxilla 23.8+/-2.2 N cm, mandible 45.0+/-7.9 N cm) (P=0.280 and 0.193, respectively). Again, no correlations could be found between the resonance frequency analysis, the bone mineral density (maxilla 259.2+/-124.8 mg/cm(3), mandible 349.8+/-113.3 mg/cm3), BV/TV (maxilla 19.7+/-8.8%, mandible 34.3+/-6.0%) and the TBPf (maxilla 2.39+/-1.46 mm-1, mandible -0.84+/-3.27 mm-1) (P=0.140 and 0.602, respectively). However, the resonance frequency analysis values did correlate with bone-to-implant contact of the oral aspect of the specimens (maxilla 12.6+/-6.0%, mandible 35.1+/-5.1%) and with the height of the crestal cortical bone penetrated by the implants in the oral aspect of the implant sites (maxilla 2.1+/-0.7 mm, mandible 5.1+/-3.7 mm) (P=0.024 and 0.002, respectively). The Periotest values showed a correlation with the height of the crestal cortical bone penetrated by the implants in the buccal aspect of the implant sites (maxilla 2.5+/-1.2 mm, mandible 5.4+/-1.2 mm) (P=0.015). The resonance frequency analysis revealed more correlations to the histomorphometric parameters than the Periotest measurements. However, it seems that the noninvasive determination of implant stability has to be improved in order to give a more comprehensive prediction of the bone characteristics of the implant site.

Bone contact, growth, and density around immediately loaded implants in the mandible of mini pigs.

The aim of the study was to compare the bone mineral apposition rate (BMAR) of immediately loaded implants with an unloaded control during the early healing phase in the partially edentulous mandible. In seven mini pigs, three premolars and the first molar were removed in the left mandible. Three months later, five implants were installed. Four implants received a fixed provisional restoration and were loaded immediately. The most anterior implant was used as unloaded control. Polychromatic fluorescence labelling was performed to assess the BMAR. After 4 months, the implants were retrieved together with the adjacent bone. Histological specimens were prepared and subjected to a fluorescence microscopic and histomorphometric analysis. Two provisional restorations were found partially lost at the end of the observation period. One implant that had lost the splinting fixation showed soft connective tissue healing. The BMAR did not differ statistically significantly between loaded and unloaded implants and within the single groups during the observation period (BMARloaded days 14-42=1.8+/-0.2 microm/d, BMARloaded days 42-70=1.8+/-0.1 microm/d, BMARloaded days 70-98=1.6+/-0.1 microm/d, pBMARloaded days 14-42/42-70/70-98 =0.156, BMARunloaded days 14-42=1.7+/-0.1 microm/d, BMARunloaded days 42-70=1.8+/-0.2 microm/d, BMARunloaded days 70-98=1.6+/-0.4 microm/d, pBMARunloaded days 14-42/42-70/70-98=0.368, pBMARloaded/unloaded days 14-42=0.073, pBMARloaded/unloaded days 42-70=0.098, pBMARloaded/unloaded days 70-98=0.262). Four months after implant placement, the bone-to-implant contact was 77.8+/-17.3% for the loaded and 78.0+/-5.8% for the unloaded implants (P=0.753). Immediate loading does not affect the bone mineral apposition rate when compared with unloaded implants. Rigid splinting seems to be the crucial factor for implant success. Uncontrolled masticatory forces can cause failure after partial loss of the provisional restoration.

Open reading frame UL26 of human cytomegalovirus encodes a novel tegument protein that contains a strong transcriptional activation domain.

A selection strategy, the activator trap, was used in order to identify genes of human cytomegalovirus (HCMV) that encode strong transcriptional activation domains in mammalian cells. This approach is based on the isolation of activation domains from a GAL4 fusion library by means of selective plasmid replication, which is mediated in transfected cells by a GAL4-inducible T antigen gene. With this screening strategy, we were able to isolate two types of plasmids encoding transactivating fusion proteins from a library of random HCMV DNA inserts. One plasmid contained the exon 3 of the HCMV IE-1/2 gene region, which has previously been identified as a strong transcriptional activation domain. In the second type of plasmid, the open reading frame (ORF) UL26 of HCMV was fused to the GAL4 DNA-binding domain. By quantitative RNA mapping using S1 nuclease analysis, we were able to classify UL26 as a strong enhancer-type activation domain with no apparent homology to characterized transcriptional activators. Western blot analysis with a specific polyclonal antibody raised against a prokaryotic UL26 fusion protein revealed that two protein isoforms of 21 and 27 kDa are derived from the UL26 ORF in both infected and transfected cells. Both protein isoforms, which arise via alternative usage of two in-frame translational start codons, showed a nuclear localization and could be detected as early as 6 h after infection of primary human fibroblasts. By performing Western blot analysis with purified virions combined with fractionation experiments, we provide evidence that pUL26 is a novel tegument protein of HCMV that is imported during viral infection. Furthermore, we observed transactivation of the HCMV major immediate-early enhancer-promoter by pUL26, whereas several early and late promoters were not affected. Our data suggest that pUL26 is a novel tegument protein of HCMV with a strong transcriptional activation domain that could play an important role during initiation of the viral replicative cycle.