Periodontal ligament on pulp-free root slices ­ an in vitro model for early tooth (re)integration. An exploratory study

Reintegration of freshly extracted healthy teeth is very successful, most likely due to the regenerative capacity of their roots' residual periodontal ligament (PDL). We hypothesised that in vitro cultures of the consecutive slices of a sectioned root will represent the entity of PDL cell types engaged in tooth-sided reintegration. For confirmation, apex and pulp from human premolars were removed and roots cut into 6 to 9 about 1 mm thick slices. These were immobilised to separate wells and cultured for 20 days, under daily inspection for the initiation of cell outgrowth (ICO). ICO and the distribution of vital slices along, the cell growth around as well as the expansion of outgrown cells off the root axes after 20 days were displayed for each tooth as 3D-like profiles. Of the 81 slices from 11 teeth, 55 showed ICO; 64% within one week and 96% within two weeks. Such dynamics compare to the early (day 2­5) and the intermediate (day 9­14) integration phase reported for PDL cells in vivo. Experimental phase contrast images of a single slice showed at ICO few fibroblast- and stem/progenitor- like cells. Four and five days later at the same site cells had grown in number and changed in shape and space over time. This exploratory study indicates that in root slice cultures PDL cells behave similarly to those during reintegration in vivo. It favours our hypothesis, which is now to be adequately verified. Eventually, the model may facilitate the identification of outgrowing cells and cellular changes over time, as triggered by tissue rupture. It may further allow for emulating cellular interactions between the root surface and alveolar bone or engineered constructs, natural or engineered scaffolds, or other tissue, in an in vivo-like situation.

A sunscreen's labeled sun protection factor may overestimate protection at temperate latitudes: a human in vivo study.

We lack comparative data on sunscreens with comparable sun protection factors (SPFs), but with different levels of UVA protection, especially against cumulative erythema from repeated suberythemal exposure. Thus, we compared the protection from cumulative sunburn with two sunscreens labeled SPF 6, but with different UVR-absorbing properties, one that absorbs the UVB waveband and the other that absorbs UVB+UVA wavebands. We simulated sunlight typical of temperate latitudes to expose skin daily to suberythemal doses for 13 consecutive days. The study population consisted of eight fair-skinned sun-sensitive healthy young adults. Erythema was assessed by eye and objectively, and the SPF of each sunscreen was modeled with changes in solar UVR with time of day and latitude. The broad-spectrum sunscreen gave much better protection against cumulative erythema than the UVB sunscreen. The explanation for this is that UVA makes a greater contribution toward sunburn at temperate latitudes than under the laboratory conditions in which SPF is tested and assigned. The data support the current trend toward broad-spectrum sunscreen protection. They also show that labeled SPF is much more reliable with broad-spectrum sunscreens because SPF with primarily UVB sunscreens is dependent on time of day and latitude.

Assessment of the photo-degradation of UV-filters and radical-induced peroxidation in cosmetic sunscreen formulations.

Photo-instability of common UV-filters is a well documented phenomenon. This study develops a method for concomitant measurement of photostability and photo-induced ROS generation in cosmetic formulations. Oil-in-water formulations containing three common UV filters (OMC, BMDBM, EHT), individually or combined, were further supplemented with phosphatidylcholine and exposed to UVA. All filters show spectral decrease after UVA exposure. OMC and EHT do not induce significant lipid-peroxidation (as measured by TBARS production) while BMDBM does. In the latter case, this is reduced when BMDBM is combined with OMC but not with EHT. Neither OMC nor EHT stabilize BMDBM with respect to loss of absorbance. ROS-generation assessed via TBARS formation was supported by EPR experiments. The UV-induced changes in UV-filter performance, as monitored in the model formulations and in commercial sunscreens, demonstrate that this is a simple and effective method for stability assessment of sunscreen filters under conditions of use.

Hes1, a new target for interleukin 1beta in chondrocytes.

OBJECTIVES: To investigate the effects of interleukin 1beta (IL1beta) treatment on the Notch1/Hes1 pathway in chondrocytes in vitro. METHODS: Mouse articular chondrocytes in primary culture were challenged with IL1beta, alone or combined with Notch1 and IL1beta pathway inhibitors. Notch1 and Hes1 expressions were investigated by immunocytochemistry, western blot and real-time quantitative (q)PCR. IL1beta-responsive genes were assessed by real-time qPCR and a specific siRNA against Hes1 was used to identify Hes1 target genes. RESULTS: Notch1 labelling remained nuclear and stable in intensity irrespective of treatment, suggesting a steady state activation of this pathway in our model. IL1beta transiently increased Hes1 mRNA (2.5-fold) and protein expression in treated versus naive chondrocytes. Hes1 mRNA level then decreased below control and its cyclic pattern of expression was lost. This was associated with nuclear translocation of the cytoplasmic Hes1 protein. IL1beta induced increase in Hes1 mRNA was transcriptional, occurred through nuclear factor (NF)kappaB activation and appeared to be associated with downregulation by its own protein. Hes1 induction was insensitive to the gamma-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), which suggested its independence from novel Notch1 activation. Hes1 expression was efficiently silenced by a specific siRNA. This experiment revealed that Hes1 did not mediate IL1beta-induced downregulation of Sox9, type II collagen and aggrecan transcription but mediated IL1beta induction of matrix metalloproteinase (MMP)13 and ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5). The Hes1-related repressor Hey1 was expressed at a very low level and was not inducible by IL1beta. CONCLUSION: Hes1 is a novel IL1beta target gene in chondrocytes which influences a discrete subset of genes linked to cartilage matrix remodelling and/or degradation.

Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy.

The pathological changes in osteoarthritis--a degenerative joint disease prevalent among older people--start at the molecular scale and spread to the higher levels of the architecture of articular cartilage to cause progressive and irreversible structural and functional damage. At present, there are no treatments to cure or attenuate the degradation of cartilage. Early detection and the ability to monitor the progression of osteoarthritis are therefore important for developing effective therapies. Here, we show that indentation-type atomic force microscopy can monitor age-related morphological and biomechanical changes in the hips of normal and osteoarthritic mice. Early damage in the cartilage of osteoarthritic patients undergoing hip or knee replacements could similarly be detected using this method. Changes due to aging and osteoarthritis are clearly depicted at the nanometre scale well before morphological changes can be observed using current diagnostic methods. Indentation-type atomic force microscopy may potentially be developed into a minimally invasive arthroscopic tool to diagnose the early onset of osteoarthritis in situ.

Surface-induced modulation of human mesenchymal progenitor cells. An in vitro model for early implant integration.

Clinical experience indicates that the surface architecture of dental implants has an important impact on their integration. This has been related to the finding that differentially treated substrates can modulate the expression of osteogenic markers in various bone-related cell lines and primary cells. Here, we investigated the influence of surface architecture on the differentiation of human mesenchymal progenitor cells (HMPC) from adult bone marrow, i. e. the cells likely involved in initial bone synthesis at the bone-implant interface. Cells were seeded on machine surfaced (MS) or sandblasted/acid etched (SE) titanium discs in agarose-coated dishes, and on polystyrene (PS) controls. On all substrates cell densities did not change between days 7 and 14. Cell numbers were higher on SE, likely due to increased attachment to the rougher material. Alkaline phosphatase activity (ALP) was similar on all substrates, whereas mRNA expression of bone sialoprotein (BSP) at day 14 was about tenfold higher on SE (p < 0.05%). The SE-related increase of BSP in progenitor cells indicates an earlier differentiation of immigrated cells and could thus explain earlier implant integration and shorter time to functional loading observed in the clinic. The in vitro model and BSP quantification could be used to screen for changes in osteogenic cell differentiation induced by specific implant surfaces, with potential relevance on the prediction of bone-implant integration.

UV-Filter combinations under UV-A exposure: concomitant quantification of over-all spectral stability and molecular integrity.

Efficient UV-absorbing molecules are designed to protect against UV-light over-exposure. However, upon UV exposure they may change spectral performance or act as photooxidants via generation of free radicals and reactive oxygen species alone or in combination with others. Therefore, information about their photointegrity which comprises (i) stable absorbance and (ii) absence of UV-induced molecular breakdown, is fundamental. In this study, seven commonly used UV-A, UV-B and broad spectrum UV-AB filters and their combinations, were incorporated into phosphatidylcholine (PC)-based liposomes and exposed to UV-A (275 kJ/m(2)). Spectral integrity, evaluated by recording UV-absorbance spectra of the extracted filter molecules and molecular integrity, assessed indirectly via quantification of UV-A induced PC peroxidation, revealed that spectral stability of filter molecules alone or in combination (e.g. trianilino p-carboxyethylhexyl triazine, EHT plus ethylhexyl p-methoxycinnamate, OMC) does not necessarily imply absence of radical generation and that spectral lability does not necessarily have to lead to radical generation and molecular decay (e.g. OMC). This simple system capable of discriminating between essentially photostable and photounstable UV-absorbing molecules alone and in mixtures, might be useful for determining the influence of UV-protection as well as of photostability of UV-absorbers with regard to UV-induced genotoxic/phototoxic and photoageing-related, radical-based processes.

Dynamic elastic modulus of porcine articular cartilage determined at two different levels of tissue organization by indentation-type atomic force microscopy.

Cartilage stiffness was measured ex vivo at the micrometer and nanometer scales to explore structure-mechanical property relationships at smaller scales than has been done previously. A method was developed to measure the dynamic elastic modulus, |E(*)|, in compression by indentation-type atomic force microscopy (IT AFM). Spherical indenter tips (radius = approximately 2.5 microm) and sharp pyramidal tips (radius = approximately 20 nm) were employed to probe micrometer-scale and nanometer-scale response, respectively. |E(*)| values were obtained at 3 Hz from 1024 unloading response curves recorded at a given location on subsurface cartilage from porcine femoral condyles. With the microsphere tips, the average modulus was approximately 2.6 MPa, in agreement with available millimeter-scale data, whereas with the sharp pyramidal tips, it was typically 100-fold lower. In contrast to cartilage, measurements made on agarose gels, a much more molecularly amorphous biomaterial, resulted in the same average modulus for both indentation tips. From results of AFM imaging of cartilage, the micrometer-scale spherical tips resolved no fine structure except some chondrocytes, whereas the nanometer-scale pyramidal tips resolved individual collagen fibers and their 67-nm axial repeat distance. These results suggest that the spherical AFM tip is large enough to measure the aggregate dynamic elastic modulus of cartilage, whereas the sharp AFM tip depicts the elastic properties of its fine structure. Additional measurements of cartilage stiffness following enzyme action revealed that elastase digestion of the collagen moiety lowered the modulus at the micrometer scale. In contrast, digestion of the proteoglycans moiety by cathepsin D had little effect on |E(*)| at the micrometer scale, but yielded a clear stiffening at the nanometer scale. Thus, cartilage compressive stiffness is different at the nanometer scale compared to the overall structural stiffness measured at the micrometer and larger scales because of the fine nanometer-scale structure, and enzyme-induced structural changes can affect this scale-dependent stiffness differently.

Immunofluorescence and confocal laser scanning microscopy of chronic myeloproliferative disorders on archival formaldehyde-fixed bone marrow.

Spatial analysis of the histoarchitecture and photographic documentation at high resolution are the principal advantages of confocal laser scanning microscopy (CLSM) over conventional fluorescence microscopy (CFM) if combined with appropriate software. Restrictions for the use of CFM and CLSM, on the other hand, include nonspecific background fluorescence, fading of photolabile fluorochromes, and both tissue-specific and fixation-induced autofluorescence. Most of those shortcomings can now be avoided. Autofluorescence, the most limiting factor of high-resolution CLSM, was recently controlled also for paraffin sections of archival formaldehyde-fixed tissues. This allowed the present study on cytoskeletal fibers and extracellular matrix proteins in both neoplastic cells of myeloproliferative disorders and in medullary stromal cells using CLSM under proper autofluorescence control. By multiple fluorescence labeling, we found that the intracellular smooth muscle alpha-actin (SMA) fibers and the two extracellular adhesive matrix proteins tenascin and fibronectin vary in their presence in stromal and/or myeloid cells according to the degree of bone marrow fibrosis in chronic myeloproliferative disorders (CMPDs). CLSM offers further insight in our attempts to understand a complex interplay between the two cellular compartments.

Repetitive versus monomeric antigen presentation: direct visualization of antibody affinity and specificity.

The concept of presenting antigens in a repetitive array to obtain high titers of specific antibodies is increasingly applied by using surface-engineered viruses or bacterial envelopes as novel vaccines. A case for this concept was made 25 years ago, when producing high-titer antisera against ordered arrays of gp23, the major capsid protein of bacteriophage T4 (Aebi et al., Proc. Natl. Acad. Sci. USA, 74 (1977) 5514-5518). In view of the current interest in this concept we thought it useful to employ this system to directly visualize the dependence of antibody affinity and specificity on antigen presentation. We compared antibodies raised against T4 polyheads, a tubular variant of the bacteriophage T4 capsid, which have gp23 hexamers arranged in a crystalline lattice (gp23(repetitive)), with those raised against the hexameric gp23 subunits (gp23(monomeric)). The labeling patterns of Fab-fragments prepared from these antibodies when bound to polyheads were determined by electron microscopy and image enhancement. Anti-gp23(repetitive) bound in a monospecific, stoichiometric fashion to the gp23 units constituting the polyhead surface. In contrast, anti-gp23(monomeric) decorated the polyhead surface randomly and with a 40-fold lower occupancy. These results concur with the difference in titers established by ELISA for the antisera against the repetitively displayed form of antigen (anti-gp23(repetitive)) and the randomly presented antigen (gp23(monomeric)), and they constitute a compelling visual documentation of the concept of repetitive antigen presentation to elicite a serotype-like immune response.