No effect in combining chondrogenic predifferentiation and mechanical cyclic compression on osteochondral constructs stimulated in a bioreactor.

Traumatic and degenerative osteochondral lesions are a common problem in orthopaedic surgery. The concept of tissue engineering represents the possibility of a promising therapeutical approach. The purpose of this study has been to improve the characteristics of osteochondral grafts consisting of a human certified collagen I-bone hybrid matrix seeded with human bone marrow stromal cells and stimulated in a custom-made biomechanoreactor. This study was undertaken as a follow-up to our prior studies. Based on our established system, we added chondrogenic growth factors (IGF-1 and TGF-beta(2)) and evaluated their effect on chondrogenic differentiation. Constructs were stimulated for 14, 21 and 28 days respectively by different protocols, including cyclic mechanical stimulation, hormonal stimulation or a combination of both. More than 70% of the cells were viable throughout the entire experimental period. Histological analysis revealed a homogeneous distribution of cells in a cartilage-like matrix organization. Immunohistological collagen II staining was positive irrespective of stimulation manner and time. Levels of DNA and glycosaminoglycans, having been normalized to DNA, did not change. Analysis of the biomechanical stiffness after 14 days showed increased stiffness in the hormonally and mechanically stimulated group compared to the static group. Stimulation time did not have a significant influence. The media supplements to foster the quality of the tissue tested here did not show any progress in our system. We conclude that cyclic compression enhances matrix stiffness, but stimulation time should be kept short and growth factors should be left out in this system with regard to clinical applicability and financial concerns.

Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture.

Until now, there has been no in vitro model that duplicates the environment of bone marrow. The purpose of this study was to analyze proliferation and differentiation of human bone marrow stromal cells (hBMSC) under the influence of continuous perfusion and cyclic mechanical loading. hBMSC of seven individuals were harvested, grown in vitro, and combined. 10(6) hBMSC were seeded on a bovine spongiosa disc and incubated in a bioreactor system. Cell culture was continued using three different conditions: Continuous perfusion (group A), 10% cyclic compression at 0.5Hz (group B) and static controls (group C). After 24h, 1, 2, and 3 weeks, we determined cell proliferation (MTS-assay) and osteogenic differentiation (osteocalcin ELISA, Runx2 mRNA). Tenascin-C mRNA was quantified to exclude fibroblastic differentiation. In groups A and B, proliferation was enhanced after 2 weeks (48.6+/-19.6x10(3) (A) and 44.6+/-14.3 x 10(3) cells (B)) and after 3 weeks (46.6+/-15.1 x 10(3) (A) and 44.8+/-10.2 x 10(3) cells (B)) compared with controls (26.3+/-10.8 x 10(3) (2 weeks) and 17.1+/-6.5 x 10(3) cells (3 weeks), p<0.03). Runx2 mRNA was upregulated in both stimulated groups after 1, 2, and 3 weeks compared to control (group A, 1 week: 5.2+/-0.7-fold; p<0.01, 2 weeks: 4.4+/-1.9-fold; p<0.01, 3 weeks: 3.8+/-1.7-fold; p=0.013; group B, 1 week: 3.6+/-1.1-fold, p<0.01, 2 weeks: 4.2+/-2.2-fold, p<0.01; 3 weeks: 5.3+/-2.7-fold, p<0.01). hBMSC stimulated by cyclic compression expressed the highest amount of osteocalcin at all time points (1 week: 294.5+/-88.4 mg/g protein, 2 weeks: 294.4+/-73.3mg/g protein, 3 weeks: 293.1+/-83.6 mg/g protein, p0.03). The main stimulus for cell proliferation in a 3-dimensional culture of hBMSC is continuous perfusion whereas mechanical stimulation fosters osteogenic commitment of hBMSC. This study thereby contributes to the understanding of physical stimuli that influence hBMSC in a 3-dimensional cell culture system.

Oncofoetal fibronectin--a tumour-specific marker in detecting minimal residual disease in differentiated thyroid carcinoma.

Supposedly, thyrocyte-specific transcripts such as thyroglobulin (Tg) and thyroid-stimulating hormone receptor (TSH-R) were proposed to be useful for the diagnosis of circulating tumour cells in patients suffering from differentiated thyroid carcinoma (DTC). However, several research groups reported blood-borne Tg transcripts in healthy individuals. This study determines in particular the origin of Tg mRNA in nucleated blood cells and analyses whether other tumour-associated sequences are absent in leukocytes, but widely expressed in DTC. Therefore, expression analyses for Tg, TSH-R, cytokeratin 19 (CK 19), human telomerase reverse transcriptase (hTERT) and oncofoetal fibronectin (onfFN) were carried out using cDNAs derived from (1) leukocyte fractions, (2) 18 follicular thyroid carcinomas (FTCs) and 48 papillary thyroid carcinomas (PTCs), and (3) leukocytes of two thyrocyte-depleted individuals treated for C-cell carcinoma of the thyroid. Expression of onfFN was additionally analysed by semiquantitative RT-PCR and by quantitative fluorescence-based real-time PCR. Tg and TSH-R expression was demonstrated not only in both athyroid individuals, but in all leukocyte subgroups tested, while hTERT was absent in resting CD4+ cells and only weakly expressed in the CD8+ group. CK 19 was notable in each leukocyte population except for resting CD14(+), as well as for activated and resting CD19+ cells. All blood cell fractions proved negative for onfFN mRNA, whereas its presence in thyroid carcinoma was 78/98% (FTC/PTC). Threshold cycle values were calculated at: porphobilinogen deaminase (PBGD) = 25.95+/-0.73 (FTC)/24.55+/-5.43 (PTC) (P = 0.2878); onfFN = 25.48+/-3.15 (FTC)/21.44+/-3.44 (PTC) (*P = 0.0001). Finally, onfFN transcripts were detected in blood samples of six out of nine patients with known DTC metastases, demonstrating a reliable assay functionality. We propose that real-time RT-PCR of onfFN mRNA is superior to other markers in monitoring minimal residual disease in DTC with regard to both assay sensitivity and specificity.

Novel perylene chromophores obtained by a facile oxidative cyclodehydrogenation route.

New perylene chromophores, phenyl-substituted diindeno[1,2,3-cd:1',2',3'-lm]perylenes 5a,b and 4,4',7,7'-tetraphenyldiacenaphtho[1,2-k:1',2',k']diindeno[1,2,3-cd:1',2',3'-me]perylenes 22a,b, have been synthesized from substituted fluoranthene derivatives 3a,b and 4a,b by means of a surprisingly simple oxidative cyclodehydrogenation reaction. The resulting chromophores, when substituted with alkyl chains at the periphery, show good solubility in organic solvents, and a full characterization of the novel red, green, and blue dyes by field-desorption mass spectrometry, UV/Vis and 1H and 13C NMR spectroscopy becomes possible.

Synthesis and self-assembly of functionalized hexa-peri-hexabenzocoronenes.

Monolayers of hexa-alkyl substituted derivatives of hexa-peri-hexabenzocoronene (HBC) 1b have previously been investigated by scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS). It is expected that different functional groups (electron donating or withdrawing) connected to the aromatic core will influence the packing pattern and possibly the current-voltage characteristics as well. In order to provide suitable model systems, a new synthetic approach to synthesize functionalized HBC derivatives has been developed. This was accomplished by [4 + 2]-cycloaddition of suitably bromo-substituted diphenylacetylenes and 2,3,4,5-tetraarylcyclopenta-2,4-dien-1-ones followed by an oxidative cyclodehydrogenation with iron(III) chloride/nitromethane. Using this strategy three different substitution patterns were synthesized: 2-bromo-5,8,11.14,17-pentadodecylhexa-pecri-hexabenzocoronene (2a), 2,5-dibromo-8,11,14,17-pentadodecylhexa-peri-hexabenzocoronene (2b), and 2,11-dibromo5,8,14,17-pentadodecylhexa-peri-hexa-benzocoronene (2c). These bromo-substituted HBC derivatives were subjected to palladium catalyzed coupling reactions to give donor (alkoxy, amino) as well as acceptor (ester, cyano) substituted derivatives. The self-assembly of these new HBC derivatives was studied in the bulk as well as at an interface. DSC, optical microscopy, and X-ray diffraction revealed the existence of columnar mesophases. The bulk structure in the mesophase is largely insensitive to changes of the substitution pattern; however, in situ scanning tunneling microscopy at the solid-fluid interface between an organic solution of the HBC derivative and highly oriented pyrolytic graphite reveals different packing patterns of the first adsorbed monolayer.