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-β2) 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.

Knorpel- und Knochendegeneration stellen ein großes volkswirtschaftliches und individualspezifisches Problem dar, mit deren Lösung sich die Methoden der regenerativen Medizin befassen. Das Ziel dieser Studie war es, den Einfluss von einem chondrogenen Differenzierungsmedium und/oder einer mechanischen Stimulation auf Primärstabilität und morphologischen Aufbau eines osteochondralen Transplantates zu untersuchen. Während unfallchirurgischen Eingriffen wurden stromale Zellen aus dem Knochenmark (BMSCs) des Beckenkamms gewonnen, die isoliert, passagiert und für sieben Tage mithilfe eines u.a. TGF-β2- und IGF-1-haltigen Differenzierungsmediums in zweidimensionaler Kultur chondrogen vordifferenziert wurden. Anschließend erfolgte der Transfer in eine biologische Hybridmatrix (CaReS Gel, Arthrokinetics, Esslingen, Germany und Tutobone, Tutogen Medical GmbH, Neunkirchen a. Br., Germany), die in einer speziell entwickelten Glasapparatur für 24 h durch eine Kombination von Druck- und Zugkräften komprimiert wurde. Die so entstandenen osteochondralen Konstrukte wurden verschiedenen Stimulationsgruppen zugeteilt, die sich in Stimulationsdauer, Stimulationsart (dynamisch im Bioreaktor oder statisch) und Anwesenheit der Wachstumsfaktoren unterschieden. Die Auswertung erfolgte dreigliedrig in Nasschemie (DNA- und GAG-Gehalt), Histologie (immunhistochemische Bestimmung der Matrixbestandteile) und Biomechanik, bei der unter Bedingungen der constraint compression die Steifigkeit errechnet wurde. Histologisch zeigte sich eine homogene und vitale Zellverteilung (Live/Dead Assay: >70% vitale Zellen). Die noch ausstehenden immunhistochemischen Ergebnisse werden genauere Informationen über die Morphologie der Konstrukte liefern. Mit der biomechanischen Auswertung konnte ein statistisch signifikanter Anstieg (p<0,05) der Konstruktsteifigkeit durch die mechanische Stimulation gezeigt werden, während weder durch Veränderung der Stimulationsdauer noch durch Hinzunahme der Wachstumsfaktoren ein signifikanter Einfluss auf die Steifigkeit gezeigt werden konnte. Die statisch stimulierten Konstrukte wiesen nach drei und vier Wochen einen signifikant (p<0,05) höheren Glycosaminoglycan-Gehalt auf als die mechanisch stimulierten Gruppen. Bei der Erstellung eines Protokolls für die Produktion eines osteochondralen Konstruktes mit Hilfe von humanen mesenchymalen Stammzellen in einer Hybridmatrix stellte sich die mechanische Stimulation als vorteilhaft in Hinblick auf die mechanische Stabilität der Konstrukte heraus. Obwohl Wachstumsfaktoren einen bekannten Stellenwert in der chondrogenen Zelldifferenzierung besitzen, konnte in dieser Studie kein signifikanter Einfluss der Hinzunahme von Mediumzusätzen gezeigt werden. Das Herauslassen dieser Faktoren aus dem Protokoll sowie eine Verkürzung der Stimulationsdauer kann diskutiert werden, um Vorteile bezüglich der Kosten und der klinischen Anwendbarkeit zu erzielen.

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. 106 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 0.5 Hz (group B) and static controls (group C). After 24 h, 1, 2, and 3 weeks, we determined cell proliferation (NITS-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 +/- 19.6 x 10(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 in RNA 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 +/- 88.4mg/g protein, 2 weeks: 294.4 +/- 73.3 mg/g protein, 3 weeks: 293.1 +/- 83.6 mg/g protein, p <= 0.03).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. (c) 2008 Elsevier Ltd. All rights reserved.

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.

Matrix metalloproteinases (MMPs) are central to tissue remodelling; however, little is known about the temporal pattern and differential regulation of hepatic MMP expression in the course of chronic human liver disease. Using quantitative reverse transcription-PCR ELISA assays, we studied hepatic mRNA expression of MMP-1, -2, -3, -7, -9, -10, -11, -13 and -14 in patients with chronic hepatitis C and hepatitis C virus-induced end-stage liver cirrhosis and controls. Results were compared with histology, hepatic expression of tissue inhibitor of metalloproteinases (TIMP)-1, -2 and -3, procollagen types I and IV, laminin, and with circulating protein levels of hyaluronate, TIMP-1 and -2 and MMP proenzymes, as measured by ELISA. The impact of the MMP-3(-1171) promoter polymorphism on hepatic MMP-3 expression was analysed. Hepatic mRNA expression data identified differentially regulated groups of MMPs during the course of chronic hepatitis C, showing either steadily increasing mRNA expression with disease progression (MMP-1, -2, -7 and -14) or transiently elevated expression (MMP-9, -11 and -13). The first group closely correlated to the parameters of fibrogenesis. Hepatic MMP-3 expression was unrelated to disease stage, but was determined by the MMP-3(- 1171) promoter polymorphism. In conclusion, MMP expression during the course of chronic hepatitis C appears to be a closely regulated process, with different clusters of coordinately regulated MMP genes being identified.