Characterization of primary chondrocytes harvested from hips with femoroacetabular impingement.

OBJECTIVE: Acetabular chondral lesions are common in patients with femoroacetabular impingement (FAI) syndrome. The aim of this study was (1) to evaluate the proliferation potential of primary human chondrocytes (hC) derived from both acetabular and femoral site and (2) to validate cellular differentiation during three-dimensional (3D) cultivation as a prerequisite for autologous matrix-assisted cartilage regeneration of the hip joint. METHODS: hC were isolated from cartilage samples obtained from N = 6 patients during offset reconstruction. Proteoglycan content was assessed by Safranin-O staining. Proliferation and cell viability were quantified by microscopic cell counting and Trypan Blue exclusion. Messenger ribonucleic acid (mRNA) expression levels of collagen type 1 and 2, aggrecan (ACAN), and interleukin-1ß (IL-1ß) genes were assessed upon monolayer cultivation, after 48 h/4-10°C - transport simulation and after 14 days of 3D hydrogel cultivation. RESULTS: Primary hC from acetabular and femoral damaged sites were viable. No significant intergroup differences were observed concerning cell viability (>95%) after monolayer cultivation and transport simulation. Harvest yields from acetabular and femoral cartilage samples were comparable to that known from knee joints (mean ± standard deviation (SD), 13.4 × 10(6) ± 5 × 10(6) cells per culture vs 20 × 10(6) cells). Redifferentiation was induced during 3D hydrogel cultivation as observed by increased levels of collagen II (1000-fold) and ACAN (10-fold) gene vs monolayer cultivation (P < 0.001). CONCLUSION: hC derived from damaged acetabular and femoral site are qualified for autologous matrix-assisted cartilage transplantation paving the way for cell-based cartilage regeneration in FAI patients.

[Cell-based therapy to treat stress urinary incontinence: which cell type at what cost?]. / Zellbasierte Therapie der Belastungsinkontinenz: Welche Zelle zu welchen Kosten?

In Germany, 6-8 million woman and men suffer urinary incontinence, which represents 12.5 % of the population. It is estimated that by the middle of this century, it will increase to almost 30 %. The primary reason will be primarily related to the aging population but also to patient awareness and seeking a solution. In addition to the cost which is covered by the health insurance, the patient will spend more than half a billion euro/year out-of-pocket, not to mention the social stigma associated with urinary incontinence. The current common treatment options are symptomatic but do not restore functionality. One option might be tissue engineering or stem cell therapy. This article describes the likelihood that this therapy will change the approach in treating stress urinary incontinence. Boundaries and legal aspects are highlighted as well as approximated cost. These treatment costs might be currently higher than the standard treatment options, but the investment to reduce these costs are paid indirectly by society.

Genomic chondrocyte culture profiling by array-CGH, interphase-FISH and RT-PCR.

OBJECTIVE: In vitro expansion is an important step to acquire sufficient cells in human tissue engineering technologies. The high number of chondrocytes needed for human articular cartilage implants requires in vitro expansion of the primary cells, bearing a theoretical risk of in vitro induced changes in the genomes. To gain more insights into this situation, model cultures were prepared and analyzed. DESIGN: 25 chondrocyte cell DNA samples from nine donors were analyzed by array comparative genomic hybridization (aCGH) on whole genome level and 28 chondrocyte cell samples from 16 individuals were analyzed by fluorescence in situ hybridization (FISH) on single cell level. The expanded cells were further characterized upon the chondrocytic mRNA phenotype by reverse-transciptase polymerase chain reaction (RT-PCR). RESULTS: The molecular karyotyping results revealed autosomal stability, but all male samples analyzed by aCGH displayed a variable loss of the Y-chromosome. These data were confirmed by FISH-experiments and suggest an age dependant effect toward the loss of the Y-chromosome in cultured chondrocytes. RT-PCR data for the mRNAs from collagen types I, II, and aggrecan and the pro-inflammatory cytokine interleukin-1ß (IL-1ß) did not reveal any correlation of transcriptional activity in cultures with Y-chromosome losses, nor were there statistically significant differences between cells from female and male donors. CONCLUSIONS: While cells of male origin may suffer from an age-related loss of the Y-chromosome, there was no indication of a functional impairment. The data suggest some caution toward applying proliferative steps when considering chondrocytes from elderly male patients for tissue engineering approaches.

In vitro phenotypic modulation of chondrocytes from knees of patients with osteochondritis dissecans: implications for chondrocyte implantation procedures.

We attempted to characterise the biological quality and regenerative potential of chondrocytes in osteochondritis dissecans (OCD). Dissected fragments from ten patients with OCD of the knee (mean age 27.8 years (16 to 49)) were harvested at arthroscopy. A sample of cartilage from the intercondylar notch was taken from the same joint and from the notch of ten patients with a traumatic cartilage defect (mean age 31.6 years (19 to 52)). Chondrocytes were extracted and subsequently cultured. Collagen types 1, 2, and 10 mRNA were quantified by polymerase chain reaction. Compared with the notch chondrocytes, cells from the dissecate expressed similar levels of collagen types 1 and 2 mRNA. The level of collagen type 10 message was 50 times lower after cell culture, indicating a loss of hypertrophic cells or genes. The high viability, retained capacity to differentiate and metabolic activity of the extracted cells suggests preservation of the intrinsic repair capability of these dissecates. Molecular analysis indicated a phenotypic modulation of the expanded dissecate chondrocytes towards a normal phenotype. Our findings suggest that cartilage taken from the dissecate can be reasonably used as a cell source for chondrocyte implantation procedures.

Widespread hypomethylation occurs early and synergizes with gene amplification during esophageal carcinogenesis.

Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined "CpG islands," but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1) in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery.

Suppression of adverse angiogenesis in an albumin-based hydrogel for articular cartilage and intervertebral disc regeneration.

An injectable polyethylene glycol-crosslinked albumin gel (AG) supplemented with hyaluronic acid as a matrix for autologous chondrocyte implantation was evaluated with regard to its impact on angiogenesis. Healthy articular cartilage and intervertebral discs (IVD) are devoid of blood vessels, whereas pathological blood vessel formation augments degeneration of both theses tissues. In contrast to human endothelial cells, primary human articular chondrocytes encapsulated in the AG retained their viability. Endothelial cells did not adhere to the gel surface to a significant extent nor did they proliferate in vitro. The AG did not release any diffusible toxic components. Contrary to Matrigel employed as positive control, the AG prevented endothelial chemoinvasion in Transwell filter assays even in the presence of a chemotactic gradient of vascular endothelial growth factor. In ovo, the AG exhibited a barrier function for blood vessels of the chick chorioallantoic membrane. Subcutaneous implantation of human IVD chondrocytes enclosed in the albumin gel into immunodeficient mice revealed a complete lack of angiogenesis inside the gel after two weeks. At the same time, the IVD chondrocytes within the gel remained vital and displayed a characteristic gene expression pattern as judged from aggrecan, collagen type I and type II mRNA levels. In summary, aiming at articular cartilage and IVD regeneration the albumin gel promises to be a beneficial implant matrix for chondrocytes simultaneously exhibiting non-permissive properties for adverse endothelial cells.

Gelatin-based haemostyptic Spongostan as a possible three-dimensional scaffold for a chondrocyte matrix?: an experimental study with bovine chondrocytes.

The gelatin-based haemostyptic compound Spongostan was tested as a three-dimensional (3D) chondrocyte matrix in an in vitro model for autologous chondrocyte transplantation using cells harvested from bovine knees. In a control experiment of monolayer cultures, the proliferation or de-differentiation of bovine chondrocytes was either not or only marginally influenced by the presence of Spongostan (0.3 mg/ml). In monolayers and 3-D Minusheet culture chambers, the cartilage-specific differentiation markers aggrecan and type-II collagen were ubiquitously present in a cell-associated fashion and in the pericellular matrix. The Minusheet cultures usually showed a markedly higher mRNA expression than monolayer cultures irrespective of whether Spongostan had been present or not during culture. Although the de-differentiation marker type-I collagen was also present, the ratio of type-I to type-II collagen or aggrecan to type-I collagen remained higher in Minusheet 3-D cultures than in monolayer cultures irrespective of whether Spongostan had been included in or excluded from the monolayer cultures. The concentration of GAG in Minusheet cultures reached its maximum after 14 days with a mean of 0.83 +/- 0.8 microg/10(6) cells; mean +/-, SEM, but remained considerably lower than in monolayer cultures with/without Spongostan. Our results suggest that Spongostan is in principle suitable as a 3-D chondrocyte matrix, as demonstrated in Minusheet chambers, in particular for a culture period of 14 days. Clinically, differentiating effects on chondrocytes, simple handling and optimal formability may render Spongostan an attractive 3-D scaffold for autologous chondrocyte transplantation.

Phase-sensitive X-ray imaging of synovial joints.

OBJECTIVE: To test the efficacy of phase-sensitive X-ray imaging for intact synovial joints, whereby refraction effects, along with the attenuation of conventional radiography, can be exploited. DESIGN: Intact cadaveric human knee joints were imaged, in the computed tomographic mode, using an analyzer-based X-ray system at the National Synchrotron Light Source, Brookhaven National Laboratory. A collimated fan beam of 51 keV X-rays was prepared by a silicon [1,1,1 reflection] double-crystal monochromator. The X-ray beam transmitted through the specimen was imaged after diffraction in the vertical plane by means of the analyzer crystal with the analyzer crystal tuned to its half-reflectivity point (6.5 microrad). A two-dimensional filtered backprojection (FBP) algorithm was used for reconstructing transverse slices of images. RESULTS: The resulting images demonstrate simultaneous soft tissue and bone contrast at a level that has not been achieved previously. Identifiable structures include articular cartilage, cruciate ligaments, loose connective tissue, menisci, and chondrocalcinosis. CONCLUSION: Phase-sensitive X-ray imaging using an analyzer-based system renders exceptionally high quality images of soft and hard tissues within synovial joints, with high contrast and resolution, and thus holds promise for the eventual clinical utility.

Expression of ion channels of the TRP family in articular chondrocytes from osteoarthritic patients: changes between native and in vitro propagated chondrocytes.

The maintenance of a differentiated chondrocyte phenotype is influenced by several factors of which signal transduction of extracellular stimuli through the cell membrane is of major interest. One important group of membrane-bound proteins which are involved in transmembrane signal transduction are ion channels. Human articular chondrocytes were obtained from osteoarthritic femoral condyles. Cells were released from the surrounding matrix and cultivated under standard conditions. We investigated gene expression of 12 members of the TRP ion channel family of freshly prepared (passage 0; P0) and in vitro propagated human articular chondrocytes (passage 2; P2) using conventional and real-time PCR (RT-PCR). In addition, the protein appearance of four TRP channels was demonstrated by immunofluorescence and western blotting. Chondrocyte differentiation was monitored by quantification of collagen type-II, type-I, and aggrecan gene expression. By conventional PCR, 8 channels could be detected, of which some displayed a heterogeneous PCR pattern. RT-PCR quantification revealed that TRPC1 was expressed on the same level in P0 and P2 chondrocytes while gene expression of TRPC3 and TRPC6 was elevated in passage 2 cells. TRPM5, TRPM7, and TRPV1 displayed an enhanced gene expression in freshly isolated chondrocytes. Immunofluorescence signal intensity of all four investigated TRP proteins was consistent with the corresponding gene expression data. In the present study, a correlation between the appearance of some members of the TRP ion channel family and the state of de-differentiation of osteoarthritic articular chondrocytes was shown. A possible direct involvement in the process of chondrocyte de-differentiation has to be investigated in further studies.

Respiratory Deleted in Malignant Brain Tumours 1 (DMBT1) levels increase during lung maturation and infection.

Deleted in Malignant Brain Tumours 1 (DMBT1) is a secreted scavenger receptor cysteine-rich protein that binds and aggregates various bacteria and viruses in vitro. Studies in adults have shown that DMBT1 is expressed mainly by mucosal epithelia and glands, in particular within the respiratory tract, and plays a role in innate immune defence. We hypothesized that respiratory DMBT1 levels may be influenced by various developmental and clinical factors such as maturity, age and bacterial infection. DMBT1 levels were studied in 205 tracheal aspirate samples of 82 ventilated preterm and full-term infants by enzyme-linked immunosorbent assay. Possible effects of various clinical parameters were tested by multiple regression analysis. DMBT1 levels increased significantly with lung maturity (P < 0.0001 for both gestational and postnatal age) and in small-for-gestational-age infants (P = 0.0179). An increase of respiratory DMBT1 levels was detected in neonatal infections (P < 0.0001). These results were supported by Western blotting. Immunohistochemical analyses of archived newborn lung sections (n = 17) demonstrated high concentrations of DMBT1 in lungs of neonates with bacterial infections. Our data show that preterm infants are able to up-regulate DMBT1 in infection as an unspecific immune reaction.

DMBT1 as an archetypal link between infection, inflammation, and cancer

Los estudios epidemiológicos y moleculares indican vínculosentre infección, inflamación y cáncer, que parece que convergena nivel molecular en mecanismos asociados con la inmunidadinnata. Aquí, presentamos un resumen del conocimientosobre la proteína secretada "scavenger receptor cysteine-rich(SRCR)" Deleted in Malignant Brain Tumors 1 (DMBT1), tambiénconocida como glicoproteína-340 o aglutinina de la saliva. DMBT1se expresa diferencialmente en varios tipos de cáncer, en muchoscasos disminuyendo su regulación. Como proteína secretada allumen, tiene funciones en la defensa innata contra los patógenos,y la regulación de la inflamación. En contraste, podría inducir ladiferenciación epitelial y de células madre, como proteína de lamatriz extracelular. Su amplia respuesta a estímulos patofisiológicossugiere un papel general en la protección celular y tisular,probablemente uniendo la defensa contra patógenos y la regulaciónde la respuesta inflamatoria a procesos regenerativos. Existensimilitudes muy interesantes con las funciones de otras proteínasSRCR presentes en metazoos primitivos, como las esponjasy los erizos de mar. Esto sugiere que sus diferentes funcionespodrían basarse en un principio antiguo y simple, que seríala mediación diferencial de adhesión y anti-adhesión. De manerasimilar a las vías de señalización de NF-κB, que también estánreguladas indirectamente por DMBT1, el conocimiento actualindica que DMBT1 no sólo podría tener funciones de prevenciónde enfermedad, sino probablemente también funciones generadorasde enfermedad. En resumen, DMBT1 podría representarun paradigma del vínculo arquetípico entre infección, inflamación,y cáncer. La comprensión de su complejo modo de acciónpromete nuevos puntos de vista sobre el origen y las bases molecularesde las grandes enfermedades humanas

Epidemiological and molecular studies have pointed to linksbetween infection, inflammation and cancer, which appear to convergeat the molecular level in mechanisms associated with innateimmunity. Here, the present knowledge about the secreted scavengerreceptor cysteine-rich (SRCR) protein Deleted in MalignantBrain Tumors 1 (DMBT1), also known as glycoprotein-340or salivary agglutinin, is summarized. DMBT1 is differentially expressed in various cancer types with most of these displayinga downregulation. As a lumenally secreted protein, it exerts functionsin innate pathogen defense and the regulation of inflammation.By contrast, it may trigger epithelial and stem cell differentiationas an extracellular matrix protein. Its broad responsivenessto pathophysiological stimuli points to a general role incell and tissue protection, which possibly is best circumscribedby linking pathogen defense and regulation of the inflammatoryresponse to regenerative processes. Compelling similaritiesto the functions of SRCR proteins in primitive metazoa such assponges and sea urchins exist, which support that its various functionsmay rely on an ancient and simple principle, i.e. the differentialmediation of adhesion and anti-adhesion. Similar to NF-κB signaling pathways, which are also indirectly regulated byDMBT1, the present state of the art indicates that DMBT1 not onlycould exert disease-preventing, but probably also disease-promotingfunctions. Taken together, DMBT1 may represent a paradigmfor an archetypal link between infection, inflammation, andcancer. Understanding its complex mode of action promises novelinsights into the origin and the molecular basis of major humandiseases

[Intra-articular pain during gonarthrosis: a case report about an extremely increased cathepsin expression in chondrocytes]. / Intraartikulärer Schmerz bei Gonarthrose: Ein Fallbericht über eine stark erhöhte Expression der Cathepsine in Chondrozyten.

AIM AND BACKGROUND: Cysteine proteases as cathepsins K and L as well as matrix metalloproteases are considered to be basically involved in collagen turnover. Degenerative joint diseases such as gonarthrosis are characterised by massive cartilage degradation mediated by increased activities of these proteases. These enzymes are, therefore, interesting targets for the treatment of painful arthritic joint diseases. The aim of these studies was to reconsider the hypothesis that cathepsin activities are enhanced in patients suffering from osteoarthritis. METHOD AND RESULTS: We report on a 69-year-old female suffering from severe pain due to predominant retropatellar arthrosis. The clinical symptoms of this patient did not significantly differ from that of 30 other patients who were involved in this study. All patients undergone an endoprosthetic knee joint replacement. During the operation we harvested cartilage probes and isolated the chondrocytes from the joint cartilage for determination of the mRNA and the activities of cathepsins B, H, K and L. Compared to chondrocytes isolated from the control patients we found the activity of cysteine proteases to be extremely enhanced in chondrocytes of this patient. Moreover, the concentration of cystatin c, an endogenous inhibitor of cathepsins, was not detectable. CONCLUSION: The results raise doubts on the predominant role of cysteine proteases in severe cartilage destruction.

[Ankle chondrocytes are more resistant to Interleukin-1 than chondrocytes derived from the knee]. / Sprunggelenkchondrozyten besitzen eine höhere Interleukin-1-Resistenz als Kniegelenkchondrozyten.

BACKGROUND: The incidence of degenerative changes and osteoarthritis is lower in the ankle than in the knee joints. This cannot be explained exclusively with differences in anatomy and biomechanical properties of these two synovial joints. Previous studies have indicated distinct differences in the biochemical composition of the extracellular matrix of articular cartilage from knee and ankle joints. The aim of this study was to identify potential metabolic differences between knee and ankle joint chondrocytes using isolated cells to distinguish the secondary effects of the resident extracellular matrix from the primary matrix-independent effects of cellular differentiation. METHOD: Isolated knee and ankle chondrocytes from the same human donor were cultured in alginate beads and subsequently exposed to a three-day pulse of the catabolic cytokine interleukin-1 (IL-1) as a model of an inflammatory episode. The metabolism of proteoglycans (PG's) was analyzed as expressed changes in 35S-sulfate incorporation into glycosaminoglycans (GAG's). RESULTS: The presence of IL-1 induced an inhibition of PG synthesis in knee and ankle articular chondrocytes. The 50% inhibitory concentration (IC50) of IL-1 was about 5 times lower for knee than for ankle chondrocytes. CONCLUSION: Ankle chondrocytes are more resistant to IL-1 induced inhibition of PG synthesis than chondrocytes from the knee.

Molecular characterisation of non-absorptive and absorptive enterocytes in human small intestine.

BACKGROUND AND AIMS: Perturbation of differentiation of the crypt-villus axis of the human small intestine is associated with several intestinal disorders of clinical importance. At present, differentiation of small intestinal enterocytes in the crypt-villus axis is not well characterised. SUBJECTS AND METHODS: Expression profiling of microdissected enterocytes lining the upper part of crypts or the middle of villi was performed using the Affymetrix X3P arrays and several methods for confirmation. RESULTS: A total of 978 differentially expressed sequences representing 778 unique UniGene IDs were found and categorised into four functional groups. In enterocytes lining the upper part of crypts, cell cycle promoting genes and transcription/translation related genes were predominantly expressed, whereas in enterocytes lining the middle of villi, high expression of cell cycle inhibiting genes, metabolism related genes, and vesicle/transport related genes was found. CONCLUSION: Two types of enterocytes were dissected at the molecular level, the non-absorptive enterocyte located in the upper part of crypts and the absorptive enterocyte found in the middle of villi. These data improve our knowledge about the physiology of the crypt-villus architecture in human small intestine and provide new insights into pathophysiological phenomena, such as villus atrophy, which is clinically important.

Qualitative evaluation of titanium implant integration into bone by diffraction enhanced imaging.

Diffraction enhanced imaging (DEI) uses refraction of x-rays at edges, which allows pronounced visualization of material borders and rejects scattering which often obscures edges and blurs images. Here, the first evidence is presented that, using DEI, a destruction-free evaluation of the quality of integration of metal implants into bone is possible. Experiments were performed in rabbits and sheep with model implants to investigate the option for DEI as a tool in implant research. The results obtained from DEI were compared to conventional histology obtained from the specimens. DE images allow the identification of the quality of ingrowth of bone into the hydroxyapatite layer of the implant. Incomplete integration of the implant with a remaining gap of less than 0.3 mm caused the presence of a highly refractive edge at the implant/bone border. In contrast, implants with bone fully grown onto the surface did not display a refractive signal. Therefore, the refractive signal could be utilized to diagnose implant healing and/or loosening.

Collagen degradation products modulate matrix metalloproteinase expression in cultured articular chondrocytes.

Destruction of collagen within osteoarthritic cartilage depends in part on collagen-degrading matrix metalloproteases (MMP). Degradative fragments of type II collagen (Col II) occur in normal and in osteoarthritic cartilage, and may contribute to regulation of matrix turnover by interfering with normal cell-matrix communication pathways. Therefore, the effects of different types of collagen fragments on mRNA and protein levels of MMP-2, MMP-3, MMP-9, and MMP-13 in cultured bovine articular knee chondrocytes and explants were examined. Primary chondrocytes and explants were incubated with fragments from whole cartilage collagen matrix (Colf) and from purified type II collagen (Col2f), or with a synthetic 29-mer peptide representing the amino-terminal domain of type II collagen (Ntelo). Gelatin zymography revealed increases of proMMP-2, a shift towards active MMP-2 and increases in proMMP-9, depending on the type of fragment. In situ hybridization of cartilage sections displayed MMP-3 mRNA in virtually all cells. Moderate to strong increases in MMP-2, MMP-3, MMP-9, and MMP-13 mRNA levels were detected by quantitative PCR. The results demonstrate stimulating effects of collagen fragments on both mRNA and/or protein from MMP -2, -3, -9, and -13, and suggest a novel mechanism of MMP induction and activation that includes a particular role for N-telo in controlling catabolic pathways of matrix turnover.

Osteointegration of hydroxyapatite-titanium implants coated with nonglycosylated recombinant human bone morphogenetic protein-2 (BMP-2) in aged sheep.

Osteointegration of metal implants into aged organisms can be severely compromised due to reduced healing capacity of bone, lack of precursor cells for new bone formation, or osteoporosis. Here, we report on successful implant healing in a novel model of aged sheep in the presence of nonglycosylated bone morphogenetic protein 2 (BMP-2). Ewes of 8 to 12 years with significant radiologic and histologic signs of osteoporosis and adipocytic bone marrow received a cylindrical hydroxyapatite-titanium implant of 12 x 10 mm. BMP-2 has been produced as a bacterial recombinant fusion protein with maltose-binding protein and in vitro generation of mature BMP-2 by renaturation and proteolytic cleavage. A BMP-2 inhibition ELISA was developed to measure the in vitro release kinetics of bioactive human BMP-2 from immersed solid implant materials by using Escherichia coli expressed and biotinylated recombinant human BMP-2 receptor IA extracellular domain (ALK-3 ECD). The implants were placed laterally below both tibial plateaus, with the left leg implant carrying 380 microg BMP-2. Both implant types became integrated within the following 20 weeks. The control implant only integrated at the cortical bone, and little new bone formation was found within the pre-existing trabecular bone or the marrow cavity. Marrow fat tissue was partially replaced by unspecific connective tissue. In contrast, BMP-2-coated implants initiated significant new bone formation, initially in trabecular arrangements to be replaced by cortical-like bone after 20 weeks. The new bone was oriented towards the cylinder. Highly viable bone marrow appeared and filled the lacunar structures of the new bone. In mechanical tests, the BMP-2-coated implants displayed in average 50% higher stability. This animal model provided first evidence that application of nonglycosylated BMP-2 coated on solid implants may foster bone healing and regeneration even in aged-compromised individuals.

DMBT1 expression and glycosylation during the adenoma-carcinoma sequence in colorectal cancer.

The gene DMBT1 (deleted in malignant brain tumour-1) has been proposed to play a role in brain and epithelial cancer, but shows unusual features for a classical tumour-suppressor gene. On the one hand, DMBT1 has been linked to mucosal protection, whereas, on the other, it potentially plays a role in epithelial differentiation. Thus its function in a particular tissue is of mechanistic importance for its role in cancer. Because the former function requires secretion to the lumen and the latter function may depend on its presence in the extracellular matrix, we decided to investigate DMBT1 expression, location and its mode of secretion during malignant transformation in colorectal cancer. Using human colorectal PC/AA cell lines and tissue sections from individual patients, we have examined the expression of DMBT1 and its glycosylation in the adenoma-carcinoma sequence leading to the adenocarcinoma phenotype.

[Indications and implementation of recommendations of the working group "Tissue Regeneration and Tissue Substitutes" for autologous chondrocyte transplantation (ACT)]. / Indikations- und Durchführungsempfehlungen der Arbeitsgemeinschaft "Geweberegeneration und Gewebeersatz" zur Autologen Chondrozyten-Transplantation (ACT).

For the treatment of full-thickness articular cartilage lesions of the knee joint, as a result of trauma or osteochondritis dissecans, a variety of biological reconstruction techniques have been developed. Different studies, some of which were performed as randomised, prospective clinical studies, showed that the autologous chondrocyte transplantation (ACT) provides the most satisfying and reliable method of cartilage reconstruction in the adult when applied to defects exceeding 4 cm (2). Based on these results, ACT seems to be of economic benefit, as the risk of developing osteoarthritis correlates significantly with the size of the cartilage defect, when not treated properly and in time. Surveying the studies on basic scientific aspects of ACT, cartilage defect animal models and clinical studies, it can be concluded that clinical results of ACT depend on a variety of factors. In this review, published by the joined advisory board of the German Societies of Traumatology (DGU) and Orthopaedic Surgery (DGOOC), we summarize the current knowledge available and the state of the art concerning ACT. Especially we discuss the advantages of different procedures, methods for treating knee cartilage defects and factors that influence the outcome of the different treatment regimens, with the aim to develop guidelines for the correct indication and application of the ACT.

DMBT1 expression is down-regulated in breast cancer.

BACKGROUND: We studied the expression of DMBT1 (deleted in malignant brain tumor 1), a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle. METHODS: Sections from 17 benign lesions and 55 carcinomas were immunostained with anti DMBT1 antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions. DMBT1 gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients. RESULTS: Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments. DMBT1 protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas (3/55 positive carcinomas versus 33/42 positive normal/hyperplastic epithelia; p = 0.0001). In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive. CONCLUSIONS: The redistribution and up-regulation of DMBT1 in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation.