[Organoids for the advancement of intraoperative diagnostic procedures]. / Organoide zur Weiterentwicklung der intraoperativen Diagnostik.

In the context of cancer surgery, there is always a trade-off between oncological safety and preservation of function. This is especially true in pelvic surgery due to the close relationship to the pelvic floor muscles, blood supply and nerves. Currently, risk models, preoperative imaging, the surgeon's assessment, and the intraoperative frozen section serve as the basis for decision-making. New imaging techniques and standardization in frozen section have significantly improved this in recent years. However, limitations remain due to time delays as well as more difficult correct anatomical assignment in the follow-up. Alternative intraoperative techniques may overcome this limitation in the future. Patient-derived organoids have emerged as an important new research vehicle in recent years. They are based on tumor stem cells that, under special culture conditions, form three-dimensional replicas of the original tissue. This makes them ideally suited for testing individual system therapies but also as a validation technique for new intraoperative diagnostic procedures. The Research Training Group 2543/I, which is funded by the German Research Foundation, is researching the potential of new diagnostic methods in an interdisciplinary team regarding validation in addition to intraoperative frozen sections.

Loss of spatial organization and destruction of the pericellular matrix in early osteoarthritis in vivo and in a novel in vitro methodology.

OBJECTIVES: Current repair procedures for articular cartilage (AC) cannot restore the tissue's original form and function because neither changes in its architectural blueprint throughout life nor the respective biological understanding is fully available. We asked whether two unique elements of human cartilage architecture, the chondrocyte-surrounding pericellular matrix (PCM) and the superficial chondrocyte spatial organization (SCSO) beneath the articular surface (AS) are congenital, stable or dynamic throughout life. We hypothesized that inducing chondrocyte proliferation in vitro impairs organization and PCM and induces an advanced osteoarthritis (OA)-like structural phenotype of human cartilage. METHODS: We recorded propidium-iodine-stained fetal and adult cartilage explants, arranged stages of organization into a sequence, and created a lifetime-summarizing SCSO model. To replicate the OA-associated dynamics revealed by our model, and to test our hypothesis, we transduced specifically early OA-explants with hFGF-2 for inducing proliferation. The PCM was examined using immuno- and auto-fluorescence, multiphoton second-harmonic-generation (SHG), and scanning electron microscopy (SEM). RESULTS: Spatial organization evolved from fetal homogeneity, peaked with adult string-like arrangements, but was completely lost in OA. Loss of organization included PCM perforation (local micro-fibrillar collagen intensity decrease) and destruction [regional collagen type VI (CollVI) signal weakness or absence]. Importantly, both loss of organization and PCM destruction were successfully recapitulated in FGF-2-transduced explants. CONCLUSION: Induced proliferation of spatially characterized early OA-chondrocytes within standardized explants recapitulated the full range of loss of SCSO and PCM destruction, introducing a novel in vitro methodology. This methodology induces a structural phenotype of human cartilage that is similar to advanced OA and potentially of significance and utility.

[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.

[Stem cell therapy and tissue engineering in regenerative urology]. / Stammzelltherapie und "Tissue Engineering" in der regenerativen Urologie.

BACKGROUND: So far there is no clinically established, effective tissue engineering therapy for dysfunction or defects of the lower urinary tract. The concentration of experimental data, initial clinical studies and individual case reports underlines that stem cell treatment for bladder storage and voiding problems, erectile dysfunction and other urothelial defects of the lower urinary tract could close the gap between individualized therapy and potential biomedical applications. RESULTS: As a result of fundamental research work over the last decade a characterization of various stem cell populations and evaluation of different urological therapy options could be performed. Thereby, aspects of optimal administration, migration, secretion of bioactive factors and stage of differentiation of stem cells with respect to an improved efficiency of treatment were investigated. Because successful tissue regeneration depends on angiogenesis and innervation, particular attention was paid to these important factors. CONCLUSIONS: Various clinical indications for stem cell treatment and tissue reconstruction that may be required after radical prostatectomy, such as stress urinary incontinence, urethral reconstruction and erectile dysfunction have materialized and are currently being verified in preclinical studies and phase I trials.

Transforming growth factor beta1 and laminin-111 cooperate in the induction of interleukin-16 expression in synovial fibroblasts from patients with rheumatoid arthritis.

OBJECTIVES: In synovial tissues of patients with rheumatoid arthritis (RA), strong expression of laminins and integrins co-localises with increased expression of inflammatory cytokines. Synovial fibroblasts (SF) contribute to the pathogenesis of RA through increased expression of cytokines and chemoattractant factors, one of which is interleukin-16 (IL16). A study was undertaken to investigate the regulatory pathways of IL16 in SF from patients with RA (RA-SF) and osteoarthritis (OA-SF). METHODS: SF were seeded in laminin-coated flasks and activated by the addition of cytokines. The expression of IL16 was investigated by quantitative RT-PCR, immunoblotting and ELISA; its biological activity was determined by a cell migration assay. Cell-matrix interactions were investigated by cell binding and attachment assays. Relevant intracellular signalling pathways were studied by immunoblotting and with pharmacological blocking reagents. RESULTS: Stimulation of SF with transforming growth factor beta(1)(TGF-beta(1)) and growth on laminin-111 (LM-111) significantly increased the expression of IL16. Binding to LM-111 induced significantly more IL16 mRNA in RA-SF than in OA-SF (p<0.05). The IL16 cytokine was detected in supernatants of TGF-beta(1)-activated and in LM-111+TGF-beta(1)-activated RA-SF (38 to 62 pg/ml), but not in supernatants of OA-SF. This IL16 regulation involved p38MAPK, ERK1/2 and SMAD2 signalling, but not NFkappaB. CONCLUSIONS: Binding of RA-SF to LM-111 in the presence of TGF-beta(1) triggers a significant IL16 response and thus may contribute to the infiltration of CD4+ lymphocytes into synovial tissues. This mode of IL16 induction represents a novel pathway leading to IL16 production in RA-SF but not in OA-SF, which operates independently of NFkappaB signalling.

Hypoxia reduces the inhibitory effect of IL-1beta on chondrogenic differentiation of FCS-free expanded MSC.

OBJECTIVE: Mesenchymal stromal cells (MSC) are a promising tool for tissue engineering of the intervertebral disc (ID). The IDs are characterized by hypoxia and, after degeneration, by an inflammatory environment as well. We therefore investigated the effects of inflammation induced with interleukin (IL)-1beta and of hypoxia (2% O(2)) on the chondrogenic differentiation of MSC. METHODS: Bone-marrow-derived MSC (bmMSC) were cultured in a fetal-calf-serum-free medium and characterized according to the minimal criteria for multipotent MSC. Chondrogenic differentiation of MSC was induced following standard protocols, under hypoxic conditions, with or without IL-1beta supplementation. After 28 days of differentiation, micromasses were analyzed by histochemical staining and immunohistochemistry and by determining the mRNA level of chondrogenic marker genes utilizing quantitative RT-PCR. RESULTS: Micromasses differentiated under IL-1beta supplementation are smaller and express less extracellular matrix (ECM) protein. Micromasses differentiated under hypoxia appear larger in size, display a denser ECM and express marker genes comparable to controls. The combination of hypoxia and IL-1beta supplementation improved chondrogenesis compared to IL-1beta supplementation alone. Micromasses differentiated under standard conditions served as controls. CONCLUSION: Inflammatory processes inhibit the chondrogenic differentiation of MSC. This may lessen the regenerative potential of MSC in situ. Thus, for the cell therapy of IDs using MSC to be effective it will be necessary to manage the inflammatory conditions in situ. In contrast, hypoxic conditions exert beneficial effects on chondrogenesis and phenotype stability of transplanted MSC, and may improve the quality of the generated ECM.

[Laminin-dependent inflammatory response in synovial fibroblasts of rheumatoid arthritis patients]. / Laminin-modulierte Entzündungsreaktionen in synovialen Fibroblasten von Rheumapatienten.

Elevated expression of matrix-metalloproteinases (MMP) contributes to cartilage destruction in rheumatoid arthritis. We report on a novel pathway of inflammatory activation of synovial fibroblasts that is induced by TGF-beta and laminin (extracellular matrix) and leads to increased expression of the proteases MMP-3 and MMP-10. Neither costimulation by the central inflammatory cytokines TNF-alpha and IL-1beta nor NFkB signalling is needed for this pathway.

Co-activation of synovial fibroblasts by laminin-111 and transforming growth factor-beta induces expression of matrix metalloproteinases 3 and 10 independently of nuclear factor-kappaB.

We showed previously that the attachment of synovial fibroblasts to laminin (LM)-111 in the presence of transforming growth factor-beta induces significant expression of the matrix metalloproteinase (MMP)-3. Here we go on to investigate the regulation of additional MMPs and their specific tissue inhibitors of matrix proteases (TIMPs). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative reverse transcription-polymerase chain reaction. Production of MMPs was monitored by a multiplexed immunoarray. Signal transduction pathways were studied by immunoblotting. Attachment of synovial fibroblasts to LM-111 in the presence of transforming growth factor-beta induced significant increases in MMP-3 mRNA (12.35-fold, p < 0.001) and protein (mean 62 ng/ml, sixfold, p < 0.008) and in expression of MMP-10 mRNA (11.68-fold, p < 0.05) and protein (54 ng/ml, 20-fold, p > or = 0.02). All other TIMPs and MMPs investigated failed to show this LM-111-facilitated transforming growth factor-beta response. No phosphorylation of nuclear factor-kappaB was observed. We conclude that co-stimulation of synovial fibroblasts by LM-111 together with transforming growth factor-beta suffices to induce significant expression of MMP-3 and MMP-10 by synovial fibroblasts and that this induction is independent of nuclear factor-kappaB phosphorylation.

[Expression analysis of different collagens and cytokines in cartilage cells derived from arthrotic hip and knee joints]. / Expressionsanalyse verschiedener Kollagene und Zytokine in Knorpelzellen aus arthrotisch veränderten Hüft- und Kniegelenken.

AIM: Osteoarthritis (OA) is characterized by an irreversible destruction of articular cartilage. This is associated with a multiplicity of factors, causing an increased catabolic metabolism in cartilage. However, the prevalence of the OA is very variable in different joints. Therefore , we conducted a comparative analysis of chondrocytes derived from knee and hip joints with respect to their expression of inflammatory factors, such as IL-1beta, IL-1beta-receptorantagonist, iNOS, components of cartilage matrix (collagen I, II, and VI) as well as vimentin. METHODS: Different cytokines and proteins were detected by immune-histochemical staining of cartilage samples ex vivo. Further, chondrocytes were isolated from OA knee and hip joints, expanded in vitro and gene expression patterns were investigated by quantitative RT-PCR. RESULTS: Chondrocytes from knee and hip joints of OA patients express collagenes I, II and VI, IL-1beta and IL-1beta-RA, iNOS as well as Vimentin. A significant difference in gene expression patterns was not found in chondrocytes from the hip joints versus the knee joint ex vivo or in primary culture cells in vitro. However, in vitro the expression of type I collagen exceeded the expression of type II collagen. The IL-1beta-expression was high ex vivo, remained low during primary culture but was significantly elevated after primary culture in hip chondrocytes. CONCLUSION: Osteoarthritic gene expression patterns in cells derived from hip or knee joints ex vivo and in primary culture were not significantly different. We conclude that the rather frequent occurrence of OA in these joints in comparison to the ankle joint may be associated with a close physiological relation of cells in these joints. However, future studies which will include ankle cartilage must be investigated in further detail.

[Characterisation of human meniscus cells]. / Charakterisierung von humanen Meniskuszellen.

AIM: The meniscus of the human knee joint has an important function for the shock absorption, stability and power transmission from the upper to the lower leg. After meniscus injury often a partial or complete resection is necessary. Only injuries in the outer third may heal spontaneously or upon primary suture due to the vascularisation in these segments. After partial or total meniscectomy osteoarthritis of the knee joint is common in a large number of patients. The goal of our investigations was to establish meniscus cell cultures and to characterise the fibrochondrocytes (meniscus cells) in vitro. METHODS: We examined the expression of different growth factors, cytokines and proteins in human menisci from surgical preparations using immunohistochemistry and RT-PCR analysis. RESULTS: Human meniscus cells express the collagens I , II, III, and VI, the matrix metalloproteinases-1, -2, -3, -8, and -13, BMP-2, and -4, TGFbeta1, VEGF, IGF-I, and -II, FGF-2, endostatin, iNOS, vimentin, TIMP-1, and -2, aggrecan, IL-1beta, IL-6, and IL-18. Staining with the monoclonal antibody AS.02 in all examined cells confirmed their mesenchymal origin. CONCLUSION: New strategies for the treatment of meniscus damage can be derived from these results and further advances for the tissue engineering of meniscus tissue can be obtained.

Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures.

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-beta and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.

[Frequency of terminally differentiated fibroblasts in the synovial membrane of rheumatoid arthritis patients]. / Häufigkeit terminal differenzierter Fibroblasten in der Synovialmembran von Rheumapatienten.

The metabolic activation of synovial fibroblasts (SF) and their expression of matrix degrading enzymes and inflammatory cytokines contributes to the pathology of rheumatoid arthritis (RA). It is remarkable that SF of RA patients do not proliferate at higher rates when compared to SF of other patients, but they are resistant to apotposis inducing signals. The chronic inflammation in RA causes fibrosis of the synovial tissue and fibrosis has been associated with terminal differentiation. Therefore we investigated if there are increased numbers of terminally differentiated fibroblasts in the RA synovium and if there is a correlation between terminal differentiation of SF and increased levels of expression of interleukins and matrix metalloproteinases. We analyzed specimen of four RA patients, two patients with osteoarthritis (OA) and two healthy donors suffering from joint injuries. By use of RT-PCR techniques we examined mRNA expression of two genes in SF which are associated with terminal differentiation, p16INK4a and p21-cip. In addition, we labelled differentiated fibroblasts using the SA-beta-galaktosidase assay and investigated differences in protein expression patterns of factor PIVa and the tropomyosin 1 and 2 molecules. We report that the number of terminally differentiated fibrolasts are not increased in the synovial membrane of RA patients. On the contrary we show that the synovia of the much younger patients has higher levels of terminally differentiated fibroblasts. Consequently, the fibrosis of synovial tissues in RA patients at later stages of disorder is not associated with proliferation and differentiation of the fibroblasts but rather a consequence of chronic inflammation.

Metabolic activation stimulates acid secretion and expression of matrix degrading proteases in human osteoblasts.

BACKGROUND: Both cellular and matrix components of healthy bone are permanently renewed in a balanced homoeostasis. Osteoclastic bone resorption involves the expression of vacuolar-type ATPase proton pumps (vATPase) on the outer cell membrane and the secretion of matrix degrading proteases. Osteoblasts modulate the deposition of bone mineral components and secrete extracellular matrix proteins. OBJECTIVES: To investigate the ability of osteoblasts and osteosarcoma to secrete acid and express matrix degrading proteases upon metabolic activation. To examine also the potential contribution of vATPases to proton secretion expressed on osteoblasts. METHODS: Osteoblasts were isolated from trabecular bone and characterised by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Proton secretion was analysed by a cytosensor microphysiometer. RESULTS: Osteoblasts not only express matrix degrading proteases upon stimulation with tumour necrosis factor or with phorbol ester but they also secrete protons upon activation. Proton secretion by osteoblasts is associated partially with proton pump ATPases. CONCLUSION: These data suggest that, in addition to monocyte derived osteoclasts, cytokine activated mesenchymal osteoblasts and osteosarcoma cells may contribute to the acidic milieu required for bone degradation.

p53 in rheumatoid arthritis synovial fibroblasts at sites of invasion.

OBJECTIVE: To analyse the functional response of p53 in rheumatoid arthritis synovial fibroblasts (RASF) in vitro and in vivo and to investigate whether activation of p53 modulates the destructive process of RASF. METHODS: RASF and controls grown on chamber slides were either directly examined with DO7 anti-p53 antibodies by immunofluorescence or irradiated with 10 Gy x rays and analysed time dependently for the expression of p53. The percentage of positive cells was evaluated by a quantitative scoring system. RASF and normal (N) SF cultured in vitro were co-implanted with human cartilage in SCID mice for 60 days. Consecutively, the invasion score was evaluated, and the number of p53 positive cells was determined at the sites of invasion by immunohistochemistry. In addition, synovial tissues from RA, osteoarthritis, and normal synovia were stained with DO7 antibodies. RESULTS: In vitro the rate of expression of p53 in RASF was low (<5%), but transiently inducible by ionising irradiation (50%). In vitro low p53 expressing RASF disclosed, when invading articular cartilage, a nuclear p53 signal in 20% of the cells, indicating the induction of p53 in a distinct population of RASF during the invasive process. CONCLUSIONS: These data suggest an inductive p53 response at sites of cartilage invasion during the destructive process driven by activated RASF.

[Establishment of an in vitro model for rheumatoid arthritis as test system for therapeutical substances]. / Etablierung eines in vitro Testsystems für die rheumatoide Arthritis zur Testung therapeutisch relevanter Wirkstoffe.

In our Tissue Engineering group a 3D in vitro model for rheumatoid Arthritis (in vitro pannus) was established with the aim to develop a standardized drug-screening test to analyze the effects of drugs and different biological substances. The advanced model consists of chondrocyte pellet cultures interacting with rheumatoid arthritis (RA) synovial cell cultures. To establish interactive 3D co-cultures defined rheumatoid arthritis synovial cell populations were centrifuged directly on chondrocyte pellet cultures. Histochemical stainings during time of co-culture revealed obvious invasion by RA synovial cell populations into the chondrocyte matrix. Gene expression analysis showed a downregulation of collagen type II expression in chondrocytes within 2 weeks after co-culture with RA synovial cells. Those interactive co-cultures allow the study of single cell populations as well as the cellular interactions in this system under in vitro conditions. Thus, the established co-culture model may be suitable for routine screening tests, which can be useful in supplementing animal experiments in basic research and drug testing.

Biocompatibility correlation of polymeric materials using human osteosarcoma cells.

Metal implants are the preferred materials to generate articular prostheses, plates, or bone pegs in orthopedic surgery. Although titanium and titanium alloys show a relatively good biocompatibility, clinical experience revealed that coating of the metallic implant surface may increase the biocompatibility. In a search for optimum bone implant surfaces, we determined polarity and contact angle parameters of a variety of polymers and substances and correlated the findings in a biocompatibility assay using an in vitro bone cell model. We report that an optimum adherence of SAOS-2 cells to such surfaces and a good vitality for polymers are characterized by water-based contact angles of 80 degrees and 20 degrees for advancing and receding probes, respectively.

Metabolic activation stimulates acid production in synovial fibroblasts.

OBJECTIVE: In rheumatoid arthritis (RA), synovial fibroblasts express proteases such as collagenases or cathepsins and inflammatory cytokines at elevated levels and so contribute to the inflammatory degradation process. Extracellular matrix degradation and cathepsin activity is dependent upon the presence of an acidic milieu. We examined whether activated synovial fibroblasts secrete acidic components. METHODS: Synovial fibroblasts were isolated and immortalized to study the mechanisms of metabolic activation. Naïve and immortalized fibroblasts were activated with different cytokines. The responses were investigated by immunoblot to detect Egr-1 and by a cytosensor microphysiometer analysis to evaluate acid secretion. Basic gene expression patterns were investigated in naïve and immortalized cells by RT-PCR analysis. RESULTS: We found RA synovial fibroblasts respond to different cytokines associated with the pathomechanisms of RA including interleukin 1, basic fibroblast growth factor, platelet derived growth factor, and tumor necrosis factor-alpha, with metabolic activation and enhanced secretion of acidic components. In addition, naive and SV40 TAg immortalized fibroblasts rapidly release acidic components after stimulation with phorbol ester or ionomycin as well. CONCLUSION: Activated synovial fibroblasts not only express inflammatory cytokines and matrix degrading proteases that are associated with the pathomechanisms of RA, but upon stimulation may release acidic components that lower pH and consequently enhance cathepsin activity and collagen solubilization.

Expression of sentrin, a novel antiapoptotic molecule, at sites of synovial invasion in rheumatoid arthritis.

OBJECTIVE: Sentrin, a novel antiapoptotic molecule, has been shown to interact with the signal-competent form of Fas/APO-1 and tumor necrosis factor receptor I (TNFRI), and thereby, to protect cells against anti-Fas/APO-1- and TNF-induced cell death. Since reduced apoptosis in the synovial lining is supposed to contribute to synovial hyperplasia in rheumatoid arthritis (RA), we searched for the expression of sentrin-1 messenger RNA (mRNA) in synovium from patients with RA. METHODS: The expression of sentrin-1 mRNA was examined by in situ hybridization on snap-frozen sections of normal and RA synovial tissues as well as on paraffin-embedded RA synovial specimens, including the interface of cartilage-bone and invading synovium. Immunohistochemical double labeling after in situ hybridization was performed to further characterize sentrin-1 mRNA-expressing cells. In addition, quantitative analysis of sentrin-1 mRNA expression in RA synovial fibroblasts (RASF), osteoarthritis synovial fibroblasts (OASF), and normal fibroblasts was performed by quantitative real-time polymerase chain reaction. Expression levels were standardized to the expression of GAPDH. The in vivo maintenance of sentrin expression in RASF aggressively invading human cartilage was explored in the SCID mouse model of RA. RESULTS: A marked expression of sentrin-1 mRNA could be seen in all RA synovial specimens, predominantly in SF of the lining layer and at sites of invasion of RA synovium into cartilage. In normal synovial tissues, no sentrin-1 mRNA was detectable. RASF showed a maximum 32.5-fold (mean +/- SD 14.9 +/- 11.6) increase of sentrin-1 mRNA expression compared with normal fibroblasts and a maximum 31.4-fold (mean +/-SD 14.3 +/- 10.9) increase compared with OASF. When coimplanted with normal human cartilage in the SCID mouse model, invading RASF maintained their sentrin-1 mRNA expression for at least 60 days in vivo. CONCLUSION: The marked expression of sentrin in rheumatoid synovial tissue, but not in normal or OA synovial tissue, may contribute to the modulation of Fas- and TNFR-mediated apoptosis in RA synovium, and thereby extend the lifespan of invasive, cartilage-destructive SF.