Gentherapie in der Orthopädie

Gentherapie in der Orthopädie wird intensiv im Rahmen verschiedener vererbbarer und nichtvererbbarer orthopädischer Krankheiten untersucht. Der experimentelle Fortschritt auf diesem Gebiet ist durch die Komplexität von Vektorauswahl und -herstellung, Gentransfertechnik, Applikationsweg in geeigneten Tier-Modellen sowie dem Nachweis auf struktureller und funktioneller Ebene gekennzeichnet. Die ersten klinischen Studien zur Gentherapie der chronischen Polyarthritis haben bereits ihre praktische Durchführbarkeit demonstriert. Es ist wahrscheinlich, dass genbasierte Verfahren zur Erweiterung und Verbesserung bestehender orthopädisch-chirurgischer Therapien führen werden. Schlüsselwörter Gentherapie · Gentransfer · Orthopädie · Klinische Studie

Enhanced expression of the central survival of motor neuron (SMN) protein during the pathogenesis of osteoarthritis

The identification of new components implicated in the pathogenesis of osteoarthritis (OA) might improve our understanding of the disease process. Here, we investigated the levels of the survival of motor neuron (SMN) expression in OA cartilage considering the fundamental role of the SMN protein in cell survival and its involvement in other stress-associated pathologies. We report that SMN expression is up-regulated in human OA compared with normal cartilage, showing a strong correlation with the disease severity, a result confirmed in vivo in an experimental model of the disease. We further show that the prominent inflammatory cytokines (IL-1β, TNF-α) are critical inducers of SMN expression. This is in marked contrast with the reported impaired levels of SMN in spinal muscular atrophy, a single inherited neuromuscular disorder characterized by mutations in the smn gene whereas OA is a complex disease with multiple aetiologies. While the precise functions of SMN during OA remain to be elucidated, the conclusions of this study shed light on a novel pathophysiological pathway involved in the progression of OA, potentially offering new targets for therapy

Tissue-Engineering zur Knorpelreparatur verbessert durch Gentransfer : aktuelle Forschungsergebnisse und Literaturübersicht

Tissue-Engineering ist die Züchtung von präformierten Geweben aus dreidimensionalen Zellverbänden. Werden Chondrozyten mit Trägersubstanzen assoziiert und im Bioreaktor kultiviert, so entwickelt sich Knorpelgewebe. Wir wollten verstehen, wie der humane insulinartige Wachstumsfaktor I (IGFI) die Chondrogenese in diesen Neoknorpelkonstrukten reguliert. Hierzu wurden IGF-Itransfizierte Chondrozyten in Geweben aus Polyglykolsäurefasern ausgesät und in rotierenden Bioreaktoren kultiviert. Die Transgenexpression nach lipidbasiertem Gentransfer hielt mehr als 5 Wochen im Neoknorpel an. Nach 4-wöchiger Kultivierung in rotierenden Bioreaktoren besitzen die IGF-I-Konstrukte deutlich mehr Chondrozyten und Proteoglykane als Kontrollkonstrukte aus nichtmodifizierten oder mit dem Markergen lacZ modifizierten Konstrukten. Diese strukturellen Verbesserungen resultierten in signifikant verbesserten biomechanischen Eigenschaften der IGF-I-Konstrukte. Transplantation dieser Neoknorpelkonstrukte in osteochondrale Defekte im Kaninchenmodell verbesserte die strukturellen Eigenschaften des Reparaturgewebes im Vergleich zu lacZ-Konstrukten. Diese Studien belegen erstmals die Möglichkeit einer auf der Kombination von Gentransfer und Tissue-Engineering basierenden molekularen Therapie von Knorpeldefekten

Analysis of novel nonviral gene transfer systems for gene delivery to cells of the musculoskeletal system

The aim of the present study was to evaluate the efficacy of novel nonviral gene delivery systems in cells of musculoskeletal origin. Primary cultures of lapine skeletal muscle cells, lapine articular chondrocytes, human cells from fibrous dysplasia and cell lines established from human osteosarcoma (SAOS-2), chondrosarcoma (CS-1), murine skeletal myoblasts (L8) and fibroblasts (NIH 3T3)were transfected with the P. pyralis luc or the E. coli lacZ genes using Nanofectin 1 and 2, Superfect, JetPEI, Gene-Jammer, Effectene, TransPass D2, FuGENE 6, Lipofectamine 2000, Dreamfect, Metafectene, Escort III, and calcium phosphate. Maximal transfection efficiency in lapine skeletal muscle cells was of 60.8 ± 21.2% using Dreamfect, 38.9 ± 5.0% in articular chondrocytes using Gene Jammer, 5.2 ± 8.0% in human cells from fibrous dysplasia using Lipofectamine 2000, 12.7 ± 16.2% in SAOS-2 cells using FuGENE 6, 29.9 ± 3.5% in CS-1 cells using Lipofectamine 2000, 70.7 ± 8.6% in L8 cells using FuGENE 6, and 48.9 ± 13.0% in NIH 3T3 cells using Metafectene. When the cells were transfected with a human IGF-I gene, significant amounts of the IGF-I protein were secreted. These results indicate that relatively high levels of transfection can be achieved using novel nonviral gene transfer methods. Keywords Gene transfer - Nonviral - Musculoskeletal cells - IGF-I - Transfection efficienc

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

Objective. Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. Methods. We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. Results. Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. Conclusion. These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans

A corpus-based study of Spanish L2 mispronunciations by Japanese speakers

In a companion paper (Carranza et al.) submitted to this conference we discuss the importance of collecting specific L1-L2 speech corpora for the sake of developing effective Computer Assisted Pronunciation Training (CAPT) programs. In this paper we examine this point more deeply by reporting on a study that was aimed at compiling and analysing such a corpus to draw up an inventory of recurrent pronunciation errors to be addressed in a CAPT application that makes use of Automatic Speech Recognition (ASR). In particular we discuss some of the results obtained in the analyses of this corpus and some of the methodological issues we had to deal with. The corpus features 8.9 hours of spontaneous, semi-spontaneous and read speech recorded from 20 Japanese students of Spanish L2. The speech data was segmented and transcribed at the orthographic, canonical-phonemic and narrow-phonetic level using Praat software [1]. We adopted the SAMPA phonemic inventory for the phonemic transcription adapted to Spanish [2] and added 11 new symbols and 7 diacritics taken from X-SAMPA [3] for the narrow-phonetic transcription. Non linguistic phenomena and incidents were also annotated with XML tags in independent tiers. Standards for transcribing and annotating non-native spontaneous speech ([4], [5]), as well as the error encoding system used in the project will be addressed. Up to 13410 errors were segmented, aligned with the canonical-phonemic tier and the narrow-phonetic tier, and annotated following an encoding system that specifies the type of error (substitutions, insertion and deletion), the affected phone and the preceding and following phonemic contexts where the error occurred. We then carried out additional analyses to check the accuracy of the transcriptions by asking two other annotators to transcribe a subset of the speech material. We calculated intertranscriber agreement coefficients. The data was automatically recovered by Praat scripts and statistically analyzed with R. The resulting frequency ratios obtained for the most frequent errors and the most frequent contexts of appearance were statistically tested to determine their significance values. We report on the analyses of the combined annotations and draw up an inventory of errors that should be addressed in the training. We then consider how ASR can be employed to properly detect these errors. Furthermore, we suggest possible exercises that may be included in the training to improve the errors identified

The effect of insulin-loaded chitosan particle-aggregated scaffolds in chondrogenic differentiation

Osteochondral defect repair requires a tissue engineering approach that aims at mimicking the physiological properties and structure of two different tissues (cartilage and bone) using a scaffold–cell construct. One ideal approach would be to engineer in vitro a hybrid material using a single-cell source. For that purpose, the scaffold should be able to provide the adequate biochemical cues to promote the selective but simultaneous differentiation of both tissues. In this work, attention was paid primarily to the chondrogenic differentiation by focusing on the development of polymeric systems that provide biomolecules release to induce chondrogenic differentiation. For that, different formulations of insulin-loaded chitosan particle–aggregated scaffolds were developed as a potential model system for cartilage and osteochondral tissue engineering applications using insulin as a potent bioactive substance known to induce chondrogenic differentiation. The insulin encapsulation efficiency was shown to be high with values of 70.37!0.8%, 84.26!1.76%, and 87.23!1.58% for loadings of 0.05%, 0.5%, and 5%, respectively. The in vitro release profiles were assessed in physiological conditions mimicking the cell culture procedures and quantified by Micro-BCA! protein assay. Different release profiles were obtained that showed to be dependent on the initial insulin-loading percentage. Further, the effect on prechondrogenic ATDC5 cells was investigated for periods up to 4 weeks by studying the influence of these release systems on cell morphology, DNA and glycosaminoglycan content, histology, and gene expression of collagen types I and II, Sox-9, and aggrecan assessed by real-time polymerase chain reaction. When compared with control conditions (unloaded scaffolds cultured with the standard chondrogenic-inducing medium), insulin-loaded scaffolds upregulated the Sox-9 and aggrecan expression after 4 weeks of culture. From the overall results, it is reasonable to conclude that the developed loaded scaffolds when seeded with ATDC5 can provide biochemical cues for chondrogenic differentiation. Among the tested formulations, the higher insulin-loaded system (5%) was the most effective in promoting chondrogenic differentiation.The authors would like to acknowledge the Portuguese Foundation for Science and Technology for the Ph. D. Grant to Patricia B. Malafaya (SFRH/BD/11155/2002). This work was partially supported and carried out under the scope of the European STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and European NoE EXPERTISSUES (NMP3CT-2004-500283). The authors also like to acknowledge the Life and Health Sciences Research Institute (ICVS), University of Minho, for the use of their facilities, namely, to Luis Martins for histological sections slicing and H&E stain processing

Effect of transforming growth factor-ß1 (TGF-ß1) released from a scaffold on chondrogenesis in an osteochondral defect model in the rabbit

Articular cartilage repair might be stimulated by the controlled delivery of therapeutic factors. We tested the hypotheses whether TGF-ß1 can be released from a polymeric scaffold over a prolonged period of time in vitro and whether its transplantation modulates cartilage repair in vivo. Unloaded control or TGF-ß1 poly(ether-ester) copolymeric scaffolds were applied to osteochondral defects in the knee joints of rabbits. In vitro, a cumulative dose of 9 ng TGF-ß1 was released over 4 weeks. In vivo, there were no adverse effects on the synovial membrane. Defects treated with TGF-ß1 scaffolds showed no significant difference in individual parameters of chondrogenesis and in the average cartilage repair score after 3 weeks. There was a trend towards a smaller area (42.5 %) of the repair tissue that stained positive for safranin O in defects receiving TGF-ß1 scaffolds. The data indicate that TGF-ß1 is released from emulsioncoated scaffolds over a prolonged period of time in vitro and that application of these scaffolds does not significantly modulate cartilage repair after 3 weeks in vivo. Future studies need to address the importance of TGF-ß1 dose and release rate to modulate chondrogenesis

Local stimulation of articular cartilage repair by transplantation of encapsulated chondrocytes overexpressing human fibroblast growth factor 2 (FGF-2) in vivo

Background Defects of articular cartilage are an unsolved problem in orthopaedics. In the present study, we tested the hypothesis that gene transfer of human fibroblast growth factor 2 (FGF-2) via transplantation of encapsulated genetically modified articular chondrocytes stimulates chondrogenesis in cartilage defects in vivo. Methods Lapine articular chondrocytes overexpressing a lacZ or a human FGF-2 gene sequence were encapsulated in alginate and further characterized. The resulting lacZ or FGF-2 spheres were applied to cartilage defects in the knee joints of rabbits. In vivo, cartilage repair was assessed qualitatively and quantitatively at 3 and 14 weeks after implantation. Results In vitro, bioactive FGF-2 was secreted, leading to a significant increase in the cell numbers in FGF-2 spheres. In vivo, FGF-2 continued to be expressed for at least 3 weeks without leading to differences in FGF-2 concentrations in the synovial fluid between treatment groups. Histological analysis revealed no adverse pathologic effects on the synovial membrane at any time point. FGF-2 gene transfer enhanced type II collagen expression and individual parameters of chondrogenesis, such as the cell morphology and architecture of the new tissue. Overall articular cartilage repair was significantly improved at both time points in vivo. Conclusions The data suggest that localized overexpression of FGF-2 enhances the repair of cartilage defects via stimulation of chondrogenesis, without adverse effects on the synovial membrane. These results may lead to the development of safe gene-based therapies for human articular cartilage defects

Clinical potential and challenges of using genetically modified cells for articular cartilage repair

Articular cartilage defects do not regenerate. Transplantation of autologous articular chondrocytes, which is clinically being performed since several decades, laid the foundation for the transplantation of genetically modified cells, which may serve the dual role of providing a cell population capable of chondrogenesis and an additional stimulus for targeted articular cartilage repair. Experimental data generated so far have shown that genetically modified articular chondrocytes and mesenchymal stem cells (MSC) allow for sustained transgene expression when transplanted into articular cartilage defects in vivo. Overexpression of therapeutic factors enhances the structural features of the cartilaginous repair tissue. Combined overexpression of genes with complementary mechanisms of action is also feasible, holding promises for further enhancement of articular cartilage repair. Significant benefits have been also observed in preclinical animal models that are, in principle, more appropriate to the clinical situation. Finally, there is convincing proof of concept based on a phase I clinical gene therapy study in which transduced fibroblasts were injected into the metacarpophalangeal joints of patients without adverse events. To realize the full clinical potential of this approach, issues that need to be addressed include its safety, the choice of the ideal gene vector system allowing for a long-term transgene expression, the identification of the optimal therapeutic gene(s), the transplantation without or with supportive biomaterials, and the establishment of the optimal dose of modified cells. As safe techniques for generating genetically engineered articular chondrocytes and MSCs are available, they may eventually represent new avenues for improved cell-based therapies for articular cartilage repair. This, in turn, may provide an important step toward the unanswered question of articular cartilage regeneration