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

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

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

Outcomes and Tendon Integrity After Arthroscopic Treatment for Articular-Sided Partial-Thickness Tears of the Supraspinatus Tendon: Results at Minimum 2-Year Follow-Up

Background: The best surgical treatment option for symptomatic moderate- to high-grade articular-sided partial-thickness rotator cuff tears (PTRCTs) is still controversial. Purpose/Hypothesis: The purpose of this study was to evaluate patient-reported and clinical outcomes and tendon integrity after arthroscopic debridement or repair for PTRCTs at a minimum of 2 years postoperatively. We hypothesized that the overall outcomes would be positive, showing pain relief, good shoulder function, and high tendon integrity. Study Design: Cohort study; Level of evidence, 3. Methods: We evaluated 30 patients (16 men, 14 women; mean age, 51 years) who underwent arthroscopic treatment for symptomatic PTRCTs (Ellman grades 2 and 3). Debridement was performed in 15 patients, and arthroscopic tendon repair was performed in the remaining 15 patients. Patients completed the Constant score; American Shoulder and Elbow Surgeons (ASES) shoulder score; Western Ontario Rotator Cuff Index; Simple Shoulder Test; and visual analog scale (VAS) for pain, function, and satisfaction. In addition, patients were examined clinically (range of motion, impingement tests, rotator cuff tests, and tests for the long head of the biceps tendon), and morphologic assessment of rotator cuff integrity was performed using direct magnetic resonance arthrography and was classified according to Sugaya. Results: The mean follow-up period was 55 months. The patient-reported outcome measures showed high patient satisfaction, reduction in persistent pain, and good shoulder function. Linear regression analysis showed that the debridement group had significantly better results on the Constant (bias-corrected and accelerated [BCa] 95% CI, 4.20-26.30), ASES (BCa 95% CI, 5.24-39.26), and VAS (pain: BCa 95% CI, 0.13-3.62; function: BCa 95% CI, 1.04-4.84; satisfaction: BCa 95% CI, 0.14-6.28) scores than did the repair group. At follow-up, there was no significant difference between the groups in clinical testing results. Good supraspinatus tendon integrity was seen in most patients: Sugaya classification grade 1 in 13 patients, grade 2 in 11 patients, and grade 3 in 6 patients. Conclusion: Midterm results after arthroscopic debridement and repair for PTRCTs showed high patient satisfaction, good shoulder function, and high tendon integrity for both procedures. Patients who underwent arthroscopic debridement had higher Constant, ASES, and VAS scores compared with patients who underwent tendon repair

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

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

Time-dependent screening of a positive charge distribution in metals: Excitons on an ultra-short time scale

Experiments determining the lifetime of excited electrons in crystalline copper reveal states which cannot be interpreted as Bloch states [S. Ogawa {\it et al.}, Phys. Rev. B {\bf 55}, 10869 (1997)]. In this article we propose a model which explains these states as transient excitonic states in metals. The physical background of transient excitons is the finite time a system needs to react to an external perturbation, in other words, the time which is needed to build up a polarization cloud. This process can be probed with modern ultra-short laser pulses. We calculate the time-dependent density-response function within the jellium model and for real Cu. From this knowledge it is possible within linear response theory to calculate the time needed to screen a positive charge distribution and -- on top of this -- to determine excitonic binding energies. Our results lead to the interpretation of the experimentally detected states as transient excitonic states.Comment: 24 pages, 9 figures, to appear in Phys. Rev. B, Nov. 15, 2000, issue 2