[Tissue engineering for therapy of osteochondral cartilage lesions]. / Tissue Engineering zur Therapie von osteochondralen Knorpelläsionen.

Cartilage defects still represent an unsolved problem in joint surgery. The intrinsic healing capacity of cartilage is insufficient and at best leads to reparative tissue like fibrous cartilage or cartilage like tissue regardless of the therapy applied. Cell culture techniques and generation of tissue specific matrix tread new paths to treat traumatic cartilage lesions. This technology referred to as tissue engineering allows for formation of constructs consisting of chondrocytes capable of production of cartilage specific matrix in combination with three-dimensional cell carriers. Polymers such as polylactid, co-polymers like polydioxanon with polyglactin and lyophilized dura have been used successfully to create such constructs with chondrocytes of different animal species. Cartilage specific compounds can be detected by histological and immunohistochemical techniques. To apply these constructs in humans, the distinguishing characteristics and problems of cell culture with human chondrocytes have to be considered. A further improvement of the artificially created tissue is conceivable using growth factors even including genetic manipulation of the applied cells.

[Current aspects of tendon healing]. / Aktuelle Aspekte der Sehnenheilung.

This review describes structure, function and healing of tendinous tissue and discusses new biologically based treatment options to modulate tendon healing. The repair process after tendon rupture results in a morphologically different and biomechanically inferior structure compared to a normal tendon. The collagen fibril diameters are decreased months after the traumatic lesion and show also different phenotypes. We know that cytokines and growth factors are key components for normal tissue development and regulate wound healing processes. Some growth factors have been detected to influence tenocytes by promoting cell proliferation and matrix synthesis. Application of the adequate growth factors at certain periods during the repair process might improve the healing result after tendon rupture. However, most of these growth factors are proteins which are rapidly metabolized by the organism. Transfer of growth factor genes into tenocytes might eliminate this problem by a continuous local release of growth factors at the healing site.

[TGF-beta-1 gene transfer in joint cartilage cells. Stimulating effect in extracellular matrix synthesis]. / TGF beta-1-Gentransfer in Gelenkknorpelzellen. Stimulierende Wirkung in der extrazellulären Matrixsynthese.

TGF beta-1 has been shown to upregulate matrix synthesis in articular chondrocytes. TGF beta-gene transfer to chondrocytes has the potential to increase the local production of this key component within regenerating cartilage after trauma and could support the repair process in articular cartilage lesions. Primary rabbit articular chondrocytes were cultured and retrovirally transfected with the experimental TGF beta-1 and the lacZ marker gene for control purposes. After radioactive labeling of new synthesized matrix proteins results were compared with normal primary chondrocytes. After TGF beta-1 gene transfer the endogenous growth factor concentration was doubled compared to normal chondrocytes and decreased in the lacZ control group. The proteoglycan synthesis in TGF beta-1 transfected chondrocytes showed a 96% increase compared to the basal production of normal chondrocytes. The LacZ transfected group revealed the opposite effect by a 44% decrease. The collagen synthesis of TGF beta-1 transfected chondrocytes was 304% compared to normal chondrocytes, predominantly type II collagen. The lacZ group collagen production was reduced by 35%. We conclude that TGF beta-1 gene transfer overcomes the decreasing effect observed by transfection with the LacZ marker gene and increases matrix synthesis in articular chondrocytes. Genetically altered chondrocytes might improve the repair of cartilage lesions by stimulating matrix synthesis and supporting the expression of the hyaline phenotype.

Transfer of lacZ marker gene to the meniscus.

BACKGROUND: Lesions in the avascular two-thirds of the meniscus do not heal well and are of concern clinically. Various growth factors promote the synthesis of matrix by meniscal cells and thus have the potential to augment healing. However, their clinical application is severely hindered by problems with delivery. An attractive approach to overcoming such problems is to transfer genes that encode the growth factors in question to the site of the injury. As a prelude to this, we evaluated methods for delivering genes to the meniscus. METHODS: Gene transfer was evaluated in vitro and in vivo with a lacZ marker gene, which expresses the enzyme beta-galactosidase. Two types of vectors were tested: an adenovirus and a retrovirus. Monolayers of lapine, canine, and human meniscal cells, as well as intact lapine and human menisci, were used for the in vitro studies. Lesions were created in the menisci of rabbits and dogs for the in vivo studies. Gene transfer to the sites of the experimental meniscal lesions in vivo was accomplished in two ways. In the lapine model, a suspension of adenovirus carrying the lacZ marker gene was mixed with whole blood and the clot was inserted into the lesion. In the canine model, retrovirally transduced allogenic meniscal cells carrying the lacZ marker gene were embedded in collagen gels and transferred to the defects. The animals were killed at various time-points, and gene expression was evaluated by histological examination of sections stained with 5-bromo-4-chloro-indolyl-beta-D-galactose (X-gal), from which a blue chromagen is released in the presence of beta-galactosidase. RESULTS: Monolayer cultures of lapine, canine, and human meniscal cells were susceptible to genetic transduction by both adenoviral and retroviral vectors. In vitro gene transfer to intact human and lapine menisci proved possible both by direct, adenoviral, delivery and indirect, retroviral, delivery. Gene expression persisted for at least twenty weeks under in vitro conditions. With regard to the in vivo studies, gene expression persisted within the clot and in some of the adjacent meniscal cells for at least three weeks in the lapine defect model. In the canine defect model, gene expression persisted within the transplanted, transduced meniscal cells for at least six weeks. CONCLUSIONS: It is possible to transfer genes to sites of meniscal damage and to express them locally within the lesion for several weeks.

[Gene transfer in the treatment of arthritis]. / Gentherapeutische Ansätze in der Arthrosebehandlung.

Current concepts in treating arthritis by gene transfer are described, including different vector systems and strategies of gene transfer into target cells. Promissing antiarthritic gene products are a variety of growth factors which facilitate increased matrix synthesis and mitogensis in articular chondrocytes. Furthermore, rheumatoid joint destruction can be treated genetically by the transfer of certain antiinflammatory cytokine genes, which provide locally high concentrations of the antiinflammatory gene product. First clinical trails using the IRAP gene (interleukin I receptor antagonist protein) to eliminate the inflammatory reaction caused by interleukin I in rheumatoid joints are on its way. In order to investigate potential improvement in cartilage regeneration retroviral TGF-beta gene transfer in rabbit articular chondrocytes has been carried out. The TGF-beta group showed an in vitro increase in collagen type II neosynthesis by 304%, compared to normal chondrocytes.

Bone tunnel enlargement after anterior cruciate ligament reconstruction: fact or fiction?

Radiographic enlargement of bone tunnels following anterior cruciate ligament (ACL) reconstruction has been recently introduced in the literature; however, the etiology and clinical relevance of this phenomenon remain unclear. While early reports suggested that bone tunnel enlargement is mainly the result of an immune response to allograft tissue, more recent studies imply that other biological as well as mechanical factors play a more important role. Biological factors associated with tunnel enlargement include foreign-body immune response (against allografts), non-specific inflammatory response (as in osteolysis around total joint implants), cell necrosis due to toxic products in the tunnel (ethylene oxide, metal), and heat necrosis as a response to drilling (natural course). Mechanical factors contributing to tunnel enlargement include stress deprivation of bone within the tunnel wall, graft-tunnel motion, improper tunnel placement, and aggressive rehabilitation. Graft-tunnel motion refers to longitudinal and transverse motion of the graft within the bone tunnel and can occur with various graft types and fixation techniques. Aggressive rehabilitation programmes may contribute to tunnel enlargement as the graft-bone interface is subjected to early stress before biological incorporation is complete. Further basic research is required to verify the effect of the various proposed factors on the etiology of bone tunnel enlargement. We recommend that routine follow-up examinations after ACL reconstruction should include the measurement of bone tunnel size in order to contribute to a better understanding of the incidence, time course, and clinical relevance of this phenomenon. Improved and more anatomical surgical fixation techniques may be useful for the prevention of bone tunnel enlargement.

[Improvement of the amplification rate of human chondrocytes with IGF-I and RGD]. / Verbesserung der Amplifikationsrate humaner Chondrozyten unter dem Einfluss von IGF-I und RGD.

Human chondrocytes were incubated under following conditions: Group 1 (control group): Incubation in 25 cm2 cell culture flasks (Costar) with RPMI-medium (6%-AB-serum, L-Glutamin, Hepes-buffer and antibiotics); Group 2: Different concentrations of IGF-I (1 ng/ml, 10 ng/ml) were added to the RPMI-medium; Group 3: Incubation (like control group) with additional coating of the cell culture flasks with different concentrations of RGD (5 mg/ml; 7.5 mg/ml; 10 mg/ml; 20 mg/ml); Group 4: Combination of coating with RGD (5 mg/ml; 10 mg/ml) and addition of IGF-I (1 ng/ml; 10 ng/ml) to the medium. The cells of the control group could be doubled within 2 weeks. The amplification rate of the groups 2 and 3 was improved in comparison to group 1 with the following maxima: Group 2 (5 mg/ml RGD) 3.1 times and group 3 (1 ng/ml IGF-I) 2.6 times of the number of the cells in the beginning. Group 4 (RGD and IGF-I) showed additive effects, for 4.1 times of the number of the cells in the beginning could be counted after 14 days. RGD and IGF-I (groups 2 to 4) made possible an earlier dedifferentiation and adhesion of the cells to the bottom of the cell culture flasks. By using both growth factors (RGD and IGF-I), the number of the cells could be enhanced more than 2 times in comparison to the control group within the same time. So less than half of the autologous patient's cartilage is necessary for cultivation of hyaline cartilagee.

Collagen fibril organization in the patellar tendon autograft after posterior cruciate ligament reconstruction. A quantitative evaluation in a sheep model.

We replaced the posterior cruciate ligament in 30 skeletally mature sheep with a patellar tendon autograft using the central third of the ipsilateral patellar tendon. The healing autograft was compared with the contralateral posterior cruciate ligament and the patellar tendons and posterior cruciate ligaments of nonoperated animals. The collagen fibril diameters were analyzed using transmission electron photomicrographs of fibril cross sections taken at six periods during the 2 years after surgery. The patellar tendon and posterior cruciate ligament were characterized by a broad, nongaussian distribution of collagen fibril diameters. The autografts shifted to a unimodal distribution by an increase of small-diameter collagen fibrils. The frequency of small-diameter fibrils measuring up to 100 nm was 99% after 2 years. At that time, these small-diameter fibrils represented 91.6% of the area covered by collagen fibrils. The mean diameter of the collagen fibrils in the autografts significantly decreased to 45% of the controls at Week 26 and remained at this level until the end of this study. The percentage of area covered by collagen fibrils per 1 micron 2 was 78% of the controls 2 years post-operatively. This study suggests that the patellar tendon autograft could not reproduce the collagen fibril organization of the posterior cruciate ligament. This may be a biologic factor responsible for inconsistent results in posterior cruciate ligament replacement.