Muscle cell-mediated gene delivery to the rotator cuff.

Rotator cuff lesions are one of the most common causes of upper extremity disability. Surgical therapy addresses mostly the extrinsic etiology, but not intrinsic factors such as aging, structural changes, low vascularity, and inflammatory processes. In this study, genetically engineered, highly purified muscle-derived cells (MDCs) were characterized and injected into the supraspinatus tendons of nude rats. The injected cells were monitored for 3 weeks. In vitro, the engineered, highly purified MDCs do not express vimentin; 98% of them are positive for the beta-galactosidase marker gene, and 99% hybridize with the specific pancentromeric mouse probe. beta-Galactosidase marker gene expression of the injected cells was detected up to 21 days. From day 7 after injection, the cell nuclei became spindle shaped, cells were integrated into the tendon collagen bundles, and the cells showed differentiation into vimentin-expressing fibroblastic cells. The results indicate that the rotator cuff tendon matrix and its original cellular components modulated the injected MDCs toward a fibroblastic phenotype. The compatibility and ability of MDCs to differentiate into other cell lineages, such as fibroblasts, might have high potential utility in tissue-engineering applications for tendon healing. This approach facilitates the application of muscle-derived progenitor cells and ex vivo gene therapy for the treatment of rotator cuff lesions.

Autologous chondrocyte implantation and osteochondral cylinder transplantation in cartilage repair of the knee joint. A prospective, comparative trial.

BACKGROUND: Current methods used to restore the joint surface in patients with localized articular cartilage defects include transplantation of an autologous osteochondral cylinder and implantation of autologous chondrocytes. The purpose of this study was to evaluate the clinical and histological outcomes of these two techniques. METHODS: We performed a prospective clinical study to investigate the two-year outcomes in forty patients with an articular cartilage lesion of the femoral condyle who had been randomly treated with either transplantation of an autologous osteochondral cylinder or implantation of autologous chondrocytes. Biopsy specimens from representative patients of both groups were evaluated with histological staining, immunohistochemistry, and scanning electron microscopy. RESULTS: According to the postoperative Lysholm score, the recovery after autologous chondrocyte implantation was slower than that after osteochondral transplantation at six months (p < or = 0.015), twelve months (p < or = 0.001), and twenty-four months (p < or = 0.012). On the basis of the Meyers score and the Tegner activity score, the results were equally good with the two methods two years after treatment. Histomorphological evaluation of biopsy specimens within two years after autologous chondrocyte implantation demonstrated a complete, mechanically stable resurfacing of the defect in all patients. The tissue consisted mainly of fibrocartilage, while localized areas of hyaline-like regenerative cartilage could be detected close to the subchondral bone. Although a gap remained at the site of the transplantation in all five biopsy specimens examined as long as two years after osteochondral cylinder transplantation, histomorphological analysis and scanning electron microscopy revealed no differences between the osteochondral transplants and the surrounding original cartilage. CONCLUSIONS: Both treatments resulted in a decrease in symptoms. However, the improvement provided by the autologous chondrocyte implantation lagged behind that provided by the osteochondral cylinder transplantation. Histologically, the defects treated with autologous chondrocyte implantation were primarily filled with fibrocartilage, whereas the osteochondral cylinder transplants retained their hyaline character, although there was a persistent interface between the transplant and the surrounding original cartilage. Limitations of our study included the small number of patients, the relatively short (two-year) follow-up, and the absence of a control group.

[Gene therapy for cartilage repair]. / Gentherapie von Knorpelgewebe.

AIM: Articular cartilage has very limited intrinsic healing capacity. Although numerous attempts to repair full-thickness articular cartilage defects have been conducted, no methods have successfully regenerated long-lasting hyaline cartilage. One of the most promising procedures for cartilage repair is tissue engineering accompanied by gene therapy. METHOD: With gene therapy, genes encoding for therapeutic growth factors can be expressed at a high level in the injured site for an extended period of time. Chondrocytes have been intensively studied for cell transplantation in articular cartilage defects. RESULTS: However, recent studies have shown that chondrocytes are not the only candidate for cartilage repair. Muscle-derived cells have been found capable of delivering genes and represent a good vehicle to deliver therapeutic genes to improve cartilage repair. More importantly, recent studies have suggested the presence of pluripotent stem cells in muscle-derived cells. CONCLUSION: New techniques of cell therapy and molecular medicine for the treatment of cartilage lesions are currently undergoing clinical trials. This paper will summarize the current status of gene therapy for cartilage repair and its future application.

Muscle-based gene therapy and tissue engineering.

The development of new biological approaches based on cell and gene therapies, in combination with tissue engineering, may create innovative ways to treat various tissues of the musculoskeletal system. It is vital for practicing orthopaedic surgeons to understand the terminology, fundamental concepts, and current research in this burgeoning field so that they may practice their discipline in its fullest form. Such techniques, coupled with advances in cell biology and polymer chemistry, are resulting in novel approaches to treating musculoskeletal disorders in which surgeons, who have traditionally used the tools of excision and reconstruction to treat patients, may now serve as surgical gardeners who create microenvironments that are conducive for tissue regeneration. Gene therapy and tissue engineering applications for bone healing, articular disorders, and skeletal muscle diseases and injuries are currently being explored. This review is intended to update readers on the principles and current advances in muscle-based gene therapy and tissue engineering for the musculoskeletal system.

New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering.

STUDY DESIGN: Human intervertebral disc cells were cultured in a new three-dimensional "pellet culture" system as an alternative to conventional alginate bead microspheres. Histologic, biochemical, and immunohistologic assays were performed to characterize this new culturing method for disc cells. The feasibility of using the pellet culture system to study effects of gene therapy was also assessed. OBJECTIVES: To characterize a new and simpler, three-dimensional culture system for human intervertebral disc cells and to assess the feasibility of its use for gene therapy and tissue engineering studies. SUMMARY OF BACKGROUND DATA: The alginate microsphere three-dimensional culture system has been the most utilized culture method for disc cells, but it is technically difficult and has some disadvantages. Recently, the "pellet culture" method, a simpler three-dimensional culture system, was described for bone marrow stromal cells. This simpler method might be useful for in vitro and in vivo studies of disc cells as well as for delivery of exogenous genes. METHODS: Isolated human intervertebral disc cells were centrifuged at low speeds to form aggregates and allowed to grow as pellets for up to 3 weeks. At various times these pellet cultures were analyzed grossly, histologically, and immunohistologically. Their ability to incorporate [35S]sulfate in their response to TGF-beta1 was also analyzed. The ability of these pellets to deliver and express exogenous genes in vivo was analyzed by implantation of pellet cultures in muscles of SCID mice. RESULTS: Within several days the intervertebral disc cells were able to form mature three-dimensional aggregates that were each well encapsulated by a fibrous capsule. These pellets successfully synthesized proteoglycan and collagen Type II matrix as determined by histology and immunohistochemistry. In response to TGF-beta1, the pellets increased synthesis of proteoglycan and collagen Type II. When implanted into thigh muscles of SCID mice, the pellets remained aggregated and expressed the beta-galactosidase marker gene in vivo for up to 2 weeks. CONCLUSIONS: The pellet culture system is a new and technically simple method to culture human intervertebral disc cells. The majority of the human intervertebral disc cells retained their native phenotype in this three dimensional system and expressed a marker gene both in vitro and in vivo. Thus, this pellet system might be useful for in vitro and in vivo biochemical studies as well as for studies involving gene therapy and tissue engineering.

Effect of bone morphogenetic protein-2-expressing muscle-derived cells on healing of critical-sized bone defects in mice.

BACKGROUND: Cells that express bone morphogenetic protein-2 (BMP-2) can now be prepared by transduction with adenovirus containing BMP-2 cDNA. Skeletal muscle tissue contains cells that differentiate into osteoblasts on stimulation with BMP-2. The objectives of this study were to prepare BMP-2-expressing muscle-derived cells by transduction of these cells with an adenovirus containing BMP-2 cDNA and to determine whether the BMP-2-expressing muscle-derived cells would elicit the healing of critical-sized bone defects in mice. METHODS: Primary cultures of muscle-derived cells from a normal male mouse were transduced with adenovirus encoding the recombinant human BMP-2 gene (adBMP-2). These cells (5 yen 10(5)) were implanted into a 5-mm-diameter critical-sized skull defect in female SCID (severe combined immunodeficiency strain) mice with use of a collagen sponge as a scaffold. Healing in the treatment and control groups was examined grossly and histologically at two and four weeks. Implanted cells were identified in vivo with use of the Y-chromosome-specific fluorescent in situ hybridization (FISH) technique, and their differentiation into osteogenic cells was demonstrated by osteocalcin immunohistochemistry. RESULTS: Skull defects treated with muscle cells that had been genetically engineered to express BMP-2 had >85% closure within two weeks and 95% to 100% closure within four weeks. Control groups in which the defect was not treated (group 1), treated with collagen only (group 2), or treated with collagen and muscle cells without adBMP-2 (group 3) showed at most 30% to 40% closure of the defect by four weeks, and the majority of the skull defects in those groups showed no healing. Analysis of injected cells in group 4, with the Y-chromosome-specific FISH technique showed that the majority of the transplanted cells were located on the surfaces of the newly formed bone, but a small fraction (approximately 5%) was identified within the osteocyte lacunae of the new bone. Implanted cells found in the new bone stained immunohistochemically for osteocalcin, indicating that they had differentiated in vivo into osteogenic cells. CONCLUSIONS: This study demonstrates that cells derived from muscle tissue that have been genetically engineered to express BMP-2 elicit the healing of critical-sized skull defects in mice. The cells derived from muscle tissue appear to enhance bone-healing by differentiating into osteoblasts in vivo. CLINICAL RELEVANCE: Ex vivo gene therapy with muscle-derived cells that have been genetically engineered to express BMP-2 may be used to treat nonhealing bone defects. In addition, muscle-derived cells appear to include stem cells, which are easily obtained with muscle biopsy and could be used in gene therapy to deliver BMP-2.

[Correction of malunited distal radius fractures by osteotomy and interposition of iliac crest bone spain in the radio-palmar position. A retrospective study of 42 patients]. / Korrektur fehlverheilter distaler Radiusfrakturen durch Osteotomie und Interposition eines Beckenkammspans von radiopalmar. Eine retrospektive Untersuchung an 42 Patienten.

The retrospective study included 42 patients who have been treated with a palmar opening wedge osteotomy after malunion of distal radius fracture. 31 patients could be reexamined clinically and radiologically. The corrective osteotomy on the distal radius was done only radiopalmar with an insertion of a corticocancellous iliac graft. Radiological and functional findings as well as the subjective judgement of the patient have been commented pre- and after surgically according to the assessment scheme of Pechlaner and were then compared with the contra lateral wrist. The evaluation of the x-ray pictures showed an improvement from 3.7 to 1.9 points, the functional improvement from 3.7 to 2.6 points. The improvement of the subjective complaints results in a diminution of 3.3 to 2.2 points.

[Tissue engineering and gene therapy of the musculoskeletal system with muscle cells]. / Tissue Engineering und Gentherapie des Bewegungsapparates mit Muskelzellen.

AIM: Muscle-based somatic gene therapy is a novel way to alleviate a biochemical deficiency. METHOD: Muscle-derived cells are very promising in the field of gene therapy and tissue engineering. First, most muscle tissue is accessible by injection. Second, muscle tissue consists of multinucleated, postmitotic myofibers, which enable a long-term expression of the transduced gene. Third, muscle tissue can be biopsied easily. It is available in abundance and the biopsy does not compromise the health and function of the patient. Finally, muscle tissue is highly vascularized, which makes systemic delivery feasible. RESULTS: Muscle-derived cells can promote muscle healing and bone healing. Implanted cells maintain a long-term transgene expression of therapeutic proteins. Isolated, muscle-derived stem cells can differentiate in osteoblasts. CONCLUSION: Based on these characteristics, we present four possible applications: inherited muscular diseases, muscle injury, bone healing, and intraarticular disorders.

[Osteochondral transplantation versus autogenous chondrocyte transplantation. A prospective comparative clinical study]. / Knorpelknochentransplantation versus autogene Chondrocytentransplantation. Eine prospektive vergleichende klinische Studie.

INTRODUCTION: The treatment of full-thickness cartilage defects still represents a problem that has not yet been solved satisfactorily. Current methods used to cover defects in the knee joint are osteochondral cylinder transplantation (OCT) and autologous chondrocyte transplantation (ACT). METHODS: With a prospective clinical investigation, at the time being with 2-year results, we have examined ACT in comparison to OCT in 20 patients with regard to clinical and histomorphological (histology, immunohistochemistry, RES) outcome. RESULTS: We found equally good results with both methods in Lysholm, Meyers and Tegner Activity Scores. Histomorphologic evaluation of biopsies obtained by arthroscopy after ACT showed a defect filling in all cases, mainly with fibrous cartilage, while localized areas of hyalinelike regenerative cartilage were documented near the base. We did not see any histomorphologically visible change in the transplants after OCT. CONCLUSION: At the time we prefer OCT instead of ACT given the correct indication.

Ex vivo gene therapy to produce bone using different cell types.

Gene therapy and tissue engineering promise to revolutionize orthopaedic surgery. This study comprehensively compares five different cell types in ex vivo gene therapy to produce bone. The cell types include a bone marrow stromal cell line, primary muscle derived cells, primary bone marrow stromal cells, primary articular chondrocytes, and primary fibroblasts. After transduction by an adenovirus encoding for bone morphogenetic protein-2, all of the cell types were capable of secreting bone morphogenetic protein-2. However, the bone marrow stromal cell line and muscle derived cells showed more responsiveness to recombinant human bone morphogenetic protein-2 than did the other cell types. In vivo injection of each of the cell populations transduced to secrete bone morphogenetic protein-2 resulted in bone formation. Radiographic and histologic analyses corroborated the in vitro data regarding bone morphogenetic protein-2 secretion and cellular osteocompetence. This study showed the feasibility of using primary bone marrow stromal cells, primary muscle derived cells, primary articular chondrocytes, primary fibroblasts, and an osteogenesis imperfecta stromal cell line in ex vivo gene therapy to produce bone. The study also showed the advantages and disadvantages inherent in using each cell type.

Spirometric gated quantitative computed tomography of the lung in healthy smokers and nonsmokers.

RATIONALE AND OBJECTIVES: The authors investigated the influence of cigarette smoking on healthy, asymptomatic smokers and nonsmokers with the help of spirometric triggered quantitative computed tomography. In our prospective study, the authors compared conventional lung function parameters with the computed tomography values (lung attenuation, lung area). METHODS: The study group comprised 40 healthy volunteers consisting of 20 smokers and nonsmokers (20 females and 20 males). The corresponding groups have been matched concerning their age, height, body mass, (cigarette) pack years. Computer tomography scans were triggered at 35%, 50%, 70% and 95% of vital capacity at a defined apical and a basal level. RESULTS: Functional residual capacity (FRC), total lung capacity and airway resistance showed close correlations to lung parenchymal attenuation values especially at full inspiration and expiration. For example, the authors found a correlation coefficient of r = -0.845 (P < or = 0.001) concerning the FRC and lung attenuation values in the apical lung at 35% of vital capacity in male smokers. Male smokers proved to have a significantly higher pulmonary lung density at all inspiratory states than the other groups (P < or = 0.05; Student's t test). Although male smokers had a higher vital capacity they showed a smaller cross-sectional area increase of the lung during inspiration than nonsmokers. This phenomenon is a result of the decreasing compliance of the smoker's lung, due to small airways disease and hypoxic vasoconstriction. CONCLUSIONS: Spirometric-triggered quantitative computed tomography has proved to be a sensitive diagnostic device for the investigation of early pathomorphologic changes in healthy, asymptomatic cigarette smokers.