Wound-healing risk factors after open reduction and internal fixation of calcaneal fractures: does correction of Bohler's angle alter outcomes?

The study reviewed in this article evaluated a group of patients who underwent surgical therapy for calcaneal fractures at a Level I trauma center. One group of patients was treated after outpatient referral to the center, whereas the other group was admitted to, and underwent surgery at, the center. This study attempted to determine which patient risk factors or injury characteristics might lead to an increased rate of wound-healing complications. Bohler's angle is a classic radiographic method of determining the severity of calcaneal injury in this group of patients. The question posed by the authors of this study was: Does a drastic correction in Bohler's angle lead to an increased incidence of wound-healing complications? The authors do not recommend undercorrection of Bohler's angle but urge avoidance of overcorrection and stress the importance of early surgical fixation after lateral skin wrinkling is found.

Increased matrix synthesis following adenoviral transfer of a transforming growth factor beta1 gene into articular chondrocytes.

Monolayer cultures of lapine articular chondrocytes were transduced with first-generation adenoviral vectors carrying lacZ or transforming growth factor beta1 genes under the transcriptional control of the human cytomegalovirus early promoter. High concentrations of transforming growth factor beta1 were produced by chondrocytes following transfer of the transforming growth factor beta1 gene but not the lacZ gene. Transduced chondrocytes responded to the elevated endogenous production of transforming growth factor beta1 by increasing their synthesis of proteoglycan, collagen, and noncollagenous proteins in a dose-dependent fashion. The increases in collagen synthesis were not accompanied by alterations in the collagen phenotype; type-II collagen remained the predominant collagen. Transforming growth factor beta1 could not, however, rescue the collagen phenotype of cells that had undergone phenotypic modulation as a result of serial passaging. These data demonstrate that chondrocytes can be genetically manipulated to produce and respond to the potentially therapeutic cytokine transforming growth factor beta1. This technology has a number of experimental and therapeutic applications, including those related to the study and treatment of arthritis and cartilage repair.

Using gene therapy to protect and restore cartilage.

Numerous gene products have the potential to help protect cartilage from degradation and to repair cartilage that has become damaged as a result of disease or injury. The genes that encode these products thus may serve as chondroprotective and chondroregenerative medicines. To bring these agents into clinical use, it is necessary to screen candidate genes for efficacy under in vitro and in vivo conditions, to determine the best cells to target, and to develop appropriate gene transfer technologies. As discussed in the current review, progress has been made in each of these areas. Various viral and nonviral vectors are able to deliver genes to synoviocytes, articular chondrocytes, and mesenchymal stem cells. There also is evidence to suggest that ex vivo and in vivo approaches can be used for gene transfer to articular cartilage, synovium, and meniscus. Moreover various cytokine antagonists and growth factors have been shown to protect cartilage and stimulate chondrogenesis. In vivo methods and strategies that target synovium may be useful in a chondroprotective mode but because they do not increase the number of chondrogenic cells within lesions, they may be ill-equipped to repair large defects. Ex vivo methods however, provide cells and genes. It also is important to distinguish the treatment of isolated lesions occurring as a result of injury from the treatment of lesions resulting from underlying disease processes. Additional development of these approaches should result in clinically useful genetic methods for the protection and regeneration of cartilagenous tissues.

Gene therapy for meniscal injury: enhanced synthesis of proteoglycan and collagen by meniscal cells transduced with a TGFbeta(1)gene.

Objective To determine whether meniscal cells can express a TGFbeta(1)transgene delivered by a retroviral vector, and respond to the gene product by increasing matrix synthesis. Methods Monolayer cultures of human and canine meniscal cells were infected with retroviruses carrying either a human TGFbeta(1)cDNA or marker genes. Conditioned media were assayed for the presence of TGFbeta(1). Biosynthesis assays using radiolabeled precursors were employed to determine the effects of the transgenes on the synthesis of proteoglycan, collagen and noncollagenous proteins. Collagen phenotyping was performed by SDS-PAGE. Results Media conditioned by canine and human meniscal cells transduced with the TGFbeta(1)gene, accumulated several nanograms/10(6)cells of TGFbeta(1)during a 48 h incubation. Media conditioned by control cells contained very little TGFbeta(1). Transduction with the TGFbeta(1)gene, but not marker genes, increased the synthesis of collagen and proteoglycan by 8-15-fold. The synthesis of noncollagenous proteins was enhanced more modestly. Monolayers of meniscal cells synthesized types I, III, V and VI collagen. The TGFbeta(1)gene increased the synthesis of all types of collagen without altering the ratios between them. Conclusions Meniscal cells are readily transduced by retroviral vectors and respond to the transfer of a TGFbeta(1)cDNA by greatly increasing matrix synthesis. These findings encourage the further development of genetic approaches to the healing of meniscal lesions.

Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1.

OBJECTIVE: To determine whether articular chondrocytes express growth factor genes delivered by adenoviral vectors and whether expression of these genes influences matrix synthesis in the presence and absence of interleukin-1 (IL-1). METHODS: Monolayer cultures of rabbit articular chondrocytes were infected with recombinant adenovirus carrying genes encoding the following growth factors: insulin-like growth factor 1 (IGF-1), transforming growth factor beta1 (TGFbeta1), and bone morphogenetic protein 2 (BMP-2). As a control, cells were transduced with the lac Z gene. Cultures were also treated with each growth factor supplied as a protein. Levels of gene expression were noted, and the synthesis of proteoglycan, collagen, and noncollagenous proteins was measured by radiolabeling. Collagen was typed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The effects of growth factor gene transfer on proteoglycan synthesis in the presence of IL-1 were also measured. RESULTS: The expression of all transgenes was high following adenoviral transduction. Proteoglycan synthesis was stimulated approximately 8-fold by the BMP-2 gene and 2-3-fold by the IGF-1 gene. The effects of BMP-2 and IGF-1 genes were additive upon cotransduction. Synthesis of collagen and noncollagenous proteins, in contrast, was most strongly stimulated by the IGF-1 gene. In each case, collagen typing confirmed the synthesis of type II collagen. IL-1 suppressed proteoglycan synthesis by 50-60%. IGF-1 and TGFbeta genes restored proteoglycan synthesis to control levels in the presence of IL-1. The BMP-2 gene, in contrast, elevated proteoglycan synthesis beyond control levels in the presence of IL-1. CONCLUSION: Transfer of growth factor genes to articular chondrocytes can greatly increase matrix synthesis in vitro, even in the presence of the inflammatory cytokine IL-1. This result encourages the further development of gene therapy for the repair of damaged cartilage.

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.

A composite regulatory element in the first intron of the estrogen-responsive very low density apolipoprotein II gene.

During periods of egg laying in the chicken, when circulating levels of estrogen are increased, the liver-specific estrogen-dependent very low density apolipoprotein II (apoVLDLII) gene is expressed. This expression takes place primarily at the level of transcription, driven by two estrogen response elements that reside in the promoter. In transient transfection assays, expression is increased fourfold when the first intron is added to the promoter construct, indicating that 75% of the regulation comes from intron A. Using in vitro DNase I footprinting, six protein-binding sites were revealed throughout the first intron. The functional significance of these binding sites was evaluated by mutation and transient transfection. Two of the protein-binding regions were shown to increase transcription. Site-specific mutations introduced at either the +66 to +86 or +112 to +129 sites disrupted trans-factor binding and reduced the estrogen-dependent expression by 45% and 34%, respectively. A plasmid containing both mutations resulted in a 43% decrease in expression, indicating that the contributions of these regions are not additive. Competition with known sequences in electrophoretic mobility shift assays suggested that the +66 to +86 site binds a chicken member of the nuclear receptor transcription factor family.