Friedrich F. Hennig

Chondromodulin 1 stabilizes the chondrocyte phenotype and inhibits endochondral ossification of porcine cartilage repair tissue

OBJECTIVE To investigate the effect of chondromodulin 1 on the phenotype of osteochondral progenitor cells in cartilage repair tissue. METHODS Self-complementary adeno-associated virus (AAV) vectors carrying chondromodulin 1 complementary DNA (AAV-Chm-1) were applied to cartilage lesions in the knee joints of miniature pigs that were treated by the microfracture technique. Alternatively, isolated porcine osteochondral progenitor cells were infected with AAV-Chm-1 or with AAV-GFP control vectors ex vivo prior to being transplanted into cartilage lesions in which the subchondral bone plate was left intact. The quality of the repair tissue and the degree of endochondral ossification were assessed by histochemical and immunohistochemical methods. The effects of chondromodulin 1 overexpression were also analyzed by angiogenesis assays and quantitative reverse transcriptase-polymerase chain reaction. RESULTS AAV-Chm-1-infected cells efficiently produced chondromodulin 1, which had strong antiangiogenic effects, as verified by the inhibition of tube formation of endothelial cells. Gene expression analyses in vitro revealed the cell cycle inhibitor p21WAF1/Cip1 as one target up-regulated by AAV-Chm-1. Direct application of AAV-Chm-1 vectors into microfractured porcine cartilage lesions stimulated chondrogenic differentiation of ingrowing progenitor cells, but significantly inhibited terminal chondrocyte hypertrophy, the invasion of vessel structures, and excessive endochondral ossification, which were otherwise observed in untreated lesions. Indirect gene transfer, with infection of porcine osteochondral progenitor cells by AAV-Chm-1 ex vivo, also supported chondrogenic differentiation of these transplanted cells. AAV-Chm-1-infected cells maintained a chondrocyte-like phenotype and formed a hyaline-like matrix that was superior to that formed by uninfected or AAV-GFP-infected cells. CONCLUSION Our findings indicate that the antiangiogenic factor chondromodulin 1 stabilizes the chondrocyte phenotype by supporting chondrogenesis but inhibiting chondrocyte hypertrophy and endochondral ossification.

Quantitative T2 mapping during follow-up after matrix-associated autologous chondrocyte transplantation (MACT): full-thickness and zonal evaluation to visualize the maturation of cartilage repair tissue.

The purpose of this article was to evaluate the potential of in vivo zonal T2‐mapping as a noninvasive tool in the longitudinal visualization of cartilage repair tissue maturation after matrix‐associated autologous chondrocyte transplantation (MACT). Fifteen patients were treated with MACT and evaluated cross‐sectionally, with a baseline MRI at a follow‐up of 19.7 ± 12.1 months after cartilage transplantation surgery of the knee. In the same 15 patients, 12 months later (31.7 ± 12.0 months after surgery), a longitudinal 1‐year follow‐up MRI was obtained. MRI was performed on a 3 Tesla MR scanner; morphological evaluation was performed using a double‐echo steady‐state sequence; T2 maps were calculated from a multiecho, spin‐echo sequence. Quantitative mean (full‐thickness) and zonal (deep and superficial) T2 values were calculated in the cartilage repair area and in control cartilage sites. A statistical analysis of variance was performed. Full‐tickness T2 values showed no significant difference between sites of healthy cartilage and cartilage repair tissue (p < 0.05). Using zonal T2 evaluation, healthy cartilage showed a significant increase from the deep to superficial cartilage layers (p < 0.05). Cartilage repair tissue after MACT showed no significant zonal increase from deep to superficial cartilage areas during baseline MRI (p > 0.05); however, during the 1‐year follow‐up, a significant zonal stratification could be observed (p < 0.05). Morphological evaluation showed no significant difference between the baseline and the 1‐year follow‐up MRI. T2 mapping seems to be more sensitive in revealing changes in the repair tissue compared to morphological MRI. In vivo zonal T2 assessment may be sensitive enough to characterize the maturation of cartilage repair tissue.

Molecular differentiation between osteophytic and articular cartilage – clues for a transient and permanent chondrocyte phenotype

OBJECTIVE To identify the molecular differences between the transient and permanent chondrocyte phenotype in osteophytic and articular cartilage. METHODS Total RNA was isolated from the cartilaginous layer of osteophytes and from intact articular cartilage from knee joints of 15 adult human donors and subjected to cDNA microarray analysis. The differential expression of relevant genes between these two cartilaginous tissues was additionally validated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry. RESULTS Among 47,000 screened transcripts, 600 transcripts were differentially expressed between osteophytic and articular chondrocytes. Osteophytic chondrocytes were characterized by increased expression of genes involved in the endochondral ossification process [bone gamma-carboxyglutamate protein/osteocalcin (BGLAP), bone morphogenetic protein-8B (BMP8B), collagen type I, alpha 2 (COL1A2), sclerostin (SOST), growth arrest and DNA damage-induced gene 45ß (GADD45ß), runt-related transcription factor 2 (RUNX2)], and genes encoding tissue remodeling enzymes [matrix metallopeptidase (MMP)9, 13, hyaluronan synthase 1 (HAS1)]. Articular chondrocytes expressed increased transcript levels of antagonists and inhibitors of the BMP- and Wnt-signaling pathways [Gremlin-1 (GREM1), frizzled-related protein (FRZB), WNT1 inducible signaling pathway protein-3 (WISP3)], as well as factors that inhibit terminal chondrocyte differentiation and endochondral bone formation [parathyroid hormone-like hormone (PTHLH), sex-determining region Y-box 9 (SOX9), stanniocalcin-2 (STC2), S100 calcium binding protein A1 (S100A1), S100 calcium binding protein B (S100B)]. Immunohistochemistry of tissue sections for GREM1 and BGLAP, the two most prominent differentially expressed genes, confirmed selective detection of GREM1 in articular chondrocytes and that of BGLAP in osteophytic chondrocytes and bone. CONCLUSIONS Osteophytic and articular chondrocytes significantly differ in their gene expression pattern. In articular cartilage, a prominent expression of antagonists inhibiting the BMP- and Wnt-pathway may serve to lock and stabilize the permanent chondrocyte phenotype and thus prevent their terminal differentiation. In contrast, osteophytic chondrocytes express genes with roles in the endochondral ossification process, which may account for their transient phenotype.

Cell‐based resurfacing of large cartilage defects: Long‐term evaluation of grafts from autologous transgene‐activated periosteal cells in a porcine model of osteoarthritis

OBJECTIVE To investigate the potential of transgene-activated periosteal cells for permanently resurfacing large partial-thickness cartilage defects. METHODS In miniature pigs, autologous periosteal cells stimulated ex vivo by bone morphogenetic protein 2 gene transfer, using liposomes or a combination of adeno-associated virus (AAV) and adenovirus (Ad) vectors, were applied on a bioresorbable scaffold to chondral lesions comprising the entire medial half of the patella. The resulting repair tissue was assessed, 6 and 26 weeks after transplantation, by histochemical and immunohistochemical methods. The biomechanical properties of the repair tissue were characterized by nanoindentation measurements. Implants of unstimulated cells and untreated lesions served as controls. RESULTS All grafts showed satisfactory integration into the preexisting cartilage. Six weeks after transplantation, AAV/Ad-stimulated periosteal cells had adopted a chondrocyte-like phenotype in all layers; the newly formed matrix was rich in proteoglycans and type II collagen, and its contact stiffness was close to that of healthy hyaline cartilage. Unstimulated periosteal cells and cells activated by liposomal gene transfer formed only fibrocartilaginous repair tissue with minor contact stiffness. However, within 6 months following transplantation, the AAV/Ad-stimulated cells in the superficial zone tended to dedifferentiate, as indicated by a switch from type II to type I collagen synthesis and reduced contact stiffness. In deeper zones, these cells retained their chondrocytic phenotype, coinciding with positive staining for type II collagen in the matrix. CONCLUSION Large partial-thickness cartilage defects can be resurfaced efficiently with hyaline-like cartilage formed by transgene-activated periosteal cells. The long-term stability of the cartilage seems to depend on physicobiochemical factors that are active only in deeper zones of the cartilaginous tissue.

Balser plate stabilization: an alternate therapy for traumatic sternoclavicular instability

Traumatic instability of the sternoclavicular joint is a rare condition. It can be treated by surgical cerclage fixation, which necessitates postoperative immobilization, an approach preventing early postoperative functional rehabilitation. Balser plate stabilization is a therapeutic alternative that does not require extended periods of immobilization. From January 1, 1996, to December 31, 2000, a total of 10 trauma patients with unstable sternoclavicular joints (Allman grade III) requiring surgical management were treated with Balser plate stabilization to allow early physiotherapy. The population included 7 patients with anterior dislocations, 2 with posterior dislocations, and 1 with medial epiphysiolysis in addition to posterior dislocation. Implants were removed from 9 patients after 3 months and 1 patient after 2 months. One year or longer after the procedure, 9 of 10 patients were available for follow-up; 1 patient had moved. The results achieved with this alternative treatment are excellent. There were no cases of redislocation. The only surgical complication was a seroma that required surgical drainage. One patient had arthrosis develop. Outcome was assessed with Constant (range, 84-100; mean, 90.2 +/- 6.6) and DASH (disabilities of the arm, shoulder, and hand) (range, 4.1-16.6; mean, 8.4 +/- 1.4) scores. For the rare case of sternoclavicular joint dislocation requiring open surgical reduction and stabilization, the Balser plate technique is reliable, permits early movement, has good postoperative results, and compares favorably with alternative methods.

Role of hypoxia-inducible factor 1alpha in the integrity of articular cartilage in murine knee joints

IntroductionChondrocytes have to withstand considerable hypoxic conditions within the avascular articular cartilage. The present study investigated the effects of inhibiting or stabilizing hypoxia-inducible factor (HIF)-1α by 2-methoxyestradiol or dimethyloxaloylglycine on the progression of osteoarthritis in murine knee joints.Methods2-Methoxyestradiol was injected six times over a period of 2 weeks into the left knee joint of Balb/C mice. Joints were assessed by histochemical and immunohistochemical methods, 3 weeks and 12 weeks following the first injection. Dimethyloxaloylglycine, an inhibitor of HIF-degrading prolyl-hydroxylases, was injected into the left knee joints of STR/ORT mice once a week over the entire period of 12 weeks. Right knee joints that received a saline solution served as controls. In addition, the effects of dimethyloxaloylglycine on HIF-1 target gene expression and on collagen metabolism were analyzed in vitro.ResultsInjection of 2-methoxyestradiol led to osteoarthritic changes in the treated knee joints of Balb/C mice. The first signs of osteophyte formation were observed in the knee joints after 3 weeks, followed by progressive destruction of the articular cartilage at 12 weeks that was not, however, accompanied by inflammatory reactions. Injection of dimethyloxaloylglycine could not prevent severe osteoarthritis that spontaneously developed in the knee joints of STR/ORT mice. In chondrocyte cultures, administration of dimethyloxaloylglycine resulted in an upregulation of Sox9 expression. Such a stimulatory effect was not observed, however, for the expression of type II collagen, which might be the indirect consequence of intracellular collagen retention observed by immunofluorescence or of increased expression of IL-1β and IL-6.ConclusionsInduction of osteoarthritis by 2-methoxyestradiol demonstrates the importance of HIF-1 in maintaining the integrity of hypoxic articular cartilage. Stabilization of HIF-1 by dimethyloxaloylglycine, however, was not of therapeutic value, since this nonselective prolyl-hydroxylase inhibitor also interferes with proper collagen metabolism and induces the expression of catabolic cytokines

Chondrogenic differentiation of growth factor-stimulated precursor cells in cartilage repair tissue is associated with increased HIF-1α activity

OBJECTIVE To investigate the chondrogenic potential of growth factor-stimulated periosteal cells with respect to the activity of Hypoxia-inducible Factor 1alpha (HIF-1alpha). METHODS Scaffold-bound autologous periosteal cells, which had been activated by Insulin-like Growth Factor 1 (IGF-1) or Bone Morphogenetic Protein 2 (BMP-2) gene transfer using both adeno-associated virus (AAV) and adenoviral (Ad) vectors, were applied to chondral lesions in the knee joints of miniature pigs. Six weeks after transplantation, the repair tissues were investigated for collagen type I and type II content as well as for HIF-1alpha expression. The functional role of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling on BMP-2/IGF-1-induced HIF-1alpha expression was assessed in vitro by employing specific inhibitors. RESULTS Unstimulated periosteal cells formed a fibrous extracellular matrix in the superficial zone and a fibrocartilaginous matrix in deep zones of the repair tissue. This zonal difference was reflected by the absence of HIF-1alpha staining in superficial areas, but moderate HIF-1alpha expression in deep zones. In contrast, Ad/AAVBMP-2-stimulated periosteal cells, and to a lesser degree Ad/AAVIGF-1-infected cells, adopted a chondrocyte-like phenotype with strong intracellular HIF-1alpha staining throughout all zones of the repair tissue and formed a hyaline-like matrix. In vitro, BMP-2 and IGF-1 supplementation increased HIF-1alpha protein levels in periosteal cells, which was based on posttranscriptional mechanisms rather than de novo mRNA synthesis, involving predominantly the MEK/ERK pathway. CONCLUSION This pilot experimental study on a relatively small number of animals indicated that chondrogenesis by precursor cells is facilitated in deeper hypoxic zones of cartilage repair tissue and is stimulated by growth factors which enhance HIF-1alpha activity.

Treatment of posterior epiphyseal disruption of the medial clavicle with a modified Balser plate

BACKGROUND Acute posterior dislocation of the sternoclavicular joint is a rare but dangerous injury that is often difficult to diagnose. Because of late closure of the medial clavicular epiphysis, epiphyseal disruption must be taken into consideration in patients up to 25 years of age with an apparent diagnosis of posterior sternoclavicular dislocation. We developed a novel method of treating epiphyseal disruptions with a modified Balser plate. METHODS This method was used in a 19-year-old patient with a posterior epiphyseal disruption of the left medial clavicle. After molding of the plate, the hook was introduced into the manubrium sterni and the plate was fixed on the clavicle with screws. RESULTS The new method could be applied safely and achieved a good functional result without any external immobilization. The postoperative course was uneventful. CONCLUSION Fixation of the posterior epiphyseal disruption of the medial clavicle with a modified Balser plate is feasible and may be an alternative to traditional methods.

Paracrine effect of transplanted rib chondrocyte spheroids supports formation of secondary cartilage repair tissue

The studys objective was to investigate if transplanted chondrocyte or periosteal cell spheroids have influence on ingrowing bone marrow‐derived cells in a novel cartilage repair approach in miniature pigs. Autologous rib chondrocytes or periosteal cells were cultured as spheroids and press‐fitted into cavities that were milled into large, superficial chondral lesions of the patellar joint surface. Within the milled cavities, the subchondral bone plate was either penetrated or left intact (full‐thickness or partial‐thickness cavities). The transplantation of chondrocyte spheroids into full‐thickness cavities induced the formation of additional secondary repair cartilage that exceeded the original volume of the transplanted spheroids. The resulting continuous tissue was rich in proteoglycans and stained positive for type II collagen. Cell labeling revealed that secondarily invading repair cells did not originate from transplanted spheroids, but rather from arroded bone marrow. However, secondary invasion of repair cells was less pronounced following transplantation of periosteal cells and absent in partial‐thickness cavities. According to in vitro analyses, these observations could be ascribed to the ability of chondrocyte spheroids to secrete relevant amounts of bone morphogenetic protein‐2, which was not detected for periosteal cells. Transplanted chondrocyte spheroids exert a dual function: they provide cells for the repair tissue and have a stimulatory paracrine activity, which promotes ingrowth and chondrogenesis of bone marrow‐derived cells.

[The vacuum-assisted closure (V.A.C.) and instillation dressing: limb salvage after 3 degrees open fracture with massive bone and soft tissue defect and superinfection].

We report the case of a 17-year-old boy who was hit by a high velocity train. The polytraumatized patient suffered a 3 degrees open femur defect fracture with a substantial loss of the lateral femoral muscles and significant disruption of the soft tissue of the lower leg. The enormous wound areas on the thigh and the lower leg were infected by Pseudomonas aeruginosa, Enterobacter cloacae, and Stenotrophomonas maltophilia. The enormous tissue defects and the superinfection did not leave any hope for saving the limb from amputation. After rapid aggressive debridement and pulsatile lavage, we covered the wounds as a last resort with a new technique of vacuum-assisted closure (V.A.C) and instillation (V.A.C. Instill(R)) dressings. In sequences of 1 min we instilled Lavasept, kept it for 20 min on the wound surface, and exhausted the liquid. We repeated this for 6 consecutive days and then changed the dressing. In the follow-up examinations the number of germs was significantly reduced. During follow-up care we used the V.A.C. treatment without instillation and finally we transplanted skin onto the clean wound surface and were able to save the leg of this young patient. We discharged him with a good function of his lower leg. This technique of V.A.C. Instill seems to offer great possibilities in critically infected wound situations.