Matrix-Induced Autologous Chondrocyte Implantation (MACI) Using a Cell-Seeded Collagen Membrane Improves Cartilage Healing in the Equine Model.

BACKGROUND: Autologous chondrocyte implantation (ACI) using a collagen scaffold (matrix-induced ACI; MACI) is a next-generation approach to traditional ACI that provides the benefit of autologous cells and guided tissue regeneration using a biocompatible collagen scaffold. The MACI implant also has inherent advantages including surgical implantation via arthroscopy or miniarthrotomy, the elimination of periosteal harvest, and the use of tissue adhesive in lieu of sutures. This study evaluated the efficacy of the MACI implant in an equine full-thickness cartilage defect model at 1 year. METHODS: Autologous chondrocytes were seeded onto a collagen type-I/III membrane and implanted into one of two 15-mm defects in the femoral trochlear ridge of 24 horses. Control defects either were implanted with cell-free collagen type-I/III membrane (12 horses) or were left ungrafted as empty defects (12 horses). An additional 3 horses had both 15-mm defects remain empty as nonimplanted joints. The repair was scored by second-look arthroscopy (12 weeks), and necropsy examination (53 weeks). Healing was assessed by arthroscopic scoring, gross assessment, histology and immunohistology, cartilage matrix component assay, and gene expression determination. Toxicity was examined by prostaglandin E2 formation in joint fluid, and lymph node morphology combined with histologic screening of organs. RESULTS: MACI-implanted defects had improved gross healing and composite histologic scores, as well as increases in chondrocyte predominance, toluidine blue-stained matrix, and collagen type-II content compared with scaffold-only implanted or empty defects. There was minimal evidence of reaction to the implant in the synovial membrane (minor perivascular cuffing), subchondral bone, or cartilage. There were no adverse clinical effects, signs of organ toxicity, or evidence of chondrocytes or collagen type-I/III membrane in draining lymph nodes. CONCLUSIONS: The MACI implant appeared to improve cartilage healing in a critical-sized defect in the equine model compared with collagen matrix alone. CLINICAL RELEVANCE: These results indicate that the MACI implant is quick to insert, provides chondrocyte security in the defect, and improves cartilage healing compared with ACI.

RNA Interference Mediated Interleukin-1ß Silencing in Inflamed Chondrocytes Decreases Target and Downstream Catabolic Responses.

Posttraumatic activation of the catabolic cascade plays a major role in degradation of cartilage. Interleukin-1ß (IL-1ß), a primary instigator in the catabolic axis, is upregulated in chondrocytes following injury. IL-1ß activates key degradative enzymes, including MMPs and aggrecanases, and other proinflammatory mediators such as PGE2 which contribute to ECM breakdown. Posttranscriptional silencing of IL-1ß by RNA interference (RNAi) may drive a reduction in IL-1ß. We hypothesized that transduction of chondrocytes using rAAV2 expressing a short hairpin RNAi motif targeting IL-1ß (shIL-1ß) would significantly decrease IL-1ß expression and, in turn, decrease expression of other catabolic enzymes. Chondrocyte cultures were transduced with rAAV2-tdT-shIL-1ß in serum-free media. The fluorescent protein, tdTomato, was used to determine transduction efficiency via flow cytometry and fluorescent microscopy. Cells were stimulated with lipopolysaccharide (LPS) 48 hours following transduction. After 24-hour stimulation, supernatants were collected for cytokine analysis, and cells lysed for gene expression analysis. IL-1ß knockdown led to significantly decreased expression of IL-1ß, TNF-α, and ADAMTS5. PGE2 synthesis was also significantly downregulated. Overall, effective silencing of IL-1ß using rAAV2 vector expressing a short hairpin IL-1ß knockdown sequence was shown. Additionally, significant downstream effects were evident, including decreased expression of TNF-α and ADAMTS5. Targeted silencing of catabolic cytokines may provide a promising treatment avenue for osteoarthritic (OA) joints.

Gene therapy in musculoskeletal repair.

Local and regional gene therapy has improved healing in preclinical trials of articular and other muculoskeletal conditions. Combinations of cell supplementation and cells overexpressing growth factor genes have shown promising results for improving cartilage repair, enhancing delayed union of fractures, and driving organized tendon repair. Proof of concept has been developed using viral vectors, predominantly adenovirus, to deliver growth factor genes, such as BMP-2, TGF-beta1, and IGF-I. Integrating vectors, such as retrovirus and lentivirus, have improved the duration of gene-induced repair, however, increased risk factors have limited broad application. Cartilage repair can be improved using chondrocyte or stem cell transplantation with cells expressing IGF-I, BMP-2, or FGF-2. In cartilage injury and secondary osteoarthritis models, a combination of IL-1 knockdown and growth factor supplementation has restored cartilage matrix and stabilized the osteoarthritic process. Ultimately, nonviral vectors may provide similar control of catabolic activity in cartilage and synovial structures, which may further improve outcome after chondrocyte or mesenchymal stem cell (MSC) implantation. MSCs derived from bone marrow, fat, or other connective tissues provide a multipotent cell source that may be privileged vectors for skeletal gene therapy. MSCs expressing BMP-2, TGF-beta1, LMP-1, IGF-I, or GDF-5 have enhanced cartilage, bone, and tendon repair. Overall, the field of orthopedic gene therapy for enhanced tissue repair has made significant preclinical advances. Combining existing cell transplant technology to deliver differentiated cells in a minimally invasive way, with genes that improve matrix formation, provides a manageable protocol for a persisting anabolic impact.

Effects of gpx4 haploid insufficiency on GPx4 activity, selenium concentration, and paraquat-induced protein oxidation in murine tissues.

Selenium-dependent glutathione peroxidase-4 (GPx4) catalyzes the reduction of phospholipid hydroperoxides. Because a full gpx4 knockout is embryonic lethal, we examined the effect of deletion of one copy of gpx4 on the activities of three selenoperoxidases (GPx1, GPx3, and GPx4), selenium concentrations, and pro-oxidant-induced protein oxidation in various tissues of mice. A total of 32 gpx4 hemizygous (GPx4+/-) and wild-type (WT) mice (8- to 10-weeks old; 16 males and 16 females) were fed a selenium-adequate diet and given an intraperitoneal injection of paraquat (PQ; 24 mg/kg body wt) or phosphate-buffered saline (PBS). All mice were euthanized 4 hrs after injection to collect tissues for analyses. In PBS-treated mice, GPx4 activities in lung, liver, kidney, and testes of GPx4+/- mice were 24-39% lower (P < 0.05) than in WT mice. Among PQ-treated mice, only testis GPx4 activity in GPx4+/- mice was significantly lower (54% P < 0.05) than WT mice. Selenium concentration in testes, but not in other tissues, was reduced (34% P < 0.05) in GPx4+/- mice compared with WT mice, irrespective of treatment. Tissue GPx1 activities and plasma GPx3 and alanine aminotransferase (ALT) activities were unaffected by PQ treatment or gpx4 hemizygosity. Total protein carbonyl was elevated (73% P < 0.05) by PQ only in lung, and this effect of PQ was independent of genotypes. In conclusion, gpx4 haploid insufficiency reduced GPx4 activities and/or selenium concentrations, but had no effect on pro-oxidant-induced protein oxidation in various tissues of mice.

Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics.

Analysis of 16S rRNA gene sequences has become the primary method for determining prokaryotic phylogeny. Phylogeny is currently the basis for prokaryotic systematics. Therefore, the validity of 16S rRNA gene-based phylogenetic analyses is of fundamental importance for prokaryotic systematics. Discrepancies between 16S rRNA gene analyses and DNA-DNA hybridization and phenotypic analyses have been noted in the genus Helicobacter. To clarify these discrepancies, we sequenced the 23S rRNA genes for 55 helicobacter strains representing 41 taxa (>2,700 bases per sequence). Phylogenetic-tree construction using neighbor-joining, parsimony, and maximum likelihood methods for 23S rRNA gene sequence data yielded stable trees which were consistent with other phenotypic and genotypic methods. The 16S rRNA gene sequence-derived trees were discordant with the 23S rRNA gene trees and other data. Discrepant 16S rRNA gene sequence data for the helicobacters are consistent with the horizontal transfer of 16S rRNA gene fragments and the creation of mosaic molecules with loss of phylogenetic information. These results suggest that taxonomic decisions must be supported by other phylogenetically informative macromolecules, such as the 23S rRNA gene, when 16S rRNA gene-derived phylogeny is discordant with other credible phenotypic and genotypic methods. This study found Wolinella succinogenes to branch with the unsheathed-flagellum cluster of helicobacters by 23S rRNA gene analyses and whole-genome comparisons. This study also found intervening sequences (IVSs) in the 23S rRNA genes of strains of 12 Helicobacter species. IVSs were found in helices 10, 25, and 45, as well as between helices 31' and 27'. Simultaneous insertion of IVSs at three sites was found in H. mesocricetorum.

Absence of photoreceptor rescue with D-cis-diltiazem in the rd mouse.

PURPOSE: Because of a previous report suggesting that D-cis-diltiazem slows retinal degeneration in rd mice, this study was undertaken to examine the effect of D-cis-diltiazem on photoreceptor structure and function in this line of mice. METHODS: Mice were randomly assigned to daily intraperitoneal injections of D-cis-diltiazem or saline between postnatal days 9 and 24. On postnatal day 26 or 27, retinal function was assessed by recording dark-adapted bright-flash ERGs in all animals. Retinal morphology was examined in fixed sections and in immunolabeled frozen sections. Examiners were masked to the treatment group assignment. RESULTS: On postnatal days 26 and 27, diltiazem- and saline-treated mice had only one row of remaining photoreceptor cells throughout most of the central retina. Cone cells in the periphery had remnants of inner segments. Total cell counts and separate counts of rod and cone photoreceptor cells by immunostaining were similar in the diltiazem- versus saline-treated mice. Both groups of mice had, on average, comparable subnormal ERG amplitudes. CONCLUSIONS: D-cis-Diltiazem had no detectable effect on preservation of photoreceptor structure and function in rd mice.