Oscar Caballero

Matrix metalloproteinase inhibitors prevent a decrease in the mechanical properties of stress-deprived tendons: an in vitro experimental study.

Background An increase in matrix metalloproteinases (MMPs) and the resulting degradation of the extracellular matrix have been implicated in the pathogenesis of tendinopathy. Studies have documented the beneficial effects of MMP inhibitors used to treat pathologic conditions in which MMP activity has had a negative effect on connective tissues. Hypothesis Matrix metalloproteinase inhibitors will prevent the decrease in material properties associated with tendon stress deprivation by inhibiting MMP activity. Study Design Controlled laboratory study. Methods Rat tail tendons were subjected to 7 days of in vitro stress deprivation with and without the addition of 1 of 2 broad-spectrum MMP inhibitors (doxycycline and ilomastat). The material properties (ultimate tensile stress, strain, and tensile modulus) of the tendons were compared with each other and with fresh control tendons. In addition, tendons from each group were evaluated for MMP-13 messenger RNA expression, MMP-13 protein synthesis, MMP-13 activity, and pericellular matrix morphology. Results Both MMP inhibitors resulted in a statistically significant reduction in MMP activity in 7 day stress-deprived tendons when compared with nontreated, stress-deprived tendons. Similarly, tendons treated with either ilomastat or doxycycline had significantly improved material properties. MMP-13 messenger RNA expression and protein synthesis were not significantly affected by either MMP inhibitor. Both MMP inhibitors were able to maintain the integrity of the pericellular matrix when compared with nontreated, stress-deprived tendons. Conclusion Matrix metalloproteinase inhibitors prevented the activation of MMP-13 and significantly inhibited pericellular matrix degeneration and the loss of material properties associated with stress deprivation. Clinical Relevance Matrix metalloproteinase inhibitors may play a supportive role in the treatment of tendinopathy by limiting the MMP-mediated degradation of the extracellular matrix.

Platelet-rich fibrin constructs elute higher concentrations of transforming growth factor-β1 and increase tendon cell proliferation over time when compared to blood clots: A comparative in vitro analysis

OBJECTIVE To compare the concentration of a representative growth factor (transforming growth factor-beta [TGF-β]1) eluted from a platelet-rich fibrin matrix (PRFMatrix), a platelet-rich fibrin membrane (PRFMembrane), and a whole blood clot (BC) over time, and to compare the mitogenic effect of the eluents from each construct. STUDY DESIGN In vitro study. SAMPLE POPULATION PRFMatrix, PRFMembrane, and BC (n=4/construct/time point). METHODS Each construct was placed in tissue culture wells containing media for 7 days. The media was collected and replenished on days 1, 3, 5, and 7 and the concentration of eluted TGF-β1 was measured by enzyme-linked immunosorbent assay. Canine tendon cells were subjected to additional aliquots of the conditioned media and the amount of cell proliferation compared. RESULTS The media from both PRFM (PRFMatrix and PRFMembrane) constructs contained significantly more (P≤.026) TGF-β1 at days 1 and 3 and produced a significant increase (P≤.044) in cell proliferation at all time points compared with the BC. The PRFMembrane media contained significantly more (P≤.05) TGF-β1 at days 1 and 3 and produced a significant increase (P≤.002) in cell proliferation at all time points compared with the PRFMatrix. CONCLUSIONS Both PRFM constructs are comprised of a dense fibrin scaffold that contains increased concentrations of TGF-β1 and are capable of increasing tendon cell proliferation over time when compared with a BC. CLINICAL RELEVANCE The sustained increase in growth factor availability in PRFM constructs may be beneficial in the healing of biologically compromised tissues.

The effect of stress-deprivation and cyclic loading on the TIMP/MMP ratio in tendon cells: An in vitro experimental study

Purpose. To determine the effect of stress deprivation and cyclic loading on TIMP-1/MMP-13 mRNA expression ratio in rat tail tendon (RTT) cells. Method. Adult RTTs were stress-deprived for 0, 24, 48, or 72 hours in the presence or absence of a MMP inhibitor (ilomastat), or subjected to 1%, 3%, or 6% strain for 24 h under tissue culture conditions. TIMP-1 and MMP-13 (rat interstitial collagenase) mRNA expression were measured using quantitative PCR and TIMP/MMP ratios were calculated for each group. Results. The ratio of TIMP-1 to MMP-13 in control RTTs was 3.73:1 ± 0.73. Stress deprivation for 24 h significantly decreased the TIMP-1/MMP-13 ratio (0.25:1 ± 0.04) and MMP-13 expression continued to increase significantly with time of stress deprivation. Inhibition of MMP-13 mRNA expression with ilomastat in stress-deprived samples did not alter TIMP-1 expression when compared to normal controls. Cyclic loading significantly increased TIMP-1/MMP-13 expression at all strain levels examined. Conclusions. RTTs normally have a positive TIMP-1/MMP-13 expression ratio. While cyclic loading increased the TIMP-1/MMP-13 ratio, loss of cellular homeostatic tension inversed this ratio through a significant increase in MMP-13 mRNA expression rather than a decrease in TIMP expression. A negative TIMP/MMP ratio has been implicated in the pathogenesis of tendinopathy. Increasing the TIMP/MMP ratios in these patients through exercise may be beneficial in the management of tendinopathy.

A Gene Related to Yeast HOS2 Histone Deacetylase Affects Extracellular Depolymerase Expression and Virulence in a Plant Pathogenic Fungus

A gene, HDC1, related to the Saccharomyces cerevisiae histone deacetylase (HDAC) gene HOS2, was isolated from the filamentous fungus Cochliobolus carbonum, a pathogen of maize that makes the HDAC inhibitor HC-toxin. Engineered mutants of HDC1 had smaller and less septate conidia and exhibited an ∼50% reduction in total HDAC activity. Mutants were strongly reduced in virulence as a result of reduced penetration efficiency. Growth of hdc1 mutants in vitro was normal on glucose, slightly decreased on sucrose, and reduced by 30 to 73% on other simple and complex carbohydrates. Extracellular depolymerase activities and expression of the corresponding genes were downregulated in hdc1 mutant strains. Except for altered conidial morphology, the phenotypes of hdc1 mutants were similar to those of C. carbonum strains mutated in ccSNF1 encoding a protein kinase necessary for expression of glucose-repressed genes. These results show that HDC1 has multiple functions in a filamentous fungus and is required for full virulence of C. carbonum on maize.

Evaluation of the use of an autologous platelet-rich fibrin membrane to enhance tendon healing in dogs

OBJECTIVE To examine effects of an autologous platelet-rich fibrin (PRF) membrane for enhancing healing of a defect of the patellar tendon (PT) in dogs. ANIMALS 8 adult dogs. PROCEDURES Defects were created in the central third of the PT in both hind limbs of each dog. An autologous PRF membrane was implanted in 1 defect/dog, and the contralateral defect was left empty. Dogs (n = 4/time period) were euthanized at 4 and 8 weeks after surgery, and tendon healing was assessed grossly and histologically via a semiquantitative scoring system. Cross-sectional area of the PTs was also compared. RESULTS Both treated and control defects were filled with repair tissue by 4 weeks. There was no significant difference in the histologic quality of the repair tissue between control and PRF membrane-treated defects at either time point. At both time points, the cross-sectional area of PRF membrane-treated tendons was significantly greater (at least 2.5-fold as great), compared with that of sham-treated tendons. At 4 weeks, the repair tissue consisted of disorganized proliferative fibrovascular tissue originating predominantly from the fat pad. By 8 weeks, the tissue was less cellular and slightly more organized in both groups. CONCLUSIONS AND CLINICAL RELEVANCE A PRF membrane did not enhance the rate or quality of tendon healing in PT defects. However, it did increase the amount of repair tissue within and surrounding the defect. These results suggested that a PRF membrane may not be indicated for augmenting the repair of acutely injured tendons that are otherwise healthy.

The Location in Cartilage of Infectious Retrovirus in Cats Infected with Feline Leukemia Virus

BACKGROUND Previous studies have suggested that articular cartilage allografts were not likely to transmit infectious retrovirus since viral DNA could not be isolated from chondrocytes of infected individuals. However, the ability of the extracellular matrix of articular cartilage to harbor and transmit a retrovirus has not been examined. We hypothesized that articular cartilage fragments, but not isolated chondrocytes, from cats systemically infected with feline leukemia virus (FeLV) are capable of transmitting infectious retrovirus. METHODS Fresh cartilage segments and chondrocytes isolated from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cocultured with segments of cartilage, isolated chondrocytes, or fragments of cortical bone from each infected cat. The FeLV p27 antigen was measured in the coculture media by enzyme-linked immunosorbent assay. In addition, FeLV proviral nucleic acids were quantified by real-time quantitative polymerase chain reaction with use of DNA extracted from feline embryonic fibroblast cell cocultures as well as isolated chondrocytes. Immunohistochemistry was used to assess for FeLV p27 antigen in both intact cartilage fragments and isolated chondrocytes. RESULTS Feline embryonic fibroblast cells cocultured with cartilage fragments from each of the five FeLV-infected cats all demonstrated high levels of proviral DNA, indicating transmission of infective virus. In addition, media from all cocultures of feline embryonic fibroblast cells and chondral fragments became positive for p27 antigen, indicating active viral replication. In contrast, cocultures of feline embryonic fibroblast cells and isolated chondrocytes from all FeLV-infected cats were negative for proviral DNA and p27 antigen. Likewise, no proviral nucleic acids could be detected in isolated chondrocytes from any infected cats. Cocultures of feline embryonic fibroblast cells with cortical bone fragments were positive for proviral DNA and p27 antigen. Immunohistochemical staining of cartilage fragments from FeLV-infected cats demonstrated the presence of p27 antigen throughout the extracellular matrix, but the p27 antigen was not detected in isolated chondrocytes. CONCLUSIONS Articular cartilage fragments can readily transmit infectious retrovirus, but isolated chondrocytes were likely not the source of the infectious virus because they did not harbor proviral DNA or p27 antigen.