Marla J. Steinbeck

Activation of bovine neutrophils by recombinant interferon-γ

Abstract The effect of recombinant bovine interferon-γ (r-IFN-γ) on neutrophil functions was investigated and compared to the effects of an unpurined lymphokine preparation. Incubation of purified bovine neutrophils with r-IFN-γ or antigen-induced lymphokine for 2.5 hr at 37 °C resulted in impairment of the ability of neutrophils to migrate under agarose, and an enhancement of their ability to mediate antibody-dependent and antibody-independent cell-mediated cytotoxicity against chicken erythrocytes. Neither the lymphokine preparation nor the r-IFN-γ had any influence on Staphylococcus aureus ingestion, or iodination by neutrophils. The lymphokine preparation enhanced cytochrome c reduction by neutrophils and was weakly chemotactic, whereas the r-IFN-γ had neither of these effects. Only 5 min of r-IFN-γ preincubation with neutrophils were needed to trigger protein synthesis by the neutrophils resulting in inhibition of random migration. Therefore, recombinant interferon-γ acts as a neutrophil migration inhibition factor and a neutrophil activation factor resulting in enhanced neutrophil-mediated antibody-dependent and -independent cell-mediated cytotoxicity. Many, but not all, of the in vitro effects of an unpurified lymphokine preparation on neutrophil function can be attributed to the interferon-γ contained in the lymphokine.

Active caspase-3 is required for osteoclast differentiation

Based on our earlier observation that caspase‐3 is present in osteoclasts that are not undergoing apoptosis, we investigated the role of this protein in the differentiation of primary osteoclasts and RAW264.7 cells (Szymczyk KH, et al., 2005, Caspase‐3 activity is necessary for RANKL‐induced osteoclast differentiation. The Proceedings of the 8th ICCBMT). We noted that osteoclast numbers are decreased in long bones of procaspase‐3 knockout mice and that receptor activator of NF‐κB ligand (RANKL) does not promote differentiation of isolated preosteoclasts. In addition, after treatment with inhibitors of caspase‐3 activity, neither the wild‐type primary nor the RAW264.7 cells express TRAP or became multinucleated. We found that immediately following RANKL treatment, procaspase‐3 is cleaved and the activated protein is localized to lipid regions of the plasma membrane and the cytosol. We developed RAW264.7 procaspase‐3 knockdown clonal cell lines using RNAi technology. Again, treatment with RANKL fails to induce TRAP activity or multinucleation. Finally, we evaluated NF‐κB in procaspase‐3 silenced cells. We found that RANKL treatment prevented activation and nuclear translocation of NF‐κB. Together these findings provide direct support for the hypothesis that caspase‐3 activity is required for osteoclast differentiation. J. Cell. Physiol. 209: 836–844, 2006.

Chlorination of Pyridinium Compounds POSSIBLE ROLE OF HYPOCHLORITE, N-CHLORAMINES, AND CHLORINE IN THE OXIDATION OF PYRIDINOLINE CROSS-LINKS OF ARTICULAR CARTILAGE COLLAGEN TYPE II DURING ACUTE INFLAMMATION

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H2O2), hypochlorous acid/hypochlorite (HOCl/OCl−), and singlet oxygen (O2(1Δg)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B6, which share the same chemical structure and spectral properties of Pyd cross-links. Neither H2O2 (125–500 μm) nor O2(1Δg) (10–25 μm) significantly changed the spectral properties of pyridoxamine or vitamin B6. Reaction of HOCl/OCl− (12.5–50 μm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of anN-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B6, which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl2/HOCl/OCl− reacted with both pyridoxamine and vitamin B6. Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl2/HOCl/OCl− on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5.0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.

Mast cells and hypoxia drive tissue metaplasia and heterotopic ossification in idiopathic arthrofibrosis after total knee arthroplasty

BackgroundIdiopathic arthrofibrosis occurs in 3-4% of patients who undergo total knee arthroplasty (TKA). However, little is known about the cellular or molecular changes involved in the onset or progression of this condition. To classify the histomorphologic changes and evaluate potential contributing factors, periarticular tissues from the knees of patients with arthrofibrosis were analyzed for fibroblast and mast cell proliferation, heterotopic ossification, cellular apoptosis, hypoxia and oxidative stress.ResultsThe arthrofibrotic tissue was composed of dense fibroblastic regions, with limited vascularity along the outer edges. Within the fibrotic regions, elevated numbers of chymase/fibroblast growth factor (FGF)-expressing mast cells were observed. In addition, this region contained fibrocartilage and associated heterotopic ossification, which quantitatively correlated with decreased range of motion (stiffness). Fibrotic, fibrocartilage and ossified regions contained few terminal dUTP nick end labeling (TUNEL)-positive or apoptotic cells, despite positive immunostaining for lactate dehydrogenase (LDH)5, a marker of hypoxia, and nitrotyrosine, a marker for protein nitrosylation. LDH5 and nitrotyrosine were found in the same tissue areas, indicating that hypoxic areas within the tissue were associated with increased production of reactive oxygen and nitrogen species.ConclusionsTaken together, we suggest that hypoxia-associated oxidative stress initiates mast cell proliferation and FGF secretion, spurring fibroblast proliferation and tissue fibrosis. Fibroblasts within this hypoxic environment undergo metaplastic transformation to fibrocartilage, followed by heterotopic ossification, resulting in increased joint stiffness. Thus, hypoxia and associated oxidative stress are potential therapeutic targets for fibrosis and metaplastic progression of idiopathic arthrofibrosis after TKA.

Management of Stiffness Following Total Knee Arthroplasty

Stiffness following total knee arthroplasty is a disabling complication1-7. Although some predisposing factors have been identified, in most cases the exact etiology of the stiffness cannot be discerned. The reported prevalence of this complication has ranged widely from 1.3% to 12%1,8-10. The difference in rate may be due in part to varied definitions of stiffness11. Several factors affecting the postoperative range of motion that have been identified include the preoperative range of motion, contracture of the extensor mechanism and capsular structure, the preoperative diagnosis, personality of the patient, lack of patient compliance with the rehabilitation protocol, and the patients threshold for pain12-18. Technical factors, such as overstuffing the patellofemoral joint, mismatch of the flexion and extension gaps, inaccurate ligament balancing, component malpositioning, use of oversized components, joint-line elevation, excessive tightening of the extensor mechanism, and underresection of the patella have also been implicated1,19. Various management protocols have been proposed to address this complication. This exhibit presents our institutional experience with the management of stiffness following total knee arthroplasty. We report the findings of a case-control study that was designed to predict the factors responsible for stiffness after total knee arthroplasty. In addition, the results of an ongoing basic-science study attempting to unravel the molecular mechanism of arthrofibrosis following total knee arthroplasty are presented. We also provide the outline of our current treatment strategy for the management of stiffness following total knee arthroplasty. Fig. 1 Demographic distribution of patients in the study and control groups. TKA = total knee arthroplasty, and BMI = body-mass index. ### Case-Control Study The purpose of this study was to identify factors that predispose patients to stiffness following total knee arthroplasty. With use of an institutional computerized database, the outcome of …

Bone cell survival in microgravity: evidence that modeled microgravity increases osteoblast sensitivity to apoptogens.

Abstract: Studies were performed to evaluate the effects of modeled microgravity on the induction of osteoblast apoptosis. MC3T3‐E1 osteoblast‐like cells were cultured in alginate carriers in the NASA‐approved high aspect ratio vessel (HARV). This system subjects the cells to a time‐averaged gravitational field (vector‐averaged gravity) to simulate low gravity conditions. Cells were cultured in the HARV for five days, and then examined for apoptosis. In simulated microgravity, the cells remained vital, although analysis of expressed genes indicated that there was loss of the mature osteoblast phenotype. Additionally, we noted that there was a loss of the mitochondrial membrane potential, a low level of the antiapoptotic protein Bcl‐2, as well as Akt protein, and the redox status of the cells was disturbed. All of these parameters indicated that vector‐averaged gravity disrupts mitochondrial function, thereby sensitizing osteoblasts to apoptosis. We then used a challenge assay to evaluate the apoptotic sensitivity of the cells subjected to vector‐averaged gravity. When challenged with staurosporine, cells subjected to vector‐averaged gravity evidenced elevated levels of cell death relative to control cell populations. Another objective of the study was to improve upon conventional carriers by using alginate encapsulation to support cells in the HARV. We have demonstrated that the alginate carrier system affords a more robust system than surface‐seeded carriers. This new system has the advantage of shielding cells from mechanical damage and fluid shear stresses on cells in the HARV, permitting carefully controlled studies of the effects of vector‐averaged gravity.

Skeletal cell differentiation is enhanced by atmospheric dielectric barrier discharge plasma treatment.

Enhancing chondrogenic and osteogenic differentiation is of paramount importance in providing effective regenerative therapies and improving the rate of fracture healing. This study investigated the potential of non-thermal atmospheric dielectric barrier discharge plasma (NT-plasma) to enhance chondrocyte and osteoblast proliferation and differentiation. Although the exact mechanism by which NT-plasma interacts with cells is undefined, it is known that during treatment the atmosphere is ionized generating extracellular reactive oxygen and nitrogen species (ROS and RNS) and an electric field. Appropriate NT-plasma conditions were determined using lactate-dehydrogenase release, flow cytometric live/dead assay, flow cytometric cell cycle analysis, and Western blots to evaluate DNA damage and mitochondrial integrity. We observed that specific NT-plasma conditions were required to prevent cell death, and that loss of pre-osteoblastic cell viability was dependent on intracellular ROS and RNS production. To further investigate the involvement of intracellular ROS, fluorescent intracellular dyes Mitosox (superoxide) and dihydrorhodamine (peroxide) were used to assess onset and duration after NT-plasma treatment. Both intracellular superoxide and peroxide were found to increase immediately post NT-plasma treatment. These increases were sustained for one hour but returned to control levels by 24 hr. Using the same treatment conditions, osteogenic differentiation by NT-plasma was assessed and compared to peroxide or osteogenic media containing β-glycerolphosphate. Although both NT-plasma and peroxide induced differentiation-specific gene expression, neither was as effective as the osteogenic media. However, treatment of cells with NT-plasma after 24 hr in osteogenic or chondrogenic media significantly enhanced differentiation as compared to differentiation media alone. The results of this study show that NT-plasma can selectively initiate and amplify ROS signaling to enhance differentiation, and suggest this technology could be used to enhance bone fusion and improve healing after skeletal injury.

The effect of simulated microgravity on osteoblasts is independent of the induction of apoptosis

Bone loss during spaceflight has been attributed, in part, to a reduction in osteoblast number, altered gene expression, and an increase in cell death. To test the hypothesis that microgravity induces osteoblast apoptosis and suppresses the mature phenotype, we created a novel system to simulate spaceflight microgravity combining control and experimental cells within the same in vitro environment. Cells were encapsulated into two types of alginate carriers: non‐rotationally stabilized (simulated microgravity) and rotationally stabilized (normal gravity). Using these specialized carriers, we were able to culture MC3T3‐E1 osteoblast‐like cells for 1–14 days in simulated microgravity and normal gravity in the same rotating wall vessel (RWV). The viability of cells was not affected by simulated microgravity, nor was the reductive reserve. To determine if simulated microgravity sensitized the osteoblasts to apoptogens, cells were challenged with staurosporine or sodium nitroprusside and the cell death was measured. Simulated microgravity did not alter the sensitivity of C3H10T‐1/2 stem cells, MC3T3‐E1 osteoblast‐like cells, or MLO‐A5 osteocyte‐like cells to the action of these agents. RT‐PCR analysis indicated that MC3T3‐E1 osteoblasts maintained expression of RUNX2, osteocalcin, and collagen type I, but alkaline phosphatase expression was decreased in cells subjected to simulated microgravity for 5 days. We conclude that osteoblast apoptosis is not induced by vector‐averaged gravity, thus suggesting that microgravity does not directly induce osteoblast death. J. Cell. Biochem. 102: 483–495, 2007.

Retrieval Analysis of a ProDisc-L Total Disc Replacement

Study Design We retrieved a functioning ProDisc-L total disc replacement and associated tissues at 16 months of service life. Objective To analyze a previously unreported mode of implant malpositioning, wear mechanisms, and polyethylene locking mechanism, and to study retrieved periprosthetic tissues. Summary of Background Data The clinical performance of polyethylene in the context of total disc replacements remains poorly understood. In the ProDisc-L, the polyethylene core is fixed to the inferior metal endplate through a mechanical interference locking mechanism similar to those used in tibial total knee components. This case represents the third report of an explanted ProDisc-L prosthesis, and the first reported case of posterior malpositioning with this device. Methods The implant was removed via a transperitoneal approach. Its polyethylene core was evaluated for burnishing, fracture, third-body abrasion, and permanent deformation. An identical, never-implanted set of polyethylene and endplate components served as controls for the microscopic evaluation of wear. Two tissue samples were collected from a region adjacent to the failed implant to evaluate tissue morphology and inflammation. Hematoxylin and eosin-stained tissue sections were also evaluated for the presence of polyethylene debris by polarized light microscopy. Results The implant was removed without serious incident, although there were incidental venotomies. The patient went on to solid arthrodesis. We found minimal wear, oxidation, and periprosthetic tissue reaction, as might be expected given the short-term duration of implantation and its reason for revision. No evidence was found of malfunction or improper deployment of the locking mechanism. Burnishing seemed to be the result of short-term impingement. Some areas of the tissue matrix showed evidence of early cell degeneration, and some of these areas contained polyethylene particles identified by polarized light microscopy. Conclusions A larger series of implant retrievals will be needed to investigate possible wear and the biologic response to increased particle generation.

Characteristics of highly cross-linked polyethylene wear debris in vivo†

Despite the widespread implementation of highly cross-linked polyethylene (HXLPE) liners to reduce the clinical incidence of osteolysis, it is not known if the improved wear resistance will outweigh the inflammatory potential of HXLPE wear debris generated in vivo. Thus, we asked: What are the differences in size, shape, number, and biological activity of polyethylene wear particles obtained from primary total hip arthroplasty revision surgery of conventional polyethylene (CPE) versus remelted or annealed HXLPE liners? Pseudocapsular tissue samples were collected from revision surgery of CPE and HXLPE (annealed and remelted) liners, and digested using nitric acid. The isolated polyethylene wear particles were evaluated using scanning electron microscopy. Tissues from both HXLPE cohorts contained an increased percentage of submicron particles compared to the CPE cohort. However, the total number of particles was lower for both HXLPE cohorts, as a result there was no significant difference in the volume fraction distribution and specific biological activity (SBA; the relative biological activity per unit volume) between cohorts. In contrast, based on the decreased size and number of HXLPE wear debris there was a significant decrease in total particle volume (mm(3)/g of tissue). Accordingly, when the SBA was normalized by total particle volume (mm(3)/gm tissue) or by component wear volume rate (mm(3)/year), functional biological activity of the HXLPE wear debris was significantly decreased compared to the CPE cohort. Indications for this study are that the osteolytic potential of wear debris generated by HXLPE liners in vivo is significantly reduced by improvements in polyethylene wear resistance.