A novel two-step method for the formation of tissue-engineered cartilage by mature bovine chondrocytes: the alginate-recovered-chondrocyte (ARC) method.

Most attempts to tissue-engineer cartilage have involved seeding of cultured cells into a biological or synthetic scaffold. We have developed a novel two-step culture approach that makes possible the in vitro formation of cartilaginous-like tissue by mature adult bovine chondrocytes without the aid of a synthetic matrix. The first step consists of culturing chondrocytes under conditions that maintain their rounded shape and their molecular phenotype as assessed by type II collagen and aggrecan production. This step was accomplished by culturing the isolated chondrocytes in alginate beads until the cells have reestablished a proteoglycan-rich cell-associated matrix (CM). The second step consists of culturing the cells with their CM, after recovery from the beads, on a tissue culture insert with a porous membrane. In this study, young adult bovine articular chondrocytes were cultured in alginate beads in the presence of 10% or 20% fetal bovine serum (FBS). After 7 days of culture, the alginate beads were dissolved by incubating the beads for 20 min in sodium citrate buffer, a calcium chelator. Following a brief centrifugation, the cells with their CM were recovered, resuspended in medium containing 10% or 20% FBS and seeded onto a tissue culture insert. After 1 week of culture on the insert, the individual cells with their CM progressively became incorporated into a mass of cartilaginous tissue. Culture with 20% FBS resulted in the best formation of tissues. These tissues, easily recovered from the insert, were then subjected to biochemical and histological analyses. The biochemical results showed that the chondrocytes remain phenotypically stable in the tissues. The de novo tissue has a relatively high ratio of PG/collagen. Histological examination of the tissue revealed it contained a cartilage-like matrix strongly stained with toluidine blue. This scaffold-free system appears ideal to study, in vitro, the development of transplantable cartilaginous tissue.     

Repair of large midline incisional hernias with polypropylene mesh: Comparison of three operative techniques

Polypropylene mesh is widely used for the reconstruction of incisional hernias that cannot be closed primarily. Several techniques have been advocated to implant the mesh. The objective of this study was to evaluate, retrospectively, early and late results of three different techniques, onlay, inlay, and underlay. The records of 53 consecutive patients with a large midline incisional hernia — 25 women and 28 men, mean age 60.4 (range 28–94) — were reviewed. Polypropylene mesh was implanted using the onlay technique in 13 patients, inlay in 23 patients, and underlay in 17 patients. Either the greater omentum or a polyglactin mesh was interponated between the mesh and the viscera. The records of these 53 patients were reviewed with respect to: size and cause of the hernia, pre- and postoperative mortality and morbidity, with special attention to wound complications. Patients were invited to attend the outpatient clinic at least 12 months after implantation of the mesh for physical examination of the abdominal wall. Postoperative complications occurred in 14 (26.4%) patients. The onlay technique had significantly more complications, as compared to both other techniques. Reherniation occurred in 15 (28.3%) patients. The reherniation rate of the inlay technique was significantly higher than after the underlay technique (44% vs 12%, P=0.03) and tended to be higher than the onlay technique (44% vs 23%, P=0.22). Repair of large midline incisional hernias with the use of a polypropylene mesh carries a high risk of complications and has a high reherniation rate. The underlay technique seems to be the better technique.     

Nonheme iron in sickle erythrocyte membranes: association with phospholipids and potential role in lipid peroxidation

Previous studies documented the abnormal association of heme and heme proteins with the sickle RBC membrane. We have now examined RBC ghosts and inside-out membranes (IOM) for the presence of nonheme iron as detected by its formation of a colored complex with ferrozine. Sickle ghosts have 33.8 +/- 18.2 nmol nonheme iron/mg membrane protein, and sickle IOM have 4.3 +/- 3.0 nmol/mg. In contrast, normal RBC ghosts and IOM have no detectable nonheme iron. The combination of heme and nonheme iron in sickle IOM averages nine times the amount of membrane- associated iron in normal IOM. Kinetics of the ferrozine reaction show that some of this nonheme iron on IOM reacts slowly and is probably in the form of ferritin, but most (72% +/- 18%) reacts rapidly and is in the form of some other biologic chelate. The latter iron compartment is removed by deferoxamine and by treatment of IOM with phospholipase D, which suggests that it represents an abnormal association of iron with polar head groups of aminophospholipids. The biologic feasibility of such a chelate was demonstrated by using an admixture of iron with model liposomes. Even in the presence of tenfold excess adenosine diphosphate, iron partitions readily into phosphatidylserine liposomes; there is no detectable association with phosphatidylcholine liposomes. To examine the bioavailability of membrane iron, we admixed membranes and t-butylhydroperoxide and found that sickle membranes show a tenfold greater peroxidation response than do normal membranes. This is not due simply to a deficiency of vitamin E, and this is profoundly inhibited by deferoxamine. Thus, while thiol oxidation in sickle membranes previously was shown to correlate with heme iron, the present data suggest that lipid peroxidation is related to nonheme iron. In control studies, we did not find this pathologic association of nonferritin, nonheme iron with IOM prepared from sickle trait, high-reticulocyte, postsplenectomy, or iron-overloaded individuals. These data provide additional support for the concept that iron decompartmentalization is a characteristic of sickle RBCs.     

Topical high-molecular-weight hyaluronan and a roofing barrier sheet equally inhibit postlaminectomy fibrosis.

BACKGROUND CONTEXT: The relevance of epidural fibrosis to failed back surgical outcomes remains controversial. Previous studies on the correlation between epidural fibrosis and clinical outcome after laminectomy are inconclusive, and clinical approaches applied to reduce postlaminectomy spinal canal scarring have produced mixed outcomes. PURPOSE: Improved preclinical models are required to address the fundamental question of the relationship between postlaminectomy fibrosis and chronic pain. This study is directed at establishing small animal postlaminectomy models characterized by significantly reduced scar within the spinal canal postoperatively. Such preclinical models are offered as a platform for future studies to explore the potential relationship between postlaminectomy epidural fibrosis and persistent neuropathy with its potential for altered spinal mechanisms for pain processing, so-called spinal facilitation. Such experiments could be constructed in these models for comparison of pain behavior and its underlying neurochemistry both in the presence and absence of extensive postlaminectomy epidural scar. STUDY DESIGN/SETTING: A modified rat laminectomy model was employed to assess epidural fibrosis using a quantitative biochemical collagen assessment approach along with correlative histology. This group served as the control for comparison with groups in which antifibrotic measures were employed. We compared antifibrotic efficacy of a bioabsorbable roofing barrier sheet placed over the laminectomy defect with topical high-molecular-weight hyaluronan (HMW HA) gel, each applied postoperatively to prevent proliferative epidural scarring. Routine biomechanical tensile strength testing was employed to assess wound-healing strength. METHODS: A bilateral laminectomy (L5 and L6) with associated unilateral disc injury (L5-L6) was performed in 98 male Harlan Sprague-Dawley rats. The laminectomy models described incorporated a unilateral disc injury at L5-L6 because herniated disc material has been shown to contribute proinflammatory cytokines in the postoperative wound. Five groups were employed for the study: 1) normal controls without surgery; 2) a laminectomy-disc injury group without treatment; 3) a laminectomy-disc injury group treated with topical HMW HA gel; 4) a laminectomy-disc injury group treated with 0.2-mm thick bioabsorbable roofing barrier sheet in which a protected space was maintained between overlying paraspinous muscles and the dura and 5) a 0.02-mm thin barrier sheet treatment group in which the sheet was placed directly on the dura. The animals were sacrificed at 3- and 8-week postoperative intervals for analysis. The dissected specimens were studied biochemically for hydroxyproline content to estimate total collagen within the canal and on the dura between L4 and L7. Additional specimens were prepared histologically and stained with Masson-Goldner Trichrome stain to confirm presence of proliferative collagen and to describe the presence or absence of wound-healing scar adherence to the dura. The surgical incisions were studied biomechanically by uniaxial tensile testing to determine ultimate force, strain and prefailure stiffness. Statistics were performed using analysis of variance. RESULTS: Gross appearance and histology studies showed that the untreated laminectomy group demonstrated postoperative scar formation that is adherent between the wound and the dorsum of the dura mater in both 3- and 8-week groups. Proliferative scar was substantially increased grossly between the 3- and 8-week intervals. By gross observation there was adherence of the L5 spinal nerve to the underlying disc and adjacent pedicle on the disc injury side. Gross observation of treatment groups, in contrast, disclosed that both the 0.2-mm thick roofing barrier sheet and topical HMW HA gel each prevented scar attachment to the dural sleeve at both the 3- and 8-week postoperative intervals. Furthermore, both the HMW HA gel and 0.2-mm thick roofing barrier sheet treatment groups had significant reduction of total collagen content in the laminectomy specimens measured biochemically at the two time periods compared with the untreated controls. Histologically, the HMW HA gel and the 0.2-mm thick barrier sheet findings were consistent with the gross observations concerning lack of adherence between scar of the overlying wound and the dura. Notably, both the 0.2- and the 0.02-mm barrier sheets became enveloped by a fibrotic envelope consistent with a foreign body reaction. In the group in which the 0.02-mm thin sheet was placed within the canal on top of the dura, there was an increase of fibrosis around the sheet within the canal leading to a space-occupying mass within the canal. Although the 0.2-mm thick roofing barrier placed external to the canal became enveloped by scar, it appeared to attract proliferative scar away from the epidural space, leaving the dura relatively free of scarring or adherence to overlying tissues. The mechanical properties of the incisional wound increased significantly between 3 and 8 weeks. The ultimate strength, stress, strain and stiffness of the several groups were similar at each time point. CONCLUSION: These results provide two preclinical rat laminectomy models of potential usefulness for the future study of the relevance of epidural fibrosis to behaviorally defined pain states, and for the study of the potential of an altered neurochemical signature in postlaminectomy pain conditions. Such preclinical models have become standard in studies of pain behavior and its neurochemistry in preclinical sciatic nerve and spinal nerve injury models, and should be of utility in the studies of postlaminectomy fibrosis. There was progressive scar proliferation and maturation in the untreated postlaminectomy group in the postoperative interval between 3 and 8 weeks. HMW HA gel applied topically and a 0.2-mm thick bioabsorbable Macropore sheet used as a roofing barrier each significantly reduced postlaminectomy proliferative scar without affecting the integrity of incisional wound healing. However, if the 0.02-mm thin barrier sheet used in this study is placed within the canal in contact with the dura and adjacent to the pedicles, the process of reabsorption results in a fibrotic mass within the canal. The preferred barrier sheet placement for this model is clearly in a roofing position bridging over the open epidural space. It must be placed in a manner to block off the paraspinous muscle healing response and still leave a gap between the sheet and the dura.     

Female and male partner age and menotrophin requirements influence pregnancy rates with human menopausal gonadotrophin therapy in combination with intrauterine insemination

This study analyses the influence of female and male patient age and human menopausal gonadotrophin (HMG) requirements on clinical pregnancy rates and live birth rates with ovulation stimulation using HMG in combination with intrauterine insemination (IUI). In this study, 363 consecutive HMG/IUI treatment cycles in 184 patients carried out at a university fertility centre were analysed in a retrospective fashion. The main outcomes measured were clinical pregnancy rates and live birth rates. Increased female partner age (> or = 35) and male partner age (> or = 40) were found to negatively influence pregnancy rates with HMG/ IUI therapy. In addition, this study demonstrated a critical threshold of HMG requirements beyond which pregnancy did not occur. No pregnancies occurred in treatment cycles requiring > 25 ampoules (1875 IU) of menotrophins to achieve follicular maturity, irrespective of patient age. In conclusion, female partner age, male partner age, and HMG requirements all significantly influence pregnancy rates with HMG/IUI therapy.      

The intron 4c allele of the NOS3 gene is associated with ischemic stroke in African Americans

Background  

Ischemic stroke is the most common cause of disability in North America and in addition to the generally accepted risk factors, there is increasing evidence for the potential pathophysiological role of genes. One of these genes, the endothelial nitric oxide synthase gene (NOS3) has been reported as a genetic risk factor for ischemic stroke. To independently confirm and extend the results of these previous reports, we investigated this gene as a risk factor for stroke in an ethnically diverse study population.