Comparison of two PCR protocols for the detection of Neospora caninum DNA in rodents.

The objective of the present study was, due to increasing interest in the epidemiological role of small mammals as potential reservoir of Neospora caninum, to compare two different PCR protocols for the diagnosis of N. caninum in rodents. We tested tissue samples from 50 house mice (Mus musculus), 50 rats (Rattus norvegicus) and 35 field mice (Apodemus sylvaticus). Two different PCR protocols based on primer pairs, Np4-Np7 and Np6plus-Np21plus, were used for diagnosis on these samples. While there were not mismatches between the results of both PCR from rats or field mice, 49 out of 50 samples from house mice gave positive results with Np4-Np7 primer set. However after cloning and sequencing the PCR products, only six of these were confirmed to be N. caninum, while all the other 43 amplicons matched with house mice DNA sequence from clone RP23-14F5 on chromosome 11 sequence. Our results evidence that Np4-Np7 PCR could not be reliable in diagnosis of N. caninum in rodents.

Evidence of Neospora caninum DNA in wild rodents.

Seventy-five house mice (Mus musculus), 103 rats (Rattus norvegicus) and 55 field mice (Apodemus sylvaticus) from North-West Italy were PCR analysed for Neospora caninum infection. Brain, kidney and muscle tissues collected from the above mentioned animals were tested by PCR using Np6 and Np21 primers. The brain tissue from 2 house mice and 2 rats, the kidney from 4 rats, 1 house mouse and 1 field mouse and muscle from 10 rats, 8 house mice and 1 field mouse were tested positive for N. caninum. Sequencing showed a 96-97% identity of PCR products with N. caninum NC1 sequence. Our findings support previous report on house mouse and rat, and for the first time, provides the evidence of the infection also in field mice. Based on our data, it could be hypothesized that mice can act as a reservoir of N. caninum, and they can play a role in maintaining/spreading N. caninum infection also in the sylvatic cycle. The possibility that dogs could be infected by eating infected house mice suggests new opportunities for N. caninum prophylaxis and control.

Porous structure of ion exchange membranes investigated by various techniques.

A comparative review of various techniques is provided: mercury intrusion porosimetry, nitrogen sorption porosimetry, differential scanning calorimetry (DSC)-based thermoporosimetry, and standard contact porosimetry (SCP), which allows determining pore volume distribution versus pore radius/water binding energy in ion-exchange membranes (IEMs). IEMs in the swollen state have a labile structure involving micro-, meso- and macropores, whose size is a function of the external water vapor pressure. For such materials, the most appropriate methods for quantifying their porosity are DSC and SCP. Especially significant information is given by the SCP method allowing measuring porosimetric curves in a very large pore size range from 1 to 105nm. Experimental results of water distribution in homogeneous and heterogeneous commercial and modified IEMs are presented. The effect of various factors on water distribution is reviewed, i.e. nature of polymeric matrix and functional groups, method for membrane preparation, membrane ageing. A special attention is given to the effect of membrane modification by embedding nanoparticles in their structure. The porosimetric curves are considered along with the results of electrochemical characterization involving the measurements of membrane conductivity, as well as diffusion and electroosmotic permeability. It is shown that addition of nanoparticles may lead to either increase or decrease of water content in IEMs, different ranges of pore size being affected. Hybrid membranes modified with hydrated zirconium dioxide exhibit much higher permselectivity in comparison with the pristine membranes. The diversity of the responses of membrane properties to their modification allows for formation of membranes suitable for fuel cells, electrodialysis or other applications.

Biomechanical analysis of an interference screw and a novel twist lock screw design for bone graft fixation.

BACKGROUND: Malpositioning of an anterior cruciate ligament graft during reconstruction can occur during screw fixation. The purpose of this study is to compare the fixation biomechanics of a conventional interference screw with a novel Twist Lock Screw, a rectangular shaped locking screw that is designed to address limitations of graft positioning and tensioning. METHODS: Synthetic bone (10, 15, 20lb per cubic foot) were used simulating soft, moderate, and dense cancellous bone. Screw push-out and graft push-out tests were performed using conventional and twist lock screws. Maximum load and torque of insertion were measured. FINDINGS: Max load measured in screw push out with twist lock screw was 64%, 60%, 57% of that measured with conventional screw in soft, moderate and dense material, respectively. Twist lock max load was 78% and 82% of that with conventional screw in soft and moderate densities. In the highest bone density, max loads were comparable in the two systems. Torque of insertion with twist lock was significantly lower than with conventional interference screw. INTERPRETATION: Based on geometric consideration, the twist lock screw is expected to have 35% the holding power of a cylindrical screw. Yet, results indicate that holding power was greater than theoretical consideration, possibly due to lower friction and lower preloaded force. During graft push out in the densest material, comparable max loads were achieved with both systems, suggesting that fixation of higher density bone, which is observed in young athletes that require reconstruction, can be achieved with the twist lock screw.

Ameloblastic fibroma in an alpine chamois (Rupicapra rupicapra).

Spontaneous odontogenic tumors are neoplasms characterized by a mixed odontogenic ectomesenchymal and odontogenic epithelial origin; they are rare in both humans and animals. A 3-year-old male Alpine Chamois (Rupicapra rupicapra) was found dead in north-west Italy, and was referred for the necropsy to the Department of Veterinary Sciences of the University of Turin (Italy). At the external examination a 10 × 8 cm, exophytic, red-pink, smooth, firm and ulcerated mass was observed on the inferior lip. Histologically the tumor was characterized by spindle shaped cells arranged in bundles in an abundant hyaline matrix. Multifocal and rare chords of odontogenic epithelium mixed with rare melanocytes that penetrate the neoplasia were visible. Immunohistochemistry showed a clear cytokeratin positivity of epithelial clusters. Macroscopical, histological and immunohistochemical findings were consistent with a diagnosis of locally infiltrative ameloblastic fibroma. To our best knowledge, this is the first report of this tumor in a wild ungulate and in Alpine Chamois.

Chondrocytes isolated from mature articular cartilage retain the capacity to form functional gap junctions.

The distribution, expression, and functionality of gap junctions was examined in bovine chondrocytes (BCs) isolated from mature articular cartilage. BC cells displayed immunoreactivity for connexin 43 (Cx43), a specific gap junction protein. Cx43 protein expression was confirmed by Western blot analysis, and Cx43 mRNA was detected by nuclease protection assay. Additionally, BCs were shown to be functionally coupled, as revealed by dye transfer studies, and octanol, a gap junction uncoupler, greatly attenuated coupling. Furthermore, confocal microscopy of fluo-3 loaded BC cells revealed that deformation-induced cytosolic Ca2+ ion (Ca2+) signals propagated from cell-to-cell via gap junctions. To our knowledge, this is the first evidence suggesting that chondrocytes isolated from adult articular cartilage express functional gap junctions.

Clonal origin of dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans (DFSP) is a malignant tumor originating in the dermis. Although it is known to be locally aggressive, it only rarely metastasizes and will recur unless completely excised. The exact cell responsible for the development of a DFSP has been a matter of controversy for several decades; however, most histochemical and electron microscopic studies support a fibroblastic origin, with the tumor cells staining uniformly for vimentin and containing active endoplasmic reticulum synthesizing collagen. Cytogenetic analysis of some of these tumors has demonstrated at least two specific chromosomal abnormalities in DFSP and suggested that this tumor may be polyclonal in origin. To further address the clonal origin of this locally invasive, mesenchymal tumor, we analyzed DNA from two female patients by restriction fragment length polymorphisms and methylation analysis. Our data strongly support the concept that DFSP is monoclonal in origin and that this tumor mass reflects the clonal expansion of a single cell.

Extracellular matrix protein gene expression of bovine chondrocytes cultured on resorbable scaffolds.

It has been demonstrated that using cultured chondrocytes that have been seeded onto various biomatrices can enhance the quality of the articular cartilage repair tissue. As tissue-engineering becomes increasingly more complex there is a need to understand how a specific biomaterial may influence gene expression. In this study several commonly used scaffold materials for cartilage tissue engineering were evaluated with respect to their influence on matrix gene expression. Primary cultures of bovine chondrocytes were established in monolayer then seeded onto polylactic acid (PLLA), polyglycolic acid (PGA), collagen matrices. The induction of collagen type I, collagen type II, and aggrecan was observed at various time points on these biomaterials using RT-PCR. The collagen type I gene was upregulated on collagen scaffolds throughout the culture period. PLLA and PGA showed initial induction followed by downregulation. Monolayer culture did not induce collagen I message. Collagen II genes were selectively upregulated after 72 and 96 h post seeding depending the scaffold material. Monolayer culture had strong induction of collagen II. The aggrecan protein was consistently expressed in all scaffold materials cultures and monolayer.

Fibroblast growth factor in chick osteogenesis.

In vivo effects of FGF on osteogenesis in the chick embryo were evaluated. Day-11 embryos were injected with FGF followed by radiotracers. 3H-thymidine labelling of osteogenic cells was significantly higher following FGF administration; 3H-proline labelling over bone matrix was greater in the controls. Cartilage cells and matrix were sparsely labelled indicating a low level of metabolic activity. These data provide the first in vivo evidence that FGF stimulates osteogenic cell proliferation, while inhibiting production of bone matrix collagen. A role for FGF in embryonic osteogenesis and in fracture repair is suggested.

Tgf-Beta promotes the growth of bovine chondrocytes in monolayer culture and the formation of cartilage tissue on three-dimensional scaffolds.

We have investigated the ability of transforming growth factor beta (TGF-beta) to promote the growth and differentiation of chondrocytes in monolayer and on three-dimensional scaffolds. Treatment of chondrocytes with TGF-beta and ascorbate individually stimulated the proliferation of bovine articular chondrocytes about 2-fold when cells were grown in monolayer culture: the combination of TGF-beta and ascorbate resulted in a 3-fold increase in cell number over a 72-h period. Peak stimulation with TGF-beta occurred at about 1.0 ng/ml: bFGF was slightly inhibitory in these assays. TGF-beta led to an increase in glycosaminoglycan synthesis as detected by Western blotting using anti-chondroitin sulfate antibodies. No significant change in collagen type II mRNA or protein was observed. When cells were grown on grown on three-dimensional scaffolds composed of polyglyocolic acid, TGF-beta treatment led to an increase in the size of the cartilage-like constructs produced. This was accompanied by increases in collagen and glycosaminoglycan deposition; immunohistochemical staining showed that the predominant collagen was type II. These results indicate that TGF-beta is capable of increasing the proliferation rate of chondrocytes in monolayer as well as increasing cartilage production on three-dimensional scaffolds and may find utility in the in vitro engineering of cartilage tissue.

Repair of articular cartilage defects with collagen-chondrocyte allografts.

This study was designed to evaluate the potential use of a prototype collagen-chondrocyte allograft in the repair of full-thickness articular cartilage defects. Articular cartilage was harvested from young donor New Zealand White rabbits, enzymatically digested, cultured in monolayer, and passed into a three-dimensional porous type I collagen sponge (American Biomaterials). The composite grafts were incubated for 1 week. (Phase I) Twenty adult NZW rabbits underwent bilateral knee arthrotomies. Three-millimeter full-thickness articular cartilage defects were made in the trochlea of the distal femur. A 4-mm circular punch from the composite cell-seeded grafts was press-fit into the right knee defects. The left knee served as a control (collagen sponge alone or ungrafted defect). Animals were allowed free activity postoperatively and were killed in groups of five at 4, 8, 12, and 24 weeks postoperatively. Defect areas were harvested. Sections were cut at 5-microm thickness and stained with hematoxylin and eosin. The degree as well as quality of healing were assessed and scored with a grading system modified from Salter and O'Driscoll for cartilage repair. (The maximum score was 24 points.) Safranin-O staining as well as polarized light examination of representative sections was undertaken to assess the proteoglycan content and structural characteristics of the repair matrix. (Phase II) An additional 15 NZW rabbits underwent the above procedure but with the addition of fibroblast growth factor (FGF) (100 ng/ml) and insulin (5 microg/ml) to the growth medium of the composite grafts as stimulators of chondrocyte proliferation and proteoglycan synthesis. Control specimens in phase I and II (collagen sponge alone or ungrafted defects) healed with a primarily fibrous or fibrocartilagenous matrix. Defects grafted with cell-seeded collagen sponges demonstrated enhanced healing at all time points examined when compared to controls. There was a strong tendency toward a hyaline appearing matrix with increased Safranin-O staining and birefringence under polarized light more closely resembling the normal native cartilage. Mean histologic score for grafted defects was 18.4 (+/-3.1). Mean scores for collagen sponge alone and ungrafted defects in phase I were 12.7 (+/-4) and 12.7 (+/-3.1) (P<0.01). The addition of FGF and insulin to the growth medium (phase II) resulted in a significantly enhanced repair matrix when compared to the non-FGF-enhanced grafts, with a greater percentage of hyaline appearing tissue at all time points examined (4,8, and 12 weeks). Organization of the chondrocytes was improved at all time points examined as well. Mean histologic score for the FGF-grafted defects was 21.1 (+/-3.0). Mean scores for collagen sponge alone and ungrafted defects in phase II were 14.9 (+/-2.9) and 15.5 (+/-1.9) (p<0.01).

Repair of articular cartilage defects using mesenchymal stem cells.

Degeneration of articular cartilage in osteoarthritis is a serious medical problem. We have isolated a population of cells from the connective tissue of mammals termed mesenchymal stem cells (MSCs) for their apparent unlimited growth potential and their ability to differentiate into several phenotypes of the mesodermal lineage, including cartilage and bone. These qualities make them ideal candidates for cartilage repair. We isolated MSCs from adult rabbit muscle and cultured them in vitro into porous polyglycolic acid polymer matrices. The matrices were implanted into 3-mm-diameter full thickness defects in rabbit knees with empty polymer matrices serving as the contralateral controls. The implants were harvested 6 and 12 weeks postop. At 6 weeks, the controls contained fibrocartilage while the experimentals seemed to contain undifferentiated cells. By 12 weeks postop, the controls contained limited fibrocartilage and extensive connective tissue, but no subchondral bone. In contrast, the implants containing MSCs had a surface layer of cartilage approximately the same thickness as normal articular cartilage and normal-appearing subchondral bone. There was good integration of the implant with the surrounding tissue. Implantation of MSCs into cartilage defects appears to effect repair of both the articular cartilage and subchondral bone. Studies are ongoing to further characterize the use of MSCs for cartilage repair.

Subcutaneous island pedicle flaps.

The subcutaneous island pedicle flap is a useful and reliably successful means of closing small- to medium-sized cutaneous excisional defects. It is especially useful in areas where primary closure could result in distortion of critical features. The technique is conceptually straightforward and offers advantages over skin grafting and transposition flaps. We describe our experience with 60 consecutive, successful subcutaneous island pedicle flaps.

Surgical treatment of rhinophyma with the Shaw scalpel.

We present our experience with the Shaw scalpel in the treatment of a patient with extensive rhinophyma. The heated blade of the scalpel provides rapid maintenance of hemostasis with minimal tissue damage and thus allows for rapid and accurate removal of tissue. After removing the bulk of tissue with the Shaw scalpel, the carbon dioxide laser and dermabrader are used to refine nasal contours.

The repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation.

Using the knee joints of New Zealand White rabbits, a baseline study was made to determine the intrinsic capability of cartilage for healing defects that do not fracture the subchondral plate. A second experiment examined the effect of autologous chondrocytes grown in vitro on the healing rate of these defects. To determine whether any of the reconstituted cartilage resulted from the chondrocyte graft, a third experiment was conducted involving grafts with chondrocytes that had been labeled prior to grafting with a nuclear tracer. Results were evaluated using both qualitative and quantitative light microscopy. Macroscopic results from grafted specimens displayed a marked decrease in synovitis and other degenerative changes. In defects that had received transplants, a significant amount of cartilage was reconstituted (82%) compared to ungrafted controls (18%). Autoradiography on reconstituted cartilage showed that there were labeled cells incorporated into the repair matrix.

Chondrocyte-fibrin matrix transplants for resurfacing extensive articular cartilage defects.

Cartilage resurfacing by chondrocyte implantation, with fibrin used as a vehicle, was examined in large (12 mm) full-thickness articular cartilage defects in horses. Articular chondrocytes, isolated from a 9-day-old foal, were mixed with fibrinogen and injected with thrombin, in a 1:1 mixture, into 12 mm circular defects on the lateral trochlea of the distal femur of eight normal horses. The contralateral femoropatellar (knee) joint served as a control in which the defect was left empty. Synovial fluid from the femoropatellar joints was sampled on days 0, 4, 7, 30, 120, and 240 postoperatively. Groups of four horses were killed at 4 or 8 months postoperatively, and the repair tissue was evaluated by gross and histologic examination with use of hematoxylin and eosin and safranin O staining and by autoradiography. Biochemical analyses included quantitation of proteoglycan, total collagen, and type-II collagen in the repair tissue. Grossly, grafted defects had improved filling of the cartilage lesions; histologically, these areas consisted of differentiated chondrocytes in the deep and middle zones. The cellular arrangement in these zones resembled that of hyaline cartilage. The control defects contained poorly attached fibrous tissue throughout. Grafted tissue at 8 months had increased proteoglycan synthesis evident by both safranin O staining and autoradiography. Glycosaminoglycan quantitation by dye-binding assay confirmed a significantly elevated glycosaminoglycan content in grafted defects (58.8 micrograms/mg of dry weight) compared with control defects (27.4 micrograms/mg; p < 0.05). Similarly, the levels of chondroitin sulfate/dermatan sulfate was significantly elevated in the grafted defects, and this was the predominant glycosaminoglycan epitope present. There was a statistically significant (p < 0.05) increase in type-II collagen in the grafted tissue at 8 months (61.2% grafted; 25.1% control). This resurfacing attempt with use of allograft chondrocytes, secured in large full-thickness articular defects with polymerized fibrin, resulted in an improved cartilage surface in comparison with the control defects, a significantly greater aggrecan level, and a significantly higher proportion of type-II collagen.

Mechanically induced calcium waves in articular chondrocytes are inhibited by gadolinium and amiloride.

Chondrocytes in articular cartilage utilize mechanical signals from their environment to regulate their metabolic activity. However, the sequence of events involved in the transduction of mechanical signals to a biochemical signal is not fully understood. It has been proposed that an increase in the concentration of intracellular calcium ion ([Ca2+]i) is one of the earliest events in the process of cellular mechanical signal transduction. With use of fluorescent confocal microscopy, [Ca2+]i was monitored in isolated articular chondrocytes subjected to controlled deformation with the edge of a glass micropipette. Mechanical stimulation resulted in an immediate and transient increase in [Ca2+]i. The initiation of Ca2+ waves was abolished by removing Ca2+ from the extracellular media and was significantly inhibited by the presence of gadolinium ion (10 microM) or amiloride (1 mM), which have previously been reported to block mechanosensitive ion channels. Inhibitors of intracellular Ca2+ release (dantrolene and 8-diethylaminooctyl 3,4,5-trimethoxybenzoate hydrochloride) or cytoskeletal disrupting agents (cytochalasin D and colchicine) had no significant effect on the characteristics of the Ca2+ waves. These findings suggest that a possible mechanism of Ca2+ mobilization in this case is a self-reinforcing influx of Ca2+ from the extracellular media, initiated by a Ca2+-permeable mechanosensitive ion channel. Our results indicate that a transient increase in intracellular Ca2+ concentration may be one of the earliest events involved in the response of chondrocytes to mechanical stress and support the hypothesis that deformation-induced Ca2+ waves are initiated through mechanosensitive ion channels.

Teaching prevention through electives.

The Liaison Committee on Medical Education (LCME) accreditation standards affirm that the medical school curriculum should include elective courses to supplement the required courses and provide opportunities for students to pursue individual academic interests. The breadth of opportunities in preventive medicine and population health is extensive as students seek rotations at health departments, rural and urban community health centers, community agencies, occupational health sites, schools, and abroad. A growing number of students choose to participate in MD/MPH dual-degree programs. This article describes four prototypes that foster student learning in preventive medicine: population health, international health, American Medical Student Association opportunities, and public health degree programs. These four types of electives enable students to participate in the front lines of preventive services through experiential learning in: community and population health both at home and abroad, continuous quality improvement, organization and behavioral change, interprofessional teamwork, and health care policy. For those with particular interests in population health and preventive medicine, an increasing number of medical schools offer dual MD/MPH programs, either in conjunction with schools of public health or in graduate programs in public health.

Tissue engineered bone repair of calvarial defects using cultured periosteal cells.

Periosteum has been demonstrated to have cell populations, including chondroprogenitor and osteoprogenitor cells, that can form both cartilage and bone under appropriate conditions. In the present study, periosteum was harvested, expanded in cell culture, and used to repair critical size calvarial defects in a rabbit model. Periosteum was isolated from New Zealand White rabbits, grown in cell culture, labeled with the thymidine analog bromodeoxyuridine for later localization, and seeded into resorbable polyglycolic acid scaffold matrices. Thirty adult New Zealand White rabbits were divided into groups, and a single 15-mm diameter full-thickness calvarial defect was made in each animal. In group I, defects were repaired using resorbable polyglycolic acid implants seeded with periosteal cells. In group II, defects were repaired using untreated polyglycolic acid implants. In group III, the defects were left unrepaired. Rabbits were killed at 4 and 12 weeks postoperatively. Defect sites were then studied histologically, biochemically, and radiographically. In vitro analysis of the cultured periosteal cells indicated an osteoblastic phenotype, with production of osteocalcin upon 1,25(OH)2 vitamin D3 induction. In vivo results at 4 weeks showed islands of bone in the defects repaired with polyglycolic acid implants with periosteal cells (group I), whereas the defects repaired with untreated polyglycolic acid implants (group II) were filled with fibrous tissue. Collagen content was significantly increased in group I compared with group II (2.90 +/- 0.80 microg/mg dry weight versus 0.08 +/- 0.11 microg/mg dry weight, p < 0.006), as was the ash weight (0.58 +/- 0.11 mg/mg dry weight versus 0.35 +/- 0.06 mg/mg dry weight, p < 0.015). At 12 weeks there were large amounts of bone in group I, whereas there were scattered islands of bone in groups II and III. Radiodensitometry demonstrated significantly increased radiodensity of the defect sites in group I, compared with groups II and III (0.740 +/- 0.250 OD/mm2 versus 0.404 +/- 0.100 OD/mm2 and 0.266 +/- 0.150 OD/mm2, respectively, p < 0.05). Bromodeoxyuridine label, as detected by immunofluorescence, was identified in the newly formed bone in group I at both 4 and 12 weeks, confirming the contribution of the cultured periosteal cells to this bone formation. This study thus demonstrates a tissue-engineering approach to the repair of bone defects, which may have clinical applications in craniofacial and orthopedic surgery.

Temporal matrix synthesis and histologic features of a chondrocyte-laden porous collagen cartilage analogue.

Cartilage resurfacing by chondrocyte transplantation, using porous collagen matrices as a vehicle to secure the cells in cartilage defects, has been used experimentally in animals. This in vitro study evaluated the temporal morphologic features and proteoglycan synthesis of chondrocyte-laden collagen matrices. Forty-two porous collagen disks were implanted with a minimum of 6 x 10(6) viable chondrocytes, covered by a polymerized collagen gel layer, and 6 disks were harvested after 0, 3, 7, 10, 14, 18, or 22 days of incubation in supplemented Ham's F12 medium at 37 C and 5% CO2. Histologic and histochemical evaluation of formalin-fixed segments of the cultured disks indicated that the chondrocytes proliferated in the implant, producing small groups and linear segments of cells by day 14. The collagen framework remained intact over the course of the study with thick areas attributable to depositions of matrix material after day 10. Alcian blue-stained matrix was evident in the pericellular region of chondrocytes in sections of disks harvested on days 14, 18, and 22. Glycosaminoglycan (GAG) assay by dimethylmethylene blue dye binding after papain digestion of the disk segments revealed negligible amounts of GAG at day 0. Significant (P < or = 0.0001) increase in total GAG content was observed by day 3 (0.329 micrograms/mg of disk) and further increases were observed until a plateau in GAG quantity was seen on day 14. Mean peak GAG content was 0.553 +/- 0.062 micrograms/mg. Secondary treatment of the papain-digested implants with keratanase and chondroitinase ABC yielded similar trends in chondroitin sulfate (CS) and keratan sulfate (KS) concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)