Stiffness of the Healing Medial Collateral Ligament of the Mouse

The knee joints of mice can serve as a model for studying knee ligament properties. The goal of our study was to measure the structural stiffness of the medial collateral ligament (MCL) of the murine knee. A tensile test was developed for this purpose. First 84 femur-MCL-tibia complexes of 11-week-old C57Black6 mice were tested. Of four groups (n = 14 per group) the right MCL was ruptured. The mice were sacrificed at 1.5, 3, 6, and 9 weeks after the operation. The other two groups served as controls at 0 and 9 weeks after the operation. Absolute values of the structural stiffness of the healed MCLs at 1.5 weeks were initially significantly lower than their unoperated controls, but were not different from normal values at three, six, and nine weeks of healing. The structural stiffness of the unoperated controls increased by 11% at 20 weeks compared to 11 weeks of age.     

An Evaluation of the Quasi-Linear Viscoelastic Properties of the Healing Medial Collateral Ligament in a Goat Model

The viscoelastic properties of the healing medial collateral ligament (MCL) at 12 weeks after isolated injury were investigated in a goat model. The stress-strain relationships, static and cyclic stress-relaxation behaviors of the healing MCL up to 5% strain were determined experimentally using a femur-MCL-tibia complex. These experimental data were used in combination with the quasi-linear viscoelastic (QLV) theory of Fung (1972) to characterize the reduced relaxation function, G(t) (described by constants C, tau1, and tau2) and the elastic response, sigmae(epsilon) (described by constants A and B) of this tissue. It was found that the percentage of stress relaxation for the healing MCLs was significantly greater than those for sham-operated controls (49.0 +/- 12.1% vs. 26.5 +/- 8.1%, respectively; p < 0.05). The product of constants A x B, i.e. the initial slope of the stress-strain curves, was found to be significantly lower for healing MCLs compared to those for sham-operated controls (32.9 +/- 15.8 MPa vs. 118.8 +/- 48.3 MPa; p < 0.05). The dimensionless constant C, i.e. the magnitude of the viscous response, was nearly three times greater for healing MCLs, while constant tau1 was found to be similar between the two groups (0.80 +/- 0.43 s vs. 0.89 +/- 0.52 s, respectively). Constant tau2 for the healing MCL was significantly less than the controls (1269 +/- 38 s vs. 1845 +/- 431 s; p < 0.05) indicating that the stress relaxation reached a plateau earlier. These constants of the QLV theory used to describe the healing MCL were validated for the strain level utilized in this experiment (approximately equal to 4.5%) by predicting the peak stresses during a cyclic stress-relaxation experiment. The theoretically determined values closely matched the experimentally measured values. Thus, this study demonstrates that the QLV theory could be successfully used to describe the viscoelastic behavior of the MCL during the early phases of healing.     

Microstructural Morphology in the Transition Region Between Scar and Intact Residual Segments of a Healing Rat Medial Collateral Ligament

This study used a rat model to investigate the microstructural organization of collagen through the transition from scar to intact residual segments of a healing medial collateral ligament (MCL). Twenty-two male retired breeder Sprague-Dawley rats were randomly separated into two groups. Eleven underwent surgical transections of both MCLs and were allowed unrestricted cage activity until euthanized two weeks post surgery. The remaining eleven rats were used as normal controls. All 44 MCLs were harvested including intact femoral and tibial insertions and prepared for scanning electron microscopy (SEM) imaging. At harvest the scar region in the healing ligaments was more translucent than the normal tissue. Ligaments were viewed from femoral to tibial insertions at magnifications of 100X through 20,000X. Tissue away from the scar region in the transected MCLs was indistinguishable from normal tissue in uninjured ligaments. Collagen fibers and fibrils in these tissues were more aligned along the longitudinal axis of the ligament than in the scar tissue. Continuity of collagen fibers and fibrils were consistently observed from the residual portions of the transected ligament through the scar region. Bifurcations/fusions, but no anastomoses, in fibers and fibrils were observed in both normal and scar tissues of ligaments. Qualitatively, bifurcations were encountered more frequently in scar tissue. In the transition region, larger diameter fibers from the residual tissue bifurcated into smaller diameter fibrils in the scar. This connection between larger diameter and smaller diameter fibers and fibrils indicates that bifurcations/fusions are likely to be the dominant way in which force is transmitted from a region with larger fibrils (residual ligament) into and through a region with smaller fibrils (scar).     

Determination of thein situ forces and force distribution within the human anterior cruciate ligament

Thein situ forces and their distribution within the human anterior cruciate ligament (ACL) can clarify this ligament's role in the knee and help to resolve controversies regarding surgical treatment of ACL deficiency. We used a universal force-moment sensor (UFS) to determine the magnitude, direction, and point of application of thein situ forces in the ACL in intact human cadaveric knees. Unlike previous studies, this approach does not require surgical intervention, the attachment of mechanical devices to or near the ACL, ora priori assumptions about the direction ofin situ force. Anterior tibial loads were applied to intact knees, which were limited to 1 degree of freedom at 30^o flexion. Thein situ forces developed in the ACL were lower than the applied force for loads under 80 N, but larger for applied loads of more than 80 N. The direction of the force vector corresponded to that of the anteromedial (AM) portion of the ACL insertion on the tibial plateau. The point of force application was located in the posterior section of the anteromedial portion of the tibial insertion site. The anterior and posterior aspects of the anteromedial portion of the ACL supported 25% and 70% of thein situ force, respectively, with the remainder carried by the posterolateral portion. We believe that the data obtained with this new UFS methodology improves our understanding of the role of the ACL in knee function, and that this methodology can be easily extended to study the function of other ligaments.     

Investigation of the Collagen Fibril Distribution in the Medial Collateral Ligament in a Rat Knee Model

This study compared the collagen fibril diameter distribution among six anatomical sites of the rat medial collateral ligament (MCL). Ultrathin MCL sections from 4 male Sparague-Dawley rats were examined electron microscopically. With an automated quantitation method, 41,638 fibrils were measured and compared among the periphery and core regions of the femoral, middle, and tibial portions of the MCL. Results demonstrated significant difference ( p < .0033) in mean fibril diameter distribution among the six sites. The mass-averaged diameters of the core and peripheral fibrils were between 175.53 to 190.82 nm and 88.47 to 109.18 nm, respectively, with the peripheral fibrils more homogeneous in size. The fibrils occupied 36.7% to 57.1% of the cross-sectional area of the ligament. About 50% of the fibrils had an oblique factor of 0.8-1.0, implying that most fibrils were aligned longitudinally. This study has provided a detailed profile of the collagen fibril distributions in rat MCL.     

Effects of Herbal Application on the Ultrastructural Morphology of Repairing Medial Collateral Ligament in a Rat Model

The present study investigated the effects of an external herbal application on the ultrastructural morphology of repairing medial collateral ligament (MCL) in a rat model. Eight MCL-transected/herb-treated rats (group 1) were compared with 8 MCL-transected/placebo-treated controls (group 2) and 8 MCL-intact/placebo-treated rats (group 3). At 3 and 6 weeks posttransection, MCL specimens were examined under electron microscope. With analysis of 370,709 collagen fibrils, all herb-treated animals had significantly larger fibrils than the controls (p <. 001) at 6 weeks postinjury. The mean diameter of those peripheral collagen fibrils of group 1 was larger than the sham group (p <. 001). The mass-averaged diameters of group 1 (50.49 to 143.07 nm) and group 3 (59.69 to 188.88 nm) were larger than group 2 (50.59 to 121.94 nm). The area coverage by collagen fibrils ranged from 46.86 to 94.97% for group 1 and 43.70 to 68.08% for group 3 as compared with 40.01 to 50.77% for group 2. Mode obliquity was 0.56 to 0.84 among groups. We concluded that herbal remedy increases collagen fibril size of healing rat MCLs homogeneously at 6 weeks posttransection.     

Identification of the homologue of the bovine Rob(1;29) in a captive gaur (Bos gaurus)

Robertsonian translocations have been well documented in domestic cattle, with the most commonly occurring fusion involving chromosomes 1 and 29. The widespread nature of this translocation is indicative of its ancient origin. The gaur (Bos gaurus) is one of many wild cattle species currently listed as vulnerable or endangered. Due to the small founder stock and 50 years of restricted breeding, the captive herd is showing signs of inbreeding and reduced fertility. Recent cytogenetic analysis of a female gaur at Toronto Zoo found that the individual contained 2n=57 chromosomes instead of the normal 2n=58, with an extra submetacentric and the loss of two acrocentric chromosomes being observed. This study was undertaken to identify the translocation in this individual and to examine the karyotype of immediate family members. Chromosome analysis of fibroblast cell cultures was carried out using GTG-banding, C-banding and FISH (bovine 1 and 29 paints) techniques to characterize the translocation. Results from the GTG-banding and FISH analyses confirm that the two autosomes involved in the translocation are the bovine homologues 1 and 29. A monocentric centromere was observed by C-banding. Chromosome abnormalities have not been detected in other gaur tested to date. This study demonstrates the importance of cytogenetic analysis for the establishment of screening protocols for the assessment of reproductive potential in this and other exotic bovinae.     

Chromosomal locations of four minor rDNA loci and a marker microsatellite sequence in barley

Four minor rDNA loci have been mapped physically to barley (Hordeum vulgare L.) chromosomes 1 (7I), 2 (2I), 4 (4I), and 5 (1I) by a two-stepin situ hybridization procedure including a GAA microsatellite sequence. Reprobing with the microsatellite resulted in a distinct banding pattern, resembling the C-banding pattern, which enabled unequivocal chromosome identification. This study suggests that gene mapping accuracy may be improved by using probes with well-characterized and narrow hybridization sites as cytological markers which are situated close to the gene locus. One of the rDNA loci is located about 54% out on the short arm of chromosome 4 and it has not previously been reported in barley. We have disignated the new locusNor-16. rDNA loci on homoeologous group 4 chromosomes have not yet been reported in otherTriticeae species. The origin of these 4 minor rDNA loci is discussed in relation to their equilocal distribution on the chromosomes.     

Reciprocal chromosome painting between a New World primate, the woolly monkey, and humans

We employed fluorescence-activated chromosome sorting (FACS) to construct chromosome paint sets for the woolly monkey (Lagothrix lagotricha) and then FISH to reciprocally paint human and woolly monkey metaphases. Reciprocal chromosome painting between humans and the woolly monkey allowed us to assign subchromosomal homologies between these species. The reciprocal painting data between humans and the woolly monkey also allow a better interpretation of the chromosomal difference between humans and platyrrhines, and refine hypotheses about the genomic rearrangements that gave origin to the genome of New World monkeys. Paints of woolly monkey chromosomes were used to paint human metaphases and forty-five clear signals were detected. Paints specific to each human chromosome were used to paint woolly monkey metaphases. The 23 human paints gave 39 clear signals on the woolly monkey karyotype. The woolly monkey chromosomes painted by human paints produced 7 associations of segments homologous to human chromosomes or human chromosome segments: 2/16, 3/21, 4sol;15, 5/7, 8/18, 10/16 and 14/15. A derived translocation between segments homologous to human chromosomes 4 and 15 is a synapomorphic marker linking all Atelines. These species may also be linked by fragmentation of homologs to human 1, 4, and 15.     

High <Emphasis Type="BoldItalic">PRL-3</Emphasis> expression in human gastric cancer is a marker of metastasis and grades of malignancies: an in situ hybridization study

Phosphatase of regenerating liver (PRL)-3, encoding a 22-kD low molecular weight tyrosine phosphatase, has been reported to be associated with metastasis of colorectal carcinoma. We assessed the levels of PRL-3 mRNA expression to know whether its up-regulation was involved in progression and metastasis of gastric carcinoma. Levels of PRL-3 expression in 94 human gastric adenocarcinomas and 54 matched lymph node metastases were detected by in situ hybridization and compared with clinicopathological characteristics including prognosis. High PRL-3 expression was detected in 36.2% of primary gastric carcinoma (with nodal metastasis, 55.6%; without nodal metastasis, 10%; P < 0.001) and in 74.1% of lymph node metastases. The incidence of high PRL-3 expression in lymph node metastasis was significantly higher than in primary tumors (P < 0.044). Moreover, high expression of PRL-3 was closely associated with tumor size, lymphatic invasion, venous invasion, extent of lymph node metastasis, and tumor stage. These results suggest that high PRL-3 expression may participate in the progression and metastasis of gastric carcinoma. PRL-3 might be a novel molecular marker for aggressive gastric cancer.     

Post-zygotic modifications and intra- and inter-individual nucleolar organizing region variations in fish: report of a case involvingLeporinus friderici

Silver nitrate staining, a rapid and efficient method, has proven to be excellent for nucleolar organizing region (NOR) studies in fish. Some fish appear to have only two NOR-bearing chromosomes in their karyo-type, whereas others probably have several. In the present study we analyzed the NORs ofLeporinus friderici, a species that, on the basis of previous studies, has been considered as representative of species with NORs carried by a single chromosome pair. The analyses were performed by a combination of three methods,i.e. silver nitrate staining, staining with the GC-specific fluorochrome chromomycin A₃, andin situ hybridization with digoxigenin-labeled probes. The results showed that, although more frequent and conspicuous in a single chromosome pair, the NORs of this species are present in multiple chromosomes. Intra- and inter-individual variations observed by the three methods strongly suggest the occurrence of post-zygotic modifications involving NORs. NOR identification in fish, almost exclusively performed by the silver nitrate method, is currently being re-evaluated by methods such as chromomycin A₃ staining andin situ hybridization, which may provide important information leading to a better understanding of chromosome evolution in these animals.     

Analysis of NORs and NOR-associated heterochromatin in the mussel Mytilus galloprovincialis Lmk

The chromosomes of the mussel Mytilus galloprovincialis were analysed by means of chromomycin A3 (CMA), distamycin A/DAPI (DA/DAPI), DAPI/actinomycin D (DAPI/AMD) and chromomycin A3/distamycin A/DAPI(CDD) fluorescence banding techniques, C-banding, silver staining, N-banding and in situ hybridization with 18S+28S rDNA and telomere probes. 18S+28S rDNA clusters were located on the telomeres of two pairs of submeta/subtelocentric chromosomes. The nucleolar organizing regions (NORs) were associated with bright CMA fluorescence, dull DAPI fluorescence and C- and N-positive bands, but not all four NOR-associated heterochromatin bands showed bright CMA fluorescence in a given cell; intra- and interindividual variability was found in this character. Additional non-ribosomal C-bands did not show any differential fluorescent behaviour.This revised version was published online in November 2005 with corrections to the Cover Date.     

Meiotic instability of chicken ultra-long telomeres and mapping of a 2.8 megabase array to the W-sex chromosome

The objective of this research was to study the meiotic stability of a subset of chicken telomere arrays, which are the largest reported for any vertebrate species. Inheritance of these ultra-long telomere arrays (200 kb to 3 mb) was studied in a highly homozygous inbred line, UCD 003 (F ≥ 99.9). Analysis of array transmission in four families indicated unexpected heterogeneity and non-Mendelian segregation including high-frequency-generation of novel arrays. Additionally, the largest array detected (2.8 Mb) was female-specific and correlated to the most intense telomeric DNA signal on the W-sex chromosome by fluorescence in situ hybridization (FISH). These results are discussed in regard to the potential functions of the ultra-long telomere arrays in the chicken genome including generation of genetic variation through enhanced recombination, protection against erosion by providing a buffer for gene-dense regions, and sex-chromosome organization.     

Nature of B chromosomes in the harvest mouse Reithrodontomys megalotis by fluorescence in situ hybridization (FISH)

Using fluorescence in situ hybridization, we examined the characteristics of two types of B chromosomes in harvest mice of the genus Reithrodontomys. B chromosomes were interrogated with rDNA, telomeric repeat, LINE element and centromeric heterochromatin probes. The two types of B chromosomes share the following features: (a) telomeres present on the ends of both arms; (b) hybridization to LINE probes; (c) absence of hybridization to the ribosomal gene probes; (d) C-band-positive centromeric regions; and (e) euchromatic arms. They differ as follows: (a) the larger B element hybridizes to the centromeric heterochromatin (pMeg-1) probe whereas the smaller B element does not; (b) the amount of C-band-positive material is reduced in the smaller B chromosome relative to that present on the larger B chromosome; and (c) the smaller element is reduced in size by about a third. It is concluded that the larger B chromosome arose as a leftover centromere from centric fusion, whereas the smaller element has a different origin — perhaps as an intact fragment or as an amplified region from the A chromosomes. The presence of euchromatic regions on B chromosomes may account for their survival in the karyotype.This revised version was published online in November 2005 with corrections to the Cover Date.     

Prevalence and Prognostic Significance of Neuroendocrine Differentiation in Colorectal Carcinomas

The prognostic significance of neuroendocrine differentiation in colorectal carcinoma is uncertain. We analyzed 289 moderately differentiated (grades II and III) colorectal carcinomas for neuroendocrine differentiation by immunohistochemistry andin situ hybridization. The tumors were divided into three groups based on the presence of and the numbers of neuroendocrine cells, with group I having no neuroendocrine cells, group II having <1 positive cell/mm², and group III with >1 positive cell/mm².In situ hybridization with probes for chromogranin A and B detected almost twice as many neuroendocrine cells as did immunostaining with an antibody for chromogranin A. There was no prognostic difference associated with the presence or absence of neuroendocrine differentiation in this group of moderately differentiated carcinomas. These results indicate that the presence of neuroendocrine cells detected by expression of chromogranin protein or mRNA does not influence prognosis in moderately differentiated colorectal carcinomas.     

Microhardness of starch based biomaterials in simulated physiological conditions

In this work the variation of the surface mechanical properties of starch-based biomaterials with immersion time was followed using microhardness measurements. Two blends with very distinct water uptake capabilities, starch/cellulose acetate (SCA) and starch/poly(epsilon-caprolactone) (SPCL), were immersed in a phosphate buffer solution (PBS) at 37.5 degrees C for various times. The microhardness of the blends decreased significantly ( approximately 50% for SPCL and approximately 94% for SCA), within a time period of 30 days of immersion, reflecting the different hydrophilic character of the synthetic components of the blends. The dependence of microhardness on the applied loading time and load was also analysed and showed a power law dependency for SCA. Water uptake and weight loss measurements were performed for the same immersion times used in the microhardness experiments. The different swelling/degradation behaviour presented by the blends was related to the respective variation in microhardness. Moreover, complementary characterization of the mechanical properties of SCA and SPCL was accomplished by dynamic mechanical analysis (DMA) and creep measurements. Microhardness measurements proved to be a useful technique for characterizing the mechanical behaviour near the surface of polymeric biomaterials, including in simulated physiological conditions.