bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological role of the second alcohol oxidase gene MOD2 in the methylotrophic growth of Pichia methanolica
Authors: Nakagawa Tomoyuki , Mizumura Tasuku , Mukaiyama Hiroyuki , Miyaji Tatsuro , Yurimoto Hiroya , Kato Nobuo , Sakai Yasuyoshi , Tomizuka Noboru
Published: September 2002

The methylotrophic yeast Pichia methanolica has nine multiple alcohol oxidase (AOD) isozymes, which can be detected on native electrophoretic polyacrylamide gel and are encoded by two genes, MOD1 and MOD2. The aim of this work is to reveal the physiological roles of these AOD subunits, especially that of Mod2p, encoded by the second AOD‐encoding gene, MOD2. A strain expressing only MOD2 showed severe growth inhibition with a low concentration of methanol (0.1%), but its growth was restored with an increase in the methanol concentration (up to 3%). The expression of MOD2 using the CbAOD1 promoter in the Candida boidinii alcohol oxidase‐depleted strain was more advantageous for methylotrophic growth with high methanol concentrations than that of MOD1. The expression of MOD2 was not observed under derepression conditions (0% methanol), and the expression level increased with an increase in the methanol concentration used for induction. The expression of MOD1 was observed under derepression conditions and was rather constant throughout the tested methanol concentration range. Therefore, the ratio of Mod2p to Mod1p in an active AOD octamer was proved to be mainly controlled by changes in the MOD2 mRNA level. These and other results show that Mod2p is a unique AOD subunit more adapted to methylotrophic growth with high methanol concentrations (3%) than Mod1p. Copyright © 2002 John Wiley & Sons, Ltd.
ARL1 and membrane traffic in Saccharomyces cerevisiae
Authors: Rosenwald Anne G. , Rhodes Mary Ann , Van Valkenburgh Hillary , Palanivel Vikram , Chapman George , Boman Annette , Zhang Chun‐jiang , Kahn Richard A.
Published: September 2002

To examine the functions of the Arf‐like protein, Arl1p, in Saccharomyces cerevisiae, a null allele, arl1Δ::HIS3, was constructed in two strains. In one background only, loss of ARL1 resulted in temperature‐sensitive (ts) growth (suppressed on high‐osmolarity media). Allelic variation at the SSD1 locus accounted for differences between strains. Strains lacking ARL1 exhibited several defects in membrane traffic. First, arl1Δ strains secreted less protein as measured by TCA‐precipitable radioactivity found in the media of [35S]‐labelled cells. A portion of newly synthesized carboxypeptidase Y (CPY) was secreted rather than correctly targeted to the vacuole. Uptake of the fluid‐phase marker, lucifer yellow, was reduced. All these phenotypes were exacerbated in an ssd1 background. The ts phenotype of the arl1Δssd1 strain was suppressed by YPT1, the yeast Rab1a homologue, suggesting that ARL1 and YPT1 have partially overlapping functions. These findings demonstrate that ARL1 encodes a regulator of membrane traffic. Copyright © 2002 John Wiley & Sons, Ltd.
Characterization of Ypr1p from Saccharomyces cerevisiae as a 2‐methylbutyraldehyde reductase
Authors: Ford Gordon , Ellis Elizabeth M.
Published: September 2002

The metabolism of aldehydes and ketones in yeast is important for biosynthetic, catabolic and detoxication processes. Aldo‐keto reductases are a family of enzymes that are able to reduce aldehydes and ketones. The roles of individual aldo‐keto reductases in yeast has been difficult to determine because of overlapping substrate specificities of these enzymes. In this study, we have cloned, expressed and characterized the aldo‐keto reductase Ypr1p from the yeast Saccharomyces cerevisiae and we describe its substrate specificity. The enzyme displays high specific activity towards 2‐methylbutyraldehyde, as well as other aldehydes such as hexanal. It exhibits extremely low activity as a glycerol dehydrogenase. The enzyme functions over a wide pH range and uses NADPH as co‐factor. In comparison to other mammalian and yeast aldo‐keto reductases, Ypr1p has relatively high affinity for D,L‐glyceraldehyde (1.08 mM) and hexanal (0.39 mM), but relatively low affinity for 4‐nitrobenzaldehyde (1.07 mM). It displays higher specific activity for 2‐methylbutyraldehyde than does yeast alcohol dehydrogenase and has a Km for 2‐methyl butyraldehyde of 1.09 mM. The enzyme is expressed during growth on glucose, but its levels are rapidly induced by osmotic and oxidative stress. Yeast in which the YPR1 gene has been deleted possess 50% lower 2‐methylbutyraldehyde reductase activity than the wild‐type strain. This suggests that the enzyme may contribute to 2‐methyl butyraldehyde reduction in vivo. It may therefore play a role in isoleucine catabolism and fusel alcohol formation and may influence flavour formation by strains of brewing yeast. Copyright © 2002 John Wiley & Sons, Ltd.
Vectors designed for efficient molecular manipulation in Candida albicans
Authors: Park Nok‐Hyun , Choi Wonja
Published: September 2002

Functional studies on genes of Candida albicans have been hampered by the fact that few vectors are available for efficient cloning and expression in C. albicans, in contrast to Saccharomyces cerevisiae. Here we report that six vectors were constructed for molecular manipulation in C. albicans. All of them contained the autonomous replicating sequence ARS2 and the uracil gene as a selective marker. Introduction of multicloning site (MCS) facilitated directional cloning into various convenient restriction sites is discussed. Distal to the MCS, the additions of sequences encoding yeast‐enhanced green fluorescent protein 3 (yEGFP3) and the terminator of chitin synthase 2 (TCHS2) enabled us to express an open reading frame (ORF) with its own promoter as a GFP fusion protein, so that its intracellular localization could be easily determined. A vector of 7.4 kb was also constructed to express a cloned ORF as a GFP fusion protein under the control of an inducible MET3 promoter (PMET3) located proximal to the MCS. Since this vector was relatively large in size for expressing ORFs, two additional vectors of 6.7 kb were constructed by inserting PMET3 and TCHS2 proximal and distal to the MCS of the above vector containing MCS only, respectively. These six vectors made it possible to study C. albicans in greater detail. They can be used in identification of a promoter, intracellular localization of a protein, and in the induction of lethal genes. Copyright © 2002 John Wiley & Sons, Ltd.
Genetic interactions link ARF1, YPT31/32 and TRS130
Authors: Zhang Chun‐jiang , Bradford Bowzard J. , Greene Michelle , Anido Aimee , Stearns Katherine , Kahn Richard A.
Published: September 2002

A genetic screen for synthetic lethal interactions with arf1− identified a recessive mutation in TRS130, one of 10 components in the trafficking protein particle (TRAPP) complex (Sacher et al., 2000). As TRS130 is an essential gene, the synthetic lethal allele (trs130‐101) is a novel one that requires ARF1 for viability. This allele was found to exhibit no defects in secretory function, i.e. processing of carboxypeptidase Y or invertase. YPT31 and YPT32 were identified in a subsequent screen as high‐copy suppressors of arf1−trs130‐101. Increasing the gene dosage of YPT31/32 also suppressed lethality resulting from deletion of TRS130 or TRS120 but not three other essential TRAPP subunit‐encoding genes. Although unable to suppress defects in several alleles of ARF1, increasing the gene dosage of YPT31/32 suppressed the cold sensitivity of gcs1−, an Arf GTPase‐activating protein (GAP). Thus, these genetic interactions provide initial evidence for linkage of Arf and TRAPP signalling and for Ypt31/32 proteins functioning downstream of both components in the TRAPP complex and of Arf signalling via the Gcs1 Arf GAP. Copyright © 2002 John Wiley & Sons, Ltd.
Mutational analysis of HOXD13 and HOXA13 genes in the triphalangeal thumb—brachyectrodactyly syndrome
Authors: Pérez‐Cabrera A. , Kofman‐Alfaro S. , Zenteno J. C.
Published: August 2002

The triphalangeal thumb‐brachyectrodactyly syndrome is a very rare autosomal dominant disorder of unknown etiology characterized by an unusual pattern of limb malformations: triphalangeal thumbs and brachyectrodactyly in the hands, and ectrodactyly in the feet. In a previous report, we described the clinical and radiographical features of three related subjects with the disease and suggest that due to the unusual combination of limb defects and to its phenotypic similarity with the limb malformative pattern induced by disrupting the Hoxd13 gene in mouse, the triphalangeal thumb‐brachyectrodactyly syndrome might be caused by mutations in a HOX gene. After sequencing the entire coding region of HOXD13 and the highly conserved homeodomain encoding region of HOXA13, we do not detect any deleterious mutation in any of the patients excluding that alterations at these sequences are responsible for the disease. Mutations in regulatory regions of these genes or in other genes involved in limb development might be responsible for the disease.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Insulin‐like growth factor‐I improves cellular and molecular aspects of healing in a collagenase‐induced model of flexor tendinitis
Authors: Dahlgren Linda A. , van der Meulen Marjolein C. H. , Bertram John E. A. , Starrak Greg S. , Nixon Alan J.
Published: August 2002

Flexor tendinitis is a common and debilitating injury of elite and recreational athletes. Healing may be improved through intratendinous injection of insulin‐like growth factor‐I (IGF‐I), which has been shown in vitro to stimulate mitogenesis and enhance tendon matrix production. This study investigated the effects of intratendinous injection of IGF‐I on tendon healing in an equine model of flexor tendinitis. Collagenase‐induced lesions were created in the tensile region of theflexor digitorum superficialistendon of both forelimbs of eight horses. Treated tendons were injected with 2 μg rhIGF‐I intralesionally every other day for 10 injections, while controls received 0.9% NaCl. Tendon fiber deposition and organization were evaluated serially using ultrasonography throughout the 8 week trial period. Following euthanasia, the tendons were harvested and DNA, hydroxyproline, and glycosaminoglycan content determined, mechanical strength and stiffness evaluated, gene expression and spatial arrangement of collagen types I and III assessed by northern blot and in situ hybridization, and tendon fiber architecture assessed by polarized light microscopy. Local soft tissue swelling was reduced in the IGF‐I treated limbs. Similarly, lesion size in IGF‐I treated tendons was smaller 3 and 4 weeks after initiation of treatment. Cell proliferation and collagen content of the IGF‐I treated tendons were increased compared to controls. Mechanically, IGF‐I treated tendons showed a trend toward increased stiffness compared to saline treated controls. Considered together with the decreased soft tissue swelling and improved sonographic healing, these data support the potential use of intralesional IGF‐I for treatment of debilitating tendon injuries.
Rib cage surgery for the treatment of scoliosis: a biomechanical study of correction mechanisms
Authors: Gréalou L. , Aubin C.‐É. , Labelle H.
Published: August 2002

Rib shortening or lengthening are surgical options that are used to address the cosmetic rib cage deformity in scoliosis, but can also alter the equilibrium of forces acting on the spine, thus possibly counteracting in a mechanical way the scoliotic process and correcting the spinal deformities. Although rib surgeries have been successful in animal models, they have not gained wide clinical acceptance for mechanical correction of scoliosis due to the lack of understanding of the complex mechanisms of action involved during and after the operation. The objective of this study was to assess the biomechanical action of different surgical approaches on the rib cage for the treatment of scoliosis using a patient‐specific finite element model of the spine and rib cage. Several unilateral and bilateral rib shortening/lengthening procedures were tested at different locations on the ribs (convex/concave side of the thoracic curvature; at the costo‐transverse/costo‐chondral joint; 20 and 40 mm adjustments). A biomechanical analysis was performed to assess the resulting geometry and load patterns in ribs, costo‐vertebral articulations and vertebrae. Only slight immediate geometric variations were obtained. However, concave side rib shortening and convex side rib lengthening induced important loads on vertebral endplates that may lead to possible scoliotic spine correction depending on the remaining growth potential. Convex side rib shortening and concave side rib lengthening produced mostly cosmetic rib cage correction, but generated inappropriate loads on the vertebral endplates that could aggravate vertebral wedging. This study supports the concept of using concave side rib shortening or convex side rib lengthening as useful means to induce correction of the spinal scoliotic deformity during growth, though the effects of growth modulation from induced loads must be addressed in more detail to prove the usefulness of rib shortening/lengthening techniques.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Retention of 125I‐labeled recombinant human bone morphogenetic protein‐2 by biphasic calcium phosphate or a composite sponge in a rabbit posterolateral spine arthrodesis model
Authors: Louis‐Ugbo John , Kim Hak‐Sun , Boden Scott D. , Mayr Matthew T. , Li Ronald C. , Seeherman Howard , D'Augusta Darren , Blake Cara , Jiao Aiping , Peckham Steve
Published: August 2002

The purpose of this study was to characterize the retention kinetics of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) applied to two calcium‐based delivery matrices. Biphasic calcium phosphate (BCP) and a composite containing BCP in an absorbable collagen sponge (BCP/ACS) were evaluated using a spinal fusion model in rabbits. rhBMP‐2 labeled with radioactive iodine (125I) was used as a tracer to assess in vivo retention of rhBMP‐2 in the presence of these materials (nine animals per material studied). Over a 36 day study period, animals were assessed for the following: percent administered dose retained at the implant site as measured by scintigraphic imaging (counting) with a gamma camera (all animals), radiography of the implant site (all animals), radioactivity in blood and plasma (all animals), and radioactivity in the urine and feces (three animals for each material). Radioactivity data were corrected for the decay of 125I and the attenuation between the implant in vivo and the gamma camera.

Differences observed between the two materials for the area under the retention vs. time profile (AUC; 988%day for BCP vs. 1070%day for BCP/ACS, p = 0.57) and the mean residence time (MRT; 10.2 days for BCP vs. 7.6 days for BCP/ACS, p = 0.06) were not statistically significant. Initial retention/incorporation of rhBMP‐2 was slightly higher for rhBMP‐2/BCP/ACS than for rhBMP‐2/BCP (96.8% vs. 86.0%, p lt; 0.05). Animals receiving rhBMP‐2/BCP showed a longer terminal retention half‐life (t1/2) than did those receiving rhBMP‐2/BCP/ACS (7.5 vs. 4.5 days, p < 0.05). The urinary radioactivity recovery data supported the data obtained by scintigraphy. Over the 36 day collection period, essentially complete recovery of radioactivity (dose) in urine was observed for rhBMP‐2/BCP and rhBMP‐2/BCP/ACS and the majority of the radioactivity (approximately 95%) was soluble in trichloroacetic acid, suggesting extensive catabolism of rhBMP‐2 before renal excretion. Fecal recovery of radioactivity was low, approximately 2–3%.

In conclusion, rhBMP‐2 was retained at the implant site when delivered with either BCP or BCP/ACS based on mean residence time and area under the retention curve vs. time profile. Use of these matrices resulted in detectable rhBMP‐2 levels at the surgical site for over a week in contrast to data reported with several other matrices that lasted less time. Systemic catabolism and elimination of the rhBMP‐2 was extensive and systemic presence of the protein was negligible.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Vascular physiology and long‐term healing of partial ligament tears
Authors: Bray Robert C. , Leonard Catherine A. , Salo Paul T.
Published: August 2002

Functional outcomes of anterior cruciate ligament (ACL) injury are generally poorer than those of medial collateral ligament (MCL) tears. Following ligament damage, all phases of ligament healing require an adequate blood supply. We hypothesized that the differences in healing properties of the ACL and MCL would reflect their vascular responses to joint injury. This paper examines the long‐term changes in blood flow and vascular volume of rabbit knee ligaments after direct injury, and under conditions of chronic joint instability induced by section of the posterior cruciate ligament (PCL).

Standardized injuries were surgically induced in adult rabbit knee ligaments: partial MCL transection, partial ACL transection, or complete PCL transection (joint instability). Sixteen weeks later the blood flow and vascular volume of the ACL and MCL were measured and compared to control and sham‐operated animals.

Direct ligament injury induced significant increases in standardized blood flow and vascular volume of both ACL and MCL after 16 weeks; however, the vascular volume of the ACL was not higher than the control levels in the MCL. We conclude that direct injury to both the anterior cruciate and MCLs induces long‐term physiological responses.

Joint laxity is a common sequel to PCL injury. Chronic joint laxity failed to induce adaptive vascular responses in the ACL, while the MCL shows significant amplification of blood supply. Although both MCL and ACL showed increased weight after PCL transection, the lack of a long‐term vascular response in the ACL may be a major factor in its the diminished healing potential.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Particle size and morphology of UHMWPE wear debris in failed total knee arthroplasties—a comparison between mobile bearing and fixed bearing knees
Authors: Huang Chun‐Hsiung , Ho Fang‐Yuan , Ma Hon‐Ming , Yang Chan‐Tsung , Liau Jiann‐Jong , Kao Hung‐Chan , Young Tai‐Horng , Cheng Cheng‐Kung
Published: August 2002

Osteolysis induced by ultrahigh molecular weight polyethylene wear debris has been recognized as the major cause of long‐term failure in total joint arthroplasties. In a previous study, the prevalence of intraoperatively identified osteolysis during primary revision surgery was much higher in mobile bearing knee replacements (47%) than in fixed bearing knee replacements (13%). We postulated that mobile bearing knee implants tend to produce smaller sized particles. In our current study, we compared the particle size and morphology of polyethylene wear debris between failed mobile bearing and fixed bearing knees. Tissue specimens from interfacial and lytic regions were extracted during revision surgery of 10 mobile bearing knees (all of the low contact stress (LCS) design) and 17 fixed bearing knees (10 of the porous‐coated anatomic (PCA) and 7 of the Miller/Galante design). Polyethylene particles were isolated from the tissue specimens and examined using both scanning electron microscopy and light‐scattering analyses. The LCS mobile bearing knees produced smaller particulate debris (mean equivalent spherical diameter: 0.58 μm in LCS, 1.17 μm in PCA and 5.23 μm in M/G) and more granular debris (mean value: 93% in LCS, 77% in PCA and 15% in M/G).

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Multilineage mesenchymal differentiation potential of human trabecular bone‐derived cells
Authors: Nöth Ulrich , Osyczka Anna M. , Tuli Richard , Hickok Noreen J. , Danielson Keith G. , Tuan Rocky S.
Published: August 2002

Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast‐related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase‐pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow‐derived mesenchymal progenitor cells. High‐density pellet cultures of hOB maintained in chemically defined serum‐free medium, supplemented with transforming growth factor‐β1, were composed of morphologically distinct, chondrocyte‐like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high‐density pellet cultures were surrounded by a sulfated prote‐oglycan‐rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase‐positive cells, that expressed osteoblast‐related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, β‐glycerophosphate, and bone morphogenetic protein‐2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte‐specific genes, such as lipoprotein lipase and peroxisome proliferator‐activated receptor γ2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3‐isobutyl‐l‐methylxanthine. Taken together, these results show that cells derived from collagenase‐treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Polyethylene and methyl methacrylate particle‐stimulated inflammatory tissue and macrophages up‐regulate bone resorption in a murine neonatal calvaria in vitro organ system
Authors: Ren Weiping , Wu Bin , Mayton Lois , Wooley Paul H.
Published: August 2002

There is considerable evidence that orthopaedic wear debris plays a crucial role in the pathology of aseptic loosening of joint prostheses. This study examined the effect of inflammatory membranes stimulated with methyl methacrylate and polyethylene on bone resorption, using the murine air pouch model. The capacity of RAW 264.7 mouse macrophages exposed to polymer particles to produce factors affecting bone metabolism was also studied. Neonatal calvaria bones were co‐cultured with either pouch membranes or conditioned media from activated macrophages. Bone resorption was measured by the release of calcium from cultured bones, and the activity of tartrate‐resistant acid phosphatase in both bone sections and culture medium was also assayed. Results showed that inflammatory pouch membrane activated by methyl methacrylate and polyethylene enhanced osteoclastic bone resorption. Conditioned media from particles stimulated mouse macrophages also stimulated bone resorption, although this effect was weaker than resorption induced by inflammatory pouch membranes. The addition of the particles directly into the medium of cultured calvaria bones had little effect on bone resorption. Our observations indicate that both inflammatory tissue and macrophages provoked by particles can stimulate bone resorption in cultured mouse neonatal calvaria bones. This simple in vitro bone resorption system allows us to investigate the fundamental cellular and molecular mechanism of wear debris induced bone resorption and to screen potential therapeutic approaches for aseptic loosening.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
High intratendinous lactate levels in painful chronic Achilles tendinosis. An investigation using microdialysis technique
Authors: Alfredson Håkan , Bjur Dennis , Thorsen Kim , Lorentzon Ronny , Sandström Patrick
Published: August 2002

In this investigation the microdialysis technique was used to study the concentrations of lactate in Achilles tendons with painful chronic tendinosis and in normal pain‐free tendons. In four patients (mean age 40.7 years) with a painful thickening localized at the 2–6 cm level in the Achilles tendon (chronic Achilles tendinosis) and in five controls (mean age 37.2 years) with normal Achilles tendons, the local concentrations of lactate were registered under resting conditions. All tendons were examined using ultrasonography. In the tendons with tendinosis the painful thickening corresponded to a widened tendon and structural tendinosis changes. Normal tendons showed no widening and a normal structure. A standard microdialysis catheter was inserted into the Achilles tendon under local anesthesia. Samplings were done every 15 min during a 4 h period.

The results showed significantly higher mean concentrations of lactate in tendons with tendinosis compared to normal tendons (2.15 mmol/l vs. 1.14 mmol/l). The lactate concentrations in the tendons with tendinosis were stable, and approximately twofold higher than in the normal tendons during the whole 4 h investigation period.

In conclusion, the higher concentrations of lactate in Achilles tendons with painful tendinosis indicate that there are anaerobic conditions in the area with tendinosis. The importance of this finding for the pathogenesis and pain mechanisms in this chronic condition needs to be further investigated.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Chondropathy after meniscal tear or partial meniscectomy in a canine model
Authors: Wyland Douglas J. , Guilak Farshid , Elliott Dawn M. , Setton Lori A. , Vail Thomas P.
Published: August 2002

A primary goal in considering treatment for meniscal injuries is the preservation of the health of the articular cartilage. However, the chondroprotective effects of various techniques for meniscal injury treatments are unknown. We used a canine model to quantify articular cartilage degeneration in the medial compartment of the canine knee, resulting from a surgically created tear or a partial meniscectomy (PM) of the posterior region of the medial meniscus (each group, n = 10). After sacrifice at 12 weeks, the development of gross chondropathy and the changes in cartilage tensile stiffness were quantified, and correlations between these measurements were examined. Both treatment surgical treatment groups caused significantly greater gross chondropathy as compared to the unoperated contralateral controls. Cartilage tensile stiffness was significantly lower than unoperated controls by nearly 28% in both experimental groups. However, there were no significant differences observed between the gross chondropathy or the cartilage mechanical property changes between the experimental groups. Importantly, the severity of gross chondropathy was found to significantly correlate with the decrement in tensile stiffness properties of the articular cartilage. These findings indicate that significant degeneration of canine articular cartilage develops to a similar degree in the presence of a partially healed meniscus tear or a PM of the knee. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Fibroblast orientation to stretch begins within three hours
Authors: Neidlinger‐Wilke C. , Grood E. , Claes L. , Brand R.
Published: August 2002

Most connective tissue cells align in response to stretch. Previous studies have shown these responses occur within 12–14 h of initiation of stretch, but do not identify the time at which this orientation occurs, nor whether the orientation continues after cessation of stretch. To ascertain the earliest times at which fibroblast orientation occurs, we cultured primary human fibroblasts on deformable culture dishes and stretched (1 Hz, 8% uniaxial strain) them for up to 24 h. We photographed the cells at 0.5, 1–6, 8, 10, 12, 14, 16, and 24 h. Similarly cells were photographed at 1–3, or 4 h after cessation of stretch for stretch durations of 1, 2, and 3 h. Orientation of cells were ascertained by an interactive computer program. The fibroblasts began to orient by 2–3 h and orientation appeared nearly complete by 24 h. Cultures stretched for 2 or 3 h continued to exhibit greater degrees of orientation (compared to controls) for 2 or 3 h respectively after cessation of stretch. We conclude fibroblasts begin to orient within 3 h of initiation of stretch, and that they continue to orient for several hours after cessation of stretch.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Effects of growth factors on cell proliferation and matrix synthesis of low‐density, primary bovine chondrocytes cultured in collagen I gels
Authors: Chaipinyo Kanda , Oakes Barry W. , van Damme Marie‐Paule I.
Published: August 2002

Low cell density cell numbers and dedifferentiation are two major problems of human chondrocyte culture associated with articular cartilage repair. Bovine chondrocytes seeded at low density (3.5 x 104 cells/ml of gels) in three‐dimensional collagen type I gels do proliferate and maintain their phenotype as shown by cell counts, morphology and matrix synthesis. The combination of three growth factors (3GFs; 10 ng/ml TGF‐β1 + 100 ng/ml IGF‐I + 10 ng/ml b‐FGF) added to serum‐free culture medium in this culture system enhances the mitotic activity of bovine chondrocytes similar to 20% foetal calf serum (FCS). At day 21, cells proliferated by 41 fold in gels–FCS and 37 fold in gels–3GFs. Protein synthesis by gels–3GFs cultures was similar to 20% PCS when cultured for 3 weeks but much less proteoglycan was synthesized. The matrix deposition as observed by light and electron microscopy was quite different. More small diameter branching collagen fibrils and a denser matrix were presented in gels‐FCS culture whilst loosely arranged larger diameter collagen fibrils were observed in gels‐3GFs.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Denervation impairs healing of the rabbit medial collateral ligament
Authors: Ivie T. J. , Bray R. C. , Salo P. T.
Published: August 2002

Little is known about the contribution of innervation to ligament healing after traumatic disruption, although there is good evidence of an important role for the peripheral nervous system in the healing of fractures and skin injuries.

Tissues such as ligament, with a low resting blood supply, are dependent on substantial increases in blood flow and vascular volume during the initial stages of repair. We hypothesized that this initial healing response would be strongly promoted by neurogenic inflammation. Since the saphenous nerve (a major sensory branch of the femoral nerve) supplies the medial half of the knee joint, we elected to use femoral nerve transection as a model to determine the role of sensory and autonomic innervation in the initial outcome of repair of the injured medial collateral ligament. Twelve adult, female NZW rabbits underwent right medial collateral ligament transection. Of these, six rabbits underwent right femoral nerve transection to disrupt the somatic sensory and autonomic nerve supply to the knee joint and six were kept neurologically intact (controls). At six weeks post‐injury, the animals were assessed by laser Doppler perfusion imaging (LDI) to determine the local blood flow, at both the injury site and at the uninjured contralateral ligament. The animals were then killed, the knee joints were removed and the biomechanical characteristics of the healing bone–median collateral ligament (MCL)–bone complexes assessed. In a separate cohort of 16 rabbits, vascular volumes of the injured ligaments were measured by infusion of a carmine red/gelatin solution.

At six weeks post‐injury, in vivo measurement of perfusion with LDI revealed that normally innervated ligaments had an almost three‐fold higher average blood flow. Carmine red/gelatin infusion revealed a 50% higher density of blood vessels as compared to denervated ligaments. The force required for ultimate failure was found to be 50% higher in normally innnervated MCL's as compared to denervated MCL's: 153.14 ± 20.71 N versus 101.29 ± 17.88 N (p < 0.05). Static creep was increased by 66% in denervated MCL's: 2.83 ± 0.45% versus 1.70 ± 0.12% (p < 0.05). Total creep was increased by 45% in denervated MCL's: 5.29 ± 0.62% compared to 3.64 ± 0.31% in innervated MCL's (p < 0.05).

We conclude that intact innervation makes a critical contribution to the early healing responses of the MCL of adult rabbits.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Achilles tendon healing: Long‐term biomechanical effects of postoperative mobilization and immobilization in a new mouse model
Authors: Palmes D. , Spiegel H. U. , Schneider T. O. , Langer M. , Stratmann U. , Budny T. , Probst A.
Published: August 2002

The aim of the study was to investigate the long‐term effects of postoperative immobilization as opposed to mobilization on the biomechanical attributes of healing Achilles tendons in a new experimental mouse model.

In 114 Balb‐C‐mice the left Achilles tendon was transected and sutured by the Kirchmayr–Kessler technique. The tendons healed either under postoperative immobilization effected by fixing the upper ankle joint in equinus position or under mobilization through a limited range of movement. The contralateral Achilles tendons served as internal control. All tendons were tested biomechanically at short intervals up to the 112th postoperative day in terms of load to failure [N], tendon deflection [mm] and tendon stiffness [N/mm], and were evaluated histologically after 8 and 112 days.

Postoperative mobilization resulted in a continuous and significantly more rapid restoration of load to failure in comparison to the immobilization group. Tendon deflection was decreased by postoperative mobilization, whereas under immobilization it paradoxically increased still further in the later course. After 112 days the tendons of the mobilization group had regained their original tendon stiffness, whereas the tendons after immobilization reached only about half the values seen in the control tendons. Histologically, postoperative mobilization led to increased immigration of inflammatory cells in the early phase. In the late phase, as compared to immobilization, tendon structure was more mature, with fibre bundles arranged in parallel and interposed tendocytes.

Tensile loading of the healing tendon by postoperative mobilization leads to fundamental changes in the biological process of tendon healing resulting in accelerated restoration of load to failure and reduced tendon deflection.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Effects of femoral tunnel placement on knee laxity and forces in an anterior cruciate ligament graft
Authors: Markolf Keith L. , Hame Sharon , Monte Hunter D. , Oakes Daniel A. , Zoric Bojan , Gause Paul , Finerman Gerald A. M.
Published: August 2002

The purpose of this study was to measure the effects of variation in placement of the femoral tunnel upon knee laxity, graft pretension required to restore normal anterior–posterior (AP) laxity and graft forces following anterior cruciate ligament (ACL) reconstruction. Two variants in tunnel position were studied: (1) AP position along the medial border of the lateral femoral condyle (at a standard 11 o'clock notch orientation) and (2) orientation along the arc of the femoral notch (o'clock position) at a fixed distance of 6–7 mm anterior to the posterior wall. AP laxity and forces in the native ACL were measured in fresh frozen cadaveric knee specimens during passive knee flexion‐extension under the following modes of tibial loading: no external tibial force, anterior tibial force, varus–valgus moment, and internal–external tibial torque. One group (15 specimens) was used to determine effects of AP tunnel placement, while a second group (14 specimens) was used to study variations in o'clock position of the femoral tunnel within the femoral notch. A bone‐patellar tendon‐bone graft was placed into a femoral tunnel centered at a point 6–7 mm anterior to the posterior wall at the 11 o'clock position in the femoral notch. A graft pretension was determined such that AP laxity of the knee at 30 deg of flexion was restored to within 1 mm of normal; this was termed the laxity match pretension. All tests were repeated with a graft in the standard 11 o'clock tunnel, and then with a graft in tunnels placed at other selected positions. Varying placement of the femoral tunnel 1 h clockwise or counterclockwise from the 11 o'clock position did not significantly affect any biomechanical parameter measured in this study, nor did placing the graft 2.5 mm posteriorly within the standard 11 o'clock femoral tunnel. Placing the graft in a tunnel 5.0 mm anterior to the standard 11 o'clock tunnel increased the mean laxity match pretension by 16.8 N (62%) and produced a knee which was on average 1.7 mm more lax than normal at 10 deg of flexion and 4.2 mm less lax at 90 deg. During passive knee flexion‐extension testing, mean graft forces with the 5.0 mm anterior tunnel were significantly higher than corresponding means with the standard 11 o'clock tunnel between 40 and 90 deg of flexion for all modes of constant tibial loading. These results indicate that AP positioning of the femoral tunnel at the 11 o'clock position is more critical than o'clock positioning in terms of restoring normal levels of graft force and knee laxity profiles at the time of ACL reconstruction.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Activation of stress‐activated protein kinases (SAPK) in tendon cells following cyclic strain: the effects of strain frequency, strain magnitude, and cytosolic calcium
Authors: Arnoczky Steven P. , Tian Tao , Lavagnino Michael , Gardner Keri , Schuler Paul , Morse Patrick
Published: August 2002

Cyclic strain has been shown to benefit tendon health. However, repetitive loading has also been implicated in the etiology of tendon overuse injuries. Recent studies demonstrated that in several cell lines cyclic strain was associated with an activation of stress‐activated protein kinases (SAPKs). These SAPKs, in turn, were shown to be important upstream regulators of a variety of cell processes including apoptosis. To examine the effect of repetitive loading on SAPK activation in tendon cells in vitro, canine patellar tendon cells were cyclically strained, and the cellular stress response evaluated by measuring c‐Jun N‐terminal kinase (JNK) activation. The effects of strain frequency and strain magnitude as well as the role of calcium signaling in this mechanotransduction mechanism were also examined. Cyclic strain resulted in an immediate activation of JNK, which peaked at 30 min and returned to resting levels by 2 h. This activation was regulated by a magnitude‐dependent but not frequency‐dependent response and appeared to be mediated through a calcium‐dependent mechanotransduction pathway. While transient JNK activation is associated with normal cell processes, persistent JNK activation has been linked to the initiation of the apoptotic cascade. A similar mechanism could be responsible for initiating the pathological events (localized cell death) seen in tendon overuse injury.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The effects of bone density and disc degeneration on the structural property distributions in the lower lumbar vertebral endplates
Authors: Grant J. P. , Oxland Thomas R. , Dvorak Marcel F. , Fisher Charles G.
Published: August 2002

In this study, we hypothesized that vertebral bone density and disc degeneration would affect the structural property distributions of the lower lumbar vertebral endplates (L3–L5). The results may have implications for improving interbody implant designs to better resist subsidence. A 3 mm diameter hemispherical indenter was used to perform indentation tests at 0.2 mm/s to a depth of 3 mm at 27 standardized locations in 55 bony endplates of intact human lumbar vertebrae (L3–L5). The resulting load–displacement curves were used to extract the failure load and stiffness of each test site. Bone density was measured using lateral DEXA scans. Disc condition was determined using a four‐point grading scale. Three‐way analyses of variance were used to analyze the relationships between the data. The overall failure load decreased with bone mineral density (BMD) in the superior (p = 0.0001) and inferior (p = 0.011) lumbar endplates. In both endplates, the posterolateral regions were significantly stronger than more central regions. With increasing BMD, this difference became more pronounced in the superior endplates only (p = 0.005). Increased disc degeneration was associated with an overall failure load decrease in the inferior lumbar endplates (p = 0.002). The strength in the central regions of the superior endplates was reduced with increasing degeneration, but this was not observed peripherally (p = 0.001). Stiffness magnitude or distribution was not significantly affected by BMD or disc degeneration. The locations of the strongest regions of the endplate did not change with either bone density or disc degeneration. This implies that implant shapes designed using the basic structural property maps for the L3–L5 endplates are appropriate for use in patients with a wide range of pathologies, even though overall failure loads are generally lower in patients with reduced bone density and greater degrees of disc degeneration.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Releasing the A3 pulley and leaving flexor superficialis intact increases pinch force following the Zancolli lasso procedures to prevent claw deformity in the intrinsic palsied finger
Authors: Valero‐Cuevas Francisco J. , Hentz Vincent R.
Published: August 2002

Objective estimates of fingertip force magnitude following surgery to prevent digital metacarpophalangeal (MCP) hyperextension (clawing) in cases of paralysis of the hand's intrinsic muscles will assist clinicians in setting realistic expectations for post‐operative pinch strength. We used a cadaveric/optimization approach to predict and confirm the maximal biomechanically possible fingertip force in the intrinsic palsied hand before and after two popular tendon transfer methods to the volar plate of the MCP joint. Both surgeries were also evaluated after release of the A3 pulley—a modification predicted by our published computer model of the forefinger to increase fingertip force magnitude. We predicted maximal static fingertip force by mounting eight fresh cadaveric hands on a frame, placing their forefinger in a functional posture (neutral abduction, 45° of flexion at the MCP and proximal inter‐phalangeal joints, and 10° at the distal interphalangeal joint) and pinning the distal phalanx to a 3D dynamometer. We pulled on individual tendons with tensions up to 25% of maximal isometric force of their associated muscle and measured fingertip force and torque output. Using these measurements, we predicted the optimal combination of tendon tensions that maximized palmar force (analogous to pinch force, directed perpendicularly from the midpoint of the distal phalanx, and in the plane of finger flexion‐extension) for four cases: (i) the non‐paretic case (all muscles available), (ii) intrinsic palsied hand (no intrinsic muscles functioning), (iii) transfer of flexor superficialis tendon to the volar plate of the MCP (Zancolli lasso) in the intrinsic palsied hand, and (iv) leaving flexor superficialis intact and transferring a tendon of comparable strength to the volar plate of the MCP in the intrinsic palsied hand. Lastly, we applied these optimal combinations of tension to the cadaveric tendons and measured fingertip output. With the A3 pulley intact, the maximal palmar force in cases (ii)–(iv) averaged 48 ± 23% SD (non‐paretic = 100%; case (iv) (61 ± 25%) > cases (ii) and (iii) (43 ± 23% and 39 ± 19%, respectively), p < 0.05). Releasing the A3 pulley significantly increased the average palmar force in cases (ii)–(iv) (73 ± 42%, p < 0.05), with no significant differences among them. Thus, releasing the A3 pulley may improve palmar force magnitude when it is necessary to transfer the digit's own flexor superficialis tendon to the volar plate of the MCP to prevent clawing in the intrinsic palsied hand.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The effect of mechanical stability on local vascularization and tissue differentiation in callus healing
Authors: Claes Lutz , Eckert‐Hübner Kerstin , Augat Peter
Published: August 2002

To investigate the influence of the stability of an osteotomy fixation on the local vascularization and tissue differentiation in callus healing, a transverse osteotomy of the right metatarsal with a gap size of 2 mm was performed in 10 sheep and stabilized with an external fixator. This fixator permitted a defined axial movement. Two groups of 5 sheep were each operated upon to allow 0.2 mm (group A) or 1 mm (group B) of axial movement. Nine weeks after surgery, the callus was dissected and histological sections prepared. The type of tissue and the vessel distribution were determined.

Larger interfragmentary movements led to significantly more fibrocartilage (small axial movement A: 6.2%, large axial movement B: 21.6%) and significantly less bone formation (A: 38.2%, B: 26.3%). On average, and particularly close to the periosteum the number of vessels in the callus healing area was greater in the group with smaller movements than in the group with larger movements. There was a significant difference between the distribution of small (<20 μm) and large (>40 μm) vessels across the whole healing area for both groups. Whereas the large vessels showed maximum density in the medullary cavity, the small vessels showed the highest frequency in the peripheral part of the periosteal callus.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Visual linearization of the display of digital radiographs.
Authors: Li G , Yoshiura K , Welander U , Sällstrom P , McDavid W D
Published: September 2002

OBJECTIVES

To derive and test a method to linearize the visual response of the display of digital radiographs so that equal steps in gray levels will be perceived as equal steps in brightness.

METHOD

A mathematical analysis was performed and expressions for visual linearization were derived. Twenty-four test images were computer generated to confirm that visual linearization may be achieved. Each image had three groups of square areas of different size placed in three rows. The left and right squares in each row were given different gray levels to simulate various contrast levels. The middle squares were initially given the same gray level value as one of the outer squares. The images were examined by ten observers who could change the gray levels of the middle squares so that the step in brightness between the middle square and the outer squares become subjectively equal. The test was performed three times employing two different monitors.

RESULTS

The experimental test confirmed that visual linearization could be achieved. Linear regression analyses gave determination coefficients of 0.9926 amd 0.9942 for monitors with gamma-values of 1.93 and 2.50. respectively. The mean data from the ten observers perfectly fitted those theoretically calculated.

CONCLUSION

Visual linearization of gray levels can be achieved but further clinical research is needed to determine if this improves diagnosis.