bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Er: YAG laser holes on osteoinduction in demineralized rat calvarial allografts
Authors: O'Donnell Richard J. , Deutsch Thomas F. , Flotte Thomas J. , Lorente Carol A. , Tomford William W. , Mankin Henry J. , Schomacker Kevin T.
Published: February 2005

Massive cortical autografts and allografts have been found to incorporate into host bone very slowly and thus are subject to complications such as fatigue fracture and infection. In order to understand and improve the process of osteogenesis in these types of bone grafts, a new experimental model was developed using bone discs from rat calvaria prepared by demineralization and drilling of 0.5 mm diameter holes with a pulsed, 2.94 μm wavelength Erbium: Yttrium‐Aluminum‐Garnet laser. Four types of bone discs were analyzed: untreated (Type I), demineralized (Type II), laser‐ablated (Type III), and laser‐ablated then demineralized (Type IV). The discs were transplanted into a subcutaneous site in adult Sprague‐Dawley rats and followed for as long as 6 weeks. Histologic analysis of the discs at weekly intervals with use of hematoxylin and eosin staining confirmed the presence of new bone growth in Type‐II and Type‐IV discs. The amount of new bone growth in each disc was estimated by determining the mineral x‐ray attenuation coefficient, which is proportional to mineral density, from digitized radiographs of the discs. The results showed that the processes of demineralization (p < 0.001) and laser ablation with demineralization (p < 0.05) were both significant in enhancing new bone growth in this model. This study demonstrated that osteoinduction can be fostered in cortical bone through the processes of demineralization and laser ablation. To the extent that laser ablation may allow maintenance of structural integrity while altering the surface geometry in such a way as to promote ingrowth of new bone, this experimental model represents an advance in understanding how osteogenesis in cortical bone grafts might be improved.
Myofibroblasts in the healing lapine medial collateral ligament: Possible mechanisms of contraction
Authors: Faryniarz D. A. , Chaponnier C. , Gabbiani G. , Yannas I. V. , Spector M.
Published: February 2005

The specific objective of this study was to determine the chronology of the appearance of the myofibroblast in the healing ligament. The overall goal of our work is to elucidate the cellular mechanism of contraction in this tissue. The myofibroblast has been found to be responsible for wound contraction in many tissues and to be the cause of the contracture in several pathological conditions. This cell type contains the actin isoform previously thought to be unique to smooth muscle cells and displays certain characteristic features at the ultrastructural level. In 26 New Zealand White male rabbits, the right medial collateral ligament was transected, whereas the left medial collateral ligament received a sham operation. The central third of the ligament (ligament scar tissue) was evaluated at 2, 3, 6, 8, 10, and 12 weeks postoperatively by immunohistochemical techniques, transmission electron microscopy, and Western blot analyses. Three other rabbits served as anatomic controls. During the early reparative phase (2 and 3 weeks after transection), there was an increase in the number of cells containing α‐smooth muscle actin as well as augmentation of the α‐smooth muscle actin content within each cell—a finding attributed to smooth muscle cells and pericytes associated with neovascularity. No myofibroblasts were detected at this stage, immediately postoperatively, or in the sham‐operation controls. Ligaments in the remodeling phase of healing (6, 8, 10, and 12 weeks) exhibited α‐smooth muscle actin in fibroblasts (myofibroblasts) as well as in vascular pericytes and smooth muscle cells. During this stage of healing, transmission electron microscopy demonstrated an increase in the number of cells displaying myofibroblastic features. It was estimated that at 12 weeks of healing 10% of the cells at the site of injury were myofibroblasts. This is the first definitive finding of myofibroblasts in the injury site of the healing ligament, to our knowledge. The appearance of myofibroblasts in the 6–12 week healing period, the interval during which the ligament has been shown to contract in studies by other investigators, is a rationale for a hypothesis that a cellular contractile apparatus comprising α‐smooth muscle actin (i.e., the myofibroblast) may contribute to the recovery of original ligament length (and normal in situ strain).
Basic fibroblast growth factor stimulates articular cartilage enlargement in young rats in vivo
Authors: Shida Jun‐Ichi , Jingushi Seiya , Izumi Toshihiro , Iwaki Akira , Sugioka Yoichi
Published: February 2005

Basic fibroblast growth factor is a potent mitogen for chondrocytes and influences the protein synthesis of their extracellular matrix in vitro. To investigate its effect on normal developing articular cartilage in vivo, we injected basic fibroblast growth factor once into the knee joints of 4‐week‐old rats. Phosphate buffered saline was similarly injected into the contralateral knee joints as controls. A histological analysis showed that an injection of basic fibroblast growth factor induced enlargement of the articular cartilage area, especially in the condylar ridge region on day 7 after the injection. The extent of the enlargement was dose‐dependent. The localization and amount of proliferating cells in the articular cartilage were analyzed immunohistochemically by the detection of proliferating cell nuclear antigen. On day 1 after the injection, the number of cells positive for proliferating cell nuclear antigen increased significantly in the joints that were injected compared with the controls, and Northern blot analysis showed that the level of messenger RNA for α(II) procollagen was lower in these joints than in the controls. The message in the joints that had been injected increased on day 7, and it was greater than that in the controls. This suggests that proliferating chondrocytes in developing articular cartilage respond to basic fibroblast growth factor with a resulting proliferation of chondrocytes followed by enlargement of cartilage.
Articular chondrocyte tenascin‐C production and assembly into de Novo extracellular matrix
Authors: Savarese J. J. , Erickson H. , Scully S. P.
Published: February 2005

Tenascin‐C is an oligomeric glycoprotein of the extracellular matrix that is expressed in a variety of processes including development, tissue remodeling, wound healing, cell adhesion/antiadhesion, and cell/matrix interactions. Tenascin has recently been acknowledged as a component of the extracellular matrix of articular cartilage, but its function remains unclear. In this study, bovine articular chondrocytes were grown in alginate beads for 35 days to examine the kinetics of tenascin synthesis and incorporation into de novo extracellular matrix. During the culture period, 6 harvest days were established in which culture medium was recovered, alginate beads were dissociated with an EDTA solution, and chondrocytes were collected and lysed by sonication. Total DNA determination performed on the cell lysates demonstrated chondrocyte survival and proliferation. Western blotting performed on the medium, EDTA/alginate, and lysate samples demonstrated the production of both the 220 and 320 kDa tenascin size variants and their differential compartmentalization within the culture system. Tenascin was incorporated into the alginate bead matrix at a constant rate of 3.8 μg/day. The 320 kDa variant was produced in higher quantity, but the 220 kDa fragment was twice as likely to be incorporated into the de novo matrix. Methylene blue/acid fuchsin staining and tenascin immunohistochemistry demonstrated the incorporation of tenascin into a progressively expanding matrix surrounding the chondrocytes. The results suggest a role for tenascin in the assembly of the chondrocyte matrix and as a soluble mediator of chondrocytes with possible diverse functions for the tenascin size variants.
Anterior glenohumeral stabilization factors: Progressive effects in a biomechanical model
Authors: Malicky David M. , Soslowsky Louis J. , Blasier Ralph B. , Shyr Yu
Published: February 2005

The aim of this study was to evaluate the anterior stabilizing factors of the glenohumeral joint over a range of translations. The stabilizers examined included the capsular ligaments, the coracohumeral ligament, the rotator cuff muscles and the long head of the biceps. Simulated muscle forces were applied to eight shoulder specimens to produce 90° of total elevation of the arm in the scapular plane. Stability, defined as the force required to reach a specified subluxation, then was evaluated under varying configurations of capsule cuts, humeral rotation, and muscular loads. The overall force‐displacement relationship of the subluxation was found to increase exponentially in external rotation to 239 N at 10 mm of displacement and to level off in neutral rotation to 172 N at 10 mm of displacement. Among the muscles, the biceps was the most important stabilizer in neutral rotation, providing more than 30 N of stabilization: the subscapularis provided the greatest degree of stabilization in external rotation, increasing to approximately 20 N. The subscapularis and supraspinatus were the most consistently important stabilizers in both types of rotation. In external rotation, the superior, middle, and inferior glenohumeral ligaments were the most effective ligamentous stabilizers, and all provided progressively more stabilization as higher displacements were reached. The stability provided by some of the ligaments reached nearly 50 N at 10 mm of displacement.
Activating mutations of Gs protein in monostotic fibrous lesions of bone
Authors: Alman Benjamin A. , Greel Debra A. , Wolfe Hubert J.
Published: February 2005

Activating mutations of the alpha chain of the heterotrimeric signal transducer Gs disrupt the inherent guanosine triphosphatase activity of the alpha chain, stimulate adenylyl cyclase, and can result in independent cell proliferation. Such mutations are identified in a number of endocrine disorders, including McCune‐Albright syndrome, which is a triad of endocrinopathy, café au lait spots, and polyostotic fibrous dysplasia. The mutation in this syndrome is a missense point mutation in exon 8 that results in the substitution of either histidine or cysteine for arginine at position 201. Monostotic fibrous dysplasia is a nonhereditary isolated bone lesion. Other isolated bone lesions that share some cytologic and clinical similarities to fibrous dysplasia are osteofibrous dysplasia and aggressive fibromatosis involving bone. Four cases of monostotic fibrous dysplasia, four cases of aggressive fibromatosis involving bone, and one case of osteofibrous dysplasia were studied to determine if a mutation was present in exon 8 of the alpha chain of Gs. A missense mutation was present in all of the fibrous dysplasias. The other fibrous lesions and uninvolved tissue did not contain a mutation. Somatic activating mutations of Gs differentiate fibrous dysplasia from the other lesions and may be responsible for the loss of control of local proliferation and growth factor expression.
Effect of human adrenomedullin on vascular resistance of the canine tibia
Authors: Kato Teiji , Bishop Allen T. , Wood Michael B. , Adams Mary L.
Published: February 2005

An ex vivo model of a perfused canine tibia was used to investigate the effect of human adrenomedullin, a novel peptide with known vasodilator properties, on the vascular resistance of bone. Human adrenomedullin has a potent and long‐lasting vasodilator effect in the canine tibia following precontraction of vascular smooth muscle by infusion of prostaglandin F2α. A 0.1 ml bolus injection of 10−5 M human adrenomedullin suppressed the pressor response of the canine tibia preparation to an infusion of norepinephrine by 43–52% for a duration of 100 minutes. An injection of 10−6 adrenomedullin suppressed the pressor response to an infusion of norepinephrine by 22–23% for a duration of 40 minutes. These data suggest that human adrenomedullin may be a potent and long‐acting vascular smooth‐muscle relaxant in bone.
Effect of link protein concentration on articular cartilage proteoglycan aggregation
Authors: Tang Lih‐Heng , Buckwalter Joseph A. , Rosenberg Lawrence C.
Published: February 2005

Previous work has shown that alterations in proteoglycan aggregates are among the first changes detected with aging, disuse, and degeneration of articular cartilage, yet the cause or causes of these alterations remain unknown. To determine if differences in link protein concentration can explain alterations in the assembly, size, and stability of articular cartilage proteoglycan aggregates, we isolated proteoglycan monomer (aggrecan) and link protein from adult bovine articular cartilage and then assembled proteoglycan aggregates from aggrecan and 0.8% hyaluronan relative to aggrecan weight, in the presence of 0, 2, 4, 6, 8, 10, 15, and 20% concentrations of link protein relative to aggrecan weight. We determined the amount, sedimentation coefficient, and stability of the aggregates by analytical ultracentrifugation and measured their dimensions by electron microscopy with use of the monolayer technique. Increased aggregate size, as determined by ultracentrifugation, was directly correlated with an increased number of aggrecans per aggregate and with increased hyaluronan length, as determined by electron microscopy. The concentration of link protein significantly influenced aggregation: concentrations of 6–8% produced maximum aggregation, aggregate stability, and uniformity of aggrecan spacing; concentrations greater than 10% led to the formation of superaggregates (aggregates with sedimentation velocities greater than 100 S that may result from linking two or more hyaluronan filaments) but decreased aggregate stability; and concentrations of less than 4% link protein significantly decreased aggregation, the size and stability of aggregates, and the regularity of aggrecan spacing. The latter observations suggest that a decline in the concentration of link protein could decrease the organization and stability of the articular cartilage matrix.
Transforming growth factor‐β1 enhances bone healing to unloaded tricalcium phosphate coated implants: An experimental study in dogs
Authors: Lind Martin , Overgaard Søren , Søballe Kjeld , Nguyen Tue , Ongpipattanakul Boonsri , Bünger Cody
Published: February 2005

Growth of bone into cementless prosthetic components is compromised after revision of failed joint prostheses and by osteoporosis, gaps, and micromotion. We studied the effects of recombinant human transforming growth factor‐β1 adsorbed on ceramic coated implants on the improvement of mechanical fixation and bone growth on the implant. Unloaded cylindrical grit‐blasted titanium alloy implants were inserted bilaterally into both the medial and lateral femoral condyles of 10 skeletally mature mongrel dogs. The implants measured 10 mm in length and 6 mm in diameter and were initially surrounded by a 2 mm gap. One implant had an uncoated titanium surface and three implants were coated with tricalcium phosphate and 0, 0.3, or 3.0 μg of recombinant human transforming growth factor‐β1. The dogs were killed at 6 weeks. Mechanical testing showed a 3‐fold increase in fixation for the 0.3 μg dose of recombinant human transforming growth factor‐β1 and a 2‐fold increase for the 3.0 μg dose. Histological analysis of bone growth on the implant demonstrated that maximal stimulation occurred with the 0.3 μg dose, but bone volume in the gap was maximally stimulated by the 3.0 μg dose and increased 2‐fold over control values. The majority of tricalcium phosphate was resorbed after the 6‐week observation period. This study suggests that recombinant human transforming growth factor‐β1 adsorbed onto implants coated with tricalcium phosphate ceramic can enhance mechanical fixation and bone growth on the implant. The use of transforming growth factor‐β1 on ceramic coated prosthetic components may help to improve the functional outcome of cementless total joint replacements.
Expression of syndecan‐3 and tenascin‐C: Possible involvement in periosteum development
Authors: Koyama Eiki , Shimazu Atsushi , Leatherman Judith L. , Golden Eleanor B. , Nah Hyun‐Duck , Pacifici Maurizio
Published: February 2005

The development of cartilaginous elements of long bone during embryogenesis and postnatal bone repair processes is a complex process that involves skeletal cells and surrounding mesenchymal periosteal cells. Relatively little is known of the mechanisms underlying these processes. Previous studies from this and other laboratories have suggested that the extracellular matrix protein tenascin‐C is involved in skeletogenesis. Using in situ hybridization and immunofluorescence, we extended those studies by comparing the expression of tenascin‐C with that of syndecan‐3, which belongs to a family of cell surface receptors with which tenascins are known to interact. We found that syndecan‐3 transcripts at first were very abundant in the presumptive periosteum surrounding the diaphysis of early chondrocytic skeletal elements in chick limb. As the elements developed further syndecan‐3 gene expression decreased in the diaphyseal periosteum, whereas it became stronger around the early epiphysis and within the forming articular cells. However, as the diaphyseal periosteum initiated osteogenesis and gave rise to the intramembranous bone collar, syndecan‐3 gene expression increased again. At early stages of skeletogenesis, the tenascin‐C gene exhibited patterns of expression that were similar to, and temporally followed, those of the syndecan‐3 gene. At later stages, however, tenascin‐C gene expression was markedly reduced during intramembranous osteogenesis around the diaphysis. In addition, although syndecan‐3 gene expression was low in osteoblasts and osteocytes located deep into trabecular bone, tenascin‐C gene expression remained strong. Thus, tenascin‐C and syndecan‐3 display distinct temporal and spatial patterns of expression in periosteum and during the development of long bone. Given their multidomain structure and specific patterns of expression, these macromolecules may regulate site‐specific skeletal processes, including interactions between developing periosteum and chondrocytes and delineation of the early cartilaginous skeletal elements.
Zone‐specific cell biosynthetic activity in mature bovine articular cartilage: A new method using confocal microscopic stereology and quantitative autoradiography
Authors: Wong M. , Wuethrich P. , Eggli P. , Hunziker E.
Published: February 2005

A new methodology was developed to measure spatial variations in chondrocyte/matrix structural parameters and chondrocyte biosynthetic activity in articular cartilage. This technique is based on the use of a laser scanning confocal microscope that can “optically” section chemically fixed, unembedded tissue. The confocal images are used for morphometric measurement of stereologic parameters such as cell density (cells/mm3), cell volume fraction (%), surface density (1/cm), mean cell volume (μm3), and mean cell surface area (μm2). Adjacent pieces of tissue are simultaneously processed for conventional liquid emulsion autoradiography, and a semiautomated grain counting program is used to measure the silver grain density at regions corresponding to the same sites used for structural measurements. An estimate of chondrocyte biosynthetic activity in terms of grains per cell is obtained by dividing the value for grain density by that for cell density. In this paper, the newly developed methodology was applied to characterize the zone‐specific behavior of adult articular cartilage in the free‐swelling state. Cylinders of young adult bovine articular cartilage were labelled with either [3H]proline or [35S]sulfate, and chondrocyte biosynthesis and structural parameters were measured from the articular surface to the tidemark. The results showed that chondrocytes of the radial zone occupied twice the volume and surface area of the chondrocytes of the superficial zone but were 10 times more synthetically active. This efficient and unbiased technique may prove useful in studying the correlation between mechanically induced changes in cell form and biosynthetic activity within inhomogeneous tissue as well as metabolic changes in cartilage due to ageing and disease.
Topographic variation in biglycan and decorin synthesis by articular cartilage in the early stages of osteoarthritis: An experimental study in sheep
Authors: Little C. B. , Ghosh P. , Bellenger C. R.
Published: February 2005

Articular cartilage contains large molecular weight proteoglycans that aggregate with hyaluronic acid (aggrecan) and small species, particularly biglycan (dermatan sulphate proteoglycan‐1) and decorin (dermatan sulphate proteoglycan‐2). that do not. Mechanical stresses have been shown to profoundly influence the metabolism of aggrecan by articular chondrocytes; however, there are limited corresponding data on the metabolism of dermatan sulphate proteoglycans 1 and 2. The objective of this study was to examine the metabolism of aggrecan, biglycan, and decorin in articular cartilage from different weight‐bearing areas of normal ovine stifle joints and in joints 6 months after meniscectomy, a procedure that has been shown to induce early osteoarthritic changes. [35S]proteoglycans synthesised by cartilage explants from eight different weight‐bearing regions of unoperated and meniscectomised ovine stifle joints during 48 hours of culture were separated by size‐exclusion chromatography, hydrophobic chromatography, and sodium dodecyl sulfatepolyacrylamide gel electrophoresis and were quantitated by phosphor‐screen autoradiography. The synthesis and degradation of the proteoglycans were expressed relative to the DNA content of the explants. In control joints, the cartilage exposed to high contact stress synthesised significantly less proteoglycan overall and more decorin than joint regions bearing less stress. Explants from high stress regions also released significantly greater amounts of resident proteoglycans (dimethylmethylene blue positive) into media during culture. After lateral meniscectomy, the lateral tibial and femoral cartilages showed elevated biosynthesis of both 35S‐dermatan sulphate proteoglycans 1 and 2. This chondrocyte biosynthetic response was accompanied by increased catabolism of aggrecan and the release of its degradation products into culture media. These experiments revealed, in normal joints, a topographic variation in proteoglycan synthesis by articular cartilage that was related to the mechanical stress to which the tissues were subjected in vivo. This biosynthetic pattern changed when the load distribution of the joint was altered by unilateral meniscectomy. These data suggest that an altered chondrocyte phenotypic expression of proteoglycans in response to abnormal mechanical loading is an early event in osteoarthritis.
Role of aspartate in ischemic spinal cord damage
Authors: Ogata T. , Nakamura Y. , Tsuji K. , Okumura H. , Kataoka K. , Shibata T.
Published: February 2005

To study the potentially different roles of the excitatory amino acids glutamate and aspartate in the development of ischemic injury of the spinal cord, we measured their release from cultured neurons and glial cells under ischemic conditions. We also examined changes in intracellular Ca2+ concentration and the damage elicited in cultured neurons by glutamate and aspartate. Hypoxic‐hypoglycemic treatment (in vitro ischemia) elicited a rapid release of the excitatory amino acids from cultured spinal cord neurons and glial cells, but the release was greater from glial cells than from neurons. The ischemia‐induced glutamate release from glial cells was transient: the aspartate release lasted longer, although the peak level was smaller than that of glutamate. In cultured neurons, a remarkable elevation in intracellular Ca2+ concentration was induced by glutamate but not by a lower concentration (10 μM) of aspartate, which is below the neurotoxic dose. At the higher concentration (100 μM), both excitatory amino acids induced a marked elevation in intracellular Ca2+ concentration and neuronal death. These results indicate that aspartate is less potent than glutamate in eliciting excitatory neurotransmission under normal physiological conditions. However, under pathological conditions such as ischemia, the increased release of aspartate from glial cells may add to the damage to neighboring neurons.
Primary immunolocalization of estrogen and progesterone target cells in the human anterior cruciate ligament
Authors: Liu Stephen H. , Al‐Shaikh Raad , Panossian Vahé , Yang Rong‐Sen , Nelson Scott D. , Soleiman Neptune , Finerman Gerald A. M. , Lane Joseph M.
Published: February 2005

To identify estrogen and progesterone target cells in the human anterior cruciate ligament, immunohistochemical localization of both estrogen and progesterone receptors was performed in 17 specimens of human anterior cruciate ligament. All ligament specimens were obtained at surgery. Thirteen specimens were from women, and four were from men: the average age was 57 years (range, 18–78 years). Eleven specimens (from nine women and two men) came from total knee replacements for osteoarthritis of the knee; three (from two women and one man). from reconstructions of the anterior cruciate ligament; two (both from women). from medial meniscectomies: and one (from a man), from an amputation secondary to chondrosarcoma of the pelvis. An immunoperoxidase method using monoclonal antibodies to the estrogen and progesterone receptors was employed to identify estrogen and progesterone target cells in the anterior cruciate ligament. Staining of both receptors was demonstrable in 14 specimens, and in the remaining three specimens less than 15% of the cells were stained. Both estrogen and progesterone receptors were localized to synoviocytes in the synovial lining, fibroblasts in the anterior cruciate ligament stroma, and cells in the blood vessel walls of the ligament. This demonstration of receptors for estrogen and progesterone in the cells of anterior cruciate ligament suggests that female sex hormones may have an effect on its structure and composition.
Cell cycle analysis of proliferative zone chondrocytes in growth plates elongating at different rates
Authors: Wilsman Norman J. , Farnum Cornelia E. , Green Eric M. , Lieferman Ellen M. , Clayton Murray K.
Published: February 2005

Regulation of postnatal growth of long bones occurs in multiple levels of chondrocytic activity, including stem cell proliferation, proliferative zone cycling, and regulation of changes in chondrocytic shape during hypertrophy. The differentiation sequence of chondrocytes is the same in all growth plates, but rates of elongation at a single point in time and over a period of time differ widely among individual growth plates, which suggests that the rates of sequential gene activation and suppression in this phenotypic pattern can vary. The purpose of this study was to investigate, directly and in vivo, parameters of the cell cycle of proliferative chondrocytes in growth plates growing at widely different rates at a single point in time in order to analyze the relationship between cell cycle time, including the duration of each phase of the cell cycle (G1, S, G2, and M), and the rate of growth. The experimental design used repeated pulse labeling with bromodeoxyuridine and was analyzed using a regression model of time of pulse label with increasing labeling index. Total cell cycle time was calculated as the inverse of the slope of the relationship of the labeling index and the time between labels. The y intercept was the calculated labeling index at time zero. Multiple comparison contrasts were used to test for individual differences among four growth plates with growth rates ranging from approximately 50 to 400 μm per 24 hours from 28‐day‐old rats. The estimate of total cell cycle time for the proximal tibial growth plate was 30.9 hours. Cell cycle times for the other three growth plates were 34.0, 48.7, and 76.3 hours for the distal radius, distal tibia and proximal radius, respectively. Although the times for the proximal tibia and distal radius did not differ significantly, all other times were significantly different (p < 0.05) Almost all differences in total cell cycle time were attributable to significant differences in the length of the G1 phase. The S phase was estimated at 3.4–6.1 hours; the G2 phase, at 3.0 hours; and the M phase, at 0.5‐0.6 hours. The current study suggests that regulation through cell cycle parameters, specifically in the G1 phase, may be involved in overall regulation of differential postnatal long bone growth. It has previously been established that increase and shape change of cellular volume during hypertrophy may be regulated at the level of individual growth plates and that both are significant in understanding differential growth of long bone at this level. By demonstrating that chondrocytes in the proliferating zone have different cell cycle times that are regulated primarily through differences in the duration of G1, this study suggests that, in addition to systemic controls of chondrocyte proliferation, local controls may modulate rates of proliferation of individual growth plates and thus may be another locally mediated regulator of differential growth.
Acceleration of experimental endochondral ossification by biophysical stimulation of the progenitor cell pool
Authors: Aaron Roy K. , Mc Ciombor Deborah K.
Published: February 2005

Endochondral ossification can be modulated by a number of biochemical and biophysical stimuli. This study uses the experimental model of decalcified bone matrix‐induced endochondral ossification to examine the effect of one biophysical stimulus, an electromagnetic field, on chondrogenesis, calcification, and osteogenesis. A temporal acceleration and quantitative increase in sulfate incorporation, glycosaminoglycan content, and calcification suggests that the stimulation of endochondral ossification is due to an increase in extracellular matrix synthesis. The locus of that stimulation is identified in the mesenchymal stage of endo‐chondral bone development, and stimulation at this stage is essential for accelerated bone formation. The data suggest that enhanced differentiation of mesenchymal stem cells present at this stage is most likely responsible for the increase in extracellular matrix synthesis and bone maturation.
Osteoinductive ability of confluent Saos‐2 cells correlates with enhanced expression of bone morphogenetic proteins
Authors: Raval Prafulla , Hsu Howard H. T. , Anderson H. Clarke
Published: February 2005

Implants of defatted, freeze‐dried Saos‐2 human osteosarcoma cells grown to confluency induce de novo bone formation in athymic mice. These cells are also richly endowed with bone morphogenetic proteins and express mRNA for bone morphogenetic proteins 1, 2, 3, 4, and 6, as well as for transforming growth factor‐β1. Our aim was to study whether the ability to induce bone formation is related to the level of expression of bone morphogenetic protein. We studied the osteoinductive abilities and levels of expression of bone morphogenetic protein of Saos‐2 cells both during the growth phase and after confluency was reached. Subconfluent cells were at least 70% less effective in their osteoinductive ability than confluent cells. Comparison of bone morphogenetic protein mRNA expression in confluent and subconfluent cells revealed that the latter had lower expression of all the mRNAs studied. The expression of bone morphogenetic protein‐1, bone morphogenetic protein‐2, and bone morphogenetic protein‐6 mRNAs was 2, 3, and 6 to 10‐fold lower, respectively, in subconfluent cells. These results suggest that the ability of Saos‐2 cells to induce de novo bone formation may be correlated with the relative expression of these proteins; the expression of bone morpho‐genetic proteins in Saos‐2 cells also may be dependent on the cell cycle.
Adaptiive finite element modeling of long‐term polyethylene wear in total hip arthroplasty
Authors: Maxian Tina A. , Brown Thomas D. , Pedersen Douglas R. , Callaghan John J.
Published: February 2005

Adaptive remeshing capability was addded to an existing sliding‐distance‐coupled finite element model of polyethylene wear in total hip arthroplasty. This augmentation allowed earlier postoperative wear simulation to be extended to the clinically more significant long‐term regimen (as long as 20 years). Loads and femoral head excursions were taken from a physically validated gait analysis model of a patient with an instrumented total hip replacement. For otherwise identical 22, 28, and 32 mm components, the least volumetric wear but the most linear wear occurred for the 22 mm head. When the polyethylene thickness in a 22 mm component was reduced to the same as that in a 32 mm component, the volumetric wear rate for the 22 mm component was still much less than that for the larger component, indicating that sliding distance (head size). rather than polyethylene liner thickness, was primarily responsible for the difference in rates, A “28 mm” series, for which head sizes were varied across the range of currently accepted industrial tolerances, showed that although initial wear rates were greatest for the least congruent articulations, the long‐term volumetric wear was nearly the same, regardless of initial clearance.
Contents and distributions of the proteoglycans decorin and biglycan in normal and osteoarthritic human articular cartilage
Authors: Poole A. Robin , Rosenberg Lawrence C. , Reiner Agnes , Ionescu Mirela , Bogoch Earl , Roughley Peter J.
Published: February 2005

The study was designed to determine the contents and distributions of the proteoglycans decorin and biglycan in adult human femoral condylar cartilage and whether these may change in osteoarthritis. New radioimmunoassays were established using peptides representing the amino‐terminal 21 amino acid sequence of each proteoglycan (to which a tyrosine was added for radioiodination) and antibodies in a rabbit antiserum raised to both these molecules. Cartilage was extracted with 4 M guanidine HCl to determine total content, and extracts were analyzed by chromatography to determine molecular sizes. Frozen sections were cut parallel to the articular surface and were extracted to determine distribution within the tissue. Gel chromatography on Sepharose CL‐2B under dissociative conditions revealed molecules with a partition coefficient of 0.7–0.75 in both normal and osteoarthritic cartilage. In normal adult cartilage, the mean contents of the core proteins of biglycan and decorin were calculated to be approximately 0.34 and 0.48 mg per gram wet weight, respectively. These represented molar contents similar to that of aggrecan. In osteoarthritic cartilage, there were no overall significant changes in the content and distribution of these molecules. There was, however, considerable individual variation in both distribution and content. Analyses indicated that there was a trend in osteoarthritic cartilage toward a loss of biglycan and decorin from the more superficial layers of intact cartilage, where both these molecules are normally more concentrated. This was accompanied by maintenance of proteoglycan content deeper in the cartilage, regardless of the degree of degeneration.
Autocrine/paracrine mechanism of insulin‐like growth factor‐1 secretion, and the effect of insulin‐like growth factor‐1 on proteoglycan synthesis in bovine intervertebral discs
Authors: Osada Ryusuke , Ohshima Hiroshi , Ishihara Hirokazu , Yudoh Kazuo , Sakai Kiyoshi , Matsui Hisao , Tsuji Haruo
Published: February 2005

The present study was undertaken to investigate the effect of insulin‐like growth factor‐1 on proteoglycan synthesis and the autocrine/paracrine mechanisms involving insulin‐like growth factor‐1 in the bovine coccygeal intervertebral disc. Insulin‐like growth factor‐1 stimulated proteoglycan synthesis in cultured cells of the nucleus pulposus of bovine intervertebral discs in a dose‐dependent manner, and the effect was inhibited by an anti‐insulin‐like growth factor‐1 monoclonal antibody. In situ hybridization histochemistry revealed the expression of insulin‐like growth factor‐1 mRNA in the cultured cells, and its production in these cells was demonstrated by radioimmunoassay. Insulin‐like growth factor‐1 receptor in the cultured cells was also demonstrated immunohistochemically. Scatchard analysis using an [125I]insulin‐like growth factor‐1 binding assay showed that the cells cultured in monolayer had a single type of insulin‐like growth factor‐1 receptor, whose affinity and number were estimated to be 7.38 × 108/M and 9.27 × 104/cell, respectively. These results suggest that insulin‐like growth factor‐1 stimulates proteoglycan synthesis in cells of the nucleus pulposus and that these cells in culture have an insulin‐like growth factor‐1 autocrine/paracrine mechanism. The expressions of insulin‐like growth factor‐1 mRNA and insulin‐like growth factor‐1 receptor in disc tissue were greater in cells of the nucleus pulposus of fetal bovine intervertebral discs than in those of the adult discs. These findings suggest that the action of autocrine/paracrine insulin‐like growth factor‐1 is more active in cells of the young nucleus pulposus than in cells of mature subjects.
Interaction of tobramycin and pH in cultured chick tibiae
Authors: Murakami Takanori , Murakami Hiroko , Ramp Warren K. , Hanley Edward N.
Published: February 2005

The toxicity of tobramycin at concentrations released from antibiotic‐impregnated polymethylmeth‐acrylate beads was determined using cultured embryonic chick tibiae. Because previous results from this laboratory have shown that osteoblast metabolism is inhibited at low pH and because of the potential for a low local pH in infected bone, the antibiotic was tested in medium with pH values from 6.8 to 7.4. Bone metabolism was evaluated by measuring the rates of glycolysis (lactate production), protein synthesis ([3H]proline uptake), and collagen synthesis ([3H]proline hydroxylation). Tobramycin at the concentrations released from the beads (1.0–1.5 mg/ml) contributed to lowering the pH of the medium. At pH 7.4, the antibiotic produced as much as a 30, 39, and 48% decrease in glycolysis, protein synthesis, and collagen synthesis, respectively. Tibiae exposed to tobramycin for 3 days, followed by 2 days without the antibiotic, showed only a partial recovery from its toxic effects. Although tobramycin was relatively less toxic in an acidic environment, the overall metabolic activity of the bones was poorest when the tobramycin concentration was high (2.0 mg/ml) and pH was low (6.8). The results of this study support the following conclusions: (a) tobramycin at high concentrations is toxic to bone, (b) a combination of high tobramycin concentration and low pH in the bone microenvironment may greatly inhibit bone metabolism, and (c) treatment and prevention of osteomyelitis by means of tobramycin‐impregnated beads may be augmented by preventing the pH from decreasing in traumatized areas.
In vitro fatigue behavior of the equine third metacarpus: Remodeling and microcrack damage analysis
Authors: Martin R. B. , Gibson V. A. , Stover S. M. , Gibeling J. C. , Griffin L. V.
Published: February 2005

We studied remodeling and microcrack damage in specimens of Thoroughbred racehorse third metacarpal bone that had been subjected to monotonic or fatigue failure. We asked three questions. What effects does mechanical loading have on histologically observable microcrack damage? Are there regional variations in remodeling of the equine cannon bone, and do these variations correlate with mechanical properties? To what extent are remodeling and microcrack damage age‐dependent? Machined beams from the medial, lateral, and dorsal cortices were loaded to fracture in four‐point bending monotonically, or cyclically at a load initially producing 10,000 microstrain. Specimens were then bulk‐stained in basic fuchsin, and cross sections were prepared from loaded and load‐free regions of each beam. Current and past remodeling, porosity, and microcrack density and length were determined histomorphometrically. Stained and unstained microcracks were observed. Unstained cracks were associated with regions of woven bone and appeared to be damaged Sharpey's fibers. Their density (approximately 30/mm2) did not increase after failure, but their length (approximately 25 μm) did, especially near the surfaces of the beam. Stained cracks were wider and longer than unstained cracks and were located primarily near the fracture surface and on the compressed side of the beam. Stained cracks after failure were more numerous in those beams having a higher elastic modulus, a shorter fatigue life, or greater deformation at failure. The extent of past remodeling increased with age, especially in the medial region; the rate of current remodeling generally declined with age, but not in the dorsal region, which has the best fatigue resistance. In summary, while remodeling varied with age and region, its effects on bone structure did not appear to influence microdamage. Basic fuchsin staining of damage in fractured equine bone was independent of age and region and confined to near the fracture surfaces. Distributed microdamage consisted only of what appeared to be subtle disruptions of Sharpey's fibers.
Exposure to low‐intensity ultrasound increases aggrecan gene expression in a rat femur fracture model
Authors: Yang Kyu‐Hyun , Parvizi Javad , Wang Shyu‐Jye , Lewallen David G. , Kinnick Randall R. , Greenleaf James F. , Bolander Mark E.
Published: February 2005

The effects of ultrasound stimulation on various parameters of bone repair after diaphyseal injury were assessed in a standard rat femur fracture model. Bilateral closed femoral fractures were made in 79 skeletally mature male Long‐Evans rats. An ultrasound signal consisting of a 200 microsecond burst sine wave of 0.5 MHz repeating at 1 kHz, with an intensity of 50 or 100 mW/cm2 spatial and temporal average, was applied to one fracture in each animal. The contralateral fracture was not exposed to ultrasound and served as a control. Mechanical testing of the healing fracture was performed 3 weeks after injury. In fractures treated with a 50 mW/cm2 ultrasound signal, the average maximum torque (223.5 ± 50.5 Nmm compared with 172.6 ± 54.9 Nmm, p = 0.022, paired t test) and average torsional stiffness (13.0 ± 3.4 Nmm/° compared with 9.5 ± 2.9 Nmm/°, p = 0.017) were significantly greater in treated than in control fractures. In animals treated with a 100 mW/cm2 ultrasound signal, the average maximum torque and torsional stiffness were greater in treated than in control fractures, but this trend did not reach statistical significance. Biochemical analysis of callus in ultrasound‐treated and control fractures failed to demonstrate significant differences in cell number, collagen content, or calcium content. Evaluation of gene expression in fractures treated with 50 mW/cm2 ultrasound demonstrated a shift in the expression of genes associated with cartilage formation; aggrecan gene expression was significantly higher on day 7 after fracture and significantly lower on day 21 (p = 0.033 and 0.035, respectively). αl (11) procollagen gene expression was similarly modified, but this trend did not reach statistical significance. Expression of genes coding for bone‐related proteins, including α1(I) procollagen, bone γ‐carboxyglutamic acid protein, alkaline phosphatase, and transforming growth factor‐β1, did not differ between ultrasound‐treated and control fractures. These data suggest that ultrasound stimulation increased the mechanical properties of the healing fracture callus by stimulating earlier synthesis of extracellular matrix proteins in cartilage, possibly altering chondrocyte maturation and endochondral bone formation.
Correlation of bony ingrowth to the distribution of stress and strain parameters surrounding a porous‐coated implant
Authors: Qin Yi‐Xian , McLeod Kenneth J. , Guilak Farshid , Rubin Clinton T. , Chiang Fu‐Pen
Published: February 2005

The ability of shear strains to inhibit bony ingrowth was investigated by use of a transcortical porouscoated cylindrical plug implant in a functionally isolated turkey ulna model in which the mechanical loading environment could be accurately controlled and rigorously defined. The distribution of ingrowth at the bone‐implant interface was quantified following 8 weeks of in vivo loading consisting of 100 seconds per day of a 20 Hz sinusoidal stimulus sufficient to cause a local peak strain of approximately 100 microstrain in the cortex at the bone‐implant interface in four turkeys. A nonuniform but repeatable pattern of bony ingrowth, from 33 ± 6 to 72 ± 6% (mean ± SE), was observed. The mechanical environment in the vicinity of the bone‐implant interface was calculated using a three‐dimensional elastic orthotropic finite element model. The general stress‐strain state of the bone as predicted by the finite element model was validated in two additional turkeys using four three‐element rosette strain gauges, while high resolution moiré interferometry was used to determine the mechanical state of the region immediately adjacent to the implant itself. Shear strains and stresses were evaluated at the interface and correlated to the pattern of bony ingrowth circumscribing the implant interface. Linear regressions between ingrowth and both shear strain and shear stress were negative, with the values of R = −0.75 and R = −0.78 (p < 0.001), respectively, indicating significant inhibition of ingrowth where shear components were maximal. These results suggest that the minimization of shear stress and strain components is a major determinant in achieving successful ingrowth of bone into a prosthesis.
Novel animal model for studying the molecular mechanisms of bacterial adhesion to bone‐implanted metallic devices: Role of fibronectin in Staphylococcus aureus adhesion
Authors: Fischer Béat , Vaudaux Pierre , Magnin Marc , El Mestikawy Yousti , Vasey Harold , Lew Daniel P. , Proctor Richard A.
Published: February 2005

Infection around metallic implants is a rare but severe complication of orthopaedic surgery. A novel animal model mimicking conditions of internal fixation devices was developed to evaluate the role of host proteins adsorbed on metallic devices in promoting adhesion and colonization of the material surfaces by Staphylococcus aureus. Small plates made of pure titanium were either fixed (three screws per plate) onto the iliac bones of guinea pigs or implanted into their subcutaneous space as controls. Five to 6 weeks after surgery, the plates and screws were removed from the previously killed animals, carefully rinsed in buffer, and tested in an in vitro assay of S. aureus adhesion to metallic surfaces. To evaluate the role of fibronectin in staphylococcal adhesion to explanted plates and screws, a mutant of S. aureus that is specifically defective in fibronectin adhesion due to decreased expression of the fibronectin adhesin was compared with its isogenic parental strain. A significant reduction in adhesion of the fibronectin adhesin‐defective mutant compared with the parental strain occurred on both the subcutaneously implanted and bone‐implanted metallic plates. The results of this specific biological assay suggest that fibronectin is present on bone‐implanted metallic devices and promotes attachment of S. aureus to their surfaces. This novel experimental model should help to characterize several parameters of bacterial adhesion to metallic orthopaedic devices and to develop novel anti‐adhesive strategies for preventing such infections.