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Biomedical subjects

W H Akeson

Publications and source records attributed to W H Akeson.

At least 19 recordsLinked to original sources
PubMed2002

Integrative articular cartilage repair: dependence on developmental stage and collagen metabolism.

OBJECTIVES: The objectives of this research were to determine whether the integrative repair of bovine cartilage explants was dependent on developmental stage, and whether observed differences in integration with developmental stage were related to deposition of newly synthesized collagen and lysyl oxidase-mediated collagen cross-linking. METHODS: Pairs of fetal, newborn calf, and adult bovine cartilage blocks were cultured in partial apposition for 2 weeks in medium supplemented with serum, ascorbate, and [3H]proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength in a single-lap shear configuration. Formation and stabilization of newly synthesized protein and collagen was investigated by determination of [3H]proline and [3H]hydroxyproline in tissue digests and guanidine extracts. RESULTS: Calf cartilage exhibited a relatively high integrative repair phenotype, achieving an adhesive strength that was three--four-fold that of adult or fetal specimens. The low and high integrative repair phenotypes appeared related in part to different levels of collagen biosynthesis, which was approximately four--five-fold higher in calf cartilage samples than in the adult. However, fetal cartilage also exhibited a high level of biosynthesis. The different integrative repair phenotypes were not associated with marked differences in the kinetics of chemical stabilization of newly synthesized collagen, as the proportion of incorporated [3H]proline and newly-formed [3H]hydroxyproline that was resistant to extraction by 4M guanidine-HCl following culture was similar for cartilage from all developmental stages. Integration of calf cartilage appeared to depend on lysyl oxidase-mediated collagen cross-link formation, since inclusion of beta-aminopropionitrile (BAPN) in the culture medium completely eliminated development of adhesive strength. BAPN treatment also increased the percentage of newly synthesized protein in the guanidine extracts from 10% to 36% of the total, and that of newly synthesized collagen from 2% to 20%, while having only slight inhibitory effects on overall protein and collagen biosynthesis. CONCLUSION: The finding that cartilage exhibits enhanced integrative repair at a certain developmental stage suggests that it may ultimately be possible to enhance repair when needed in clinical situations.

Animals↗
PubMed2001

Differences in mesenchymal tissue repair.

Variations in certain mesenchymal tissue healing processes are not widely recognized. The current review summarizes key differences in healing mechanisms and healing potential after injury to soft tissues having different healing outcomes.

Animals↗
PubMed2000

Titanium particles inhibit osteoblast adhesion to fibronectin-coated substrates.

To illuminate the effect of titanium particles on osteoblast function, we compared the adhesion force of neonatal rat calvarial osteoblasts on fibronectin-coated glass after incubation with titanium particles (80% had diameters of less than 5 microm). The cells were incubated with the particles for 1.5-72 hours. Using a micropipette single-cell manipulation system, we showed that the adhesion force of the osteoblasts to fibronectin-coated glass (1.0 microg/ml) was significantly affected by the presence of particulate debris. The adhesion force of the cells incubated with titanium particles for less than 4 hours was not significantly affected by exposure to the particles; after 4 hours, however, it was significantly reduced relative to that of controls. Aspiration of particle-challenged osteoblasts into the micropipette demonstrated that the particles were not stripped from the cell surface and therefore confirmed that the osteoblasts had ingested them. During aspiration, the particles traveled through the cytoplasm rather than on the cell surface. When the osteoblasts were exposed to the particles and cytochalasin D, they exhibited much lower adhesion forces than did the controls or the cells exposed to titanium particles only; this indicates an important role of actin filaments in the osteoblastic response to particles. Staining for F-actin also indicated an influence of internalized titanium particulate on cytoskeletal arrangement and cell spreading. Furthermore, with standard Northern blotting techniques, levels of mRNA for collagen type I and fibronectin were significantly reduced as early as 4 hours after exposure to particles compared with levels in controls, and this effect continued to 72 hours. These data indicate that direct exposure of osteoblasts to titanium particles, which we propose to be ingested by the osteoblasts, can significantly decrease osteoblast adhesion force; this may lead to decreased cellular activity and gene expression of fibronectin and collagen type I in the presence of titanium wear debris.

Actins↗
PubMed2000

Time-dependent increases in type-III collagen gene expression in medical collateral ligament fibroblasts under cyclic strains.

Numerous studies have demonstrated the capacity of mechanical strains to modulate cell behavior through several different signaling pathways. Understanding the response of ligament fibroblasts to mechanically induced strains may provide useful knowledge for treating ligament injury and improving rehabilitation regimens. Biomechanical studies that quantify strains in the anterior cruciate and medial collateral ligaments have shown that these ligaments are subjected to 4-5% strains during normal activities and can be strained to 7.7% during external application of loads to the knee joint. The objective of this study was to characterize the expression of types I and III collagen in fibroblast monolayers of anterior cruciate and medial collateral ligaments subjected to equibiaxial strains on flexible growth surfaces (0.05 and 0.075 strains) by quantifying levels of mRNA encoding these two proteins. Both cyclic strain magnitudes were studied under a frequency of 1 Hz. The results indicated marked differences in responses to strain regimens not only between types I and III collagen mRNA expression within each cell type but also in patterns of expression between anterior cruciate and medial collateral ligament cells. Whereas anterior cruciate ligament fibroblasts responded to cyclic strains by expression of higher levels of type-I collagen message with almost no significant increases in type-III collagen, medial collateral ligament fibroblasts exhibited statistically significant increases in type-III collagen mRNA at all time points after initiation of strain with almost no significant increases in type-I collagen. Furthermore, differences in responses by fibroblasts from the two ligaments were detected between the two strain magnitudes. In particular, 0.075 strains induced a time-dependent increase in type-III collagen mRNA levels in medial collateral ligament fibroblasts whereas 0.05 strains did not. The strain-induced changes in gene expression of these two collagens may have implications for the healing processes in ligament tissue. The differences may explain, in part, the healing differential between the anterior cruciate and medial collateral ligaments in vivo.

Adult↗
PubMed2000

Novel method for the quantitative assessment of cell migration: a study on the motility of rabbit anterior cruciate (ACL) and medial collateral ligament (MCL) cells.

A novel method of quantitating cell migration has been proposed for the potential utilization of tissue engineered scaffolds. Applying Alt's conservation law to describe the motion of first passage ACL and MCL cells, we have developed a quantitative method to assess innate differences in the motility of cells from these two ligamentous tissues. In this study, first passage ACL and MCL cells were cultured from four mature New Zealand white rabbits. One side of the cell monolayer was scraped completely away to create a wound model. The cell moved into the cell-free area, and cell density profiles were analyzed at 6 h and 12 h. Values of the random motility coefficient (mu) were then estimated by curve fitting the 6 h and 12 h data to a mathematical model, derived from the conservation law of cell flux. During 6 h of incubation in medium supplemented with 1% FBS, MCL cells (mu(MCL) = 4.63 +/- 0.65 X 10(-6) mm(2)/sec) were significantly (p < 0.05) more mobile than ACL cells (mu(ACL) = 2.51 +/- 0.31 X 10(-6) mm(2)/sec). At 12 h, the MCL cells also appeared to move faster (mu(ACL) = 4.39 +/- 0.63 X 10(-6) mm(2)/sec, mu(MCL) = 6.59 +/- 1.47 X 10(-6) mm(2)/sec), but the difference was not statistically significant (p = 0.18). Exposure of the cells to growth factors PDGF-BB or bFGF for 6 h had no significant effect on the migration of the ACL and MCL cells. However, exposure of the ACL cells (p < 0.05) and the MCL cells (p = 0.19) to 1 ng/mL of PDGFBB for 12 h enhanced their migration. Incubation with a high concentration (100 ng/mL) of PDGF-BB or bFGF at concentrations tested (1 or 100 ng/mL) for 12 h, produced little or no migratory stimulation on these ligament cells. Our findings support the previous qualitative observations made by numerous investigators. The novel methodology developed in this study may provide a basis for tissue engineering, and the results may be applied to tissue reconstruction techniques of the knee ligaments.

Animals↗
PubMed2000

Adhesive force of chondrocytes to cartilage. Effects of chondroitinase ABC.

Chondrocyte transplantation is a clinical procedure for cartilage repair. Transplanted cells may have difficulty attaching to the surface of chondral lesions because of the anti-adhesive properties of the proteoglycan rich matrix. This study used micromanipulation methods to determine if pretreatment of cartilage with chondroitinase ABC affects chondrocyte adhesion to cartilage and if chondrocytes adhere preferentially to the superficial, middle, or deep layers of cartilage. Bovine chondrocytes were transplanted in vitro on articular cartilage sections cut perpendicular to the articular surface. At various times between 15 and 75 minutes after seeding, a micropipette micromanipulation system was used to measure the adhesion force of individual chondrocytes to cartilage. The chondrocyte adhesion force increased with chondroitinase ABC treatment and seeding time but generally was similar for the different regions of articular cartilage (superficial, middle, deep layer) to which the cells were attached. For normal cartilage, the adhesion force increased from 1.29 +/- 0.24 mdyne after 15 to 30 minutes seeding to 5.29 +/- 0.25 mdyne after 60 to 75 minutes. Treatment with chondroitinase ABC at certain concentrations and durations (1.0 U/mL for 5 minutes or 0.5 or 1 U/mL for 15 minutes) led to an increase in adhesion force, whereas relatively low concentration or treatment time (0.25 U/mL for 15 minutes or 0.5 U/mL for 5 minutes) had little or no detectable effect. The increase in adhesion attributable to chondroitinase ABC treatment appeared most marked (+144% to +292%) for short (15 to 30 minutes) seeding durations but was still significant (+46%) for the longest seeding period (60 to 75 minutes) studied after the 1 U/mL for 15 minute treatment condition. These results provide direct biomechanical evidence that enzymatic treatment of a cartilage surface can enhance chondrocyte adhesion.

Analysis of Variance↗
PubMed1999

Adhesion strength differential of human ligament fibroblasts to collagen types I and III.

Fibroblasts embedded in the amorphous healing tissue matrix of ligaments migrate into damaged sites during the inflammatory process that precedes the formation of new connective tissue after ligament injury. Cell motility involved in this migration is strongly influenced by cellular adhesion to proteins of the extracellular matrix. The adhesion mechanism of interest in this study is the attachment of fibroblasts from the anterior cruciate and medial collateral ligaments to types I and III collagen, two fibrillar collagens secreted by fibroblasts during tissue repair. Types I and III collagen constitute a major portion of these ligaments and are assembled by fibroblasts into long cable-like fibrils in the extracellular space. In this study, a micropipette aspiration technique was used to measure the force required to separate fibroblasts of the anterior cruciate and medial collateral ligaments from substrates composed of type I or III collagen, each at a concentration of 2 or 5 microg/ml. Approximately 1,200 fibroblasts from the anterior cruciate ligament and 1,600 from the medial collateral ligament were used, and the adhesion force and area of these cells were determined. Fibroblasts from the anterior cruciate ligament exhibited greater adhesion force than did those from the medial collateral ligament for all concentrations of types I and III collagen. In addition, the adhesiveness of fibroblasts from both ligaments was dependent on seeding time for all experimental conditions. To determine the adhesiveness per unit area, defined here as the adhesion strength, the adhesion force was normalized by the adhesion area. At early seeding times (15-45 minutes), fibroblasts from the anterior cruciate ligament exhibited greater adhesion strength on surfaces coated with type-I collagen than did those from the medial collateral ligament. However, for both collagen substrates, adhesion strength for fibroblasts from the anterior cruciate ligament decreased with seeding time whereas that for fibroblasts from the medial collateral ligament remained relatively constant for all seeding periods (15-75 minutes).

Adult↗
PubMed1999

Articular cartilage transplantation. Clinical results in the knee.

Between December 1983 and August 1991, 55 consecutive patients (55 knees) who underwent articular cartilage transplantation to their damaged knees were enrolled in the study. Average followup was 75 months (range, 11-147 months). Eight-two percent were younger than 45 years of age. Patients were evaluated through an 18-point scale, with 6 points each allocated to pain, range of motion, and function. An excellent knee was pain free, had full range of motion, and permitted unlimited activity. A good knee allowed full time employment and moderate activity. Eleven of 15 (73%) allografts transplanted 10 or more years ago were still good or excellent at the time of last followup. Overall, 45 of 55 (76%) knees that received the transplants were rated good or excellent. Specifically, 36 of 43 (84%) patients with unipolar transplants regained normal use of their resurfaced knee. The results after bipolar resurfacing were less encouraging, with only six of 12 (50%) knees rated good or excellent. The described technique of osteochondral shell allograft resurfacing of the knee capitalize on the different healing potentials of bone and cartilage by transplanting the viable articular cartilage organ in its entirety along with just enough of the underlying bone to allow for graft incorporation through creeping substitution. The results support the use of fresh osteochondral shell allograft transplantation for the treatment of large, full thickness articular cartilage defects to the medial or lateral femoral condyles and to the patella.

Adolescent↗
PubMed1999

Collagen fibril diameter distributions in rabbit anterior cruciate and medial collateral ligaments: changes with maturation.

This study presents morphometric analyses of the collagen fibril diameters of rabbit anterior cruciate and medial collateral knee ligaments of New Zealand White rabbits (young, age two months and adult, age thirty-six to forty months). Measurements were made from transmission electron micrographs of transverse ligament sections of approximately 50,000x magnification. Statistically significant differences in the mean fibril diameters were found between the anterior cruciate and medial collateral ligaments of the thirty-six to forty month old animals (.069 +/- .005, .092 +/- .016 mm, p < .1); however, no statistical significance was found for differences between these ligaments in two month old animals (.077 +/- .006, .082 +/- .009, p > .1). These data support the idea that known differences in fibril distributions of adult rabbit anterior cruciate and medial collateral ligaments develop with maturation, and may reflect both the cellular environment in which the fibrocytes of these ligaments are subject to, as well as the developmental genetic program of these cell populations.

Animals↗
PubMed1998

Growth factor expression in healing rabbit medial collateral and anterior cruciate ligaments.

Exogenously administered growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta) and basic fibroblast growth factor (bFGF) have been shown to affect connective tissue healing in vivo, but their intrinsic role in the healing response has not been established. In the present study, immunohistochemistry with antibodies directed against these growth factors showed that expression of PDGF, TGF-beta 1 and bFGF was increased in and around the wound site in the rabbit medial collateral ligament (MCL) seven days following surgical injury. The strong expression of PDGF correlated with the observed increased cellularity consistent with this growth factor's mitogenic and chemotactic properties. Expression of these growth factors was also increased in wounded rabbit anterior cruciate ligaments (ACL) at seven days following surgical injury, but such expression was limited to the edge of the ACL injury site and was of lesser intensity relative to the MCL. This study suggests that PDGF and TGF-beta 1, and to a lesser extent bFGF, are actively involved during the early stage of MCL healing, but have a more limited presence in the injured rabbit ACL.

Animals↗
PubMed1997

A curriculum for the ideal orthopaedic residency. Academic Orthopaedic Society.

The Academic Orthopaedic Society met in April 1994 to discuss manpower issues in orthopaedics. The members developed an approach using the Delphi system to define and obtain consensus on the characteristics of the ideal residency. Six categories of educational attributes were included: General; Clinical Management; Skills and Technical Aspects; Rehabilitation; Basic Science and Research; and Educational Environment. The following year a questionnaire was sent to more than 125 programs in an attempt to have residents and staff anonymously self score their residencies according to the standards defined by the Delphi panels. The results obtained from the 745 responders from 73 programs validate effectively the characteristics of the ideal program and also show the variation among the programs.

Clinical Competence↗
PubMed1997

Decrease in fibronectin occurs coincident with the increased expression of its integrin receptor alpha5beta1 in stress-deprived ligaments.

Stress deprivation secondary to immobilization leads to atrophic changes in periarticular soft tissues. The changes in ligaments include a disorganization of collagen and cellular ultrastructure with varied biochemical alterations resulting in a functionally weaker tissue. This study tests the hypothesis that alterations in fibronectin (Fn) and the expression of its integrin receptor alpha5beta1 in ligament fibroblasts accompany the extracellular matrix remodeling which occurs in stress-deprived knee ligaments. The left knees of eighteen New Zealand white rabbits were surgically immobilized in acute flexion. Fibroblasts within three nine week and three twelve week stress-deprived anterior cruciate ligaments (ACLs) and medial collateral ligaments (MCLs) demonstrated markedly increased immunostaining for the beta1 and alpha5 integrin subunits, as compared to fibroblasts in the contralateral unoperated control ligaments. The effects of stress deprivation on the concentration of Fn was measured by competitive ELISA on the remaining twelve rabbits. Decreases in Fn of 54.0 percent and 63.7 percent occurred in the ACL after nine and twelve weeks of stress deprivation when compared to contralateral controls. The MCL had less of a decrease, losing 37.7 percent and 41.7 percent at nine and twelve weeks, respectively. These results suggest an important role for the Fn-specific integrin receptor alpha5beta1 in remodeling stress-deprived periarticular ligamentous tissue, and the importance of maintaining normal stresses on periarticular ligaments to prevent the degradation of extracellular matrix components such as Fn.

Animals↗
PubMed1996

The differential adhesion forces of anterior cruciate and medial collateral ligament fibroblasts: effects of tropomodulin, talin, vinculin, and alpha-actinin.

We have determined the effects of tropomodulin (Tmod), talin, vinculin, and alpha-actinin on ligament fibroblast adhesion. The anterior cruciate ligament (ACL), which lacks a functional healing response, and the medial collateral ligament (MCL), a functionally healing ligament, were selected for this study. The micropipette aspiration technique was used to determine the forces needed to separate ACL and MCL cells from a fibronectin-coated surface. Delivery of exogenous tropomodulin, an actin-filament capping protein, into MCL fibroblasts significantly increased adhesion, whereas its monoclonal antibody (mAb) significantly decreased cell adhesiveness. However, for ACL fibroblasts, Tmod significantly reduced adhesion, whereas its mAb had no effect. mAbs to talin, vinculin, and alpha-actinin significantly decreased the adhesion of both ACL and MCL cells with increasing concentrations of antibody, and also reduced stress fiber formation and cell spreading rate as revealed by immunofluorescence microscopy. Disruption of actin filament and microtubule assembly with cytochalasin D and colchicine, respectively, also significantly reduced adhesion in ACL and MCL cells. In conclusion, both ACL and MCL fibroblast adhesion depends on cytoskeletal assembly; however, this dependence differs between ACL and MCL fibroblasts in many ways, especially in the role of Tmod. These results add yet another possible factor in explaining the clinical differences in healing between the ACL and the MCL.

Actinin↗
PubMed1996

Signal pathways and ligament cell adhesiveness.

The influence of signal pathways involved in the adhesion of fibroblasts from the anterior cruciate and medial collateral ligaments to fibronectin was investigated. Specific emphasis was paid to the cyclic adenosine monophosphate and Ca2+/phospholipid pathways to determine the signaling mediated by integrin receptors during cell binding and spreading on a fibronectin-coated glass surface and to compare the roles of these two pathways in integrin-mediated adhesion in fibroblasts from the two ligaments. Individual cell adhesion strengths were determined using a micropipette-micromanipulation system after the cells were treated with signal pathway inhibiting agents. Adhesion in fibroblasts from the medial collateral ligament was significantly reduced by inhibiting agents for Gi protein, protein kinase A, protein kinase C, protein kinase G, phospholipase C, and calmodulin, which suggests a crucial role for cyclic adenosine monophosphate and Ca2+/phospholipid signaling in integrin-mediated adhesion of these fibroblasts. Adhesion in fibroblasts from the anterior cruciate ligament, however, was reduced only by a protein kinase C inhibiting agent and was increased by inhibiting agents for protein kinase A, protein kinase G, and calmodulin, which suggests only a partial role of Ca2+/phospholipid signaling in integrin-mediated adhesion of these fibroblasts. On the basis of additional parallel studies on the role of intracellular calcium in integrin-mediated adhesion, medial collateral ligament and anterior cruciate ligament fibroblast adhesion was calcium dependent throughout the 60 minute time course of adhesion experiments. Fibroblasts from the medial collateral ligament demonstrated a 2.2-fold increase in cytosolic free calcium upon binding to fibronectin, whereas fibroblasts from the anterior cruciate ligament demonstrated no significant increase in calcium. Overall, the study of the intrinsic differences between anterior cruciate ligament and medial collateral ligament fibroblasts in their signal pathways upon binding to fibronectin may reveal information important for further explaining the lack of functional healing response in the anterior cruciate ligament after injury.

Adult↗
PubMed1995

Adhesiveness of human ligament fibroblasts to laminin.

The adhesiveness of fibroblasts from the human anterior cruciate and medial collateral ligaments to the laminin molecule was studied, with particular emphasis on the intrinsic differences between fibroblasts from the two ligaments. Cellular adhesion strength, adhesion area, laminin concentration, and seeding time were examined. Cell adhesion to laminin anchored with poly-D-lysine to a cleaned cover glass was measured with a micropipette micromanipulation system after seeding. The adhesion strength of fibroblasts from the anterior cruciate ligament to laminin was greater than and significantly different from that of fibroblasts from the medial collateral ligament, depending on the laminin concentration. Fibroblasts from the anterior cruciate ligament also exhibited an increase in adhesion strength, dependent on laminin concentration of as much as 30 micrograms/ml, at which the laminin receptors were thought to be saturated. Fibroblasts from the medial collateral ligament did not show such an increase except at laminin concentrations of 5-10 micrograms/ml. There was no significant difference in adhesion area between fibroblasts from the two ligaments except after 45 minutes at a laminin concentration of 40 micrograms/ml. For both, the adhesion to laminin showed little correlation to seeding time during periods of as long as 60 minutes. Measurements of adhesion area also failed to show a significant correlation to seeding time for fibroblasts from either ligament at laminin concentrations of 20 and 40 micrograms/ml. Adhesion strength normalized by adhesion area had no correlation to seeding time.(ABSTRACT TRUNCATED AT 250 WORDS)

Anterior Cruciate Ligament↗
PubMed1995

Integrin display increases in the wounded rabbit medial collateral ligament but not the wounded anterior cruciate ligament.

The differential capacities of the anterior cruciate and medial collateral ligaments to heal may be related to differences in cellular function. This study tested the hypothesis that differential expression of integrins occurs in these ligaments after injury. The integrins are a family of cell surface receptors that mediate adhesion, migration, and other cellular functions critical to the healing of a wound. A similar complement and amount of the beta 1 subfamily of integrins are known to be present on the unperturbed anterior cruciate and medial collateral ligaments in humans and rabbits. A partial laceration was surgically created in these two ligaments in 12 anesthetized New Zealand White rabbits. Immunohistochemistry was performed on sections from the ligaments at 1, 3, 7, and 10 days after injury, using monoclonal antibodies directed against the integrin subunits beta 1, alpha 5, alpha 6, and alpha v. Between 3 and 7 days, the wounded medial collateral ligament demonstrated a striking increase in staining for the beta 1, alpha 5, and alpha v subunits on the fibroblasts, within the repair site, and on capillary endothelium. Increased staining was most marked for the beta 1 subunit and less marked for the alpha 5 and alpha v subunits. The alpha 6 subunit stained exclusively vascular structures within the healing medial collateral ligament. In marked contrast, the anterior cruciate ligament, which does not mount an effective repair response, demonstrated no comparable alteration of integrin expression from baseline levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗