James H. Kimura

Influence of nonenzymatic glycation on biomechanical properties of cortical bone

In this study, the influence of nonenzymatic glycation (NEG) on the mechanical properties of bone and bone collagen were investigated. Bovine cortical bone specimens were incubated in ribose to cause collagen cross-links in vitro, and nondestructive mechanical testing was used to determine tensile and compressive elastic modulus as a function of incubation time. Mechanical properties associated with yield, postyield, and final fracture of bone were determined at the end of the incubation period. The stiffness of the collagen network was measured using stress relaxation tests of demineralized bone cylinders extracted periodically throughout the incubation period. It was found that accumulation of nonenzymatic glycation end-products in cortical bone caused stiffening of the type I collagen network in bone (r2 = 0.92; p < 0.001) but did not significantly affect the overall stiffness of the mineralized bone (p = 0.98). The ribosylated group had significantly more NEG products and higher yield stress and strain than the control group (p < 0.05). Postyield properties including postyield strain and strain energy were lower in the ribosylated group but were not significantly different from the control group (p = 0.24). Compared with the control group, the ribosylated group was characterized by significantly higher secant modulus and lower damage fraction (p < 0.05). Taken together, the results of this study suggest that collagen in bone is susceptible to the same NEG-mediated changes as collagen in other connective tissues and that an increased stiffness of the collagen network in bone due to NEG may explain some of the age-related increase in skeletal fragility and fracture risk.

Chondrocyte differentiation is modulated by frequency and duration of cyclic compressive loading.

AbstractAs part of a program of research aimed at determining the role of mechanical forces in connective tissue differentiation, we have developed a model for investigating the effects of dynamic compressive loading on chondrocyte differentiation in vitro. In the current study, we examined the influence of cyclic compressive loading of chick limb bud mesenchymal cells to a constant peak stress of 9.25 kPa during each of the first 3 days in culture. Cells embedded in agarose gel were subjected to uniaxial, cyclic compression at 0.03, 0.15, or 0.33 Hz for 2 h. In addition, load durations of 12, 54, or 120 min were evaluated while holding frequency constant at 0.33 Hz. For a 2 h duration, there was no response to loading at 0.03 Hz. A significant increase in chondrocyte differentiation was associated with loading at 0.15 Hz, and an even greater increase with loading at 0.33 Hz. Holding frequency constant at 0.33 Hz, a loading duration of 12 min elicited no response, whereas chondrocyte differentiation was enhanced by loading for either 54 or 120 min. Although not statistically significant from the 120 min response, average cartilage nodule density and glycosaminoglycan synthesis rate were highest in the 54 min duration group. This result suggests that cells may be sensitive to the level of cumulative (nonrecoverable) compressive strain, as well as to the dynamic strain history.

Proteoglycans and glycosaminoglycan fine structure in the mouse tail tendon fascicle

The isolated mouse tail tendon fascicle, a functional and homogenous volume of tendon extracellular matrix, was utilized as an experimental system to examine the structure–function relationships in tendon. Our previous work using this model system demonstrated relationships between mean collagen fibril diameter and fascicle mechanical properties in isolated tail tendon fascicles from three different groups of mice (3‐week and 8‐week control and 8‐week Mov13 transgenic) K.A. Derwin, L.J. Soslowsky, J. Biomech. Eng. 121 (1999) 598–604. These groups of mice were chosen to obtain tendon tissues with varying collagen fibril structure and/or biochemistry, such that relationships with material properties could be investigated. To further investigate the molecular details of matrix composition and organization underlying tendon function, we report now on the preparation, characterization, and quantitation of fascicle PGs (proteoglycans) from these three groups. The chondroitin sulfate/dermatan sulfate (CS/DS)‐substituted PGs, biglycan and decorin, which are the abundant proteoglycans of whole tendons, were also shown to be the predominant PGs in isolated fascicles. Furthermore, similar to the postnatal maturation changes in matrix composition previously reported for whole tendons, isolated fascicles from 8‐week mice had lower CS/DS PG contents (both decorin and biglycan) and a higher collagen content than 3‐week mice. In addition, CS/DS chains substituted on PGs from 8‐week fascicles were shorter (based on a number average) and richer in disulfated disaccharide residues than chains from 3‐week mice. Fascicles from 8‐week Mov13 transgenic mice were found to contain similar amounts of total collagen and total CS/DS PG as age‐matched controls, and CS/DS chain lengths and sulfation also appeared normal. However, both decorin and biglycan in Mov13 tissue migrated slightly faster on sodium dodecyl sulfate polyacrylamide gel electorphoresis (SDS‐PAGE) than the corresponding species from 8‐week control, and biglycan from the 8‐week Mov13 fascicles appeared to migrate as a more polydisperse band, suggesting the presence of a unique PG population in the transgenic tissue. These observations, together with our biomechanical data [Derwin and Soslowsky, 1999] suggest that compensatory pathways of extracellular matrix assembly and maturation may exist, and that tissue mechanical properties may not be simply determined by the contents of individual matrix components or collagen fibril size.

Determination of bone volume by osteocyte population

During development and growth, biological tissues and organisms can control their size and mass by regulating cell number (Raff, 1992 ; Conlon and Raff, 1999 ). Later in life both cell number and organ mass decrease (Buetow, 1985 ). We demonstrate that the number density of bone cells buried in the calcified matrix (osteocyte lacunar density) predicts extracellular matrix volume for both cancellous and cortical bone in a broad cross‐section of the population (males and females, age range 23–91 years, r2 = 0.98). Our hypothesis is that bone mass is determined by the control of osteocyte number, and that this is a particular instance of the control of organ size through the social controls on cell survival and death (Raff, 1992 ; Conlon and Raff, 1999 ). Anat Rec 267:292–295, 2002.

Spontaneous and experimental osteoarthritis in dog: similarities and differences in proteoglycan levels.

The unilateral canine model is the most commonly used model of experimental osteoarthritis (OA). In this model, the anterior cruciate ligament (ACL) of one knee is transected and the contralateral joint is usually used as a control. However, dogs, similar to humans, can develop OA spontaneously with old age. Additionally, certain breeds of dogs are genetically predisposed to OA and can develop symptoms at a young age. The goal of this study was to compare the pathological changes of proteoglycans in OA cartilage from dogs that developed OA spontaneously to those that underwent ACL transection. For this reason, biglycan, decorin and fibromodulin levels and degradation patterns were compared by Western blot hybridization, and aggrecan contents were quantified by dimethylmethylene blue assay. The changes in proteoglycan levels in the cartilage of dogs with spontaneous OA, regardless of their age, were very similar to those published for human OA cartilage. However, when OA developed as a result of ACL‐surgery, the changes in proteoglycans were different from those of slowly developing spontaneous OA. Therefore, these differences should be taken into consideration when the ACL‐transection model is used.

A newly identified enhancer element responsible for type II collagen gene expression

Type II collagen is a major component of cartilage where it is present at a high concentration, which is essential for the functional maintenance of the tissue. Therefore, any fundamental understanding of the physiology of cartilage tissue must include an understanding of the mechanism that allows the high level of expression of type II collagen gene, Col2a1, by chondrocytes. To this end, we developed a new reporter assay system based on the co-transfection of candidate enhancer elements and reporter construct into Swarm rat chondrosarcoma chondrocytes that allowed their stable expression. Using this system, we screened more than 70 kb of the Col2a1 gene and found an enhancer domain that is responsible for the regulation of its expression level. The domain is localized in intron 7, and consists of an 800-bp region that contains within it a previously unidentified domain, ∼140 bp in size.