Hans Oxlund

Biomechanical properties of keratoconus and normal corneas

The uniaxial tensile strength was determined for six keratoconus and seven normal corneas. Strip specimens were stretched at a constant speed in a materials testing machine while recording load and deformation. Load-deformation curves were transformed into load-strain and stress-strain curves. The following parameters were derived from the load-strain and stress-strain curves: the maximum load and stress values and the corresponding strain value, the values for maximum stiffness, and the relative energy absorption of the specimens during testing. With the exception of the strain value at maximum load or stress, these parameters were smaller in the keratoconus group than in the normal group. The load and stress values at corresponding strain values were less in keratoconus corneas than in normal corneas both for the initial and for the linear parts of the curves. Biochemically, no difference was found in the concentration of uronic acid in the two groups. Similarly no difference was found in the concentration of hydroxyproline in the two groups. The solubility of pepsin treated collagen in the keratoconus corneas was greater than that in the normal corneas, however, the solubility was less than that in tendons.

Intermittent Parathyroid Hormone (1–34) Treatment Increases Callus Formation and Mechanical Strength of Healing Rat Fractures

The influence of intermittent parathyroid hormone (PTH(1–34)) administration on callus formation and mechanical strength of tibial fractures in rats was investigated after 20 and 40 days of healing. A dose of 60 μg of PTH(1–34)/kg/day and 200 μg of PTH(1–34)/kg/day, respectively, was administered during the entire periods of healing, and control animals with fractures were given vehicle. The dose of 200 μg of PTH(1–34)/kg/day increased the ultimate load and the external callus volume of the fractures by 75% and 99%, respectively, after 20 days of healing and by 175% and 72%, respectively, after 40 days of healing. The dose of 60 μg of PTH(1–34)/kg/day did not influence either ultimate load or external callus volume of the fractures after 20 days of healing, but the ultimate load was increased by 132% and the external callus volume was increased by 42% after 40 days of healing. During the healing period, the callus bone mineral content (BMC) increased in all groups. After 40 days of healing, the callus BMC was increased by 108% in the 200 μg of PTH(1–34)/kg/day group and by 76% in the 60 μg of PTH(1–34)/kg/day group. Both doses of PTH(1–34) steadily augmented the contralateral intact tibia BMC (20 days and 40 days: 60 μg of PTH (1–34)/kg/day 9% and 19%, respectively; 200 μg of PTH (1–34)/kg/day 12% and 27%, respectively) and bone mineral density (20 days and 40 days: 60 μg of PTH(1–34)/kg/day 11% and 12%, respectively; 200 μg of PTH(1–34)/kg/day 11% and 15%, respectively).

Reduced concentrations of collagen cross-links are associated with reduced strength of bone

The known cross-links of bone collagen are derived from lysine and hydroxylysine. The first step in the enzymatic cross-linking process is a deamination by lysyl oxidase producing an aldehyde which then may condense with a lysyl or hydroxylysyl residue of a neighbouring collagen molecule. Some of the resulting divalent aldimine and oxo-imine cross-links may later on be incorporated in trivalent hydroxylysyl-pyridinoline and lysyl-pyridinoline cross-links. In bone collagen prepared from the cancellous bone of vertebral bodies of osteoporotic individuals we found a reduced stability towards acetic acid and pepsin, and a substantial reduction in the concentration of the divalent collagen cross-links compared with sex- and age-matched controls. To what extent do the collagen cross-links influence the mechanical properties of bone? beta-amino-propionitrile (BAPN) irreversibly inhibits the enzyme lysyl oxidase and therefore, the formation of cross-links between the collagen molecules. In the present study female rats, 70 days old, injected subcutaneously two times daily with BAPN (333 mg/kg/day) for 1 month and saline injected control rats were studied. The concentration of the hydroxypyridinium cross-links of femoral mid-diaphyseal cortical bone was determined by HPLC with fluorescence detection and the mechanical properties of the rat femoral diaphyses were analyzed by a materials testing machine. The BAPN injections resulted in a 45% reduction in the concentration of the hydroxypyridinium cross-links and a 31% decrease in the stability of the bone collagen towards acetic acid and pepsin compared with the control rats. No changes were found in ash or collagen concentrations of the cortical bone.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of glycation cross-links in diabetic vascular stiffening.

SummaryPrevious studies have shown that biomechanical analysis of aorta from diabetic subjects reveals a marked increase in stiffness compared to aorta from age-matched control subjects. In the present paper we have proposed that this increased stiffness can be attributed to glycation-induced inter-molecular cross-links based on a direct analysis of the two known glycation cross-links, the fluorescent pentosidine and the non-fluorescent NFC-1. There was a significant difference in the increase in concentration of both cross-links with increasing age for both the intima (p<0.0025) and the media (p<0.0005) from the diabetic compared to the control subjects, but no correlation with the mature enzymic cross-link hydroxylysyl-pyridinoline. Finally, we have obtained a significant correlation of stiffness with both glycation cross-links (NFC-1, r=0.86; p<0.005 and pentosidine r=0.75, p<0.05), but the concentration of NFC-1 is about 50 times greater than that of pentosidine, indicating that it is the major glycation cross-link responsible for the stiffening of the aorta.

Changes in biomechanical properties, composition of collagen and elastin, and advanced glycation endproducts of the rat aorta in relation to age

During ageing and senescence the aorta becomes stiffer and its elasticity is reduced. The mechanism causing this increased stiffness of the aortic wall was studied using a rat model. Ring-shaped samples were prepared from the thoracic aorta of three groups of rats aged 4.5, 14 and 27 months, representing young, adult and old animals. Analysis of the static biomechanical properties showed increased diameter (2.20 +/- 0.03 mm) and increased stiffness (4.0 +/- 0.2 mN) of aortic samples from old rats compared with adult rats (1.82 +/- 0.02 mm and 3.0 +/- 0.1 mN, respectively). The total hydroxyproline and elastin content per sample was not changed. However, the hydroxyproline content/mm2 of the aortic wall was reduced by 20% and the elastin content/mm2 of the aortic wall was reduced by 19% comparing the old with the adult rats. No differences were found in the pyridinoline concentrations between old and adult rats. The collagen- and elastin-associated fluorescence was determined as a marker of advanced glycation endproducts (AGE). Both parameters were increased in the old rats compared with the adult rats by 42% and 17%, respectively, and positively correlated with stiffness at physiological loads. A positive correlation between collagen-associated fluorescence and maximum stiffness was found as well. In conclusion, the age-related increase in stiffness of the aorta was associated with increased diameter, reduced collagen and elastin contents/mm2 of the aortic wall, increased fenestration of elastic laminae and accumulation of fluorescent material in collagen and elastin.

Reduced concentration of collagen reducible cross links in human trabecular bone with respect to age and osteoporosis

The decrease of bone strength in relation to age and osteoporosis is more pronounced than would be expected from the relative deficit in the amount of bone. Besides bone mass, the mechanical properties of cancellous bone also depend on the microarchitecture and possibly on the molecular structure of inorganic and organic components. The present study examines the bone collagen, especially the collagen cross links, in relation to age and osteoporosis. Samples of vertebral trabecular bone were taken at autopsy from 43 normal individuals, aged 15-90 years. Eleven of these served as sex- and age-matched controls for similar samples from 11 osteoporotic individuals, 70-90 years. The volume of each trabecular bone sample was estimated. After removal of the marrow, the trabeculae were ground to powder and decalcified. The extractability of the bone collagen was studied by repeated extractions with acetic acid and pepsin. The divalent reducible collagen cross-links, dehydro-dihydroxylysinonorleucine (DHLNL) and dehydro-hydroxylysinonorleucine (HLNL), were determined by reducing the bone collagen with tritiated potassium borohydride followed by ion-exchange chromatography. The mature trivalent pyridinium cross links were determined by reverse-phase HPLC with fluorescence detection. The extractability of collagen prepared from the vertebral trabecular bone of control individuals was increased with age. Bone collagen of osteoporotic individuals showed increased extractability and a substantial decrease in the concentration of the divalent reducible collagen cross links (DHLNL reduced by 30% and HLNL by 24%) compared with the sex- and age-matched controls. No alterations were observed in the concentration of the pyridinolines. The divalent reducible cross-links are the most frequent known cross links in bone (2-4 times the concentration of the pyridinium cross links). These changes would therefore be expected to reduce the strength of the bone trabeculae and could explain why the osteoporotic individuals had bone fractures even though the collagen density (mg/cm3) did not differ from that of the sex- and age-matched controls. The microarchitecture of the cancellous bone was not assessed. The osteoporotic and control individuals seemed to have the same amount of trabecular bone, but the quality of the osteoporotic bone collagen was reduced.

Local injection of TGF-β increases the strength of tibial fractures in the rat

The effect of Transforming Growth Factor beta (TGF-beta) administered locally around the fracture line of healing rat tibial fractures was investigated after 40 days of healing. TGF-beta in a dose of 4 ng or 40 ng was injected every second day during the healing period. The strength, stiffness, energy absorption and deflection of the fractures were measured in a materials-testing machine. Compared with placebo-treated animals, the ultimate load of the fractures increased in the group injected with 40 ng of TGF-beta, but not in those injected with 4 ng. TGF-beta induced a dose-dependent increase in the cross-sectional area of the callus and bone at the fracture line. Consequently, local treatment of fractures with TGF-beta increases the callus formation and strength. The energy absorption and deflection capacities of the healing fractures are preserved.

THE ROLE OF ELASTIN IN THE MECHANICAL PROPERTIES OF SKIN

The elastin fibers of rat skin samples were degraded by the use of a purified preparation of elastase to which soybean inhibitor was added, preventing the collagenolytic activity of the elastase on collagen. Control experiments ascertained degradation of elastin and no effect on collagen. The mechanical properties of the skin samples were studied before and after the enzymatic treatment and differences ascribed to the degraded elastin fibers. Elastin plays a role in the mechanical behaviour of rat skin at small stress values and small deformations. Especially, the elastin fibers are responsible for the recoiling mechanism after a stress or deformation has been applied.

Inhibition of cross-links in collagen is associated with reduced stiffness of the aorta in young rats.

Collagen and elastin fibres are of major importance in providing the aorta with tensile strength and elasticity. The presence of cross-links in collagen and elastin is essential for the mechanical stability of collagen and elastin fibres. beta-aminopropionitrile (BAPN) reduces the formation of cross-links by inhibiting the enzyme lysyloxidase. Young rats were injected with BAPN to inhibit the formation of cross-links, and the changes in the biomechanical and biochemical properties of the thoracic aorta were studied. The biomechanical analyses of aortic samples from BAPN-treated rats showed a significantly increased diameter (1.64 +/-0.02 mm), a significantly reduced maximum load (1.08+/-0.08 N), and a significantly reduced maximum stiffness (3.34+/-0.10 N) compared with controls (1.57+/-0.02 mm, 1.55+/-0.04 N and 4.49 +/-0.14 N, respectively). No changes in the concentrations of collagen and elastin were found. The content of pyridinoline, a mature collagen cross-link, was significantly decreased by 49% in the BAPN-treated group compared with controls. No changes in the concentration of desmosine + isodesmosine, the major cross-links of elastin. were found. The present study shows that cross-links are essential in providing mechanical stability of the aorta. Even a partial inhibition of the cross-linking processes results in a destabilisation of the aortic wall with increased diameter and reduced strength and stiffness.

Increased aortic stiffness in patients with Type 1 (insulin-dependent) diabetes mellitus

SummaryThe biomechanical properties of aortic samples from patients with Type 1 (insulin-dependent) diabetes mellitus and age- and sex-matched control subjects were analysed using a materials testing machine. The specimens were prepared from tissue outside areas of visible atherosclerosis in order to discriminate between primary Type 1 diabetic alterations in the aortae and secondary changes due to increased atherosclerosis. We paid special attention to the correction of biomechanical parameters for differences in wall thickness and registration of specimen length values. In the Type 1 diabetic aortae a marked reduction was found in the extensibility and an increase in their stiffness. The reduced extensibility was correlated significantly to the duration of Type 1 diabetes. The pronounced alterations in the mechanical properties could not be explained by the increase in the wall thickness which was observed among the Type 1 diabetic patients and the alterations could not be correlated to the grade of atherosclerosis in the thoracic aorta. The results of the present study, therefore, strongly suggest that Type 1 diabetic patients develop alterations in the arterial connective tissue independent of the presence of atherosclerosis. Such primary alterations in the vessel wall may play a role in the pathogenesis of large vessel disease among these patients.