Mechanism of bone collagen degradation due to KOH treatment.

BACKGROUND: The mechanisms underlying the effect of alterations in type I collagen on bone mechanical properties are not well defined. In a previous study, male and female emu tibiae were endocortically treated with 1M potassium hydroxide (KOH) solution for 1-14days. This treatment resulted in negligible mass loss (0.5%), collagen loss (0.05%), no differences in geometrical parameters but significant changes in mechanical properties. The objective of this study was to determine the mechanism of collagen degradation due to KOH treatment in order to explain the previously observed mechanical property changes. METHODS: Bone mineral was assessed using x-ray diffraction (XRD), microhardness and backscattered electron imaging (BSE). Bone collagen was assessed using α-chymotrypsin digestion, differential scanning calorimetry (DSC), gel electrophoresis (SDS-PAGE) and polarized light microscopy (PLM). RESULTS: BSE, microhardness and XRD revealed no changes in bone mineral due to KOH treatment. DSC showed an altered curve shape (lower and broader), indicating a change in collagen organization due to KOH treatment. Decreased α-chain band intensity in 14-day KOH treated groups detected using SDS-PAGE indicated α-chain fragmentation due to KOH treatment. PLM images revealed differences in collagen structure in terms of pattern distribution of preferentially oriented collagen between the periosteal and endocortical regions. CONCLUSION: These results suggest that endocortical KOH treatment causes in situ collagen degradation, which explains the previously reported altered mechanical properties. GENERAL SIGNIFICANCE: Compromising the organic component of bone contributes to an increase in bone fragility.

The fatigue resistance of rabbit tibiae varies with age from youth to middle age.

UNLABELLED: Young adults are at risk of stress fractures. Risk is higher in younger and female individuals. Stress fractures occur due to repeated loading of the bone (fatigue). We modeled this with rabbit tibiae. Age increased fatigue resistance which correlated with bone mineral density. A sex difference was not detected. INTRODUCTION: Younger adults who engage in intense physical activity with a sudden increase in intensity level (military recruits/college athletes) are at risk of bone stress fractures. Risk is greater in females and diminishes with aging. Stress fractures may be the result of fatigue damage, which is not repaired rapidly enough to avoid fracture. It was hypothesized that the fatigue resistance of whole rabbit tibiae would be less in female specimens but greater as animal age increased. METHODS: Rabbit tibiae were harvested from three age groups (4, 7, and ≥ 12 months (females only)). The tibiae were scanned with dual energy X-ray absorptiometry to determine bone mineral density (BMD), computed tomography to quantify geometry, and then fatigue tested in three-point bending. RESULTS: In the ≥ 12-month group, BMD was approximately 20% higher, while the fatigue resistance was found to be approximately ten times higher than the other age groups. Sex was not a factor in the 4- and 7-month groups. Multiple linear regression revealed that fatigue life was negatively correlated with applied stress range and positively correlated with BMD (adjusted r (2) = 0.69). CONCLUSIONS: A difference in fatigue behavior due to sex was not detected, but there was a large increase in fatigue resistance with age. This correlated with increased BMD and parallels a reduced risk of stress fracture due to age in military recruits. Skeletal "maturation" may play an important role in determining stress fracture risk. Increased risk in females may be due to mechanisms other than those that determine material behavior.

A new tool to assess the mechanical properties of bone due to collagen degradation.

Current clinical tools for evaluating fracture risk focus only on the mineral phase of bone. However, changes in the collagen matrix may affect bone mechanical properties, increasing fracture risk while remaining undetected by conventional screening methods such as dual energy x-ray absorptiometry (DXA) and quantitative ultrasound (QUS). The mechanical response tissue analyzer (MRTA) is a non-invasive, radiation-free potential clinical tool for evaluating fracture risk. The objectives of this study were two-fold: to investigate the ability of the MRTA to detect changes in mechanical properties of bone as a result of treatment with 1 M potassium hydroxide (KOH) and to evaluate the differences between male and female bone in an emu model. DXA, QUS, MRTA and three-point bending measurements were performed on ex vivo emu tibiae before and after KOH treatment. Male and female emu tibiae were endocortically treated with 1 M KOH solution for 1-14 days, resulting in negligible collagen loss (0.05%; by hydroxyproline assay) and overall mass loss (0.5%). Three-point bending and MRTA detected significant changes in modulus between days 1 and 14 of KOH treatment (-18%) while all values measured by DXA and QUS varied by less than 2%. This close correlation between MRTA and three-point bending results support the utility of the MRTA as a clinical tool to predict fracture risk. In addition, the significant reduction in modulus contrasted with the negligible amount of collagen removal from the bone after KOH exposure. As such, the significant changes in bone mechanical properties may be due to partial debonding between the mineral and organic matrix or in situ collagen degradation rather than collagen removal. In terms of sex differences, male emu tibiae had significantly decreased failure stress and increased failure strain and toughness compared to female tibiae with increasing KOH treatment time.