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Anlage n 2011 pdf

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Permeability is also related to the amount of compression, which causes a decrease in the pore size and an increase in FCD. The collagen integrity increases with development and is higher on the surfaces. Discussion We previously showed marked differences between the mechanical properties of talar cartilage anlage and mature articular cartilage Jeremi Leasure: Your browser does not support JavaScript or it is turned off.

Pdf anlage n 2011

The hydraulic permeability, a measure of the ease of fluid transport through the solid matrix, is dependent physically on the porous structure of the matrix, and chemically on proteoglycans through various mechanisms 3. Fourier transform infrared FTIR spectroscopy is a powerful tool for simultaneous assessment of composition and microstructure of biological tissues such as bone and cartilage The frequency at which a molecule absorbs infrared radiation is sensitive to conformation and can be used to obtain information on protein structure and when polarized, on orientation.

FTIR spectroscopy has been used to characterize the spectral signatures of collagen and proteoglycans as well as structural parameters of the collagen network including collagen orientation, integrity COL Int , and level of collagen cross-linking Many studies describe the mechanics, composition and structure of adult articular cartilage 4 , 14 , 15 , but only few studies focus on prenatal articular cartilage 5 , 16 — 18 , early postnatal articular cartilage 19 , 20 , and growth plate cartilage In more recent studies of postnatal development of the collagen network in articular cartilage, differences between immature and mature collagen network have been revealed 22 — In other work, the developmental changes in PG concentration, monomer and aggregate size, and side chains in articular cartilage and different zones of growth plate cartilage of long bones, as well as the changes associated with calcification have been investigated 26 — Additionally, prior studies have only focused on a single fetal stage in comparison to young postnatal and adult specimens, without evaluation of different gestational stages to focus on prenatal development.

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While the general development of anlagen and their ossification are well documented, much remains to be elucidated on the temporal and spatial sequences for replacing primitive i. Knowledge of the changes that anlagen undergo during development may provide a paradigm for guidance constructs tissue engineered from primitive mesenchymal stem cells, particularly those intended for cartilage repair or replacement.

Furthermore, relationships between cartilage compositional and mechanical properties may provide for helpful markers for assessment of mechanical quality of engineered cartilage. An improved understanding of such relationships might also aid in development of more realistic theoretical models of developing cartilage.

Spatial mapping of tissue contents at various ages at high resolution achievable by FT-IRIS techniques provides easily interpretable visual clues into development. Our objectives were thus to 1 characterize the mechanical properties of human talar cartilage anlagen in the middle to late fetal period, 2 assess the molecular composition of the same tissue specimen using FTIR analysis and obtain high resolution spatial mappings of these parameters, and 3 determine whether relations existed between the molecular composition and mechanical properties, as well as assess the variation of compositional and mechanical properties with age and with respect to one another.

Fresh talus anlagen were obtained from seven still-born fetuses with gestational ages of 20, 25, 28, 30, 31, 33 and 36 weeks as determined from crown-to-rump length measurements Fig. Each talus was prepared for two purposes: The three stages involved in preparation of the samples for testing: A Dorsal view of an isolated fetal talus. The scale units are millimeters. C A cylindrical sample of fetal anlage used for testing. One half of the talus was sliced into 1.

In the younger specimens 20, 25 and 28 weeks of gestation no ossific nucleus was detected, while in the older specimen a well developed ossific nucleus was present Fig. Two samples were obtained from slices of each age 14 total using a 3.

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On the day of testing, samples were thawed at room temperature and allowed 1. Each sample was first tested in confined compression, allowed to recover for 1. Samples were submerged in normal saline solution containing enzyme inhibitors during testing and recovery time between the tests. Prior to data collection, a preconditioning load of 3.

The loading protocol was the same for both configurations. In a custom-written code in MATLAB, the permeability parameters k 0 and M were determined by finding the best fit to the stress-time data of the confined compression tests using the nonlinear biphasic theory 38 , and the permeability equation proposed by Lai and Mow Further detail can be found in our previous report Following mechanical testing, the specimens were kept frozen. The primary mid-IR vibrations of collagen and PG in the tarsal anlagen samples were monitored simultaneously.

Integrated areas of mid infrared absorbance bands are proportional to the quantity of a specific component present. A graph of a typical spectrum is displayed in Fig. The region of interest of the spectra of the youngest and oldest specimens is shown in Fig. The region of interest in the FTIR spectra obtained from talus cartilage anlagen of the youngest a and oldest b specimens.

The absorbance bands utilized to calculate collagen, proteoglycan and collagen integrity are labeled. Tissues were processed for paraffin embedding through a series of steps that involved decalcification, dehydration and proteoglycan containment. Sagittal sections were cut at seven micron thickness and mounted onto KBr IR windows. The absorbance bands were baseline-corrected and then integrated. Information on distribution and dry weight content of collagen and proteoglycan, and spatial mapping of collagen integrity was obtained based on integrated areas or area ratios of infrared absorbance bands, as described in the previous paragraph.

Transmission light microscopy TLM images of histologically stained slices from around the talar dome region articulation with the tibia in the 20 week youngest and 36 week oldest specimens were also obtained.

Mean values of mechanical and compositional quantities measured at each developmental stage were analyzed by bivariate correlations for detecting a linear association with age. Pearson correlation coefficients r and significance levels of effects p were calculated. Univariate linear regression was utilized to obtain models of mechanical and compositional properties as a function of age, with the exception of H A for which a second order polynomial was used.

Linear mixed model analysis based on a maximum likelihood method was performed to study relationships between each of the mechanical properties and each of the compositional parameters. The intercept and regression coefficients were treated as fixed effects while age was used for subject grouping total of seven groups and considered a random effect. This allowed for the specimenss belonging to the same fetus two per fetus to be considered dependent while measurements from different fetuses are independent.

There were no repeated measures. The regression coefficients and significance levels of effects p were calculated. Changes in mechanical properties with age. C Permeability and D strain dependence coefficient of permeability as determined from confined compression. Age is the number of weeks from conception as estimated from the crown-rump length.

Tissues became less compliant and less permeable with maturation, and permeability became more compaction dependent. Two specimens were harvested from each fetus; each was tested only once. Changes in compositional properties with age. Compositional parameters were determined via FTIR analysis by calculating areas of corresponding absorbance bands: A Collagen dry weight content Amide I band.

With maturation collagen dry weight content and integrity increased while PG dry weight content decreased. Models of mechanical properties as functions of the measured compositional quantities were obtained Figs. Relationships between stiffness moduli and tissue composition. Compositional parameters were determined via FTIR analysis by calculating areas of corresponding absorbance bands as specified in the following parentheses.

Regression lines were obtained using a linear mixed model approach to include the effect of dependence of observations within each fetal age group 2 specimens from each of the 7 age groups — each specimen tested once mechanically followed by FTIR analysis.

Relationships between permeability parameters and tissue composition. Asterisks mark the cases where no significant relationship existed between the two quantities permeability and PG, M and COL. FT-IRIS images of sagittal slices of anlagen showed a continuous increase in the collagen content per dry weight with gestational age in all regions Fig. Consistently across ages, the spatial maps also showed a very thin layer of higher collagen dry weight content on the surfaces followed by a region of lower collagen dry weight content right beneath the surface, continued by a higher and rather uniform distribution throughout the rest of the cartilage.

This band decreased in width and almost entirely disappeared by full term. PG dry weight content decreased with development Fig. In the older samples 30, 33, and 36 weeks regions of higher PG concentration seemed to form near the articulation surfaces. The collagen integrity parameter increased with age Fig. In the 28 week specimen COL Int was distinctively higher on the outer surface than on the interior regions of the cartilage.

This difference lessened with development; however, the slightly higher values for this parameter were still observed on most of the outer surface as compared to the inside. Growth of the ossific nucleus can be appreciated in all images as the sample age increases. Tissue sections were obtained from five different human anlage samples, ages 25, 28, 30, 34 and 36 weeks, respectively.

Pdf 2011 anlage n

Red and blue on the color scale represent the highest and lowest contents respectively. Collagen dry weight content increased with development. An area of lower collagen concentration right below the articulation regions is noted by arrows.

This feature disappeared by the 36 th week. The ossific nucleus was partially masked in the 30 and 34 week samples to facilitate image comparison. Red and blue on the color scale represent the highest and lowest values respectively. Decrease in PG dry weight content is observed with maturation the dark red region in the 36 week sample at the location of the ossific nucleus is a saturation artifact.

Tissue sections were obtained from five different anlage samples, ages 25, 28, 30, 34 and 36 weeks, respectively. The collagen integrity increases with development and is higher on the surfaces. TLM images showed enlargement of chondrocytes from the youngest to the oldest specimen Fig. A band of notably lower cell density was observed near the surface in both images.

The width of this band was larger in the more mature sample. Transmission light microscopy image of human cartilage anlage near and including talar dome, obtained from the 20 and 36 week fetuses.

The images show an area of low chondrocyte density near the surface. Cell enlargement seen with maturation can be a sign of hypertrophy as the tissue eventually calcifies and forms bone. We previously showed marked differences between the mechanical properties of talar cartilage anlage and mature articular cartilage In the present study, changes in mechanical properties and molecular composition of human talar anlagen during the mid to late fetal stages and the relationships between the two were determined.

Only a small fraction of the talar cartilage anlage located at the articulating surfaces evolves into articular cartilage while most of it used here for testing and FTIR undergoes hypertrophy and calcification, and is eventually replaced by bone. It has been demonstrated that PGs have an inhibitory effect on matrix calcification and apparently need to be removed prior to the onset of calcification The increase in the collagen content indicated by our results has been previously reported 5 , Paralleled with this growth in the collagen network we found an increase in both stiffness moduli H A and E s.

Although the collagen network has been commonly believed to primarily contribute to the tensile properties of cartilage, its role in defining the compressive behavior has been highlighted more in recent studies 2 , 3 , 34 , Several different factors might explain this observation. It is known that the swelling pressure of the GAG molecules depends on their fixed charge density FCD - amount of negative charges per free fluid volume which depends on the volume of the extrafibrillar space where they reside.

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In this context, it is the amount of the negative charges that contribute to the compressive stiffness and not the PG mass content itself. Additionally, another way in which the PGs enhance the structural rigidity of the matrix is by their bulk mass and immobilization of their large aggregates by the collagen network 3. It is expected that this form of contribution of PGs to stiffness is lessened by the decline in PG amounts as tissue develops. These results suggest a more prominent role for the collagen network than has been suggested by many articles, with key functional consequences.

Finally, there is a possibility of overestimation of PG content with FTIR in samples with an inherently low PG content due to lack of specificity of this technique 33 , A similar issue has been reported for estimation of collagen content With these data together, we can be confident that there is indeed a real decrease in PG content per dry weight during anlagen development, as expected.

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The current analysis did not attempt to distinguish between different types of collagen. Noting that hydraulic permeability is related to the matrix pore structure, and the pore size and connectivity, the decrease with age in this parameter can be explained by augmentation of the collagen network size as development progresses.

Permeability is also related to the amount of compression, which causes a decrease in the pore size and an increase in FCD. This effect is captured by the coefficient M. We observed an increase in M with age, in line with findings of Williamson et al. This suggests that as the tissue matures, the permeability becomes more sensitive to the degree of compaction it experiences.

The decrease in porosity with maturation may also explain the inverse relationship found here between permeability and the collagen dry weight content. This suggests an active tissue repair response manifested by heightened levels of collagen synthesis early in the development of osteoarthritis.

Although this parameter has been used previously to examine normal versus degenerative articular cartilage, the finding of our study is consistent with those reports in that the integrity parameter exhibits a rise with age, possibly attributable to collagen synthesis. The tested fetal cartilage anlage mostly precursor to bone, not articular cartilage is soft and its collagen network lacks the organized fibril arrangement and high cross-linking governing the tensile behavior present in adult articular cartilage, and thus has greater tendency for lateral expansion e.

Increase of this parameter was previously reported over a wider age range spanning fetal to adult humeral head articular cartilage Further investigation focusing on collagen orientation and the degree of cross-linking will help explain these results. The valley observed in the spatial mapping of collagen content per dry weight right below the surface corresponds well and can be explained with the TLM images.

The state of hypertrophy in the chondrocytes can be inferred from the enlargement of cells. It seems that less collagen synthesis is occurring due to lower concentration of chondrocytes underneath the surface as compared to the rest of the tissue, explaining the observed valley in the collagen content per dry weight in those areas.

Understanding the origin and composition-function relationships in this valley is of great value, and remains to be elucidated.

Despite this factor, the resulting standard deviations were comparable to literature values. Our observations have potential applications in functional tissue engineering. Mechanical and compositional properties are currently used mainly to assess the viability of tissue engineered cartilage end-product.

However, one might envision inspecting intermediate states of tissue-engineered cartilage to ensure its development is along the path taken by native tissue. The use of FTIR imaging of native and tissue-engineered cartilage has been reviewed 13 and parameters such as those utilized in our study have been highlighted as appropriate measures of the development of engineered cartilage 1 , The type of data we report may also present a potential in the future for elucidating the mechanisms underlying normal cartilage maintenance as well as the etiology of diseases such as osteoarthritis and congenital deformities.

The authors would like to gratefully acknowledge Dr. Brand, the Editor-in-Chief of Clinical Orthopaedics and Related Research for his kind help with conception and design of the study, and revision of the article; and Dr. Jeremi Leasure: Anlage N - KB anlage-n.

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