Mechanical Modulation of Cartilage Structure and Function during Embryogenesis in the Chick

Transcription

1 Annals of Biomedical Engineering, Vol. 32, No. 1, January 2004 ( 2004) pp Mechanical Modulation of Cartilage Structure and Function during Embryogenesis in the Chick BORJANA MIKIC, 1 ARIN LYNN ISENSTEIN, 2 and ABHINAV CHHABRA 3 1 Picker Engineering Program, Smith College, Northampton, MA; 2 University of California at San Francisco, San Francisco, CA; and 3 Department of Orthopaedic Surgery, University of Virginia Health Sciences Center, Charlottesville, VA (Received 30 June 2003; accepted 9 September 2003) Abstract The mechanical behavior of cartilage is intimately related to its biochemical composition, and tissue composition is known to be influenced by its local mechanical loading environment. Although this phenomenon has been well-studied in adult cartilage, few investigations have examined such structure function relationships in embryonic cartilage. The goal of this work was to elucidate the role of mechanical loading on the development of cartilage composition during embryogenesis. Using an embryonic chick model, cartilage from the tibiofemoral joints of immobilized embryos was compared to that of controls. The normal time course of changes in glycosaminoglycan/dna and hydroxyproline/dna were significantly influenced by loading history, with the most pronounced effects observed between days 9 and 14 during the period of most rapid increase in motility in control embryos. Stress-relaxation tests conducted on samples from day 14 indicate that the effects of embryonic immobilization on cartilage matrix composition have direct consequences for the mechanical behavior of the tissue, resulting in compromised material properties (e.g. 50% reduction in E inst ). Because embryogenesis provides a unique model for identifying key factors which influence the establishment of functional biomechanical tissues in the skeleton, these data suggest that treating mechanical loading as an in vitro culture variable for tissue engineering approaches to cartilage repair is likely to be a sound approach. Keywords Cartilage, Embryogenesis, Immobilization, Mechanical loading, Development. INTRODUCTION tissue have demonstrated that changes in mechanical loading history can affect the biosynthesis of these matrix constituents, 2,4,10,12,15,17 19,28 30,32 with subsequent effects on the mechanical behavior of the tissue (the so-called structure function relationship). 23 However, the extent to which mechanical loading environment affects cartilage composition and (consequently) mechanical behavior during embryonic development remains largely unknown. The primary goal of this work was to determine how embryonic immobilization affects cartilage composition by analyzing cartilage plugs taken from normal and immobilized embryonic chick bone ends. We hypothesized that immobilization would result in decreased levels of cartilage matrix constituents and an associated compromise in mechanical behavior of the developing embryonic tissue. The importance of this work rests on the fact that development provides a unique model for identifying the key factors which influence the establishment of functional biomechanical tissues in the skeleton. As the goal of cartilage tissue engineering is ultimately to produce tissue with sufficient biomechanical integrity to withstand multiple cycles of daily functional loading, investigating the mechanical modulation of cartilage during embryogenesis has important implications for understanding the relevance of treating mechanical loading as a potential in vitro culture variable for tissue engineering approaches to cartilage repair. The postnatal function of cartilage is largely mechanical in nature, and the biomechanical behavior of this tissue is intimately related to its biochemical composition. 27,34 Each of the two primary solid matrix constituents (collagen and proteoglycans) play important yet distinct mechanical roles, with the negatively charged proteoglycan molecules interacting with water to provide compressive resilience, and collagen fibers imparting tensile strength to the tissue. Numerous experimental studies using postnatal Address correspondence to Borjana Mikic, PhD, Picker Engineering Program, Smith College, 51 College Lane, Northampton, MA Electronic mail: bmikic@ .smith.edu /04/ /1 C 2004 Biomedical Engineering Society 18 METHODS Immobilization Model Immobilization was induced in chick embryos using the neuromuscular blocking agent decamethonium bromide (DMB; Sigma, St. Louis, MO), thereby causing the limbs to develop in the absence of normal mechanical loading. 9,24,25 Starting at day 6 of incubation, 0.25 ml of 0.02% DMB (20 mg per 100 ml) in Hank s Balanced Saline Solution (HBSS) was dropped onto the chorioallantoic membrane twice daily. Control embryos received equal volumes of HBSS. On days 9, 10, 11, 12, 14, and 17, embryos were

2 sacrificed, the femorotibial joint was cut free, and all connective tissue and periosteum were removed under a dissecting microscope. All appropriate Institutional Animal Care and Use Committee guidelines were followed. In the chick, the cartilage ends of the long bones sit like a cone within the periosteal bone sleeve. Each cone (femoral and tibial) was further dissected into two regions the upper third, representing primarily the round cell zone, and the lower two-thirds which encompasses the flat and hypertrophic cell zones (Fig. 1). This subdivision was performed to avoid confounding results due to the known differences in matrix production per cell in different regions of the developing tissue. 33 For each embryo, one sample consisted of the pooled left and right, tibial and femoral plugs taken from either the upper or lower portions of the cartilage cone. Biochemical Composition At each time point, 5 10 samples per location were digested in papain (1 ml of 125 µg/ml in 1 Phosphate Buffered EDTA (PBE), ph 6.5 (Sigma, St. Louis, MO) for 18 h at 60 C. DNA content was determined using the Hoechst Dye assay 16 with calf thymus DNA as a standard, and glycosaminoglycan (GAG) content was determined using the dimethylmethylene blue (DMMB) assay 8 with chondroitin sulfate as standard. Subsequent to acid hydrolysis in 6N HCl for 24 h at 110 C, hydroxyproline (HPro) content (an indicator of total collagen) was determined using a dimethylaminobenzaldehyde (DMBA) color reaction 5 with purified hydroxyproline as standard. All samples and standards were processed in duplicate and average values were used for analysis. Immobilization and Embryonic Chick Cartilage 19 Mechanical Testing Because of the small size and softness of the embryonic tissue with which we were working, mechanical testing could not be reliably performed prior to day 14. Thus, this single time point was chosen for the purposes of comparison. Uniform cylindrical samples with flat ends were cored from the tibias of 10 control and 10 immobilized embryos at this stage immediately following sacrifice using a dermal punch. Approximate sample dimensions were 1-mm high 2 mm in diameter (exact dimensions were measured in triplicate for each specimen using a dissecting microscope with reticulated eye-piece and average values used for calculations). Each specimen was subjected to a time-dependent stress-relaxation test in unconfined compression in a 1 PBS solution. A tare load of N was initially applied to establish contact between the specimen and impermeable platens. A ramped strain was applied at a rate of %/s 1 to 15% strain, which was then held for 480 s using a desktop mechanical testing system (Instron Model 5542, Instron Corp., Canton, MA). Stress and strain were recorded as a function of time, and a peak instantaneous modulus and final relaxed modulus were calculated FIGURE 1. (A) Histological section through the proximal tibiotarsus of a normal day 13 chick embryo depicting the upper and lower zones used for compositional analysis; (B) whole mount of a femorotibial joint showing the two skeletal elements (femur and tibiotarsus) from which cartilage samples were harvested. as the ratio of peak stress divided by strain at peak stress, and final stress divided by final strain, respectively. Effect of DMB on Cartilage Biosynthesis To determine whether DMB alone was capable of affecting cartilage biosynthesis, uniform cylindrical plugs of bovine articular cartilage were extracted, washed in PBS containing 50 µg/ml gentamycin, and cultured in

3 20 MIKIC et al. temperature- and CO 2 -equilibrated DMEM/F12 (1:1) to which 2 mm L-glutamine, 0.01% BSA, 0.35 mm proline, 50 µg/ml gentamycin, 20 µg/ml ascorbate, 1 mm cysteine, and 1 mm pyruvate were added. Disks were cultured for 3 days, followed by 3 days in media containing 0, 0.004, 0.02, or 0.1% DMB with six samples per group. During the last 18 h of the experiment, explants were labeled in medium containing DMB and 10 µci/ml ( Bq/ml) of [ 35 S]sulfate (Amersham International, Little Chalfont, England). After labeling, disks were rinsed for 30 min and chased for 2 hrs in temperature- and CO 2 -equilibrated media. To assess the incorporation of radiolabel, cartilage disks were first weighed wet, lyophilized, and weighed dry. Subsequent to lyophilization, each sample was digested overnight at 60 C in papain (125 µg/ml in sterile PBE, Sigma, St. Louis, MO), and subsequently counted on a liquid scintillation counter. Total sulfated GAG content was also measured using the DMMB assay to normalize CPM obtained from scintillation counting. Statistical Analyses Dependent variables from the biochemical assays (GAG/DNA and HPro/DNA) were analyzed using a threefactor ANOVA with loading history (control or immobilized), location (upper or lower), and embryonic age as the three factors. Where significant effects were detected (p < 0.05), comparisons between groups were made with the Fisher s PLSD post hoc test. Mechanical testing data were analyzed with a one-factor ANOVA with loading as the independent factor. Biosynthesis results were also analyzed using a one-factor ANOVA with dose as the independent variable and GAG-normalized incorporation (CPM/µg GAG) as the dependent variable. All statistical analyses were performed using the statistical software package StatView 5.0 (SAS Institute Inc., Cary, NC). RESULTS At sacrifice, all of the embryos had a heart beat and no discernable limb motion was detected in the DMB-treated group. All major skeletal muscle groups were obviously decreased in size in the immobilized embryos. Effect of DMB on Cartilage Biosynthesis At no dose in the range tested (including the dose of 0.02% used in ovo) did DMB affect the level of newly synthesized GAG in cartilage explants as indicated by radiolabeled sulfate incorporation normalized to total GAG content (3370 ± 530 CPM/µg GAG at 0.02% DMB vs ± 830 CPM/µg GAG for 0% DMB, p > 0.05). FIGURE 2. Glycosaminoglycan content (GAG/DNA) for control and immobilized chick cartilage samples as a function of embryonic age in the upper (A) and lower (B) regions of the cartilage. Effect of Immobilization on Glycosaminoglycan Content In the upper regions of the cartilage cone, embryonic age significantly influenced glycosaminoglycan content in control cartilage, with a dramatic increase of over 160% between days 11 and 14 (Fig. 2(A)). In addition, the timedependent changes in glycosaminoglycan content in the upper portion of the cartilage cone were significantly affected by loading history: that is, the interaction between loading and embryonic age was statistically significant. In embryos which experienced normal skeletal muscle contractions, proteoglycan levels increased dramatically between days 11 and 14 of incubation. Proteoglycan content in immobilized embryos was comparable to that of controls until approximately day 12, at which point immobilized GAG values were consistently lower than that of control cartilage. As with the upper regions of the cartilage, the timedependent changes in glycosaminoglycan content in the lower portion of the tissue were also significantly affected by loading history, with immobilized levels being comparable to controls until day 12, at which point immobilized GAG values were consistently lower than that of control cartilage (Fig. 2(B)).

4 Immobilization and Embryonic Chick Cartilage 21 Effect of Immobilization on Mechanical Properties at Day 14 Four of the ten DMB-treated samples developed a small fissure prior to achieving a peak strain of 15%, leaving a final sample size of n = 6 for the immobilized group. From the standpoint of both proteoglycan and collagen content, immobilized tissue exhibited significantly lower levels of both matrix constituents by day 14 of development. Not surprisingly, then, the mechanical behavior of the tissue was also significantly affected (Fig. 4(A)). When the material properties were calculated, the instantaneous modulus of the tissue was 50% lower in the immobilized tissue compared to controls (Figure 4(B)). Similarly, the relaxed modulus was 30% lower, although this difference was not statistically significant because of the high degree of scatter in the immobilized data (Fig. 4(C). DISCUSSION FIGURE 3. Hydroxyproline content (HPro/DNA), indicative of total collagen content, for control and immobilized chick cartilage samples as a function of embryonic age in the upper (A) and lower (B) regions of the cartilage. Effect of Immobilization on Collagen Content Shown in Fig. 3(A) is the time-dependent profile of hydroxyproline content (indicative of total collagen content) in the upper regions of cartilage taken from control and immobilized embryos. The characteristic profile of a dip in total collagen per DNA occurring at days followed by an increase of over 250% between days 11 and 14 in control cartilage is consistent with the data published by Stocum and colleagues regarding time-depending changes in collagen synthesis. 33 As with proteoglycan content, the time-dependent changes in hydroxyproline were significantly affected by loading history. Immobilized values of hydroxyproline content were significantly lower than controls between days 11 and 14. In contrast to the proteoglycan data, however, immobilized values of hydroxyproline were also significantly lower than control values prior to day 11. Whereas control cartilage demonstrates a dip in collagen content during this period, immobilized values start off low and remain relatively constant until day 12. Similar trends were seen in the data for HPro per DNA in the lower portion of the cartilage cone as shown in Fig. 3(B). In this study, we (1) document the time-dependent changes in proteoglycan and collagen content in developing chick cartilage; (2) demonstrate that these changes are significantly affected by embryonic immobilization; and (3) show that the effects of embryonic immobilization on cartilage matrix composition have direct consequences for the mechanical behavior of the tissue, resulting in compromised material properties. Spontaneous, rhythmic muscular contractions begin in the chick at roughly 3.5 days of incubation. 13,14 At this stage, each cycle of movement consists of a short activity phase, followed by a longer phase of quiescence. At 6.5 days, movement of the wings and legs begins, and the frequency and duration of muscle contractions increase as development progresses. The percentage of time spent in active movement rises steadily from less than 10% at 3.5 days to approximately 80% by day 13 (Fig. 5). 14 A plateau is then maintained at this peak level until day 17, with motility subsequently decreasing prior to hatching (approximately day 21). When vibrational recordings were used to characterize the magnitude of movements during development, Llusa- Perez and colleagues found that, prior to day 7, movements were of low intensity. 21 Between days 8 and 9, intensity increased, and continued to increase steadily until day 14, at which point a plateau was reached until days Thus, the intensity of embryonic movements in the chick appears to follow roughly the same pattern as the increase in movement duration shown in Fig. 5. The relative frequency of movements at different intensities as a function of gestational age was not reported, however. It is interesting to note that the time course of changes in both proteoglycan and collagen content in control embryos obtained in this study follows roughly the same pattern as the changes in activity duration depicted in Fig. 5 (as well

5 22 MIKIC et al. FIGURE 4. (A) Representative stress-relaxation curves for control and immobilized day 14 samples; (B) Instantaneous moduli (defined as peak stress divided by strain at peak stress); (C) Relaxed moduli (defined as end stress divided by strain at end stress). as the changes in magnitude of movements as described by Llusa-Perez 21 ), thus suggesting that the normal changes in embryonic cartilage composition over time are related to changes in mechanical loading of the developing tissue. If such a relationship were causal rather than correlative, one would then expect to see the most pronounced effects of embryonic immobilization on cartilage composition occurring during the period of most rapid increase in embryonic motility in normal control embryos, i.e. sometime between days 9 and 14. Indeed, it is precisely during this interval that immobilized tissue composition begins to lag significantly behind that of controls (Figs. 2 and 3), thus further supporting the hypothesis that mechanical loading leads to changes in matrix composition in embryonic tissue. What remains unclear, however, is the specific nature of the mechanical (or associated electrical and/or chemical) stimulus that is

6 Immobilization and Embryonic Chick Cartilage 23 FIGURE 5. Mean duration of activity and inactivity phases and length of cycle, in seconds, at different embryonic stages in normal chick embryos. Reproduced with permission from Hamburger et al. 14 Reprinted by permission of Wiley Liss, Inc., a subsidiary of John Wiley & Sons, Inc. most significant in affecting changes in embryonic chondrocyte biosynthetic activity: in other words, which combinations of frequency, amplitude, and duration are most relevant? In postnatal tissue, it is quite clear that chondrocytes respond to their local mechanical loading environment, although the nature of the response depends on a variety of factors, including the specific loading protocol. In general, static compression of cartilage in vitro can induce suppression of proteoglycan and protein sythesis, 2,10,12,15,30,32 whereas dynamic compressive loads can elicit increased matrix synthesis. 4,17 19,28 30 The dynamic response is fairly complex, however, as different responses are seen with different compression frequencies and amplitudes. 30 In essence, embryonic muscle contractions can be thought of as a dynamic applied load to the developing joint. Thus, to truly understand the connection between mechanical loading and changes in embryonic cartilage matrix composition over time, it is clear that a more detailed characterization of the loading history will need to be developed. Because of its small size, the embryonic chick model presents a unique challenge for accurately characterizing in ovo joint loading, and new approaches will need to be developed to achieve this aim. Our mechanically tested specimens spanned the upper and lower portions of the cartilage cones that were analyzed separately for the purposes of assessing biochemical composition. When GAG/DNA differences in the upper and lower regions of the tissue are averaged together for day 14, immobilized GAG content is roughly 30% lower than that of control samples. Similar values are obtained for HPro/DNA ( 36%). From these numbers, one might expect that the difference in relaxed moduli should be on the order of 30 35%, and that the difference in instantaneous moduli would be greater than 35%. Indeed, our measured values correspond well to these estimates, with E relaxed being 30% lower and E inst, 50% lower in the immobilized tissue. Because of the high degree of scatter present in the immobilized data, however, as well as the small final sample size, the difference in relaxed modulus was not found to be statistically significant. It is important to keep in mind that one of the limitations of this study is that only the bulk biochemical composition of the developing tissue was examined, and thus definitive

7 24 MIKIC et al. statements cannot be made at this time about the effects of embryonic immobilization on synthesis or degradation of key matrix components. Further insight into the mechanisms behind the mechanical modulation of cartilage composition during embryogenesis will be gained by studying both the anabolic and catabolic components of cartilage matrix metabolism. In addition, our pilot explant data suggest that DMB alone does not have an adverse effect on matrix biosynthesis in the dose range used for this study. To more definitively address this possibility, however, explants should be taken from embryonic chick cartilage and tested at multiple time points and for durations longer than 3 days. Such detailed characterizations were beyond the scope of this study, however. A second limitation of this study is the relatively simple approach taken to compare the mechanical behavior between immobilized and control tissue samples. The aim of these tests was to provide a comparison between the two groups at one point in time and not to precisely determine the biphasic constitutive coefficients for embryonic chick cartilage. Thus, a simple stress-relaxation protocol was followed on the basis of previously published unconfined compression tests in human embryonic tissue. 1 The protocol of ramped compressions followed by oscillatory displacements 20,34 was not used, nor were the stress time data fitted to the well-known biphasic model. 26 Nonetheless, we believe that the approach used in this study represents a valid comparison of the mechanical behavior of immobilized and control embryonic chick cartilage. Very few studies in the literature have reported material properties for embryonic cartilage, and the range of ages, species, sample harvest locations, testing protocols, and modeling assumptions makes it difficult to compare results directly. Brown and Singerman obtained an equilibrium modulus for human stillborn femoral head cartilage of MPa. 1 Williamson et al. reported a fetal bovine free swelling compressive modulus (H A0 ) of 0.11 ± 0.03 MPa, and H A (ε = 15%) of 0.15 ± 0.01 MPa. 34 This same group also reported a fetal bovine free swelling compressive modulus (E 0 ) from unconfined compression tests of ± MPa for surface layers and ± for middle layers. 20 Despite the differences in testing methods, species, and other parameters, our results for day 14 control embryonic chick cartilage are not inconsistent with these fetal bovine data. Now that the embryonic chick model has been established as a useful one for examining mechanical modulation of cartilage compositional changes during embryogenesis, a logical next step will be to invoke a more rigorous constitutive characterization of normal and immobilized tissue at multiple time points using a two-phase porous hydrated material representation. One final limitation of this study is the relatively small sample size used for mechanical testing. Originally, sample sizes of 10 per group were used, however, four of the immobilized samples failed by fissuring prior to achieving peak strain values. These failures are consistent with the known biochemical composition of the immobilized tissue: the lower levels of collagen measured in the day 14 immobilized samples possibly resulted in a tissue that was less able to withstand the tensile stresses associated with compression, and would thus be more prone to splitting. It is likely that lower peak strain levels (i.e. <15%), or the use of multiple smaller steps would have resulted in fewer lost specimens, and such modifications should be fully explored in any future studies designed to rigorously characterize the constitutive behavior of these tissues. Because the peak instantaneous modulus depends on permeability, water content, and equilibrium modulus, future studies should also characterize these additional parameters as well as examine the correlation between equilibrium modulus and water content. Despite these limitations, this study supports the hypothesis that mechanical factors play an important role not only in postnatal functional adaptation of cartilage, but also during embryonic development. In recent years, functional tissue engineering approaches to generating neocartilage have begun to utilize mechanical bioreactors in an effort to create replacement tissues with enhanced biomechanical properties. 3,6,7,22,23,31 Embryogenesis provides a unique model for identifying the key factors which influence the establishment of functional biomechanical tissues in the skeleton. 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