Effects of Cyclic Compressive Loading on Chondrogenesis of Rabbit Bone-Marrow De - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:38 |只看该作者 |倒序浏览 |打印

a Research Service and Geriatrics Research, Education, and Clinical Center, Veterans Affairs Medical Center, Miami, Florida, USA;0 L1 [# a! F' @# c. x
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b Department of Biomedical Engineering, University of Miami, Coral Gables, Florida, USA

Key Words. Bone marrow ? Chondrogenesis ? Mesenchymal stem cells ? TGF-?1 ? Compressive loading
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Herman S. Cheung, M.D., Research Service, Miami VA Medical Center, 1201 NW 16th Street, Miami, Florida 33125, USA. Telephone: 305-575-3388; Fax: 305-575-3365; e-mail: hcheung@med.miami.edu# W$ T; W# G7 L# ~. `0 v

ABSTRACT

Injured articular cartilage has a limited capacity of self-repair provided damage does not extend beyond the subchondral bone. However, when injuries penetrate the subchondral bone, mesenchymal stem cells (MSCs) from the bone marrow migrate toward the injured area and form new cartilage-like reparative tissue . This clinical finding indicates that local biomechanical and/or biochemical stimuli at the injured site of articular cartilage can induce chondrogenic differentiation of MSCs.
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Recent in vitro studies demonstrated that chondrogenesis of bone marrow-derived MSCs (BM-MSCs) can be induced with the treatment of cytokines such as transforming growth factor-? (TGF-?) and bone morphogenetic protein (BMP) . While combining BMP and TGF-? treatments, proteoglycan biosynthesis can be upregulated during chondrogenesis of BM-MSCs . In addition, O’Driscoll et al. demonstrated that continuous passive motion helped to heal injured rabbit knee joint articular cartilage repaired with autogenous periosteal grafts containing MSCs . Recently, Wakitani et al. transplanted BM-MSCs into full-thickness cartilage defects of rabbit knee joints. They showed that 6 months after the implantation, different local mechanical environments resulted in substantial differences in mechanical properties of reparative tissues on the posterior and anterior aspects of the repair area . These previous studies suggested that chondrogenic differentiation of MSCs is influenced by mechanical stimuli.- w3 b* h" d4 G8 b4 W* `# d! M

The effects of mechanical loading on the biosynthetic activities of chondrocytes have been extensively studied using agarose and cartilage explant cultures. Compressive loading has been shown to modulate the cartilage-specific macromolecule biosynthesis and pericellular matrix deposition of mature chondrocytes . Additionally, static and dynamic compressive loadings were found to promote chondrogenic differentiation of embryonic limb-bud mesenchymal cells . More recently, Angele et al. showed that cyclic hydrostatic pressure enhanced the extracellular matrix deposition of human BM-MSCs, which underwent chondrogenesis in pellet cultures . However, the effects of physical stimuli associated with the mechanical environment of articular cartilage on chondrogenic differentiation of BM-MSCs still remain unclear. The hypothesis of this study is that dynamic compressive loading can promote chondrogenesis of BM-MSCs. Therefore, the objective of this study is to examine the effects of cyclic compressive loading on chondrogenic differentiation of rabbit BM-MSCs in agarose cultures.
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MATERIALS AND METHODS6 e4 j3 s( J% I' i2 J* X$ L
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Based on the calculations from the biphasic model, the mechanical responses of the agarose disks can be considered to reach equilibrium after 200 cycles of loading in the configuration described in Figure 1B. The predicted axial strain, axial fluid flow, and fluid pressure of the three zones within the agarose disks, shown in Figure 2, are at the peak of the two hundredth cycle. Generally, the distributions of the strain, fluid flow, and fluid pressure were not uniform throughout the specimen. The axial strain (in compression) increased from about 4% at the region attached to the porous filter to 14% at the region attached to the impermeable platen (Fig. 2A) while the radial strain (in tension) increased from 1% to 7%. The axial fluid velocity decreased with increasing radius and decreasing depth (from the porous filter side to the impermeable platen side) with a maximal velocity of 9 μm/second (Fig. 2B), while the radial fluid velocity was near zero except near the edge. The profile of fluid pressure decreased with increasing radius and had a maximal pressure of 0.1 kPa. This profile remained consistent along each depth, except at the region close to the porous filter (Fig. 2C).
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Figure 2. The predictions of the (A) axial strain, (B) fluid flow, and (C) fluid pressure for three zones within the agarose disk at the peak of the two hundredth cycle in the unconfined compression test.+ C! `# S: q1 {# b
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Chondrogenic potential of rabbit BM-MSCs was confirmed in pellet cultures, as illustrated in Figure 3. After a 21-day culture, the TGF-?1-treated specimens exhibited stronger expressions of collagen type II and aggrecan than the specimens without the treatment of TGF-?1 (Fig. 3A). The specimens of the TGF-?1-treated group exhibited stronger alcian blue staining of proteoglycans (Fig. 3B–3E), while the immunohistochemical assay revealed intense dark brown staining of collagen type II on the TGF-?1-treated specimens (Fig. 3F–3I).- y# T5 o; |4 [
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Figure 3. A) Typical gene expressions of pellet cultures with and without the treatment of TGF-?1 after a 21-day culture. B-E) Typical alcian blue staining of proteoglycans and (F-I) the immunohistochemical staining of collagen type II protein for pellet cultures without (B, C, F, G) and with (D, E, H, I) the treatment of TGF-?1 after a 21-day culture. Bar = 150 μm in (B, D, F, H); 20 μm in (C, E, G, I). Immunopositive reaction for collagen type II protein illustrated by dark brown staining is only seen in (H) and (I). The TGF-?1-treated specimen and nuclei (arrow) were counterstained using hematoxylin. The more deposition of proteoglycans illustrated by strong blue staining was found in (D) and (E). The TGF-?1-treated specimen and nuclei (arrow) were counterstained using nuclear fast red.3 t+ x$ K! X5 |. |  z7 j

Figure 4 shows the comparison of DNA content among the four groups at each time period. The DNA content of the specimens increased an average of 40% after the 14-day experiment. The TGF-?1-treated specimens tended to have more DNA content than the specimens without the treatment. However, no significant differences were found in the DNA content between the four groups at each of the three time periods.
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Figure 4. Comparison of normalized DNA content among the four experimental groups at three time periods.
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The typical gene expressions of the four groups at the three time periods are shown in Figure 5. Generally, the control group exhibited weaker chondrogenic gene expressions than the other three groups at all time periods. The collagen II gene expression of the TGF-?, loading, and TGF-? loading groups slightly increased with increasing time of culture, whereas their aggrecan gene expressions remained similar for every time period. Four groups expressed the collagen type I expression at a similar level except the 14-day experiment in which the collagen type I expressions of the TGF-?, loading, and TGF-? loading groups were decreased and were weaker than that of the control group. However, none of the experimental group exhibited the expression of collagen type X at all time periods.3 {5 ~! |7 h3 a3 |
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Figure 5. Typical RT-PCR analyses of chondrogenic genes for the four experimental groups at three time periods.$ |7 O3 q( W7 C# q& ~- ]# O  x

Statistical analyses of the chondrogenic gene expressions of the control and experimental groups are shown in Figures 6. Significant differences were found in both collagen II and aggrecan expressions among the four groups at each time period, with the control group having the lowest production of both chondrogenic genes while compressive loading and TGF-?1 were similar in their abilities to induce chondrogenesis. For the 14-day experiment, compressive loading in the presence of TGF-?1 promoted the collagen type II expression better than TGF-?1 alone. After five extra days of culture in serum-free medium following each of the three experiments, the chondrogenic gene expressions of the TGF-?, loading, and TGF-? loading groups only slightly decreased, whereas the specimens of the control groups still exhibited less production of collagen type II and aggrecan than those of the other groups in which no significant differences were found (Fig. 7).

Figure 6. Comparison of gene expression of (A) collage type II and (B) aggrecan among the four experimental groups at three time periods.2 H4 G( V9 {/ F3 K% U1 g# @
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Figure 7. Comparison of gene expression of (A) collage type II and (B) aggrecan among the four experimental groups after five extra days of culture in serum-free medium following each of the three experiments.. h7 e% n- k8 l- }+ c0 d
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Figure 8 shows typical gene expressions of TGF-?1 in all 4 groups, as well as its statistical comparison among the groups, for each time period. The control group significantly exhibited less TGF-?1 gene expression than the other three groups for the 3-day and 7-day experiments, whereas no significant difference was found for the 14-day experiment. The TGF-?1 gene expression of the TGF-?, loading, and TGF-? loading groups appeared to be inversely related to the time of culture.

Figure 8. A) Typical RT-PCR analysis of TGF-?1 gene for the four experimental groups at three time periods. B) Comparison of TGF-?1 gene expression among the four experimental groups at three time periods.

The histological and immunohistochemical analyses demonstrated that the rabbit BM-MSCs of the TGF-? group, the loading group (Fig. 9), and the TGF-? loading group were able to proliferate and deposit more extracellular macromolecules (proteoglycan and collagen type II protein) to form larger cellular aggregates than those of the control group., F' H0 H& @: X. H

Figure 9. Typical immunohistochemical staining of collagen type II protein (A and B) and alcian blue staining (C and D) of proteoglycans on the cell-agarose constructs of the control (A and C) and loading (B and D) groups, which were cultured for two extra weeks after a 14-day experiment (bar = 20 μm). The rabbit BM-MSCs were able to proliferate and deposit more extracellular matrix to form larger cellular aggregates (arrow) within the agarose constructs of the loading group (B and D) than the control group (A and C). The loading group exhibited deposition of collagen type II protein (brown staining in B) and proteoglycans (strong blue staining in D). Nuclei (arrow) were counterstained using (blue-violet) in (A) and (B) and nuclear fast red (red-pink) in (C) and (D).
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This work was supported by a NIH grant AR 38421 and a VA Merit Review Grant. The authors would like to thank Dr. Paul M. Reuben and Mr. Felix Soto for their technical assistance with RT-PCR, histological and immunohistochemical analyses, and cell harvest.0 f. ]) `3 G& _+ X) V8 ^

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