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Chondrocyte Proliferation of the Cranial Base Cartilage upon in vivo Mechanical StressesSkeletal Tissue Engineering Laboratory, Rm 237, Department of Orthodontics and Bioengineering, Univ. of Illinois at Chicago MC 841, 801 South Paulina Street, Chicago, IL 60612-7211, USA; Correspondence: * corresponding author, jmao2{at}uic.edu
Whereas the growth of the cranial base cartilage is thought to be regulated solely by genes, epiphyseal growth plates are known to respond to mechanical stresses. This disparity has led to our hypothesis that chondrocyte proliferation is accelerated by mechanical stimuli above natural growth. Two-Newton tensile forces with static and cyclic waveforms were delivered in vivo to the premaxillae of actively growing rabbits for 20 min/day over 12 consecutive days. The average number of BrdU-labeled chondrocytes in the proliferating zone treated with cyclic forces was significantly higher than both static forces of matching peak magnitude and sham controls representing natural chondral growth. Cyclic forces also evoked greater area of the proliferating zone than both static forces and sham controls. Thus, chondrocyte proliferation is enhanced by mechanical stresses in vivo, especially those with oscillatory waveform. Analysis of these data suggests that genetically coded chondral growth is up-regulated by mechanical signals.
Key Words: cartilage • chondrocyte • matrix • stress • mechanical
Longitudinal skeletal growth takes place by the conversion of growth cartilage into mineralized bone, while chondral growth continues to maintain the presence of the growth plate. Eventually, chondrocytes undergo apoptosis, followed by complete osseous replacement of growth cartilage (Martin et al., 1998). Although chondrocytes likely are programmed to undergo a definite number of mitotic cycles before they degenerate, there is increasing evidence that chondrocyte proliferation is modulated by mechanical stresses (Grodzinsky et al., 2000; Elder et al., 2001). However, most previous evidence of mechanical modulation of chondrocyte differentiation and proliferation has been obtained either in vitro or without specification of the precise characteristics of the applied mechanical stresses. For the effects of mechanical forces on chondral growth to be determined, the precise characteristics of mechanical stimuli—such as magnitude, frequency, and duration—must be known. Chondral growth of the cranial base, like epiphyseal plates, relies on the differentiation, proliferation, and maturation of chondrocytes in addition to concurrent synthesis of extracellular matrices. The spheno-occipital synchondrosis is of particular importance, due to its late ossification in adolescent humans (Ingervall and Thilander, 1972). The growth of the cranial base cartilage (CBC) is considered to be controlled by predetermined cycles of mitosis and apoptosis, both of which are genetically coded in chondrocytes, and with little contribution from mechanical stimuli (Scott, 1958; Baume, 1970; Copray et al., 1986; Peltomaki et al., 1997; Proffit et al., 2000). However, this notion of sole genetic control has been questioned, along with the speculation that the growth of the CBC also depends upon gradual enlargement of the growing brain, implying that chondral growth is enabled by the continuous presence of tensile forces (Enlow, 1990; Alberius and Friede, 1992; Dixon et al., 1997). Recently, chondral growth of the CBC was found to be accelerated upon delivery of brief cyclic mechanical forces as compared with both natural chondral growth and static forces (Wang and Mao, 2002). However, chondrocyte proliferation of the CBC during either natural growth or upon mechanical stimulation is not well-understood. Chondrocyte proliferation and associated matrix synthesis may be responsible for many of the histomorphometric changes that were reported in our prior work (Wang and Mao, 2002). The hypothesis of the present work is that chondrocyte proliferation is modulated by in vivo mechanical stimuli. Our data demonstrate that application of cyclic mechanical stimuli for 1200 cycles per day is sufficient to up-regulate chondrocyte proliferation and to increase the total area of the proliferating zone of the CBC, more than static stimuli of matching peak amplitude and natural chondral growth.
Animal Model and in vivo Delivery of Mechanical Stimuli Twenty six-week-old male New Zealand White rabbits with a mean body weight of 1.1 kg (range, 0.9-1.3 kg) were randomly allocated to sham control (N = 7), static force (N = 6), and cyclic force (N = 7) groups. The rabbits were housed in a temperature- and light-controlled room (23-25°C, 14 hrs light/day), permitted cage activities, and given standard daily amounts of food and water. The animal protocol was approved by the Animal Care Committee of the University of Illinois at Chicago.
Delivery of mechanical stimuli followed procedures described in Wang and Mao (2002). Under general anesthesia induced by intramuscular injection of 90% ketamine (100 mg/kg; Aveco CO, Fort Dodge, IA, USA) and 10% xylazine (20 mg/kg; Mobay, Shawnee, KS, USA), rabbits were placed in a supine position in custom-made resin body holders. We secured the premaxilla tightly to the resin body holder by restraining the oral commissure with stainless steel wires wrapped in plastic sheath, which reverted the upward movement of the cranium. An O ring connected the maxillary incisors to a servohydraulic system (Minibionix 853, MTS, Eden Prairie, MN, USA) enabled tensile mechanical forces to be applied with the direction and point of application illustrated in Fig. 1A
Tissue Harvest and Immunolabeling with BrdU Bromodeoxyuridine (BrdU), a thymidine analogue, was used to label actively dividing chondrocytes. While the rabbits were under general anesthesia, 5-bromo-2'-deoxyuridine (Sigma, St. Louis, MO, USA) was dissolved in sterile saline and injected intraperitoneally (40 mg/kg) 30 min before the last episode of force application in treated groups, and 50 min prior to death of all groups. Within 15 min of death by pentobarbital overdose (300 mg/kg, i.v.), the CBC with its subchondral bone was harvested with a fine surgical saw, resulting in a 6 x 6 x 4 mm3 tissue block. The tissue block was fixed in 10% paraformaldehyde, demineralized in equal volumes of 20% sodium citrate and 50% formic acid, sectioned along the midsagittal plane into left and right halves, and embedded in paraffin. Serial 8-µm-thick sections were cut in the parasagittal plane and stained with either hematoxylin and eosin (H&E) or safranin O and fast green. Safranin O, a cationic dye, binds to polyanions such as chondroitin sulfate and keratan sulfate (Rosenberg, 1971; Lammi and Tammi, 1988). Specimens were stained with safranin O and fast green under the same conditions, with timed changes of the staining solutions prepared in a standard fashion (cf. Mao et al., 1998). After deparaffinization and rehydration, adjacent parasagittal histologic sections were subjected to immunolabeling with BrdU antibodies. Endogenous peroxidase was blocked by incubation with 3% H2O2. Sections were further digested with 0.1% trypsin, which revealed halogenated nucleotides incorporated into the nuclei. The sections were incubated with 5% normal goat serum, which blocked non-specific binding. A monoclonal anti-BrdU antibody (Sigma, St. Louis, MO, USA) was diluted 1:200 in PBS. Negative control sections were incubated with non-related mouse IgG. Both BrdU-treated and control sections were then incubated overnight at 4°C. The sections were incubated with biotinylated rabbit anti-mouse antibody (Vector Laboratories, Burlingame, CA, USA) containing 1% normal rabbit serum, followed by treatment with peroxidase-conjugated streptavidin. Diaminobenzidine (DAB) was used for the visualization of BrdU-labeled cells. The sections were counterstained with hematoxylin, cleared with xylene, and mounted on glass slides for computerized image analysis.
Computerized Histomorphometric Analysis, Cell Counting, and Statistical Analysis
Upon confirmation of a normal data distribution, we applied analysis of variance (ANOVA) with Bonferroni tests to the average total area of the proliferating zone and BrdU labeling indices among sham control, static force, and cyclic force groups to determine whether proliferating zone areas and BrdU labeling indices differed significantly among groups. P < 0.05 was considered to indicate statistical significance.
Despite the same magnitude of 2 N, static forces maintained a straight line of peak force magnitude without any substantial oscillation (Fig. 1B  ), whereas cyclic forces oscillated at 1 cycle/sec, totaling 10 cycles in the representative 10-second time course (Fig. 1C). This difference in force waveforms induced different chondral growth responses. BrdU labeling of chondrocytes in the proliferating zone was demonstrated in Fig. 2 . Whereas minimum background labeling was present in the negative control (Fig. 2A), proliferating chondrocytes were labeled with BrdU in sham controls (Fig. 2B) and mechanically treated conditions (Figs. 2C, 2D). There was marked proliferation of chondrocytes in the proliferating zone treated with both static and cyclic forces (Figs. 2C and 2D, respectively), in comparison with sham control (Fig. 2B). Quantification of proliferating chondrocytes by automatic cell counting revealed that although both cyclic force (43% ± 0.04; mean ± SD) and static force (41% ± 0.02) induced more chondrocyte proliferation than sham controls (24% ± 0.06) (p < 0.01), cyclic forces also induced more chondrocyte proliferation than static forces (p < 0.05) (Fig. 4A).
The total area of the proliferating zone treated with cyclic forces (Figs. 3C, 3C' ) was markedly greater than those of static forces (Figs. 3B, 3B') and sham controls (Figs. 3A, 3A'). Chondral matrix reacted positively to safranin O, shown in representative sections in sham control (Fig. 3A'), static force (Fig. 3B'), and cyclic force (Fig. 3C') specimens, confirming the presence of abundant chondroitin-sulfate and keratan-sulfate binding proteoglycans, most likely aggrecan, in the cartilage matrix. Quantitatively, the average total area of the proliferating zone treated with cyclic forces was significantly greater than that of both static forces and sham controls (p < 0.05 and 0.01, respectively) (Fig. 4B).
The present data provide experimental evidence of mechanical modulation of chondrocyte proliferation in vivo, representing the first demonstration of the capacity of the cranial base growth plate to enhance its growth responses to mechanical stimuli. Chondrocyte proliferation has been shown to be up-regulated by in vitro mechanical stimuli applied to tissue explants and chondrocyte culture of the appendicular growth plate cartilage (Grodzinsky et al., 2000; Elder et al., 2001). It is interesting to note that mechanical stimuli applied to long bones are primarily compressive and shear, whereas the present mechanical stimuli are tensile. Despite these differences, there is no a priori reason not to assume that the presently observed chondrocyte proliferation upon in vivo mechanical stimuli may apply to growth plate chondrocytes under tensile mechanical stresses in general, and is not specific to the cranial base growth plate.
The present application of macroscale 2-N tensile forces against the rabbit premaxilla is likely transmitted as microscale mechanical strain toward the cranial base cartilage, based on our bone strain recordings posterior to the cranial base growth plate in human skulls (Mao et al., 1999). The magnitude of this microscale mechanical stress measured at the cranial base cartilage is even smaller than the equivalency of exogenous forces (Mao et al., 1999). This noninvasive model of delivery of micromechanical tensile stresses to growth cartilage differs from conventional physeal distraction models in which distraction forces are often large, e.g., up to 20 N, and are applied directly adjacent to the growth plates with invasively implanted pin distractors (Porter, 1978; De Bastiani et al., 1986; Wilson-MacDonald et al., 1990; Apte and Kenwright, 1994). In addition, continuous static forces that have been used in previous physeal distraction models more likely evoke physical separation of growth plates. The present delivery of cyclic forces for 20 min/day over 12 consecutive days likely provides the growth plate with an opportunity for biological growth responses. Mechanical stretch probably takes place transiently during the daily 20-minute loading episode ( Chondrocyte proliferation in association with transient cyclic mechanical stimuli for 20 min/day over 12 consecutive days in the present model represents approximately 1% of total daily time and therefore is of further interest. Normal cell cycles of chondrocyte proliferation range from 31 to 76 hrs (Farnum and Wilsman, 1993; Vanky et al., 1998). If one assumes that this timing applies to the cranial base cartilage, it is then probable that, upon cyclic mechanical stimuli for 20 min/day, multiple cycles of chondrocyte proliferation have occurred after each episode of force delivery. BrdU labeling of chondrocytes was within 30 min after the last episode of mechanical stimulation on the day of the animals’ death, which was sufficient to have induced significantly higher chondrocyte counts upon cyclic mechanical stimuli than static stimuli with the same peak magnitude and natural chondral growth. It is likely, however, that several cell cycles of chondrocyte proliferation have been completed over the total 12-day duration of mechanical stimuli. Proliferation of chondrocytes up-regulated by both static and cyclic mechanical forces in the present experiment indicates that chondrocytes of the cranial base cartilage are sensitive to exogenous mechanical stimuli. The present findings should be interpreted with several caveats, which therefore are areas of improvement in our continuing investigations. First, only a single force magnitude of 2 N was used. More force magnitudes are being examined as investigators search for optimal mechanical stimuli for anabolic chondral growth responses. Second, subchondral osteogenesis will be examined with fluorescent labeling of new bone formation in addition to the present demonstration of up-regulated chondrogenesis. Third, biochemical markers will be used to characterize mechanotransduction pathways that are responsible for the up-regulated chondrocyte proliferation. Nonetheless, the present data are consistent with the notion that genetically coded chondral growth can be up-regulated by mechanical signals.
We are indebted to Professors Carla Evans, Jon Daniel, and especially Moneim Zaki for their constructive comments on earlier versions of the manuscript. We thank two anonymous reviewers for their suggestions that have substantially improved the quality of the manuscript. Verna Brown is gratefully acknowledged for processing histology samples. We thank Tejas Joshi, Agnes Knobloch, Naama Lewis, and especially Jikun Shen for their technical assistance. This research was supported in part by the Wach Fund, a Biomedical Engineering Research Grant from the Whitaker Foundation, and by USPHS Research Grants DE13964 and DE13088 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892. Received for publication March 26, 2002. Revision received July 29, 2002. Accepted for publication August 6, 2002.
Journal of Dental Research, Vol. 81, No. 10,
701-705 (2002)
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ABSTRACT
INTRODUCTION
1% of total daily time), but the physical effects of mechanical stretch likely would not persist for the remaining 99% of the daily time. The present chondral growth response is evidenced by both chondrocyte proliferation and increased total proliferating zone area concurrently evoked to higher levels by cyclic forces than by static forces, albeit both at the same peak magnitude of 2 N, suggesting that the oscillatory component of mechanical forces further accelerates chondral growth, beyond the peak force magnitude. The strong reaction of the cartilage matrix to safranin O in samples treated with cyclic force further indicates biological, anabolic chondral growth, rather than mechanical stretch (