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Chondrocyte Proliferation of the Cranial Base Cartilage upon in vivo Mechanical Stresses

Skeletal 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;

* corresponding author, jmao2{at}uic.edu

	ABSTRACT

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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

	INTRODUCTION

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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.

	MATERIALS & METHODS

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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. Static and cyclic forces were both delivered with the same peak magnitude of 2 Newtons (N) for 20 min/day over 12 consecutive days, representing approximately 1% of total daily time. Static forces had a frequency of 0 Hz (Fig. 1B), whereas cyclic forces had a frequency of 1 Hz and oscillated from 0.5 to 2 N (Fig. 1C). The rationale for the delivery of 2-N tensile forces was based on both our previously observed enhancement of chondrogenesis upon cyclic mechanical stimuli (Wang and Mao, 2002) and the observation that 2 N represents a small fraction of masticatory forces in the rabbit (Weijs and de Jongh, 1977). All procedures were applied in sham controls except for the application of exogenous forces.

View larger version (17K): [in this window] [in a new window]   Figure 1. Structure of the rabbit cranial base cartilage and force waveforms delivered to the rabbit skull. (A) Schematic diagram of the rabbit cranial base cartilage (CBC) or the spheno-occipital synchondrosis. The horizontal arrow indicates 2-Newton tensile forces applied to the maxillary incisors. The CBC, schematically represented as a rectangle, is located between the sphenoid and occipital bones. (B,C) Traces of exogenous forces with the same peak magnitude of 2 Newtons but with two different waveforms: static force (B) and cyclic force (C). The static force failed to induce any substantial oscillation in force magnitude over the representative 10-second time course (B). In contrast, the cyclic force of the same peak magnitude oscillated at 1 cycle per sec, totaling 10 cycles in the representative 10-second time course (C).

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
We performed quantitative histomorphometric analysis to calculate the total area of the proliferating zone of the CBC in all groups. Histological sections stained with both H&E and safranin O/fast green were imaged under a research microscope (Nikon Eclipse E800) with a digital video camera (SPOT RT-II, Diagnostic Instruments, Sterling Heights, MI, USA). Once the borders of the proliferating zone of the CBC on the occipital side were manually defined by being separated from the reserve and hypertrophic zones (cf. Fig. 3A) by image analysis (ImagePro Plus, Media Cybernetics, Silver Spring, MD, USA), the total area of the proliferating zone was automatically calculated. For cell counting, a standard grid system with 100-µm²-grid block size was overlaid on 20x images of the proliferating zones of BrdU-treated sections. Eight standard, continuous grid blocks laid over the proliferating zone were used for cell counting. Cell counts from all 8 grid blocks were averaged per rabbit and subjected to statistical analyses. BrdU-labeled chondrocytes were identified by assignment of a uniform brown scale of DAB-stained nuclei to all proliferating chondrocytes in the S phase, and automatically counted. Upon assignment of a uniform gray scale that labeled the nuclei of all chondrocytes present in the proliferating zone, the total numbers of chondrocytes in the proliferating zones of all groups were automatically counted. The BrdU labeling index was calculated as the percentage of BrdU-labeled nuclei of chondrocytes undergoing active mitosis over the total number of chondrocytes present in the proliferating zone.

View larger version (99K): [in this window] [in a new window]   Figure 3. Proliferating zone area of sham control and mechanical stimulation. Representative photomicrographs of the cranial base cartilage (CBC) showing sham control (A,A'), static force (B,B'), and cyclic force (C,C'). A, B, and C were stained with hematoxylin and eosin; A', B', and C' were stained with safranin O and fast green. The total proliferating zone area treated with cyclic forces (C,C'), manually isolated by computerized image analysis, showed marked increase in comparison with static forces (B,B') and sham controls (A,A'). Reaction to safranin O confirmed the presence of abundant chondroitin-sulfate and keratan-sulfate binding proteoglycans, most likely aggrecan, in the cartilage matrix. Scale bar: 150 µm.

	RESULTS

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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).

View larger version (150K): [in this window] [in a new window]   Figure 2. BrdU labeling of growth plate chondrocytes. Representative photomicrographs of chondrocyte proliferation labeled with bromodeoxyuridine (BrdU) in the proliferating zones of the cranial base cartilage (CBC). (A) Negative control (no BrdU antibody). (B,C,D) Samples treated with BrdU antibodies. B, sham control; C, static force; D, cyclic force. There are marked increases in BrdU-labeled proliferating chondrocytes in association with cyclic force (D), in comparison with natural chondral growth (B) and static force (C). Arrows indicate the nuclei of proliferating chondrocytes. Scale bar: 40 µm.

View larger version (27K): [in this window] [in a new window]   Figure 4. Quantification of BrdU labeling and histomorphometry of the proliferating zone. (A) The average BrdU labeling index of the proliferating zone treated with cyclic force (N = 7) was significantly greater than that of static force (N = 6) (p < 0.05) and sham control (N = 7) (p < 0.01). (B) The average total area of the proliferating zone treated with cyclic force was significantly greater than those of static force (p < 0.05) and sham control (p < 0.01).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
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 (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 (Rosenberg, 1971; Lammi and Tammi, 1988; Mao et al., 1998).

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.

	ACKNOWLEDGMENTS

 
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 March 26, 2002; Last revision July 29, 2002; Accepted August 6, 2002

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This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (14) Citing Articles via Google Scholar Google Scholar Articles by Wang, X. Articles by Mao, J.J. Search for Related Content PubMed PubMed Citation Articles by Wang, X. Articles by Mao, J.J.