J Oral Maxillofac 48:276-282.
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The Effects of Mechanical Strain on Osteoblasts In Vitro MICHAEL J. BUCKLEY, DDS, MS,* ALBERT J. BANES, PHD,t RICHARD D. JORDAN, DDSt
AND
The effect of mechanical strain on bone is important to the field of oral and maxillofacial surgery. Oral and maxillofacial surgeons are involved on a daily basis with problems including alveolar ridge resorption, implant stability, craniofacial growth, and bone resorption related to trauma and pathology. To understand and control these effects on bone, it is important to examine the effects of mechanical strain on the osteoblasts. This series of experiments provides the changes in alkaline phosphatase, collagen synthesis, and protein synthesis in osteoblast-like cell subjected to mechanical strain The effect of stress on bone as a tissue has been studied since the inception of Wolff s law.’ Bone tissue is known to respond to strain, but little is known of the response of osteoblasts within bone to strain. Recently, Frost put forth a theory on the importance of mechanical strain to the pathophysiology of bone mass changes.’ He pointed out that since 1930 the vast proportion of research on bone and bone cells involved nonmechanical agents; this view concludes that circulating agents affect osteoclasts/osteoblasts, which results in bone loss. Frost proposed a new concept that differed fundamentally from the previous scheme:
imposed on bone. The bone responds to a minimum essential strain (MES mechanisms), and this in turn regulates the modeling and remodeling capabilities of bone. In this scheme, the nonmechanical agents effect the MES mechanism and adjust or alter the response. Minimal effective strain (MES) is the amount of strain or threshold that the bone tolerates. Any strain greater than that is responded to by active changes. The MES for mammalian bone modeling is believed to be 1500 to 2500 microstrains. This is in contrast to a bone fraction strain of 25,000 microstrains. Remodeling, however, is believed to involve much less strain, in the range of 100 to 300 microstrains. Frost concluded that “when a normal biological system fulfills its mechanical requirements for function, the mechanical environment controls the biologic response directly.“’ Therefore, because bone mass always responds proportionately to strain (except in disease and trauma), the demands of mechanical loading should control bone mass. Also, biological systems only respond to error, not unlike a mechanical thermostat, so that the MES system is able to distinguish acceptable from unacceptable errors that require correction. Frost refers to this as the “mechanostat theory.“’
MU + bone + MES mechanism + t (nonmechanical agents) modeling/remodeling
+ bone mass
In this scheme, MU is the sum of mechanical forces * Former resident and AAOMS Research Fellow, University of North Carolina; presently. Assistant Professor, The University of Iowa, Iowa City. t Associate Professor, University of North Carolina at Chapel Hill. Supported in part by the American Association of Oral and Max&ofacial Surgeons-Research Fellowship, AFDH. AM30952, AM 30478. and AR 38121. and Dr Ronald Baker and the University of North Carolina Department of Oral and Maxillofacial Surgery. Address correspondence and reprint requests to Dr Buckley: Department of Hospital Dentistry, Oral and Maxillofacial Surgery, The University of Iowa Hospital and Clinics, Iowa City, IA 52242. 0 1990 American Association of Oral and Maxillofacial
Mechanical Strain In Vitro The literature is sparse with information on the effects of mechanical strain on individual tissues or cells within the organ system. In 1975, Rodan et al, using the embryonic chick and rat long bone, reported a 50% reduction in glucose consumption and a stimulation of thymidine incorporation into DNA
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during bone compression in vitro.3 Similarly, they found an 8% decrease in extracellular fluid, but no effect on sodium equilibrium, and an increase in thymidine incorporation.3 They noted the importance in realizing the difference between a mechanical stimulus and a chemical signal response that, in turn, stimulated cell proliferation.3 Similarly, information about the results of mechanical strain on individual cells in vitro has been limited. Changes in many facets of cellular activity, including protein and collagen production,4,5 mitotic figures,” collagenases,’ second messengers,* prostaglandins,’ and matrix production’ have been shown in a variety of cells stimulated by many different mechanical devices that produce mechanical strain. The effects of cyclic compressive deformation on cell expression in cartilagenous long bone metatarsal rudiments have been studied by Klein-Nulend and coworkers. *’ They examined the effects of constant versus intermittent compressive force to modulate calcification. Discontinuous hydraulic compressive force, 13% above ambient pressure at 0.3 Hz, invoked a greater mineralization response than did other regimens as evidenced by increasing 45Ca and 32P incorporation. lo Of all the tissue systems and cell types thus far examined, endothelial cells and aortic smooth muscle cells have claimed the most attention. Ives et al, using human and bovine endothelial cells, found that the cells responded differently to various types of strain.” The cells oriented themselves parallel to the direction of shear strain induced by fluid flow, but perpendicular to the axis of mechanical deformation on a cyclically stretched Mitrathane (Mitral Medical, Denver, CO) polyurethane membrane.” In a separate series of experiments using a vaccum-operated strain unit (Flexercell Strain Unit, Flexcell Corp, McKeesport. PA), cyclic mechanical strain was shown by Sumpio et alI2 to stimulate endothelial cell proliferation. The bovine aortic endothelial cells were cultured on the flexiblebottomed culture dishes and were exposed to up to 24% elongation at 3 cycles per minute. He found a significant increase in cell number and ‘H-thymidine incorporation. I2 Similarly, Sumpio and coworkers reported a coordinate increase in collagen and noncollagen protein synthesis in smooth muscle cells isolated from porcine aorta using the same system and regimen of cyclic strain.13 Previous work with osteoblast-like cells stimulated by cyclic mechanical strain have shown an increase in growth rate, increase in thymidine incorporation, and alignment perpendicular to the strain vector. I4 Similarly, these osteoblasts, when stressed, mineralized the matrix under conditions
that ordinarily do not allow for mineralization (ie, no added P-glycerophosphate, and within 72 hours after initiating cyclic strain). The mineralization was confirmed by the results of histochemical staining for calcium and calcium phosphate, as well as by the results of back-scatter electron microscopy and infrared analysis. Collagen Production The organic matrix of bone is composed of collagen and noncollagen proteins.‘5-” Type I collagen, the most abundant type in mammalian bone, is a heterotrimer composed of two CY - 1 (I) chains and one 01- 2 (I) chain. These are synthesized by osteoblasts in bone as precursor procollagen molecules containing both amino terminal and carboxy terminal globular extensions. 18*19The difference between the type I collagen seen in bone and that of skin is due to a posttranslational modification. The mineral that is deposited on collagen is done in one of three compartments: 1) within fibrils, 2) on the surface of fibrils, and 3) between the fibrils.20 The deposition varies between lamellar bone and woven bone, and between newly formed bone and mature bone. It is of importance, however, that in all studied tissues, collagen deposition is necessary before deposition of mineral. Alkaline Phosphatase Alkaline phosphatase activity is an established phenotypic marker of bone formation. The exact function of this enzyme is yet to be determined, but it is thought to play a role in the mineralization of bone. The enzyme activity is regulated by many factors: alkaline phosphatase is inhibited by parathyroid hormone, isoproterenol, and 1,25 (OH), D ‘6,2’ Alkaline phosphatase activity is increased b;’ treatment with diphosphonates and glucocorticoids.‘2 We report in this study the effects of defined mechanical strain in vitro on the expression of alkaline phosphatase, a known phenotypic marker of osteoblasts, collagen, and noncollagen protein synthesis, as well as the 2-D gel protein profile of osteoblasts subjected to cyclic mechanical strain in culture. Materials and Yethods CELL
ISOLATION
Osteblast-like cells from calvaria of 4-week-old chicks were isolated under sterile conditions and
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subjected to sequential enzyme hydrolysis (modified from Wong and Cohn3’) with collagenase and trypsin. I4 Periosteum was removed and placed in Eagles Minimal Essential Medium (MEM; Sigma) at 37°C in a 5-mL-capacity culture tube and mechanically agitated by rolling at 2 RPM for 20 minutes. The tissue was then rinsed with calcium- and magnesium-free Hanks (CMFH) solution. The dissociated cells and fluid were decanted and discarded, and then the tissue was treated with 0.25% trypsin at 37°C and mechanically agitated as previously for 20 minutes. The dissociated cells and fluid were discarded. The final digestion was accomplished by rinsing as before, mincing the tissue, then digesting in 0.5% collagenase for 4 hours. This digest was then centrifuged at 900 x g for 5 minutes and the supematant fluid discarded. The pellet was resuspended in 2 mL of CMFH and recentrifuged. The pellet was then resuspended in culture medium (MEM, 10% fetal calf serum [FCS]) and plated in 60-mm culture dishes.
sures, and a vacuum baseplate and gasket upon which specifically built, flexible-bottomed culture plates are placed (FLEX I culture plates, Flexcell Corp) Control of frequency, strain rate, and degree of elongation of the deformation regimen are achieved by regulating the rate of evacuation (vacuum level) and rate of air influx to the plate bottoms. The culture plates consisted of six-well, flexiblebottomed culture plates (FLEX I plates) with a hydrophilic surface capable of up to 200% stretch. Control plates (FLEX II culture plates) had nondeformable surfaces made with the same substrate material present in the flexible-bottomed plates. The deformation regimen used for these studies involved three cycles per minute (0.05 Hz), ie, 10 seconds of a maximum 24% elongation followed by 10 seconds of relaxation, verified after timing the recording from a strip-chart recorder. At a vacuum level equivalent to 5 inches of mercury, the empirically measured tension on the surface of a Flex I plate followed an exponential curve from 0 to 24% elongation along a radius from the well center to the edge (Banes and coworkers, manuscript in preparation). Real-time video analyses of Flex I plates indicated that when a flexible membrane was stretched, the adherent cells also stretched and remained adherent, demonstrating that the deformation to the flexible membrane was transiated to the cells.
UMR-106 CLONAL CELLS UMR-106 cells, a rat osteosarcoma cell line (originally isolated by T.J. Martin), were obtained from the University of North Carolina Cancer Center repository. These are a clonal, osteoblast-like cell line that has been shown to respond to parathyroid hormone (PTH) and prostaglandins, and is capable of forming a ground substance and mineralizing it.23 This particular cell line was used because it is rich in alkaline phosphatase.23 CELL CULTURE
Osteoblast-like cells obtained using the isolation procedure previously described, or UMR-106, were maintained in MEM containing 10% fetal calf serum, 20 mol/L glutamine, 10 mmoYL pyruvate, 0.5 mmoYL ascorbic acid, 20 mmol/L HEPES, pH 7.2, and antibiotics (100 U sodium penicillin, 100 mg streptomycin, and 2 ug amphopteracin B per milliliter). Culture dishes were maintained at 37°C in a humidified incubator with 5% carbon dioxide. Cells were passaged after incubation with 0.01% trypsin for 20 minutes and used from passes one to three. IN VITRO APPLICATION OF STRAIN TO CULTURED DISHES
Cells were subjected to mechanical strain with a FlexcelI Strain Unit. The instrument comprises a computer system controller and monitor, a control module that regulates negative and positive pres-
ONOSTEOBLASTS
QUANTITATION OF COLLAGEN AND NONCOLLAGEN PROTEIN SYNTHESIS
To investigate collagen and noncollagen protein synthesis, calvarial osteoblast-like cells were grown in six-well Flex I plates (200,000 tells/35-mm* well) and allowed to attach for 24 hours before experimentation. Twenty-four hours before harvesting the cells or supematant fluids, the cells were cultured in 2 mL of serum-free medium containing 50 $i L(3H) proline, an amino acid that is hydroxylated at specific sites in collagen, and supplemented with ascorbic acid (0.5 mmoYL final concentration), 0.2 mmoYL r_-glutamine, and antibiotics. The radioisotope was lyophilized before use. On days 1 and 3, the media and new cells of the static (n = 6 wells per day) and experimental (n = 6 wells per day) groups were collected, transferred to chilled tubes, and processed separately. Calf serum (50 p.L) was added as carrier protein, the suspension then was mixed and precipitated with trichloroacetic acidtannic acid (final concentration 5% TCA # 0.125% TA). Precipitates were washed with TCA-TA until radioactivity in 1 mL of a lo-mL wash was less than 100 cpm, then the samples were sedimented and
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lyophilized. Hydrochloric acid (Pierre, amino acid hydrolysis grade) was added to each pellet to a final concentration of 6 N, the tubes were sealed, and the samples were hydrolyzed overnight at 100°C. The tubes were then opened, and the samples were collected and filtered through a fine-glass filter. Hydroxyproline was quantitated by a chemical assay based on the oxidation of hydroxyproline to a pyrrole derivative which produces a chromogen with Ehrlich’s reagent.‘4 The absorbance was recorded at 565 nm, and a standard curve was prepared. Calculation of collagen and noncollagen protein synthesis per culture was based on quantitation of hydroxyproline and proline. Appropriate corrections were made for efficiency of counting, volume counted, volume of homogenate hydrolyzed, and amount of hydroxyproline in collagen (12.8%). To calculate the amount of noncollagen protein present, 14.7% of the radioactivity in collagen (proline radioactivity) was subtracted from total proline radioactivity, and that value was divided by 0.041 to yield the value for radioactivity of noncollagen protein presentZ5 PREPARATION OF 2-D GELS
Calvarial osteoblast-like cells plated at 100,000 cells per well were subjected to cyclic strain using a Flexercell Strain Unit at 3 cycles per minute at a maximum elongation of 24%, for 4 days. At this time the cells were near confluency. The cells were then rinsed two times with CMFH. They were then incubated with 400 pCi methionine S35 in 2 mL of MEM with 1% FCS for 24 hours. At this time the strain regimen was ended, and the plates were immediately placed on ice. Cell sheets were then rinsed three times with ice-cold CMFH to remove excess radioisotope and serum proteins. Preheated sodium dodecyl sulfate (SDS) was then added at a ratio of 100 1.~L/400,000 cells per well. DNA RNASE was then added to a ratio of 10 pl # 100 t.~l of SDS. The cell sheets were then scraped with a rubber policeman and immersed in liquid nitrogen and stored at -70°C. The samples were then shipped to Protein Database Inc, of Long Island, NY, for two-dimensional gel analysis using isoelectrofocusing followed by separation on a 12.5% acrylamide slab gel with a broad-range pH 3-10. Autoradiograms were prepared, and spot analysis was performed with the use of a P D Quest computer analysis system. Statistical analysis used was the f test and log transformed data with P 5 .05 (n = 3). ALKALINE PHOSPHATASE ASSAY
Alkaline phosphatase activity was assayed using homogenized cells at 37°C by a modified version of
Lowry’s method.26 The assay mixture contained 0.1 mmol/L 2-amino-2-methyl- 1-propanol, 2 mmol/L MgCl,. and 2 mmol/L disodium p-nitrophenyphosphate (Na,PNPP). The reaction was stopped after 15 minutes with 0.8 mL of 1 N NaOH, and the absorbance was measured at 410 nm. Standard curves were prepared using p-nitrophenol. Protein concentrations were determined using Coomassie blue as described by Spector.27 Results
The results in Table 1 represent synthesized cellassociated collagen and noncollagen protein, as well as the amount of secreted noncollagenous and collagenous protein (amount present in the supernatant fluid). There was a significant elevation in cell-associated collagen and noncollagen protein at day 3, whereas day 1 showed no significant change. Similarly, synthesis of secreted collagen and noncollagen proteins in the stressed cells was significantly decreased at day I, but increased on day 3. The changes in alkaline phosphatase with time are expressed in Fig 1. The UMR-106 cells were plated at 250 X lo3 cells per well and maintained in cultures for 48 hours before beginning the strain regimen (3 cpm, maximum 24% elongation). Alkaline phosphatase specific activity increased between 8 and 24 hours and was 1.67-fold higher by 48 hours in stressed cells (Fig 1). The control cells showed a slight increase in activity consistent with the increase seen as the cultures reached confluency . The two-dimensional gel electrophoresis results showed the separation of 847 spots on the control cell gels. The stressed cell profile showed 749 spots, for an elimination of 98 proteins. Statistical analysis of the 112 proteins decreased using the t test and log Table 1.
Collagen and Noncollagen Protein
Day
Sample
1
Control cell Stress cell Control supemate Stress supemate Control cell Stress cell Control supemate Stress supemate
I 3 3
Noncollagen (dpmkell x 10’) 17,320 16,470 2,880* 2.290* 8,730* 11,000* 3,070* 3.720*
Collagen (dpmkell x 10’) 2,580 2,840 189* 152* I ,410*
1.870* 264 283
Collagen and noncollagen proteins are shown for both the cellassociated and secreted fractions. Control and stressed cells were incubated with 50 uCi H,-proline for 24 hours before assay. * P G .os.
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EFFECTS OF MECHANICAL
L
I
0
4
I
I
I
I3 12
I
I
16 20 24
I
I
I
26
32
36
I
I
40 44
I
48
Tlmeh
FIGURE 1. Change in alkaline phosphatase specific activity with time (hour). Alkaline phosphatase is presented as micromole-per-hour milligrams of protein. Solid line represents the control cell, and the broken line represents the stressed cells.
transformed data (P G .05) showed that 29 proteins were significantly decreased with stress. Three of these proteins are known: 80, 90, and 100 kd heat shock proteins. Table 2 shows the proteins that were significantly increased in the strained cells. Six of these proteins are known: calmodulin (2.2fold), o-tubulin (8.7-fold), vimentin (22.5fold), 73kd heat shock protein, vinculin @.dfold), and phosphorylase B (18.2-fold). Similarly, three proteins were detected in stressed cells that were absent from the controls. Discussion
The increase in organic matrix seen in these osteoblast-like cells from chick calvaria is critical to the cascade of events that lead to the production of bone. Cells stressed for 24 hours had a nonsignificant increase of 10% in cellular collagen synthesis. Table 2. Synthesis of Known Osteoblast Proteins Affected by Cyclic Deformation
Calmodulin u-Tubulin Vimentin HSP 80 HSP 90 HSP 100 Actin HSP 73 Vinculin Phosphorylase B
kd
pI
Fold Change
Control (dpm/spot)
Stress (dprn/spot)
17 57.4 61 80 90 100 42 73 130
4 5.21 5.16 5.19 5.19 5.09 5.42 5.5 6.07
2.12* 8.7* 22.54 0.77* 0.063 0.500 5.62*
2,620 2,516 260 1,466 161 1,135 17,54030.1
5,827 + 21,861+ 5,838+ 1,1357235,055 2,787+ 169+
20
6.96
18.2*
55.5
1,009+
Results of the two-dimensional gel electrophoresis. change is represented as the fold increase or decrease. Abbreviations: kd, kilodalton; p1, isoelectic constant. *PC .05.
The
STRAIN ON OSTEOBLASTS
The amount of secreted noncollagen protein on day 1 was decreased by 20% and 19.5%, respectively. On day 3, synthesis of both noncollagen proteins and collagen showed an increase of 26% and 33%, respectively. Secretion at day 3 was increased for noncollagen proteins, but no significant change was noted in collagen secretion. Although much work must be done to substantiate the effects of mechanical strain on the rate of procollagen mRNA synthesis, translation, and posttranslational modifications, one can speculate that the initial decrease in secretion without a change in collagen synthesis could be attributed to alterations in posttranslational modifications and therefore delayed secretion.5 Similarly, although the cellular events involved in the transduction of this mechanical signal to alter DNA, RNA, or protein synthesis remain unknown, the increase in cellular collagenous and noncollagenous proteins seen at day 3 may be a transcriptional event. Further study is required before the mechanism can be elucidated. Activity of alkaline phosphatase, an established osteoblast phenotypic marker, increases in response to the mechanical signal. Although the role of alkaline phosphatase is yet be completely established, all evidence points to its importance in mineralization. One widely held view is that it provides an additional source of phosphate for mineralization by hydrolysis of organic phosphate.” An opposite view holds that alkaline phosphatase is present for the removal of inhibitors to mineralization such as diphosphonates.29*30 Our data show that an increase in alkaline phosphatase activity in the first 24 hours precedes the mineralization that occurs (data not shown). The elevation again relates the two events (alkaline phosphatase activity with mineralization) in a timely fashion. It does not, however, help us differentiate between the two possible roles of alkaline phosphatase.28-30 The protein profile of stressed cells compared with non-stress controls shows that 98 proteins that are present in the controls are absent from the stressed cells. This may be explained in the terms of luxury proteins or, put another way, proteins that are being produced at a basal level in noncommitted cells are turned off when the cell and its cellular functions are called on to perform a distinct function or respond to an outside element. Of the decreased synthesis in 112 proteins, the most striking result was the decrease in 80-kd, 90kd, and lOO-kd heat shock proteins. These “stress” proteins are of particular interest because they are induced by elevated temperature or thermal stress.3’ In the mechanical strain system, knowledge of the roles of 73-, BO-, 90-, and lOO-kd stress proteins may help distinguish their functions.” It
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may be that mechanical strain induces one heat shock protein which functions in a protective manner, while other stresses (ie, heat) invoke other protective mechanisms. One may speculate that the presence of three proteins in the stressed cells that are absent in the controls may, in light of the increase in growth rateI and induction of mineralization, be related to production of a growth factor or mineralization induction factor. Adhesion and spreading of cells on substrate is necessary for anchorage-dependent cells to grow in culture. Anchorage-dependent cells cultured in suspension can show a drastic inhibition of DNA, RNA, and protein synthesis. The exact parameters needed for cells to proliferate are unknown, but may be linked to focal contact proteins at the membrane level and the cytoskeleton.32 Vinculin, a 130kd focal contact protein, was increased 5.6-fold with this regimen of stress. These and other focal contact proteins play not only an obvious role in cell substrate interface and attachment but also a role in cell mitogenic function.3’ The cytoskeletal proteins vimentin, actin, and a-tubulin showed 22.5, OS-, and 8.7-fold increases, respectively, in strained cells. Recent evidence has strongly favored an interaction between matrix, cell receptors, and cytoskeleton.33 Direct evidence of a matrix-cytoskeletal connection has been provided by ultrastructural studies demonstrating transmembrane association of extracellular fibronectin fibers in intracellular actin filaments.34 The role of the cytoskeleton in modulating DNA synthesis, and hence gene expression, has been substantiated by a number of investigators.35-37 This increase in critical focal contact and cytoskeletal proteins to mechanical strain and the concurrent changes in phenotypic markers (ie, alkaline phosphatase, collagen and noncollagen proteins) gives further strength to the role of the cytoskeleton in modulating gene expression. Furthermore, the matrix-receptor-cytoskeleton may be the mechanism by which the signal from the external environment is transmitted in the cell and be the mechanism of Frost’s “Mechanostat Theory. “’ Summary Osteoblast-like cells, when subjected to mechanical stress, increase their synthesis of collagen and noncollagen proteins at day 3. On day 1, secretion of both proteins is decreased, whereas secretion of cellular proteins is unchanged. Alkaline phosphatase activity, when examined in UMR-I06 (rat osteosarcoma cells), showed a marked increase at 24 hours, which complements induction of mineral-
ization. The dimensional many luxury tal and focal
protein profile as determined by twogel electrophoresis shows a decrease in proteins and an increase in cytoskelecontact proteins.
References 1. Wolff J: Das Gesete der Transformation 2.
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der Knochen. Berlin, Hirshwald, 1892 Frost HM: The mechanostat: A proposed pathogenic mechanism of osteoporosis and the bone mass effects of mechanical and nonmechanical agents. Bone Mineral 2:73, 1987 Rodan GA, Mensi T, Harvey A: A quantitative method for the application of compressive forces to bone in tissue cult&e. Calcif Tissue ties 18:125. 1975 Leunc! DY. Glaeor S. Mathews MB. A new in vitro svstem for-studying cell response to mechanical stimulation: Exp Cell Res 109:285, 1977 Meikle M, Reynolds J, Sellers H, et al: Rabbit cranial sutures in vitro: A new experimental model for studying the response of fibrous joints to mechanical stress. Calcif Tissue Int 28:137, 1979 Curtis AS, Seehar GM: The control of cell division by tension or diffusion. Nature 274:52, 1978 Meikle M, Sellers A, Reynolds J: Effect of tensile mechanical stress on the stynthesis of metalloproteinases by rabbit coronal sutures in vitro. Caicif Tissue Int 30:77, 1980 Hare11 A, Dekel S, Binderman I: Biochemical effect of mechanical stress on cultured bone cells. Calcif Tissue Res 22:202, 1977 Dewitt M, Handley CJ, Oaks B, et al: In vitro response of chondrocytes to mechanical loading: The effect of short term mechanical tension. Connect Tissue Res 12:97. 1984 Klein-Nulend J, Veldhuijzen J, Burger E: Increased calcification of growth plate cartilage as a result of compressive force in vitro. Arthritis Rheum 29:1002, 1986 Ives C, Eskin S, M&tire C: Mechanical effects on endothelial cell morphology: In vitro assessment. In Vitro Cell Dev Bioi 22500, 1986 Sumpio B, Banes A, Levine L, et al: Mechanical stress stimulates aortic endothelial cells to proliferate. J Vast Surg 61252, 1987 Sumpio B, Banes A, Link W, et al: Enhanced collagen production by smooth muscle cells during repetitive mechanical stretching. (in press) Buckley MJ, Banes AJ, Levine LG, et al: Osteoblasts increase their rate of division and align in response to cyclic mechanical tension in vitro. Bone Mineral 4:225, 1988 Glimcher MJ: Composition, structure and organization of bone and other mineralized tissue and the mechanism of calcification, in Greep RO, Astwood EB (eds): Handbook of Physiology, vol 7. Washington, DC, American Physiology Society, 1976, p 25 Kane SM: Genetic and acquired disorders of collagen deposition, in Piez KA, Reddi AH (eds): Extracellular Matrix. Biochemistry. New York, NY, Elsevier 1984, p 413 Nim ME: Collagen: Structure, function and metabolism in normal and fibrotic tissue. Semin Arthritis Rheum 13:1, 1983 Prockop DJ, Kiririkko KI: Heritable disease of collagen. N Engl J Med 311:376, 1984 Prockop DJ, Kiririkko KI, Tuderman L, et al: The biosynthesis of collagen and its disorders. N Engl J Med 301: 13, 1979 Robinson RA, Dotz SB, Cooper RR: Electron microscopy of mammalian bone, in Sipkin I (ed): Biological Mineralization. New York, NY, tiiley, 1973, p 257 Luber RA. Wang GL. Cohr DV: Endocrinoloev 99:526. 1976 Felix R, FleischH: Increase in alkaline phosphatase aciivity in calvaria cells cultured with diphosphonates. Biochem J 183:73, 1979 Pantridge NC. Alcom D, Michelangeli V. ef al: Morpholog-
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31. Lindquist S: The heat shock response. Ann Res Biochem 55:1151, 1986 32. Burridge K: Substrate adhesion in normal and transformed fibroblasts. Cancer Res 4:18, 1986 33. Hay ED: Cell Biology of Extracellular Matrix. New York, NY, Plenum, 1982 34. Singer 11: The fibronexus: A transmembrane association in fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human tibroblasts. Cell 16:675.
26. Lowry OH, Rubeuts NR, Wu ML, et al: The quantitative histochemistry of brain. II. Enzyme measurements. J Biol Chem 207: 19, 1954 27. Spector T: Refinement of the Coomassie blue method of protein quantitation. Anal Biochem 86:142, 1978
35. Zannetti NC, Solursh M: Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskelton. J Cell Biol99:115, 1984 36. Term M. Bartholomew JC. Bissell MJ: Svnereism between aitimicrotubule agents and growth stimulants in enhancement of cell cycle traverse. Nature 268:739, 1977 37. Friedkin M, Legg A, Rozergrot E: Antitubulin agents enhance the stimulation of DNA synthesis by polypeptide growth factors in 3T3 mouse fibroblasts. Proc Nat1 Acad Sci USA 76:3902, 1979 38. Wong G, Cohn DV: Formation of parathyroid hormone and calcitonin. Sensitive cells from responsive bone cells. Nature New Biol 252:713, 1974
28. Russell R, Caswell A, Heam P, et al: Calcium in mineralized tissues and pathological calcification. Br Med Bull 42:439, 1986 29. Meyer JC: Can biological calcification occur in the presence of pyrophosphate? Arch Biochem Biophys 23 1: 1, 1984 30. Russell R: Metabolism of inorganic phosphate. Rheum 19:465, 1976
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Discussion The Effects of Mechanical Strain on Osteoblasts In Vitro David L. Cochran,
DDS, MS, PhD, MMSc
Medical College of Virginia, Richmond
In the preceding article by Buckley, Banes, and Jordan, several effects of cyclic mechanical strain have been examined on cells derived from 4-week-old chick calvaria. These investigators point out that bone undergoes varied mechanical strain under many circumstances on a daily basis. They further stipulate that the consequence of such strain is directed at the cellular level, and they have investigated these effects on bone-derived cell cultures that should be enriched in osteoblast cells. These investigators have used a cyclic stress force on the cell cultures, and they indicate that cyclic strain may be very different from a constant stress or, for that matter, thermal or chemical stress. One must be constantly mindful that even in the most appropriate of all the mechanical stress model systems described for cultured cells, the investigations may not mimic in vivo stress. It is well documented that in vivo bone coupling occurs so that bone formation and bone resorption occur simultaneously. Because these are linked processes, analysis of only one aspect of this process without the influence of the other may lead to results not compatible with what occurs in vivo. Recent data’ suggest that transforming growth factor-beta may act as a coupling agent between bone formation and bone resorption, and may allow one to better define model systems that reflect in vivo conditions more accurately. In the study by Buckley et al, bone cells were derived from chick calvaria which were sequentially digested with collagenase and trypsin. This is a method used by several
laboratories and was originally described by Wong and Cohn.’ Buckley et al digested the calvaria three times, with the first two incubations lasting 20 minutes and the last one 4 hours. Many investigators digest calvaria live to six times for 10 to 20 minutes and combine the last two to three digestions to obtain osteoblast-enriched populations. Our experience has been that the purity of the collagenase and the length of time of each digestion are critical for the enrichment of specific cell populations. An absolute requirement in any of these preparative procedures is the characterization of the resultant cell cultures. Several common methods are available to characterize such cell populations: collagen phenotype; response to hormone treatment including PTH, vitamin D, and calcitonin; as well as the measurement of enzymatic activities, including cyclic AMP, alkaline phosphatase, and measurement of osteocalcin levels. Perhaps the best characterization of putative osteoblast-like cells is to determine if the cell cultures undergo mineralization. No data on mineralization are presented, yet the authors state in the summary that the increase in alkaline phosphatase activity in the transformed cell line complemented induction of mineralization. Without analysis of mineralization or data on the induction of mineralization, one can never be sure that the isolated cultures are functional osteoblast cells. Osteoblast-enriched cultures may mean that 20% of the cells are osteoblasts, or perhaps 70%; but the question remains, are the cells functionally osteoblast-like, and how much must the culture be enriched to have significant results for bone-forming cells? Because fibroblasts may be a contaminating cell type, control assays should be performed on fibroblast cultures for comparison, in addition to the biochemical and functional characterization of the osteoblast-like cultures.
Surg
1990
The Effects of Mechanical Strain on Osteoblasts In Vitro MICHAEL J. BUCKLEY, DDS, MS,* ALBERT J. BANES, PHD,t RICHARD D. JORDAN, DDSt
AND
The effect of mechanical strain on bone is important to the field of oral and maxillofacial surgery. Oral and maxillofacial surgeons are involved on a daily basis with problems including alveolar ridge resorption, implant stability, craniofacial growth, and bone resorption related to trauma and pathology. To understand and control these effects on bone, it is important to examine the effects of mechanical strain on the osteoblasts. This series of experiments provides the changes in alkaline phosphatase, collagen synthesis, and protein synthesis in osteoblast-like cell subjected to mechanical strain The effect of stress on bone as a tissue has been studied since the inception of Wolff s law.’ Bone tissue is known to respond to strain, but little is known of the response of osteoblasts within bone to strain. Recently, Frost put forth a theory on the importance of mechanical strain to the pathophysiology of bone mass changes.’ He pointed out that since 1930 the vast proportion of research on bone and bone cells involved nonmechanical agents; this view concludes that circulating agents affect osteoclasts/osteoblasts, which results in bone loss. Frost proposed a new concept that differed fundamentally from the previous scheme:
imposed on bone. The bone responds to a minimum essential strain (MES mechanisms), and this in turn regulates the modeling and remodeling capabilities of bone. In this scheme, the nonmechanical agents effect the MES mechanism and adjust or alter the response. Minimal effective strain (MES) is the amount of strain or threshold that the bone tolerates. Any strain greater than that is responded to by active changes. The MES for mammalian bone modeling is believed to be 1500 to 2500 microstrains. This is in contrast to a bone fraction strain of 25,000 microstrains. Remodeling, however, is believed to involve much less strain, in the range of 100 to 300 microstrains. Frost concluded that “when a normal biological system fulfills its mechanical requirements for function, the mechanical environment controls the biologic response directly.“’ Therefore, because bone mass always responds proportionately to strain (except in disease and trauma), the demands of mechanical loading should control bone mass. Also, biological systems only respond to error, not unlike a mechanical thermostat, so that the MES system is able to distinguish acceptable from unacceptable errors that require correction. Frost refers to this as the “mechanostat theory.“’
MU + bone + MES mechanism + t (nonmechanical agents) modeling/remodeling
+ bone mass
In this scheme, MU is the sum of mechanical forces * Former resident and AAOMS Research Fellow, University of North Carolina; presently. Assistant Professor, The University of Iowa, Iowa City. t Associate Professor, University of North Carolina at Chapel Hill. Supported in part by the American Association of Oral and Max&ofacial Surgeons-Research Fellowship, AFDH. AM30952, AM 30478. and AR 38121. and Dr Ronald Baker and the University of North Carolina Department of Oral and Maxillofacial Surgery. Address correspondence and reprint requests to Dr Buckley: Department of Hospital Dentistry, Oral and Maxillofacial Surgery, The University of Iowa Hospital and Clinics, Iowa City, IA 52242. 0 1990 American Association of Oral and Maxillofacial
Mechanical Strain In Vitro The literature is sparse with information on the effects of mechanical strain on individual tissues or cells within the organ system. In 1975, Rodan et al, using the embryonic chick and rat long bone, reported a 50% reduction in glucose consumption and a stimulation of thymidine incorporation into DNA
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during bone compression in vitro.3 Similarly, they found an 8% decrease in extracellular fluid, but no effect on sodium equilibrium, and an increase in thymidine incorporation.3 They noted the importance in realizing the difference between a mechanical stimulus and a chemical signal response that, in turn, stimulated cell proliferation.3 Similarly, information about the results of mechanical strain on individual cells in vitro has been limited. Changes in many facets of cellular activity, including protein and collagen production,4,5 mitotic figures,” collagenases,’ second messengers,* prostaglandins,’ and matrix production’ have been shown in a variety of cells stimulated by many different mechanical devices that produce mechanical strain. The effects of cyclic compressive deformation on cell expression in cartilagenous long bone metatarsal rudiments have been studied by Klein-Nulend and coworkers. *’ They examined the effects of constant versus intermittent compressive force to modulate calcification. Discontinuous hydraulic compressive force, 13% above ambient pressure at 0.3 Hz, invoked a greater mineralization response than did other regimens as evidenced by increasing 45Ca and 32P incorporation. lo Of all the tissue systems and cell types thus far examined, endothelial cells and aortic smooth muscle cells have claimed the most attention. Ives et al, using human and bovine endothelial cells, found that the cells responded differently to various types of strain.” The cells oriented themselves parallel to the direction of shear strain induced by fluid flow, but perpendicular to the axis of mechanical deformation on a cyclically stretched Mitrathane (Mitral Medical, Denver, CO) polyurethane membrane.” In a separate series of experiments using a vaccum-operated strain unit (Flexercell Strain Unit, Flexcell Corp, McKeesport. PA), cyclic mechanical strain was shown by Sumpio et alI2 to stimulate endothelial cell proliferation. The bovine aortic endothelial cells were cultured on the flexiblebottomed culture dishes and were exposed to up to 24% elongation at 3 cycles per minute. He found a significant increase in cell number and ‘H-thymidine incorporation. I2 Similarly, Sumpio and coworkers reported a coordinate increase in collagen and noncollagen protein synthesis in smooth muscle cells isolated from porcine aorta using the same system and regimen of cyclic strain.13 Previous work with osteoblast-like cells stimulated by cyclic mechanical strain have shown an increase in growth rate, increase in thymidine incorporation, and alignment perpendicular to the strain vector. I4 Similarly, these osteoblasts, when stressed, mineralized the matrix under conditions
that ordinarily do not allow for mineralization (ie, no added P-glycerophosphate, and within 72 hours after initiating cyclic strain). The mineralization was confirmed by the results of histochemical staining for calcium and calcium phosphate, as well as by the results of back-scatter electron microscopy and infrared analysis. Collagen Production The organic matrix of bone is composed of collagen and noncollagen proteins.‘5-” Type I collagen, the most abundant type in mammalian bone, is a heterotrimer composed of two CY - 1 (I) chains and one 01- 2 (I) chain. These are synthesized by osteoblasts in bone as precursor procollagen molecules containing both amino terminal and carboxy terminal globular extensions. 18*19The difference between the type I collagen seen in bone and that of skin is due to a posttranslational modification. The mineral that is deposited on collagen is done in one of three compartments: 1) within fibrils, 2) on the surface of fibrils, and 3) between the fibrils.20 The deposition varies between lamellar bone and woven bone, and between newly formed bone and mature bone. It is of importance, however, that in all studied tissues, collagen deposition is necessary before deposition of mineral. Alkaline Phosphatase Alkaline phosphatase activity is an established phenotypic marker of bone formation. The exact function of this enzyme is yet to be determined, but it is thought to play a role in the mineralization of bone. The enzyme activity is regulated by many factors: alkaline phosphatase is inhibited by parathyroid hormone, isoproterenol, and 1,25 (OH), D ‘6,2’ Alkaline phosphatase activity is increased b;’ treatment with diphosphonates and glucocorticoids.‘2 We report in this study the effects of defined mechanical strain in vitro on the expression of alkaline phosphatase, a known phenotypic marker of osteoblasts, collagen, and noncollagen protein synthesis, as well as the 2-D gel protein profile of osteoblasts subjected to cyclic mechanical strain in culture. Materials and Yethods CELL
ISOLATION
Osteblast-like cells from calvaria of 4-week-old chicks were isolated under sterile conditions and
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subjected to sequential enzyme hydrolysis (modified from Wong and Cohn3’) with collagenase and trypsin. I4 Periosteum was removed and placed in Eagles Minimal Essential Medium (MEM; Sigma) at 37°C in a 5-mL-capacity culture tube and mechanically agitated by rolling at 2 RPM for 20 minutes. The tissue was then rinsed with calcium- and magnesium-free Hanks (CMFH) solution. The dissociated cells and fluid were decanted and discarded, and then the tissue was treated with 0.25% trypsin at 37°C and mechanically agitated as previously for 20 minutes. The dissociated cells and fluid were discarded. The final digestion was accomplished by rinsing as before, mincing the tissue, then digesting in 0.5% collagenase for 4 hours. This digest was then centrifuged at 900 x g for 5 minutes and the supematant fluid discarded. The pellet was resuspended in 2 mL of CMFH and recentrifuged. The pellet was then resuspended in culture medium (MEM, 10% fetal calf serum [FCS]) and plated in 60-mm culture dishes.
sures, and a vacuum baseplate and gasket upon which specifically built, flexible-bottomed culture plates are placed (FLEX I culture plates, Flexcell Corp) Control of frequency, strain rate, and degree of elongation of the deformation regimen are achieved by regulating the rate of evacuation (vacuum level) and rate of air influx to the plate bottoms. The culture plates consisted of six-well, flexiblebottomed culture plates (FLEX I plates) with a hydrophilic surface capable of up to 200% stretch. Control plates (FLEX II culture plates) had nondeformable surfaces made with the same substrate material present in the flexible-bottomed plates. The deformation regimen used for these studies involved three cycles per minute (0.05 Hz), ie, 10 seconds of a maximum 24% elongation followed by 10 seconds of relaxation, verified after timing the recording from a strip-chart recorder. At a vacuum level equivalent to 5 inches of mercury, the empirically measured tension on the surface of a Flex I plate followed an exponential curve from 0 to 24% elongation along a radius from the well center to the edge (Banes and coworkers, manuscript in preparation). Real-time video analyses of Flex I plates indicated that when a flexible membrane was stretched, the adherent cells also stretched and remained adherent, demonstrating that the deformation to the flexible membrane was transiated to the cells.
UMR-106 CLONAL CELLS UMR-106 cells, a rat osteosarcoma cell line (originally isolated by T.J. Martin), were obtained from the University of North Carolina Cancer Center repository. These are a clonal, osteoblast-like cell line that has been shown to respond to parathyroid hormone (PTH) and prostaglandins, and is capable of forming a ground substance and mineralizing it.23 This particular cell line was used because it is rich in alkaline phosphatase.23 CELL CULTURE
Osteoblast-like cells obtained using the isolation procedure previously described, or UMR-106, were maintained in MEM containing 10% fetal calf serum, 20 mol/L glutamine, 10 mmoYL pyruvate, 0.5 mmoYL ascorbic acid, 20 mmol/L HEPES, pH 7.2, and antibiotics (100 U sodium penicillin, 100 mg streptomycin, and 2 ug amphopteracin B per milliliter). Culture dishes were maintained at 37°C in a humidified incubator with 5% carbon dioxide. Cells were passaged after incubation with 0.01% trypsin for 20 minutes and used from passes one to three. IN VITRO APPLICATION OF STRAIN TO CULTURED DISHES
Cells were subjected to mechanical strain with a FlexcelI Strain Unit. The instrument comprises a computer system controller and monitor, a control module that regulates negative and positive pres-
ONOSTEOBLASTS
QUANTITATION OF COLLAGEN AND NONCOLLAGEN PROTEIN SYNTHESIS
To investigate collagen and noncollagen protein synthesis, calvarial osteoblast-like cells were grown in six-well Flex I plates (200,000 tells/35-mm* well) and allowed to attach for 24 hours before experimentation. Twenty-four hours before harvesting the cells or supematant fluids, the cells were cultured in 2 mL of serum-free medium containing 50 $i L(3H) proline, an amino acid that is hydroxylated at specific sites in collagen, and supplemented with ascorbic acid (0.5 mmoYL final concentration), 0.2 mmoYL r_-glutamine, and antibiotics. The radioisotope was lyophilized before use. On days 1 and 3, the media and new cells of the static (n = 6 wells per day) and experimental (n = 6 wells per day) groups were collected, transferred to chilled tubes, and processed separately. Calf serum (50 p.L) was added as carrier protein, the suspension then was mixed and precipitated with trichloroacetic acidtannic acid (final concentration 5% TCA # 0.125% TA). Precipitates were washed with TCA-TA until radioactivity in 1 mL of a lo-mL wash was less than 100 cpm, then the samples were sedimented and
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lyophilized. Hydrochloric acid (Pierre, amino acid hydrolysis grade) was added to each pellet to a final concentration of 6 N, the tubes were sealed, and the samples were hydrolyzed overnight at 100°C. The tubes were then opened, and the samples were collected and filtered through a fine-glass filter. Hydroxyproline was quantitated by a chemical assay based on the oxidation of hydroxyproline to a pyrrole derivative which produces a chromogen with Ehrlich’s reagent.‘4 The absorbance was recorded at 565 nm, and a standard curve was prepared. Calculation of collagen and noncollagen protein synthesis per culture was based on quantitation of hydroxyproline and proline. Appropriate corrections were made for efficiency of counting, volume counted, volume of homogenate hydrolyzed, and amount of hydroxyproline in collagen (12.8%). To calculate the amount of noncollagen protein present, 14.7% of the radioactivity in collagen (proline radioactivity) was subtracted from total proline radioactivity, and that value was divided by 0.041 to yield the value for radioactivity of noncollagen protein presentZ5 PREPARATION OF 2-D GELS
Calvarial osteoblast-like cells plated at 100,000 cells per well were subjected to cyclic strain using a Flexercell Strain Unit at 3 cycles per minute at a maximum elongation of 24%, for 4 days. At this time the cells were near confluency. The cells were then rinsed two times with CMFH. They were then incubated with 400 pCi methionine S35 in 2 mL of MEM with 1% FCS for 24 hours. At this time the strain regimen was ended, and the plates were immediately placed on ice. Cell sheets were then rinsed three times with ice-cold CMFH to remove excess radioisotope and serum proteins. Preheated sodium dodecyl sulfate (SDS) was then added at a ratio of 100 1.~L/400,000 cells per well. DNA RNASE was then added to a ratio of 10 pl # 100 t.~l of SDS. The cell sheets were then scraped with a rubber policeman and immersed in liquid nitrogen and stored at -70°C. The samples were then shipped to Protein Database Inc, of Long Island, NY, for two-dimensional gel analysis using isoelectrofocusing followed by separation on a 12.5% acrylamide slab gel with a broad-range pH 3-10. Autoradiograms were prepared, and spot analysis was performed with the use of a P D Quest computer analysis system. Statistical analysis used was the f test and log transformed data with P 5 .05 (n = 3). ALKALINE PHOSPHATASE ASSAY
Alkaline phosphatase activity was assayed using homogenized cells at 37°C by a modified version of
Lowry’s method.26 The assay mixture contained 0.1 mmol/L 2-amino-2-methyl- 1-propanol, 2 mmol/L MgCl,. and 2 mmol/L disodium p-nitrophenyphosphate (Na,PNPP). The reaction was stopped after 15 minutes with 0.8 mL of 1 N NaOH, and the absorbance was measured at 410 nm. Standard curves were prepared using p-nitrophenol. Protein concentrations were determined using Coomassie blue as described by Spector.27 Results
The results in Table 1 represent synthesized cellassociated collagen and noncollagen protein, as well as the amount of secreted noncollagenous and collagenous protein (amount present in the supernatant fluid). There was a significant elevation in cell-associated collagen and noncollagen protein at day 3, whereas day 1 showed no significant change. Similarly, synthesis of secreted collagen and noncollagen proteins in the stressed cells was significantly decreased at day I, but increased on day 3. The changes in alkaline phosphatase with time are expressed in Fig 1. The UMR-106 cells were plated at 250 X lo3 cells per well and maintained in cultures for 48 hours before beginning the strain regimen (3 cpm, maximum 24% elongation). Alkaline phosphatase specific activity increased between 8 and 24 hours and was 1.67-fold higher by 48 hours in stressed cells (Fig 1). The control cells showed a slight increase in activity consistent with the increase seen as the cultures reached confluency . The two-dimensional gel electrophoresis results showed the separation of 847 spots on the control cell gels. The stressed cell profile showed 749 spots, for an elimination of 98 proteins. Statistical analysis of the 112 proteins decreased using the t test and log Table 1.
Collagen and Noncollagen Protein
Day
Sample
1
Control cell Stress cell Control supemate Stress supemate Control cell Stress cell Control supemate Stress supemate
I 3 3
Noncollagen (dpmkell x 10’) 17,320 16,470 2,880* 2.290* 8,730* 11,000* 3,070* 3.720*
Collagen (dpmkell x 10’) 2,580 2,840 189* 152* I ,410*
1.870* 264 283
Collagen and noncollagen proteins are shown for both the cellassociated and secreted fractions. Control and stressed cells were incubated with 50 uCi H,-proline for 24 hours before assay. * P G .os.
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L
I
0
4
I
I
I
I3 12
I
I
16 20 24
I
I
I
26
32
36
I
I
40 44
I
48
Tlmeh
FIGURE 1. Change in alkaline phosphatase specific activity with time (hour). Alkaline phosphatase is presented as micromole-per-hour milligrams of protein. Solid line represents the control cell, and the broken line represents the stressed cells.
transformed data (P G .05) showed that 29 proteins were significantly decreased with stress. Three of these proteins are known: 80, 90, and 100 kd heat shock proteins. Table 2 shows the proteins that were significantly increased in the strained cells. Six of these proteins are known: calmodulin (2.2fold), o-tubulin (8.7-fold), vimentin (22.5fold), 73kd heat shock protein, vinculin @.dfold), and phosphorylase B (18.2-fold). Similarly, three proteins were detected in stressed cells that were absent from the controls. Discussion
The increase in organic matrix seen in these osteoblast-like cells from chick calvaria is critical to the cascade of events that lead to the production of bone. Cells stressed for 24 hours had a nonsignificant increase of 10% in cellular collagen synthesis. Table 2. Synthesis of Known Osteoblast Proteins Affected by Cyclic Deformation
Calmodulin u-Tubulin Vimentin HSP 80 HSP 90 HSP 100 Actin HSP 73 Vinculin Phosphorylase B
kd
pI
Fold Change
Control (dpm/spot)
Stress (dprn/spot)
17 57.4 61 80 90 100 42 73 130
4 5.21 5.16 5.19 5.19 5.09 5.42 5.5 6.07
2.12* 8.7* 22.54 0.77* 0.063 0.500 5.62*
2,620 2,516 260 1,466 161 1,135 17,54030.1
5,827 + 21,861+ 5,838+ 1,1357235,055 2,787+ 169+
20
6.96
18.2*
55.5
1,009+
Results of the two-dimensional gel electrophoresis. change is represented as the fold increase or decrease. Abbreviations: kd, kilodalton; p1, isoelectic constant. *PC .05.
The
STRAIN ON OSTEOBLASTS
The amount of secreted noncollagen protein on day 1 was decreased by 20% and 19.5%, respectively. On day 3, synthesis of both noncollagen proteins and collagen showed an increase of 26% and 33%, respectively. Secretion at day 3 was increased for noncollagen proteins, but no significant change was noted in collagen secretion. Although much work must be done to substantiate the effects of mechanical strain on the rate of procollagen mRNA synthesis, translation, and posttranslational modifications, one can speculate that the initial decrease in secretion without a change in collagen synthesis could be attributed to alterations in posttranslational modifications and therefore delayed secretion.5 Similarly, although the cellular events involved in the transduction of this mechanical signal to alter DNA, RNA, or protein synthesis remain unknown, the increase in cellular collagenous and noncollagenous proteins seen at day 3 may be a transcriptional event. Further study is required before the mechanism can be elucidated. Activity of alkaline phosphatase, an established osteoblast phenotypic marker, increases in response to the mechanical signal. Although the role of alkaline phosphatase is yet be completely established, all evidence points to its importance in mineralization. One widely held view is that it provides an additional source of phosphate for mineralization by hydrolysis of organic phosphate.” An opposite view holds that alkaline phosphatase is present for the removal of inhibitors to mineralization such as diphosphonates.29*30 Our data show that an increase in alkaline phosphatase activity in the first 24 hours precedes the mineralization that occurs (data not shown). The elevation again relates the two events (alkaline phosphatase activity with mineralization) in a timely fashion. It does not, however, help us differentiate between the two possible roles of alkaline phosphatase.28-30 The protein profile of stressed cells compared with non-stress controls shows that 98 proteins that are present in the controls are absent from the stressed cells. This may be explained in the terms of luxury proteins or, put another way, proteins that are being produced at a basal level in noncommitted cells are turned off when the cell and its cellular functions are called on to perform a distinct function or respond to an outside element. Of the decreased synthesis in 112 proteins, the most striking result was the decrease in 80-kd, 90kd, and lOO-kd heat shock proteins. These “stress” proteins are of particular interest because they are induced by elevated temperature or thermal stress.3’ In the mechanical strain system, knowledge of the roles of 73-, BO-, 90-, and lOO-kd stress proteins may help distinguish their functions.” It
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may be that mechanical strain induces one heat shock protein which functions in a protective manner, while other stresses (ie, heat) invoke other protective mechanisms. One may speculate that the presence of three proteins in the stressed cells that are absent in the controls may, in light of the increase in growth rateI and induction of mineralization, be related to production of a growth factor or mineralization induction factor. Adhesion and spreading of cells on substrate is necessary for anchorage-dependent cells to grow in culture. Anchorage-dependent cells cultured in suspension can show a drastic inhibition of DNA, RNA, and protein synthesis. The exact parameters needed for cells to proliferate are unknown, but may be linked to focal contact proteins at the membrane level and the cytoskeleton.32 Vinculin, a 130kd focal contact protein, was increased 5.6-fold with this regimen of stress. These and other focal contact proteins play not only an obvious role in cell substrate interface and attachment but also a role in cell mitogenic function.3’ The cytoskeletal proteins vimentin, actin, and a-tubulin showed 22.5, OS-, and 8.7-fold increases, respectively, in strained cells. Recent evidence has strongly favored an interaction between matrix, cell receptors, and cytoskeleton.33 Direct evidence of a matrix-cytoskeletal connection has been provided by ultrastructural studies demonstrating transmembrane association of extracellular fibronectin fibers in intracellular actin filaments.34 The role of the cytoskeleton in modulating DNA synthesis, and hence gene expression, has been substantiated by a number of investigators.35-37 This increase in critical focal contact and cytoskeletal proteins to mechanical strain and the concurrent changes in phenotypic markers (ie, alkaline phosphatase, collagen and noncollagen proteins) gives further strength to the role of the cytoskeleton in modulating gene expression. Furthermore, the matrix-receptor-cytoskeleton may be the mechanism by which the signal from the external environment is transmitted in the cell and be the mechanism of Frost’s “Mechanostat Theory. “’ Summary Osteoblast-like cells, when subjected to mechanical stress, increase their synthesis of collagen and noncollagen proteins at day 3. On day 1, secretion of both proteins is decreased, whereas secretion of cellular proteins is unchanged. Alkaline phosphatase activity, when examined in UMR-I06 (rat osteosarcoma cells), showed a marked increase at 24 hours, which complements induction of mineral-
ization. The dimensional many luxury tal and focal
protein profile as determined by twogel electrophoresis shows a decrease in proteins and an increase in cytoskelecontact proteins.
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35. Zannetti NC, Solursh M: Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskelton. J Cell Biol99:115, 1984 36. Term M. Bartholomew JC. Bissell MJ: Svnereism between aitimicrotubule agents and growth stimulants in enhancement of cell cycle traverse. Nature 268:739, 1977 37. Friedkin M, Legg A, Rozergrot E: Antitubulin agents enhance the stimulation of DNA synthesis by polypeptide growth factors in 3T3 mouse fibroblasts. Proc Nat1 Acad Sci USA 76:3902, 1979 38. Wong G, Cohn DV: Formation of parathyroid hormone and calcitonin. Sensitive cells from responsive bone cells. Nature New Biol 252:713, 1974
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Discussion The Effects of Mechanical Strain on Osteoblasts In Vitro David L. Cochran,
DDS, MS, PhD, MMSc
Medical College of Virginia, Richmond
In the preceding article by Buckley, Banes, and Jordan, several effects of cyclic mechanical strain have been examined on cells derived from 4-week-old chick calvaria. These investigators point out that bone undergoes varied mechanical strain under many circumstances on a daily basis. They further stipulate that the consequence of such strain is directed at the cellular level, and they have investigated these effects on bone-derived cell cultures that should be enriched in osteoblast cells. These investigators have used a cyclic stress force on the cell cultures, and they indicate that cyclic strain may be very different from a constant stress or, for that matter, thermal or chemical stress. One must be constantly mindful that even in the most appropriate of all the mechanical stress model systems described for cultured cells, the investigations may not mimic in vivo stress. It is well documented that in vivo bone coupling occurs so that bone formation and bone resorption occur simultaneously. Because these are linked processes, analysis of only one aspect of this process without the influence of the other may lead to results not compatible with what occurs in vivo. Recent data’ suggest that transforming growth factor-beta may act as a coupling agent between bone formation and bone resorption, and may allow one to better define model systems that reflect in vivo conditions more accurately. In the study by Buckley et al, bone cells were derived from chick calvaria which were sequentially digested with collagenase and trypsin. This is a method used by several
laboratories and was originally described by Wong and Cohn.’ Buckley et al digested the calvaria three times, with the first two incubations lasting 20 minutes and the last one 4 hours. Many investigators digest calvaria live to six times for 10 to 20 minutes and combine the last two to three digestions to obtain osteoblast-enriched populations. Our experience has been that the purity of the collagenase and the length of time of each digestion are critical for the enrichment of specific cell populations. An absolute requirement in any of these preparative procedures is the characterization of the resultant cell cultures. Several common methods are available to characterize such cell populations: collagen phenotype; response to hormone treatment including PTH, vitamin D, and calcitonin; as well as the measurement of enzymatic activities, including cyclic AMP, alkaline phosphatase, and measurement of osteocalcin levels. Perhaps the best characterization of putative osteoblast-like cells is to determine if the cell cultures undergo mineralization. No data on mineralization are presented, yet the authors state in the summary that the increase in alkaline phosphatase activity in the transformed cell line complemented induction of mineralization. Without analysis of mineralization or data on the induction of mineralization, one can never be sure that the isolated cultures are functional osteoblast cells. Osteoblast-enriched cultures may mean that 20% of the cells are osteoblasts, or perhaps 70%; but the question remains, are the cells functionally osteoblast-like, and how much must the culture be enriched to have significant results for bone-forming cells? Because fibroblasts may be a contaminating cell type, control assays should be performed on fibroblast cultures for comparison, in addition to the biochemical and functional characterization of the osteoblast-like cultures.
