Martinez,. D. A., M. W. Orth, K. E. Cam, R. Vanderby,. Jr., and A. C. Vailas. Cortical bone growth and maturational changes in dwarf rats induced by recombinant ...
Cortical bone growth and maturational changes in dwarf rats induced by recombinant human growth hormone D. A. MARTINEZ, M. W. ORTH, K. E. CARR, R. VANDERBY, JR., AND A. C. VAILAS Biodynamics Laboratory, University of Wisconsin-Madison, Madison 53706, and Department of Orthopaedic Surgery, University of Wisconsin-Madison, Madison, Wisconsin 53792 Martinez, D. A., M. W. Orth, K. E. Cam, R. Vanderby, Jr., and A. C. Vailas. Cortical bone growth and maturational changes in dwarf rats induced by recombinant human growth hormone. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E51E59, 1996.-The growth hormone (GH)-deficient dwarf rat was used to investigate recombinant human (rh) GH-induced bone formation and to determine whether rhGH facilitates simultaneous increases in bone formation and bone maturation during rapid growth. Twenty dwarf rats, 37 days of age, were randomly assigned to dwarf plus rhGH (GH; n = 10) and dwarf plus vehicle (n = 10) groups. The GH group received 1.25 mg rhGHYkg body wt two times daily for 14 days. Biochemical, morphological, and X-ray diffraction measurements were performed on the femur middiaphysis. rhGH stimulated new bone growth in the GH group, as demonstrated by significant increases (P < 0.05) in longitudinal bone length (6%), middiaphyseal cross-sectional area (20%), and the amount of newly accreted bone collagen (28%) in the total pool of middiaphyseal bone collagen. Cortical bone density, mean hydroxyapatite crystal size, and the calcium and collagen contents (ug/mm”) were significantly smaller in the GH group (P < 0.05). Our findings suggest that the processes regulating new collagen accretion, bone collagen maturation, and mean hydroxyapatite crystal size may be independently regulated during rapid growth. collagen cross-links; hydroxyapatite; gen accretion; extracellular matrix
density;
porosity;
colla-
REGULATION OF BONE MASS is obtainedbybalancing the processes of bone formation and bone resorption. In the growing skeleton, higher bone turnover is associated with increased bone formation, due to the small amount of bone resorption during this time period (31). In young pituitary-deficient animals, cortical bone mass increases are significantly less than in normal young growing rodents (22, 27). However, bone mass can be augmented significantly by the administration of exogenous human (h) growth hormone (GH) in hypophysectomized rats (15, 27, 35). Therefore, the rapidly growing rat offers the opportunity for scientists to study specific processes associated with bone formation and the effects that these processes have on bone mass, density, morphology, and maturation. However, limitations exist when interpreting endocrine effects on bone metabolism obtained from the hypophysectomized rat model, due to the absence of abundant synergistic hormones, antagonistic hormones, and releasing hormones normally secreted by the hypothalamus. Because many pituitary hormones have an effect on the growth of bone, it is difficult to substantiate which hormones provide the primary stimulus for growth and maintenance of cortical bone during rapid growth. Therefore, an animal model that is selectively deficient THE
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Copyright
o 1996
in an anabolic hormone, such as GH, could be used to investigate GH-stimulated bone formation and to determine whether this pharmacological approach upregulates bone formation and bone maturation. Recently, a GH-deficient (dwarf) rat model has been developed with reduced pituitary GH levels that are ~5% of normal and with plasma levels of GH that are barely detectable (6, 8). Using this unique animal model, scientists can view the downregulation of skeletal and whole body metabolism due to GH insuEciency and, upon resupplementation with GH, can accelerate the rate at which new bone is formed. The interrelationships between rapid bone growth and bone maturation are not well understood. It has been suggested from earlier work in our laboratory on slowly growing hypophysectomized rats that cortical bone maturation is partially dependent on pituitary hormones (22). In contrast, accelerated bone growth through recombinant human (rh) GH supplementation may affect the processes of bone maturation, causing changes in the microorganization of new bone, and, due to the rapidity at which it is being formed, the new bone may be less optimal. Because GH is an anabolic agent and is being used in humans to augment longitudinal bone growth, it is important that scientists explore the organizational aspects of growing bone when challenged by an endocrine stimulus, such as GH. The primary purpose of this investigation is to use rhGH supplementation in a GH-deficient dwarf rat to analyze the changes in the extracellular matrix and inorganic mineral in cortical bone during growth. We will also investigate whether the maturation of cortical bone simultaneously increases at a time when there is an upregulation of cortical bone formation. MATERIALS
AND
METHODS
Animals Twenty male Lewis mutant dwarf rats were used in this study (n = 20 rats). The mutation, inherited as an autosomal recessive mutation, arose spontaneously in a breeding colony and is established at Simonsen Laboratories, South San Francisco, CA. The dwarf rats arrived at 30 days of age and initially weighed 57 IT 2 g. The rats were individually housed in a climate-controlled room (25°C 14:10-h light-dark cycle) in stainless steel cages (25 X 18 X 25 cm). The rats were allowed to acclimatize to vivarium conditions for 7 days and were given water and the American Institute of Nutrition’s AIN-76A diet for rats, ad libitum (Teklad, Madison, WI). The mineral mixture included a fixed amount of calcium (0.5%) and phosphorus (0.4%). Twenty rats were randomly assigned to the following experimental groups: dwarf plus vehicle (vehicle, n = 10) and dwarf plus rhGH (GH, n = 10). Biochemical, morphological, and X-ray diffraction analyses were performed on cortical bone samples procured from each the American
Physiological
Society
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GH EFFECTS
ON CORTICAL
BONE GROWTH AND MATURATION
animal in the study (n = 20). The rats were 37 days of age and averaged 83 t 2 g body wt at the beginning of the study. GH and Vehicle Administration Subcutaneous injections of rhGH (Genentech, South San Francisco, CA) and vehicle (bacteriostatic sterile water containing 0.9% benzyl alcohol) were given to the appropriate groups two times daily at 0730 and 1630. Bacteriostatic dH20 injections based on body weight were administered subcutaneously to the vehicle group. The GH group was given rhGH injections for 14 days totaling 2.5 mgekg body wttl=day-? The rhGH is best administered in two equal doses (1.25 mg/kg body wt SC) to simulate the pulsatile release that occurs in vivo (8, 15, 16). The dose of rhGH given per day ranged from 280 ug/day at the beginning of the experiment to 434 ug/day at the end of the experiment based on body weight gain. Death The rats were killed by exsanguination after 14 days. After sedation with pentobarbital sodium (50 mg/kg body wt), -3-5 ml whole blood were collected in a preheparinized syringe via cardiac puncture. The blood samples were immediately centrifuged in nalgene test tubes [room temperature, 2,500 revolutions/min (rpm), 20 min], and the plasma was removed and frozen at -70°C for future free proline and free [3H] proline analyses. Bone Morphology Length and thickness measurement. The length and thickness of each femur middiaphyseal cross section were measured to compare size differences between groups. Femur length was measured from the proximal end of the femoral head to the distal medial condyle. All measurements were obtained with a digital micrometer caliper. Each measurement was repeated three times, and the average value is reported. Morphological dimensions of the femur were obtained using video imaging and computer-enhanced digitizing programs described in an earlier publication from our laboratory (40). Middiaphyseal cross sections (1 and 2 mm) were cut orthogonal to the longitudinal axis of the bone on a microprecision lathe. Also, a 5-mm cross section was obtained from the opposite femur, stored at -70°C and was used to analyze femur mean hydroxyapatite (HAP) mineral crystal size. The computer tracings resulted in a measurement precision of to.001 mm and a coefficient of variation of 0.82%. The specific morphological data collected were total, cortical, and medullary cross-sectional areas, periosteal and medullary perimeters, and regional cortical thicknesses. Density determination. Cortical bone specimens were hydrated in a physiological buffer (50 mM potassium phosphate buffer, pH 7.4) for 24 h. Density was determined using Archimedes’ Principle. Weights for volume determination were measured using a density determination kit (Mettler Instruments, Highstown, NJ). Density was calculated by using the formula: density = (A/A - B) X P, where A is the weight of the hydrated bone out of water, B is the weight of the hydrated bone submerged in water, P is the density of distilled water of known temperature for each weighing period, and A - B is the difference in weight, equivalent to the weight of the volume of water displaced by the bone, which in turn is equivalent to the volume of the bone according to Archimedes’ Principle. This method and apparatus have been proven successful and have been published elsewhere (18).
Biochemistry Sample preparation. Small samples (4-6 mg) of the femoral middiaphysis were hydrolyzed in 6 M HCl for 24 h at 110” in vaccuo. Hydrolyzed samples were dried, redissolved in 1.0 ml dHBO, and filtered through a nylon 66 membrane, 0.45 urn pore size (Gelman Sciences, Ann Arbor, MI). An aliquot (300 ul) was redissolved in 0.4 ml of 1% n-heptafluorobutyric acid (HFBA; Pierce Chemical, Rockford, IL) and centrifuged for 5 min through a 1.5-ml microfilterfuge tube, 0.22 urn pore size (Rainin Instrument, Woburn, MA). The filtrate was then transferred to brown glass Wheaton autosampler vials and was immediately applied to the chromatography system for cross-link analysis. Nonreducible collagen cross-link analysis. The collagen cross-link analysis (hydroxylysylpyridinoline, HP; and lysylpyridinoline, LP) was performed by the ion-paired Cl8 reversed-phase high-pressure liquid chromatography (HPLC) method used by our laboratory (40). Elution of the cross-links was performed isocratically using 15% acetonitrile (EM Science, Gibbstown, NJ) in 0.01 M HFBA at 1.0 ml/min with a 16-min retention time. Standardization of our system was performed by applying a HP standard purified by our laboratory. The recovery was determined by applying a pure collagen source (bovine achilles tendon, Elastin Products, Owensville, MO) and was shown to be 99%. The samples were run in duplicate, and the average area under the peak was used for subsequent calculations. The collagen cross-links are expressed as mole cross-link per mole collagen, Calcium and phosphorus. Determination of femur middiaphyseal calcium was performed by atomic absorption spectrophotometry (Perkin-Elmer model 403). An aliquot (200 ul) from a dilution (1:40) from the original redissolved hydrolysate was added to 1.5 ml lanthanum hydrochloride stabilizing solution (1% lanthanum, 5% HCl, Sigma Chemicals, St. Louis, MO) and 1.3 ml dHzO. Samples were analyzed in triplicate, quantified to a relative known calcium standard (EM Science), and read at 424 nm. Phosphorus concentration was determined by taking an aliquot (100 ul) out of a dilution (1:40) from the original l-mm hydrolysate. Phosphorus samples were measured in triplicate according to previous colormetric methods described by our laboratory (22) and were read at 660 nm. Hydroxyproline and proline determination. Amino acid analyses of hydroxyproline and proline were performed using the precolumn derivatizing Edman reagent phenylisothiocyanate (PITC; Pierce Chemicals, Rockford, IL) before analysis according to Vailas et al. (40). Hydroxyproline is a specific marker for all phenotypes of collagen, representing 14% of collagen’s mass. An aliquot (400 ul) was taken out of the original redissolved hydrolysate and was evaporated to dryness. PITC derivatized amino acid samples were eluted isocratically (140 mM sodium acetate-tetraethylammoniumEDTA buffer solution) at a l.O-ml/min flow rate using a 10 cm X 4.6 mm Cl8 PICO-TAG column (Waters, Milford, MA) and were monitored on a Waters model 490 absorbance detector at 254 nm. The retention times for hydroxyproline and proline were 2.6 and 8.0 min, respectively. Amino acid chromatograms were quantified relative to a known amount of derivatized hydroxyproline and proline (Sigma Chemicals) standard, and the recovery for the amino acid analyses was 95%. New collagen accretion determination. The deposition of newly synthesized collagen in cortical bone, a metabolic index of bone formation, was measured by the in vivo single-pulse labeling methods previously published (10). This method determines the relative amount of [“HI hydroxyproline incor-
GH EFFECTS
ON CORTICAL
BONE GROWTH AND MATURATION
poration into insoluble bone collagen. This method requires a time lag after a single pulse injection of radiolabeled proline to guarantee systemic extracellular equilibration and tissue deposition into newly synthesized insoluble and soluble collagen, measured by the specific activity of hydroxyproline (10, 25, 29). Measurement of newly labeled collagen accumulated in the extracellular matrix reflects an increase in the deposition of new insoluble collagen independent of intracellular collagen biosynthesis. Proline pools are always maintained by the animal’s endogenous proline sources. Osteoblasts and osteocytes are uniformly supplied with an adequate amount of proline, and fluctuations in the systemic extracellular compartment of proline does not affect the deposition rate of newly secreted collagen (21, 29). A single pulse of L-[2,3,4, 5-3H]proline (sp act = 103 Ci/mmol; 1.0 uCi/g body wt; Amersham, Arlington Heights, IL) was administered intraperitoneally 72 h before death. To obtain a pure bone collagen source, the pieces of cortical bone were demineralized for 24 h in a diluted HCl solution and were agitated every 6 h to ensure complete saturation and mixing. Samples were rinsed with dH20 and lyophilized again to obtain a demineralized dry weight. Sample preparation and amino acid analyses were performed using previously stated amino acid methods, with a few modifications. Fractions (1.0 ml) of effluent containing the [3H] hydroxyproline and [“Hlproline amino acids were collected and mixed with 18 ml scintillation cocktail (Ecoscint, ICN Biochemicals, Irvine, CA), adapted to the dark for 24 h, and counted for 100.0 min in a static scintillation counter. The counts per minute for each sample were converted to disintegrations per minute per micromole hydroxyproline and disintegrations per minute per micromole proline, using the hydroxyproline and proline values determined from HPLC amino acid analysis. [3H]hydroxyproline incorporation was used to evaluate new bone collagen deposition. Plasma free proline and free [3H]proline. At the time of death, blood samples were taken to measure free proline and free [“Hlproline levels to ensure that both groups of animals had the same systemic access to labeled and unlabeled proline. An aliquot (1.0 ml) of rat plasma was added to 1.0 ml 24% trichloroacetic acid, vortexed, and spun for 20 min at 2,500 rpm to remove proline-bound proteins. The amount of supernatant recovered (1.6 ml), containing the free proline and free [3H]proline amino acids, was frozen at -70°C for future analysis. The supernatant was thawed, and an aliquot (800 ul) of supernatant containing the free labeled and unlabeled proline was dried and prepared using amino acid sample preparations previously mentioned. Samples were applied to a solid-phase extraction medium (Sep-Pak Light, Millipore Waters) and derivatized, resuspended in 400 ul of sample diluent, and filtered. A loo-u1 aliquot of the prepared plasma was applied to the HPLC. The free amino acids were separated and quantified relative to known proline standard (Sigma Chemical). The proline recovery from this procedure was 95%. The effluent from the detector was collected (1.0 ml/test tube) and mixed with 18.0 ml scintillation cocktail (Ecoscint). Vials containing labeled proline were counted for 100.0 min in a static scintillation counter (Pharmacia LKB Nuclear, Gaithersburg, MD). Counting efficiency for 3H on our system was 60.3%. X-ray powder diffraction. Middiaphyseal cortical bone pieces (5 mm) from left femurs were cleaned of muscle tissue and bone marrow and were immediately stored at -70°C. Each bone specimen was lyophilized (20 h) and then powdered using a Spex freezer mill (Spex Industries, Metuchen, NJ) for 5 min. The bone powder was transferred to nalgene cryotubes and was stored at -70°C for powder analysis. Cortical bone
E53
X-ray diffraction procedures were performed according to the method of Grynpas et al. (12). Samples of powdered cortical bone were analyzed on a Nicolet 12N X-ray powder diffractometer equipped with a diffracted beam graphite monochrometer using CuKa (copper, K alpha) radiation. Highly crystalline fluorapatite was used as a standard. The crystallinity of the HAP can be determined by measuring the crystal length along its c-axis, as measured by the 002 X-ray diffraction peak, and the crystal width and thickness, as measured by the 130 X-ray diffraction peak. The values of B1,Z(002) and B1,2(1301,and the width at one-half the maximum height of the HAPoo2 and HAP130 reflections were measured using a step scanning procedure with increments of 0.04” over each peak. Each sample was done in duplicate. The measured halfwidths were corrected for instrumental broadening by subtracting the square of p 002 and p13o for fluorapatite from the square of the bone values and taking the square root of the difference. D 002 and D130, which are related to crystal size and perfection in the long and cross-dimensions of the apatite crystal, were calculated from the corrected Poo2 and (313o values (p1i2) using the Scherrer equation (20) D=
KX radian P l/2
case
where X is the X-ray wavelength, 131/zis the breadth at half the height of the 002 and 130 peaks, and 8 the diffraction angle for a given reflection (002 = 25”, 130 = 39”). K is a constant that varies with crystal habit and was chosen as 0.9 for the elongated crystals of bone. Statistical
Analysis
An unpaired two-tailed Student’s t-test (SAS, SAS Analysis System, Cary, NC) was used to detect differences between groups when viewing biochemical, morphological, and X-ray diffraction data. Statistical significance was set at the P < 0.05 probability level. RESULTS
Body Weight Changes Daily body weight gains are shown in Fig. 1. The two groups of rats, rhGH group and vehicle group (37 days), showed no significant differences in mean body mass (83 t 2 g) at the beginning of the experiment (day 0). By the 2nd day of injections, the rhGH group was significantly larger (P -=c0.05) than the vehicle group. The final body mass was significantly greater (35%) in the rhGH group vs. the vehicle group after 14 days of rhGH supplementation (P < 0.05). The average body mass gain per day for the 14-day period was 7.0 g/day for the rhGH group and 3.7 g/day for the vehicle group. Bone Morphology The dwarf animals receiving rhGH showed significant alterations in long bone growth and in radial width (Table 1). There was a significant increase (P < 0.05) in the femur length in the rhGH group, resulting in a 1,500~urn longer bone compared with the vehicle group. The total cross-sectional area and periosteal perimeter of the rhGH group middiaphysis were significantly larger (P < 0.05) compared with vehicle controls. The cortical cross-sectional area was 20% larger (P < 0.05) in the rhGH group, representing a 37.24~um2/day
E54
GH
EFFECTS
ON
CORTICAL
200
BONE
GROWTH
AND
MATURATION
181 3-5 *
* 2640 zk 136 2750
160 2500 2056 & 162
2250 Start
of rhGH
2000 80 1750 14 0 Vehicle Animal
Age (days)
Fig. 1. Growth curves for male dwarf rats receiving recombinant human growth hormone (rhGH) and vehicle. Male dwarf rats (37 days old) received either rhGH treatment (dwarf + hGH; 0) or vehicle weights in dwarf + rhGH (dwarf-tvehicle; a> for 14 days. *Body group were significantly larger than in dwarf + vehicle group 2 days after beginning injections, and body weights continued to be significantly larger until end of experiment (P < 0.05).
rhGH
Fig. 2. Femur middiaphyseal new collagen accretion. Amount of new bone collagen in middiaphyseal cross section is expressed as specific activity of hydroxyproline [disintegrations min 1 (dpm) umol Hyp-i] . Values represent means + SE. *Dwarf + rhGH (rhGH) group is significantly larger than dwarf + vehicle (vehicle) group (P < 0.05). l
l
lar matrix. Plasma levels of free proline (ug/ml; rhGH group 15.98 5 1.15 vs. vehicle group 16.87 t 2.90) and free [“HI proline (disintegrations. mini umolll proline; rhGH group 4,465 t 297 vs. vehicle group 6,225 t 1,187) were not statistically different 72 h after the administration of the radiolabeled proline, indicating that both groups had an adequate supply of precursor labeled and unlabeled proline within their bloodstream. l
greater rate of radial growth than the vehicle group. The medullary cross-sectional area and the medullary perimeter showed no significant differences from the vehicle control group. Regional thicknesses were 3-37% larger in the rhGH group in contrast to vehicle controls. The medial thickness in the rhGH group was significantly larger (37%; P < 0.05); however, nonsignificant increases in thickness were noted in the lateral (ll%), anterior (3%), and posterior (9%) regions of the middiaphyseal cross section (P > 0.05). Cortical Bone New Collagen Accretion and Plasma Levels of Labeled and Unlabeled Proline In Fig. 2, the amount of newly accreted collagen, as shown by the specific activity of [3H]hydroxyproline in the femur middiaphysis, was significantly greater (P < 0.05) in the rhGH group compared with the vehicle controls. This represents a 28% increase in the amount of new collagen deposited into the insoluble extracelluTable 1. Femur length and middiaphyseal cross-sectional morphology
Left femur length, mm Total cross-sectional area, mm2 Cortical cross-sectional area, mm2 Medullary cross-sectional area, mm2 Periosteal perimeter, mm Medullary perimeter, mm Anterior thickness, mm Posterior thickness, mm Medial thickness, mm Lateral thickness, mm
Dwarf
+ Vehicle
Dwarf
+ rhGH
26.39 6.02 2.66 3.36 9.39 7.60 0.299 0.453 0.435 0.311
k 0.19 + 0.11 t 0.07 t 0.09 t 0.10 -+ 0.14 t 0.009 + 0.033 + 0.025 t 0.017
27.89 6.68 3.18 3.49 10.18 7.55 0.309” 0.494 0.598 0.344
t 0.18* 2 0.16* 7fI 0.10* ‘-t 0.09 + 0.18* + 0.16 0.007 + 0.027 t 0.046* + 0.010
Values represent means t SE. *Dwarf growth hormone (rhGH) group is significantly vehicle group (P < 0.05).
+ recombinant different from
human dwarf +
Matrix
Biochemistry and Bone Density
Cortical bone composition measurements are found in Table 2. Total contents (ug) of calcium, phosphorus, and collagen were calculated per unit volume of bone (mm”). Results indicate that calcium content and collagen content (reflecting the total pool of new and old collagen) per unit volume of bone were significantly smaller in the rhGH group (P < 0.05). There was no significant difference in the content of phosphorus per unit volume in the cortical bone. Total mineral density, estimated by the summation of calcium and phosphorus content, was 5% less in the rhGH group but was not statistically significant. The calcium-to-collagen ratio, an index of the amount of mineralized femoral bone collagen, showed no significant difference between Table 2. Femur middiaphyseal
biochemistry
Dwarf
Total collagen content, ug/mm3 Total calcium content, ug/mm3 Total phosphorus content, &mm3 Calcium-to-collagen ratio Total mineral density, ug/mm3 Cortical bone density, mg/mm3
+ Vehicle
262.64 + 3.88 434.76 + 7.67 327.38 + 9.83 1.67 + 0.02 762.14 + 15.67 1.776 ‘-t 0.01
Dwarf
235.39 413.34 313.56 1.77 726.915 1.734
+ rhGH
Ih 7.46* 2 6.15* 2 9.07 ? 0.05 12.48 5 0.01*
Values are means + SE. Collagen and mineral contents are expressed as mass of material (ug) per unit volume of bone (mm3>. Total mineral density was estimated by summation of total calcium and total phosphorus. *Dwarf + rhGH group is significantly different from dwarf + vehicle group (P < 0.05).
GH
Table 3. Femur
middiaphyseal
HP content, mole HP/mole LP content, mole LP/mole Total pyridinoline content, cross-link/mole collagen Values dinoline. significant
EFFECTS
collagen collagen mole
ON
collagen
CORTICAL
BONE
cross-links
Dwarf
+ Vehicle
Dwarf
+ rhGH
0.163 0.210
+ 0.005 + 0.009
0.165 0.205
t 0.006 + 0.011
0.374
t 0.012
0.370
5 0.016
are means ‘r SE. HP, hydroxylysylpyridinoline; LP, lysylpyriTotal pyridinoline content is sum of HP + LP. There were no differences between groups (P > 0.05).
groups. The specific gravity of cortical bone was analyzed to document the effects of rhGH supplementation on bone mass. The middiaphyseal bone density (Table 2) was significantly smaller in the rhGH group in contrast to vehicle controls (P < 0.05). The maturation of the extracellular matrix in bone was analyzed by measuring the nonreducible collagen cross-links. As shown in Table 3, the contents of the HP cross-link, the LP cross-link, and total cross-links (HP+LP) did not differ between rhGH and vehicle groups. HAP Mean Mineral Crystal Size The results for the X-ray diffraction analysis of the cortical bone powder are found in Table 4. The bone powder scans of the DO02and DISOreflections demonstrate a significantly smaller (P < 0.05) mean crystal size and perfection, indicating a decrease in the crystallinity of the HAP mineral in the rhGH group in contrast to the vehicle controls. The integral width of the diffraction peak (p) is smaller in the vehicle group, which results in a sharper peak, signifying an improved crystallinity as shown by the l&/Z values in Table 4. DISCUSSIBN
Bone growth. A main objective of this study was to use exogenous rhGH treatments to accelerate the processes of osteogenesis in the slowly growing dwarf rat. Precise measures of longitudinal bone growth and middiaphyseal radial width have proven to be good indexes of increased bone growth. The results from this study show that dwarf rat bone was responsive to the 14-day rhGH treatment. Longitudinal bone growth of the femur was significantly greater in the rhGHtreated dwarf rats. These data are in agreement with experiments studying the direct or indirect actions of GH on bone growth in hypophysectomized rats (27,34), dwarf mice (Z), and dwarf rats (9). The exact mechanisms by which GH influences bone elongation are Table 4. Hydroxyapatite Crystallite
Size
mean mineral crystal size
Dwarf
+ Vehicle
Dwarf
+ rhGH
Peak
Do02
A P1/2(002)
125.94 + 3.75 0.657
114.67 + 3.28* 0.715
65.115 2.42 1.380
57.64 ‘-t 2.04* 1.433
D130 Peak
A
h2(130)
Values represent means rhGH group is significantly
(PcO.05).
t SE. See text for definitions. *Dwarf + different from dwarf + vehicle group
GROWTH
AND
MATURATION
E55
unclear. However, GH has been shown to bind directly to GH receptors (3, 26) and influence bone growth indirectly by local and systemic insulin-like growth factors (IGF) I and II (14). In addition to the elongation of bone, growth occurs simultaneously at the middiaphysis. The increase in the middiaphyseal bone crosssectional width occurs through the process of intramembranous-like bone formation. New bone is formed through the production of bone matrix at some surfaces and resorption of that matrix at other sites without a cartilage precursor. In the rapidly growing rat, the net result is bone gain due to a disassociation between osteoblast matrix production and osteoclastic resorption, favoring matrix production. The femoral total cross-sectional area and cortical cross-sectional areas were significantly larger after treatment in the GH group compared with vehicle controls. In addition, the periosteal perimeter was significantly larger in the rhGH group with no changes in the medullary (endosteal) cross-sectional area or medullary perimeter, suggesting that new bone was formed through direct periosteal apposition. This is in agreement with GHtreated animals in tetracycline labeling experiments (1, 13) and by direct morphometry (17). Harris and colleagues (13) injected hGH for 12 wk into adult dogs and found an increase in tibia1 bone mass, increased periosteal bone apposition, and a decrease in endosteal resorption. Female rats (75-95 days) given hGH injections for 20 days had increases in periosteal bone formation and significantly reduced endosteal growth in femoral cortical bone compared with controls (1). Jorgensen (17) injected female rats (42-49 days) with hGH for 90 days and demonstrated significantly larger periosteal diameters and no changes in the endosteal perimeter in the tibia and femur diaphysis vs. controls. These studies confirm our findings of bone apposition at the middiaphysis and suggest that there may be local and regional differences in the cellular response to the systemic administration of rhGH in rats. Cortical bone thicknesses measured regionally indicated a significantly greater thickness in the medial region in the rhGH group. All other regions were larger (anterior 3%, posterior 9%, lateral 11%) in the rhGH group compared with vehicle controls but were not statistically significant. However, it remains unclear whether rhGH supplementation alters the specific regional bone turnover of growing bone. In normal growing rats, it has been shown that the femur grows in length and diameter but also has a transversal drift of the midshaft geometries (22,37). This transversal drift is greatest in the middle of the femur and decreases in the direction of the distal end of the femur (37). Another explanation for the altered distribution of cortical mass may be reflected by the alterations in muscle mass and/or activation suggested by Vailas et al. (39), which could affect the periosteal surface, causing localized changes in bone formation. Further work is needed regarding the distribution of cortical mass and the influence that load, in combination with hormone supplementation, has upon regional geometries.
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GH EFFECTS
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BONE GROWTH AND MATURATION
Bone composition. There are numerous factors that influence cortical bone density such as the amount of inorganic mineral, the amount of organic matrix accretion, and how the bone mass is distributed in a given volume of bone. Cortical bone densities in the rat have been shown to range from 1.4 to 1.6 mg/mm3 in immature rats (22) and to 2.24 mg/mm3 in adult rats (4). The fern oral cortical bone density was significantly less in the rhGH-treated group compared with the vehicle controls, suggesting that GH has less of an initial effect on increasing bone density. Similar findings also have been reported by J$rgensen (17), who showed an 11% decrease in femoral cortical bone density in rats treated with hGH for 90 days. In contrast, downregulated bone growth in young slowly growing hypophysectomized rats exhibited a 16% increase in cortical bone density compared with normal growing rats of the same age, suggesting that pituitary hormones may control normal tissue turnover rates, thus influencing bone density (22). In a recent study, female Lewis dwarf rats receiving 200 ug rhGWday for 4 wk showed an increase in total bone mineral density (whole body trabecular and cortical bone) measured by dual-energy X-ray absorptiometry and an increase in trabecular bone volume (41). In our study, the rhGH middiaphyseal bone was smaller in mass and larger in circumference compared with the vehicle group. Our study suggests that acute rhGH supplementation may initially involve changes in the microorganization of cortical bone, which resulted in a decreased cortical bone density, but also could reflect changes in other structural elements such as increased cortical bone porosity (personal communication). Future studies on longer-duration GH supplementation and its effects on cortical bone density are needed. Many of the experiments concerning bone growth or GH-stimulated bone growth have used hydroxyproline concentration as an index of collagen concentration. Bone collagen mass in the rat increases linearly from birth to 8 wk of age, as demonstrated by an increasing concentration of hydroxyproline during growth (42). This increase in collagen mass is thought to be a function of a high turnover of calcified type I collagen (19). Other experiments involving exogenous GH supplementation have shown a varied response in bone collagen concentration. Shaar et al. (35) showed an increase in the femoral hydroxyproline concentration after 7 days of hGH treatment in young hypophysectomized rats. Ninety days of hGH treatment at different doses (0.16-8.33 mg/day) did not show an effect on femoral collagen concentration in growing rats (17). In a previous study from our laboratory, growing intact rats had a smaller total collagen content in a given volume of bone vs. age-matched hypophysectomized rats (22). In this study, the total collagen content in the femur middiaphysis was significantly smaller in the rhGH group after 14 days of rhGH supplementation. The mechanism for a decreased collagen content in the GH-supplemented animals has yet to be elucidated. It is proposed that an altered hormonal state may in-
crease collagen deposition (22, 28). The regulation of the extracellular matrix during growth and development requires the coordination of systems controlling accretion and resorption of collagen to maintain normal physiological turnover. Dwarfism in the rat has been shown to downregulate musculoskeletal metabolism (9, 36) caused by a primary reduction in the endogenous levels of circulating GH. GH insufficiency in dwarfism may reduce the adaptive response of cortical bone by decreasing the collagen turnover rate, as shown in pituitary-deficient hypophysectomized rats (22). In the growing rat skeleton, another factor contributing to changes in bone mass is the simultaneous change in inorganic mineral chemistry with increasing age. Newly formed HAP has a low calcium-to-phosphorus ratio, low carbonate content, and high hydrogen phosphate content, as well as high content of bound water and a very poor X-ray diffraction, revealing a very small mean crystal size (7). X-ray diffraction, infrared spectroscopy, and chemical analysis have revealed that the mineral component of bone is similar to that of ideal HAP Ca10(P0&(OH)2 (4,5). It has been shown in many bone growth/aging studies that biological HAP stoichiometry approaches that of ideal HAP, and the bone mineral crystal size increases while the strain (atomic ordering and arrangement) becomes increasingly organized during aging and maturation in calcified tissue (5, 24, 32). It is thought that an improvement in crystallinity constitutes crystal growth and/or an improvement in the crystal strain (32). In this experiment, the mean HAP crystallite size in the rhGH femur middiaphysis was significantly smaller compared with vehicle controls. To date, this is the first study that has investigated rhGH-stimulated bone growth and the effect of accelerated growth on mean HAP mineral crystal size. The current literature is sparse with regard to the regulation of HAP crystal size during rapid bone growth. Crystallinity seems to be a sensitive indicator of bone mineral maturation, and changes are more apparent in younger bone material. In the rat bone, the calcium-to-phosphorus ratio and the calcium and phosphate content, density, percentage of mineral, and the relative amount of collagen extracellular matrix increased with age, whereas the HAP X-ray diffraction pattern showed an increasing integrated intensity and a decreasing peak, broadening in rats 4-22 wk of age (5). It has also been shown in rats from 2 wk to 1 yr of age that the crystal formation process with age includes a decrease in lattice imperfections and an increased crystal size (24). Our results suggest that accelerated bone growth may have influenced the mean HAP crystal size in the rhGH group. rhGH treatment in this experiment produced no significant differences in the biochemical determinants of mineralization. Calcium and phosphorus contents in the present study corroborate findings of earlier studies that GH does not affect the processes of mineralization (17, 22). Downregulated cortical bone growth in rats induced by hypophysectomy (22) and 90 days of hGH supplementation given to normal growing rats
GH EFFECTS
ON CORTICAL
BONE GROWTH AND MATURATION
(17) did not change the mineral concentrations or ash weight of femoral cortical bone. The present data show that the calcium-to-collagen ratio, an index of the amount of mineralized collagen, was not significantly different after rhGH treatment, thus supporting this observation. The microorganization of mineral and matrix shows that the calcium per unit volume and total mineral density, estimated by the summation of calcium and phosphorus contents, and the total collagen content (new collagen + old collagen) were significantly smaller in the rhGH-treated animals. Similar mineral density findings were previously shown in normal growing intact rats vs. hypophysectomized rats from our laboratory (22). This reaffirms the hypothesis that rhGH may n.ot infl uence the precesses of mineralization in cortical bone. Bone collagen accretion and maturation. The extracellular domain of collagen is a function of collagen secretion and accumulation (accretion) compared with collagen resorption (extracellular degradation). Secreted collagen deposited in a growing matrix is suggested to be deposited until the death of the animal, and the fraction of newly accreted collagen that is resorbed during this time span is very small (21). Increases in the amount of extracellular collagen would be most likely explained by increased exocytosis of precursor protein, either due to increased numbers of collagen-producing cells or to increased synthesis per cell (21). Our study provides direct in vivo evidence that rhGH stimulates new bone collagen accretion using [3H]hydroxyproline as an indicator. The evidence for GH-stimulated increases in collagen production are scant. The short-term effect of exogenous GH on in vivo tibia1 bone total protein synthesis was shown 40 min after the administration of a single dose (60 ug) of rat GH (23). Immature hypophysectomized rats were given rat GH and demonstrated an in vivo increase in the radiolabeled incorporation of [“HI thymidine and [3H]proline, both indirect markers for cell proliferation and intracellular collagen synthesis (34). Wright and colleagues (41) measured serum alkaline phosphatase (AP), a marker for bone formation and serum IGF-I that is also known to potentiate bone growth through paracrine and endocrine pathways, in female dwarf rats receiving rhGH for 4 wk. Their results showed significant elevations in serum levels of AP and IGF-I compared with dwarf controls, suggesting that rhGH treatment may directly or indirectly influence bone matrix formation (41). The mechanism by which GH directly stimulates matrix production and matrix secretion is unclear; however, it is suggested that GH may act through GH receptors located on the osteoblast cell surface (26,33) or indirectly through stimulation of the local production of IGF-I by osteoblasts, which can upregulate cell function (38). The final analyses of this study involved the interrelationship between bone collagen maturation and bone growth induce d by rhGH supple ment ‘ation. Mature intermo lecular and intramol .ecular cross-links function to stabilize the molecular matrices of the collagen
E57
fibrils by increasing the tissue’s mechanical tensile strength, increasing the tissue’s resistance to proteolytic resorption, decreasing the tissue’s solubility, and providing proper spacing of the bone collagen a-chains for normal physiological function (l&28). The nonreducible mature trifunctional cross-link HP is found in many tissue types containing different collagen phenotypes, with the exception of skin collagen (11). The LP cross-link has been found in significant amounts only in calcified tissues, such as bone, dentine, and hypertrophic calcifying cartilage (11, 30). HP and LP have been reported to be regulated by a number of posttranslational modifications and tissue turnover rates and are thought to reflect the physiological age of the collagen fibrils rather than the biological age of the animal (11, 22, 28, 30). In mineralized tissue, the impact of pituitary hormone modulation on cortical bone maturation in hypophysectomized rats was studied in our laboratory. The data indicate that norm al growth and maturation of collagen in cortical bone (HP cross-link content) are dependent on pituitary hormones (22). These data also suggest that hormonal mechanisms associated with a suppression of bone growth may enhance the probability of HP cross-link formation. Unlike hypophysectomized rats, dwarf rats have an intact hypothalamus and possess hormonal modulators that are known to regulate connective tissue metabolism (e.g., thyroidstimulating hormone and glucocorticoids). Our present study shows that accelerated bone formation in the rhGH-treated dwarf rats did not alter the content of the mature HP and LP cross-links in femoral cortical bone vs. vehicle-treated dwarfs. These results suggest that rhGH does not enhance bone collagen maturation. At this time, it is unclear whether the presence or absence of other endogenous humoral factors specifically impact bone collagen maturation. Future in vivo studies isolating known modulators of musculoskeletal metabolism, such as pituitary hormones and/or growth factors, may provide more information on the maintenance and regulation of extracellular matrix maturation in bone. In summary, our experiments have shown that rhGH stimulated cortical bone growth at the middiaphysis and increased the total femur length. The increased specific activity of collagen in the rhGH-treated animals provides evidence that there may be a direct relationship between new collagen accretion and the organization of the extracellular matrix milieu. The smaller cortical bone density and the larger bone volumes (greater periosteal circumference) in the rhGHtreated animals suggest that the processes regulating new bone formation may also impact other structural elements such as porosity. rhGH did not facilitate the simultaneous acceleration of new collagen accretion, mature cross-link formation, and enlargement of the mean HAP min era1 tryst #als, suggesting that maturational processes in young growing cortical bon e are not simultaneously regulated over time. GH-deficient dwarf rats supplemented with rhGH produce new bone initially; however, the bone quality may be altered due to the observed decreases in density, mean HAP size, and
E58
GH
EFFECTS
ON
CORTICAL
BONE
the decreased amount of calcium and collagen per unit volume, which may result in an increased bone compliance. Our results have further implications with regard to exogenous GH treatments and bone growth .. In the acute phase, GH treatment sti .mulates new bone growth, but a subsequent lag period (no treatment) may be required before the next series of GH treatments to facilitate the accretion of new bone matrix, which would increase cortical bone density. The recombinant human growth hormone and dwarf male rats were supplied by Drs. Mike Cronin and Ross Clark, Genentech, South San Francisco, CA. We are grateful to Dr. Doug Powell, Department of Chemistry, Univ. Wisconsin-Madison for technical expertise related to the X-ray diffraction measurements and to Dr. Michael J. Zuscik for critiquing this manuscript. Funding for this research was provided through National Aeronautics and Space Administration Grant NAG2-568. Address for reprint requests: D. A. Martinez, Univ. of Rochester School of Medicine and Dentistry, Department of Orthopaedics, Musculoskeletal Research Unit, 601 Elmwood Ave., Box 665, Rochester, NY 14642. Received
8 March
1995;
accepted
in final
form
7 August
1995.
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