The biphasic response of porcine tendon to recombinant porcine growth hormone

The biphasic response of porcine tendon to recombinant porcine growth hormone

Growth Hormone & IGF Research 15 (2005) 39–46 The biphasic response of porcine tendon to recombinant porcine growth horm...

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Growth Hormone & IGF Research 15 (2005) 39–46

The biphasic response of porcine tendon to recombinant porcine growth hormone Valerie E. Choy a, Antonios Kyparos b, Arthur C. Vailas b, Thomas D. Crenshaw c, Daniel A. Martinez b,* a

Biodynamics Laboratory, Department of Kinesiology, University of Wisconsin-Madison, Madison, WI 53706, USA b Connective Tissue Physiology Laboratory, Department of Biology and Biochemistry, University of Houston, 369 Science and Research Bldg II, Houston, TX 77204-5001, USA c Department of Animal Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA Received 7 July 2004; revised 12 November 2004; accepted 23 November 2004

Abstract The purpose of this study was to investigate the effect of recombinant porcine growth hormone (rpGH) administration on the growth and maturational changes of the calcanean tendon in male pigs. Twenty-four orchidectomized crossbred (Duroc X Large White X Landrace) pigs were randomly assigned to 2 months of rpGH-treatment (2mo-rpGH), 3 months of rpGH-treatment (3mo-rpGH), or saline-treated control (Control) groups. Saline or rpGH (10 mg/mL given as a constant dose throughout the experiment) was administered twice weekly via 1 mL intramuscular injections. Following the 2mo-rpGH treatment, tendon concentrations of proteoglycan (uronic acid) significantly decreased, non-reducible collagen cross-link content (HP) significantly increased, and hydroxyproline (Hyp) concentrations remained unchanged, with a concomitant significant increase in tendon DNA concentrations, suggesting an up-regulation of cell proliferation. In the 3mo-rpGH treated animals, a decrease in tendon DNA concentration, an increase in proteoglycan and hydroxyproline concentrations, as well as a decrease in HP cross-links were found, suggesting accretion and differentiation of the extracellular matrix components. These findings support the idea that calcanean tendon responds temporally to rpGH treatment, affecting both cell division and tendon metabolism. Responsiveness of the tendon collagen to rpGH may be influenced by the onset and/or the duration of the exogenous growth hormone treatment. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Recombinant porcine somatotropin; Tendon; Extracellular matrix; DNA; Pigs

1. Introduction Tendon, a dense fibrous connective tissue that connects bone and muscle, plays a crucial role in skeletal tissue homeostasis and muscle force transformation. The ability of the tendon to adapt as a result of local and systemic factors is important to maintain normal function. Although the structure of the tendon and its extracellu*

Corresponding author. Tel.: +1 713 743 2662; fax: +1 713 743 2636. E-mail address: [email protected] (D.A. Martinez). 1096-6374/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ghir.2004.11.003

lar matrix components have been well characterized [1,2], research on the role of growth hormone (GH) on tendon metabolism is scarce. Recently our laboratory explored the anabolic actions GH has on the extracellular matrix of ligament and tendon from mutant dwarf rats [3]. Knowledge of the GH effects on tendon metabolism may increase our understanding in tendon adaptation and wound healing responses, which may prove useful as countermeasures to offset the atrophic effects during injury repair. Findings from studies using animal models [4] and in vitro systems [5,6] have shown GH and insulin-like


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growth factor I (IGF-I) play substantial roles in regulating collagen synthesis and metabolism. Chronic administration of GH to young pigs resulted in increased protein accretion and decreased fat deposition [7,8]. Growing pigs treated with porcine somatotropin for two months had enhanced collagen deposition in the skin, head and viscera, whereas non-collagen protein deposition and collagen maturation were increased in the carcass [8]. These findings suggest that GH administration has differential effects on various components of the body, resulting primarily in either new soluble collagen or the deposition of insoluble collagen depending upon the anatomical location. Time, frequency and duration of the treatment may be factors in which GH administration affects protein metabolism. Whereas protein accretion in GH-treated pigs has been well established, little information is available with respect to the effect of GH on collagen metabolism and much less regarding the underlying regulatory mechanism(s) of tendon growth and maturation. Therefore, the objective of the present study was to investigate effects of recombinant porcine growth hormone (rpGH) on growth and maturational changes of the calcanean tendon in male pigs. More specifically, we examined the tendon extracellular matrix and cell density alterations as well as the responsiveness of the calcanean tendon to different time courses of GH administration.

2. Materials and methods 2.1. Animals and treatments All experimental procedures were in accordance with institutional and national guidelines for animal experiments. A total of twenty four (n = 24) orchidectomized crossbred (Duroc X Large White X Landrace) pigs were used in the study. The animals, housed at the University of Wisconsin Swine Research Center, had free access to a standard swine diet and water throughout the experiment. The recombinant porcine growth hormone used in the study (rpGH) was obtained from Pitman-Moore, Inc. (Terre Haute, IN). The rpGH solutions were diluted to 10 mg/mL with carbonate buffer (0.025 M NaHCO3

and 0.025 M Na2CO3) and frozen. The carbonate buffer was handled in the same way as the rpGH solutions and was used as the injection vehicle for the saline-treated control groups. Vials for each day of injections were allowed to thaw at 4 °C before injections. The pigs were randomly assigned to three treatment groups as follows: (a) saline-treated control group (Control); (b) recombinant porcine growth hormone (rpGH)-treated for 2 months group (2mo-rpGH); and (c) recombinant porcine growth hormone (rpGH)-treated for 3 months group (3mo-rpGH) as shown in detail in Table 1. Saline or rp GH (10 mg/mL) was administered via 1mL intramuscular injections in the dorsal neck region twice weekly. The rpGH dosages and the number of injections are also represented in Table 1. The weight of the animals was recorded weekly. Four pigs (two females and two males) were in each pen with four pens per treatment group, but the tissue samples from only the males were used for the assessment of tendons. Pigs were killed at a commercial slaughter plant when their average pen weight was approximately 140 kg. Thus, to attain the two month treatment, injections were delayed until pigs were 75 kg. 2.2. Sample collection Tissue samples and carcass measurements were collected after an overnight chill on the carcass. The common calcanean tendon was dissected from the right hindlimb and stored at 70 °C until further analysis. To assess back-fat thickness, cross-sectional cuts of the longissimus muscle cranial and caudal to the tenth rib were made. Using a ruler, fat thickness from the perimeter of the longissimus muscle to the skin was measured to the nearest 0.1 cm at approximately 5 cm off the midline. A plastic transparent sheet was placed directly on the muscle cross-section and the perimeter of the muscle carefully traced. These were later digitized to obtain the muscle cross-sectional area. 2.3. Sample preparation A tendon tissue specimen approximately 0.5 cm thickness was procured from the part of the tendon located 1.5 mm cranial to the bifurcation of the common

Table 1 Detailed experimental design of rpGH administration, number of treatment days, group initial and final body weights, and pigs per pen Treatment




mg of rpGH injected per 1 mL Dose of rpGH given during treatment based on body weight Months and number of treatments of placebo [saline] or (rpGH) Weight (kg) initial at first injection Sacrifice weight (kg) Number of pigs/pen

0 0 3 [28] 48.0 ± 1.0 139.0 ± 1.8 4

10 133 lg/kg BW 2 (19) 75.0 ± 1.1 141.0 ± 2.7 4

10 212 lg/kg BW 3 (28) 47.0 ± 1.0 144 ± 4.0 4

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calcanean and lateral tendon. Samples were lyophilized at 55 °C for 21 h to a constant dry weight and stored at 70 °C until further analysis. 2.4. Papain digestion for uronic acid and DNA preparation For preparation of uronic acid (UA) and DNA, the method of Vailas et al. [9] was used. Approximately 6–13 mg of tissue were weighed and placed into pyrex glass screw top test tubes. For each 5 mg of tissue, 1 mL of a papain (Sigma–Aldrich Chemical Co., St. Louis, MO; P-3125) solution was added (1 mL = 28 mg of protein; 33 U of enzyme/mg protein, therefore 924 U/ mL of papain enzyme were used to digest tendon samples). This solution contained 0.001 M cysteine-hydrochloride, 0.01 M EDTA, 200 lL/10 mL digest of papain suspension (type III, Sigma, St. Louis, MO) in a 0.01 M phosphate buffer (pH 6.5). In addition, chondroitin sulfate and DNA standards (Sigma, St. Louis, MO) were subject to the same treatment to determine recoveries for the uronic acid and DNA, respectively. Samples were heated in an oven at 65 °C for 48 h. Digests were extracted with chloroform in a chloroform:digest volume ratio of 10:1, and centrifuged at 1000 rpm (500g) for 10–15 min. Samples were frozen at 20 °C until analyzed. The recoveries for DNA and chondroitin S were typically between 93% and 97% for each assay as reported using similar methodology [10]. 2.5. Hydroxyproline and hydroxylysylpyridinoline cross-link preparation Small samples (2–10 mg lyophilized tissue) were placed into 2 mL Wheaton ampules, flame sealed and hydrolyzed in 6 M HCL for 24 h at 110 °C in vaccuo. Hydrolyzed samples were then dried under vacuum in a covered dessicator. Standard L -hydroxyproline (Sigma, St. Louis, MO) underwent the same treatment to estimate recovery. For hydroxyproline (Hyp) measurements, the samples were resuspended in 2.0 mL of 0.065 M, pH 2.0 sodium citrate buffer (filtered and degassed). The resuspended hydrolyzates were filtered with 0.5 lm Millipore filters (Rainin Co., Milford, MA) and were frozen at 70 °C until analyzed.


Louis, MO) were added. The tube was vortexed and read on the Beckman 520 spectrophotometer at 520 nm within 3 min after chromagen addition. The standard curve was linear from 0.5 to 5.0 mg. Proteoglycan concentration was expressed per unit cell (lg UA/lg DNA). 2.7. DNA analysis The estimation of the tendon cell density was achieved by determining the amount of DNA using a modified method of Bonting and Jones [12]. A stock solution of 0.1 mg NaDNA (type I, Sigma, St. Louis, MO) per mL dH2O with the addition of a thymol crystal was prepared. DNA assay samples were made by diluting 100 lL of the sample extracted with chloroform with 300 lL of 0.1 N NaOH. To this, 400 lL of the indole:HCl (50:50 w/v) reagent was added and briefly vortexed. Samples were heated for 20 min at 100 °C, cooled to room temperature, and read with a Beckman Spectrophotometer 520 at 490 nm. The standard curve showed linearity up to 22 lg DNA. 2.8. Hydroxyproline analysis Hydroxyproline analysis was performed spectrophotometrically according to the WoessnerÕs method [13] of micro-quantitation of amino acids. Briefly, a stock solution of 1 mg/mL L -hydroxyproline was diluted to a 0.01 mg/mL solution with dH2O and aliquots were taken to prepare standards of 1, 2 and 4 lg/mL hydroxyproline. Aliquots of 25 lL of the resuspended samples were diluted with dH2O and analyzed in triplicate. To both the standards and samples a 1.2% chloramine-T solution, 19% perchloric acid and 20% p-dimethylamino-benzalhehyde were added and set in a dry heating bath for 20 min at 60 °C. Samples were then cooled to room temperature and read at 557 nm (Beckman Spectrophotometer 520). Hydroxyproline comprises approximately 14% of the collagen molecule and thus was used as an indicator of collagen concentration (lg Hyp/mg dry wt). Hyp was also expressed per unit cell derived from DNA concentration measurements (lg Hyp/lg DNA). 2.9. Hydroxylysylpyridinoline crosslink analysis

2.6. Uronic acid analysis Total uronic acid (UA) content was determined using the method by Blumenkrantz and Asboe-Hansen [11] to determine proteoglycan concentration. One milliliter of 0.0125 M sodium tetraborate/sulfuric acid was added to 200 lL of the standard or sample of papain extract and heated for 10 min at 100 °C. After samples were cooled to room temperature, 10 lL of meta-hydroxybiphenyl (0.15% solution in 0.5% NaOH, Sigma, St.

Dried tissue hydrolyzates were resuspended in 0.5 mL of 1% n-heptafluourobutyric acid (HFBA; Pierce Chemical, Rockford, IL) and vortexed. Samples were centrifuged for 5 min via 1.5 mL microfilterfuge tubes, 0.20 lm nylon-66 pore size membrane (Rainin Instrument Company, Woburn, MA). Using 15 ll of the filtrate, cross-links were measured by a modified method of Eyre (1980, 1984) using the Waters HPLC system (Walters Corp., Milford, MA). Pyridoxamine-2-HCL


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was used as a relative standard. Excitation and emission were set at 290 and 395 nm, respectively. Chromatographs were normalized and hydroxylysylpyridinoline (HP) cross-link fluorescence was determined on a Waters 470 fluorescence detector (Waters Corp., Milford, MA). HP cross-link samples were expressed as moles of HP cross-link per mole of collagen. 2.10. Statistical analysis To evaluate the differences among groups for each dependent variable, one-way analysis of variance (ANOVA) was applied. When the univariate F tests were significant, TukeyÕs post hoc test was used to further identify differences between group pairs. Statistical significance level is set at P < 0.05.

Fig. 2. Back fat thickness of the 10th rib. Values are expressed in centimeters (cm) and represent means ± SE. Fat thickness of both 2mo-rpGH and 3mo-rpGH groups was significantly smaller than control group (P < 0.05).

3. Results No differences (P > 0.05) in mean body mass were detected between the control and the experimental groups at the beginning of the experiment (Table 1). The rate of growth in each of the three groups was not significantly altered by the rpGH twice weekly injections as shown in Fig. 1. Fat thickness at the tenth rib differed (P < 0.05) among groups (Fig. 2). Both rpGH-treated groups had less fat at the tenth rib region than the control group (2.36 ± 0.23 and 2.79 ± 0.38 cm for 2mo-rpGH and 3mo-rpGH, respectively, vs. 3.40 ± 0.20 cm for controls). The longissimus muscle area was significantly (P < 0.05) larger in 2mo-rpGH (41.5 ± 0.90 cm2) and 3mo-rpGH (42.5 ± 0.97 cm2) groups compared to the control (39.1 ± 0.90 cm2) group (Fig. 3). Significant differences among groups were found in tendon DNA concentration (Fig. 4). The 2mo-rpGH group (7.77 ± 1.01 lg/mg dry weight) had significantly (P < 0.05) larger DNA concentration in the calcanean tendon compared to either the 3mo-rpGH

Fig. 1. Weekly pig weights (kg) are represented as means ± SE. There were no significant differences in mean body weights between groups (P > 0.05).

Fig. 3. Longissimus muscle area. Values are expressed in square centimeters (cm2) and represent means ± SE. Muscle area of both 2mo-rpGH and 3mo-rpGH groups was significantly larger than control group (P < 0.05).

Fig. 4. Deoxyribonucleic acid (DNA) concentration. Values are expressed as lg of DNA per mg tissue dry weight and represent means ± SE. DNA concentration in the calcanean tendon of the 2morpGH group was significantly larger, while that of the 3mo-rpGH group was significantly smaller than control group (P < 0.05). The 3mo-rpGH group DNA concentration was also significantly smaller than that of the 2mo-rpGH group (P < 0.05).

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(4.82 ± 0.56 lg/mg dry weight) or the control (6.22 ± 0.39 lg/mg dry weight) group. In addition, the DNA value of the 3mo-rpGH group was significantly (P < 0.05) smaller than the controls. Extracellular matrix measurements (UA and Hyp) were normalized per unit cell derived from DNA concentration measurements. The uronic acid concentration of the 3mo-rpGH group (0.44 ± 0.05 lg/lg DNA) was significantly (P < 0.05) greater than the 2mo-rpGH (0.29 ± 0.05 lg/lg DNA) and the control (0.35 ± 0.20 lg/lg DNA) groups (Fig. 5). In addition, the concentration of the 2mo-rpGH group was significantly (P < 0.05) lower that the controls. There were significant (P < 0.05) differences in Hyp per lg of total DNA among groups (Fig. 6). Calcanean

Fig. 5. Proteoglycan concentration per unit cell. Values are expressed as lg of uronic acid per lg DNA and represent means ± SE. Uronic acid concentration in the calcanean tendon of the 2mo-rpGH group was significantly smaller, while that of the 3mo-rpGH group was significantly larger than Control group (P < 0.05). The 3mo-rpGH group uronic concentration was also significantly larger than that of the 2mo-rpGH group (P < 0.05).

Fig. 6. Total collagen concentration per cell. Amount of hydroxyproline (HYP) normalized to the content of DNA per lg dry wt tissue (lg Hyp/lg DNA) and represents means ± SE. The amount of total Hyp per unit cell in the calcanean tendon of 3mo-rpGH group was significantly larger than either the 2mo-rpGH or the Control group (P < 0.05).


Fig. 7. Non-reducible collagen cross-links. Values are expressed as mole of hydroxylysylpyridinoline (HP) per mole collagen and represent means ± SE. The calcanean tendon content of HP cross-links in the 2mo-rpGH group was significantly larger than either the 3mo-rpGH or the Control group (P < 0.01). No significant difference was found between the 3mo-rpGH and the Control group.

tendon Hyp concentration of the 3mo-rpGH group (5.35 ± 0.65 lg/lg DNA) was significantly (P < 0.05) greater than either the 2mo-rpST (3.25 ± 0.41 lg/lg DNA) or the control (3.93 ± 0.27 lg/lg DNA) group. No differences were found between the 2mo-rpGH group and the controls. Finally, in Fig. 7 the amount of non-reducible collagen cross-links in the calcanean tendon, as shown by HP content, was significantly (P < 0.05) greater in the 2morpGH group (0.41 ± 0.08 M/M collagen) compared to either the 3mo-rpGH (0.20 ± 0.09 M/M collagen) or the control (0.17 ± 0.06 M/M collagen) group.

4. Discussion This study was designed to examine the effect of rpGH administration on the growth and maturational changes of the calcanean tendon in orchidectomized pigs. Responses to exogenous GH varies among tissues. A general effect of GH is a change in body composition. This response may be accomplished by: (a) direct action of GH on adipocytes to decrease lipogenesis and increase lipolysis; and (b) action mediated by IGF-I to increase protein synthesis [14]. The combined effects result in a change in body composition, even without differences in overall body weight. Caperna et al. [7,8] showed that deposition rates of protein were markedly higher, while deposition rates of fat were markedly lower in rpGH-treated growing pigs. Exogenous rpGH caused hypertrophy [15,16] as well as increased shear force (Solomon et al. [17]) of the longissimus muscle in young pigs. The findings of the present study are in agreement with the general changes expected after GH administration. Although muscle weight was not specifically assessed, the longissimus muscle cross sectional area was greater in the rpGH-treated pigs. Additionally, signifi-


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cant decreases in the backfat thickness of the rpGHtreated animals were observed. However, the main purpose of the current study was to determine if adaptations in calcanean tendon collagen occurred concomitantly with metabolic changes in body composition after rpGH administration. The biphasic response of tendon to rpGH administration is first noted by DNA concentration, a marker of cell density, observed in the rpGH treatment groups. The 2mo-rpGH pigs had a 25% increase in the tendon DNA concentration, while 3mo-rpGH pigs showed a 22.5% decrease compared to control values. We hypothesize that rpGH stimulated tendon fibroblast proliferation in 2mo-rpGH group followed by the production of extracellular matrix collagen by tendon fibroblasts resulting in a decreased cellularity per dry weight of tendon in the 3mo-rpGH group. Studies analyzing various connective tissues demonstrated higher amounts of DNA with acute GH or IGF treatment. Franchimont and Bassleer [18] and Franchinmont et al. [19] used a tridimensional in vitro model to demonstrate that incubation of human chondrocytes with GH induced a significant increase of DNA and proteoglycan release into the culture medium. Similar findings were reported in animal cell cultures by Madsen et al. [20,21]. Growth hormone stimulated DNA synthesis in cultured chondrocytes isolated from rat rib growth cartilage and rabbit ear. This action was postulated to be mediated through the stimulation of the proteoglycan core-protein synthesis. The increase in cell density found in the present study for the 2morpGH treatment may not be attributed to a decrease in extracellular matrix components but rather a probable up-regulation of mitosis. The findings of this study suggest that there is a biphasic response of the tendon DNA concentration to rpGH treatment. The observed responses are consistent with GH treatment initially stimulating mitosis followed by an up-regulation of extracellular matrix synthesis. In this aspect, tendon treated for three months may have fewer cells to support a greater matrix. Furthermore, the tendon may increase in size without a change in concentration of matrix components. The Achilles tendon of growing rabbits showed decreased cellularity and ground substance with a concomitant increase in collagen concentration, collagen crosslinks and collagen fibril diameter [22]. Similar results have also been reported by other research groups. In mechanically stressed chickens, Hitchcock et al. [23] reported tendon hypertrophy without alterations in collagen or proteoglycan concentration. Following strenuous exercise in chickens, Curwin et al. [10] found a significant increase in Achilles tendon collagen deposition without any changes in DNA, proteoglycan, collagen concentration or tendon dry weight. The DNA concentration was consistently unaltered in growing chickens across all ages examined, namely 6, 8 and 12 weeks

[24]. The above findings suggest a greater matrix-collagen turnover resulting in reduced maturation of tendon collagen. In the present study, considering that proteoglycan per DNA was decreased while collagen per unit DNA remained unchanged in tendon, there was no anabolic effect on proteoglycan collagen metabolism in 2morpGH pigs. Proteoglycans initially decreased in the 2mo-rpGH group followed by an increase in 3mo-rpGH animals compared to controls. Accordingly, tendon collagen concentration per unit cell was greater in the 3morpGH group compared to 2mo-rpGH, while there was no difference between 2mo-rpGH and the control group. The increased hydroxyproline production per number of cells after 3 months of GH treatment suggests the GH up-regulates extracellular matrix collagen in the tendon. These findings also suggest that there is a time delay effect of GH on the tendon collagen accumulation, or a ‘‘lag’’ period where proliferation takes place before differentiation occurs. We hypothesize that initially both regulatory pathways that control collagen protein formation and collagen degradation during the remodeling process are in equilibrium to maintain an optimal tendon collagen concentration. At a later stage, we suggest that the balance in collagen turnover changes in favor of the up-regulatory pathways resulting in an increase in total collagen concentration as a result of GH supplementation. The proposed biphasic response of tendon to rpGH treatment is further supported by the contents of the non-reducible collagen cross-link hydroxylysylpyridinoline (HP). The 3mo-rpGH group had reduced HP crosslink content compared to the 2mo-rpGH group. HP collagen cross-links are traditionally associated with non-reducible mature collagen and they are significantly reduced in tissues containing immature or newly synthesized collagen [25,26]. Increased relative amounts of reducible collagen cross-links were shown in skin collagen of rats treated with GH suggesting an increased collagen synthesis [27]. We have also recently shown [3] that HP cross-links content in dense fibrous connective tissues were significantly smaller while new collagen accretion indicated by 3H-Hyp production was greater in the GH-treated Dwarf rats compared to ambulatory controls. Our findings suggest that GH up-regulation of ligament and tendon collagen production does not always correlate with an increase in collagen maturation [3]. Limitations of the study. There were some limitations in this study that should receive attention. The two groups of animals receiving hormone treatment: (1) did not receive rpGH treatment at the same starting body weights; (2) rpGH dosage was not altered as animal body weights increased, thus, although the concentration of rpGH administered remained constant, the dosages of rpGH per unit body weight decreased as

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the animals grew larger. The shorter exposure and decreased dosage of rpGH in the 2mo-rpGH group would hypothetically have influenced the overall musculoskeletal metabolism in the 2mo-rpGH group less since the group was exposed to lower dosages than that of the 3mo-rpGH treated group. Based on our findings, the 2mo-rpGH calcanean tendon ECM characteristics changed in response to the rpGH treatment despite fewer exogenous rpGH injections. At this time, it is unclear whether the length of time and/or the dose of rpGH is responsible for the biphasic response in calcanean tendon ECM characteristics of in our study. In conclusion, the results of this study indicate that calcanean tendon responds biphasically to rpGH treatment. In the first phase, 2 months of GH treatment resulted in an increased cellularity (DNA) and collagen cross-links (HP) per mole of collagen, suggesting an increase in tendon mitosis. A second phase is characterized by differentiation and synthesis of the extracellular matrix components, namely proteoglycans (uronic acid) and total collagen content (Hyp), in the 3mo-rpGH treated pig tendon. Growth and maturation of collagen in pig tendon in response to GH administration may not occur simultaneously, but rather through specific time dependent events, which produce optimal tissue strength and structure. The evidences in the literature with respect to the stimulatory effects of acute administration of GH on collagen extracellular matrix and DNA concentration are becoming increasingly persuasive. However, the knowledge of the effect of prolonged GH treatment on connective tissue content are currently limited and the underlying mechanism(s) have yet to be determined. The elucidation of such mechanisms would give insight into designing more effective therapeutic modalities in repairing of injured or dysfunctional tendon.

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