Systemic administration of IGF-I enhances healing in collagenous extracellular matrices: evaluation of loaded and unloaded ligaments.

BACKGROUND: Insulin-like growth factor-I (IGF-I) plays a crucial role in wound healing and tissue repair. We tested the hypotheses that systemic administration of IGF-I, or growth hormone (GH), or both (GH+IGF-I) would improve healing in collagenous connective tissue, such as ligament. These hypotheses were examined in rats that were allowed unrestricted activity after injury and in animals that were subjected to hindlimb disuse. Male rats were assigned to three groups: ambulatory sham-control, ambulatory-healing, and hindlimb unloaded-healing. Ambulatory and hindlimb unloaded animals underwent surgical disruption of their knee medial collateral ligaments (MCLs), while sham surgeries were performed on control animals. Healing animals subcutaneously received systemic doses of either saline, GH, IGF-I, or GH+IGF-I. After 3 weeks, mechanical properties, cell and matrix morphology, and biochemical composition were examined in control and healing ligaments. RESULTS: Tissues from ambulatory animals receiving only saline had significantly greater strength than tissue from saline receiving hindlimb unloaded animals. Addition of IGF-I significantly improved maximum force and ultimate stress in tissues from both ambulatory and hindlimb unloaded animals with significant increases in matrix organization and type-I collagen expression. Addition of GH alone did not have a significant effect on either group, while addition of GH+IGF-I significantly improved force, stress, and modulus values in MCLs from hindlimb unloaded animals. Force, stress, and modulus values in tissues from hindlimb unloaded animals receiving IGF-I or GH+IGF-I exceeded (or were equivalent to) values in tissues from ambulatory animals receiving only saline with greatly improved structural organization and significantly increased type-I collagen expression. Furthermore, levels of IGF-receptor were significantly increased in tissues from hindlimb unloaded animals treated with IGF-I. CONCLUSION: These results support two of our hypotheses that systemic administration of IGF-I or GH+IGF-I improve healing in collagenous tissue. Systemic administration of IGF-I improves healing in collagenous extracellular matrices from loaded and unloaded tissues. Growth hormone alone did not result in any significant improvement contrary to our hypothesis, while GH + IGF-I produced remarkable improvement in hindlimb unloaded animals.

The biphasic response of porcine tendon to recombinant porcine growth hormone.

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 (10mg/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.

Hindlimb unloading alters ligament healing.

We investigated the hypothesis that hindlimb unloading inhibits healing in fibrous connective tissue such as ligament. Male rats were assigned to 3- and 7-wk treatment groups with three subgroups each: sham control, ambulatory healing, and hindlimb-suspended healing. Ambulatory and suspended animals underwent surgical rupture of their medial collateral ligaments, whereas sham surgeries were performed on control animals. After 3 or 7 wk, mechanical and/or morphological properties were measured in ligament, muscle, and bone. During mechanical testing, most suspended ligaments failed in the scar region, indicating the greatest impairment was to ligament and not to bone-ligament insertion. Ligament testing revealed significant reductions in maximum force, ultimate stress, elastic modulus, and low-load properties in suspended animals. In addition, femoral mineral density, femoral strength, gastrocnemius mass, and tibialis anterior mass were significantly reduced. Microscopy revealed abnormal scar formation and cell distribution in suspended ligaments with extracellular matrix discontinuities and voids between misaligned, but well-formed, collagen fiber bundles. Hence, stress levels from ambulation appear unnecessary for formation of fiber bundles yet required for collagen to form structurally competent continuous fibers. Results support our hypothesis that hindlimb unloading impairs healing of fibrous connective tissue. In addition, this study provides compelling morphological evidence explaining the altered structure-function relationship in load-deprived healing connective tissue.

Extracellular matrix maturation in the left ventricle of normal and diabetic swine.

The main objective of this study is to determine the transmural distribution of extracellular matrix (ECM) collagen and maturation in non-diabetic and diabetic hearts. The Yucatan miniature swine heart ECM was analyzed in eight streptozotocin (STZ) induced diabetic pigs (Diabetic-Swine) and age matched normal control pigs (Nondiabetic-Swine). After 12 weeks of STZ induced diabetes, transmural biopsies were obtained from the left ventricular free wall divided into subendocardial, mid- and subepicardial layers. Collagen concentration and maturation were measured by RP-HPLC determination of hydroxyproline (Hyp) and content of hydroxylysylpyridinoline (HP) cross-links, respectively. Results showed a significant elevation in arterial glucose (P<0.05) and reduction in arterial plasma insulin levels in the Diabetic-Swine. Hyp concentration was significantly greater (P<0.05) in the subendocardial layers in both the Diabetic and Nondiabetic animals. The HP cross-link content was significantly greater (17%) in the Diabetic-swine subendocardial layer compared to Nondiabetic-Swine (P<0.05), but not in other layers. In summary, the accumulation and/or increase in HP cross-link content in the Diabetic-Swine subendocardial layer suggests that myocardial fibrosis may be greater in this specific region.

Growth and maturational changes in dense fibrous connective tissue following 14 days of rhGH supplementation in the dwarf rat.

The purpose of this study was to investigate the impact of recombinant human growth hormone (rhGH) on patella tendon (PT), medial collateral ligament (MCL), and lateral collateral ligament (LCL) on collagen growth and maturational changes in dwarf GH-deficient rats. Twenty male Lewis mutant dwarf rats, 37 days of age, were randomly assigned to Dwarf + rhGH (n = 10) and Dwarf + vehicle (n = 10) groups. The GH group received 1.25 mg rhGH/kg body wt twice daily for 14 days. rhGH administration stimulated dense fibrous connective tissue growth, as demonstrated by significant increases in hydroxyproline specific activity and significant decreases in the non-reducible hydroxylysylpyridinoline (HP) collagen cross-link contents. The increase in the accumulation of newly accreted collagen was 114, 67, and 117% for PT, MCL, and LCL, respectively, in 72 h. These findings suggest that a short course rhGH treatment can affect the rate of new collagen production. However, the maturation of the tendon and ligament tissues decreased 18-25% during the rapid accumulation of de novo collagen. We conclude that acute rhGH administration in a dwarf rat can up-regulate new collagen accretion in dense fibrous connective tissues, while causing a reduction in collagen maturation.