MrPerfect
MuscleChemistry Guru
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.
================================================
Background
Insulin-like growth factor-I (IGF-I) plays a crucial role in muscle regeneration, can reduce age-related loss of muscle function, and cause muscle hypertrophy when over-expressed [1-5]. These effects appear to be largely mediated by promoting proliferation and differentiation of satellite cells [3] as well as promoting recruitment of proliferating bone marrow stem cells to regions of muscle tissue damage [6]. Furthermore IGF-I and growth hormone (GH) are involved in a large variety of physiologic functions and are reported to promote healing and repair in bone [7,8], cartilage [9-11], gastric ulcers [12], muscle [13,14], skin [15-17], and tendon [18,19]. This action is largely mediated by the fact that GH and IGF-I directly affect cells involved in the healing response [20-30], with IGF-I having endocrine action, as well as local expression, resulting in autocrine and/or paracrine signaling that plays a role in proliferation, apoptosis, cellular differentiation, and cell migration [31-36]. Insulin-like growth factor-I also stimulates fibroblast synthesis of extracellular matrix (ECM) molecules such as proteoglycans and type I collagen [18,30,37,38], and IGF-I mRNA and protein levels are increased in healing ligaments [39] and tendons [40], respectively. As such, IGF-I is of particular interest in tissue regeneration due to its influence on cell behavior and role in type I collagen expression.
Fibrous connective tissues, such as ligament and tendon, are composed primarily of type I collagen with type III collagen levels increased during healing [41]. During development, collagen molecules organize into immature collagen fibrils that fuse to form longer fibrils [42-45]. In mature tendon and ligament these fibrils appear to be continuous and transfer force directly through the matrix [46]. In ligament, groups of fibrils form fibers and it is these fiber bundles that form fascicles; the primary structural component of the tissue. Previous studies in healing ligament have shown that disruption of the medial collateral ligament (MCL) results in substantial reduction in mechanical properties which does not return to normal after long periods of healing [47]. Such tissue behavior is likely associated with matrix flaws, reduced microstructural organization, and small diameter collagen fibrils in the scar region of the ECM [48-50]. Additionally, during normal ligament healing collagen fibrils from residual tissue fuse with collagen fibrils formed in the scar region [51]. However, in tissues which are exposed to a reduced stress environment such as joint immobilization [52] or hindlimb unloading [48] collagen fibers contain discontinuities and voids [48] which likely account for the substantial decrease in tissue strength when compared to ligaments experiencing physiologic stress during healing. Since soft tissue injuries are common and do not heal properly in a stress-reduced environment [48,52], such as is present during prolonged bed rest or spaceflight, methods to further understand tissue healing and promote tissue healing require study.
The purpose of this study is to test the hypotheses that systemic administration of IGF-I, GH, or GH+IGF-I will improve healing in a collagenous ECM. Furthermore, since the addition of GH has been shown to up-regulate IGF-I receptor [53], levels of IGF-I receptor in healing tissues were examined in order to begin to elucidate the molecular mechanism by which GH and/or IGF-I may be acting to locally to stimulate tissue repair. Since IGF-I and GH are feasible for clinical use, identifying benefits from short-term systemic administration, such as improved connective tissue healing, have great potential to improve the human condition. The hypotheses are examined in animals that are allowed normal ambulation after injury and in animals that are subjected to disuse through hindlimb unloading. The MCL was chosen as a model system since this ligament, unlike tendons, has no muscular attachment and therefore possible alterations in muscle strength after IGF-I and/or GH treatment do not impose substantial differential loads on the ligament during hindlimb unloading. Furthermore, since MCLs have two attachments/insertions into bone, and hindlimb unloading/disuse is known to reduce the mechanical properties of bone [48,54], failure location was recorded for all mechanical testing. Results indicate improved mechanical properties and collagen organization and composition of the collagenous extracellular matrix following treatment with IGF-I in both ambulatory and hindlimb unloaded animals or IGF-I+GH in hindlimb unloaded animals.
==============================================
Paolo P Provenzano1 email, Adriana L Alejandro-Osorio2 email, Kelley W Grorud1,3 email, Daniel A Martinez4,5 email, Arthur C Vailas5 email, Richard E Grindeland6 email and Ray Vanderby Jr1,3 email
1 Dept. of Biomedical Engineering, University of Wisconsin, Madison, WI, USA
2 Dept. of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA
3 Dept. of Orthopedics and Rehabilitation, University of Wisconsin, Madison, WI, USA
4 Dept. Health and Human Performance, University of Houston, Houston, TX, USA
5 Dept. of Mechanical Engineering and The Biomedical Engineering Program, University of Houston, Houston, TX, USA
6 Life Sciences Research Division, NASA-Ames Research Center, Moffett Field, CA, USA
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.
================================================
Background
Insulin-like growth factor-I (IGF-I) plays a crucial role in muscle regeneration, can reduce age-related loss of muscle function, and cause muscle hypertrophy when over-expressed [1-5]. These effects appear to be largely mediated by promoting proliferation and differentiation of satellite cells [3] as well as promoting recruitment of proliferating bone marrow stem cells to regions of muscle tissue damage [6]. Furthermore IGF-I and growth hormone (GH) are involved in a large variety of physiologic functions and are reported to promote healing and repair in bone [7,8], cartilage [9-11], gastric ulcers [12], muscle [13,14], skin [15-17], and tendon [18,19]. This action is largely mediated by the fact that GH and IGF-I directly affect cells involved in the healing response [20-30], with IGF-I having endocrine action, as well as local expression, resulting in autocrine and/or paracrine signaling that plays a role in proliferation, apoptosis, cellular differentiation, and cell migration [31-36]. Insulin-like growth factor-I also stimulates fibroblast synthesis of extracellular matrix (ECM) molecules such as proteoglycans and type I collagen [18,30,37,38], and IGF-I mRNA and protein levels are increased in healing ligaments [39] and tendons [40], respectively. As such, IGF-I is of particular interest in tissue regeneration due to its influence on cell behavior and role in type I collagen expression.
Fibrous connective tissues, such as ligament and tendon, are composed primarily of type I collagen with type III collagen levels increased during healing [41]. During development, collagen molecules organize into immature collagen fibrils that fuse to form longer fibrils [42-45]. In mature tendon and ligament these fibrils appear to be continuous and transfer force directly through the matrix [46]. In ligament, groups of fibrils form fibers and it is these fiber bundles that form fascicles; the primary structural component of the tissue. Previous studies in healing ligament have shown that disruption of the medial collateral ligament (MCL) results in substantial reduction in mechanical properties which does not return to normal after long periods of healing [47]. Such tissue behavior is likely associated with matrix flaws, reduced microstructural organization, and small diameter collagen fibrils in the scar region of the ECM [48-50]. Additionally, during normal ligament healing collagen fibrils from residual tissue fuse with collagen fibrils formed in the scar region [51]. However, in tissues which are exposed to a reduced stress environment such as joint immobilization [52] or hindlimb unloading [48] collagen fibers contain discontinuities and voids [48] which likely account for the substantial decrease in tissue strength when compared to ligaments experiencing physiologic stress during healing. Since soft tissue injuries are common and do not heal properly in a stress-reduced environment [48,52], such as is present during prolonged bed rest or spaceflight, methods to further understand tissue healing and promote tissue healing require study.
The purpose of this study is to test the hypotheses that systemic administration of IGF-I, GH, or GH+IGF-I will improve healing in a collagenous ECM. Furthermore, since the addition of GH has been shown to up-regulate IGF-I receptor [53], levels of IGF-I receptor in healing tissues were examined in order to begin to elucidate the molecular mechanism by which GH and/or IGF-I may be acting to locally to stimulate tissue repair. Since IGF-I and GH are feasible for clinical use, identifying benefits from short-term systemic administration, such as improved connective tissue healing, have great potential to improve the human condition. The hypotheses are examined in animals that are allowed normal ambulation after injury and in animals that are subjected to disuse through hindlimb unloading. The MCL was chosen as a model system since this ligament, unlike tendons, has no muscular attachment and therefore possible alterations in muscle strength after IGF-I and/or GH treatment do not impose substantial differential loads on the ligament during hindlimb unloading. Furthermore, since MCLs have two attachments/insertions into bone, and hindlimb unloading/disuse is known to reduce the mechanical properties of bone [48,54], failure location was recorded for all mechanical testing. Results indicate improved mechanical properties and collagen organization and composition of the collagenous extracellular matrix following treatment with IGF-I in both ambulatory and hindlimb unloaded animals or IGF-I+GH in hindlimb unloaded animals.
==============================================
Paolo P Provenzano1 email, Adriana L Alejandro-Osorio2 email, Kelley W Grorud1,3 email, Daniel A Martinez4,5 email, Arthur C Vailas5 email, Richard E Grindeland6 email and Ray Vanderby Jr1,3 email
1 Dept. of Biomedical Engineering, University of Wisconsin, Madison, WI, USA
2 Dept. of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA
3 Dept. of Orthopedics and Rehabilitation, University of Wisconsin, Madison, WI, USA
4 Dept. Health and Human Performance, University of Houston, Houston, TX, USA
5 Dept. of Mechanical Engineering and The Biomedical Engineering Program, University of Houston, Houston, TX, USA
6 Life Sciences Research Division, NASA-Ames Research Center, Moffett Field, CA, USA
