popo, What type of injuries responded well to igf-1? Chronic over use, acute ruptures or both?
Please read this carefuly -very interesting - I am on Lr3 right now and will make ultrasound in a few weeks to see what is going on with it.
in the below text you can see that Igf-1 have large effects on tendons and ligament healing but HGH does not -please read all-I arased some of it to fit.
BMC Physiol. 2007; 7: 2.
Published online 2007 March 26. doi: 10.1186/1472-6793-7-2. PMCID: PMC1851714
Copyright © 2007 Provenzano et al; licensee BioMed Central Ltd.
Systemic administration of IGF-I enhances healing in collagenous extracellular matrices:
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.
References BackgroundInsulin-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.
Elastic modulus values at 3 weeks (mean ± S.E.M.). The additional of IGF-I significantly improved elastic modulus levels in tissues from ambulatory healing animals when compared to tissues from ambulatory healing animals which received saline (more ...)
In accordance with data showing increased matrix deposition and organization (Figs. 4 and 5), expression of type-I collagen was increased in tissues from IGF-I treated ambulatory (p = 0.0018) and unloaded (p = 0.0006) animals and GH+IGF-I treated unloaded tissue (p = 0.0005; Figs. 6A and 6B). Densitometry analysis further confirmed an increase in type-I collagen since the ratio of type-I to type-III collagen was significantly increased in tissues from ambulatory animals treated with IGF-I (p = 0.0129) and in unloaded tissues from GH+IGF-I treated animals (p = 0.0131), with a trend of increased levels in ambulatory GH+IGF and HU + IGF tissues (Fig. 7). Since normal ligaments are primarily composed of type I collagen and the scar region of normal healing ligaments contain an increase in type III collagen [41] that is remodeled to transition to more type I collagen rich region over the healing period, changes in the type I to III ratio are a measure of healing and may indicate part of the structural mechanism resulting in the observed differences in mechanical properties following treatment.
Figure 5
Multiphoton Laser Scanning Microscopy (MPLSM) was performed on hematoxylin and eosin sections in order to evaluate the organization and structure of the collagen matrix in more detail then allowed by conventional brightfield light microscopy. The longitudinal (more ...)
Figure 6
Significantly increased expression of type-I collagen in tissues from animals treated with IGF-I. (A: Left) Immunohistochemistry shows increased staining for type I collagen in ambulatory tissues when compared to Sham, while hindlimb unloaded tissues (more ...)
Figure 7
Densitometry analysis of type I and III collagen expression. Quantification of Western blots for type I and III collagen indicated that the ratio of type I to type III collagen was significantly increased in tissues from ambulatory animals treated with (more ...)
Lastly, GH has been reported to increase levels of IGF receptors [53]. Herein, ambulatory IGF-I and GH+IGF-I treated animals showed a strong trend of increased IGF-I receptor expression, although this trend was not significant (Fig. 8). However, in hindlimb unloaded animals treated with IGF-I or GH+IGF-I, IGF-I receptor expression was significantly increased (Fig. 8). Hence, increased levels of IGF-I receptor in healing tissues may be part of the molecular mechanism by which systemic administration of IGF-I and GH+IHG-I act to locally to stimulate tissue repair.
Figure 8
Densitometry analysis of IGF-I receptor expression. Ambulatory IGF-I and GH+IGF-I treated animals showed a strong trend of increased IGF-I receptor expression, however, this trend was not significant. In hindlimb unloaded animals treated with IGF-I or (more ...)
References DiscussionPrevious work in our laboratory has shown that mechanical properties and matrix organization of MCLs are substantially reduced after injury and that this impairment is significantly compounded by stress reduction through hindlimb unloading [48]. This result is confirmed by examination of the mechanical properties in MCLs from the saline receiving control groups in this study (Sham, Amb + Sal, HU + Sal) which are not significantly different from values obtained in our previous work in tissues from sham, ambulatory healing, and hindlimb unloaded animals after three weeks of healing [48]. Furthermore, structural analysis of the matrix with MPLSM confirmed previous morphological information obtained with electron microscopy [48], elucidating changes in the structure-function relationship, such as collagen fiber misalignment and matrix voids, that help explain the reduced mechanical properties associated with tissue unloading (i.e. disuse).
Results of this study demonstrate a substantial increase in healing with the systemic application of IGF-I in ambulatory and hindlimb unloaded animals and with GH + IGF-I in hindlimb unloaded animals. Since the strong majority of tissues failed in the ligament and not by avulsion, it is clear that the mechanical properties of the ligament are being evaluated and that systemic treatment with IGF-I or GH+IGF-I improves the integrity of the collagenous matrix. This finding is in contrast with results from healing tendons following local injections of IGF-I that showed no significant improvement in the mechanical properties of treated tendons [18], indicating that local and systemic administration of IGF-I may act through different mechanisms. Furthermore, it is interesting that IGF-I alone positively influenced healing in tissues from both ambulatory and hindlimb unloaded animals, while combined GH and IGF-I only had a positive affect on tissues from HU animals. Since IGF-I increased tissue strength measures by ~60% in both ambulatory and hindlimb unloaded animals, it appears that IGF-I improves healing regardless of mechanical loading and that the affects of mechanical loading and IGF-I may be additive. Interestingly, addition of GH alone showed a trend of decreasing the mechanical properties of healing ligaments and hematomas in the healing site, further supporting the concept of a negative role for GH in ligament healing, even though combined supplementation of GH+IGF-I has been reported to increase serum IGF-I levels more than adding IGF-I alone [61,62]. Hence, the behavior reported herein is a complex phenomena superimposing the influence of mechanotransduction during tissue loading, local growth factor signaling, and endocrine hormone levels, yet implies a positive role for IGF-I and a negative role for GH in connective tissue healing through mechanisms that are, to date, not well understood.
Connective tissue atrophy and diminished levels of healing after disuse (e.g. spaceflight, hindlimb unloading, immobilization, etc.) is associated with reduced physical stimuli, however local growth factor signaling, and endocrine factors also play an important role. For instance, it is well established that microgravity or simulated microgravity disrupts pituitary GH function [58-60] and alters IGF-I expression and plasma concentration [60,63]. Interestingly, in this study the addition of GH alone, which can bind to cells via specific surface receptors activating numerous signaling pathways that direct changes in gene expression [64-67], did not offer any significant increase in mechanical properties. In contrast, the addition of IGF-I significantly improved the mechanical properties of tissues in treated animals, likely through structural improvements in the extracellular matrix as seen in Figures 4 and 5. In ambulatory animals treated with IGF-I and unloaded animals treated with IGF-I or GH+IGF-I, MCL matrix organization was greatly improved. This may be due to increased type-I collagen expression resulting in increased the matrix density, and altered cell behavior resulting in a more organized collagen matrix. Given the clear increases in matrix alignment, it appears probable that either collagen organization is initially improved during post-fibrillogenesis deposition or better organized during continued matrix remodeling, or both. Since collagen alignment is achieved before substantial tissue loading during fetal development [46,68], which is not reproduced during tissue repair in unloaded mature tissue [48], and it appears that matrix repair in adult tissue may be an imperfect reversion to processes seen in fetal development [46], addition of IGF-I may be stimulating signaling pathways similar to those seen during development. Moreover, one possible link in the molecular mechanism playing a role in increased matrix organization and collagen expression may be signals associated with IGF-I receptor signaling as indicated by increased IGFR levels in treated animals. However, IGFR activation was not examined in this study and multiple pathways associated with IGFR and matrix adhesion signaling (i.e. integrin signaling) are likely acting in concert to produce the profound improvements seen after IGF-I treatment. Hence, it is clear from these data that further work studying the systemic effects IGF-I need to be performed in order to better understand the mechanism of IGF-I administration on local tissue behavior.
It is known that soft tissue injuries do not fully recover even after long periods of healing [47] and stress reduction has a negative effect on healing in collagenous tissue, [48,52] which does not return to normal after re-establishing physiologic stress (i.e. remobilization) [69]. This pattern of healing is problematic since the injured joint often need immobilization, the growing aged population often experiences decreased activity levels or prolonged bed rest, and since prolonged space flight is becoming more feasible. Therefore, methods to improve tissue healing and counteract this negative decline during injury and/or disuse are increasing in need. The reported application of IGF-I clearly has a positive effect on tissue repair from a mechanical (functional) viewpoint, and therefore shows promise to improving normal tissue healing and to improving healing under normal or disuse conditions. Of particular note is the increase in force, stress, and elastic modulus in tissues from unloaded animals with IGF-I or GH+IGF-I, which become comparable to or surpass levels in ambulatory (Amb + Sal) animals. This improvement in tissue properties compares well with other methods to improve tissue repair, but may be more clinically feasible. For instance, application of PDGF-BB has also shown promising improvements in fibrous connective tissue healing. In two studies allowing unrestricted cage movement, Batten and co-workers [70] reported increases in maximal force up to 90% of controls, and Hildebrand and co-workers [71] reported increases in force of ~50% after PDGF-BB was administered locally immediately following injury. However, administering PDGF-BB 48 hrs post-injury resulted in decreased force values [70], while administering IGF-I post-injury herein improved tissue healing. Therefore, the results presented in this paper showing an increase in maximal force of ~60% in ambulatory animals after 3 weeks of systemic IGF-I compares favorably to the levels of improvement previously reported in the literature but treatment can begin post-injury. Yet, although short term systemic application of IGF-I or GH+IGF-I provide compelling evidence for improved healing in a collagenous matrix, potential side effects of altering GH and IGF-I levels for long periods of time have not yet been fully explored in conjunction with these data and therefore further study is required before long term use is warranted in humans.
References ConclusionIn conclusion, results support our hypothesis that systemic administration of IGF-I improves healing in collagenous extracellular matrices. 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. Interestingly, addition of IGF-I or GH + IGF-I in HU animals resulted in recovery of strength measures to a level equal to ambulatory controls, indicating that in fact IGF-I may be a plausible therapy for overcoming reduced tissue healing due to disuse from bed rest, immobilization, or microgravity. Additionally, although supplementation with IGF-I in ambulatory animals did not result in full recovery of the mechanical properties at 3 weeks, treatment resulted in an ~60% increase in tissue strength demonstrating the potential for IGF-I to improve tissue healing. These changes in tissue healing with tissue loading or IGF-I supplementation raise important questions regarding the essential role of mechanical stress for collagen matrix organization in connective tissues and the mechanisms by which systemic IGF-I or GH+IGF-I lead to improved tissue healing.
