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

Provenzano PP, Alejandro-Osorio AL, Grorud KW, Martinez DA, Vailas AC, Grindeland RE, Vanderby R - BMC Physiol. (2007)

Bottom Line: Tissues from ambulatory animals receiving only saline had significantly greater strength than tissue from saline receiving hindlimb unloaded animals.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept. of Biomedical Engineering, University of Wisconsin, Madison, WI, USA. ppproven@wisc.edu

ABSTRACT

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.

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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 axis of the ligament is from the top to bottom of each image. Tissues from sham control animals have the characteristic crimp pattern associated with normal tissue (Sham). Scar tissue from ambulatory healing animals (Amb + Sal) revealed typical scar morphology with matrix disorganization while hindlimb unloaded animals (HU + Sal) showed good fiber aggregation but possessed abnormal scar formation with misaligned collagen fibers not directed along the longitudinal axis of the tissue creating voids and defects by not connecting. Assessment of collagen matrices from GH treated animals revealed no improvement in matrix organization in tissues from ambulatory (Amb + GH) or hindlimb unloaded (HU + GH) animals. Examination of collagen structure in animals treated with IGF-I supported data shown in Fig. 4 revealing greatly increased matrix density and considerably improved matrix alignment in tissues from ambulatory (Amb + IGF) and hindlimb unloaded (HU + IGF) animals. Animals treated with GH+IGF showed no significant improvement in ambulatory tissues (Amb + GH + IGF) but unloaded tissues (HU + GH + IGF) showed substantially increased matrix density and alignment.
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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 axis of the ligament is from the top to bottom of each image. Tissues from sham control animals have the characteristic crimp pattern associated with normal tissue (Sham). Scar tissue from ambulatory healing animals (Amb + Sal) revealed typical scar morphology with matrix disorganization while hindlimb unloaded animals (HU + Sal) showed good fiber aggregation but possessed abnormal scar formation with misaligned collagen fibers not directed along the longitudinal axis of the tissue creating voids and defects by not connecting. Assessment of collagen matrices from GH treated animals revealed no improvement in matrix organization in tissues from ambulatory (Amb + GH) or hindlimb unloaded (HU + GH) animals. Examination of collagen structure in animals treated with IGF-I supported data shown in Fig. 4 revealing greatly increased matrix density and considerably improved matrix alignment in tissues from ambulatory (Amb + IGF) and hindlimb unloaded (HU + IGF) animals. Animals treated with GH+IGF showed no significant improvement in ambulatory tissues (Amb + GH + IGF) but unloaded tissues (HU + GH + IGF) showed substantially increased matrix density and alignment.

Mentions: To further examine extracellular matrix organization in histology sections, multiphoton laser scanning microscopy (MPLSM), which allows greatly enhanced imaging of collagen structure, was employed (Fig. 5). Examination of sham, ambulatory, and hindlimb unloaded tissues supports morphological information obtained with classical histology (Fig. 4) and scanning electron microscopy [48], with tissues from HU animals showing good fiber bundle formation but fiber misalignment creating matrix discontinuities and voids (Fig. 5). Confirming results obtained from viewing H&E section with light microscopy, MPLSM analysis showed improved matrix organization in tissues from Amb + IGF-I, HU + IGF, and HU + GH + IGF-I animals (Fig. 5). 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.


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

Provenzano PP, Alejandro-Osorio AL, Grorud KW, Martinez DA, Vailas AC, Grindeland RE, Vanderby R - BMC Physiol. (2007)

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 axis of the ligament is from the top to bottom of each image. Tissues from sham control animals have the characteristic crimp pattern associated with normal tissue (Sham). Scar tissue from ambulatory healing animals (Amb + Sal) revealed typical scar morphology with matrix disorganization while hindlimb unloaded animals (HU + Sal) showed good fiber aggregation but possessed abnormal scar formation with misaligned collagen fibers not directed along the longitudinal axis of the tissue creating voids and defects by not connecting. Assessment of collagen matrices from GH treated animals revealed no improvement in matrix organization in tissues from ambulatory (Amb + GH) or hindlimb unloaded (HU + GH) animals. Examination of collagen structure in animals treated with IGF-I supported data shown in Fig. 4 revealing greatly increased matrix density and considerably improved matrix alignment in tissues from ambulatory (Amb + IGF) and hindlimb unloaded (HU + IGF) animals. Animals treated with GH+IGF showed no significant improvement in ambulatory tissues (Amb + GH + IGF) but unloaded tissues (HU + GH + IGF) showed substantially increased matrix density and alignment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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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 axis of the ligament is from the top to bottom of each image. Tissues from sham control animals have the characteristic crimp pattern associated with normal tissue (Sham). Scar tissue from ambulatory healing animals (Amb + Sal) revealed typical scar morphology with matrix disorganization while hindlimb unloaded animals (HU + Sal) showed good fiber aggregation but possessed abnormal scar formation with misaligned collagen fibers not directed along the longitudinal axis of the tissue creating voids and defects by not connecting. Assessment of collagen matrices from GH treated animals revealed no improvement in matrix organization in tissues from ambulatory (Amb + GH) or hindlimb unloaded (HU + GH) animals. Examination of collagen structure in animals treated with IGF-I supported data shown in Fig. 4 revealing greatly increased matrix density and considerably improved matrix alignment in tissues from ambulatory (Amb + IGF) and hindlimb unloaded (HU + IGF) animals. Animals treated with GH+IGF showed no significant improvement in ambulatory tissues (Amb + GH + IGF) but unloaded tissues (HU + GH + IGF) showed substantially increased matrix density and alignment.
Mentions: To further examine extracellular matrix organization in histology sections, multiphoton laser scanning microscopy (MPLSM), which allows greatly enhanced imaging of collagen structure, was employed (Fig. 5). Examination of sham, ambulatory, and hindlimb unloaded tissues supports morphological information obtained with classical histology (Fig. 4) and scanning electron microscopy [48], with tissues from HU animals showing good fiber bundle formation but fiber misalignment creating matrix discontinuities and voids (Fig. 5). Confirming results obtained from viewing H&E section with light microscopy, MPLSM analysis showed improved matrix organization in tissues from Amb + IGF-I, HU + IGF, and HU + GH + IGF-I animals (Fig. 5). 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.

Bottom Line: Tissues from ambulatory animals receiving only saline had significantly greater strength than tissue from saline receiving hindlimb unloaded animals.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept. of Biomedical Engineering, University of Wisconsin, Madison, WI, USA. ppproven@wisc.edu

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus