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BMC Physiology

Open AccessResearch article

Systemic administration of IGF-I enhances healing in collagenousextracellular matrices: evaluation of loaded and unloaded ligamentsPaolo P Provenzano*1, Adriana L Alejandro-Osorio2, Kelley W Grorud1'3,Daniel A Martinez4'5, Arthur C Vailas5, Richard E Grindeland6 andRay Vanderby Jr*1'3

Address: 1Dept. of Biomedical Engineering, University of Wisconsin, Madison, WI, USA, 2Dept, of Biomolecular Chemistry University ofWisconsin, Madison, WI, USA, 3Dept. of Orthopedics and Rehabilitation, University of Wisconsin, Madison, WI, USA, 4Dept. Health and HumanPerformance, University of Houston, Houston, IX, USA, 5Dept of Mechanical Engineering and The Biomedical Engineering Program, Universityof Houston, Houston, TX, USA and 6Life Sciences Research Division, NASA-Ames Research Center, Moffett Held, CA, USA

Email! Paolo P Provenzano* - [email protected]; Adriana L Alejandro-Osorio - [email protected];Kelley W Grorud - [email protected]; Daniel A Martinez - [email protected]; Arthur C Vailas - [email protected];Richard E Grindeland - [email protected]; Ray Vanderby* - vanderby® orthorehab.wisc.edu* Corresponding authors

Published: 26 March 2007 Received: 25 July 2006

&MC Physiology 2007, 7:2 dot 10.1186/1472-6793-7-2 Accepte* U March 2°07

This article is available from: http://www.biomedcentral.eom/l472-6793/7/2

© 2007 Provenzano et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.0rg/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: Insulin-like growth factor-l (IGF-I) plays a crucial role in wound healing and tissue repair. Wetested the hypotheses that systemic administration of IGF-I, or growth hormone (GH), or both (GH+IGF-I) wouldimprove healing in collagenous connective tissue, such as ligament. These hypotheses were examined in rats thatwere allowed unrestricted activity after injury and in animals that were subjected to hindlimb disuse. Male ratswere 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 receivedsystemic doses of either saline, GH, IGF-I, or GH+IGF-I. After 3 weeks, mechanical properties, cell and matrixmorphology, and biochemical composition were examined in control and healing ligaments.

Results: Tissues from ambulatory animals receiving only saline had significantly greater strength than tissue fromsaline receiving hindlimb unloaded animals. Addition of IGF-I significantly improved maximum force and ultimatestress in tissues from both ambulatory and hindlimb unloaded animals with significant increases in matrixorganizationlind type-l collagen expression. Addition of GH alone did not have a significant effect on either group,while addition of GH+IGF-1 significantly improved force, stress, and modulus values in MCLs from hindlimbunloaded animals. Force, stress, and modulus values in tissues from hindlimb unloaded animals receiving IGF-I orGH+IGF-I exceeded (or were equivalent to) values in tissues from ambulatory animals receiving only saline withgreatly improved structural organization and significantly increased type-l 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-Iimprove healing in collagenous tissue. Systemic administration of IGF-I improves healing in collagenousextracellular matrices from loaded and unloaded tissues. Growth hormone alone did not result in any significantimprovement contrary to our hypothesis, while GH + IGF-I produced remarkable improvement in hindlimbunloaded animals. .J "

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BackgroundInsulin-like growth factor-I (IGF-I) plays a crucial role inmuscle regeneration, can reduce age-related loss of musclefunction, and cause muscle hypertrophy when over-expressed [1-5]. These effects appear to be largely medi-ated by promotinR proliferation and differentiation of sat-ellite cells [3] as well as promoting recruitment ofproliferating bone marrow stem ceilstissue damage [6]. Furthermore IGF-I and growth ho?mone (GH) are involved in a large variety of physiologicfunctions and are reported to promote healing and repairin bone [7,8], cartilage [9-11], gastric ulcers [12], muscle[13,14], skin [15-17], and tendon [18,19]. This action islargely mediated by the fact that GH and IGP-I directlyaffect cells involved in the healing response [20-30], withIGF-I having endocrine action, as well as local expression,resulting in autocnne and/or paracnne signaling-thaT1

plays a role in proliferation, apoptosis, cellular differenti-ation, and cell migration [3 1-36]. Insulin-like growth fac-tor-I also stimulates fibroblast synthesis of extracellularmatrix (ECM) molecules such as proteoglycans and type Icollagen [18,30,37,38], and IGF-I mRNA and protein lev-els are increased in healing ligaments [39] and tendons[40], respectively. As such, IGF-I is of particular interest intissue regeneration^due to its influence on cell behaviorand, role in type 1 collagen expression..

Fibrous connective tissues, such as ligament and tendon,are composed primarily oi type 1 collagen with type IIIcollagen levels increased dming healing [41]. Duringdevelopment, collagen molecules organize into immaturecollagen fibrils that fuse to form longer fibrils [42-45]. Inmature tendon and ligament these fibrils appear to becontinuous and transfer force directly through the matrix[46]. In ligament, groups of fibrils form fibers and it isthese fiber bundles that form fascicles; the primary struc-tural component of the tissue. Previous studies in healingligament have shown that disruption of the medial collat-eral ligament (MCL) results in substantial reduction inmechanical properties which does not return to normalafter long periods of healing [47]. Such tissue behavior islikely associated with matrix flaws, reduced microstruc-tural organization, and small diameter collagen fibrils inthe scar region of trie HGM- [48-50]. Additionally, duringnormal ligament healing collagen fibrils from residual tis-sue fuse with collagen fibrils formed in the scar region[51]. However, in tissues which are exposed to a reducedstress'environment such as joint immobilization [52] orhindlimb unloading [48] collagen fibers contain disconti-nuities and voids [48] which likely account-for the sub-stantial decrease in tissue strength when compared toligaments experiencing physiologic stress during healing.Since soft tissue injuries are common and do not healproperly in a stress-reduced environment [48,52], such asis present during prolonged bed rest or spaceflight, meth-

ods to further understand tissue healing and promote tis-sue healing require study.

The purpose of this study is to test the hypotheses that sys-temic administration of IGF-I, GH, or GH+IGF-I willimprove healing in a rnllagenous ECM. Furthermore,'since the addition of GH has been shown to up-regulateIGP-I receptor [53]. levels of IGF-I receptor in healing tis-sues were examined in order to begin to elucidate themolecular mechanism by which GH and/or IGP-I may be"acting to locally to stimulate tissue repair. Since IGF-I andGH are feasible tor clinical use, identifying benefits fromshort-term systemic administration, such as improvedconnective tissue healing, have great potential to improvethe human condition. The hypotheses are examined inanimals that are allowed normal ambulation after injuryand in animals that are subjected to disuse through hind-limb unloading. The MCL was chosen as a model systemsince this ligament, unlike tendons, has no muscularattachment and therefore possible alterations in musclestrength after IGF-I and/or GH treatment do not imposesubstantial differential loads on the ligament during hind-limb unloading. Furthermore, since MCLs have twoattachments/insertions into bone, and hindlimb unload-ing/disuse is known to reduce the mechanical propertiesof bone [48,54], failure location was recorded for allmechanical testing. Results indicate improved mechanicalproperties and collagen organization and composition ofthe collagenous extracellular matrix following treatmentwith IGF-I in both ambulatory and hindlimb unloadedanimals or IGF-I+GH in hindiimb unloaded animals.

ResultsInitial body weights were not different between groupsand no wound infections or other apparent complicationsassociated with surgery were observed. All of the ambula-tory animals returned to normal cage activity shortly aftersurgery, and no treatment complications were observed inthe suspended animals. The hindlimb unloaded (HU)animals were not able to gain weight as rapidly as theambulatory animals, thus significant differences (p <0.0001) in body weight were observed between groupsafter 10 days of healing. The GH, IGF-I, and GH+IGF-Itreatment increased body weight in HU animals (com-pared to HU animals receiving only~salffie)~butthis differ-ence was not statistically significant (p = 0.49, 0,49, and0.26, respectively). No significant differences in bodyweight were present between the ambulatory animals (allp values > 0.38). At tissue harvest all healing ligamentsshowed a bridging of the injury gap^wltli translucent scartissue. In the ambulatory animals receiving GH, three ani-mals had hematomas and tissue adhesions in the surgicalsite. In all other animals no gross differences in the tissuesor the surgical wound site were observed.

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The location of structural failure in each ligament wasexamined, revealing that the majority of the femur-MCL-tibia complexes failed in the MCL proper, not at the liga-ment to bone insertion sites, nor by bone avulsion (Table1). Sham control ligaments failed in the tibial third of theligament during 100% of the tests. For all other groups,the primary location of failure was the scar region of theligament. The only exceptions to this were one failure inthe tibial third of the ligament in the Amb + IGF group,one tibial avulsion in the HII + Sal group, and one tibialavulsion in the HU + IGF group, indicating that a signifi-cantportion of the failure locations was in thescar region(all p^values < 0.01). These results indicate the dominanteffects in the measured mechanical properties are due tochanges in ligamentous tissue and not in the insertions.

Substantial differences in tissue mechanical propertieswere present between groups. Hindlimb unloadingadversely affected ligament healing while IGF-I orGH+IGF-I had a substantial effect on ligament healing ineither ambulatory or hindlimb unloaded animals. Maxi-mum rorce (Fig. 1) vvaa AiguIG.LcUJ.uy different between tis-sues from Sham and both Amb + Sal and HU + Sal groups(p = 0.0001 and p = 0.0001, respectively). Hindlimbunloaded animals had significantly decreased maximumforce in the MCLs when compared to ambulatory healinganimals (Amb + Sal; p = 0.03). In ambulatory animals theaddition of IGF-I significantly improved maximum forcevalues by approximately 60% when compared to ambula-tory healing animals that^cecdged^saline (p = 0.0002).Addition of IGF-I and (gg+IGF-HipKn dlimb^unloadedanimals sigriifeaatly rncreasecK^^rmuin f5Ttg5varu.es(60% and "X4%, respemvely) when compared to HU + Salanimals (p = O^OU7Tand p = 0.0013, respectively). In fact,addition of IGF-I or GH+IGF-I to hindlimb unloaded ani-mals increased force to be comparable with Amb + Salanimals. Growth hormone alone did not have a signifi-cant effect within either- the ambulatory or unloadedgroups. HQW'eVer, in the unloaded group, GH increasedmaximum force and brought it closer to values in the Amb+~Sal group, yet this increase was not statistically signifi-cant (p =JjJJij-Ultimate stress (Pig. 2) was significantlydecreased in tissues from both ambulatory healing andhindlimb unloaded healing animals when compared tosham control tissues (p = 0.0001 and p = 0.0001, respec-tively), ligaments from HU + Sal animals had ultimatestress values that were significantly lower than salinereceiving ambulatory animals (p = 0.022). Addition ofonly IGF-I to ambulatory animals significantly increasedultimate stress when compared to Amb + Sal animals (p =0.0077). Delivery of IGF-I and GH+IGF-I significantlyincreased ultimate stress in tissues from the hindlimbunloaded animals when compared to hindlimb unloaded(plus saline) animals (p = 0.0236 and p = 0.0202, respec-tively). In fact, ultimate stress values after the addition of

IGF-I or GH+IGF-I to hindlimb unloaded animals wascomparable to ultimate stress values in ambulatory ani-mals receiving saline. In ambulatory animals, no statisti-cally significant effect on ultimate stress values were seenafter GH or GH+IGF-I was administered. No significantdifferences in strain at failure were present betweengroups. Elastic modulus (Fig. 3) was statistically differentbetween IGF-I and saline treated ambulatory healing ani-mals. Addition of IGF-I in ambulatory animals resulted inan elastic modulus that had an ~49% greater mean value(p = 0.049). In hindlimb unloaded animals the additionof IGF-I or GH+IGF-I resulted in a significant increase inmodulus when compared to unloaded animals receivingsaline (p = 0.049 and p = 0.014, respectively). Growthhormone alone had no significant effect on elastic modu-lus in either group.

Representative H&E stained sections (Fig. 4) revealedhypercellularity and extracellular matrix disorganizationin injured tissues from both ambulatory and suspendedanimals. Ligaments from Sham animals demonstrated thecharacteristic crimp pattern and aligned fibroblasts associ-ated with normal ligament (Fig. 4). However, while Amb+ Sal animals revealed normal scar morphology, tissuesfrom HU + Sal animals had pockets of cell clusters, cellu-lar misalignment, and misaligned collagen bundles ori-ented in separate directions creating discontinuities andvoids within the matrix confirming a previous report [48].Furthermore, while matrices from animals treated withGH had abnormal cell dusters and no gross improvementin matrix organization in both ambulatory and unloadedtissues, tissues from animals treated with IGF-I showedsubstantially increased matrix density and collagen align-ment (Fig. 4); a morphology also present in tissues fromHU + GH + IGF-I animals.

To further examine extracellular matrix organization inhistology sections, multiphoton laser scanning micros-copy (MPLSM), which allows greatly enhanced imaging ofcollagen structure, was employed (Fig. 5). Examination ofsham, ambulatory, and hindlimb unloaded tissues sup-ports morphological information obtained with classicalhistology (Fig. 4) and scanning electron microscopy [48],with tissues from HU animals showing good fiber bundleformation but fiber misalignment creating matrix discon-tinuities and voids (Fig. 5). Confirming results obtainedfrom viewing H&E section with light microscopy, MPLSManalysis showed improved matrix organization in tissuesfrom Amb + IGF-I, HU + IGF, and HU + GH + IGF-I ani-mals (Fig. 5). In accordance with data showing increasedmatrix deposition and organization (Figs. 4 and 5),expression of type-I collagen was increased in tissues fromIGF-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

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Table I: Failure location of the bone-ligament-bone complex at 3 weeks post-injury.

Failure Location Scar Region Tibial Third of the Ligament Tibial Avulsion

Sham ControlAmb •*• Saline

Amb+GHAmb + IGF

Amb + GH + IGFHU + Saline

HU+GHHU + IGF

HLH-GH + 1GF

N/A66565656

600I00000

00000I0I0

The strong majority (>96%) of tissues failed in the ligament and not by avulsion, indicating that the measured mechanical changes are due to changesin the ligament properties and not bone.

X X x ^x _ x^ x

Figure 1Maximum force values at 3 weeks (mean ± S.E.M.). Maximum force was significantly decreased in tissues from hindlimbunloaded (HU) animals receiving saline when compared to tissues from Amb + Sal animals (p = 0.03). The addition of IGF-I sig-nificantly improved maximum force in tissues from ambulatory healing animals when compared to tissues from ambulatoryhealing animals that received saline (p = 0.0002). Addition of IGF-I and GH+IGF-I in HU animals significantly increased maxi-mum force values when compared to hindlimb unloaded animals receiving saline (p = 0.0074 and p = 0.0013, respectively), asignificantly different from Sham group; fa significantly different from Amb + Sal group; c significantly different from HU + Salgroup.

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X X X X

Figure 2Ultimate stress values at 3 weeks (mean ± S.E.M.). Ultimate stress was significantly decreased in tissues from unloaded animalsreceiving saline when compared to tissues from Amb + Sal (p = 0.022). The additional of IGF-I significantly improved ultimatestress levels in tissues from ambulatory healing animals when compared to tissues from ambulatory healing animals whichreceived saline (p = 0.0077). Addition of IGF-I and GH+IGF-I in hindlimb unloaded animals significantly increased ultimatestress values when compared to hindlimb unloaded animals receiving saline (p = 0.0236 and p = 0.0202, respectively), a signifi-cantly different from Sham group; b significantly different from Amb + Sal group; c significantly different from HU + Sal group.

confirmed an increase in type-I collagen since the ratio oftype-I to type-Ill collagen was significantly increased intissues from ambulatory animals treated with IGF-I (p =0.0129} and in unloaded tissues from GH+IGF-I treatedanimals (p = 0.0131), with a trend of increased levels inambulatory GH+IGF and HU + IGF tissues (Fig. 7). Sincenormal ligaments are primarily composed of type I colla-gen and the scar region of normal healing ligaments con-tain arTtocTease in type III collagen [41] that is remodeledTcrtranaitiorr lo more type I collagen rich re

langes in the type I to III ratio;healiiijJ ana may "indicate" part of the structural

mechanism resulting in the observed differences inmechanical properties following treatment.

Lastly, GH has been reported to increase levels of IGFreceptors [53]. Herein, ambulatory IGF-I and GH+IGF-Itreated animals showed a strong trend of increased IGF-Ireceptor expression, although this trend was not signifi-^cant (Fig. 8). However, in hindlimb unloaded animalstreated with IGF-I or GH+IGF-I, IGF-I receptor expressionwas significantly increased (Fig. 8). Hence, increased lev-els of IGF-I receptor in healing tissues may be part of the

lamsm by which systemic administration.

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Figure 3Elastic modulus values at 3 weeks (mean ± S.E.M.). The additional of IGF-I significantly improved elastic modulus levels in tis-sues from ambulatory healing animals when compared to tissues from ambulatory healing animals which received saline (p =0.049). A significant increase in elastic modulus was present in tissues from hindlimb unloaded animals after the addition of IGF-I or GH+IGF-I when compared to hindlimb unloaded animals receiving saline (p = 0.049 and p = 0.014, respectively), a signifi-cantly different from Sham group; b significantly different from Amb + Sal group; c significantly different from HU + Sal group.

of IGF-I and GH+IHG-I act to locally to stimulatejissuer e p a r n " "

DiscussionPrevious work in our laboratory has shown that mechan-ical properties and matrix organization of MCLs are sub-stantially reduced after injury and that this impairment issignificantly compounded by stress reduction throughhindlimb unloading [48]. This result is confirmed byexamination of the mechanical properties in MCLs fromthe saline receiving control groups in this study (Sham,Amb + Sal, HU + Sal) which are not significantly differentfrom values obtained in our previous work in tissues fromsham, ambulatory healing, and hindlimb unloaded ani-mals after three weeks of healing [48]. Furthermore, struc-tural analysis of the matrix with MPLSM confirmedprevious morphological information obtained with elec-

tron microscopy [48], elucidating changes in the struc-ture-function relationship, such as collagen fibermisalignment and matrix voids, that help explain thereduced mechanical properties associated with tissueunloading (i.e. disuse).

Results of this study demonstrate a substantial increase inhealing with the systemic application of IGB-I in ambula-tory and hindlimb unloaded animals and with GH + IGF-I in hindlimb unloaded animals. Since the strong majorityof tissues failed in the ligament and not by avulsion, it isdear that the mechanical properties ot the ligament arebeing evaluated and that systemic treatment with IGF-I ortSU+IGF'I impiuveh lhe hitegnly ot the collagenousmatrix. This finding is in contrast with results from heal-ing tendons following local injections ot IGt'-l tnatsh'uwed uu significant improvement in the mecnanical

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Sham

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Figure 4Representative tissue sections taken from the midsubstance region of control normal tissue (Sham) and scar tissues (Saline, GH,1GF-I, GH+IGF-f). The longitudinal axis of the ligament is from the top left to bottom right of each image (200X). Tissues fromsham control (Sham) animals have the characteristic crimp pattern and aligned fibroblasts associated with normal tissue. Exam-ination of scar tissue from ambulatory healing animals (Amb + So/) revealed typical scar morphology with matrix disorganizationand hypercellularity. In contrast to ambulatory animals, hindlimb unloaded (HU + Sal) animals showed abnormal scar formationwith pockets of cell clusters and misaligned collagen fibers creating defects and voids by not connecting. Examination of extra-cellular matrices from GH treated animals revealed no improvement in matrix organization in tissues from ambulatory (Amb +GH) or hindlimb unloaded (HU + GH) animals. However, animals treated with 1GF-1 showed extensively increased matrix den-sity and substantially improved matrix alignment in tissues from ambulatory (Amb + IGF) and hindlimb unloaded (HU + IGF) ani-mals; indicating a considerable improvement in the matrix structure in animals treated with IGF-L Animals that were treatedwith both GH and IGF-I showed similar improvement to that seen with 1GF-I in unloaded tissues (HU + GH + IGF) but showedno improvement in ambulatory tissues (Amb + GH + IGF).

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Sham

+Saline

+GH

+IGF

+GH+IGF

Figure 5Multiphoton Laser Scanning Microscopy (MPLSM) was performed on hematoxylin and eosin sections in order to evaluate theorganization and structure of the collagen matrix in more detail then allowed by conventional brightfield light microscopy. Thelongitudinal axis of the ligament is from the top to bottom of each image. Tissues from sham control animals have the character-istic crimp pattern associated with normal tissue (Sham). Scar tissue from ambulatory healing animals (Amb + Sal) revealed typ-ical scar morphology with matrix disorganization while hindlimb unloaded animals (HU + Sal) showed good fiber aggregationbut possessed abnormal scar formation with misaligned collagen fibers not directed along the longitudinal axis of the tissue cre-ating voids and defects by not connecting. Assessment of collagen matrices from GH treated animals revealed no improvementin matrix organization in tissues from ambulatory (Amb + GH) or hindlimb unloaded (HU + GH) animals. Examination of colla-gen structure in animals treated with 1GF-I supported data shown in Fig. 4 revealing greatly increased matrix density and con-siderably 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 tis-sues (HU + GH + IGF) showed substantially increased matrix density and alignment.

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Figure 6Significantly increased expression of type-l collagen in tissues from animals treated with IGF-I. (A: Left) Immunohistochemistryshows increased staining for type I collagen in ambulatory tissues when compared to Sham, while hindlimb unloaded tissuesshow no increase. In tissues from ambulatory and hindlimb unloaded animals treated with IGF-I type 1 collagen staining isincreased. GH+IGF-I also results in increased collagen expression in unloaded tissues. (A~ Right) Thresholded images utilized forquantitative analysis of expression. (B) Quantitative analysis of type I collagen expression. Ambulatory healing (Amb + Sal) tis-sue had significantly more collagen expression when compared to sham tissues (** p = 0.0002) while unloaded tissues did notGH showed no significant increase between saline treated animals. Ambulatory IGF-I treated animals had significantly (*)increased type-l collagen protein expression (p = 0.0018) when compared to ambulatory animals treated with saline. Tissuesfrom GH+1GF treated ambulatory and hindlimb unloaded animals had significantly (# p = 0.0006 and p = 0.0005, respectively)increased collagen expression when compared to hindlimb unloaded tissues given saline.

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Figure 7Densitometry analysis of type 1 and 111 collagen expression. Quantification of Western blots for type I and III collagen indicatedthat the ratio of type I to type III collagen was significantly increased in tissues from ambulatory animals treated with 1GF-I (* p= 0.0129) and in unloaded tissues from GH+IGF-1 treated animals (p = 0.0131), when compared to saline treated ambulatoryand hindlimb unloaded tissues. Note: the values for each group were normalized to the Sham group (ratio set to one) to allowcomparison of data across multiple Western blot experiments.

properties of treated tendons [18], indicating ^and systemic administration of IGP-I may act through dif-fereUL mediaiiitilusTTurfEermore, it is interesting that IGF-i alone posmveTymfluenced healing in tissues from bothambulatory and hindlimb unloaded animals, while com-bined GH and IGF-I only had a positive affect on tissuesfrom HU animals. Since IGF-I increased tissue strengthmeasures by ~60% in both ambulatory and hindlimb

unloaded animals, itappears that IGP-I improves healingregardless of mechanical loading and that the affects ofmechanical loading and IGP-I may be additive!. The

associated improvement observed only inhindlimb unloaded animals is more difficult to interpret.Nonetheless, this difference between ambulatory and HUanimals after GH+IGF-I treatment may result fromchanges in the endocrine systemjue to unloading [55-57]

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Figure 8Densitometry analysis of IGF-I receptor expression. Ambulatory IGF-I and GH+IGF-1 treated animals showed a strong trend ofincreased IGF-I receptor expression, however, this trend was not significant. In hindlimb unloaded animals treated with IGF-Ior GH+IGF-I, IGF-I receptor expression was significantly increased compared to saline treated hindlimb unloaded animals, indi-cating a possible role for localized increases in IGF-I receptor to play a role in mediating improvements seen in the structureand function of treated ligaments.

or an inhibitory role of exogenously elevated GH on IGF-I function. Decreased levels of growth hormone, andchanges in other endocrine factors, with unloading have

s allowing the influence^ ofincreased IGP-I lu jjioceed. In contrast, elevated levels ofGBHn-ambula'rory auililaTs mniDit the positive affectjjfKSF-I, iudiLatLuLg dial lit fact elevated GH in ambnlaloiyamaxalii may uuLbepeimlsslveto elevatedn^-ifunction^But thai improved healing trom. IGJr'-I addition is nol.aependenT on exogenousiy elevated GJdi levels. Interest-ingly, addition ot GH alone showed a trend of decreasing

the mechanical properties of healing ligaments andhematomas in the healing site, further supporting theconcept of a negative role for GH in liRarnent healing,even though combined supplementation of GH+IGP-I hasbeen reported to increase serum IGP-I levels more thanadding IGF-I alone [61^21- Hence, the behavior reportedherein is a complex phenomena superimposing the influ-ence of mechanotransduction during tissue loading, localgrowth factor signaling, and endocrine hormone levels,yet implies a positive role for IGF-I and a negative role forGH in connective tissue healing through mechanisms thatare, to date, not well understood.

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Connective tissue atrophy and diminished levels of heal-ing after disuse (e.g. spaceflight, hindlimb unloading,immobilization, etc.) is associated with reduced physicalstimuli, however local growth factor signaling, and endo-crine factors also play an important role. For instance, it iswell established that microgravity or simulated micro-gravity disrupts pituitary GH function [58-60] and altersIGF-I expression and plasma concentration [60,63]. Inter-estingly, in this study the addition of GH alone, which canbind to cells via specific surface receptors activatingnumerous signaling pathways that direct changes in geneexpression [64-67], did not offer any significant increasein mechanical properties. In contrast, the addition of IGF-I significantly improved the mechanical properties of tis-sues in treated animals, likely through structural improve-ments in the extracellular matrix as seen in Figures 4 and5. In ambulatory animals treated with IGF-I and unloadedanimals treated with IGF-I or GH+IGF-I, MCL matrixorganization was greatly improved. This may be due toincreased type-I collagen expression resulting in increasedthe matrix density, and altered cell behavior resulting in amore organized collagen matrix Given the dear increasesin matrix alignment, it appears probable that either colla-gen organization is initially improved during post-fibrill-ogenesis deposition or better organized during continuedmatrix remodeling, or both. Since collagen alignment isachieved before substantial tissue loading during fetaldevelopment [46,68], which is not reproduced during tis-sue repair in unloaded mature tissue [48], and it appearsthat matrix repair in adult tissue may be an imperfectreversion to processes seen in fetal development [46],addition of IGF-I may be stimulating signaling pathwayssimilar to those seen during development Moreover, onepossible link in the molecular mechanism playing a rolein increased matrix organization and collagen expressionmay be signals associated with IGF-I receptor signaling asindicated by increased IGFR levels in treated animals.However, IGFR activation was not examined in this studyand multiple pathways associated with IGFR and matrixadhesion signaling (i.e. integrin signaling) are likely act-ing in concert to produce the profound improvementsseen after IGF-I treatment Hence, it is clear from thesedata that further work studying the systemic effects IGF-Ineed to be performed in order to better understand themechanism of IGF-I administration on local tissue behav-

It is known that soft tissue injuries do not fully recovereven after long periods of healing [47] and stress reduc-tion has a negative effect on healing in collagenous tissue,[48,52] which does not return to normal after re-estab-lishing physiologic stress (i.e. remobilization) [69], Thispattern of healing is problematic since the injured jointoften need immobilization, the growing aged populationoften experiences decreased activity levels or prolonged

bed rest, and since prolonged space flight is becomingmore feasible. Therefore, methods to improve tissue heal-ing and counteract this negative decline during injuryand/or disuse are increasing in need. The reported appli-cation of IGF-I dearly has a positive effect on tissue repairfrom a mechanical (functional) viewpoint, and thereforeshows promise to improving normal tissue healing and toimproving healing under normal or disuse conditions. Ofparticular note is the increase in force, stress, and elasticmodulus in tissues from unloaded animals with IGF-I orGH+IGF-I, which become comparable to or surpass levelsin ambulatory (Amb + Sal) animals. This improvement intissue properties compares well with other methods toimprove tissue repair, but may be more dinically feasible.For instance, application of PDGF-BB has also shownpromising improvements in fibrous connective tissuehealing. In two studies allowing unrestricted cage move-ment, Batten and co-workers [70] reported increases inmaximal force up to 90% of controls, and Hildebrand andco-workers [71] reported increases in force of ~50% afterPDGF-BB was administered locally immediately follow-ing injury. However, administering PDGF-BB 48 hrs post-injury resulted in decreased force values [70], whileadministering IGF-I post-injury herein improved tissuehealing. Therefore, the results presented in this papershowing an Increase in maximal force of ~60% in ambu-latory animals after 3 weeks of systemic IGF-I comparesfavorably to the levels of improvement previouslyreported in the literature but treatment can begin post-injury. Yet, although short term systemic application ofIGF-I or GH+IGF-I provide compelling evidence forimproved healing in a collagenous matrix, potential sideeffects of altering GH and IGF-I levels for long periods oftime have not yet been fully explored in conjunction withthese data and therefore further study is required beforelong term use is warranted in humans.

ConclusionIn condusion, results support our hypothesis that sys-temic administration of IGF-I improves healing in colla-genous extracellular matrices. Growth hormone alone didnot result in any significant improvement contrary to ourhypothesis, while GH + IGF-I produced remarkableimprovement in hindlimb unloaded animals. Interest-ingly, addition of IGF-I or GH + IGF-I.in HU animalsresulted in recovery of strength measures to a level equalto ambulatory controls, indicating that in fact IGF-I mayb e a plausible therapy for overcoming reduced tissue heal-ing due to disuse from bed rest, immobilization, or micro-gravity. Additionally, although supplementation withIGF-I in ambulatory animals did not result in full recoveryof the mechanical properties at 3 weeks, treatmentresulted in an ~60% increase in tissue strength demon-strating the potential for IGF-I to improve tissue healing.These changes in tissue healing with tissue loading or IGF-

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I supplementation raise important questions regardingthe essential role of mechanical stress for collagen matrixorganization in connective tissues and the mechanisms bywhich systemic IGF-I or GH+IGF-I lead to improved tissuehealing.

MethodsAnimal model and study designThis study was approved by the institutional animal useand care committee and meets N.I.H. guidelines for ani-mal welfare. Seventy-two male Sprague-Dawley rats (248± 6 grams) were used as an animal model. These animalswere randomly divided, into nine groups each containingeight rats: 1) sham control surgery - ambulatory + saline(Sham), 2) healing - ambulatory + saline (Amb + Sal), 3)healing - hindlimb unloaded + saline (HU + Sal), 4) heal-ing - ambulatory + GH (Amb + GH), 5) healing - hind-limb unloaded + GH (HU + GH), 6) healing - Ambulatory+ IGF-I (Amb + IGF), 7) healing - hindlimb unloaded +IGF-I (HU + IGF), 8) healing - ambulatory + GH + IGF-I(Amb + GH + IGF), 9) healing - hindlimb unloaded + GH+ IGF-I (HU + GH + IGF). Each rat in the ambulatory heal-ing and hindlimb suspension (unloaded) groups under-went aseptic surgery to disrupt both knee MCLs. Rats wereanesthetized with an isofluorane anesthetic administeredby facemask using a non-rebreathing delivery system. Theincision area was clipped and prepared for surgery. A skinincision approximately 8 mm long was made on themedial side of the stifle and fascia was incised to exposethe MCL. The MCL was exposed and transected at the jointline, after which the muscles and skin were dosed withsuture. Sham control animals were subjected to identicalsurgical procedures without MCL disruption. All animalswere given an analgesic (Tylenol®/codeine) in their waterfor 72 hours post-surgery. Animals in the sham controland ambulatory healing groups were allowed unrestrictedcage movement. Hindlimb unloaded healing rats weresubjected to hindlimb unloading (24 hours after surgery),which induced substantial stress reduction by eliminatingground reaction forces, using the noninvasive tail suspen-sion protocol of the NASA-Ames center [72]. Only thehindlimbs were suspended (unloaded) and the animalswere allowed to move freely within the cages using theforelimbs while being restricted from kicking the sides ofthe cages. For GH and IGF-I delivery, recombinant humanGH or IGF-I (Genentech, South San Francisco, GA) wasdissolved in sterile saline at a concentration of 0.25 mg/mL Rats received two 0.5 mL injections daily (0830 and1530 hours) of GH, IGF-I, or GH+IGF-I subcutaneously inthe nape of the neck. The dosage of GH and IGF-I werebased upon previous studies in rats [73-75] based on ini-tial body weight and was not adjusted during the courseof the study. Combined GH and IGF-I were administeredsince previous reports have shown that increased levels ofserum IGF-I and IGF-I binding proteins are obtained after

co-injection of GH and IGF-I when compared to eitherGH or IGF-I alone [61,62]. All animals were housed at24 ° C under a 12 hr light and 12 hr dark cycle, fed Purinarat chow, and watered ad libitum. Rats were checked twicedaily for overall health, skin incision healing, food andwater consumption, and the condition of their tails (theharness should prevent slippage without restricting circu-lation). After 3 weeks the animals were euthanized.Immediately after death, animal hindlimbs were flash fro-zen and stored at -80° C until testing. Of the sixteen liga-ments per group, six ligaments were used for mechanicaltesting, six for histology and immunohisto chemistry, andfour for Western blotting.

Bfomechan/co/ testingOn the day of testing, hindlimbs were thawed at roomtemperature from -8 0 ° C. It has been shown that postmor-tem storage by freezing does not change the biomechani-cal properties of ligament [76], Tissue harvest and testingwere performed using methods similar to those previ-ously described [48,77]. Extraneous tissue was carefullydissected away to expose each MCL and the femur-MCL-tibia complex was removed. Ligaments were kept moist inPBS at 25 ° C to prevent dehydration (pH = 7,4), and crosssectional area measured optically. While remaininghydrated the femur-MCL-tibia complex was placed into acustom designed tissue bath system with special structuresto hold the femoral and tibial bone sections of the samplealong the longitudinal axis of the MCL where all fibersappear to load simultaneously (~70° flexion). For tissuestrain measurement, optical markers (impregnated car-bon grease) were placed onto the ligament tissue near theinsertion sites and the tissue bath containing the MCLsamples were inserted into our custom testing machine. Asmall preload of 0.1 N was applied in order to obtain auniform start (zero) point. The ligaments were precondi-tioned (~1% strain for 10 cycles) to allow the tissues tosettle into the grips of the testing bath and then they werepulled to failure in displacement control at 10 %/s. Tissuedeformation was examined and recorded until post-fail-ure. Digital images of each test were analyzed to assess thestructural failure location. For the case of a potential tibialavulsion the distal end of the ruptured tissue was exam-ined for bone.

Tissue displacement was obtained using video dimen-sional analysis (resolution =10 Jim). The change in dis-tance between optical markers was calculated byanalyzing digital frames from the test with N.I.H. Imageusing a custom macro to calculate the change in x-y coor-dinate center (centroid coordinates) of each marker. Force(resolution 0.005 N) was obtained, displayed on thevideo screen and synchronized with displacement. Fromdisplacement data, engineering strain was calculated

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where L0 is the initial length of the tissue at preload (0.1N) and L is the current distance between strain markers:

(1)The stretch ratio is represented by A, and is the currentlength divided by the original length. Lagrangian stresswas calculated as the current force (F) divided by the ini-tial undeformed area

(2)

Stress versus stretch-ratio curves were created and fit withthe microstructural model presented by Hurschler and co-workers [78] to obtain the elastic modulus. Biomechani-cal parameters for comparison are ultimate force, ultimatestress, strain at failure, and elastic modulus (determinedfrom the microstructural model).

Histology and immunohistochemistryImmediately following tissue harvest, samples for histol-ogy were fixed in 10% formalin. After standard histologyprocedures, sections underwent hematoxylin and eosin(H&E) staining. Slides were coverslipped and viewed withlight microscopy. For immunohistochemistry, fixed liga-ments were flash frozen in Optimal Cutting TemperatureMedia (OCT) at -70°C. Cryosections (6 Jim) of each MCLwere mounted onto lysine coated slides for staining.Mounted specimens were washed in a solution of PBS and0.1% Tween-20 (PBST) between all incubation steps.Endogenous peroxidase activity was blocked by incubat-ing slides in 3% hydrogen peroxide for 10 minutes, fol-lowed by blocking with 5% goat serum for 30 minutes atroom temperature in a moist chamber. Tissue sectionswere then incubated for one hour at room temperature ina 1:1 dilution of SP1.D8 (University of Iowa Develop-mental Studies Hybridoma Bank), a primary antibodyagainst the amino-terminal of type I procollagen. Sectionswere then incubated in rat adsorbed secondary antibody(Innovex Biosciences, Parsippany, New Jersey) for 20minutes at room temperature, followed by incubation inhorseradish peroxidase (HRP) labeled strepavidm(Innovex Biosciences, Parsippany, New Jersey) for 20minutes at room temperature. Slides were stained withDAB (3,3'-diarninobenzidine; Innovex Biosciences, Par-sippany, New Jersey) for 5 minutes at room temperature,and then washed in distilled water (twice) for 5 minutes.No counterstaining was performed and no primary anti-body and a species appropriate IgG antibody served asnegative controls. All slides were dehydrated in gradedethanol solutions, and then cleared in xylenes prior tocover-slipping. Light microscopy was used to examineeach specimen and digital images of each MCL specimen

were taken at lOOx magnification. The area of stainingwas measured using ImageJ software after establishing aconstant threshold across images.

Muttiphoton laser scanning microscopyMultiphoton Laser Scanning Microscopy (MPLSM) wasperformed on hematoxylin and eosin stained slides usingan optical workstation [79,80] to better examine collagenorganization in tissue sections. Since, MPLSM of formalinfixed paraffin embedded sections produces images of col-lagen structure and organization that far exceed the detailprovided with standard histology under brightfield illu-mination, MPLSM was employed to detect potential dif-ferences in collagen matrix organization that have beenpreviously reported in healing ligaments from ambula-tory and hindlimb unloaded animals using scanning elec-tron microscopy [48]. The excitation source was aTi:sapphrre laser (Spectra-Physics-Millermium/Tsunanii,Mountain View, CA) producing around lOOfs pulsewidths and tuned to 89 0 nm. The beam was focused ontothe sample with a Nikon 40X Plan Fluor oil-immersionlens (N.A. = 1.4).

Western Wot analysisLigaments were finely minced and boiled in 3X Laemmlibuffer for 10 minutes. Following centrifugationto removetissue debri, protein was separated by SDS-PAGE on 7.5%gel, and electro-transferred to PVDF membrane (Milli-pore, Billerica, MA), Membranes were blocked with TEST(10 mM Tris, 150 mM NaCL, 0.3% Tween-20) plus 5%Blotto (Santa Cruz Biotechnology, Santa Cruz, CA), fol-lowed by incubation with either rabbit anti-collagen type-I (Biotrend, Germany) or mouse anti-collagen type-Ill(Abeam; FH-7A) for 30 minutes at 37°C or anti-IGF-Ireceptor antibody (Cell Signaling Technology, Danvers,MA) overnight at 4 ° C. The membrane was then incubatedin secondary HKP-conjugated donkey anti-rabbit or anti-mouse antibody (Jackson ImmunoResearch Lab; 0.8 ug/ml) and visualized using the ECL Plus detection reagent(Amersham, Piscataway, NJ). For analysis of the ratio ofcollagen I to III the membranes were probed for type IIIcollagen then stripped and re-developed to confirm theabsence of residual signal, and then reprobed for type Icollagen. IGF-I receptor levels were normalized to levels ofGAPDH (anti-GAPDH; Santa Cruz Biotechnology, SantaCruz, CA). Following automated development of anumber of exposures to confirm linearity, densitometryanalysis was performed with ImageJ software. The valuesfor each group were normalized to the Sham group toallow comparison of data across multiple Western blotexperiments. Specificity of the collagen antibodies wasconfirmed by blotting purified type I and III collagen (BDBiosciences) and a panel tissues known to b e composed oftypes I and III collagen. Cross-reactivity of the antibodiesfor other collagens was negligible (data not shown). The

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anti-collagen type I antibody did not cross-react with typeIII collagen consistent with the manufacturers report thatthe antibody has minirnal cross reactivity to types II, III,IV, V, and VI collagen. The anti-collagen type III antibodydid not substantially cross-react with type I collagen con-sistent with the manufacturers data indicating that theantibody does recognize collagen types I, II, IV, V, VI andX.

Statistical analysisDue to the non-factorial nature of our experimentaldesign, statistical analysis was performed using a one-wayanalysis of variance (ANOVA) followed by pair wise com-parisons. The level of significance was set to 0.05 andanalysis was performed with SAS PROG MIXED (SASInstitute, Inc., Gary, NC). Fisher's protected least signifi-cant difference test was used for post-hoc multicompari-sons. For analyzing failure location data, the non-parametric Rruskal-Wallis test was performed.

AbbreviationsExtracellular matrix (ECM), Growth hormone (GH), Glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH), Hind-limb Unloaded (HIT), Insulin-like growth factor I (IGF-I),Insulin-like growth factor receptor (IGFR), Medial collat-eral ligament (MCL), Multiphoton Laser Scanning Micro-scopy (MPLSM), Platelet derived growth factor (PDGP);Group Abbreviations: sham control surgery - ambulatory +saline (Sham), healing - ambulatory + saline (Amb + Sal),healing - hindlimb unloaded + saline (HU + Sal), healing- ambulatory + GH (Amb + GH), healing - hindlimbunloaded + GH (HU + GH), healing - Ambulatory + IGF-I (Amb + IGF), healing - hindlimb unloaded + IGF-I (HU+ IGF), healing - ambulatory + GH + IGF-I (Amb + GH +IGF), healing - hindlimb unloaded + GH + IGF-I (HU +GH + IGF).

Authors' contributionsPPP conducted and analyzed all biomechanical testingand multiphoton microscopy experiments, performedanalysis of histology, assisted in immunohistochemistryanalysis and western blotting with AAO, and prepared themanuscript and figures. AAO additionally contributedwith manuscript preparation. KWG performed rmmuno-histochemistry experiments. REG managed animal treat-ments and tissue harvest (with DAM). DAM, REG, andACV participated in the study design (with PPP and RV),performed and/or supervised animal surgeries, and coor-dinated IGF-I and GH treatment. RV oversaw all aspects ofthe study and assisted in manuscript preparation. Allauthors assisted in critical editing the manuscript.

AcknowledgementsThis work was funded by NASA (Grant #NAG9-11S2). Growth hormoneand IGF-I were provided through a gift from Genentech Inc., South SanFrancisco, CA. The authors thank Dr. Dennis Heisey for his statistical

insight, and Kevin Hiceiri and John White at the Laboratory for Optical andComputational Instrumentation (LOCI) for assistance with multiphotonmicroscopy. In addition the authors would like to thank individuals at theNASA-Ames center who contributed to the animal model: Lisa Baer,Megan Moran, Cyra Fung, Giulia Gurun, David Garcia, Pria Seenan, WilliamWade, and Dr. Charles E Wade.

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