S. Thomopoulos
G. R. Williams
L. J. Soslowsky
1 McKay Orthopaedic Research Laboratory, University of Pennsylvania, Philadelphia, PA 19104-6081Tendon to Bone Healing:
Differences in Biomechanical,
Structural, and Compositional
Properties Due to a Range of
Activity Levels
Little knowledge exists about the healing process of the tendon to bone insertion, and hence little can be done to improve tissue healing. The goal of this study is to describe the healing of the supraspinatus tendon to its bony insertion under a variety of loading conditions. Tendons were surgically detached and repaired in rats. Rat shoulders were then immobilized, allowed cage activity, or exercised. Shoulders that were immobilized demonstrated superior structural (significantly higher collagen orientation), composi-tional (expression of extracellular matrix genes similar to the uninjured insertion), and quasilinear viscoelastic properties (A⫽0.30⫾0.10 MPa vs. 0.16⫾0.08 MPa, B⫽17.4 ⫾2.9 vs. 15.1⫾0.9, and 2⫽344⫾161 s vs. 233⫾40 s) compared to those that were ex-ercised, contrary to expectations. With this knowledge of the healing response, treatment modalities for rotator cuff tears can be developed. 关DOI: 10.1115/1.1536660兴
Introduction
Surgical repair of tendon to bone injuries is often followed by a post-operative rehabilitation protocol that consists of a period of immobilization and/or a period of exercise, with the belief that the mechanical environment plays an important role in the healing process 关1–3兴. Current repair and rehabilitation protocols, how-ever, often provide a less than desired outcome and can result in re-injury and subsequent increased disability 关4,5兴. Determining the role of the mechanical environment on the healing of tendon to bone would greatly enhance the ability of the clinician to pre-scribe a proper post repair protocol.
Tendon to bone healing has been studied in a number of animal models关6–10兴. Most studies have looked at the structural prop-erties of the healing tissue, but no studies to date have examined the viscoelastic material properties of the healing tissue. In other models, the structural properties of the injured tissue increased over time, but never reached the properties of the original site 关9,10兴. It is difficult to determine the quality of the repair tissue in these models, however, because the material properties were not measured. Additionally, the viscoelastic properties of the tendon to bone insertion site have never been studied; the time dependent behavior of the tissue has therefore never been measured. To elu-cidate the mechanisms through which changes in biomechanical properties occur, a careful analysis of the structure and the com-position of the healing tendon to bone insertion site is also necessary.
Using animal models, a number of previous studies have evalu-ated the effect of increased or decreased loading on normal ten-dons and ligaments as well as on healing tenten-dons and ligaments 关2,3,11–14兴. The properties of uninjured tendons and ligaments generally increase with increased loading, and decrease with de-creased loading 关11,12兴. Results in healing tissue show similar trends 关2,3,14兴. No studies to date have examined the effect of
loading on the healing of tendon to bone. Based on these previous studies, it was expected that increased activity in our model would result in improved properties. However, it was noted that the ef-fect of the mechanical environment may be site specific and de-pendent on the loading regime.
The Quasilinear Viscoelastic共QLV兲 Model, originally proposed by Fung关15兴, was used in our study to quantitatively determine the material properties of the healing insertion site. Through this model, viscous and elastic parameters can be extracted from stress relaxation experiments. This model has been used successfully to model a variety of soft tissues关15–19兴, but it has never been used to model the normal or the healing tendon to bone insertion site. Note that the properties reported in our study are the apparent 共i.e., average兲 properties of the insertion, and do not take into account possible variation in properties along the length of the insertion.
Histologic analysis was used to describe the tissue and cell morphology of the healing insertion site. The types of cells present, their level of activity and maturity, and the integration of the collagen into its bony interface helped to explain the differ-ences found in the biomechanical property results. Additionally, histologic analysis was coupled with polarized light microscopy to quantitatively determine the orientation of the collagen matrix. By quantitating collagen orientation, a parameter of structural organi-zation was deduced. This information was useful in explaining the differences found in the material properties from the biomechani-cal studies.
Finally, the expression of various extracellular matrix compo-nents was measured locally in order to assess the composition of the insertion site. Because the composition of the uninjured inser-tion site changes dramatically over a very short distance 共i.e., from tendon to bone兲, it is useful to evaluate intact tissue sections 共as opposed to homogenized tissue samples兲. In situ hybridization was used to make a comparative assessment of the expression of a variety of genes on whole tissue sections. Using this technique, mRNA expression was visualized throughout the complex tendon to bone transition zone关20兴.
The global hypothesis for this study was that viscoelastic bio-mechanical properties共elastic: A, B and viscous: C,1, 2) and structural properties 共collagen orientation兲 of healing tendon to
1Correspondence to: Louis J. Soslowsky, Ph.D., McKay Orthopaedic Research
Laboratory, University of Pennsylvania, 424 Stemmler Hall, Philadelphia, PA 19104-6081, 215-898-8653, 215-573-2133共fax兲, [email protected]
Contributed by the Bioengineering Division for publication in the JOURNAL OF BIOMECHANICALENGINEERING. Manuscript received January 2002; revised manu-script received August 2002. Associate Editor: L. A. Setton.
bone insertions treated with increased activity levels will be supe-rior to those of untreated healing tendon to bone insertions. Materials and Methods
Study Design. Studies were approved by the University of Pennsylvania IACUC. Sixty-five Sprague-Dawley rats 共452 ⫾60 g兲 were operated upon bilaterally to detach and repair the supraspinatus tendon at its insertion site关20兴. Post-operative ac-tivity level was controlled in three groups: cast immobilization 共IM, n⫽25), cage activity 共CA, n⫽20), and exercise 共EX, n ⫽20). For immobilization, the left limb was placed in a plaster cast at 90 deg forward elevation, 20 deg abduction, with the right shoulder free. Plaster was wrapped around the arm, shoulder, and upper torso, to completely immobilize one shoulder. The plaster surface was coated with polymethylmethacrylate to prevent the rat from clawing or chewing through the cast. Immobilization was effective in preventing gross movement of the shoulder joint. The effectiveness of the cast immobilization was verified daily by ob-servation. Cage activity animals were allowed free movement im-mediately post-operatively. For animals in the exercise group, an exercise regime was chosen based on a protocol which caused increased metabolic activity in rats关21,22兴 and was less strenuous than what is required to incite overuse injuries in rats关23兴. Rats were allowed one week of post-operative recovery before being placed on the treadmill for exercise. After this recovery period, rats in the EX group were exercised at 10 m/min, for 1 hour/day, for 5 days/week. Initial pilot studies verified that the repairs re-mained intact in all activity level groups a few days after the surgical injury and repair with no gap formation visible grossly or histologically.
Animals were sacrificed at 2, 8, or 16 week timepoints共Fig. 1兲. A separate group of 11 uninjured animals共CTL兲 served as a nor-mal, un-operated control group. Shoulders were allocated for structural, compositional, or biomechanical analysis. Note that for the CTL, CA, and EX groups, both shoulders had undergone the same treatment. Therefore, one shoulder was allocated for bio-logic and histobio-logic assays, and the contralateral was allocated for biomechanical assays. For the IM group, since only one shoulder was immobilized, separate animals were necessary for structural/ compositional assays and biomechanical assays共Fig. 1兲. Note that
the contralateral limbs of the immobilized rats were left free. This was necessary to allow the animals to feed and ambulate with ease. The data from this limb was not used in this study due to uncertainty regarding its activity level. It is likely that the animals used this limb more than the cage activity level animals. There-fore, all data regarding the free shoulder of the immobilized rats were discarded.
Structural Assays. Histologic tissue sections from 5 animals in each group at each timepoint were used for structural, in situ hybridization, and histologic analysis. Briefly, specimens were fixed in 4% paraformaldehyde, decalcified at 4°C for up to 2 weeks共using EDTA/polyvinyl pyrolidione兲, and embedded in par-affin. For structural analysis, multiple sections for each specimen were stained with 0.1% picrosirrius red and viewed under polar-ized light关24,25兴. Digital images were taken at 5 deg increments for specimen orientations between 35 deg and 145 deg. Images were then analyzed to quantitatively determine the distribution of collagen orientation at 109 points per tissue section 共validation studies demonstrated that 109 points were sufficient to effectively represent a tissue section兲. Entropy 共H兲 and second angular mo-ment of momo-mentum共M2兲 were calculated for each specimen 关26兴. Fiber distributions were compared using the Kolmogorov-Smirnov test, and H and M2 were compared using a MANOVA followed by an LSD post-hoc test.
Compositional Assays. In situ hybridization was performed on histologic sections to determine the localized expression of a variety of extracellular matrix genes共collagens: I, II, III, IX, X, XII, proteoglycans: aggrecan, decorin, biglycan, other: alkaline phosphatase兲 关20兴. Specimens were hybridized to 33P-labeled sense and antisense RNA probes (2.5⫻105cpm mm⫺1) for the genes listed above. All sense probes were hybridized to sections of the tendon to bone insertion site adjacent to those used for antisense probes. Slides were coated with Ilford K5 emulsion, developed after 10-20 days of exposure time, and stained with 2% toluidine blue before being photographed. Sections for each speci-men and each gene were examined by two investigators blinded to group and timepoint to determine the presence of mRNA at the tendon to bone insertion. Specimens were graded using a four part scale共high, intermediate, low, and no detectable levels of mRNA expression兲. Because levels of expression indicated by silver emulsion grains are dependent on exposure time and probe length, comparisons were only valid within each in situ hybridization run relative to the positive control. Experiments were designed so that each in situ run contained a complete set of injured specimens and multiple normal 共uninjured兲 specimens for each gene studied. Therefore, valid comparisons could be made between activity lev-els, and relative to control for each probe. Comparison of expres-sion levels between different genes and over time were therefore not performed.
Biomechanical Assays. The remaining animals from the 8 week timepoint共IM: n⫽10, CA: n⫽10, EX: n⫽10兲 and the CTL group共n⫽11兲 were geometrically quantified and biomechanically tested. For cross-sectional area, thickness and width were mea-sured as described previously关23兴. Briefly, the thickness of the insertion was determined using an indenter probe attached to a high resolution linear variable differential transformer. The width of the insertion was then measured using optical methods. Be-cause the insertion is relatively broad and flat, a rectangular cross section was assumed and the cross sectional area was calculated as the thickness multiplied by the width. Cross sectional area was measured at the center of the insertion共i.e., halfway between the start of bone proper and the start of tendon proper兲. This location was easily identifiable by gross inspection. For biomechanical testing, specimens were immersed in a 39°C共rat body tempera-ture兲 PBS bath and preconditioned for ten cycles. Specimens were fixed at the bony end using polymethylmethacrylate and at the tendon end using a spring loaded grip 共the tendon was first
se-Fig. 1 Tendon to bone healing study design. Note that for the CTL, CA, and EX groups, both shoulders had undergone the same treatment. Therefore, one shoulder was allocated for structural and compositional assays, and the contralateral was allocated for biomechanical assays. For the IM group, since only one shoulder was immobilized, separate animals were necessary for structuralÕcompositional assays and for biome-chanical assays„Fig. 1….
cured between two pieces of fine grit sand paper using Krazy Glue™兲. A custom testing rig was used to secure the grips. This rig allowed for accurate setting of the angle between the tendon and the bone共set to 140 deg兲. A stain line placed on the tendon within the grip allowed for verification that no slipping had oc-curred. A stress relaxation test was performed共to 5% strain at an elongation rate of 50%/s followed by 600 seconds of relaxation to equilibrium兲, followed by a constant strain rate test to failure 共0.5%/s兲. A slow strain rate was chosen for the constant strain rate test to ensure quasi-static conditions. Local tissue strain was mea-sured optically using texture correlation techniques关27兴. Geomet-ric and mechanical data were statistically compared between groups using a Multifactor Analysis of Variance followed by a Least Squares Difference post-hoc test.
To determine properties of the insertion site, the Quasilinear Viscoelastic Model共QLV兲 关15,17兴 was used to extract viscous and elastic parameters from stress relaxation experiments. The QLV model was used to model the viscoelastic behavior of the normal and healing tendon to bone insertion sites. QLV theory assumes that the stress relaxation function of the tissue is dependent on both extension and time and can be expressed as Eq.共1兲, where G共t兲 is the reduced relaxation function,e(A) is the elastic stress response, andAis the applied step increase in strain:
共A,t兲⫽G共t兲e共A兲 (1) The stress response at time t can be obtained for a general strain history through Eq.共2兲:
共t兲⫽
冕
0 t G共t⫺兲 e共兲 d (2)For the relaxation function G共t兲 in Eq. 共3兲, C,1, and2are the material coefficients describing the relaxation characteristics of the material and E1is the exponential integral:
G共t兲⫽ 1⫹C
冉
E1冉
t 2冊
⫺E1冉
t 1冊冊
1⫹C ln冉
2 1冊
(3)The elastic stress response can be given by the exponential law e
()⫽A(eB⫺1), where A and B are material coefficients de-scribing the elastic characteristics of the material.
For a specimen extended at a finite strain rate in time interval t0 and combining Eq.共2兲 and Eq. 共3兲 with the exponential law, the stress response is given by Eq.共4a兲 and Eq. 共4b兲 关15,17兴:
共t兲⫽ AB␥ 1⫹C ln
冉
2 1冊
冕
0 t再
1⫹C冋
E1冉
t⫺ 2冊
⫺E1冉
t⫺1冊册冎
eB␥d ⬍t⬍t0, (4a) 共t兲⫽ AB␥ 1⫹C ln冉
2 1冊
冕
0 t0再
1⫹C冋
E1冉
t⫺ 2冊
⫺E1冉
t⫺1冊册冎
eB␥d t0⬍t⬍⬁, (4b) where A, B, C,1, and2are the material coefficients determined by curve fitting the stress relaxation data to Eq. 共4b兲 and ␥ ⫽d/dt. The model was implemented in Matlab software 共Natick, Ma兲 using approximations described in the literature 关28兴. A non-linear least squares method was used to curve fit all five param-eters to the experimental data. The QLV model is defined by these five coefficients which describe the elastic component共A and B兲 and the viscous component共C,1, and2) of the material. Due tothe complex geometry and possible inhomogeneity at the insertion site, properties reported in this study are best described as appar-ent properties共i.e., average properties兲 and not true intrinsic prop-erties. It is also important to note that the properties reported here are for the tissue in extension. Properties of the tissue in compres-sion were not measured.
Results
Structural Results. Histologically, dramatic increases in cel-lularity and changes in cell shape共to a more rounded morphology兲 were seen in the healing tissue共immobilization: IM, cage activity: CA, and exercise: EX兲 compared to the uninjured control 共CTL兲. Subtle improvements were seen in the injured tissue with decreas-ing activity and over time.
The injured tissue was significantly less organized than the un-injured control when comparing distributions共Fig. 2兲 共based on the Kolmogorov-Smirnov statistical test兲 and when comparing the properties entropy共H兲 and second angular moment 共M2兲 共Table 1, Figs. 2, 3兲. Figure 2 schematically demonstrates the differences between groups at the 2 week and 16 week timepoints with nor-malized density plots. The distribution of each group is
approxi-Fig. 2 Normal curve fits for the distribution data at 2 weeks and 16 weeks. Note that the IM group demonstrated superior organization relative to EX. Also note that the EX group showed little improvement over time.
mated by a normal curve, making comparisons between groups easier to visualize. When comparing activity level groups, the IM group demonstrated significantly increased organization compared to either the CA or the EX group, especially at the longer time-points 共Table 1, Fig. 2兲. CA also demonstrated significantly in-creased organization compared to the EX group, again most dra-matically at the longer timepoints. There were fewer differences between the groups at the early timepoint共2 weeks兲. It is clear from these plots and tables that among the injury groups, the IM group demonstrated the highest level of organization, whereas the EX group demonstrated the lowest level of organization. In-creased activity therefore caused a decrease in the collagen orien-tation. When looking at changes over time, significant differences in the angular distribution of collagen were demonstrated in all of the groups based on the Kolmogorov-Smirnoff statistical test
共Table 1, Fig. 2兲. However, these differences were less apparent in the EX group共Table 1, Fig. 2兲. Properties H and M2 improved for the IM and for the CA group over time but were not changed for the EX group 共Table 1兲. Specifically, for the IM group, H im-proved significantly over time, while for the CA group H and M2 improved significantly over time. Therefore, improvement over time was hindered by increased activity level.
Compositional Results. The composition of the healing tis-sue共as measured qualitatively by in situ hybridization兲 was clos-est to normal in the IM group and furthclos-est from normal in the EX group. Of note are the increased levels of type I collagen and the decreased levels of type X collagen at the healing insertion site. Also of note are the increased levels of type XII collagen and aggrecan, and the decreased levels of decorin in the healing ten-don. Changes due to activity level were noted for a number of the genes studied共Table 2, Figs. 4–6兲. Expression of type I collagen, type XII collagen共Fig. 4兲, type II collagen, and aggrecan were higher in the IM group compared to the EX group at the 2 and 8 week timepoints. Expression of type III collagen was lower in the IM group compared to the EX group共Fig. 5兲. The ratio of type I to type III collagen expression共an indication of scar tissue兲 was therefore highest in the IM group compared to the EX group 共in-dicating increased scar formation in the EX group兲. Type X col-lagen was lower than CTL in all injured groups共Fig. 6兲. Note that
Table 2 Qualitative differences in gene expression due to ac-tivity level for three timepoints„2wk, 8wk, 16wk… „nd: no differ-ence….
Gene Role Gene 2wk 8wk 16wk
tendinous matrix
collagen I IM⬎EX,CA IM,CA⬎EX EX⬎IM, CA
collagen III nd EX,CA⬎IM EX⬎IM,
CA collagen XII IM⬎CA,EX IM⬎CA,EX nd cartilaginous
matrix
aggrecan IM⬎CA,EX IM⬎EX,CA nd collagen II IM⬎CA IM⬎EX,CA nd
collagen IX nd IM⬎EX,CA nd
fibrillogenesis decorin nd nd nd
biglycan nd nd nd
other collagen X nd nd nd
alk. phosphatase nd nd nd
Table 1 Polarized light analysis results for the parameters second angular moment „M2… and entropy „H…. „Mean¿Õ
Àstandard deviation,*: significant difference relative to CTL
†pË0.05‡, &: significant change from previous timepoint
„within group… †pË0.05‡, #: significant difference relative to IM
†pË0.05‡, and $: significant difference relative to CA †p
Ë0.05‡…. Group Timepoint n M2共°兲 H CTL 7 95 ⫾ 44 2.6 ⫾ 0.3 2wk 4 198 ⫾ 119 3.2 ⫾ 0.3* IM 8wk 3 81 ⫾ 67 2.4 ⫾ 0.5& 16wk 5 95 ⫾ 45 2.7 ⫾ 0.3 2wk 4 525 ⫾ 234*,# 3.7 ⫾ 0.3* CA 8wk 5 209 ⫾ 67& 3.3 ⫾ 0.3*,&,# 16wk 4 109 ⫾ 107& 2.5 ⫾ 0.7& 2wk 5 195 ⫾ 73$ 3.2 ⫾ 0.3* EX 8wk 5 255 ⫾ 135*,# 3.3 ⫾ 0.4*,# 16wk 5 221 ⫾ 157 3.2 ⫾ 0.4*,#,$
Fig. 3 Collagen orientation in the CTL„A…, IM„B…, and EX„C… groups at the 8 week timepoint. Light field images are pre-sented on the left, polarized light images are prepre-sented on the right„picrosirius red stain, 5Ãobjective…. Note the decreasing level of organization when comparing CTL„most organized, A…, IM„B…, and EX„least organized, C….
Fig. 4 Type XII collagen expression in the IM„A…and EX„B… groups at 2 weeks. Light field images are presented on the left, darkfield images are presented on the right „10Ã objective…. Note the increased levels of type XII collagen in the IM „A… specimen compared to the EX specimen„B….
comparisons over time are not appropriate because they would arise from different in situ hybridization experimental runs.
Biomechanical Results. Cross sectional area was signifi-cantly increased in all injury groups relative to CTL共Table 3, Fig. 7兲. Area was significantly decreased in the IM group compared to the CA and EX groups共Table 3, Fig. 7兲. Increased activity there-fore stimulated an increase in tissue formation. Note that these results are only for the tissue at the 8 week timepoint.
Biomechanically, there were significant changes in structural properties relative to uninjured control and in apparent viscoelas-tic properties due to injury and due to activity level共Table 3, Figs. 8 –9兲. Structural properties of the healing insertion 共peak load ‘Lpeak’, equilibrium load ‘Lequil’, and stiffness ‘k’兲 were decreased in all groups relative to CTL, but these properties were unaffected by activity level共Table 3, Fig. 8兲. For material viscoelastic prop-erties共modulus E, QLV elastic: A, B, and QLV viscous: C,1and 2), significant changes were seen in all injury groups for most properties relative to CTL共Table 3, Fig. 9兲. All viscoelastic
erties in the EX group were changed compared to CTL. All prop-erties except for1 were changed in the CA group compared to CTL. When comparing IM to CTL, changes were only seen for A, B, and 1. When activity levels were compared, changes were seen relative to IM. There was a significant decrease in A and B when comparing IM to CA. There was a significant decrease in A, B, and2when comparing IM to EX. Note that these results are only for the tissue at the 8 week timepoint.
Fig. 6 Type X collagen expression in the CTL„A… and IM 2 week„B…groups. Light field images are presented on the left, darkfield images are presented on the right„20Ã objective…. Note the decreased levels of type X collagen in the healing tissue„B…compared to the uninjured tissue„B…
Fig. 5 Type III collagen expression in the IM„A…and EX „B… groups at 16 weeks. Light field images are presented on the left, darkfield images are presented on the right„10Ã objec-tive…. Note the increased levels of type III collagen in the EX„B… specimen compared to the IM specimen„A….
Table 3 Results for geometry„cross sectional area…, structural properties„peak load ‘Lpeak’, equilibrium load ‘Lequil’, and
stiff-ness ‘k’…, and material properties„modulus E, and QLV proper-ties A, B, C,1, and2….„Mean¿ÕÀstandard deviation,*: sig-nificant difference relative to CTL †pË0.05‡, # significant difference relative to IM†pË0.05‡…. CTL IM CA EX Area (mm2) 2.2⫾0.8 12.6⫾3.1* 14.6⫾3.6* 17.3⫾4.3*,# k共N兲 55.3⫾12.7 38.0⫾12.8.2* 35.4⫾8.1* 39.2⫾9.4* Lpeak共N兲 7.0⫾2.3 3.2⫾1.6* 3.1⫾1.1* 2.9⫾0.7* Lequil共N兲 2.4⫾1.0 0.97⫾0.6* 0.81⫾0.4* 0.75⫾0.25* E共MPa兲 203.1⫾85.9 25.3⫾11.6* 21.4⫾6.9* 18.3⫾7.8* A共MPa兲 1.29⫾0.57 0.30⫾0.10* 0.13⫾0.06*,# 0.16⫾0.08*,# B 33.6⫾9.1 17.4⫾2.9* 15.1⫾0.9*,# 15.1⫾0.9*,# C 0.18⫾0.07 0.27⫾0.12 0.32⫾0.14* 0.34⫾0.17* 1共s兲 0.045⫾0.013 0.059⫾0.015* 0.055⫾0.011 0.058⫾0.015* 2共s兲 412⫾171 344⫾161 253⫾85* 233⫾40* ,#
Fig. 7 Geometry results for CTL, IM, CA, and EX. Cross sec-tional area was significantly increased in the injured groups relative to CTL. Area was significantly increased in the EX group compared to the IM group.„Mean¿ÕÀstandard devia-tion,*:significant difference relative to CTL†pË0.05‡, #
sig-nificant difference due to activity level†pË0.05‡….
Fig. 8 Results for structural properties peak load, equilibrium load, and stiffness. Properties for all injury groups were signifi-cantly decreased compared to CTL. There were no differences when comparing activity level groups.„Mean¿ÕÀstandard de-viation,*:significant difference relative to CTL†pË0.05‡, #
Discussion
A surgical injury model was utilized to study the effect of load-ing environment on the healload-ing tendon to bone insertion site. Ac-tivity level was controlled in three groups through immobilization, cage activity, or exercise. Geometric results indicated that the ex-ercised group had an increased cross sectional area compared to the immobilized group. Biomechanical structural properties were not significantly different when comparing activity level, but they were significantly decreased compared to uninjured control. When looking at material viscoelastic properties, the immobilization group demonstrated superior properties compared to the exercise group, although inferior to uninjured control. When examining the structure of the tissue, it was found that collagen organization was increased in the immobilized group compared to the exercised group. Organization improved over time in the immobilization and the cage activity groups, but did not improve over time in the exercise group. Organization was inferior in all groups relative to uninjured control. When examining the gene expression at the healing insertion site, it was found that the level of gene expres-sion for a variety of extracellular matrix proteins was affected by activity level. Notably, there was an increased ratio of type I col-lagen to type III colcol-lagen and increased expression of fibrocarti-lage genes in the immobilized group compared to the exercised group.
The biomechanical, organizational, and biologic studies each provided valuable information describing the healing tendon to bone insertion site. While the ultimate functional measure of a tissue may be its biomechanical properties, differences in these properties cannot be fully explained without further study. By combining a description of tissue organization and a description of the gene expression, a more complete understanding of the
differ-ences in biomechanical properties can be achieved. The hypoth-esis for this study was that the mechanical properties of healing tendon to bone insertion sites treated with increased activity levels would be superior to those of immobilized insertion sites. The results presented here indicate that the converse was true; healing insertion sites treated with decreased activity levels were superior to those of mobilized insertion sites. The studies measuring col-lagen orientation and gene expression fully support this biome-chanical result. Specifically, elastic parameters A and B were in-creased in the immobilized tissue relative to the exercised tissue. These material properties are an indication of the elastic quality of the tissue. The increase in collagen orientation, coupled with the increase in the ratio of type I collagen to type III collagen 共indica-tive of decreased scar兲 fully support this result. Both the structure and the composition of the tissue in the immobilized group were therefore superior to that in the exercised group. It is concluded here that the quality of the tissue is improved with decreased loading.
Other groups have verified the intuitive conclusion that the or-ganization of collagen and the composition of collagen type will alter the tissue properties关29–31兴. Literature in uninjured tissue, or in tendon and ligament midsubstance healing, has proposed that decreased loads within the tissue will lead to a decrease in the tissue properties关11,32兴. These results, however, are not entirely applicable to the healing tendon to bone insertion site. At the insertion collagen fibers must integrate into the bone and a fibro-cartilaginous transition zone must form for an optimal repair关6兴. This is a multifaceted repair process, more complex than the re-pair between two tendon or ligament ends. Two materials with dramatically different properties共bone and tendon兲 must integrate and form an effective bond. The optimal mechanical environment for this healing process has not been looked at prior to this study. In the normal uninjured insertion site, decreased loading will cause a decrease in properties共i.e., a physiologic level of load is necessary to stimulate remodeling and maintain the properties of the tissue兲 关11兴. Healing of tendon to bone may require a unique loading environment for optimal repair. Increased loading across the healing insertion may cause microdamage at the interface, and prevent integration of collagen fibers into the bone. Decreased loading may provide a protective environment which allows inte-gration to occur. This protective effect may also lead to the pro-duction of a higher quality material, albeit at a slower rate. Exer-cise was effective in increasing the production of matrix, but this matrix was largely scar tissue, with relatively low material prop-erties and poor organization. By protecting the healing interface from excess loading, the normal wound healing response共which would normally consist of scar formation兲 is directed down a dif-ferent pathway resulting in decreased scar.
When looking at the viscous properties, C,1, and2, which describe the time varying behavior of the tissue, only2 demon-strated significant differences due to activity level. The exercised tissue reached equilibrium load faster than the immobilized tissue. This can be explained by the increased expression of genes such as type II collagen and aggrecan in the immobilized group relative to the exercised group. These genes are predominantly found in cartilaginous tissues, which show significant compressive strength and viscoelastic behavior 关33,34兴. Aggrecan has been shown to dictate viscoelastic effects in these tissues due to its high affinity to water. By controlling the flow of water into and out of the tissue, the time varying behavior is influenced. This may be the case in the immobilized tissue in this study, which demonstrated increased levels of aggrecan expression relative to exercised tissue.
Immobilization was effective in promoting the expression of genes normally found in cartilage and fibrocartilage. This was manifested biomechanically as improved viscous properties in the immobilized tissue compared to the exercised tissue. The high levels of unidirectional loading in the exercised tissue may have promoted the expression of genes normally found in tendon
Fig. 9 Results for QLV parameters A, B, and2, and typical stress relaxation curve fits for IM and EX. ‘A’ was significantly decreased in injured groups relative to CTL. ‘A’ was signifi-cantly increased in the IM group compared to the EX and CA groups. ‘B’ was significantly decreased in injured groups rela-tive to CTL. ‘B’ was significantly increased in the IM group com-pared to the EX and CA groups. ‘2’ was significantly de-creased in the CA and EX groups relative to CTL. ‘2’ was significantly increased in the IM group compared to the EX groups. The IM group had a higher peak stress, and maintained this stress for a longer period of time, than the EX group.„Mean
¿ÕÀ standard deviation, *: significant difference relative to
CTL†pË0.05‡, # significant difference due to activity level†p Ë0.05‡….
共whose function is to transfer loads in tension兲. This may not be optimal in the repair of tendon to bone, due to the multidirectional nature of the stresses which are necessary for an effective tendon to bone transition zone. The ability to form a fibrocartilaginous transition zone may be enhanced by immobilization through a prevention of the high tensional loads that would normally occur across the healing interface.
The level of collagen organization was influenced negatively by increased levels of loading. These results are not explained by the biologic studies performed in this study. It was expected that the levels of biglycan and decorin, which influence collagen fibrillo-genesis, would be different between activity level groups. This was not the case, as no differences were seen. It is likely that other factors, both biologic and mechanical, had a greater influence on the organization of the tissue
An optimal loading environment may exist which will promote the development of strong material properties in healing tendon insertion sites. Early in the repair process, protection of the inter-face may be necessary, to allow integration of collagen fibers into bone. Low levels of stress in the healing tissue may also be ben-eficial by promoting an increase in the levels of proteins such as aggrecan and type II collagen. In the long term, however, higher levels of stress may be necessary to promote remodeling of the tissue. The optimal environment at the insertion site also differs along its length. Compressive stress at the forming fibrocartilagi-nous zone may be beneficial to promote the production of type X collagen, type II collagen, and aggrecan. At the tendinous end of the insertion, on the other hand, tensional stresses may be neces-sary to promote the production of type I collagen, biglycan, and decorin. The optimal loading environment at the cellular level may therefore vary locally and temporally. This complex relation-ship can only be fully understood with further study.
The biomechanical, organizational, and compositional studies performed for this study are supportive of each other. Biome-chanical studies demonstrated detrimental effects on elastic and viscous properties due to increased activity. In an independent set of studies, polarized light microscopy showed a detrimental effect on collagen organization due to increased activity. Finally, a third set of independent assays showed detrimental effects on compo-sition due to increased activity. Therefore, in this model of tendon to bone healing, detrimental effects due to increased activity were demonstrated using three independent assays. These results are consistent with a study examining the orientation of matrix mate-rial in healing rabbit medial collateral ligaments共MCL兲 关26兴. In this study, rabbits were given complete MCL midsubstance tears and either had their joints immobilized or were allowed cage ac-tivity. Collagen matrix orientation was quantified using image analysis techniques. It was found that the immobilized ligaments were better aligned than the cage activity ligaments at the three and six week timepoints. These differences were not found at the longer fourteen week timepoint. Therefore, increased activity was detrimental and delayed the healing process. The results from the current study are also supported by a study by Kamps et al.关13兴, which looked at the influence of immobilization and the influence of exercise on scar formation. In this study, it was found that the tensile modulus was decreased with increasing activity, while structural properties were unchanged with increasing activity. A qualitative improvement in collagen organization was also seen with immobilization. The initially surprising results of this study are therefore fully supported by independent assays in our rat insertion model as well as by two other injury models presented in the literature.
The results of this study lend support to the clinical practice of immobilization or decreased activity after surgical repair. In our animal model, increased activity directly after surgical repair was detrimental to the healing process. While the exercise was effec-tive in increasing the amount of tissue present, it was ineffeceffec-tive in improving the quality of the tissue present. Therefore, protec-tive immobilization may be beneficial in the clinical setting to
prevent micro-damage at the interface during early tendon to bone healing. It remains to be seen what role remobilization will have in the rehabilitation protocol after surgical repair. It is possible that healing may be optimized by an initial period of immobiliza-tion followed by a period of remobilizaimmobiliza-tion. It is also important to note here that the immobilized group does not represent a zero load group. It is expected that there is some muscle firing that occurs in the immobilized limb, providing enough load to direct the reorganization of the collagen fibers.
With any tendon to bone healing study, there is always a con-cern that the repair may not stay intact, and that a gap may form between the tendon edge and its bony insertion. However, in the rat animal model, in our hands, we did not see any gross gap formation at early or late timepoints. This visual observation was corroborated by the histologic data. Initial pilot studies verified that the repairs remained intact in all activity level groups a few days after the surgical injury and repair. Having said this, there is a slight possibility that gradual partial detachment may have oc-curred at the repair site over time which was not detectable by gross observation or histologic assessment. It is unlikely however, that such a small gap could lead to the changes that were seen in all assays in this study.
The biomechanical results from this study are for only one timepoint共8 weeks兲. A complete assessment of the healing pro-cess will require biomechanical evaluation at both early 共e.g., 2 weeks兲 and long 共e.g., 32 weeks兲 timepoints. It is expected that properties in all groups would improve over time. Structural and compositional data from our study indicate that the trends seen in the biomechanical data may remain even at 16 weeks of healing. It is expected that immobilization will at some point become det-rimental to the properties of the tissue and that remobilization will be necessary to further improve the properties.
A limitation of this study is that the activity level of the animals was not quantitatively monitored. While the environment in our model was not quantified, it was extremely well controlled. Tread-mill exercise was applied at a precisely controlled speed and du-ration. This protocol did not vary by more than 10% in any rat. Immobilization was achieved through casting, which completely immobilized the joint. The only loads present on the healing ten-don were from random isometric contractions. The effectiveness of the casting was evaluated daily by visual inspection. The only loading conditions that were not well characterized were the ones during normal cage activity. Our intention here was to provide an activity level that was intermediate between immobilization and exercise. True quantification of the activity levels would provide valuable information relating the true loading across the repair site with the healing process. This information is not available in this study due to the difficult nature of obtaining in vivo loading in tissues. Future studies will develop methods to measure in vivo loads using minimally invasive techniques.
Future directions will focus on four basic areas of study: 1兲 further elucidating the mechanisms of injury and repair, including a functional assessment of the joint 共e.g., range of motion兲, 2兲 increasing the clinical relevance of the model by developing a chronic injury model, 3兲 developing treatment modalities for the injured tissue, 4兲 quantitatively measuring and controlling the me-chanical environment, and 5兲 measuring the compressive proper-ties of the tissue.
Acknowledgments
This study was supported by the National Institutes of Health, the Aircast Foundation, and the Orthopaedic Research and Educa-tion FoundaEduca-tion. The authors thank M. Bey, M. Favata, C. Flana-gan, J. Gimbel, C. Haldis, G. Hattersley, W. Johannessen, M. Mertens, and J. Smith for their contributions.
References
Knowl-edge Update: Shoulder and Elbow, Norris, T. R., ed., American Academy of
Orthopaedic Surgeons, pp. 289–298.
关2兴 Gelberman, R. H., Woo, S. L., Lothringer, K., Akeson, W. H., and Amiel, D., 1982, ‘‘Effects of Intermittent Passive Mobilization on Healing Canine Flexor Tendons,’’ J. Hand Surg.关Am兴, 7, pp. 170–175.
关3兴 Gomez, M., Woo, S. L., Amiel, D., Harwood, F., Kitabayashi, L., and Matyas, J., 1991, ‘‘The Effects of Increased Tension on Healing Medial Collateral Ligaments,’’ Am. J. Sports Med., 19, pp. 347–354.
关4兴 Iannotti, J. P., Naranja, R. J., and Gartsman, G. M., 1994, ‘‘Surgical Treatment of the Intact Cuff and Repairable Cuff Defect: Arthroscopic and Open Tech-niques,’’ Orthopaedic Knowledge Update: Shoulder and Elbow, Norris, T. R., ed., Rosemont IL, American Academy of Orthopaedic Surgeons, pp. 151–156. 关5兴 Naranja, R. J., Iannotti, J. P., and Gartsman, G. M., 1994, ‘‘Complications in Rotator Cuff Surgery,’’ Orthopaedic Knowledge Update: Shoulder and Elbow, Norris, T. R., ed., Rosemont IL, American Academy of Orthopaedic Surgeons, pp. 157–166.
关6兴 Oguma, H., Murakami, G., Iwanaga, H., Aoki, M., and Ishii, S., 2001, ‘‘Early Anchoring Collagen Fibers at the Bone-Tendon Interface are Conducted by Woven Bone Formation: Light Microscope and Scanning Electron Microscope Observation Using a Canine Model,’’ J. Orthop. Res., 19, pp. 873– 880. 关7兴 Fujioka, H., Thakur, R., Wang, G. J., Mizuno, K., Balian, G., and Hurwitz, S.
R., 1998, ‘‘Comparison of Surgically Attached and Non-Attached Repair of the Rat Achilles Tendon-Bone Interface. Cellular Organization And Type X Col-lagen Expression,’’ Connect. Tissue Res., 37, pp. 205–218.
关8兴 Liu, S. H., Panossian, V., Al-Shaikh, R., Tomin, E., Shepherd, E., Finerman, G. A., and Lane, J. M., 1997, ‘‘Morphology and Matrix Composition During Early Tendon to Bone Healing,’’ Clin. Orthop. Relat. Res., 339, pp. 253–560. 关9兴 Rodeo, S. A., Arnoczky, S. P., Torzilli, P. A., Hidaka, C., and Warren, R. F., 1993, ‘‘Tendon Healing in a Bone Tunnel,’’ J. Bone Jt. Surg., 74A, pp. 1795– 1803.
关10兴 St. Pierre, P., Olson, E. J., Elliott, J. J., O’Hair, K. C., McKinney, L. A., and Ryan, J., 1995, ‘‘Tendon Healing to Cortical Bone Compared With Healing to a Cancellous Trough,’’ J. Bone Jt. Surg., 77A, pp. 1858 –1866.
关11兴 Woo, S. L., Gomez, M. A., Sites, T. J., Newton, P. T., Orlando, C. A., and Akeson, W. H., 1987, ‘‘The Biomechanical and Morphological Changes in the MCL of the Rabbit After Immobilization and Remobilization,’’ J. Bone Jt. Surg., 69A, pp. 1200–1211.
关12兴 Woo, S. L. Y., Gomez, M. A., Amiel, D., Ritter, M. A., Gelberman, R. H., and Akeson, W. H., 1981, ‘‘The Effects of Exercise on The Biomechanical and Biochemical Properties of Swine Digital Flexor Tendons,’’ J. Biomech. Eng., 103, pp. 51–56.
关13兴 Kamps, B. S., Linder, L. H., DeCamp, C. E., and Haut, R. C., 1994, ‘‘The Influence of Immobilization Versus Exercise on Scar Formation in the Rabbit Patellar Tendon After Excision of The Central One Third,’’ Am. J. Sports Med., 22, pp. 803– 811.
关14兴 Bray, R. C., Shrive, N. G., Frank, C. B., and Chimich, D. D., 1992, ‘‘The Early Effects of Joint Immobilization On Medial Collateral Ligament Healing in an ACL-Deficient Knee: A Gross Anatomic and Biomechanical Investigation in the Adult Rabbit Model,’’ J. Orthop. Res., 10, pp. 157–166.
关15兴 Fung, Y. C., 1972, ‘‘Stress-Strain-History Relations of Soft Tissues in Simple Elongation,’’ Biomechanics: Its Foundations And Objectives, Fung, Y. C., Per-rone, N., and Anliker, M., eds., Prentice-Hall, San Diego, CA, pp. 181–208. 关16兴 Huang, C. Y., Wang, V. M., Roh, M. S., Pawluk, R. J., Flatow, E. L., and Mow,
V. C., 1998, ‘‘Temperature Dependent Viscoelastic Properties of the Su-praspinatus Tendon,’’ Advances in Bioengineering—American Society of Me-chanical Engineering: Biomedical Engineering Division, 39, pp. 251–252. 关17兴 Kwan, M. K., Lin, T. H., and Woo, S. L., 1993, ‘‘On the Viscoelastic
Proper-ties of the Anteriomedial Bundle of the Anterior Cruciate Ligament,’’ J. Bio-mech., 26, pp. 447– 452.
关18兴 Woo, S. L., Gomez, M. A., and Akeson, W. H., 1981, ‘‘The Time and History Dependent Viscoelastic Properties of the Canine Medical Collateral Liga-ment,’’ J. Biomech. Eng., 103, pp. 293–298.
关19兴 Sauren, A. A. H. J., Hout, M. C., Steenhoven, A. A., Veldpaus, F. E., and Janssen, J. D., 1983, ‘‘Mechanical Properties of Porcine Aortic Valve Tissues,’’ J. Biomech., 16, pp. 327–338.
关20兴 Thomopoulos, S., Hattersley, G., Rosen, V., Mertens, M., Galatz, L., Williams, G. R., and Soslowsky, L. J., 2002, ‘‘The Localized Expression of Extracellular Matrix Components in Healing Tendon Insertion Sites: An In Situ Hybridiza-tion Study,’’ J. Orthop. Res., 20, pp. 454 – 463.
关21兴 Uribe, J. M., Stump, C. S., Tipton, C. M., and Fregosi, R. F., 1992, ‘‘Influence of Exercise Training on the Oxidative Capacity of Rat Abdominal Muscles,’’ Respir. Physiol., 88, pp. 171–180.
关22兴 Wegner, J. A., Lund, D. D., Overton, J. M., Edwards, J. G., Oda, R. P., and Tipton, C. M., 1987, ‘‘Select Cardiovascular and Metabolic Responses of Dia-betic Rats to Moderate Exercise Training,’’ Med. Sci. Sports Exercise, 19, pp. 497–503.
关23兴 Soslowsky, L. J., Thomopoulos, S., Tun, S., Flanagan, C. L., Keefer, C. C., Mastaw, J., and Carpenter, J. E., 2000, ‘‘Overuse Activity Injures the Su-praspinatus Tendon in an Animal Model. A Histologic and Biomechanical Study,’’ J. Shoulder Elbow Surg., 9, pp. 79– 84.
关24兴 Whittaker, P., and Canham, P. B., 1991, ‘‘Demonstration of Quantitative Fabric Analysis of Tendon Collagen Using Two-Dimensional Polarized Light Micros-copy,’’ Matrix, 11, pp. 56 – 62.
关25兴 Dickey, J. P., Hewlett, B. R., Dumas, G. A., and Bednar, D. A., 1998, ‘‘Mea-suring Collagen Fiber Orientation: A Two Dimensional Quantitative Macro-scopic Technique,’’ J. Biomech. Eng., 120, pp. 537–540.
关26兴 Frank, C., MacFarlane, B., Edwards, P., et al., 1991, ‘‘A Quantitative Analysis of Matrix Alignment in Ligament Scars: A Comparison of Movement Versus Immobilization in an Immature Rabbit Model,’’ J. Orthop. Res., 9, pp. 219– 227.
关27兴 Bey, M. J., Song, H. K., Wehrli, F. W., and Soslowsky, L. J., 2001, ‘‘A Non-Contact, Non-Destructive Method for Quantifying Intratissue Strains,’’ J. Bio-mech. Eng., in press.
关28兴 Nigul, I., and Nigul, U., 1987, ‘‘On the Algorithms of Evaluation of Fung’s Relaxation Function Parameters,’’ J. Biomech., 20, pp. 343–352.
关29兴 Woo, S. L. Y., An, K. N., Frank, C. B., et al., 2000, ‘‘Anatomy, Biology, And Biomechanics Of Tendon and Ligament,’’ Orthopaedic Basic Science, 2nd
edition, Buckwalter, J. A., Einhorn, T. A., and Simon, S. R., eds., American
Academy of Orthopaedic Surgeons, pp. 581– 616.
关30兴 Bateman, J. F., Lamande, S. R., and Ramshaw, J. A., 1996, ‘‘Collagen Super-family,’’ Extracellular Matrix, vol 2, Comper, W. D., ed., Amsterdam, Over-seas Publishers Association, pp. 23– 67.
关31兴 Sakai, H., Koibuchi, N., Ohtake, H., Tamai, K., et al., 2001, ‘‘Type I and Type III Procollagen Gene Expressions in the Early Phase of Ligament Healing in Rabbits: An In Situ Hybridization Study,’’ J. Orthop. Res., 19, pp. 132–135. 关32兴 Amiel, D., Woo, S. L., Harwoon, F. L., and Akeson, W. H., 1982, ‘‘The Effect
of Immobilization on Collagen Turnover in Connective Tissue: A Biochemical-Biomechanical Correlation,’’ Acta Scandinavica Orthopaedica, 53, pp. 325– 332.
关33兴 Fosang, A. J. and Hardingham, T. E., 1996, ‘‘Matrix Proteoglycans,’’
Extra-cellular Matrix, vol 2, Comper, W. D., ed., Amsterdam, Overseas Publishers
Association, pp. 201–229.
关34兴 Mankin, H. J., Mow, V. C., Buckwalter, J. A., Iannotti, J. P., and Ratcliffe, A., 2000, ‘‘Articular Cartilage Structure, Composition, and Function,’’
Ortho-paedic Basic Science, 2nd edition, Buckwalter, J. A., Einhorn, T. A., and
