tendon cell contraction of collagen–gag matrices in vitro: effect of cross-linking

*Correspondence address: Department of Orthopaedic Surgery,Brigham and Women's Hospital, Harvard Medical School, 75 FrancisStreet, Boston, MA 02115, USA. Tel.: #1-617-732-6702; fax: #1-617-732-6705.

E-mail address: [email protected] (M. Spec-tor).

Biomaterials 21 (2000) 1607}1619

Tendon cell contraction of collagen}GAG matricesin vitro: e!ect of cross-linking

Donna Schulz Torres!, Toby M. Freyman", Ioannis V. Yannas!, Myron Spector!,#,$,*!Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, USA

"Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, USA#Department of Orthopaedic, Surgery, Brigham and Women+s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA, USA

$Rehabilitation Engineering Research and Development, West Roxbury Veterans Administration Medical Center,West Roxbury, MA, USA

Received 26 October 1998; accepted 9 February 2000

Abstract

The contraction of connective tissue cells can play important roles in wound healing and pathological contractures. The e!ects ofthis contractile behavior on cell-seeded constructs for tissue engineering have not yet been investigated. The goal of this work was toinvestigate in vitro tendon cell-mediated contraction of collagen}glycosaminoglycan (GAG) matrices cross-linked using selectedmethods. Highly porous collagen}GAG sponges were seeded with calf tendon cells and the projected area and DNA content of thesponges measured at 3, 7, 14, and 21 days post-seeding. Immunohistochemistry was performed to determine if a-smooth muscle actin(SMA) was associated with the cell contraction of the matrices. Dehydrothermal (DHT) treatment alone was not su$cient to resistcontraction by the seeded tendon "broblasts. Cross-linking of the collagen}GAG sponges to the extent that the modulus was threetimes that of sponges treated by DHT alone was necessary to resist contraction. SMA was seen in the cytoplasm of most cells in allsponges at all time periods. The results provide a rational basis for the determination of the mechanical properties of collagen matricesrequired for engineering certain connective tissues. ( 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Tendon; Contraction; Cross-link; Collagen; Cell

1. Introduction

Porous type I collagen sponge-like matrices have beenresearched in vivo as sca!olds for regeneration of tissuesincluding skin [1}3], bone [4,5], knee meniscus [6],articular cartilage [7], esophagus [8], dura mater [9],and muscle [10]. Other studies have investigated thebehavior of collagen}glycosaminoglycan (GAG) copoly-mers as implants in skin [11,12] and bone [13]. Onespeci"c form of a collagen}GAG sca!old, serving as ananalog of extracellular matrix [14], has been used suc-cessfully as an implant to facilitate the regeneration ofdermis [15}17] in animal and human subjects and peri-pheral nerve [18] in an animal model. Recent animal

studies have also yielded favorable results in the use ofthis type I collagen}GAG matrix in defects in tendon[19] and a type II collagen}GAG sca!old in treatingdefects in articular cartilage [20].

Implantation of sca!olds in various tissues has high-lighted the di!erence in the regenerative capacity be-tween highly cellular and well-vascularized tissues suchas dermis, and avascular or poorly vascularized tissueswith low cell density and low mitotic activity, such asarticular cartilage, ligament and tendon. In these lattertissues, sca!olds alone may not induce regeneration.This, and the goal of accelerating the process of regenera-tion in tissues that have already demonstrated the poten-tial, has prompted the investigation of collagen matricesseeded with cultured cells: dermal "broblasts [21,22],keratinocytes [21,23], ligament cells [24], meniscus cells[25] and articular chondrocytes [26}29].

Culturing articular chondrocytes and meniscus cells incollagen sponges has led to recent reports of cell-me-diated contraction [25,27] of the matrix in vitro. Thecontraction of connective tissue cells can play a bene"cial

0142-9612/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 0 5 1 - X

Table 1Treatment groups

Group Collagen type Treatment

DHT I Dehydrothermal (DHT) only, 24 hUV I DHT; ultraviolet (UV) light 12 hETH I DHT; 70% ethanol (ETH) 10 min0.5G I DHT; Glutraldehyde (G), 0.25%, 30 min12G I DTH; Glutraldehyde (G), 0.25%, 12 h24G I DHT; Glutraldehyde (G), 0.25%, 24 hIIE II UV light, 16 h; 70% ethanol 10 min

role in the closure of certain types of wounds, but canalso be responsible for pathological contractures. Thee!ects of this contractile behavior on cell-seeded con-structs for tissue engineering have not yet been investi-gated. The contraction of cell-seeded sca!olds becomesproblematic when the shape and size of the implant andits pores are changed. Alterations in the pore diametermay impede migration of cells from the surroundingtissue into the sca!olds. Changes in the size and shape ofthe matrices can make them di$cult to "t to a speci"cimplant site and, when persisting in vivo, could causeseparation of the sponge from surrounding host tissue.The cell-mediated shrinkage of unseeded implants, withthe attendant problems noted above, may also occurafter implantation as endogenous cells in"ltrate thesponge and express a contractile cell phenotype. The goalof this study was to investigate in vitro the tendon cell-mediated contraction of collagen}GAG sca!olds treatedwith selected cross-linking methods to produce matricesof varying sti!ness. While previous studies have investi-gated the e!ect of cross-linking methods on tissue com-patibility [30] and the changes in mechanical propertiesduring degradation [31], none have determined thee!ects of cross-linking treatment on cell-mediated contrac-tion.

It has been well documented that certain "broblastsin vivo and in vitro can assume a contractile phenotype,as re#ected in: (1) expression of a smooth muscle actinisoform, a-smooth muscle actin (SMA) [32,33]; (2) ultra-structural features consistent with smooth muscle cells[34]; and (3) the response to pharmacological agentsknown to selectively a!ect smooth muscle cells [35].Fibroblasts meeting these criteria have been referred toas myo"broblasts [35]. Several investigators have sug-gested that the expression of the contractile SMA isoformalone can be considered the signature of this phenotype[36]. The temporal appearance of myo"broblasts withtissue contraction in vivo [37,38] led to their associationwith wound closure and certain pathological contrac-tures. In the present study a monoclonal antibody toSMA [39] was employed for the immunohistochemicalidenti"cation of the contractile actin isoform in the ten-don cells seeded in the matrices.

2. Materials and methods

2.1. Collagen matrices

A co-precipitate of bovine tendon type I collagen (In-tegra Life Sciences, Plainsboro, NJ) and chondroitin6-sulfate (Sigma Chemical Co., St. Louis, MO) was pre-pared following a previously published protocol [14].The slurry was freeze-dried to form a porous col-lagen}GAG copolymer, 2}3 mm thick. The pore charac-teristics were comparable with those of a collagen}GAG

sponge used for dermal regeneration [40]. An averagepore diameter of approximately 120 lm was estimatedfrom light microscopy of microtomed sections of glycolmethacrylate embedded samples, 5 lm thick and stainedwith aniline blue. The GAG content was approximately9% (by weight). A type II collagen}GAG matrix pre-pared from treated porcine cartilage (Chondrocell, Geist-lich Biomaterials, Wolhusen, Switzerland) was alsostudied. The type II matrix had an average pore diameterof about 90 lm and a GAG content of approximately10% [27,41].

2.2. Cross-linking treatments

All type I matrices were dehydrated in a vacuum ovenat 1053C and 30 mTorr for 24 h for dehydrothermal(DHT) cross-linking prior to any additional chemical orphysical cross-linking. Types I and II collagen matriceswere placed 30 cm from an ultraviolet (UV) lamp rated at5.3 W total output, 55.5 W/cm2 at 1 m for UV cross-linking. Type I matrices were cross-linked for 12 h, andtype II matrices were cross-linked for 16 h, with thematrices turned over midway through the cross-linkingperiod to expose each side to the same amount of UVradiation. Our prior experience with the two collagentypes indicated that the handling characteristics of thematrices were suitable when UV-cross-linked for thesetimes. Type I collagen matrices were also cross-linkedwith 0.25% glutaraldehyde in 0.05 M acetic acid for30 min, 12 h, or 24 h (0.5, 12, and 24 G, respectively). Thespecimens were then exhaustively rinsed in sterile waterto remove any traces of free aldehyde. Type II matriceswhich had been UV cross-linked and type I matriceswhich had not undergone any cross-linking other thanDHT treatment were soaked in 70% ethanol for 10 min,and then rinsed 3 times with sterile saline (ETH). Whileethanol is not a cross-linking agent for collagen, its e!ectswere investigated because it is often used as a sterilizingsolution and/or to pre-wet collagen matrices prior to cellseeding. The treatment groups are summarized inTable 1.

1608 D. Schulz Torres et al. / Biomaterials 21 (2000) 1607}1619

2.3. Mechanical testing

A testing chamber was designed to contain a col-lagen}GAG matrix specimen, 25 mm wide]85 mmlong]3 mm thick, in 373C-phosphate bu!ered salinewhile an Instron materials tester applied an tensile load.Specimens were tested at a rate of 1 mm/min until failure.A high-resolution video camera mounted over the testingchamber recorded the change in distance between paral-lel lines that had been marked to de"ne a gage length inthe center of the specimen. The engineering stress andstrain were computed.

A stress}strain plot was created for each specimen, anda second-order polynomial regression was performed inorder to "t a curve to the data. The derivative of thecurve equation was calculated in order to determine themodulus of elasticity for each specimen. The stress}strainbehavior of these highly porous matrices re#ected geo-metric changes in the pore architecture as well asdeformation of the collagen material. The slope ofthe curve might, therefore, be referred to as the&apparent modulus of elasticity'. For the purpose of thispaper, this parameter will be referred to as the modulusof elasticity. For the purpose of comparisons amongtreatment groups, moduli were determined at 5, 10, and15% strain.

2.4. Cell isolation, culture, and matrix seeding

The patellar tendon was harvested from the knee ofa young calf under sterile conditions, and the cells fromthe mid-substance of the tendon were isolated by diges-tion in trypsin-EDTA for 1 h followed by 2}3 h of diges-tion in 0.15% collagenase. Cells were cultured at 373C,5% CO

2, and 95% humidity in DMEM/F12 with 10%

heat inactivated fetal bovine serum (Hyclone Laborator-ies, Logan, UT), 1% L-glutamine, 1% amphotericin B, 2%penicillin/streptomycin, and ascorbic acid at a concentra-tion of 25 mg/ml. Cells were passaged at con#uence.

Fourth passage patellar tendon "broblasts were usedfor the in vitro seeding experiment. A suspension of2.5]105 cells in 50 ll of culture medium was pipettedonto each matrix surface of 9-mm diameter disks thatwere 3 mm thick, bringing the total number of cellsseeded onto each disk to 5.0]105. The cell seeding den-sity was selected in part on the basis of prior studies inour laboratory [25] using 9.0]105 meniscus cells per9-mm diameter collagen}GAG disk in order to yieldseeded matrices that approximated the cell density inthat tissue (2}2.6]103 cells/mm2). The lower seedingdensity of tendon cells in the current study was based onan estimate that there would be a lower range of celldensity for tendons. While the cellularity of tendons hasnot been quanti"ed, a recent study [42] reported a rangeof cell density in the human anterior cruciate ligamentfrom 0.5 to 2.5]103 cells/mm2.

Culture of the cell-seeded discs was performed in 12-well culture dishes precoated with a thin layer of sterile2% agarose (Bio-rad standard low-mr, Bio-rad, Rich-mond, WA) to prevent the cells from attaching to thebottom of the well rather than to the matrix. The cell-seeded disks were incubated in a humidi"ed chamber at373C and 5% CO

2, with medium changes every 3 days.

Cultures were terminated 3, 7, 14, and 21 days post-seeding. Unseeded matrices exposed to the same cultureconditions were employed as controls.

2.5. Measurement of the diameter of the matrices

Projected images of disks were digitized with the ma-trices appearing in the image as a dark region on a lightbackground. MATLAB code was written to analyze theimages by determining the number of pixels occupyingthe region of the sca!old. The measurements were calib-rated by imaging an object of known diameter. An aver-age diameter was calculated from the area assuminga circular cross-section. Using this procedure the dia-meter of each matrix specimen in phosphate bu!eredsaline was obtained before it was seeded with cells, orplaced into the culture medium as a non-seeded control,and then again at the time of termination of the culture.In this way each specimen served as its own time zerocontrol. The &percentage of original diameter' was cal-culated by dividing the diameter of the matrix at thetermination of the culture by its original diameter. &Cell-mediated contraction' (CMC) was computed by subtract-ing the mean value of the percentage decrease of thenon-seeded controls for a treatment group from the per-centage decrease in diameter of each seeded specimen inthe treatment group. The CMC was then normalized tothe number of cells in the matrix by dividing it by themean value for the DNA content (see below) of the groupat each termination period.

2.6. DNA assay

Six matrices from each treatment group at the 14- and21-day periods were allocated for analysis of DNA con-tent in order to provide a measure of the number of cellsin the sponges at the times when it was expected thatmost of the contraction would be occurring. Six matricesfrom each of four of the treatment groups (DHT, UV,12G, and 24G) were also allocated for determination ofDNA contents after 3 and 7 days to provide additionalinformation about the changes in cell content of thesematrices through the 21-day period. The matrices weredigested in 1 ml of 0.5% papain/bu!er solution in a 653Cwater bath. A 200-ml aliquot of the digest was combinedwith 40 ml of Hoechst dye d33258 (Polysciences Inc,Warrenton, PA), and evaluated #uorometrically (HoeferScienti"c Instruments DNA Fluorometer). The resultswere extrapolated from a standard curve using calf

D. Schulz Torres et al. / Biomaterials 21 (2000) 1607}1619 1609

thymus DNA. A standard curve constructed using ten-don cells from the same source as those seeded in thematrices was used to estimate the cell number from theDNA measurement.

2.7. Histology and immunohistochemistry

At the termination of the cultures, two specimens ofthe cell-seeded matrices in each treatment group, allo-cated for histology and immunohistochemistry, were"xed in formalin for 48 h. After "xation, specimenswere embedded in para$n and sections, 7-lm thick, weremicrotomed and "xed onto glass slides. Selected sectionswere stained with hematoxylin and eosin (H & E). TheSMA isoform was detected by immunohistochemistryusing a monoclonal antibody (Sigma Chemical, St. Louis,MO, USA). Depara$nized slides were rehydratedthrough graded ethanol solutions and then treated with0.1% trypsin (Sigma Chemical, St. Louis, MO, USA) for1 h. Endogenous peroxidase was quenched with 3% hy-drogen peroxide for 5 min. Nonspeci"c sites wereblocked using 20% goat serum (Cat. d G9023, SigmaChemical) for 30 min. The sections were then incubatedwith a 1 : 400 dilution of mouse monoclonal antibody toSMA (Cat. d A2547, Sigma Chemical) for 2 h at roomtemperature. Negative control sections, on the samemicroscope slides, were incubated with mouse serum(1 : 800 dilution; Cat. d M5905, Sigma Chemical) insteadof the primary antibody. The sections were then incu-bated with a biotinylated goat anti-mouse IgG secondaryantibody (Cat. d B7151, Sigma Chemical) diluted to1 : 200 in PBS for 1 h followed by 20 min of incubationwith a$nity puri"ed avidin (1 : 50 dilution of ExtrAvidinperoxidase; Cat. d E2886, Sigma Chemical). The label-ing was developed using the AEC chromogen kit (Cat.d AEC-101, Sigma Chemical) for 10 min. Counterstain-ing with Mayer's hematoxylin for 20 min was followed bya 20-min tap-water wash and cover-slipping withwarmed glycerol gelatin.

Light microscopy was used to analyze H & E stainedsections for the morphology and density of cells in thematrices. The morphology of cells at the edge and centerof the disks was described qualitatively as spheroid,ovoid, or fusiform based on the shape of the nucleus andestimating the cell boundaries. Cells with nuclear aspectratios (length to width) from 1 to 2 were consideredround; those with aspect ratios up to approximately4 were classi"ed as ovoid and those with greater ratios,fusiform. A grid eyepiece was used to measure the depthof cell in"ltration into the disks in H& E stained cross-sections and to count the percentage of cells stainedpositive for SMA in "ve random areas of four sectionsfrom two specimens for each group analyzed. The changein pore size of both seeded and control matrices with timewas also assessed qualitatively.

2.8. Statistical analyses

Results are reported as means and standard errors ofthe mean (SEM). One-way analysis of variance(ANOVA) was used to determine the signi"cance of thee!ect of cross-link treatment on modulus of elasticity.Two-way ANOVA was used to determine the signi"-cance of the e!ects of time in culture and whether thematrices were seeded on the percentage of the originaldiameter (i.e., a measure of matrix contraction). Two-wayANOVAs were also employed to determine the signi"-cance of the e!ects of time in culture and treatment on(a) the DNA content of the matrices, (b) the change indiameter of cell-free and cell-seeded matrices, (c) cell-mediated contraction, and (d) cell-mediated contractionnormalized to DNA. Bonferroni/Dunn post hoc testingwas used to determine the signi"cance of di!erencesbetween speci"c groups. Linear, binomial, and exponen-tial regression analyses were performed to determine themeaningfulness of the correlation of the cell-mediatedcontraction with modulus of elasticity.

3. Results

3.1. DNA contents of the matrices

Suspending the cells in a small amount of culturemedium (2.5]105 cells/50 ll) allowed the full amount tobe pipetted onto each surface of the sponge. In someinstances a portion of the cell suspension spilled o! thesponges and onto the agarose-coated wells. The swollentype II sponges appeared to absorb the suspension betterthan the type I sponges. Cells did not attach to theagarose coating.

Results from assay of the DNA contents of matrices infour of the treatment groups after three days of culture(Fig. 1) were converted to cell numbers using the stan-dard curve (approximately 6]10~4 ng DNA/cell):

Treatment Cell no.Mean$SD

DHT 323 997$11 750UV 696 898$50 14312G 589 477$33 78624G 532 827$21 236

These results demonstrate the variability in retention andproliferation of the cells in the matrices with the varioustreatments.

Despite the fact that the same number of cells wasseeded in all matrices, there was a three-fold di!erence inthe DNA content of the matrices with the most and


Fig. 1. DNA content of collagen matrices. DNA content is expressed asnanograms of DNA per 100 ll papain digest.

fewest cells after 14 days (Fig. 1). The highest DNAcontent was found in the type II matrix and the lowest inthe DHT-treated type I sca!old. Two-way ANOVA re-vealed statistically signi"cant e!ects of cross-link treat-ment (P(0.0001) and time (P(0.0001) on DNAcontent of the matrices. Bonferroni/Dunn post hoc test-ing revealed the following signi"cant di!erences betweengroups (all P(0.0001 unless noted): DHT compared tothe other groups; ETH versus the 0.5G, 12G (P(

0.0008), and IIE groups; UV compared to the 0.5G andIIE groups; 0.5G versus the 12G, 24G, and IIE treat-ments; and IIE versus the 12G and 24G groups.

Statistically meaningful increases in DNA contentfrom 14 to 21 days were found in the UV (Bonfer-roni/Dunn post hoc testing; P(0.0001) and the 12G(P(0.0001) groups. During the same interval there weresigni"cant decreases in DNA content in the DHT(P"0.0002) and ETH (P"0.0021) specimens.

3.2. Contraction of the matrices

The non-seeded matrices in several groups underwenta reduction in diameter with time in culture. Two-wayANOVA revealed signi"cant e!ects of cross-link treat-ment (P(0.0001) and time in culture (P(0.0001) on thedecrease in diameter of the non-seeded matrices (Fig. 2a).Bonferroni/Dunn post hoc testing revealed that theDHT, ETH, and IIE groups were signi"cantly di!erentfrom the UV and glutaraldehyde-treated groups. Reduc-tion in diameter of non-seeded control matrices from theDHT and ETH group was evident at 3 days (Fig. 2a).After 7 days the decrease in diameter of the controlgroups leveled o!. The glutaraldehyde-treated controlsdid not noticeably contract throughout the 21-daycourse of the experiment (Fig. 2a).

Two-way ANOVA also demonstrated the signi"cante!ect of treatment (P(0.0001) and time in culture(P(0.0001) on the decrease in diameter of the cell-seeded matrices (Fig. 2b). Bonferroni/Dunn post hoctesting revealed that the DHT and ETH groups were

signi"cantly di!erent from the other groups and that the12 G and 24 G groups were also signi"cantly di!erentfrom all of the other groups (Fig. 2b). After 21 days,tenocyte-seeded DHT matrices contracted to 37$1% oftheir original diameter, ETH matrices to 40$2% oftheir original diameter and UV cross-linked matrices to80$3% (Fig. 2b). Contraction in glutaraldehyde-treatedmatrices varied with the duration the cross-linking treat-ment. After 21 days in culture, seeded 0.5G matrices werecontracted to 72$3% of their original diameter, whilethe average diameters of the 12G and 24G matrices werereduced to 97$1 and 98$1% of their original values,respectively (Fig. 2b). While there were not remarkabledi!erences in the percentage reduction in the diameter ofseeded and non-seeded controls in several of the treat-ment groups (viz., the 12G and 24G groups), two-wayANOVA revealed signi"cant e!ects (P(0.05) of timeand whether the matrices were seeded on the decrease indiameter for each type I collagen treatment group, whenconsidered individually; two-way ANOVA revealeda signi"cant e!ect of seeding but not time on the percent-age decrease in the diameter of the type II matrix group.

The cell-mediated contraction was determined by sub-tracting the mean value for the percent contraction of thenon-seeded controls in the treatment group from thepercentage reduction in the individual cell-seeded matrixspecimens in that group. Two-way ANOVA revealedsigni"cant e!ects of time (P(0.0001) and treatment(P(0.0001) on the cell-mediated contraction. Bonfer-roni/Dunn testing indicated that when each group wascompared to the other groups all were signi"cantly di!er-ent except for the following comparisons: ETH vs. UV,ETH vs. 0.5G, ETH vs. DHT, UV vs. 0.5G, UV vs. DHT,0.5G vs. DHT, and 12G vs. 24G. The negative CMCvalues for the type II specimens re#ected the swelling ofthe cell-seeded specimens relative to the non-seeded con-trols. Of interest was the increase in the cell-mediatedcontraction of the DHT and 0.5G groups between 14 and21 days in culture.

The cell-mediated contraction was normalized to themean DNA content of the matrices, re#ecting cell num-ber, in order to display the relative e!ects of the variouscross-linking treatments on the resistance of the sca!oldsto shrinkage (Fig. 2c). Two-way ANOVA revealed signif-icant e!ects of time (P(0.0001) and treatment(P(0.0001) on the cell-medicated contraction/DNA.Bonferroni/Dunn post hoc testing revealed that eachgroup was signi"cantly di!erent from each other groupexcept for the following comparisons: UV vs. 0.5G, 12Gvs. 24G, 12G vs. IIE, and 24G vs. IIE. Although overallcontraction of seeded DHT group matrices and seededETH group matrices was similar at 21 days (approxi-mately 37 and 40% of the original diameter), when thecell mediated contraction was normalized to DNA con-tent, a two-fold di!erence was found (Fig. 2c). The UVand 0.5G treatments yielded comparable results, which


Fig. 2. (a) Change in diameter of the non-seeded matrices with time in culture (n"6, mean$SEM). Two-way ANOVA revealed signi"cant e!ects oftime in culture and treatment group. (b) Change in the diameter of the seeded matrices with time in culture (n"6, mean$SEM). Two-way ANOVArevealed signi"cant e!ects of time in culture and treatment group. (c) Cell-mediated contraction per ng DNA in the collagen matrices at 14 and 21 days.The cell-mediated contraction for each sample was divided by the mean DNA content for the group (n"6, mean$SEM). Two-way ANOVArevealed signi"cant e!ects of time in culture and treatment group.


Fig. 3. Light micrograph of typical cell distribution and morphology incollagen}GAG matrices at (a) the edge and (b) center of the matrix.H & E stained section of an ethanol (ETH)-treated "broblast-seededcollagen}GAG matrix at 14 days post-seeding.

were about one-half the contraction found with ethanol-treated matrices. Increasing the time of treatment inglutaraldehyde resulted in less shrinkage of the sca!old.Of note was that the contraction in the glutaraldehyde-treated groups (albeit small) was entirely cell mediated.However, because of the relatively small amount of con-traction, little can yet be concluded about these speci-mens. The cell-mediated contraction in the type IImatrices was represented as negative contraction (Fig. 2c)because the shrinkage in the control specimens at sometime periods was greater than that of the seeded speci-mens.

3.3. Histology and immunohistochemistry

Histologically, at 3 days post-seeding, cells were prim-arily attached to the periphery of the matrices in allgroups. Few cells were visible in the central portions ofthe sca!olds. At 7 days, the "broblasts had migratedmidway through the depth of the matrices (approxim-ately 1.0}1.5 mm), with more cells at the edges of the diskand the center still sparsely populated. Cells at the pe-riphery of all matrices were elongated along the edges.For DHT, ETH, UV, and 0.5G groups, cells in the edgeregion of the disks were primarily fusiform or ovoid. Thecells in the center region of these disks were also bothfusiform and ovoid, with a slightly higher percentage ofovoid cells.

At 14 days post-seeding, the DHT and ETH (Fig. 3)matrices in particular were surrounded with what ap-peared to be a multi-layered cell capsule, while the celllayers at the edges of the type II, and UV and glutaral-dehyde-treated type I collagen matrices were not as thickand did not completely surround the matrices. Fibro-blasts were found distributed in the center of the disks,but in the DHT, ETH (Fig. 3) and UV groups, themajority of cells in the disk were seen in the edge regions.Fibroblasts in the 12- and 24-h glutaraldehyde cross-linked matrices were primarily fusiform in both the edgeand center regions after 14 days in culture. Cell morpho-logy in type II collagen matrices was similar to themorphology in the DHT and ETH groups, with a slightlyhigher percentage of cells at early time periods whichwere spheroid in shape.

By the 21st day in culture, both the edge and centerregions of matrices from all treatment groups had be-come populated with elongated "broblasts (Fig. 4).

Pores of type II collagen, UV and glutaraldehydecross-linked matrices underwent no apparent shrinkage.In all matrices in which signi"cant overall contractionoccurred, the pores at the edges of the matrix becamecontracted before pores in the center (Figs. 5 and 6). At 14days, clear di!erences could be seen between the porediameter distributions at the edge and central regions ofthe sca!olds (Fig. 5). By 21 days the periphery of theDHT-treated matrices displayed few open pores (Fig. 6).

A notable "nding was the presence of SMA-positivecells in all treatment groups at all time periods (Fig. 6). Inpara$n sections labeled with the SMA antibody, thechromogen was a prominent reddish-brown color, andfound in the cytoplasm of cells. Control sections on thesame microscope slide that lacked the primary antibodyonly occasionally displayed a slight pink tint in cells andalso in the extracellular matrix. The percentage of SMA-containing cells in the matrices was generally greaterthan 90%. Comparable percentages of positive cells werefound in the central and peripheral regions of the ma-trices, and cells with all morphologies stained positive.There were no remarkable di!erences in the percentagesof SMA-positive cells among cross-link treatment groupsor with time in culture.

3.4. Mechanical testing and ewect of apparent modulusof elasticity on cell-mediated contraction

Each of the 3 type II specimens allocated for mechan-ical testing failed at the clamped ends. These data werenot included in the results. Data for 1 of 3 of the speci-mens in the original ETH, UV, and 12G groups were lost


Fig. 4. Light micrograph of cell distribution and morphology inglutaraldehyde-treated collagen}GAG matrices at (a) the edge and (b)the center of the matrix. H & E stained section of a 24-h glutaraldehydecross-linked "broblast-seeded collagen}GAG matrix at 21 days postseeding.

Fig. 5. Light micrograph of an H & E stained section of ETH matrix,14 days post seeding, showing the decrease in the diameter of pores atthe edge (a) due to the contraction of the cell layer on the surface of thespecimen. Pores in the central region of the sample (b) displayed lesspore contraction.

due to hardware or software malfunction. One of theoriginal three 0.5G specimens was damaged while beingplaced into the mechanical test apparatus. Additionalspecimens were prepared to increase the sample size to atleast 3 in all groups. The additional specimen for the 0.5Ggroup failed at approximately 12% strain, precluding theuse of a modulus value for 15% strain (see below). It was,therefore, excluded from the study.

The stress}strain curves for the type I collagen}GAGspecimens displayed a characteristic concave-up shapeand were bounded by the results for the DHT and 24Ggroups (Fig. 7). The "t of the curves with binomial equa-tions demonstrated high coe$cients of determination(Fig. 7). One-way ANOVA revealed a signi"cant e!ect ofcross-link treatment on modulus at 5 (P(0.002), 10(P(0.0005), and 15% (P(0.0003) strain (Fig. 8). Bon-ferroni/Dunn post hoc testing revealed signi"cant di!er-ences between the following groups: DHT versus UV at5 (P(0.0002), 10 (P(0.0004), and 15% (P(0.0004)strains; DHT compared to 24G at 5 (P(0.0002), 10(P(0.0001), and 15% (P(0.0001) strains; and ETHversus 24G at 10 (P(0.002) and 15% (P(0.001)strains.

As might be expected, cell-mediated contraction of thematrices decreased with increasing sti!ness of the scaf-folds. Linear regression analysis demonstrated a signi"-cant inverse e!ect of modulus of elasticity (at 15% strain)on cell-mediated contraction at 21 days (ANOVA,P(0.03), with a coe$cient of determination of 0.73.Regression plots using binomial and exponential rela-tionships yielded coe$cients of determination ofr2"0.94 and 0.95, respectively, for contraction data ob-tained at 21 days and the modulus calculated at 15%strain. Correlations obtained using contraction data at14 days and modulus determined at 5 and 10% strainsdid not have as high coe$cients of determination.

4. Discussion

The results demonstrated the e!ects of selected cross-linking treatments on the degree of tendon cell contrac-tion of collagen}GAG analogs of extracellular matrix invitro. A linear regression model inversely correlating thecell-mediated contraction to modulus of elasticity hada coe$cient of determination that was high enough (0.73)


Fig. 6. Light micrograph of immunohistochemical stain for SMA.(a) Positive stain and (b) negative control (lacking the primary anti-body), of the periphery of a DHT-treated matrix at 21 days. Few openpores can be found remaining in the periphery of the sample.

to be able to begin to serve as a useful relationship.Binomial and exponential regression analyses demon-strated that approximately 95% of the variation of cell-mediated contraction, normalized to DNA content,could be explained by modulus. That the 0.5G and 12Ggroups had similar moduli and yet displayed di!erentdegrees of contraction might be explained by the smallsample size or the fact that there are other factors a!ect-ing contraction. One such factor might be the cell ad-hesion to the matrix. A similar explanation could be usedfor the 0.5G and UV groups that showed similar contrac-tion despite the di!erent modulus values. When takentogether, however, these data can begin to form the basisfor selecting cross-link treatments for collagen matricesbased on the resistance to cell-mediated contraction. Itwould be of interest to further explore the relationshipbetween the contractility of the collagen matrices andmodulus using compression data. Moreover, the failureof several mechanical test specimens at the grips high-lights the di$culty in testing these highly porous, lowmodulus materials in tension.

Although the majority of the cells were found in theedge regions of the matrices after 7 days, their in"ltration

into the interior regions showed that they were able toattach to and migrate within the sca!olds. By the 14thand 21st days, cells were found histologically to havemigrated throughout the matrices and proliferation inseveral of the groups was evident from results of theDNA assays. The decrease in the DNA content of theDHT and ETH group from 14 to 21 days may have beendue to sloughing of cells into the medium, perhaps re-lated to degeneration of the matrix. The degradationbehavior of the matrices used in this study is a topic forfuture work. Moreover, it is important to note that thisinvestigation was performed with only one cell seedingdensity. The e!ects of seeding density on cell proliferationand contraction of the matrices needs to be addressed infuture work.

An unexpected and unexplained "nding was the largedi!erence in the number of cells in the various cross-linkgroups, despite the same initial number of seeded cells.There was a 3-fold di!erence in the number of cells in theDHT and 0.5G-treated type I collagen}GAG groups.Histology did not reveal noticeable numbers of dead cells(e.g., cells with pyknotic nuclei). This, coupled with the"nding of an increase in DNA content from 14 to 21 daysin several groups, indicated that the DNA content of thematrices was re#ective of the number of living cells. Thedi!erence in cell proliferation in the various matrices mayhave been due to: (1) the e!ect of the cross-linking treat-ments on chemical constituents of the matrix that serveas ligands for integrins; (2) the e!ects of contraction inreducing the pore diameter and constricting the peri-cellular space and a!ecting di!usion of nutrients to thecell; and/or (3) the e!ects of the sti!ness of the matrix infrustrating cell contraction. With respect to this lastpoint, prior studies using cell-seeded collagen gels havefound that cells do not proliferate in freely-#oating gelsundergoing cell-mediated contraction [43]. In contrast,cells proliferate in gels that are constrained from contrac-tion. Further work including the quantitative analysis ofthe regional variation of pore size needs to be performedto address these issues.

Di!erences in the distribution and morphology of thecells in the matrices may have been related to di!erencesin contraction of the sca!olds. Even at 21 days, the porestructure of the 12G and 24G cross-linked matrices didnot appear to have been contracted by the cells. The openpores facilitated cell migration throughout the matrix,resulting in a more even distribution of cells than is seenin the highly contracted matrices with closed pores.Moreover, in the sti!er matrices, cells were more elon-gated along the length of the open matrix pores at 21days. In contrast, the matrices that underwent contrac-tion contained many more cells that were ovoid in shape.This may suggest that the elongated cells on sti!er ma-trices were exerting contractile forces on the pore walls,but that the sti!ness of the matrix was high enough toresist the contractile force of the cells. The more ovoid


Fig. 7. Stress}strain behavior of dehydrothermally cross-linked collagen}GAG matrices and matrices cross-linked with 0.25% glutaraldehyde for24 h.

Fig. 8. Modulus of elasticity of type I collagen}GAG matrices atselected strains. The moduli were derived from binomial equations "t tothe stress}strain curves. P values were determined by one-way ANOVA(mean$SEM). Sample sizes are given in parentheses in the legend.

shape of the cells in the lower modulus matrices may berelated to the contracted state of the cells.

A reduction in diameter with time in culture was notedin the non-cell-seeded control as well as the cell-seededmatrices in several groups. The shrinkage of the non-seeded control matrices was most likely explained by thepresence of strains in the matrices following freeze-dry-ing. The structure of the matrix, set by the freeze-drying

process, shrinks when the matrix is immersed in an aque-ous medium because water is a mild plasticizer for col-lagen, and the strains are relieved. Cross-linking thematrices can prevent this relaxation of the matrix strains,by chemically or physically "xing the matrix structure.This is evident in the lack of shrinkage of unseededmatrices that were cross-linked with glutaraldehyde andthe slight shrinkage in the matrices cross-linked withultraviolet irradiation. The non-seeded type II collagenmatrices had smaller diameters than their cell-seededcounterparts leading to a negative percentage of cell-mediated contraction (Fig. 2c). The seeded cells may havecaused swelling of the matrices as a result of their syn-thesis of new matrix molecules and/or as a result of thedegradation of the sca!old. Additional study is requiredto clarify this "nding.

In earlier studies employing articular chondrocytes[27] and cells isolated from the knee meniscus [25] typeII collagen matrices comparable to those used in thepresent work did not contract, whereas DHT-treatedtype I collagen}GAG matrices displayed cell-mediatedcontraction. Subsequent studies [44], however, demon-strated that type II collagen sponges also could undergocell (chondrocyte)-mediated contraction. The type II


collagen}GAG matrices employed in this study dis-played handling characteristics like the lightly cross-linked type I matrices. Moreover, their fragility was suchthat they did not lend themselves to the same tensile testsperformed on the type I collagen specimens. Yet, despitethese features, the type II collagen material was notcontracted by the tendon cells. Future work needs tore-address the mechanical properties of this type ofmatrix and consider factors other than the matrix sti!-ness, including biochemical composition of the matricesthat might be responsible for the di!erence in the resist-ance to contraction.

The contractile behavior of many types of connectivetissue cells has been investigated extensively using a col-lagen gel preparation [45]. The cells are admixed withtype I collagen in soluble form prior to cross-linking[46]. The resulting gel (also referred to as lattice), con-taining a uniform distribution of cells, comprises a net-work of "brillar collagen with a nominal pore diameter of1}10 lm. The collagen "brils are approximately50}500 nm thick. Some studies suggest that the cell-mediated contraction of these gels may be due to themotility of the cells*extension and retraction of cellprocesses to produce traction forces on the collagen"brils of the gel*within the matrix and not to frankcontraction (i.e., shortening) of the constituent cells[43,47]. In the current study of tendon cells seeded ina preformed collagen sponge, there was a reduction in thediameter of the specimen by 30% after 21 days. Thesekinetics di!er from those reported for "broblast-seededgels in which a 50% decrease can be found to occurwithin 1 day [46]. Moreover, in the preformed collagensca!old, the contraction appeared to be due principallyto a cell continuous layer on the surface of the samples.This supposition is supported by the appearance of poresof smaller diameter at the periphery of the sponge. Theseobservations suggest that the contraction of collagensponges may be due to the synchronous contraction ofcells in the layer on the surface of the specimen. Thisbehavior recalls the &picture frame' model of wound con-traction in which the cells on the periphery of a woundwere considered to those principally responsible forwound closure [48].

Prior investigations have identi"ed myo"broblasts innormal and healing tendons [49}52]. The percentage ofSMA-containing "broblasts in normal tendon, however,has not yet been determined. For comparison, a recentstudy has shown that up to 50% of the cells in certainlocations in the intact human anterior cruciate ligamentcontain SMA [42]. If identi"ed in normal tendon, SMA-expressing cells may be responsible for imparting andmaintaining the architecture (viz., crimp) of the extracel-lular matrix as has been proposed as one of their roles inligament [42].

Our other recent studies have shown that while onlyabout 10% of the cells in bovine knee meniscus [53] and

canine intervertebral disk [54] contained SMA, approx-imately 90% of the isolated cells cultured on conven-tional tissue culture plates displayed the presence ofSMA after 1 day. Several other previous studies havedemonstrated the expression of SMA in "broblasts cul-tured on two-dimensional substrates [36]. Collectivelythese studies indicated that (1) the process of isolatingcells from tissue and culturing on two-dimensional sub-strates switched on the gene for the SMA isoform in cellsthat just prior to digestion of the tissue were not express-ing the gene, and/or (2) there was a preferential prolifer-ation of SMA-expressing cells in two-dimensionalculture. The former explanation may be more plausiblein that it is consistent with the "nding of the increase inthe percentage of myo"broblasts in certain connectivetissues in response to trauma [55,56]. The current studyshows for the "rst time that tendon "broblasts can alsoexpress the SMA phenotype in a three-dimensional col-lagen sponge and that these cells display contractile ca-pabilities. Future studies could employ the Western blotmethod to determine whether the cross-linking treat-ments a!ect the percentage of cells expressing SMA orthe amount of SMA per cell.

The roles of the contractile phenotype in tendon havenot yet been determined. Using the collagen}GAGsponge as an analog of extracellular matrix, our workshows that the SMA-positive tendon cells could act tocontract the extracellular matrix at a particular stage ofhealing and thus facilitate wound closure. Moreover, the"nding that tendon cells can contract a collagen spongethat might be employed for tissue engineering highlightsthe importance of certain mechanical properties of thematrix (viz., sti!ness) as design parameters.

5. Conclusions

Tendon "broblasts attach to, migrate into, and prolif-erate within collagen}GAG matrices. A large majority ofcells seeded in collagen}GAG sponges express the myo-"broblast phenotype, as re#ected in their expression ofSMA. The degree to which these cells contract the ma-trices is a function of the cross-link treatment andmodulus of elasticity. DHT treatment alone isnot su$cient to resist contraction by tendon "broblastsseeded at the density used in this experiment. Thedistribution of cells and the non-uniformity ofcontraction in the sponges vary with cross-linking treat-ment.

The similarity in modulus values for the ETH, UV, and0.5G groups, and the comparable cell-mediated contrac-tion indicates that physical cross-linking methods such asUV irradiation can be e!ectively used to control theelastic modulus and as a result, the cell-mediated con-traction of the sponges.


Acknowledgements

We gratefully acknowledge support of a NationalScience Foundation Graduate Fellowship (DST). Anyopinions, "ndings, conclusions or recommendations ex-pressed in this publication are those of the authors anddo not necessarily re#ect the views of the NationalScience Foundation. This work was supported in part bythe Brigham Orthopaedic Foundation. We appreciatethe helpful comments of Professor Lorna Gibson of theDepartment of Materials Science and Engineering at theMassachusetts Institute of Technology and the use of herInstron materials tester.

References

[1] Belmonte MM, Biagini G, Ricotti G, Zucchini C, Castaldini C,Fini M, Giardino R, Bigini M. Experimental wound dressing byheterologous collagen*a morphological}quantitative approach.J Bioact Compat Polymer 1993;8:365}82.

[2] Anselme K, Bacques C, Charriere G, Hartman DJ, Herbage D,Garrone R. Tissue reaction to subcutaneous implantation ofa collagen sponge. A histological, ultrastructural, and immunolo-gical study. J Biomed Mater Res 1990;24:689}703.

[3] De Vries HJC, Middelkoop E, Mekkes JR, Dutrieux RP, Wil-devuur CHR, Westerhof W. Dermal regeneration in native non-cross-linked collagen sponges with di!erent extracellular matrixmolecules. Wound Repair Regen 1994;2:37}47.

[4] Nevins M, Kirkerhead C, Nevins M, Wozney JA, Palmer R. Boneformation in the goat maxillary sinus induced by absorbablecollagen sponge implants impregnated with recombinant humanbone morphogenetic protein-2. Int J Periodont Rest Dent 1996;16:9}19.

[5] Joos U, Ochs G, Ries PE. In#uence of collagen#eece on boneregeneration. Biomaterials 1980;1:23}6.

[6] Stone KR, Steadman JR, Rodkey WG, Li S-T. Regeneration ofa meniscal cartilage with use of a collagen sca!old: analysisof preliminary data. J Bone Jt Surg 1997;79-A:1770}7.

[7] Speer DP, Chvapil M, Volz RG, Holmes MD. Enhancement ofhealing in osteochondral defects by collagen sponge implantation.Clin Orthop 1979;144:326}35.

[8] Natsume T, Ike O, Okada T, Takimoto N, Shimizu Y, Ikada Y.Porous collagen sponge for esophageal replacement. J BiomedMater Res 1993;27:867}75.

[9] Narotam PK, Vandellen JR, Bhoola KD. A clinicopathologicalstudy of collagen sponge as a dural graft in neurosurgery. J Neuro-surg 1995;82:406}12.

[10] van Wachem PB, van Luyn MJA, da Costa MLP. Myoblastseeding in a collagen matrix evaluated in vitro. J Biomed MaterRes 1996;30:353}60.

[11] Matsuda K, Suzuki S, Isshiki N, Yoshioka K, Okada T, Ikada Y.In#uence of glycosaminoglycans on the collagen sponge compo-nent of a bilayer arti"cial skin. Biomaterials 1990;11:351}5.

[12] Srivastava S, Gorham SD, French DA, Shivas AA, Courtney JM.In vivo evaluation and comparison of collagen, acetylatedcollagen and collagen/glycosaminoglycan composite "lmsand sponges as candidate biomaterials. Biomaterials 1990;11:155}61.

[13] PohunkovaH H, StehlmHk J, VaH chal J, Cech O, Adam M. Mor-phological features of bone healing under the e!ect of collagen-graft}glycosaminoglycan copolymer supplemented with thetripeptide Gly}His}Lys. Biomaterials 1996;17:1567}74.

[14] Yannas IV, Lee E, Orgill DP, Skrabut EM, Murphy GF. Syn-thesis and characterization of a model extracellular matrix thatinduces partial regeneration of adult mammalian skin. Proc NatAcad Sci USA 1989;86:933}7.

[15] Burke JF, Yannas IV, Quinby WC, Bondoc CC, Jung WK.Successful use of a physiologically acceptable arti"cial skinin the treatment of extensive burn injury. Ann Surg 1981;194:413}28.

[16] Stern R, McPherson M, Longaker MT. Histologic study of arti"-cial skin used in the treatment of full-thickness thermal injury.J Burn Care and Rehab 1990;11:7}13.

[17] Yannas IV. Regeneration of skin and nerves by use of collagentemplates. In: Nimni ME, editor. Collagen, vol. III: Biotechnol-ogy. Boca Raton, FL: CRC Press, 1989. p. 87}115.

[18] Chang AS, Yannas IV, Krarup C, Sethi R, Norregaard TV,Zervas NT. Polymeric templates for peripheral nerve regenera-tion. Electrophysiological study of functional recovery. Proc ACSDiv of Polymeric Mater: Sci Engng 1988;59:906}10.

[19] Louie LK, Yannas IV, Hsu H-P, Spector M. Healing of tendondefects implanted with a porous collagen}GAG matrix: histologi-cal evaluation. Tiss Engng 1997;3:187}95.

[20] Nehrer S, Breinan HA, Ramappa A, Hsu H-P, Minas T, Short-kro! S, Sledge CB, Yannas IV, Spector M. Chondrocyte-seededcollagen matrices implanted in a chondral defect in a caninemodel. Biomaterials 1998;19:2313}28.

[21] Doillon CJ, Wasserman AJ, Berg RA, Silver FH. Behaviour of"broblasts and epidermal cells cultivated on analogues of ex-tracellular matrix. Biomaterials 1988;9:91}6.

[22] Berthod F, Hayek D, Damour O, Collombel C. Collagen syn-thesis by "broblasts cultured within a collagen sponge. Bio-materials 1993;14:749}54.

[23] Butler CE, Orgill DP, Compton C, Yannas IV. E!ect of cellculturing on keratinocyte-seeded collagen}glycosaminoglycanmatrix skin replacement in full-thickness porcine wounds. SurgForum 1996;XLVII:752}4.

[24] Dunn MG, Liesch JB, Tiku ML, Zawadsky JP. Development of"broblast-seeded ligament analogs for ACL reconstruction. J Bio-med Mater Res 1995;29:1363}71.

[25] Mueller SM, Shortkro! S, Schneider TO, Breinan HA, SpectorM. Meniscus cells seeded in type I and type II collagen matricesin vitro. Biomaterials 1999;20:701}9.

[26] Grande DA, Halberstadt C, Naughton G, Schwartz R, Manji R.Evaluation of matrix sca!olds for tissue engineering of articularcartilage grafts. J Biomed Mater Res 1997;34:211}20.

[27] Nehrer S, Breinan HA, Ramappa A, Young G, Shortkro! S,Louie L, Sledge CB, Yannas IV, Spector M. Matrix collagen typeand pore size in#uence behaviour of seeded canine chondrocytes.Biomaterials 1997;18:769}76.

[28] Nixon AJ, Sams AE, Lust G, Grande D, Mohammed HO. Tem-poral matrix synthesis and histologic features of a chondrocyte-laden porous collagen cartilage analogue. Am J Vet Res 1993;54:349}56.

[29] Fujisato T, Sajiki T, Liu Q, Ikada Y. E!ect of basic "broblastgrowth factor on cartilage regeneration in chondrocyte-seededcollagen sponge sca!old. Biomatrials 1996;17:155}62.

[30] Richter HA, Buchenroth M, Handt S, Maurin N, Mittermayer C.Tissue compatibility from di!erent high cross-linked collagensponges after subcutaneous implantation in mice. In: de Putter C,de Lange GL, de Groot K, Lee AJC, editors. Implant materials inbiofunction. Amsterdam: Elsevier, 1988. p. 279.

[31] Olde Damink LHH, Dijkstra PJ, Van Luyn MJA, Van WachemPB, Nieuwenhuis P, Feijen J. Changes in the mechanical proper-ties of dermal sheep collagen during in vitro degradation. J Bio-med Mater Res 1995;29:139}47.

[32] Hirschel BJ, Gabbiani G, Ryan GB, Majno G. Fibroblasts ofgranulation tissue: immuno#uorescent staining with antismoothmuscle serum. Proc Soc Exp Biol Med 1971;138:466}9.


[33] McGrath MH, Hundahl S. The spatial and temporal quanti"ca-tion of myo"broblasts. Plast Reconstr Surg 1982;69:975}83.

[34] Gabbiani G, Ryan GB, Majno G. Presence of modi"ed "broblastsin granulation tissue and their possible role in wound contraction.Experientia 1971;27:549}50.

[35] Majno G, Gabbiani G, Hirschel BJ, Ryan GB, Statkov PR.Contraction of granulation tissue in vitro: similarity to smoothmuscle. Science 1971;173:548}50.

[36] Masur SK, Dewal HS, Dinh TT, Erenburg I, Petridou S. Myo-"broblasts di!erentiate from "broblasts when plated at low den-sity. Proc Natl Acad Sci USA 1996;93:4219}23.

[37] Gabbiani G, Hirschel BJ, Ryan GB, Statkov PR, Majno G.Granulation tissue as a contractile organ. A study of structure andfunction. J Exp Med 1972;135:719}34.

[38] Schurch W, Seemayer TA, Gabbiani G. Myo"broblast. In: Stern-berg SS, editor. Histology for pathologists. Philadelphia, PA:Lippincott-Raven Publishers, 1997. p. 129}165.

[39] Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D,Gabbiani G. A monoclonal antibody against a-smooth muscleactin: a new probe for smooth muscle di!erentiation. J Cell Biol1986;103:2787}96.

[40] Ellis DL, Yannas IV. Recent advances in tissue synthesis in vivoby use of collagen}glycosaminoglycan copolymers. Biomaterials1996;17:291}9.

[41] Nehrer S, Breinan HA, Ashkar S, Shortkro! S, Minas T, SledgeCB, Yannas IV, Spector M. Characteristics of articular chon-drocytes seeded in collagen matrices in vitro. Tiss Engng 1998;4:175}83.

[42] Murray MM, Spector M. Fibroblast distribution in the ante-romedial bundle of the human anterior cruciate ligament: thepresence of a-smooth muscle actin positive cells. J Orthop Res1999;17:18}27.

[43] Grinnell F. Fibroblasts, myo"broblasts, and wound contraction.J Cell Biol 1994;124:401}4.

[44] Breinan HA. Development of a collagen}glycosaminoglycan ana-log of extracellular matrix to facilitate articular cartilage regen-eration. Doctoral thesis, Massachusetts Institute of Technology,Cambridge, MA, 1998.

[45] Elsdale T, Bard J. Collagen substrata for studies on cell behavior.J Cell Biol 1972;54:626}37.

[46] Bell E, Ivarsson B, Merrill C. Production of a tissue-like structureby contraction of collagen lattices by human "broblasts of di!er-ent proliferative potential in vitro. Proc Natl Acad Sci USA1979;76:1274}8.

[47] Ehrlich HP. The modulation of contraction of "broblast popu-lated collagen lattices by types I, II, and III collagen. Tissue andCell 1988;20:47}50.

[48] Grillo HC, Watts GT, Gross J. Studies in wound healing-I. AnnSurg 1958;148:145}52.

[49] Ippolito E, Natali PG, Postacchini F, Accinni L, DeMartino C.Ultrastructural and immunochemical evidence of actin in thetendon cells. Clin Orthop 1977;126:282}4.

[50] Ippolito E, Natali PG, Postacchini F, Accinni L, DeMartino C.Morphological, immunochemical, and biochemical study of rab-bit achilles tendon at various ages. J Bone Jt Surg 1980;62-A:583}98.

[51] Postacchini F, Accinni L, Natali PG, Ippolito E, DeMartino C.Regeneration of rabbit calcaneal tendon: a morpho-logical and immunochemical study. Cell Tissue Res 1978;195:81}97.

[52] Postacchini F, Natali PG, Accinni L, Ippolito E, De Martino C.Contractile "laments in cells of regenerating tendon. Experimen-tia 1977;33:957}9.

[53] Mueller SM, Schneider TO, Shortkro! S, Breinan HA, SpectorM. a-smooth muscle actin and contractile behavior of bovinemeniscus cells in type I and type II collagen}GAG matrices.J Biomed Mater Res 1999;45:157}66.

[54] Schneider TO, Mueller SM, Shortkro! S, Spector M. Expressionof a-smooth muscle actin in canine intervertebral disc cells in situand in collagen}GAG matrices in vitro. J Orthop Res 1999;17:192}9.

[55] Gabbiani G. Evolution and clinical implications of the myo-"broblast concept. Cardiovasc Res 1998;38:545}8.

[56] Ghadially FN, Lalonde J-MA, Yong NK. Myo"broblasts andintracellular collagen in torn semilunar cartilages. J SubmicroscCytol 1980;12:447}55.


tendon cell contraction of collagen–gag matrices in vitro: effect of cross-linking

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