Fibroblast-like cells from tendons differ from skin fibroblasts in their: Ability to form three-dimensional structures in vitro

dissect.ed into 0.5-l cm lengths an placed inlo T25 Basks, (five pieces per flask), in Minimum Essential medium (Eagle’s modification) (MEME) supplemented with 10% fetal calf serum (FCS) and ascorbic acid (2.5 pgiml). Specimens were incubated at 37°C and fresh medium was added after 2-3 days. Cultures were maintained under these conditions until the formation of a cell monolayer at 2-5 weeks, with media changes being performed twice weekly. At confluence, cells were either used in the characterization assays detailed below or alternatively, culture was continued and the cells allowed to attain superconfluence without disturbance. The explant technique was used for ah the experiments described in this paper ether culture techniques were investigated (enzymatic digestion, dicing of tissue) but this technique was the most successful at reproducibly permitting growth of cells in suffisient numbers for the assays.

Structurally, tendons consist of bundles of collagen: orientated along the iongtitudinal axis of the tendon and only sparsely-interspersed with cells, encapsulated by an external sheath, which is denser and more cellular. There has been considerable debate as to the :-elative contributions of these two regions to tend.on healing and comparisons have been made bet-ween c&s derived from these two areas (Abrahemsson et al; 1995; Mass and Tuel, 1991; RiedererHenderson et al; 1983: Russell and Manske, 1990). As yet, there is no generally accepted phenotype denoting a ‘tendon ceil’which identifies it as a unique, specific cell type. For this reason, authors generally refer to cells derived from tendon as fabr-oblasts. In addition, tendons and ligaments ifferent anatomical sites appear to hav-e different operties and to heal at diRerent rates (Fujii et al, 1994; efti and Stall, 1995; Liu et al, 1995). Flexor tendons for example. heal poorly and much more slowly than extensor tendons. Et is unclear at present whether the variety of responses of tendons to wounding and to other stimuli such as loading is attributable to genetic differences between cells of different tendon types or to differences in their external miiieu in vivo (Abrahamsson et al, 1989; Nessler et al, 1992; Schwarz, 1996; Spindler et al: 1996; Zayas and Schwarz, i992). There is little in the literature about the biology of cells derived from tendons. To begin to address this gap in our knowledge, we have cultured samples of tendons and begun to characterize the resultant cell populations, under the standard condirions attained in vitro.

The diff”lcultyexperienced in obtaining sufi%&nt quantities of human tendons for this study prompted us to also examine the utility of rabbit tendons as a tool for investigating tendon cell biology. We therefore cultured both rabbit and human samples and subjected them to the characterization assays listed below. In addition as cells obtained from tendons appear fibroblastic and fibroblasts are a likely contaminant of primary cultures, we cultured skin samples (rabbit and human) to obtain fibroblasts and subjected them to the assays listed below.

Human tendons were obtained from patients undergoing elective surgery; rabbit tendons were obtained from animals which were ;rsed for other experimental studies not related to tendons. Cells were obtained by the explant technique. In this experimental procedure samples of flexor tendon, stripped of their external sheath, were

Ceils that grew out of the tendon :xp?art Gtures (TC) were examined by phase contrast ~~icroscopy as they 633

(134

progressed from sparse to confluent cultures. Some cuitures were stained with haematoxylin and eosin to demonstrate general morphology. Confluent cultures were trypsinized and the TC thus obtained were set up in culture to examine growth rates and to calculate population doubling times of the TC. only primary cultures were used for these assays to avoid the possibility of dedifferentiation in culture. Grox:tlz CLWW. TC were seeded at a density of 1x10” cells/well on to 24-well multiwell plates in MEME medium as above. Cultures were incubated at 37°C and maintained for 12 days in vitro. The medium was replaced every 2 days. To monitor cell proliferation, TC were trypsinized and counted in a haemacytometer at 48-hour intervals over the culture period; three replicate wells were counted for each time point. All tissue culture media were obtained from Gibco Life Technologies (Poole, UK), and all tissue culture plastics from Falcon (Becton Dickinson UK Limited, Oxford: UK) unless otherwise stated.

Some cultures were allowed to become superconfluent, which took up to 8 weeks, and any changes in cell morphology were observed. Histological sections were prepared from these cultures and stained with haematoxylin and eosin. Similar stained slides were prepared from non-confluent cultures.

Qualitative studies Cultures were examined for the synthesis of the matrix proteins collagen and proteoglycan. TC were set up on chamber slides (Lab Tek, Gibco Life Technologies) at 1xl 05 cells/ml and cultured for 7 days to allow matrix synthesis and secretion. TC were then fixed in 100/o formalin for 2 minutes and dehydrated in graded alcohols. TC were either stained by the Periodic acid/Schi.ff method for mucopolysaccharides or by the Picrosirius reaction for collagen. Stained cells were dehydrated, cleared and mounted in DPX. All stains were obtained from Sigma-Aldrich Company Limited, (Poole, UK) except where stated otherwise. Matrix proteins synthesized by TC were further identified by immunostaining. TC were cultured on chamber slides to sub-confluence. Cells were fixed in acetone (2 minutes), air dried and stained for collagen type I or III using the immunostaining techniques of Andrew et al (1992). The primary antibody (mouse anti-human collagen I or III; Sigma-Aldrich Company Ltd, (Poele, UK) was used at a dilution of l:l,OOO in 0.5’%1%§A; the secondary antibody (anti-mouse IgG, peroxidase labelled; Sigma) was used at a dilution of I : 100) in buffer containing 0.5”io BSA.

;n addition to staining TC sultures for proteins, cultures were set up in the usual culture medium to quantitate protein synthesis. Total protein synthesis in TC cultures was examined by culturing TC at a seeding density of 25x10” cells/ml in 96 well plates (Nunc, Gibco Life Technologies, Poole, UK) for up to 14 days. Cells were lysed by freeze-thawing and total protein was assayed using the biochinoic acid method (Smith et al, 1985). For total collagen synthesis, TC were seeded into 12-well plates at a seeding density of 8~10~ cells/ml and incubated for a total of 10 days. At 48-hour intervals, TC were removed from three replicate wells by trypsinisation and assayed for total collagen using a commercial kit (Sircol Collagen Assay, %iocolor Ltd, assay uses the collagen binding ability of picrosirius reagent to develop a colour reaction in solution. For glycosammoglycan (GAG) synthesis TC were seeded at 2.5~10” cells/well into 24-well plates and incubated for a total of 16 days. Media were removed every 48 hours and replaced with fresh. Conditioned media were stored at -20°C before assay. The dimethylene blue technique (Farndale et al, 1982) was used to assay the samples for GAG.

Xodi elect

te-p i% p

In addition to the colourimetric techniques empioyed to quantitatively assess specific types of dodecyl sulphate-polyacrylamide gel (SDS-PACE) was performed on conditioned media from TC cultures to examine the profile of secreted groteilx synthesized by TC. Using the method of Laemmli (1970), media were loaded onto a lO”/o ge!, run at -100 mA and silver stained using the method of Heukeshoven and Dernick (1988).

Cell

Y

The cells obtained from tendon explant ceJtures (TC) were initially fibroblastic (bipolar, spindle-shaped cells with a low cytoplasm:nucleus ratio) in shape, but as the culture developed. the normal “swirling” pattern of cell orientation seen with fibroblasts did not occur and ceils remained randomly orientated as confluence was attained (Fig la). In addition, in some cuhures it was possible to see small subgroups of noon-p~olog~~ally distinct cells, which were much more rounded than the cell type normally seen in these cultures (Fig I b). Growth curves derived from primary cultures showed different patterns of growth, depending upon both the species under examination and the source of the iiss~ue used to produce the culture. Skin fibroblasts from human

Fig 1

Phase con;rast pxxomicrog~aphs cluster of polygonal ce!Is (o;lginal

cf living TC in vitro. (a) Ceils displa)mg magnification X10)

tissue grew more rapidly than TC, reaching a higher saturation density al 8 days, when cultures set up at the same time and density disp!ayed almost twice as mal:y cells in the fibroblast cultures compared with TC (Fig 2a). This difference in cell number at 8 days was highly significant (P < 0.001). In addition, fibroblasts appeared to senesce more slowIy, as by 12 days fibroblast numbers had decreased to only 82”/0 of the number found at 8 days, wh.ereas TC numbers had decreased to only 44%. These differences in ceil number were highly significant (P < 0.001). Ceils derived from rabbit tissue showed a quite different patterri of growth to human. Rabbit TC grew much more rapidly than skin fibroblasts (Fig 2b) so that at 8 days there were almost twice as many TC as fibroblasts (5~10~ compared with 2.6~10~). Again, the difference in these cell numbers was highly significant

Srcnth 2.0

.,

0.0

L-

F1g 2

=

TC:

A=

for

Human

TC

fibrca

/ 2

asp

4

Tine

211 numbers :wre cowtec, of three replrcate cultures.

In Vitro

morphology

(oxgina:

:na~mikazion

x10); (b)

(P = 0 0012). In addition, TC seaesced much more JOWly fibroblasts, so that at 12 days TC numbers were still at 83% of the culture maximum (seen at 6 days), whereas fibrob!asts were only 38% of the culture maximum (seen at 8 days). The difference in TC an fibroblast numbers at i2 days was highly significant (P < 0.001). Growth curves were also used to calculate popciation doubling times for the different cell types. As expected, cells derived from human tissue grew more slowly than those derived from rabbit (Table 1). In addition, human fibroblasts attained a much higher celi sattlration density than TC, whereas the opposite was seen with rabbit cells.

CL;ltu::es of TC which were allowed to become superconfiuent demonstrated an interesting and unusual

& Flbroblasts

I

6

hipolar

than

7.5

0

“(a$:

Curve:;

typical

8

1

I

10

?2

0.3

+----

0

(days)

*

<;e, II

over 12 days for (a) human

!

and jb) rabbit

2 =i,brobasr

I

/

4

6

Time

In Vitro

6

I

I

IO

12

!daysi

=TC,

T”c and siia fibro3lasLs. Resuits are the !meai~b a;:d >Landard wviarions

Saturation density

9x104

1.7xlQ~

morphology. Several weeks after the initiai culture setup (4-8 weeks for rabbit and 6-8 weeks for human), the TC appeared to spontaneously condense in certain areas of the flask, with the cells becoming more rounded and losing their fibroblastic shape (Fig 3a)(arrow). This area of condensation became denser with time and a macroscopic structure began to appear at 4 weeks (Fig 3b). ithin this structure, which we have termed “tendon-?ike structure” (TLS), the cells became extremely dense and appeared to be under considerable cell-generated tension, so that fibres were orientated parallel to the longtitudina! axis of the TLS. The TLS appeared to be anchored at either end by cells projecting from the main cell mass and adherent to the cell monolayer underneath the TLS. In addition, they were also attached to the TC monolayer by other cells growing at right angles out of the mass of the TLS and attaching to the TC monolayer (Fig 3b) (large arrow). The length of TLS generated varied from =I cm to 9 cm; these longer TLS seemed to be constrained in length only by the size of the culture vessel. In some TLS, sections took on a different morphoiogy and cells appeared rounded (Fig 3c) rather than stretched out, as was seen in the part of the TLS under tension. Histological sections of tendons and of TLS showed a similar morphology when stained with haematoxylin and eosin. A tendon section (Fig 3d) displayed a structure composed mainly of parallel fibrils, interspersed very sparsely with cells (arrows). A monolayer of TC, stained

Fig 3

6X10”

2.5x10’

with haematoxyhn and eosin (Fig 3e) showed n similar structure, albeit more cellular in nature. Development of TLS was seen in cultures of tissue from both rabbit and human and did not seem to be a species-specific property.

TC cultured in chamber slides synthesized both collagen

and proteoglycans, as demonstrated by histologicei staining. TC stained positive for collagen using the Picrosirius reaction (Fig 4a) and positive for proteoglycans usin the Periodic acidiSchiff reaction (Fig 4b) unochemical stalning us!ng specific anti(arrows). strated that TC secreted Type i collager? bodies de (Fig 5) but not type IT1 collagen (data not shown); in contrast, skin fibroblasts were positive for both cohagec types (data not shown).

edia were taken from TC at 4%hour intemus over a j 4 y culture period and assayed for tota! protein secretion. The results demonstrated that total protein synthesis increased rapidly with time in culture and reached a peak at day 9 (Fig 6) although cell numbers continued to increase to day 14: when the experiment terminated.

Phase contrast micrographs of living TC. (a) TC can be seeri ~ondenring Into polygonal ceils and foim,ng n .ongtrttic;rna! (original magnification x10); (b) part of a TLS demonstrating cell-generated tension (arrowhead) and ce!ls of multilayer TLS (original magmfication x10).

bii’L~~~i:j.e:airowj at right angles to

y;~g 4

Fig 3

Phase contrast micrographs of living TC. (c) higher power micrograph (original magnification x40) demonstrating density of TLS and anchoring cells, (d) photomicrograph of section of human tendon stained with haematoxylin and eosin, (e) photomicrograph of monolqer of TC stained with haematoxylin and eosin.

Assdgi of media taken from TC for total collagen demonstrated differences between species and between TC and Gbroblasts. Human TC initially synthesized more collagen than Gbroblasts (Fig 7a): but after 4 days in cuiture both cell types were synthesizing similar amounts. However, after 10 days in culture, tibroblasts overtook TC and by 12 days collagen synthesis by TC

Photomicrographs of stained TC sialrleti ior :narrix proteins. {a) TC stained for collagen with Picro-Sirius. (original magnification x5): (b) PAS stain for mucopolysaccharides (arrow).

was only 52Y0 that of fibrob”iasts. Stakxicai analysis revealed highly significant differences in collagen synthesis Setm-een the two cell types (P. en GAG secretion xr cell was calculated, this difference in GAG secretion between the two cell types was even clearer and was not dependent upon total cell number in the population ,Fig 8b).

s Slectrophoretic analysis of total protein secretion by TC and fibroblasts clearly demonstrated that TC cu!tured at he same density as fibroblasts sec:eted higher quantities

Fig 5

Photomicrograph lagen.

0.0

of immunostamed

2.5

5.0

Time Fig 6

7.5

in vitro

TC, stained for type I col-

10.0

12.5

l5.0

[days]

TC were cultured for up to 14 days. with sampling at 48h inter~1s. Media were assayed for total protein. Results are means and standard deviations of three replicate wells. Bars represent cell number (~10~) and points represent total protein (kg).

of protein in vitro (data not shown,). The pattern of proteins secreted by the two cell types is similar, although TC appeared to secrete two proteins (<94 kDa) not secreted by the fibroblasts, This was seen clearly when the band profiles of the lanes for the two cell types are plotted (Fig 9) (arrows). This showed two peaks unique to TC, which correspond to proteins of apparent molecalar weight of around 67 kDa.

The aim of this study was to describe the pher,otype of cells derived from intrasynovial flexor tendons (here termed TC) and to contrast their cell biology with that of skin fibroblasts. In addition, possible differences in phenotype between TC from different species were explored by culturing both human and rabbit samples. The explant technique, in which cells are permitted to grow otit from small fragments of tissue; was used to

Time

In Vitro

tdijysi

=TC; h =iiDroDlast

Fig 7

(a) Human and (b) rabbit IT were cultured for 10&ys. media were collected and assayed for total coilagen. Results are the means and standard deviations of three replicate wells at each time point.

obtain tendon. ceils for this study This is Sy ?ar the :;omcelis, monest technique used iG siu ies sf tendon although Riederer-Henderson e al (1983) a?;,d others (Wojciak and Crossan, 1993) have used other methods; most notably enzymic digestion. However, such techniques not only affect the cell membrane, they also destroy the relationship of the cell of interest with its external milieu. As cell-cell and cell-matrix are now known to be of importance to a cell’s phenotype and gene expression and may act as triggers for various metabolic processes (Damsky and Bernfield, 199 1; and Farmer, 1988; Formigli et al. 1992: Garrod, 1993; Kirkpatrick and Peifer, 1995). we considered that the

0

15

10

5 Tirrie

in vitro

10

15

TC were c~~ltwed for 16 days: media samples were coilected and assayed for glycosammog1ycans. Resuits are the means alit standard !-ions of three i-e?licate wel;s at each time point. Results are expressed as (a) GAG secreted per culture and (b) as pg GAGiiW ce!is.

0

25

50

75

Position

100

(mm)

125

150

M = FB A = TC

Kistogram of‘integrated and skin fibrob!asts.

20

:eg: B?=-:c; A =:ibroblasts

optical density of bands on gel for fC

explani technique was best suited to our purposes. In addition, as it is now we!1 established that the cells from the epitenoc and cndotenon behave differently in vitro (Abrahamsson et al, 1995; Mass and Tuel, 1991) for this study the covering layer was removed and only endotenon cells were used. We have demonstrated that there are substantial differences in the metabolism in vitro of TC when compared with skin fibroblasts, both in rates of growth and in protein synthesis. llJsing human tissue, skin cells grew more rapidly in vitro ihan TC and attained a higher saturation density? suggesting that they are less densityinhibited than TC. This would accord with the

devia-

3r^ these cell types in viva? where sk;n cells grow as densely packed, multiiayered arrays, which are able to proliferate rapidly in response to cell shedding at the dermis. In contrast, TC in situ are scaltered sparsely throughout the dense collagenous matrix which comprises the tendon body and where rapid cell prohferation is not of major importance to the normal business of the cell. However, despite the highly fibrillar, acellular appearance of tendons in vivo and of our cells at high density in vitro, there was little difference in the amount of to_al collagen synthesized by the skin or tendon cells. In contrast, human TC were found to secrete significantly more glycosaminnglycan than skin fibroblasts. Analysis by SDS-PAGE of all proteins secreted by the two cell types demonstrated cieariy that TC secreted more protein per cell than skin fibroblasts. Hn addition, TC secreted two smal! proteins (IO0 and 53 KI9a) not synthesized by skin fibroblasts. We cannot compare these findings with those of others as. to our knowledge, no other studies have been one which compare skin and tendon-derived cells in this way. However, Zayas and Schwarz (1992) have reported that avian tendon cells proliferated most rapidly at a cell density of -2x!04 cellls, which accords well with our results, which showed maximurn growth at 2.S3.5~10” cells, although Matsuda (1994) found that in human tendon explant cultures, prohferat maximum at day 9. This discrepancy mav for by the fact that Matsuda was examining outgrowth from the cut ends of tendons, whereas the results reported here are from monolayer cultures. Similarly, our results showed that total collagen synthesis (both secreted and matrix) peaked at 6 days in vitro, corresponding to peak TC number. These findings accord with the results of Zayas and Schwarz (1992). However, appexance

Fig 9

5

Time in vitro [days)

[days)

Key: ES=TC, A =t;ty-~&ES:

Fig 8

0

20

610

total protein synthesis peaked at 9 days, suggesting that once the collagen matrix is laid down the secretion of other proteins is upregulated. Alternatively, a negative feedback mechanism may be occurring at this point. Other studies (Schwarz. 1996) have demonstrated in embryonic chick tendon that at high cell density, 50% of protein synthesis is procollagen, whilst later; in tissue which has become relatively acellular, procollagen is only 1% of the total protein synthesized. More generally, Abrahamsson et al (1991) examined the synthesis of matrix proteins in explant cultures and demonstrated differences with time. lmmunocytochemical staining with specific antibodies demonstrated that TC were able to synthesize only type I collagen, whereas skin fibroblasts secreted both type 1 and type III. These results are in accordance with the findings of Idler (1985) and Harper et al (1988) who also found that cells from a flexor tendon secreted almost exclusively type I collagen. It is of interest that Kobbins and Vogel (1994) also found expression of mRNA for type PI collagen in recently isolated cells from areas of tendon under compression, indicating a chondrocytic phenotype, but that areas of tendon under tension did not express type II. The most striking difference between the two cell types investigated was the development in superconfhrent cultures of TC of three-dimensional structures, (here termed TLS): which superficially resembled tendon, although the development of a sheath around the structure was not seen. Skin fibroblasts cultured under the same culture conditions never developed TLS. It must be emphasized that TLS developed spontaneously in our cultures and no external stimulants. such as growth factors (except those usually found in FCS) or mechanical stimulation were used. It is of interest that TLS appear to generate their own intercellular tensions, after a particular cell density is attained, which may well aid the formation of these structures. Previous studies (Becker and Diegelman, 1984) have shown how tendon cells and collagen fibres align in the direction of an applied tension and this effect may occur in the TLS described here. If the tension to which these cells were exposed was disturbed by, for example, transplanting them to a different culture vessel, the TLS contracted and was often unabie to reattach and re-estabhsh its internal tension. The poorly attached structures appeared to become acellular and no new growth was seen (data not shown). This effect, seen with a sudden reduction in the tension sensed by tendon cells in vitro, may be analagous to that perceived by cells within a tendon in vivo after damage or rupture to that tendon. Other studies (Wiig et al. 1996) have shown that unloading of tendons leads to progressive degradation and loss of synthetic ability. Furthermore, Yamamoto et al (1996) have shown that when unloaded tendons are again subjected to stress, their recovery is incomplete, even after prolonged re-stressing. That tendons are able to respond to their mechanical environment has been well documented (McNeilly et al,

1996) and the discovery by TaiIji et a1 (1995) and others (McNeilly el al, 1996) of gap junctions at the point of contact between cell processes may well contribute to this response. In addition, the importance of tension to basic cell functions has been described by Ingber and Folkman (1989a). The initial stages of formation of TLS, where TC appear to condense and their morphology alters from fibroblastic to polygonal may be the key stage in the development of TLS and deserves further study. Alterations in gene expression at this possibly crucial time would be of particular interest. To our knowledge, growth of such a structure in vitro as a TLS is unique to TC and has not been reported by other authors working with ceils derived from tendons: or with cells from other tissues. However, it is weI1 estabhshed that endotheiial cells in vitro are able to form tubules -which resemble blood vessels, but very specific culture conditions are required to initiate this (Folkiman and Haudenschild. 1980; ngber and a-‘olkmanj !989b) whereas TLS form on tissue culture plastic in a simple culture medium. Comparison of rabbit TC w-ith skin fibroblasts reveals differences from human samples. Rabbit TC grew faster than fibroblasts and attained a signjficantly higher saturation density; they also secreted significantly more collagen than skin fibroblasts for the whole culture period. In addition, the time of maximum collagen synthesis was reached earlier by rabbit TC compared with human (day 4 instead of day 6). ‘Thus it appears that, under the cultureconditions used in this study, there are important differences in the phenotypes of TC derived from rabbit and from human tissue. Our results with rabbit TC from explant cuhses are not directly comparable with other studies using rabbit tissue, as the latter have concentrated on mobihzation of cells from explants (~brahamsson et al; 3992) or on celi morphology in different areas of tendon (Abrahamsson et al, 1989; Rank et al, 1980). Given the differences seen between human and rabbit tendon cell biology in our culture system, we did not therefore consider it appropriate to compare the findings of this st-udy with any of the many reports in the Literature which have used tissues from other species. In summary, this report demonstrates that fibroblastlike cells derived from tendons have a distinct growth pattern which distinguishes them from fibroblasts derived from skin, not least in their unique ability to spontaneously form three-dimensional, cylindrical S’IFUCtunes in vitro. Furthermore. tendon cells from the two different species examined display different growth characteristics and therefore it may not be suitable to extrapolate results from one species on to another.

TENDON

A&D SKIN XBROBLAS-S

OIFFER

Abrahamsson S-O. Cundbo~y S, Lohqnander LS (I 989). Segmental varlatlon m mlcrosiructurc. matrix synthesis and ceil proliferation in rabbit flexor !endon. Scandaixwian Journ 5: 56-65. Lasmmli UK (1970). Cleavage of structural proteins during the assembly of the head of ihc bacteriophage T4. Nature, 227: 680-685

64i LILASH, Yallg RS. 4:.Shalkh R, Lane !M (1995). Coiiagcn m !endon. ligament and bone ihealmg A current review. Chmcai Ort\opaedics and Related ~Rescarch, 3i8: 265-278. h&ass i>P~ Tuel RJ (1991). liltrinslc healing of iiic laceration site in human superfxialis flexor tendons in vitro. Jownal of t;and Surgery, ISA: 24-30. r\,~a:,uea S (1994). A study on cell prohferatlon in cultured lhuman tendons-tune dependence and !abeling of 5.bromodeoxyiil-idii-ie. journal of the Japanese Orthopaedic Association. 6X: 961-96?. \rcYeii!y CM. Banes A3. Benjamin M. Ra!phs jR (19Y6). Tendon crlis form a three-dimensional network of cell processes li!:Xed 54 gap junctions. Journal of Anatomy. 189: 593-600. \wlel- JP. Amadio PC. Berglund LJ. An K-N (1992). Healing o!’ canine tendon m zones subjected to different mechanical forces. Journal cf Hand Surgery. 178. 561-568. Rank F. Elken 0. Bergenholtz G. Lundborg G. Erkel i.! (1980) Flexor tendon specnnens in organ cultures. Scandanavian Journal of Pi&c and Reconstructive Surgery, 14: 179-l 83 Rxdercr-Henderson MA, Gauger .A. Olson i. Robertson C. Greenlee TK (1983). Attachment and extracellular matrix differences between tendon and synouial fibroblastic cells. In Vi&o, 1Y: 127-133. Robsin, JR. Vogel KG (1994). Reglonal expression of’ mR\rA for proteoglycans and collagen in tendon. European Journal of Cell Biology, 64: X4-270. Russell JE. Manske PR (1990). Collagen synthesis during prnnate flexor tendon I-epair in vitro. journal of QrLhopaedic Research, 8: 13-X. S~hwar~ RI (1996). Modelling tendon morphogenesis in wvo based on cell density signaIling in cell culture. Journal of Ma?hematxal Brology, 35: 97-I !3. Sm!th PK, Krohn R!, Hermanson GT et ai (1985). Measurement of protein wing bicinchoninic acid. Analytical Biochemistry. LX): 7&X5. Spmdler KP. imro 4K; Mayes CE, Davidson JM (1996). Pat&r tendon and anterior cruclate ligament ha>e different mltogenic responses to plateletderived g:-owth factor and transforming grow!h fxtol- beta. Journal of Orthopaedic Research. 14: 542-546. Tan.,1 K. Shimizu T_ Satot; T, Hashnnoto S. Bonilla E (1’995). Gap junctions between fibroblasts in rat myotendon Archives of Histclogy and Cytology, 58: 97-!02. Wng M Hanff G. Abrahamsson S-O, Lohmande: LS / 19%). Di\,lsion of flexor tendons caues pl-ogressive degradation of tendon m&ix in rabbits. Acta Orthopaedica Scandanavica, 67: 4911197. Wojclak B. Crossan .iF (1993). The accumulation of inflammatory cells in synowal sheath and epitenon dung adhesion fol-mation in healing rat !lexor tendons. Clinical and Experimental Immunology. 93: 108-I 14. Yamam~to N. Hayashi K. Kuripama H. Ohno K. Vaauda K, Kaneda K (1996). Effects of restressing on the mechanical properties of stress-shielded pate!!ar tendons in rabbits. Journal of Bmmechamcdl Engineermg. 118: 2 16-220. Zayas JR, Schwarz RI (1992). Evidence supporting the role of a proteinaceous. !oosely bound extracellular molecule in the cell density signaling between tendon cells. In Vitro, 28A: 745-754.