Journal of Orthopuedic Research 13:435-441 The Journal of Bone and Joint Surgery. Inc. 0 1995 Orthopaedic Research Society

Synergistic Effect of Growth Factors on Cell Outgrowth from Explants of

Rabbit Anterior Cruciate and Medial Collateral Ligaments

Joe Lee, "Melvin H. Green, and David Amiel

Departments of Orthopaedics and *Biology, University of California San Diego, La Jolla, California, U.S.A.

Summary: Cellular migration and proliferation are integral aspects of wound healing. An in vitro assay for cellular migration and proliferation using explants of rabbit anterior cruciate and medial collateral ligaments was developed previously. This study presents the effects of serum-free culture medium supplemented with basic fibroblast growth factor, bovine insulin, transforming growth factor-pl, and platelet-derived growth factor-B, added either individually or in combination, on cell outgrowth in explants of rabbit anterior cruciate and medial collateral ligaments. Outgrowth was assessed at 3 and 6 days by counting the number of cells surrounding the tissue explants. For explants of both ligaments, cell outgrowth was dependent on the pres- ence of 10% fetal bovine serum or the combination of growth factors. Little outgrowth occurred in explants of either ligament in the presence of basic fibroblast growth factor, transforming growth factor-pl, or bovine insulin. Platelet-derived growth factor-B at concentrations of 20 and 100 ng/ml seemed to increase cell out- growth from medial collateral ligament explants at 6 days. The outgrowth from the explants of both ligaments was much greater in the presence of all four growth factors than the sum of the outgrowth with the individual factors. This synergistic effect was as much as 10-fold and 20-fold for the anterior cruciate ligament explants at days 3 and 6, respectively, but no more than 3-fold for the medial collateral ligament explants at these times. Medial collateral ligament explants exhibited greater cell outgrowth than anterior cruciate ligament explants in 10% serum and in the presence of the four growth factors.

The anterior cruciate ligament of the knee is known to heal quite poorly (4,7,12,21). However, the medial collateral ligament of the knee heals rather well fol- lowing injury, and the surgical repair of an injured medial collateral ligament may at times be unneces- sary. In contrast, surgical repair of the injured ante- rior cruciate ligament is needed if the patient wants to lead a very active life (12) and prevent further injuries.

Studies in animals and humans have revealed sev- eral differences between the anterior cruciate and medial collateral ligaments. Biomechanical, morpho- logical, biochemical, environmental (intrasynovial or extrasynovial), vascular, and cellular differences have been demonstrated (2,8,17,30). Cell culture studies of rabbit anterior cruciate and medial collateral ligament explants have shown differences in the rate of primary cell outgrowth. The medial collateral ligament had a much greater rate of cell outgrowth than the anterior

Received October 26,1993; accepted August 17,1994. Address correspondence and reprint requests to D. Amiel at

University of California San Diego, Orthopaedic Connective Tis- sue Biochemistry. 9500 Gilman Drive. Department 0630, La Jolla, CA 92093-0630, U.S.A.

cruciate ligament (20). Outgrowth is dependent on two major factors: migration of cells out of the ex- plants and proliferation of cells on the dish. These two processes are both important aspects of wound heal- ing. By examining the conditions that promote out- growth of cells from tissue explants, the poor wound healing of the anterior cruciate ligament may be bet- ter understood and possibly improved.

Growth factors are peptides that are known to pro- mote wound healing (6,18,24,28). They have been found to be mitogenic and chemotactic. They also can stimulate the formation of extracellular matrix. Al- though much research has been done on the effects of growth factors in the healing of bone, tendons, and skin (1,10,13,15,16,26), little research has been done on their effects on wound healing of knee ligaments (33). Increased levels of basic fibroblast growth fac- tor (bFGF), transforming growth factor-pl (TGF-Pl), and platelet-derived growth factor (PDGF) have been observed during wound healing of chicken tendons (14). In the present study, these three growth factors, as well as bovine insulin, were tested individually and in combination for their effect on cell outgrowth from explants of anterior cruciate and medial collateral ligaments.

435

436 J. LEE ET A L .

MATERIALS AND METHODS Assay for Cell Outgrowth from Explants

Medial collateral and anterior cruciate ligaments were har- vested in a sterile manner from the knees of seven 8-week-old New Zealand White male rabbits (mass, 1.74 t- 0.06 kg [mean i SEMI) immediately after death. Under a tissue culture hood, the liga- ments were washed with phosphate buffered saline. The femoral and tibia1 insertion sites were removed, and each ligament was trimmed to segments about 8 mm long. The periligamentous sheath on the medial collateral ligament and the synovial sheath on the anterior cruciate ligament were not removed. The segments then were subdivided further into 32-40 explant pieces (depend- ing on the size of the ligament), approximately 1 mm2 in area. Three randomly chosen explants each were placed into one of 12 (22 mm diameter) polystyrene culture wells (Corning Glass Works, Corning, NY, U.S.A.). The pieces were held down with an 18 mm circular cover glass (0.17-0.25 mm thick) (Fisher Scientific: Pitts- burgh, PA, U.S.A.), and silicone gel was used to hold the cover glass onto the well (Fig. 1). A total of 12 explants were used to determine each data point. Attachment of the explants to the culture dish has been observcd to be necessary for cell outgrowth to occur in the vicinity of the explants. One milliliter of control or experimental medium was placed in each well. The control me- dium contained Earle's minimum essential medium with 10% fetal bovine serum, 1 % Fungi-Bact (penicillin G, 10,000 Ulml; strep- tomycin sulfate, 10,000 pgiml; and Fungizone 2.5, kg/ml; lrvine Scientific. Santa Ana, CA, U.S.A.), 0.4% L-glutamate, and 1% noncssential amino acids. The experimental medium was the same as the control medium except that 10% fetal bovine serum was replaced with 1% bovine serum albumin (Fraction V Heat Shock; Boehringer, Mannheim, Germany) and various concentrations of growth factors were added. The medium was not changed during a period of 6 days.

The explants were incubated at 37°C in 5% carbon dioxide and 100% humidity. After 3 and 6 days of incubation, they were exam- ined with a phase-contrast microscope. The number of cells within the peripheral area at a distance of one low-power field (x100 magnification, 3,000 pm) from each explant was counted. Because the outgrowth was not uniform, the entire peripheral area was examined. There were two layers the cells attached: the cover glass and the surface of the culture dish. After one layer was examined, the second layer was placed in focus, and the cells were counted. When there were approximately 1,000 cells or more, the number was estimated. The estimates were modified to take into account the differences in cell densities and morphology.

The viability of cells placed in culture for 6 days in serum-free medium (with and without single growth factors) also was exam- ined. After 6 days, 81 explants were examined (six determinations of 13 different conditions and three determinations of a 14th con- dition). After 6 days, the medium was replaced with fresh serum-

FIG. 1. Schematic diagram illustrating the attach- ment of the explants to the culture dish. ACL = anterior cruciate ligament and MCL = medial col- lateral ligament.

free medium containing the original single growth factor or no growth factor for one set of 39 explants (three determinations of 13 different conditions) from two animals. The explants were ex- amined 6 days later, at day 12. This was done to determine whether there is a time delay before any outgrowth occurs. The other 42

A Mean Cell Count SE After 3 Days "1 (N=12)

0% FBS M 1 M 2 M 3 lO%FBS

Media

Mean Cell Count k SE After 6 Days ""1 ( N = 1 2 ) B

- c m Q X -

3OOO 4 ACL

t

T

O%FBS M1 M2 M3 lO%FBS Media

FIG. 2. Effect of the combinations of growth factors, serum-free medium (0% fetal bovine serum [FBS]), and 10% FBS on cell outgrowth from explants of anterior cruciate and medial collateral ligaments after 3 days (A) and 6 days (B). Medium 1 (Ml) con- tained basic fibroblast growth factor (bFGF), 3 ng/ml; transform- ing growth factor-pl (TFG-Pl), 0.12 ng/ml; platelet-derived growth factor (PDGF), 1 ngiml; and bovine insulin, 0.5 pg/ml; medium 2 (M2) contained bFGF, 20 ng/ml; TGF-01, 5.0 ng/ml; PDGF, 20 ngiml; and bovine insulin, 5.0 pgiml; and medium 3 (M3) contained bFGF, 100 ng/ml; TGF-01, 25.0 ngiml; PDGF, 100 ng/ml; and bo- vine insulin, 25.0 pg/ml. *p < 0.05 for comparison with serum-free medium.

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EFFECT OF GROWTH FACTORS ON ACL/MCL OUTGROWTH 43 7

B

Mean Cell Count k SE After 3 Days I5O0 1 ( N = 1 2 )

0 rn I+% VZm

0 3 20 100

bFGF (ng/rnl)

Mean Cell Count 2 SE After 6 Days ( N = 1 2 ) 6oo 1

ACL MCL

300

200

100

0 0 3 20 100

bFGF (nglrnl)

FIG. 3. Effect of various concentrations of basic fibroblast growth factor (bFGF) on cell outgrowth from explants of anterior cruciate and medial collateral ligaments after 3 days (A) and 6 days (B) compared with serum-free medium (0).

explants (three determinations of 14 different conditions). from two other animals, were cultured for 6 days in serum-free medium, and then the medium was replaced with fresh medium containing 10% fetal bovine serum. These explants also were examined 6 days later (day 12).

Growth Factors The concentration of growth factors used included those noted

to be effective in prior studies. For bFGF, the range was 3-100 ngiml; McAvoy and Chamberlain found that 3 ngiml stimulated half-maximal migration of lens epithelial cells (19). The range for TGF-P1 was 0.12-25 ng/ml: Orr et al. showed that half-maximal migration was stimulated with 0.12 ngiml on rat carcinosarcoma cells (22). The concentrations of PDGF-B ranged from 1-100 ngiml, as 20 ngiml has been found to cause maximal migration of monocytes ( 2 3 ) . For bovine insulin, the concentrations ranged from 0.5-25 pgiml; Abrahamsson et al. found that 5 pgiml produced maximal proliferation of flexor tendon cells (1).

Three concentrations of the four growth factors were used to determine if they act in a synergistic manner. The first combination (medium 1) contained the growth factors at the lowest concentra- tions (bFGF, 3 ng/ml; TGF-P1. 0.12 ngiml; PDGF, 1 nginil; and bovine insulin, 0.5 pg/ml). The second combination, medium 2, contained the growth factors at a middle range of concentrations (bFGF, 20 ng/ml; TGF-PI, 5.0 ngiml; PDGF. 20 ngiml; and bovine insulin, 5.0 pgiml), and the last combination. medium 3, contained the highest concentrations (bFGF. 100 ngiml; TGF-PI, 25.0 ngiml; PDGF. 100 nglml; and bovine insulin, 25.0 pglml). The synergistic effect was defined as the ratio of the mean cell count obtained in the presence of all four growth factors divided by the sum of the

mean cell counts obtained with each factor added at that concen- tration separately.

Bovine bFGF and porcine TGF-Dl were obtained from R and D Systems (Minneapolis, MN, U.S.A.), human recombinant PDGF- B was obtained from Gibco BRL (Grand Island, NY, U.S.A.), and bovine insulin was bought from Sigma Chemical (St. Louis, MO, U.S.A.).

Statistical Analysis One-way analysis of variance was performed using statistical

software (Statworks; Cricket Software, Philadelphia, PA, U.S.A.). The level of significance was set at 95% (p 0.05). The data obtained for the ligament explants in the various concentrations of each growth factor were compared with the results in serum- free medium. The results for the explants in the media with each combination of growth factors and also for those in the medium with 10% fetal bovine serum and no growth factors were com- pared with the results for explants in serum-free medium. Com- parison of the cell outgrowth from the two ligaments in the same medium conditions also was performed. The cell outgrowth is pre- sented as the mean (t SE) cell count.

RESULTS Cell outgrowth seemed dependent on the addition

of 10% serum or the combination of the four growth factors (Fig. 2). Outgrowth increased with time and also with increasing concentrations of the mixture of

Mean Cell Count f SE After 3 Days ( N = 1 2 )

6oo 1

300

100

0 0 0.12 0.5 5 25

TGF-beta 1 (ng/rnI)

Mean Cell Count f SE After 6 Days ( N = 1 2 )

6oo 1 '-i :; 300

200 1 100

0 0 0.12 0.5 5 25

TGF-beta 1 (nghl )

FIG. 4. Effect of various concentrations of transforming growth factor-pl (TGF-PI) on cell outgrowth from explants of anterior cruciate and medial collateral ligaments after 3 days (A) and 6 days (B) compared with serum-free medium (0).

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J. LEE ET A L . 438

A

B

Mean Cell Count k SE After 3 Days ( N = 1 2 )

6oo 1 m n

400

0

100-

0 0 0.5 5 25

Mean Cell Count k SE After 6 Days 6oo 1 ( N = 1 2 )

s - 0 3001 200 T

J 0 = 100-

0 0 0.5 5 25

bovine insulin (microgm/rnl)

FIG. 5. Effect of various concentrations of bovine insulin on cell outgrowth from explants of anterior cruciate and medial collateral ligaments after 3 days (A) and 6 days (B) compared with serum- free medium (0).

the four growth factors. For the anterior cruciate lig- ament, the outgrowth after 3 days was significantly greater for explants in medium 3 (highest concentra- tion of growth factors) than for those in serum-free medium. The mean cell count for explants in medium 3 was almost twice that for explants in medium with 10% serum, but no significant difference was found with this comparison. For the medial collateral lig- ament, the outgrowth from explants in media with each mixture of growth factors and in medium with 10% fetal bovine serum was significantly greater than that in serum-free medium (Fig. 2A). There was no dif- ference between the outgrowth from explants in the media with a combination of growth factors and the outgrowth from explants in medium with 10% fetal bovine serum. When the two ligaments were compared, there was more outgrowth from the medial collateral ligament explants, but the difference was significant only for explants in serum-free medium and in medium with 10% fetal bovine serum (p < 0.05).

After 6 days in medium 2 (middle range of concen- trations) and medium 3 and in medium with 10% fetal bovine serum, the explants of both ligaments had sta- tistically greater outgrowth than explants of either

ligament in serum-free medium (Fig. 2B) (p < 0.05). With the highest concentrations of growth factors (medium 3), the number of cells approximated that obtained for explants of both ligaments in medium with 10% fetal bovine serum. Under all conditions tested, cell outgrowth was greater for the medial col- lateral ligament explants than for the anterior cruciate ligament explants. When the two ligaments were com- pared, the outgrowth from the medial collateral liga- ment was significantly greater in medium 1, medium 3, serum-free medium, and medium with 10% fetal bovine serum (p < 0.05).

When the medium was supplemented with varying concentrations of either bFGF (Fig. 3), TGF-Pl (Fig. 4), or bovine insulin (Fig. 5) , the outgrowth from the ligament explants was only slightly different from that obtained for the explants in serum-free medium. PDGF-B at 20 and 100 ng/ml appeared to stimulate cell outgrowth from medial collateral ligament ex- plants at day 6 (Fig. 6); however, due to the variability in cell outgrowth, no significant difference was noted.

Because there was little outgrowth from explants in the presence of a single growth factor, there may be a time delay before any outgrowth occurs. However,

Mean Cell Count +- SE After 3 Days 6oo 7 ( N = 1 2 )

A

B

0 = 100-

0 0 1 20 100

PDGF-B (ng/rnl)

Mean Cell Count k SE After 6 Days 6oo 1 ( N = 1 2 )

T T L 0) Q - a 300

200

0 z loo

0

- s

0 1 20 100

PDGF-B (ng/ml)

FIG. 6. Effect of various concentrations of platelet-derived growth factor-B (PDGF-B) on cell outgrowth from explants of anterior cruciate and medial collateral ligaments after 3 days (A) and 6 days (B) compared with serum-free medium (0).

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EFFECT OF GROWTH FACTORS ON ACL/MCL OUTGROWTH 43 Y

A B 5000

120 W ACL 4000

v1- 100 MCL 5s 80 3000

2000 o w w -$g 40

1000 20

0 0

= K

- x

6 12 12

day day FIG. 7. Mean ( t S E ) cell count shows that individual growth fac- tors alone and serum-free medium did not promote much out- growth after 6 days in culture (n = 81). After the medium was replaced with fresh serum-free medium, there was little outgrowth even at 12 days (n = 39) (A). Following 6 days of preincubation in serum-free medium and then 6 days in fresh medium with 10% fetal bovine serum, there was a substantial amount of outgrowth at 12 days (n = 42) (B). ACL = anterior cruciate ligament and MCL = medial collateral ligament.

TABLE 1. Synergistic effect of growth factors on cell outgrowth

All 4 growth Sum of single Synergistic factors growth factors effect"

After 3 days ACL

Medium 1 44 -+ 31 4 2 2 11 Medium 2 12 ? 35 9 2 4 8 Medium 3 109 ? 37 11 5 3 10

Medium 1 157 2 48 191 t 36 1 Medium 2 175 2 45 171 + 49 1 Medium 3 183 5 29 105 2 39 2

MCL

After 6 days ACL

Medium 1 71 -t 31 18 2 8 4 Medium 2 653 2 240 32 5 13 20 Medium 3 891 2 344 13 2 24 12

Medium 1 134 i- 287 329 -t 41 2 Medium2 1,220 t 353 413 2 164 3 Medium 3 1,898 2 287 623 Z 198 3

MCL

ACL = anterior cruciate ligament and MCL = medial collateral ligament. Values are given as the mean t SE.

Medium 1 contained basic fibroblast growth factor (bFGF), 3 ng/ml; transforming growth factor-p1 (TFG-P1). 0.12 ng/ml; platelet- derived growth factor (PDGF). 1 ngiml; and bovine insulin. 0.5 pg/ml; medium 2 contained hFGF. 20 ng/ml: TGF-P1, 5.0 ng/ml; PDGF, 20 nglml; and bovine insulin, 5.0 pg/ml; and medium 3 contained bFGF. 100 ngiml; TGF-P1.25.0 ngiml; PDGF, 100 ngiml; and bovine insulin, 25.0 pg/ml.

"The synergistic effect is the ratio of the mean cell count obtained in the presence of all four growth factors divided by the sum of the mean cell counts obtained with each factor added at that concentration separately.

for the 39 explants that were cultured in serum-free medium (with or without growth factors) that was changed after 6 days, there was no increase in out- growth at day 12 (Fig. 7A). This then raised the ques- tion of whether viable cells remained in the culture wells after 6 days in culture with serum-free medium.

However, when the serum-free medium was replaced after 6 days with medium containing 10% fetal bovine serum, there was considerable outgrowth from the 42 explants of both ligaments at day 12 (Fig. 7B). These results indicated that there were still viable cells in the culture wells.

The synergistic effect of the four growth factors on cell outgrowth from the two ligaments is evident from the data presented in Table 1, which summarizes the results presented in Figs. 2-6. The synergistic effect was much greater at days 3 and 6 for the anterior cruciate ligament explants; however, there clearly was a syner- gistic effect at day 6 for the medial collateral ligament explants as well.

DISCUSSION To improve understanding of the process of wound

healing in ligamentous tissues, we have developed an assay for the outgrowth (proliferation and migration) of cells from explants of rabbit anterior cruciate and medial collateral ligaments (9). Cell outgrowth from tissue explants of these two ligaments was assessed after 3 and 6 days of culture in medium containing three concentrations of four different growth factors: bFGF, TGF-01, PDGF-B, and bovine insulin. Cell out- growth entails several factors that are thought to play an essential role in the repair of tissue damage. These factors include the migration of cells through the tis- sue, followed by proliferation. Previous results have indicated that primary cultures of the anterior cruciate and medial collateral ligaments have very similar growth rates (9,20), but medial collateral ligament cells migrate out from explants at an initial rate that is nearly 10 times greater than that of anterior cruciate ligament cells (9). This difference may provide an im- portant insight as to the basis of the greater healing response of the medial collateral ligament.

From the present work, it is clear that a combina- tion of growth factors is necessary to promote out- growth of cells from these explants. A mixture of four growth factors (bovine insulin, bFGF, TGF-P1, and PDGF-B) at various concentrations stimulated out- growth from explants of the two ligaments almost as effectively as did 10% fetal bovine serum. When the four growth factors were added individually to serum- free medium, only PDGF-B exhibited some stimula- tory effect on outgrowth from the medial collateral ligament. The synergistic stimulatory effect of these growth factors was thus much greater for the an- terior cruciate ligament explants (1 0-fold to 20-fold) than for the medial collateral ligament explants (less than 3-fold).

The effect of the growth factors seemed to last 6 days; however, at 6 days, the effects of the growth factors on cell outgrowth appeared to be less than that of 10% fetal bovine serum. Because the medium was

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440 J. LEE E T AL.

not changed over the 6 days, the growth factors may not have persisted throughout this entire period of time. Consequently, it is possible that greater out- growth could have been obtained with the growth factors by changing the medium after 3 days.

The mechanisms involved in cell outgrowth from ligamentous tissue explants have not been investi- gated previously. In the epidermal explant culture sys- tem, outgrowth begins with attachment, spreading, and migration of epidermal cells from the edges of the explant (10). This system has been used as an in vitro model for epidermal wound healing. Cell outgrowth was stimulated by both epidermal growth factor and TGF-P1. It is known that each of the four growth factors used in this study can enhance both cellular proliferation and migration under appropriate condi- tions (29). Cell division involves different phases of the cell cycle prior to mitosis. It is believed that growth factors can affect these different phases; therefore, mixtures of growth factors can synergistically affect cellular proliferation. Cellular migration is a complex process involving the interaction of cell receptors, called integrins, with specific extracellular matrix pro- teins, such as collagen and fibronectin. Integrins con- taining the and as subunits are associated with cell motility and could be “upregulated” by TGF-P1 (11). A combination of growth factors with TGF-p1 may enhance this expression or the other growth factors may increase the expression of other integrins, leading to a synergistic effect. Our laboratories have shown that the PI and a, subunits were upregulated in vivo at days 7 and 10 during the healing of the medial collateral ligament on cells adjacent to the wound and within the repair site. This stimulation was greater for the medial collateral ligament than for the anterior cruciate ligament (27).

In vitro studies with growth factors involving cel- lular migration and proliferation have been corre- lated with in vivo wound healing with growth factors. Pierce et al. (23) first showed that recombinant TGF- p l and PDGF-B had dose-response curves with in vitro chemotactic assays with monocytes and fibro- blasts that were identical to that found with the natural proteins derived from platelets. They then demonstrated that TGF-pl-treated incisional wounds in vivo had an increased number of fibroblasts and granulation tissue during the first 7 days following injury. It also was shown that PDGF-B had a chemo- tactic response in vivo through 21 days after injury. Each of these growth factors enhanced the strength required to disrupt the incisional wounds. Lynch et al. (16) showed that partially purified PDGF used in vivo to treat skin wounds can stimulate the uptake of 3H- thymidine by biopsy samples of these treated wounds in vitro. The treatment also significantly increased the width of the newly synthesized connective tissue

and epidermal layers in a dose-dependent manner. There have been very few studies of growth fac-

tors and ligaments of the knee. Recently, Conti and Dahners reported on the effects of single applica- tions of bFGF, TGF-P1, or somatomedin (insulin-like growth factor-I [IGF-I]) on transected medial collat- eral ligaments in rats (5 ) . They found that only TGF- p l increased the tensile properties of the ligaments. However, it is clear that more than one growth factor is involved in wound healing. Joyce et al. (14) found bFGF, TGF-P, and PDGF to be present during the healing process of the middle flexor profundus ten- don in chickens. Trippel et al. also demonstrated that bFGF and IGF-I together increased the incorpora- tion of thymidine into bovine growth plate chondro- cytes (28). Lynch et al. and Rutherford et a]. observed better wound healing of skin and dental tissue with the combination of PDGF-B and IGF-I (16,25). In- teractive regulation of growth factors also has been observed. For example, PDGF promoted the cellular production of TGF-p1 in a rodent macrophage cell line (24).

The results of this study suggest that the application of single growth factors alone, in the absence of se- rum, may not be as effective as combinations of four growth factors (bFGF, TGF-P1, PDGF-B, and bovine insulin) in enhancing the healing properties of the anterior cruciate and medial collateral ligaments. It should be noted, however, that growth factors may be present in ligaments following injury, possibly from serum, and the application of single growth factors may be sufficient to enhance wound healing.

Studies of the effects of growth factors on wound healing in animals are extremely costly and time- consuming. The relatively simplistic in vitro approach described herein, despite its limitations, should prove useful in gaining an understanding of some of the fundamental processes involved in the repair of con- nective tissue damage.

Acknowledgmenk This research was supported by National In- stitutes of Health Grants AR34264 and AR07484 and the Malcolm and Dorothy Coutts Institute for Joint Reconstruction and Re- search. We would also like to acknowledge the technical advice of Anna Monosov, the technical assistance of Michael Furniss, and the word processing assistance of Frances Shepherd.

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EFFECT OF GROWTH FACTORS ON ACLIMCL OUTGROWTH 441

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