Effect of basic fibroblast growth factor on cartilage regeneration

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					 Effect of basic fibroblast growth factor on cartilage regeneration
           in chondrocyte-seeded collagen sponge scaffold


    Toshia FUJISATO, Toshinobu SAJIKI, Qiang LIU, and Yoshito IKADA



           Research Center for Biomedical Engineering, Kyoto University

             53 Kawahara-cho, Shogoin, Sakyo, Kyoto 606-01, JAPAN




ABSTRACT



A chondrocyte-collagen composite was prepared in an attempt to regenerate

cartilage by its subcutaneous implantation in nude mouse. When the composite

was impregnated with basic fibroblast growth factor (bFGF) prior to

implantation, regeneration of the cartilage tissue was remarkably accelerated.

Histological staining of the implanted composites with safranin O-fast green

revealed that the cells incorporated in the composites exhibited their phenotype

and formed a new matured cartilage. A thin layer of fibrous capsule was

observed surrounding the implanted composite and the inflammatory response

of the host to the implant was mild. Specific proteoglycans were accumulated

in the composite even 1 week after implantation. At 2 weeks after implantation,

the chondrocytes regenerated the cartilage tissue, although still immatured, but

at 4 weeks almost all of the chondrocytes transferred to the matured stage. On

the contrary, such matured cartilage tissue was not noticed up to 4 weeks after

implantation, if the collagen scaffold was not impregnated with bFGF.

Moreover, the matured area was limited to only a small fraction of the

implanted composite, unless bFGF was incorporated in it.
T.Fujisato et al.                        Effect of bFGF on cartilage regeneration




Keywords: bFGF, chondrocyte, cartilage regeneration, collagen, angiogenesis, tissue

engineering



Correspondence to Professor Y.Ikada.

Fax: +81-75-751-4144




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T.Fujisato et al.                        Effect of bFGF on cartilage regeneration



INTRODUCTION



Recently much attention has been paid on the use of biodegradable polymers to

regenerate metabolic organs such as the liver1,2 and intestine3 and to

reconstruct structural tissues like cartilage4-9, bone8-10, and urothelial

structure11 by cell transplantation12-14. In clinics, cartilage replacement is also

needed, especially in maxillofacial, orthopedic, and plastic surgery. Mostly,

silicone prosthesis and autologous rib bones have been used for this purpose,

but several problems are involved in these cartilage replacements, such as

infection at the interface between the implanted material and the tissue, and

deterioration of the donor site by the filling with fibrous cartilage tissue.

Therefore, a preferable replacement would be to use the natural cartilage tissue.

The first attempt to use the cultured chondrocytes for an articular cartilage

repair was reported by Green et al.15 and it is supposed that a template is

necessary for cartilage reconstruction by chondrocytes in vivo. There are some

reports describing the seeding of collagen and other porous matrices with

chondrocytes for this purpose4-9. Itay et al. reported the repair of bone tissue

by chondrocytes incorporated in collagen gel as a template. Langer, Vacanti,

and their coworkers have developed a tissue engineering technique with the

use of chondrocytes of the target region.       The cells will be isolated from

patients, proliferated by the in vitro culture to a higher density, and then

implanted after preparing a chondrocyte-polymer composite as the core for the

cartilage reconstruction. It is reported that chondrocytes can be isolated from

tissue and grown in culture in such a way as to maintain their phenotype15-18.

       In this work, a chondrocyte-collagen composite is prepared in an attempt

to regenerate the cartilage by its implantation in mouse.        To diminish the

difficulty associated with autograft transplantation, nude mice were used for


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T.Fujisato et al.                            Effect of bFGF on cartilage regeneration



the implantation of the composite. As a scaffold for the cartilage regeneration,

a porous collagen sponge is employed. Collagen has been used as the scaffolds

for tissue regeneration19,20.        We have also reported that it is an excellent

template to regenerate skin21 and esophageal replacement22. When a bilayer

artificial skin composed of an outer layer of silicone and an inner sponge layer

of collagen was placed on the skin defect on the backs of rats, it was observed

that epidermal cells migrated from the edge of the wound between the two

layers21. An artificial esophagus with a bilayered structure made of porous

collagen sponge and silicone was studied to promote tissue regeneration by

collagen, and the collagen sponge was replaced by autologous tissue and

regeneration        of    the   “neoesophagus”   was   observed    2   weeks    after

implantation22.          In our previous work, chondrocyte-poly(lactic acid) (PLA)

complex was prepared to study its potentiality for cartilage reconstruction, but

no matured cartilage tissue was observed in 1 month after implantation23. This

suggests that supplying nutrients to the seeded cells in the early stage of

transplantation to maintain them alive and promote tissue regeneration is very

important. An effective means for this purpose may be to induce the capillary

formation around the implanted composite by giving an angiogenic factor like

basic fibroblast growth factor (bFGF)24. In addition to the angiogenesis, bFGF

is known to perform other important functions such as parenchymal cell

proliferation, differentiation25,26, and promotion of cartilage repair in vivo

although cartilage is an avascular tissue27,28. In this study, bFGF was applied

to the chondrocyte-collagen composite prior to implantaion.




MATERIALS AND METHODS




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T.Fujisato et al.                       Effect of bFGF on cartilage regeneration



Collagen sponge

       A collagen sponge as the scaffold was prepared from 0.3% hydrochloric

solution of type I atelocollagen (Cell matrix®; Nitta Gelatin Co. Ltd., Osaka,

Japan, pH=3.0). The collagen solution was stirred at 2000 rpm for 1 hr at 4 °C to

generate small bubbles and then freeze-dried.       The resulting sponge was

vacuum-dried for 24 hrs at 105 °C, immersed in 0.2% acetic acid solution of

glutaraldehyde for 24 hrs at 4 °C to introduce chemical crosslinking, and

pressed to a sheet of 3 mm thickness. The average pore size, pore volume

fraction, and density of the sponge were 86 µm, 87%, and 1.010-2 g/cm3,

respectively.       The sponge sheet was cut to have a round shape of 9 mm

diameter. Two pieces of round sheets were overlapped each other and then

sewn together with 7-0 polypropylene suture.          The lapped sponge was

immersed overnight in 70% ethanol for sterilization and then washed with

phosphate buffered saline (PBS; Nissui pharmaceutical Co. Ltd., Tokyo, Japan,

pH=7.4). Prior to implantation, the collagen sponge was impregnated with

bFGF by immersing in the 80 µg/ml PBS solution of bFGF for 24 hrs at 4 °C,

unless otherwise stated. bFGF was kindly supplied by Kaken Pharmaceutical

Co. Ltd., Tokyo, Japan. The amount of bFGF was determined by HPLC using a

heparin column.



Chondrocytes

       Chondrocytes were isolated from the costal cartilage of rats by

collagenase digestion29. Costae were removed from sacrificed rats and the

cartilage was isolated carefully from them, so that fibrous tissues were not

included. The isolated cartilage was minced by surgical scissors and immersed

in 0.25% trypsin-0.05% collagenase (Amano Pharmaceutical Co. Ltd., Osaka,

Japan) solution for 1 hr. After 3 times washing with PBS, the treated pieces of


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T.Fujisato et al.                        Effect of bFGF on cartilage regeneration



cartilage were immersed in 0.02% ethylenediaminetetraacetic acid (EDTA)

solution for 1 hr. After washing, they were put on a petri dish for tissue culture

(Corning® Type 25020; Corning Co. Ltd., NY), incubated for about 3 weeks

adjusting the level of medium so as not to float the minced tissues in the

medium, and then the migrated cells were collected by a cell harvesting

solution (0.25% trypsin-0.02% EDTA in PBS).              Eagle's MEM (Nissui

Pharmaceutical Co. Ltd.) was used as culture medium with 10% fatal bovine

serum (Bio Whittaker, Inc., Maryland).        One hundred µl of chondrocyte

suspension containing 1106 cells was carefully injected with a 27 G needle

syringe into the center of lapped collagen sponge disk. The cell-injected sponge

was stored in CO2 incubator for 2 hrs to allow the cells to adhere to the collagen

sponge before implantation as much as possible.



Cell culture

       For the in vitro study, the chondrocyte suspension or the chondrocyte-

collagen composite was put into a 24 well-micro plate for tissue culture

(Corning® Type 258201; Corning Co. Ltd.).          The cell density was 1,000

cells/well in the case of chondrocyte suspension.         Culture medium was

exchanged everyday.     After predetermined periods of time, the cells were

counted by measuring the activity of lactate dehydrogenase (LDH) in the cells

using a test kit for clinical use (LDH monotest®; Boehringer Manheim,

Germany) after      complete cell digestion by 0.1% polyoxyethylene(10)

octylphenyl ether (Triton® X-100; Wako Pure Chemical Industries, Ltd., Osaka,

Japan)30.



Implantation




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T.Fujisato et al.                       Effect of bFGF on cartilage regeneration



       Animals were carefully reared in the Research Center for Biomedical

Engineering, Kyoto University, according to the guideline of Kyoto University

for Animal Experiments. All animals were anesthetized with diethyl ether and

pentobarbital sodium for the release of suffering from the pain during

operation. Two samples of chondrocyte-collagen composite and the control

without chondrocytes were subcutaneously implanted into the back of a male

nude mouse. The mice were divided into 2 groups receiving collagen sponges

with bFGF and without bFGF. Twelve mice were employed in each group.

After predetermined periods of time, the sponges were explanted and subjected

to gross and microscopic observation and a histological study to evaluate the

inflammatory response of the host and cartilage matrix secretion. Explanted

samples were fixed with 10% formaldehyde aqueous solution, replaced with

ethanol, and embedded in paraffin. The fixed samples were sectioned to 10 µm

thickness with a microtome at 3 different distances from the surface of samples,

and stained with Mayer’s Haematoxylin-Eosin (H.E.) solution. In addition to

the conventional H.E. staining, safranin O-fast green staining was applied for

identifying the cartilage proteoglycans31.




RESULTS



bFGF impregnation

       The processes of chondrocyte-collagen composite preparation are

schematically represented in Figure 1. The collagen sponge disk was placed in

plenty of 80 µg/ml bFGF solution in PBS to incorporate bFGF into the disk.

Figure 2 shows the plot of bFGF amount adsorbed into the disk at 37 °C as a

function of time.    As is seen, the bFGF impregnation seems to come to


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T.Fujisato et al.                        Effect of bFGF on cartilage regeneration



saturation after 30 hr incubation, reaching a leveling-off value of 60 µg per mg

of collagen sponge. An addition of serum albumin (BSA) to the bFGF solution

had no significant effect on the bFGF impregnation.          The release of the

adsorbed bFGF into PBS upon immersion of the impregnated sponge in PBS at

37 °C is shown in Figure 3. The bFGF impregnation was carried out at 4 and 37

°C. Clearly, approximately 90% of the bFGF impregnated even at 4 °C for

minimizing the bFGF deactivation still remains in the interior of the collagen

sponge. The bFGF remaining in the sponge is expected to be released upon

enzymatic degradation of the crosslinked collagen when the sponge disk is

implanted in mice.



Chondrocyte seeding

       Rat chondrocytes were seeded in the collagen sponge disk after

trypsinization of the cultured cells isolated from the rat costal cartilage on a

petri dish for tissue culture with cell harvesting solution. Figure 4 shows the in

vitro growth of the chondrocytes after trypsinization on the 24 well-micro plate.

Obviously, cell confluency is obtained upon incubation for about 5 days. As a

preliminary study to determine the effective method for seeding the

chondrocytes in the matrix, cells were seeded in the collagen sponge with three

different methods; 1. injection of the cell suspension in culture medium to the

sponge with a needle at 25 °C, 2. injection of the cell suspension in culture

medium containing 0.3% collagen to the sponge with a needle at 4 °C, and 3.

immersion of the sponge disk into the cell suspension in culture medium at 25

°C in 24 well-micro plate. Two hrs after cell seeding, the disks were washed

with PBS to remove the non-seeded chondrocytes, and the number of cells was

estimated from the activity measurement of LDH.            Figure 5 shows the

percentage of the chondrocytes still remaining in the interior of the sponge


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T.Fujisato et al.                        Effect of bFGF on cartilage regeneration



disks after washing. As can be seen, about 50% of the cells remain adhered to

the collagen sponge when a needle is used, regardless of the presence of

collagen in the cell suspension. In the following study we employed the 1st

method for the cell seeding, that is, injection of the cell suspension containing

1106 cells and bFGF through a needle.

       The chondrocytes seeded in the collagen sponge were further incubated

for their stronger adhesion to the collagen surface. Figure 6 shows the result of

cell growth in the collagen sponge determined by LDH activity. It is seen that

the cell density slowly increases with the incubation time. The decreased cell

density on day 9 is probably due to the cell injury caused after reaching

confluency. The addition of bFGF to the culture medium had no effect on the in

vitro cell growth.   Figure 7 demonstrates a SEM microphotograph of the

chondrocytes attached to the collagen surface after 3 days of incubation. As is

apparent, the cell is spreading on the collagen substrate.



Composite implantation

       The chondrocyte-seeded collagen composite with or without bFGF was

subcutaneously implanted in nude mice to study the effect of bFGF addition. In

our previous work, the collagen sponge carrying neither chondrocytes nor

bFGF disappeared as a result of collagen biodegradation when implanted for

longer than 25 weeks. On the other hand, remarkable angiogenesis was noticed

around the collagen disk when bFGF had been incorporated in the sponge,

independent of the presence of chondrocyte.            A representative optical

photograph of nude mouse with implanted collagen sponges is shown in Figure

8. The sponges were implanted for 4 weeks after impregnation with bFGF. As

can be seen, the presence of implanted sponge is no more recognizable unless

the sponge is seeded with chondrocytes, whereas we can clearly notice the


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T.Fujisato et al.                         Effect of bFGF on cartilage regeneration



sponge from the outside of the mouse if chondrocytes are seeded in the

collagen sponge.

       Optical photographs of sponges implanted for 1 week with and without

bFGF are shown in Figure 9. Obviously, we cannot see any angiogenesis if the

collagen sponge contained neither bFGF nor chondrocytes, whereas seeding of

chondrocytes in the sponge induced angiogenesis even if chondrocyte was not

seeded.      Impregnation of the sponge with bFGF markedly enhanced

angiogenesis, regardless of chondrocyte seeding.        When the chondrocyte-

collagen composite was implanted for 4 weeks together with bFGF, the

formation of cartilage was clearly noticed.      An optical photograph of the

cartilage formed by 4 week implantation of the chondrocyte-collagen composite

is shown in Figure 10. The soft sponge became smaller in size and less flexible.

The size decrease became more prominent with the increasing density of

seeded cells. The size dependence on the density of seeded cells after 2 weeks

of implantation is shown in Figure 11.

       To assess the cartilage regeneration, we stained the cross-section of the

explanted collagen composites with safranin-O fast green. The result for the

chondrocyte-collagen composites implanted for 4 weeks in mice is given in

Figure 12. The area with the regenerated cartilage should be stained strongly

reddish with this dye as a result of metachromasia31.          Figure 12 clearly

indicates that only a very small fraction of the composite cross-section shows

metachromasia unless bFGF is incorporated in the collagen sponge, whereas

most of the cross-section of the composite impregnated with bFGF and seeded

with chondrocytes exhibit strong metachromasia supporting the cartilage

formation. The staining of explants with safranin O-fast green revealed that

chondrocytes could produce specific proteoglycans only after 1 week of

implantation.       However, even at 2 weeks after implantation, chondrocytes


                                       - 10 -
T.Fujisato et al.                         Effect of bFGF on cartilage regeneration



could regenerate the cartilage tissue, although immatured, and at 4 weeks after

implantation, almost all of the chondrocytes transferred to the matured stage.

Without impregnating the collagen sponge with bFGF, such a matured cartilage

tissue was not observed up to 4 weeks after implantation.

       H.E. staining of the implanted composites showed that a thin layer of

fibrous capsule was formed surrounding the implanted composites with a mild

inflammatory response of the host to the implants (Figure 12).




DISCUSSION



To regenerate tissues and maintain their biological functions, individual cells

will be probably collected from the organ or tissue of patients, followed by

attachment of the cells to a bioabsorbable polymer scaffold by a culture

technique. The resulting cell-polymer composite is generally implanted at a site

where the cells can grow and effectively express their function. Chondrocytes

are parenchymal cells like hepatocytes, but it is reported that they exhibit much

more remarkable proliferation and cartilage formation even in vitro if the

culture condition is adequate15-18. This suggests that reconstruction of the

cartilage tissue is easier than that of the liver. In a previous work, a hepatocyte-

or chondrocyte-PLA complex was prepared to study its potentiality for liver

regeneration and cartilage reconstruction23. The measurement of the protein

production from the cells as an index of cell function and expression of their

phenotypes revealed that hepatocytes did not undergo any growth and

gradually diminished their function of albumin biosynthesis on the PLA

substrate in vitro. On the contrary, chondrocytes grew well even on culture

dishes and produced type II collagen, which became maximal on the 10th day


                                       - 11 -
T.Fujisato et al.                        Effect of bFGF on cartilage regeneration



after cultivation. This is simply because the cell density increased to the highest

level by 10 day cultivation, reaching the confluent state and decreased

gradually after the 10th day. This indicates that chondrocytes produced smaller

amounts of collagen under the confluent condition than prolifelative stage. In

the case of hepatocytes, a multicellular colony was seen in the scaffold, in

contrast to the seeded chondrocytes which spread dispersively throughout the

scaffold.

       When the chondrocyte-collagen composite was implanted into nude

mice, injection of any immunosuppressive agent was not necessary even in the

xenograft implantation.     From the histological staining of the implanted

composites with safranin O-fast green which can bind to negatively charged

glycosaminoglycans in cartilage, it is obvious that the implanted cells exhibited

their phenotype in vivo and formed a new matured cartilage in addition to the

morphological characteristics (Figure 12). On the contrary, implantation of

collagen scaffolds without cultured chondrocytes did not result in formation of

new cartilage at all.

       Quick formation of capillaries around the implanted composite seems

necessary to maintain the seeded cells alive and promote tissue reconstruction.

Therefore, bFGF, a well-known angiogenic factor, was incorporated into the

collagen sponge, although cartilage is an avascular tissue. Expectedly, a lot of

small blood vessels were observed around the matrix when the collagen

scaffold was impregnated with bFGF (Figure 9). This strongly suggests that the

use of angiogenic factor is very effective for blood vessel formation around the

complex, which may promote the cartilage formation. Indeed, acceleration of

cartilage reconstruction by bFGF incorporated in the composite was noticed as

demonstrated above. It has been proposed that cartilage tissue is transformed

to bone tissue if vascular invasion occurs and chondrocytes terminally


                                      - 12 -
T.Fujisato et al.                         Effect of bFGF on cartilage regeneration



differentiate to hypertrophic chondrocytes which produce high levels of

alkaline phosphatase. During this study we did not observe any differentiation

of chondrocytes. It is interesting to point out that bFGF is reported to promote

cartilage repair in vivo27 and inhibit the terminal differentiation of

chondrocytes and calcification28. The formation of small blood vessels around

the collagen sponge became the most remarkable at 1 week after implantation

with bFGF and then diminished gradually. The histological study suggests that

chondrocytes would be in the proliferative stage in the first 2 weeks and then

transferred in the matured stage. More detailed histological and long-term

implantation are needed to follow the fate of the regenerated cartilage.

       Any significant effect of bFGF addition on the in vitro proliferation of

chondrocytes was not observed, compared with the in vivo result (Figure 9).

This is probably because bFGF did not influence the chondrocyte growth

directly, but caused angiogenesis around the implanted tissue, which in turn

affected the cartilage regeneration. Freed et al. observed neochondrogenesis

without using any angiogenic factor when chondrocytes were seeded on

fibrous polyglycolic acid and porous PLA7. We implanted hepatocyte-collagen

composites with bFGF in nude mice, but cell viability was not improved by the

bFGF addition32. This indicates that bFGF is not always effective to accelerate

the tissue regeneration. Other kinds of growth factor such as transforming

growth factor also might affect cell proliferation and differentiation. In the

present work, the in vivo release of bFGF from the collagen sponge containing

bFGF could not be determined, as the in vivo release rate was too difficult to

measure. It is very likely that the in vivo release of bFGF accompanies the

collagen biodegradation in the body.




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T.Fujisato et al.                      Effect of bFGF on cartilage regeneration



CONCLUSIONS



It may be concluded that chondrocytes seeded onto a collagen scaffold can

proliferate, express their distinct phenotype, and mature quickly, especially

when the scaffold is impregnated with bFGF.        Thus, the composite from

chondrocytes and the collagen sponge carrying bFGF is very promising for the

tissue engineering of cartilage reconstruction. More detailed histological and

long-term implantation studies are currently under way.




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T.Fujisato et al.                          Effect of bFGF on cartilage regeneration



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T.Fujisato et al.                           Effect of bFGF on cartilage regeneration



Captions to Figures



Figure 1. Preparation scheme of rat chondrocyte-collagen sponge composite

            and its subcutaneous implantation into mouse.



Figure 2. Time course of the collagen sponge impregnation with bFGF from its

            solution at 37 °C. (The initial concentration of bFGF in PBS = 80

            µg/ml)



Figure 3. In vitro release of bFGF from the impregnated collagen sponge at 37

            °C into PBS.

            (E) Incubation at 37 °C for 10 hrs prior to the release test.

            (J) Incubation at 4 °C for 8 hrs and then 37 °C for 2 hrs prior to the

            release test.



Figure 4. Growth of rat chondrocytes cultured on 24 well-micro plate for tissue

            culture.



Figure 5. Chondrocytes adhered to the collagen sponge after washing with

            culture medium when they were applied with three different

            methods. (The initial cell density = 5106 cells/sponge)

            (1) Injection of cell suspension in culture medium with a needle at 25

            °C.

            (2) Injection of cell suspension in culture medium containing 0.3%

            collagen with a needle at 4 °C.

            (3) Immersion of the sponge disk into the cell suspension in culture

            medium at 25 °C in 24 well-micro plate.


                                         - 19 -
T.Fujisato et al.                         Effect of bFGF on cartilage regeneration




Figure 6. In vitro growth of rat chondrocytes in the collagen sponge.



Figure 7. SEM of rat chondrocytes attached to the collagen sponge.                 A

            indicates chondrocyte and B collagen sponge.



Figure 8. Photograph of nude mouse 4 weeks after subcutaneous implantation

            of      the   bFGF-impregnated      composites   with       and   without

            chondrocytes.



Figure 9. Various collagen sponges after 1 week subcutaneous implantation.



Figure 10. Transformation of the chondrocyte-collagen composite to a cartilage

            lump 4 weeks after subcutaneous implantation.



Figure 11. Effect of the chondrocyte density on the volume change of the

            chondrocyte-collagen    composite      with   bFGF      2   weeks   after

            implantation.



Figure 12. Cross-sections of chondrocyte-collagen composite with and without

            bFGF 4 weeks after implantation.

            (a) without bFGF and (b) with bFGF.




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