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

Biomoterials 17 (1996) 155-162 0 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved ELSEVIER

0142-9612/96/$15.00

Effect of basic fibroblast growth factor on cartilage regeneration in chondrocyte-seeded collage ns scaffold Toshia Fujisato, Toshinobu Sajiki, Qiang Liu and Yoshito Ikada Research Center for Biomedical

Engineering,

Kyoto

University,

53 Kawahara-cho,

Shogoin,

Sekyo,

Kyoto

6&i-01,

Japan

A chondrocyte
factor

accelerated.

(bFGF)

transferred mature

after

even

incorporated Keywords:

in the composites

1 week

to the mature

area was limited

regeneration

of the implanted capsule

was observed

after

implantation.

tissue,

although

stage.

Conversely,

their

such

phenotype

surrounding Specific

At 2 weeks

after

cartilage

was not impregnated

to only

fraction

of the implanted

a small

and formed

green

revealed

a new matured

the implanted

composite were

almost

tissue

scaffold

was remarkably

O-fast

implantation,

if the collagen

by its

with basic fibroblast

proteoglycans

but at 4 weeks

mature

tissue

with Safranin

was mild.

still immature,

cartilage

with

and the

accumulated

the chondrocytes all of the chondrocytes

was not noticed

up to 4

bFGF. Moreover,

composite,

the

unless bFGF was

in it. bFGF,

chondrocyte,

Received 7 November

cartilage

regeneration,

collagen,

angiogenesis,

tissue

engineering

1994; accepted 1 June 1995

Recently, much attention has been paid to the use of biodegradable polymers to regenerate metabolic organs such as the liver12’ and intestine3 and to reconstruct structural tissues like cartilage4-‘, bones-l0 and urothelial structures’l by cell transplantation’2-‘4. In clinics, cartilage replacement is also needed, especially in maxillofacial, orthopaedic and plastic surgery. Mostly, silicone prostheses 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 Green15 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-g. Itay et ~1.~ reported the repair of bone tissue by chondrocytes incorporated in collagen gel as a template. Langer,

Correspondence

exhibited

to regenerate

was impregnated

of the cartilage

composites

of the host to the implant

the cartilage implantation

in an attempt

When the composite

to implantation,

of fibrous

response

in the composite regenerated

layer

was prepared

in nude mouse. staining

incorporated

A thin

inflammatory

weeks

prior

Histological

that the cells cartilage.

composite

implantation

Vacanti and their co-workers 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-l*. 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 the implantation of the composite. As a scaffold for the cartilage regeneration, a porous collagen sponge was employed. Collagen has been used as the scaffold for tissue regeneration’g’20. This paper reports that it is an excellent template to regenerate skin” and oesophageal replacement”. 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 layers’l. An artificial oesophagus with a bilayered structure made of porous collagen sponge and silicone was studied to

to Dr Y. Ikada. 155

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Effect of bFGF on cartilage

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promote tissue regeneration by collagen, and the collagen sponge was replaced by autologous tissue and regeneration of the ‘neo-oesophagus’ was observed 2 weeks after implantationz2. In a previous work, chondrocyte-poly(lactic acid) (PLA) complex was prepared to study its potential for cartilage reconstruction, but no mature cartilage tissue was observed 1 month after implantation’“. This suggests that supplying nutrients to the seeded cells in the early stage of transplantation to keep 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, differentiation 25.Z6 and promotion of cartilage repair in viva, although cartilage is an avascular tissuez7,‘a. In this study, bFGF was applied to the chondrocyte-collagen composite prior to implantation.

MATERIALS Collagen

AND METHODS

sponge

A collagen sponge as the scaffold was prepared from 0.3% hydrochloric acid solution of type I atelocollagen (Cell matrix ” ; Nitta Gelatin Co. Ltd, Osaka, Japan, pH = 3.0). The collagen solution was stirred at 2000rpm for 1 h at 4°C to generate small bubbles and then freezedried. The resulting sponge was vacuum-dried for 24 h at 105”C, immersed in a 0.2% acetic acid solution of glutaraldehyde for 24 h at 4°C to introduce chemical cross-linking and pressed to a sheet of 3mm thickness. The average pore size, pore volume fraction and density of the sponge were 86pm, 87% and 1.0 x lo-’ gem “, respectively. The sponge sheet was cut to have a round shape of 9mm diameter. Two pieces of round sheets overlapped each other and were then sewn together with 7-O 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 a 80pgmlp’ PBS solution of bFGF for 24 h 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 digestion’“. Costae were removed from dead 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 h. After washing three times with PBS, the treated pieces of cartilage were immersed in 0.02% EDTA solution for 1 h. After washing, they were put on a Petri dish for tissue culture (Corning” Type 25020; Biomaterials

1996, Vol. 17 No. 2

regeneration:

T. Fujisato

et al.

Corning Co. Ltd, NY, USA), 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% fetal bovine serum (Bio Whittaker, Inc., Maryland). One hundred microlitres of chondrocyte suspension containing 1 x 10’ cells were carefully injected with a 27G needle syringe into the centre of a lapped collagen sponge disc. The cellinjected sponge was stored in a CO2 incubator for 2 h 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 24well microplate for tissue culture (Corning ” Type 258201; Corning Co. Ltd). The cell density was 1000 cells per well in the case of chondrocyte suspension. Culture medium was exchanged every day. 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(I0) octylphenyl ether (Triton ” X-100; Wako Pure Chemical Industries Ltd, Osaka, Japan)““.

Implantation Animals were carefully reared in the Research Center for Biomedical Engineering (Kyoto University, Japan) according to the Guidelines of Kyoto University for Animal Experiments. All animals were anaesthetized with diethyl ether and pentobarbital sodium for the release of suffering from the pain during the 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 two 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 10pm thickness with a microtome at three different distances from the surface of samples, and stained with Mayer’s haematoxylin-eosin (HE) solution. In addition to the conventional HE staining, Safranin O-fast green staining was applied for identifying the cartilage proteoglycans”‘.

RESULTS bFGF impregnation The processes of chondrocyte-collagen preparation are schematically represented

composite in Figure I.

Effect of bFGF on cartilage

regeneration:

7. Fujisato

et a/.

_--_ Ef ,

.-_-

157

bFGF solution

6mm

9mm

Collagen sponge disk

Impregnation of the collagen sponge with bFGF

Injection of chondrocytes in culture medium into the sponge disk

Medium

Subcutaneous implantation of the composite into nude mouse

Incubation in humidified 5% COn at 37°C for 2 hrs

Figure 1

Preparation

scheme

of rat chondrocyte-collagen

sponge

The collagen sponge disc was placed in plenty of 80 pgmlll bFGF solution in PBS to incorporate bFGF into the disc. Figure 2 shows the plot of bFGF amount adsorbed into the disc at 37°C as a function of time. As is seen, the bFGF impregnation seems to come to saturation after 30 h incubation, reaching a levellingoff value of 6Opg per mg of collagen sponge. An addition of bovine 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 cross-linked collagen when the sponge disc is implanted in mice.

composite

E 0 II

and its subcutaneous

implantation

into mouse.

30

20

10 TIME (hr)

Figure 2 Time course of the collagen sponge impregnation with bFGF from its solution at 37°C. (The initial concentration of bFGF in PBS = 8Opg ml-‘.)

Chondrocyte seeding Rat chondrocytes were seeded in the collagen sponge disc 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 34-well microplate. 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; (z) 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 disc into the cell suspension in culture medium at 25°C in a M-well microplate. Two hours after cell seeding, the

discs 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 discs 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 first method for the cell seeding, that is, injection of the cell suspension containing 1 x lo6 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 Biomaterials

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3ol----6

I

0

10

20

30

40

50

TIME (hr)

Figure 3 In vitro release of bFGF from the impregnated collagen sponge at 37°C into PBS. 0, Incubation at 37°C for 10 h prior to the release test; 0, incubation at 4°C for 8 h and then 37°C for 2 h prior to the release test.

regeneration:

7. Fujisato

et a/.

clearly notice the 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 contained neither bFGF nor collagen sponge chondrocytes, whereas seeding of chondrocytes in the sponge induced angiogenesis even if chondrocytes were 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-

1oc

8C

60

40

104

0

2

4

6

20

6

TIME (day)

Figure 4 microplate

Growth of rat chondrocytes for tissue culture.

cultured

on a 24-well

0

2

1

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 i’ demonstrates an 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 disc when bFGF had been incorporated in the sponge, independent of the presence of chondrocytes. 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 longer recognizable unless the sponge is seeded with chondrocytes, whereas we can Biomaterials

1996, Vol. 17 No. 2

3

METHOD 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 x lo6 cells per 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 disc into the cell suspension in culture medium at 25°C in a 24-well microplate.

10’

106

105 0

2

4

6

6

10

TIME (day)

Figure 6 sponge.

In vitro growth

of rat chondrocytes

in the collagen

Effect of bFGF on cartilage

regeneration:

T. Fujisato

Figure 7 SEM of rat chondrocytes attached sponge. A, Chondrocyte; B, sponge.

et al.

159

to the collagen

bFGF (-) chondrocyte (-)

bFGF (+) chondrocyte (-)

bFGF (-) chondrocyte (+)

bFGF (+) chondrocyte (+)

Figure 9 Various neous implantation.

chondrocyte-collagen composite with bFGF

collagen sponge without chondrocytes

but with bF-GF

Figure 8 Photograph of implantation subcutaneous composites with and without

nude mouse 4 weeks after of the bFGF-impregnated chondrocytes.

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 metachromasia3r. Figure 12 clearly indicates that only a very small fraction of the composite cross-section shows metachromasia unless bFGF is incorporated in the

collagen

sponges

after

1 week

subcuta-

collagen sponge, whereas most of the cross-section of the composite impregnated with bFGF and seeded with chondrocytes exhibits 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 implantation, chondrocytes could after regenerate the cartilage tissue, although immature, and at 4 weeks after implantation, almost all of the chondrocytes transferred to the mature stage. Without impregnating the collagen sponge with bFGF, such a mature cartilage tissue was not observed up to 4 weeks after implantation. HE 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 probably be 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 cellpolymer 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 Biomaterials

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Effect of bFGF on cartilaae

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10

Transformation

of the chondrocytexollagen

composite

70 -

60

100

102

104

106

108

CELL DENSITY (cells/sponge)

Figure 11 Effect of the chondrocyte change of the chondrocyte-collagen weeks after implantation.

density on the volume composite with bFGF 2

the cartilage tissue is easier than that of the liver. In a previous work, a hepatocyte-PLA or chondrocytePLA complex was prepared to study its potential for liver regeneration and cartilage reconstructionz3. 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 after cultivation. This is simply because the cell density increased to the highest level by 10 days of state, and cultivation, reaching the confluent after the 10th day. This decreased gradually chondrocytes produced smaller indicates that amounts of collagen under the confluent condition than in the proliferative 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. Biomaterials

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T. Fuiisato

et al.

bFGF (+)

bFGF (-) Figure tion.

regeneration:

to a cartilage

lump 4 weeks

after

subcutaneous

implanta-

When the chondrocyte-collagen composite was into nude mice, injection of implanted any immunosuppressive agent was not necessary even in the xenograft implantation. From the histological staining of the implanted composites with Safranin Ofast 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 mature cartilage in addition to the morphological characteristics (Figure ZZ). Conversely, implantation of collagen scaffolds without cultured chondrocytes did not result in formation of new cartilage at all. around capillaries the Quick formation of 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, many 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 differentiate to chondrocytes which produce high hypertrophic 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 vivoz7 and inhibit the terminal differentiation of chondrocytes and calcificaThe formation of small blood vessels around tion”. the collagen sponge became most remarkable at 1 with bFGF and then implantation week after gradually. histological study diminished The suggests that chondrocytes would be in the proliferative stage in the first 2 weeks and then transferred in the mature stage. More detailed histological and long-term implantation are needed to follow the fate

Effect of bFGF on cartilage

H.E. staining Figure 12 Cross-sections and (b) with bFGF.

regeneration:

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Fujisato et al.

161

SafraninrG fasptgrZGGGstaining of chondrocytexollagen

composite

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. When hepatocyte-collagen composites with bFGF were implanted in nude mice, cell viability was not improved by the bFGF addition”‘. This indicates that bFGF is not always effective to accelerate the tissue regeneration. Other kinds of growth factor such as transforming growth factor might also 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 . . of bFGF accompanies collagen release Kodrgyadation in the body.

H.E. staining

with and without

bFGF 4 weeks

Safranin-0 fast green staining after

implantation.

(a) Without

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.

REFERENCES 1

2

3

Fontaine M, Hansen LK, Thompson S et al. Transplantation of genetically altered hepatocytes using cellpolymer constructs. Transplant Proc 1993; 25: 10021004. Wald HL, Sarakinos G, Lyman MD, Mikos AG, Vacanti JP, Langer R. Cell seeding in porous transplantation devices. Biomaterials 1993; 14: 270-278. Mooney DJ, Organ G, Vacanti JP, Langer R. Design and Biomaterials 1996, Vol. 17 No. 2

162

4

5

6

7

8

9

10

11

12 13

14

15

16

17

Effect fabrication of biodegradable polymer devices to engineer tubular tissues. Cell Transplant 1994; 3: 203210. Itay S, Abramovici A, Nevo Z. Use of cultured embryonal chick epiphyseal chondrocytes as grafts for defects in chick articular cartilage. Clin Orthop 1987; 220: 284-303. Puelacher WC, Kim SW, Vacanti JP, Schloo B, Mooney D, Vacanti CA. Tissue-engineered growth of cartilage: the effect of varying the concentration of chondrocytes seeded onto synthetic polymer matrices. Int J Oral Maxillofacial Surg 1994; 23: 49-53. Vacanti CA, Paige KT, Kim WS, Sakata J, Upton J, Vacanti JP. Experimental tracheal replacement using tissue-engineered cartilage. J Pediatr Surg 1994; 29: 201-205. Freed LE, Marquis JC, Nohria A, Emmanual J, Mikos AG, Langer R. Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers. JBiomed Mater Res 1993; 27: 11-23. Vacanti CA, Vacanti JP. Bone and cartilage reconstruction with tissue engineering approaches. Otolaryngol Clin North Am 1994; 27: 263-276. Vacanti CA, Kim W, Upton J et al. Tissue-engineered growth of bone and cartilage. Transplant Prac 1993; 25: 1019-1021. Thomson RC, Yaszemski MJ, Powers JM, Mikos AG. Fabrication of biodegradable polymer scaffolds to engineer trabecular bone. J Biomater Sci Polymer Edn 1995; 7: 23-38. Atala A, Freeman MR, Vacanti JP, Shepard J, Retik AB. Implantation in viva and retrieval of artificial structures consisting of rabbit and human urothelium and human bladder muscle. J Ural 1993; 150: 608612. Langer R, Vacanti JP. Tissue engineering. Science 1993; 260: 920-926. Mikos AG, Sarakinos G, Leite SM, Vacanti JP, Langer R. Laminated three-dimensional biodegradable foams for use in tissue engineering. BiomateriaJs 1993; 14: 323330. Mikos AG, Lyman MD, Freed LE, Langer R. Wetting of poly(L-lactic acid) and poly(oL-lactic-co-glycolic acid) foams for tissue culture. Biomaterials 1994: 15: 55-58. Green WT Jr. Articular cartilage repair. Behavior of rabbit chondrocytes during tissue culture and subsequent allografting. Chin Orthop 1977; 124: 237250. Binderman I, Greene RM, Pennypacker JP. Calcification of differentiating skeletal mesenchyme in vitro. Science 1979; 206(4415): 222-225. Suzuki F. Osteogenesis by chondrocytes from growth

Biomaterials

1996, Vol. 17 No. 2

18

19

20

21

22

23

24

25

26

27

28

29

30 31

32

of bFGF

on cartilage

reoeneration:

T. Fuiisato

et al.

cartilage. Tanpakushitu Kakusan Koso 1978; 23: 13031311. Suzuki F. Hormonal effects on expression of the differentiated phenotype of chondrocytes in culture. Taisha 1982; 19: 729-736. Yannas IV, Lee E, Orgill DP, Skrabut EM, Murphy GF. Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin. Proc Nat Acad Sci USA 1989; 86: 933-937. Matsuda T, Akutsu T, Kira K, Matsumoto H. Development of hybrid compliant graft: rapid preparative method for reconstruction of a vascular wall. ASAIO Trans 1989; 35: 553-555. Matsuda K, Suzuki S, Isshiki N, Ikada Y. Re-freeze dried bilayer artificial skin. Biomaterials 1993; 14: 10301035. Natsume T, Ike 0, Okada T, Takimoto N, Shimizu Y, Ikada Y. Porous collagen sponge for esophageal replacement. J Biomed Mater Res 1993; 27: 867-875. Ito K, Fujisato T, Ikada Y. Implantation of cell-seeded biodegradable polymers for tissue reconstruction. Mater Res Sot Symp Proc 1992; 252: 359-365. Broadley KN, Aquino AM, Woodward SC et al. Monospecific antibodies implicate basic fibroblast growth factor in normal wound repair. Lab Invest 1989; 61: 571-575. Slack JMW, Darlington BG, Health JK, Godsave SF. Mesoderm induction in early Xenopus embryos by heparin-binding growth factors. Nature 1987; 326: 197200. Kimelman D, Abraham JA, Haaparanta T, Palisi TM, Kirschner MW. The presence of fibroblast growth factor in the frog egg: its role as a natural mesoderm inducer. Science 1988; 242: 1053-1056. Cuevas P, Burgos J, Baird A. Basic fibroblast growth factor (FGF) promotes cartilage repair in vivo. Biochem Biophys Res Commun 1988; 156: 611-618. Kato Y, Iwamoto M. Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation. J Biol Chem 1990; 265: 5903-5909. Shimomura Y, Yoncda T, Suzuki F. Osteogenesis bv chondrocytes from growth cartilage of rat rib. Cnlci/” Tiss Res 1975; 19: 179-187. Tamada Y. Kulik EA, Ikada Y. A simple method for platelet counting. Biomaterinls 1995; 16: 259-261. Pedrini MA, Maynard JA. Pedrini VA. Pseudoachondroplasia: biochemical and histochemical studies of cartilage. JBone Joint Surg Am 1984; 66: 1408-1414. Morikawa N, Fujisato T, Tabata Y, Ikada Y. Implantation of hepatocyte-seeded polymer scaffold. Paper presented at the 23rd Symposium of Medical Polymers, 16-17 June 1994. Tokyo, Japan.