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

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Biomoterials 17 (1996) 155-162 0 1996 Elsevier Science Limited

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

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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<ollagen 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 immature, but at 4 weeks almost all of the chondrocytes

transferred to the mature stage. Conversely, such mature cartilage tissue was not noticed up to 4

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

mature area was limited to only a small fraction of the implanted composite, unless bFGF was

incorporated in it.

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

Received 7 November 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 to Dr Y. Ikada.

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

155 Biomaterials 1996, Vol. 17 No. 2

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

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 AND METHODS

Collagen 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 freeze- dried. 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 Pharmaceu- tical 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;

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 cell- injected 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 24- well 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 composite preparation are schematically represented in Figure I.

Biomaterials 1996, Vol. 17 No. 2

Effect of bFGF on cartilage regeneration: 7. Fujisato et a/. 157

_--_ 6mm .-_- , Ef

9mm

Collagen sponge disk

bFGF solution

Impregnation of the collagen sponge with bFGF

Medium

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

Injection of chondrocytes in culture medium into the sponge disk

Subcutaneous implantation of the composite into nude mouse

Figure 1 Preparation scheme of rat chondrocyte-collagen sponge composite and its subcutaneous implantation into mouse.

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 levelling- off 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.

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

E II 0 10 20 30

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-‘.)

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


158 Effect of bFGF on cartilage regeneration: 7. Fujisato et a/.

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.

104 0 2 4 6 6

TIME (day)

Figure 4 Growth of rat chondrocytes cultured on a 24-well microplate for tissue culture.

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 microphoto- graph 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

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 collagen sponge contained neither bFGF nor 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

20

0 1 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 In vitro growth of rat chondrocytes in the collagen sponge.


Effect of bFGF on cartilage regeneration: T. Fujisato et al. 159

bFGF (-)

chondrocyte (-) bFGF (+)

chondrocyte (-)

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

bFGF (-) chondrocyte (+)

bFGF (+) chondrocyte (+)

chondrocyte-collagen

composite with bFGF

collagen sponge without

chondrocytes but with bF -GF

Figure 8 Photograph of nude mouse 4 weeks after subcutaneous implantation of the bFGF-impregnated composites with and without 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

Figure 9 Various collagen sponges after 1 week subcutaneous implantation.

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 after implantation, chondrocytes could 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 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


160 Effect of bFGF on cartilaae regeneration: T. Fuiisato et al.

bFGF (-) bFGF (+)

Figure 10 Transformation of the chondrocytexollagen composite to a cartilage lump 4 weeks after subcutaneous implantation.

70 -

60

100 102 104 106 108

CELL DENSITY (cells/sponge)

Figure 11 Effect of the chondrocyte density on the volume change of the chondrocyte-collagen composite with bFGF 2 weeks after implantation.

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


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 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.

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, 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 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 vivoz7 and inhibit the terminal differentiation of chondrocytes and calcification”. The formation of small blood vessels around the collagen sponge became 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 mature stage. More detailed histological and long-term implantation are needed to follow the fate

Effect of bFGF on cartilage regeneration: T. Fujisato et al. 161

H.E. staining SafraninrG faspt grZGGGstaining H.E. staining Safranin-0 fast green staining

Figure 12 Cross-sections of chondrocytexollagen composite with and without bFGF 4 weeks after implantation. (a) Without and (b) with bFGF.

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 neochondrogen- esis 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 . .

release of bFGF accompanies collagen Kodrgyadation in the body.

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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