FGF-18 IN GRAFT TRANSPLANTATION AND TISSUE ENGINEERING PROCEDURES

Abstract

The present invention provides a new method related to regenerative medicine for the treatment of cartilage disorders, osteoarthritis and cartilage injury in particular. More particularly, it relates to an FGF-18 compound for use in tissue engineering and graft procedures, such as osteochondral or cartilage transplantation or autologous chondrocyte implantation (ACI).

Claims

1. A process for producing a transplantable cartilage material for tissue engineering, wherein said process comprises the steps of culturing chondrogenic cells, in 3D culture, in a culture medium comprising an FGF-18 compound for a time sufficient to allow the formation of a transplantable cartilage material, and wherein said FGF-18 compound is selected from the group consisting of: a) a polypeptide comprising residues 28-207 of SEQ ID NO:1, b) a polypeptide comprising residues 28-196 of SEQ ID NO:1, and c) a polypeptide comprising or consisting of SEQ ID NO:2.

2. A process for producing a transplantable cartilage material for tissue engineering, wherein said process comprises the steps of culturing chondrogenic cells, in 3D culture, in a culture medium comprising an FGF-18 compound for a time sufficient to allow the formation of a transplantable cartilage material, wherein the FGF-18 compound is added intermittently in the culture medium, for about one, two or three days per month, and wherein said FGF-18 compound is selected from the group consisting of: a) a polypeptide comprising residues 28-207 of SEQ ID NO:1, b) a polypeptide comprising residues 28-196 of SEQ ID NO:1, and c) a polypeptide comprising or consisting of SEQ ID NO:2.

3. The process according to claim 1, wherein the FGF-18 compound is added intermittently in the culture medium, for one, two or three days per week, said one-day, two-days or three-days addition being repeated each week for 2 weeks of culture, 3 weeks of culture or 4 weeks of culture.

4. The process according to claim 2, wherein the FGF-18 compound added intermittently in the culture medium, for one, two or three days per month, said one-day, two-days or three-days addition being repeated each month for 2 months of culture, 3 months of culture or 4 months of culture.

5. The process according to claim 1, wherein the chondrogenic cells are chondrocytes.

6. The process according to claim 1, wherein the chondrogenic cells are mesenchymal stem cells derived from mature tissues.

7. The process according to claim 1, wherein the chondrogenic cells are harvested from a mammal before expansion or culture.

8. The process according to claim 7, wherein the chondrogenic cells are harvested from the mammal to be treated or from a different mammal.

9. The process according to claim 7, wherein the mammal is a human.

10. Transplantable cartilage material obtained according to the process of claim 1 for use in the treatment of a cartilage disorder.

11. The transplantable cartilage material, according to claim 10, wherein the cartilage disorder is osteoarthritis, cartilage injury or osteochondral defect.

12. A process for regenerating cartilage in a mammal in an area of articular cartilage defect due to a cartilage disorder, said process comprising the steps of: (a) culturing chondrogenic cells in scaffold-free 3D culture, in a culture medium comprising an FGF-18 compound, and (b) administering to the mammal in thereof the cultured chondrogenic cells obtained from step (a), wherein the FGF-18 compound is intermittently in the culture medium, for about one day per week, said about one-day of FGF-18 compound addition being repeated each week for at least 2 weeks of culture, at least 3 weeks of culture or at least 4 weeks of culture, and wherein said FGF-18 compound is selected from the group consisting of: a) a polypeptide comprising residues 28-207 of SEQ ID NO:1, b) a polypeptide comprising residues 28-196 of SEQ ID NO:1, and c) a polypeptide comprising or consisting of SEQ ID NO:2.

13. The process according to claim 12, wherein the cartilage disorder is osteoarthritis, cartilage injury or osteochondral defect.

14. The process according to claim 12, wherein the chondrogenic cells are mesenchymal stem cells derived from mature tissues.

15. The process according to claim 12, wherein the chondrogenic cells are harvested from a mammal before expansion or culture.

16. The process according to claim 15, wherein the chondrogenic cells are harvested from the mammal to be treated or from a different mammal.

17. The process according to claim 15, wherein the mammal is a human.

Description

DESCRIPTION OF THE FIGURES

[0059] FIG. 1: Preparation of cartilage defect-repair model: (A) 8 mm cartilage plug, (B) central 4 mm defect creation, (C) insertion of cartilage into defect, and (D) long term culture of repair construct. OD means outer diameter and ID means inner diameter.

[0060] FIG. 2: (A-C) Transverse cross sections of 3D μCT reconstruction with different treatments. (D) Integration strength of the repaired defect showing increasing strength from the control to the 1+30 treatment to the 1+6 treatment. (E) Experimental setup of the push-out testing rig. Error bars are SEM.

[0061] FIG. 3: μCT scans of cartilage-to-cartilage repair constructs. Left: single μCT scan slice representative of the sample. Center: three dimensional reconstruction. Right: cross-section of the reconstruction. The μCT scans demonstrate increasing integration from control to 1+30 to 1+6 treatments

[0062] FIG. 4: Treatment of the CTAs with rhFGF-18

[0063] FIG. 5: Cell content/CTA estimated from the DNA content/CTA after 4 weeks of treatment without rhFGF-18 (CTR), in permanent presence of rhFGF-18 (perm), with rhFGF-18 the first week only (1 w) or 1 day per week (1 d/w). rhFGF-18 was use at 10 or 100 ng/mL. N=4. */*** mean significantly different from the control with p<0.05 and 0.001 respectively

[0064] FIG. 6: GAG and HPro/CTA content after 4 weeks of treatment without rhFGF-18 (CTR), in permanent presence of rhFGF-18 (perm), with rhFGF-18 the first week only (1 w) or 1 day per week (1 d/w). rhFGF-18 was use at 10 or 100 ng/mL. N=4. */**/*** mean significantly different from the control with p<0.05, 0.01 and 0.001 respectively

[0065] FIG. 7: Collagen type I, II and Sox9 expression as well as the Collagen II/I ratio were evaluated in CTAs after 4 weeks of culture without rhFGF-18 (CTR), in permanent presence of rhFGF-18 (perm), with rhFGF-18 the first week only (1 w) or 1 day per week (1 d/w). rhFGF-18 was use at 10 or 100 ng/mL. N=4. */**/*** mean significantly different from the control with p<0.05, 0.01 and 0.001 respectively

[0066] FIG. 8: Bovine primary chondrocytes were cultivated one or two weeks in monolayer in absence (CTR) or in permanent presence of rhFGF-18 100 ng/mL. Cell concentration was determined N=6. Collagen type I, II and Sox9 expression a N=4 was measured by quantitative Real-Time PCR.

[0067] FIG. 9: Cell content/CTA estimated from the DNA content/CTA after 4 weeks of treatment without rhFGF-18 (CTR), in permanent presence of rhFGF-18 (perm), with rhFGF-18 1 day per week (1 d/w). * means significantly different from the control with p<0.05.

[0068] FIG. 10: GAG content after 4 weeks of treatment without rhFGF-18 (CTR), in permanent presence of rhFGF-18 (perm), with rhFGF-18 1 day per week (1 d/w). * means significantly different from the control with p<0.05.

[0069] FIG. 11: Collagen type I, II and Sox9 expression as well as the Collagen II/I ratio were evaluated in CTAs after 4 weeks of culture without rhFGF-18 (CTR), in permanent presence of rhFGF-18 (perm), with rhFGF-18 1 day per week (1 d/w). * means significantly different from the control with p<0.05.

DESCRIPTION OF THE SEQUENCES

[0070] SEQ ID NO.1: Amino acid sequence of the native human FGF-18.

[0071] SEQ ID NO.2: Amino acid sequence of the recombinant truncated FGF-18 (trFGF-18).

EXAMPLES

Material

[0072] The recombinant truncated FGF-18 (rhrFGF18) of the present examples has been prepared by expression in E.coli, according to the technique described in the application WO2006/063362. In the following examples rhFGF-18, FGF-18 and sprifermin are used interchangeably.

Example 1

Methods

[0073] Fresh hyaline cartilage was harvested from the trochlear groove of juvenile bovine knees (3-6 months old). Cylindrical explants of 8 mm (FIG. 1A) were removed with a biopsy punch and cultured overnight in complete medium (DMEM 4.5 g/L D-Glucose and L-Glutamine, 10% FBS, 1% PSF, 1% Fungizone, 1% MEM Vitamins, 25 mM HEPES and 50 μg/ml Vitamin C). Samples were trimmed of bone and defects (4 mm diameter) were created to form a core and annulus repair construct (FIG. 1B). Both the inner core and outer annulus were cultured separately for 24 hours before the defect was filled with the original core. Samples were then cultured in complete medium, or treated with Sprifermin (rhFGF-18, 100 ng/ml). Treatments consisted of one dose of rhFGF-18 for 24 hours, applied once a week (and repeated weekly) (1+6) or one 24 hour treatment followed by 1 month of culture in complete medium (1+30 days). Samples were harvested after 4 weeks of culture. Push-out mechanical testing (n=4-6) was performed (Instron 5848, Instron, Norwood, Mass.) using a custom testing rig (FIG. 2E, [3]). Integration strength was calculated by dividing the peak force by the integration area. For 3D visualization, samples (n=6 were soaked in a modified Lugol's solution (2.5% I.sub.2 and 5% KI in dH.sub.2O) for 24 hours [4] and scanned by μCT at an energy level of 55 kV and intensity of 145 μA with a voxel size of 6 μm and 10.5 μm (μCT 35 and vivaCT 40, SCANCO Medical, Wayne, Pa.). Scans were analyzed and reconstructed using the manufacturers software, and cross sections were used to evaluate defect integration. Additional samples (n=3) were fixed overnight in 4% PFA and analyzed histologically for cell and matrix deposition at the interface.

Results

[0074] The integration strength (FIG. 2D) of control samples was the lowest (2.5±1.4 kPa), with progressively increasing properties with the 1+30 (monthly cycle) (5.0±2.4 kPa) and 1+6 (weekly cycle) (10.2±3.7 kPa) treatments. While the results are striking when comparing controls and treated groups, with the replicate numbers possible in this study, statistical significance was not achieved. μCT analysis of control constructs (FIG. 3, top left) showed a distinct dark circle, indicating separation between the outer annulus and inner core, and thus poor integration. The 1+30 treatment (FIG. 3, middle left) showed a less distinct circle, suggesting a smaller gap and greater integration, and the 1+6 treatment (FIG. 3, bottom left) showed very homogenous μCT signal across the interface, indicative of the greatest degree of integration. Evidence of this increased integration was apparent on both vertical and transverse cross sections throughout the samples.

[0075] A successful cartilage repair requires that the repair material (engineered or native) be well-integrated into the surrounding cartilage to ensure continuous load transfer (and lack of stress concentrations) across the interface. In this study we investigated the potential of Sprifermin to enhance integration of cartilage in a well-defined ex vivo (explant) cartilage repair model. Sprifermin has an established pro-proliferative effect on chondrocytes (Elthworth et al., 2002), where transient (24 hour) exposure to this biological agent elicits the most striking response. Our findings clearly demonstrate that Sprifermin improves integration strength and matrix deposition at the interface (as evidenced by contrast-enhanced μCT showing a more uniform attenuation by increase in GAG-containing proteoglycans). In this study, one 24 hour administration weekly for 4 weeks leads to an overall better outcome than one 24 hour treatment over one month This latest regimen is also be useful as, although not as good as the weekly-cycle regimen, it provides a surprising improvement compared to the control construct (i.e. in absence of sprifermin treatment). This study demonstrates for the first time that a biologic (and in particular a sprifermin) has improved the integration of cartilage surfaces in a clinically relevant repair model.

Conclusions

[0076] This study demonstrates that Sprifermin is able to improve the integration of cartilage surfaces in a model of cartilage repair. The findings implicate its potential usefulness in surgical procedures such as OATS and in tissue engineering approaches where cartilage like biomaterials will be required to successfully integrate with native cartilage in order to achieve clinical success.

Example 2

Method

[0077] Primary osteoarthritic chondrocytes were isolated from the cartilage of patients undergoing total knee replacement. Cells were cultivated for a few days in monolayer culture first and then for one week in scaffold-free 3D culture before starting the treatment. The latter consisted of the incubation with rhFGF-18 [100 ng/mL] permanently or one day/week for a total period of four weeks. Results were compared to a control culture without sprifermin. Biochemical assays, quantitative PCR (qPCR) and histology were used to characterize the 3D constructs.

Results (Data Not Shown)

[0078] To ensure phenotype maintenance, 3D scaffold-free culture was used to test the effect of sprifermin on hOA chondrocytes. In this setting rhFGF-18 [1 day/week] has been found to have a beneficial effect on the cell content and to greatly increase the size and matrix content (GAG and HPro content) of the 3D constructs. rhFGF-18 was also found to decrease Collagen I expression in comparison with untreated cells.

Conclusion

[0079] As observed in previous studies with bovine and porcine chondrocytes, sprifermin was found to have an anabolic activity in hOA chondrocytes. The findings implicate its potential usefulness in tissue engineering approaches where cartilage like biomaterials will be required to successfully integrate with native cartilage in order to achieve clinical success.

Example 3

Methods

[0080] Porcine chondrocytes were isolated from the cartilage of a femoral head of a pig hip. After dissection of the joints, the cartilage was harvested and digested 45 minutes with collagenase 0.25%. The loosened cells were discarded and the cartilage further digested overnight with collagenase 0.1% to extract the chondrocytes. Porcine chondrocytes were cultured in suspension as CTA (Cartilage Tissue Analogs) a first week without any treatment followed by one of the following treatments: 1) four weeks of culture in permanent presence of rhFGF-18 at 10 or 100 ng/mL, 2) one week of culture in presence of rhFGF-18 at 10 or 100 ng/mL and subsequently three weeks without rhFGF-18, 3) three weeks of culture with rhFGF-18 at 10 or 100 ng/mL given 1 day per week (i.e. 24 h exposure followed by 6 days without rhFGF-18) and subsequently one week without rhFGF-18 or 4) four weeks in absence of rhFGF-18, as a control (FIG. 4). At the end of the culture period, CTAs were harvested and analyzed for their GAG, hydroxyprolin and cell content. Gene expression for Collagen I, II, and Sox9 was evaluated and histology for Safranin O and Collagen type I and II was also performed.

Results—Effect of Permanent or Intermittent Exposure to rhFGF-18 on Cell Growth in CTAs

[0081] For each culture condition, CTAs were lysed and the DNA content was evaluated to calculate the number of cells/CTA (FIG. 5). In the control culture (without rhFGF-18) no proliferation was observed as the cell number (1.2 million) was similar to the inoculation density (1 million cells/CTA). However, as expected, the permanent presence of rhFGF-18 increased chondrocyte proliferation (with 2.2 and 2.49 million cells/CTA with rhFGF-18 10 and 100 ng/mL, respectively). When rhFGF-18 was given one week only and the chondrocytes further cultured 3 weeks without rhFGF-18 (1 w), no increase in the proliferation could be observed in comparison to the control. On the contrary, when rhFGF-18 was given one day per week (1 d/w), rhFGF-18 stimulated proliferation in comparison to the control but also in comparison with the permanent exposure. The cell content/CTA reached 4 million cells/CTA with rhFGF-18 100 ng/mL, one day/week, in comparison with 1.2 million in absence of rhFGF-18 or 2.49 million in permanent presence of rhFGF-18 100 ng/mL.

Results—Effect of Permanent or Intermittent Exposure to rhFGF-18 on Matrix Production in CTAs

[0082] For each culture condition, CTAs were digested with proteinase K and the GAG and hydroxyproline contents were evaluated (FIG. 6). GAG reflects the proteoglycan content whereas hydroxyproline reflects the collagen content of the CTAs. As previously observed, the permanent presence of rhFGF-18 decreased the GAG content/CTA (2.6 less GAG in comparison with the control) and also the hydroxyproline content/CTA (2.1 less hydroxyproline in comparison with the control). On the contrary when rhFGF-18 is given intermittently (1/week or 1 day/week) the GAG and the hydroxyproline content were increased. For example, when rhFGF-18 100 ng/mL was given one day per week, the GAG content was increased by 2.67 and the hydroxyproline content by 2.13 in comparison to the control.

Results—Effect of Permanent or Intermittent Exposure to rhFGF-18 on Chondrocyte Phenotype in CTAs

[0083] For each culture condition, RNA was isolated from CTAs and Collagen, type I, type II, type X and Sox9 expression was analyzed by quantitative PCR (FIG. 7). High Sox9 and Collagen type II expression are markers of the chondrocyte phenotype whereas Collagen type I is a marker of dedifferentiation and Collagen type X of chondrocyte hypertrophy. The ratio Collagen II/I has also been calculated. This ratio is commonly used to illustrate the phenotype maintenance (higher ratio) or phenotype loss (lower ratio) of chondrocyte in culture. In all conditions with rhFGF-18, with permanent or intermittent exposure, at 10 or 100 ng/mL, Collagen type I expression was decreased. This decrease was the strongest when rhFGF-18 was given one day per week at a concentration of 100 ng/mL. As an example, Collagen type I expression was decreased by 4 in comparison to the control with rhFGF-18, 100 ng/mL, permanent but by 123 with rhFGF-18, 100 ng/mL, given one day per week. Collagen type II was found to be decreased in permanent presence of rhFGF-18 but was mostly unchanged in presence of rhFGF-18 given one week (1 w) or one day per week (1 d/w). No important variations were observed in the Sox9 expression. The latter was significantly increased (×2.2) only with rhFGF-18 10 ng/mL given one week (1 w). Finally, rhFGF-18 permanent was found to have no effect on the Collagen II/I ratio but when rhFGF-18 was given one day per week this ratio was increased by 19-fold and 138-fold in comparison to the control with rhFGF-18 10 and 100 ng/mL respectively. Collagen type X was also evaluated as a marker of chondrocyte hypertrophy and was found not to be influenced by rhFGF-18 in the present culture conditions.

Results—Effect of Permanent or Intermittent Exposure to rhFGF-18 on the Morphology and Collagen II and I Content of CTAs (Data Not Shown)

[0084] Histological analysis of the CTAs after 4 weeks of treatment with different rhFGF-18 exposures revealed that in permanent presence of rhFGF-18, CTAs were thinner and the Safranin O staining less intense in comparison with other conditions. In addition, in permanent presence of rhFGF-18 a proliferative zone with a higher cell density and absence of extracellular matrix can be observed at the periphery of the constructs. On the other hand, it can also be observed that intermittent exposure to rhFGF-18 resulted in thicker constructs in comparison to the control. In all conditions, Collagen type I was not detectable (not shown) while all CTAs were strongly stained for Collagen type II.

Conclusions

[0085] Permanent exposure to rhFGF-18 stimulated chondrocyte proliferation but decreased the matrix content of the CTAs (less GAG and hydroxyprolin). Similarly both Collagen type I and II expression were decreased in comparison with the control. No significant effects of permanent exposure to rhFGF-18 10 or 100 ng/mL were observed on Sox9 after 4 weeks of treatment. The histological analyses revealed that the CTAs were smaller and displayed proliferative zone devoid of ECM at the periphery of the CTAs. All these results together indicate that in permanent presence of rhFGF-18 proliferation is advantaged over matrix production.

[0086] When CTAs are cultivated one week with rhFGF-18, 10 or 100 ng/mL, and subsequently 3 weeks without rhFGF-18, on the contrary to the permanent exposure, no stimulation of the proliferation was observed. However, the GAG and the hydroxyproline content were found to be higher than in the control. Collagen type I expression was decreased while collagen type II expression was unchanged or even slightly increased (for rhFGF-18 10 ng/mL), in comparison to the control. As a consequence, the Collagen II/I ratio was increased, indicating a better phenotype maintenance. Similarly, Sox9 was also slightly increased in comparison to the control (significance for rhFGF-18 10 ng/mL only). Histology revealed that CTAs were composed of a Safranin O and Collagen type II positive matrix, similarly to the control CTAs. In comparison to the control, these CTAs were also thicker, in accordance with the higher content of GAG and hydroxyproline.

[0087] The best results regarding proliferation and matrix content were obtained when rhFGF-18 100 ng/mL was given 1 day per week. For this condition Collagen type I was also the lowest and the ratio of Collagen II/I was the highest. However, Collagen type II and Sox9 expression remained unchanged in comparison to the control. The CTAs were Safranin O and Collagen type II positive. As well as for the one week treatment, in comparison to the control, these CTAs were also thicker, which is also in accordance with their higher content of GAG and hydroxyproline.

[0088] As a conclusion intermittent exposure potentiates the effects of rhFGF-18 and enables to achieve increased proliferation, ECM production and promotes the chondrocyte phenotype in culture with 1 day/week>1 week>control>permanent exposure. These results support a cyclic administration of rhFGF-18 for OA treatment.

Example 4

Methods

[0089] Bovine chondrocytes were obtained as reported in Examples 2 and 3. They were cultivated 1 or 2 weeks with rhFGF-18 100 ng/mL present permanently (FGF-18), or as a control in absence of FGF-18 (CTR). At the end of the culture cells were harvested and counted or lysed for RNA isolation and gene expression. Sox9, Collagen I, and II expression were evaluated by quantitative PCR.

Results

[0090] After two weeks of culture with FGF-18 permanent, the cell concentration was higher than control group. Collagen type I expression was strongly repressed in presence of rhFGF-18 whereas Collagen type II and Sox9 expression was increased (FIG. 8).

Conclusion

[0091] When chondrocytes are cultivated in monolayer, permanent exposure to rh-FGF18 100 ng/mL enables to increase cell proliferation while enabling a better phenotype maintenance (Collagen II and Sox9 expression increased and Collagen I expression decreased).

Example 5

Methods:

[0092] The cartilage from two OA patients who underwent total knee replacement has been used. The chondrocytes were isolated as described in Example 3 and were first cultivated 3-4 days at high density in monolayer. Subsequently the chondrocytes were harvested and inoculated at 1×10.sup.6 cells/200 μL in a 96 well plate and allowed to aggregate one week without any treatment to form CTAs. They were then further cultivated 4 weeks in absence or presence of rhFGF-18 100 ng/mL according to the following treatment: 1) four weeks of culture in absence of rhFGF-18 (control) 2) four weeks of culture in permanent presence of rhFGF-18 (perm) and 3) four weeks of culture with rhFGF-18 given 1 day per week (i.e. 24 h exposure followed by 6 days without rhFGF-18) (1 d/w) (see FIG. 4). At the end of the culture period, CTAs were harvested and analyzed for their GAG and cell content. With CTAs obtained from patient 2, gene expression for Collagen type I and type II and Sox9 was evaluated and histology for Safranin O and Collagen Type I and II was also performed

Results—Cell Proliferation

[0093] rhFGF-18 100 ng/mL increased the proliferation of human osteoarthritic chondrocytes in 3D culture (see FIG. 9). For the chondrocytes coming from patient 1, in the control the number of cells/CTA was lower than the initial cell number (inoculation density was 1 million cells/CTA) indicating that many cells died. However, in presence of rhFGF-18 permanent or 1 day/week 1.5 million cells/CTA could be found suggesting that these cells did not die but proliferated. For the chondrocytes coming from patient 2 a slightly increased cell number (from 1 to 1.3 million/CTA) can be observed in untreated CTAs. This was further increased in presence of rhFGF-18 permanent (from 1 to 1.9 million cells/CTA).

Results—Matrix Production

[0094] rhFGF-18 100 ng/mL increased the GAG production by human osteoarthritic chondrocytes in 3D culture (see FIG. 10). In patient 1 with rhFGF-18 permanent and 1 day/week and in patient 2 with FGF-18 permanent significantly more GAG was present in the CTAs.

Results—Gene expression

[0095] The chondrocyte phenotype is characterized by a low or absence of Collagen type I expression and an increased expression of Sox9 and Collagen II. This expression pattern is altered in osteoarthritic chondrocytes (see FIG. 11). Indeed in untreated CTAs Collagen type I expression was higher than Collage type II expression (relative abundance of 0.67 and 0.04 respectively). rhFGF-18 was able to reduce Collagen I expression while increasing Collagen II expression. As a result the ratio Collagen II/I increased 11 to 13 fold in presence of rhFGF-18. In addition, rhFGF-18 increased Sox9 expression, a marker of the chondrocyte phenotype.

Results—Histology (Data Not Shown)

[0096] In comparison to the control, the CTAs cultivated with rhFGF-18 1 day/week or permanent showed an increase Safranin O staining indicating that they contained more GAG. This is in accordance with the results presented in FIG. 10. Collagen I staining was decreased in rhFGF-18-treated cells which also corresponds well to gene expression results FIG. 11. No Collagen II staining could be seen in the control culture indicating that these human osteoarthritic (hOA) chondrocytes were not able to produce a cartilage-like matrix. However, rhFGF-18 1 day/week and permanent were both able to restore the ability of hOA chondrocytes to produce Collagen II.

Conclusion

[0097] The results obtained with chondrocytes isolated from human osteoarthritic cartilage showed that rhFGF-18 was able to promote cell growth, increase hyaline-like cartilage matrix production and favor the chondrocyte phenotype. In this experiment, rhFGF-18 permanent and one day/week performed equally concerning several parameters. However, regarding matrix production, rhFGF-18 one day/week did slightly better (increased GAG accumulation in Patient 1 and increased Collagen II expression in Patient 2).

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