Elsevier

Biomaterials

Volume 30, Issue 12, April 2009, Pages 2385-2392
Biomaterials

The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects

https://doi.org/10.1016/j.biomaterials.2009.01.016Get rights and content

Abstract

Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n = 32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.

Introduction

Chondral and osteochondral defects are difficult to treat and up to now an appropriate therapy has not been established [1]. Methods regarding the transplantation of autologous chondrocytes into chondral defects are promising [2], but the induced repair tissue is not mature hyaline cartilage but most of the time fibrocartilage. In this regard tissue engineering has attracted great attention [3]. Scaffolds like type I collagen sponges, fibrin glue, hyaluronan or composites are suitable carriers for chondrocytes [4], [5], [6], [7]. In addition, growth factors enhance the healing process in cartilage defects by stimulating cell proliferation, differentiation and matrix synthesis [1], [8]. The gene transfer of therapeutic genes represents a promising way to efficiently deliver growth factors into injured tissue [9], [10], [11]. But the success of gene therapy is largely dependent on the development of vector systems that can efficiently deliver genes into target cells, induce a stable expression and are safe [7], [8]. Gene transfer is possible by viral and non-viral vectors. Viral vectors represent a more efficient method. The delivery of foreign genes into chondrocytes or tissue using adenoviral [12], [13], adeno-associated-viral [9], lentiviral [14] or retroviral vectors [4] has been explored as a strategy for the treatment of various joint disorders. The VSV.G-pseudotyped retroviral vector Bullet achieved a transduction efficiency of more than 90% and a stable gene transfer without any selection of cells in primary articular chondrocytes and a chondrogenic cell line [4], [8]. In the clinical application the transplantation of dedifferentiated chondrocytes is the main problem. The redifferentiation of dedifferentiated chondrocytes can be induced by Bone Morphogenetic Protein 2 (BMP2), a growth factor and member of the TGFβ-superfamily [8], [15]. BMP2 is also involved in the differentiation of mesenchymal stem cells to chondroblasts and osteoblasts [16], [17]. Sellers et al. used recombinant human BMP2 for the treatment of full-thickness defects of articular cartilage in rabbits and found an accelerated formation of new subchondral bone with an improved histological appearance of cartilage but repair was not complete and half-life of BMP2 in the joint was only 5.6 days [18]. Stimulation of perichondrium-derived mesenchymal cells by adenoviral BMP2 transfer in a partial-thickness defect model showed a repair tissue comparable with hyaline cartilage, but regeneration was also not complete [19]. However, BMP2 concentration in the repair tissue was not analysed and due to the gene transfer method it is unlikely that an adequate concentration over a longer time was achieved. Experiments using a non-viral in situ transduction of BMP2, with a stability of gene expression in vitro and in vivo for 2 weeks, showed no effect on quality of repair tissue in artificial osteochondral defects [7]. Therefore, the aim of this study was to analyse if stable expression of BMP2, using the retroviral vector Bullet, is able to regenerate osteochondral defects completely in a rabbit defect model.

Section snippets

Fibrin glue components

The fibrinogen component of TISSUCOL®-Kit is a lyophilized human plasma fraction containing fibrinogen, plasma fibronectin, factor XIII, and plasminogen in the ratio 35–55 mg/1–4.5 mg/5–25 units/0.01–0.04 mg. The lyophilized thrombin component of the kit was dissolved in 40 mm calcium chloride to result in a stock solution of 500 IU thrombin/ml. When required, the fibrinogen and thrombin stock solutions were diluted with fibrinogen and thrombin dilution buffers, respectively, which are part of the

BMP2 expression in fibrin clots in vitro

Consistency of Bullet gene transduction efficiency for BMP2 into articular chondrocytes was monitored using a parallel Bullet–eGFP transduction approach with following FACS analysis as described previously [8]. Bullet transduction was consistent and comparable to this study. Cells in fibrin clots were predominantly viable (analysed by trypan-blue staining) even after Bullet transduction. The BMP2 expression of control and Bullet–BMP2-transduced articular chondrocytes was measured by BMP2

Discussion

In vivo experiments have demonstrated a positive effect of the growth factor BMP2 on cartilage remodelling [18], [19]. However, until today BMP2 did not enter clinical trials for the treatment of chondral or osteochondral lesions. So far, all BMP2 transfer methods were insufficient in achieving stable protein expression. In addition, BMP2 levels in the repair tissue were not analysed or were low after several days and a complete cartilage repair could not be demonstrated [18], [19]. To study

Conclusion

The maximal effect of the growth factor BMP2 on osteochondral remodelling was analysed. BMP2 increased the repair tissue quality of osteochondral defects but failed to induce a complete healing despite efficient transduction and stable expression. However, the used in vivo model is today the optimal system to test genes for the repair of these defects. Following, safer and controllable vectors will be needed for the application in humans.

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (MA 2454/2-1, VO 867/4-1) and Klinische Fördermittel Technische Universität München (KKF 8745151/2).

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