The restoration of full-thickness cartilage defects with BMSCs and TGF-beta 1 loaded PLGA/fibrin gel constructs

Abstract

Poly(lactide-co-glycolide) (PLGA) sponge was filled with fibrin gel, bone marrow mesenchymal stem cells (BMSCs) and transforming growth factor-β1 (TGF-β1) to obtain a construct for cartilage restoration in vivo. The PLGA sponge lost its weight steadily in vitro, but degraded much faster in the construct of PLGA/fibrin gel/BMSCs implanted in the full-thickness cartilage defects. The in vivo degradation of the fibrin gel inside the construct was prolonged to 12 wk too. The CM-DiI labeled allogenic BMSCs were detectable after transplantation (implantation) into the defects for 12 wk by small animal in vivo fluorescence imaging and confocal laser scanning microscopy. In vivo repair experiments were firstly performed by implantation of the PLGA/fibrin gel/BMSCs and PLGA/BMSCs constructs into full-thickness cartilage defects (3 mm in diameter and 4 mm in depth) of New Zealand white rabbits for 12 wk. The defects implanted with the PLGA/fibrin gel/BMSCs constructs were filled with cartilage-like tissue containing collagen type II and glycosaminoglycans (GAGs), while those by the PLGA/BMSCs constructs were filled with fibrous-like tissues. To repair the defects of larger size (4 mm in diameter), addition of growth factors was mandatory as exemplified here by further loading of TGF-β1. Implantation of the PLGA/fibrin gel/BMSCs/TGF-β1 constructs into the full-thickness cartilage defects for 12 wk resulted in full restoration of the osteochondral tissue. The neo-cartilage integrated well with its surrounding cartilage and subchondral bone. Immunohistochemical and GAGs staining confirmed the similar distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that the cartilage special genes were significantly up-regulated compared with those of the TGF-β1 absent constructs.

Introduction

The arthritis is mostly caused by cartilage deficiency. The injured cartilage can hardly repair automatically and lead to further degeneration. Some conventional methods such as debridement, microfracture, osteochondral grafting and autologous chondrocytes implantation (ACI) are thus developed and used in clinic [1]. As an alternative treatment, tissue engineering has been demonstrated a promising approach to restore the cartilage defects too. The success of this technique relies critically on the seed cells and scaffolds and thereby the structure and functions of the regenerated cartilage.

Among the various scaffolds used, the hybrid scaffold prepared by filling soft hydrogel into hard sponge is very promising for the cartilage regeneration since their advantages can be maintained while the shortcomings can be avoided [2], [3], [4], [5], [6]. Apart from the more even distribution of cells and maintenance of the cell phenotype, bioactive factors such as functional genes and growth factors are conveniently loaded into the filled hydrogel with preserved bioactivity. These factors are known to regulate the proliferation and differentiation of the seed cells, which is particularly important when stem cells are used. Actually, the use of stem cells, in particular the bone mesenchymal marrow stem cells (BMSCs) has a lot of advantages over the autologous chondrocytes, and has achieved great success in cartilage and bone repair [7], [8], [9], [10]. Particularly, both the cartilage and bone can be simultaneously repaired when the BMSCs are used, and result in better remodelling and integration with the host surface zone [11], [12].

It is known that differentiation of the BMSCs requires suitable stimuli, which can be achieved with a large variety of different growth factors. Transforming growth factor-β1 (TGF-β1) is one of the most powerful growth factors and is routinely used to induce BMSCs to chondrocytes in vitro. There are two ways to construct the hybrid scaffold which has the ability to regulate the stem cell differentiation in vivo: direct loading of the growth factors, and loading of functional genes encoding the growth factors. In a previous study, plasmid DNA encoding TGF-β1 was loaded into the fibrin gel filled poly(lactide-co-glycolide) (PLGA) sponge. In vivo experiment demonstrated that the cartilage defects were successfully restored in the rabbit knees [13]. However, the safety of the gene therapy still remains a big concern, especially for a long term application. In contrast, the biofunctions and biosafety of the growth factors are more definite with a limited function time.

In this study, recombinant protein TGF-β1 is encapsulated into the fibrin gel and then filled into the PLGA sponges to obtain a composite construct for articular cartilage repair in vivo. Attention is paid to the in vivo degradation of the PLGA and fibrin gel, the function of the fibrin gel on the cartilage repair, and the overall repair effect by the composite constructs.