Research paper
Expansion in the presence of FGF-2 enhances the functional development of cartilaginous tissues engineered using infrapatellar fat pad derived MSCs

https://doi.org/10.1016/j.jmbbm.2011.09.004Get rights and content

Abstract

MSCs from non-cartilaginous knee joint tissues such as the infrapatellar fat pad (IFP) and synovium possess significant chondrogenic potential and provide a readily available and clinically feasible source of chondroprogenitor cells. Fibroblast growth factor-2 (FGF-2) has been shown to be a potent mitotic stimulator during ex vivo expansion of MSCs, as well as regulating their subsequent differentiation potential. The objective of this study was to investigate the longer term effects of FGF-2 expansion on the functional development of cartilaginous tissues engineered using MSCs derived from the IFP. IFP MSCs were isolated and expanded to passage 2 in a standard media formulation with or without FGF-2 (5 ng/ml) supplementation. Expanded cells were encapsulated in agarose hydrogels, maintained in chondrogenic media for 42 days and analysed to determine their mechanical properties and biochemical composition. Culture media, collected at each feed, was also analysed for biochemical constituents.

MSCs expanded in the presence of FGF-2 proliferated more rapidly, with higher cell yields and lower population doubling times. FGF-2 expanded MSCs generated the most mechanically functional tissue. Matrix accumulation was dramatically higher after 21 days for FGF-2 expanded MSCs, but decreased between day 21 and 42. By day 42, FGF-2 expanded MSCs had still accumulated ∼1.4 fold higher sGAG and ∼1.7 fold higher collagen compared to control groups. The total amount of sGAG synthesised (retained in hydrogels and released into the media) was ∼2.4 fold higher for FGF-2 expanded MSCs, with only ∼25% of the total amount generated being retained within the constructs. Further studies are required to investigate whether IFP derived MSCs have a diminished capacity to synthesise other matrix components important in the aggregation, assembly and retention of proteoglycans. In conclusion, expanding MSCs in the presence of FGF-2 rapidly accelerates chondrogenesis in 3D agarose cultures resulting in superior mechanical functionality.

Introduction

MSCs derived from non-cartilaginous knee joint tissues  such as the infrapatellar fat pad (IFP) (Dragoo et al., 2003, English et al., 2007, Jurgens et al., 2009, Buckley et al., 2010a, Buckley et al., 2010b) and synovium (Sakaguchi et al., 2005, Yokoyama et al., 2005, Mochizuki et al., 2006, Pei et al., 2008, Pei et al., 2009, Sampat et al., 2011) have been shown to possess significant chondrogenic potential. One of the key challenges in cartilage tissue engineering is ensuring sufficient functionality of the construct to be implanted which is capable of withstanding the challenging mechanical environment of the joint. MSCs and chondrocytes have been shown to be phenotypically different (Segawa et al., 2009), and one implication of this may be that they synthesise different amounts of key proteoglycans such as glycosaminoglycans (GAGs) and proteins such as collagens (predominantly Type II) that provide mechanical stability. Previous investigations using MSCs isolated from various tissues have shown they generate tissues with inferior mechanical properties compared to articular chondrocytes similarly maintained in 3D culture (Mauck et al., 2006, Huang et al., 2009, Vinardell et al., 2009, Vinardell et al., 2011). Although MSCs derived from intra-articular joint tissues are more phenotypically similar to chondrocytes (Segawa et al., 2009), we have shown that functionally they are still inferior to chondrocytes (Vinardell et al., 2011). Therefore identifying expansion and differentiation conditions that promote a more chondrogenic phenotype is critical to enhancing their utility for cartilage tissue engineering applications. Differentiation conditions that have been shown to promote the chondrogenic potential of MSCs include a low oxygen (5%) microenvironment (Khan et al., 2007, Buckley et al., 2010a, Meyer et al., 2010), various combinations of growth factors (Mastrogiacomo et al., 2001, Sakimura et al., 2006, Hennig et al., 2007, Diekman et al., 2010, Buxton et al., 2011) and mechanical signals (Huang et al., 2005, Mauck et al., 2007, Huang et al., 2010a, Huang et al., 2010b, Kelly and Jacobs, 2010, Li et al., 2010, Thorpe et al., 2010, Haugh et al., in press).

The chondrogenic capacity of MSCs can also be diminished due to the in vitro expansion conditions affecting stemness and accelerating senescence (Solchaga et al., 2010). Augmenting MSC expansion conditions to enhance proliferation kinetics while maintaining multipotency is a critical obstacle to overcome in order to engineer mechanically functional tissues for clinical applications. Fibroblast growth factor-2 (FGF-2, also known as basic fibroblast growth factor) has been shown to be a potent stimulator during ex vivo expansion of both chondrocytes (Kato and Gospodarowicz, 1985, Martin et al., 1999, Martin et al., 2001, Veilleux and Spector, 2005) and MSCs (Banfi et al., 2000, Mastrogiacomo et al., 2001, Tsutsumi et al., 2001, Bianchi et al., 2003, Solchaga et al., 2005, Khan et al., 2008, Solchaga et al., 2010), as well as regulating subsequent differentiation potential. Specifically, bone marrow derived MSCs cultured in the presence of FGF-2 have been shown to be physically smaller and proliferate more rapidly during monolayer expansion (Solchaga et al., 2005). During chondrogenic differentiation, FGF-2 expanded MSCs have been shown to produce higher levels of proteoglycans with reduced deposition of collagen type I in the peripheral region of pellet cultures while promoting collagen type II formation (Akaogi et al., 2004, Solchaga et al., 2005). In addition, expansion in the presence of FGF-2 has been shown to enhance chondrogenesis of IFP derived MSCs in pellet culture models (Khan et al., 2008).

The objective of this study was to investigate the longer term effects of FGF-2 expansion on the functional development of cartilaginous tissues engineered in vitro using MSCs derived from the infrapatellar fat pad. Given that expansion in FGF-2 has been shown to enhance the chondrogenic potential of IFP derived MSCs in short-term pellet culture, we hypothesised that this would translate into enhanced functional properties in long-term culture after encapsulation in agarose hydrogels.

Section snippets

Cell isolation and expansion

Porcine IFPs were harvested from the knee joint capsule of three 4 month old porcine donors (∼50 kg) within 3 h of sacrifice. IFPs were weighed, washed thoroughly in phosphate buffered saline (PBS) and diced followed by incubation under constant rotation at 37 °C with high-glucose Dulbecco’s Modified Eagle Medium (hgDMEM, GlutaMAX™) (GIBCO, Biosciences, Ireland) containing collagenase type II (750 U/ml, Worthington Biochemical, LanganBach Services, Ireland) and 1% penicillin

Results

IFP MSCs expanded in FGF-2 supplemented medium proliferated more rapidly than the control media formulation (STD), with significant differences observed in cell yields and population doubling times at each passage (Fig. 1). MSCs expanded with FGF-2 had a reduced population doubling time of 1.7 ± 0.2 days compared to STD expanded cells of 4.8 ± 1.3 days (P<0.05).

FGF-2 expanded MSCs embedded in agarose hydrogels generated cartilaginous tissues with equilibrium moduli that were 1.9 fold and 1.5

Discussion

We have previously shown that MSCs isolated from infrapatellar fat pad tissue are a viable alternative cell source for cartilage tissue engineering (Buckley et al., 2010a, Buckley et al., 2010b, Vinardell et al., 2011). It is evident from the literature that stem cell expansion conditions can significantly influence their subsequent chondrogenic capacity (Tsutsumi et al., 2001, Bianchi et al., 2003, Solchaga et al., 2005, Khan et al., 2008, Solchaga et al., 2010). This study develops upon

Conclusions

In conclusion, expansion in the presence of FGF-2 rapidly accelerates chondrogenesis of IFP derived MSCs embedded in 3D agarose cultures resulting in superior functional properties. Future studies will focus on strategies to promote the synthesis and assembly of matrix components into fully functional cartilaginous grafts.

Acknowledgments

Funding was provided by a Science Foundation Ireland President of Ireland Young Researcher Award (PIYRA), (Grant No: SFI/08/YI5/B1336) and a European Research Council Starter Grant (StemRepair—Project number: 258463).

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