Hydrogel depots for local co-delivery of osteoinductive peptides and mesenchymal stem cells
Graphical abstract
Introduction
Hydrogels show great potential as cell vehicles for minimally invasive bone regeneration therapies. These materials form highly hydrophilic 3D networks that recreate some features of native extracellular matrices (ECM), providing adequate cellular microenvironments where the exchange of nutrients, oxygen and metabolites with the extracellular milieu is facilitated. Compared to direct bolus injection at the injury site, often associated with poor cell survival, the pre-entrapment of cells in a hydrogel delivery vehicle may improve viability by providing physical protection, biochemical and biomechanical survival signals, and scaffolding [1], [2]. It also helps localizing cells to the targeted area, increasing the chance of post-transplantation cell retention and engraftment, and affording a template for new tissue formation [1], [2]. Hydrogels can be further decorated with specific cell-instructive cues aimed at directing the phenotype of entrapped cells. Therapeutic approaches aiming at driving mesenchymal stem cells (MSCs) fate in a controlled manner, namely by promoting their differentiation into bone-forming cells through the co-delivery of osteoinductive compounds, are promising for bone healing applications [1], [2].
Strategies involving the use of small compounds, such as peptides, can be advantageous as compared to more complex biomolecules, by leading to less expensive, more stable and easily tunable biomaterial formulations [2], [3]. Up to now, different types of osteoinductive peptides have been proposed [4]. In this study, we evaluated the potential of OGP, a naturally occurring tetradeca-peptide identical to the C-terminus of histone H4 (residues 89–102, ALKRQGRTLYGFGG), which is present in plasma at micromolar concentrations [5], [6], [7], [8]. The physiologically active form of OGP, which corresponds to its C-terminal pentapeptide sequence YGFGG (OGP10–14), is generated from full-length OGP by proteolytic cleavage [5]. This fragment directly interacts with cell membrane receptors, activating the MAP kinase, Src and RhoA signaling pathways [9], [10], [11]. Upon intravenous administration, synthetic OGP and OGP10–14 were shown to promote increased bone mass and fracture healing in vivo [12], [13]. In vitro, OGP peptides were shown to increase the proliferation of osteoblastic-like cells and MSC and accelerate osteogenesis [14], [15]. In pursuit for superior osteoinductive compounds for bone regeneration therapies, OGP has provided a useful basis for engineering additional OGP analogs with enhanced bioactivity, stability and bioavailability. This includes the design of different peptide sequences to be used in free soluble forms [8], as well as more complex systems with physically or chemically immobilized OGP for local administration [16], [17], [18]. In this context, the aim of this study was to develop protease-responsive delivery systems for OGP analogs, which could simultaneously act as injectable hMSCs vehicles, for minimally invasive healing of small bone defects. Among the numerous proteases that can be selected to trigger enzyme-activated drug release, those belonging to the metalloproteinase (MMP) family are particularly attractive. MMPs actively participate in ECM remodeling and degradation, having a key role in wound healing and tissue regeneration, and some are constitutively expressed by both naïve and differentiated hMSCs [19], [20]. Here, different OGP analogs were designed, by flanking the YGFGG N-terminus with the MMP-substrate PVGLIG or its scrambled sequence GIVGPL [20]. Both were chemically grafted to alginate, a natural polysaccharide with the ability to form hydrogels in situ, which has been extensively used as a delivery vehicle for entrapped cells [21], [22], [23]. We hypothesized that in the presence of specific MMPs, in particular MMP-2 [20], [24], the bioactive YGFGG fragment would be released from the hydrogel upon enzymatic cleavage of the PVGLIG sequence, while it would remain mostly immobilized when using the scrambled linker. Once implanted, such systems may, thus, provide localized OGP delivery for variable time periods, remaining in close proximity to the targeted host cells at the injury site. If OGP-functionalized hydrogels are simultaneously used as hMSCs carriers, the released peptides might act on the transplanted cells and specifically guide their differentiation along the osteoblastic lineage. In this study, the designed OGP analogs and OGP–alginate conjugates were firstly characterized at different levels, and then used for the preparation of OGP-alginate hydrogels. The in vivo performance of hMSCs-laden hydrogels was evaluated after 4-weeks of implantation at an ectopic site in a xenograft mouse model.
Section snippets
Synthesis and characterization of OGP analogs
Different OGP-based oligopeptide sequences, hereafter designated by OGP1, OGP2 and OGP3 (full sequences and additional information are depicted in Table 1) were synthesized by solid-phase peptide synthesis (SPPS) using the Fmoc/tBu protection scheme [25]. The polymeric support selected, Fmoc-Gly-Wang resin (1 mmol/g, Iris Biotech), was an hydroxymethylated resin pre-loaded with the Fmoc-protected C-terminal amino acid; it was first deprotected by a 20% solution of piperidine (Sigma–Aldrich)
Enzymatic cleavage of free and alginate-conjugated OGP analogs
Different OGP analogs were designed and synthesized by Fmoc/tBu SPPS, where the target bioactive fragment YGFGG was either flanked by a poly-G sequence (in OGP1), the MMP-cleavable substrate PVGLIG (in OGP2) [24], or the PVGLIG-scrambled sequence GIVGPL (in OGP3). Their molecular weight and purity were assessed by LC–MS and HPLC, respectively (Table 1). Mass spectra were acquired in the positive mode, and in all cases the base peaks at m/z 1207.67 and 1207.60, respectively, were consistent with
Conclusions
This study provided proof-of-concept on the correct design of OGP–Alg conjugates with protease-sensitive linkers, and demonstrated their usefulness as a platform for the in situ co-delivery of synthetic OGP analogs and hMSCs. Two different peptides were tested and both showed interesting effects. While in vitro OGP2 presented better results probably due to increased bioavailability, a positive in vivo outcome was obtained with both OGP analogs. Importantly, we demonstrated that the local
Acknowledgements
This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade (COMPETE) and by Portuguese funds through Fundação para a Ciência e a Tecnologia (FCT), in the framework of the projects Pest-C/SAU/LA0002/2011 and BIOMATRIX (PTDC/SAU-BEB/101235/2008 and FCOMP-01-0124-FEDER-010915), and co-financed by North Portugal Regional Operational Programme (ON.2–O Novo Norte) in the framework of project SAESCTN-PIIC&DT/2011, under the National Strategic Reference Framework
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