Therapeutic bioactive microcarriers: Co-delivery of growth factors and stem cells for bone tissue engineering
Graphical abstract
Mesoporous and bioactive silica-based microspheres demonstrated excellent biocompatibility to support cell growth and tissue responses as well as long-term delivery of growth factors, suggesting a novel therapeutic microcarrier system for bone tissue engineering.
Introduction
The release of specific signaling molecules from scaffolding materials to elicit desired cellular reactions is one of the key strategies to significantly enhance the regenerative capacity of synthetic biomaterials for use in bone tissue engineering. These signaling molecules can either be eluted to the cells that are supported upon the scaffolds for ex vivo cultivation appropriate for bone engineering, or can be supplied to the surrounding bone defects when directly implanted in vivo [1]. For either case, the signaling molecules should ideally be released in a controlled manner, and for certain durations, in order to optimize cellular propagation and/or osteogenic differentiation, as well as to aid in vivo vascularization and bone formation. Hence, one of the primary requirements of scaffolds is the sustained and controllable release of therapeutic molecules, while enabling their effective and safe incorporation into the scaffold structure.
Microspherical scaffolds, namely, microcarriers, have gained great interest as three-dimensional (3-D) substrates for the 3-D cultivation and expansion of tissue cells. Frequently allowed to rotate in cell suspensions in vitro, so as to aid cellular anchorage, the isotropic 3-D spherical substrates provide homogeneous sites for cellular recognition, distribution and multiplication [2]. Subsequently, cell-loaded microcarriers can be delivered to the defects of concern, with a delivery capacity that is controllable based on the size of the microcarriers. Furthermore, the cell–carrier constructs are possible as injectable tissue engineering devices, effectively filling defects [3]. In fact, the micro-spherical particles, which may be a few to hundreds of micrometers in diameter, have been extensively researched for the delivery of therapeutic molecules, including drugs, hormones and growth factors [4], [5]. Along with this biomolecular delivery use, the cellular delivery potential has thus been of great interest for the ex vivo culture of stem cells and engineering of tissues, including bone.
Among other things, providing therapeutic roles to the microcarriers is of great merit in regulating the behavior of stem/progenitor cells to be supported, ultimately for bone tissue engineering, such as rapid cellular engulfment, increased cell population, stimulation to osteogenic lineage specification and/or achieving highly vasculature tissues [2]. When microcarriers are used for the delivery of therapeutic molecules, cells can be made to undergo osteogenesis during ex vivo cultivation prior to implantation and, further, to play beneficial roles in the regeneration process of bone tissue in vivo, after implantation.
Therefore, the current aim is to develop therapeutic microcarriers effective for bone tissue engineering that allow 3-D cultivation of stem cells in vitro, while incorporating and releasing therapeutic molecules ultimately to aid their osteogenesis and in vivo bone formation. Here, the present authors propose a “bone-bioactive” inorganic composition composed of silica-based bioactive glass (SBG). The SBG is made through a sol–gel process under room temperature and aqueous conditions [8], [9], [10], [11], [12]. Different ions could be easily incorporated into the silica sol–gel glass network, and the addition of calcium greatly improved the bioactivity and hydrolytic degradation. Natural polymers such as gelatin and collagen have also been added, to improve the mechanical properties and cellular responses [13], [14], [15]. Importantly, the sol–gel process enables the introduction of therapeutic molecules because of the mild processing conditions. Several studies have demonstrated the effectiveness of the sol–gel glass network in capturing drugs and proteins and their release for long periods [16], [17], [18], [19], [20].
Here, the SBG composition is used in the preparation of therapeutic microcarriers, which are effective for bone tissue engineering. The SBG composition has merits over polymer-based compositions, particularly for therapeutic purposes, which can limit the incorporation of biomolecules. Synthetic polymers are generally produced in organic solvents or surfactant-mediated conditions, requiring vigorous washing steps, while natural polymers require crosslink steps to stabilize the structure, although they may be processed in aqueous solutions [2]. Moreover, the SBG composition based on a sol–gel process has a number of merits compared with conventional bioceramics, such as calcium phosphates and melt-derived bioglasses, that are generally prepared at high temperatures [6], [7]. Owing to the nature of the sol–gel reaction, i.e., hydrolysis and polycondensation, sol–gel processed BG spherical particles self-harden in a structurally and chemically stable manner, eliminating further crosslinking steps.
Another intriguing and beneficial point of SBG is the sol–gel-derived mesoporous structure, where the mesoporosity and mesopore geometry are tuned to take up a large quantity of and selective therapeutic molecules and, subsequently, to control their release behavior [16], [17], [18]. Furthermore, the silanol groups present on the surface are hydrophilic and easily inducible for calcium and phosphate ions to produce calcium phosphate crystals, which are recognized as “bone-bioactive” materials [21], and therefore SBG is considered to be a proper reservoir for therapeutic molecules and 3-D substratum for cellular reactions, particularly those required for bone regeneration.
In the present study, SBG microcarriers with bone-bioactive and self-hardening properties are prepared. The ability to populate stem cells in vitro, as well as to allow favorable reactions in vivo, is briefly assessed. Model experiments on incorporating therapeutic protein molecules within the structure and releasing them sustainably, as well as the accompanying biological effects, are also described. These studies support the further use of novel therapeutic microcarriers in bone tissue engineering.
Section snippets
Preparation of SBF microcarriers
Tetraethyl orthosilicate (TEOS, C8H20O4Si, 98%, Sigma–Aldrich) (10 ml) was mixed with 0.1 M HCl (2.4 ml), with the addition of deionized water to form an acid catalyzed sol. The molar ratio of total water (including the CaCl2 and the HCl water) to TEOS was 8. The microspheres were doped with calcium ions by incorporating specific amounts of calcium chloride (CaCl2, Sigma–Aldrich) into the solution. The molar percentage of doped calcium ranged from 0 to 30. Once the sol was obtained, it was cooled
Characteristics of microcarriers
Fig. 1a presents the typical SEM morphology of the microcarriers. Spherical microparticles were successfully generated for all compositions (shown as representative images for 0Ca and 30Ca). The size distribution of microcarriers (Fig. 1b) shows the generation of microparticles primarily with diameters of hundreds of micrometers, and the incorporation of calcium slightly increased the mean diameter of microcarriers from ∼200 μm for 0Ca to ∼300 μm for 30Ca. The XRD patterns illustrate all the
Discussion
Tissue engineering scaffolds, primarily guiding 3-D physical substrate conditions to stem cells, can also provide biochemical signaling cues to regulate cellular behavior and, ultimately, to achieve a tissue mimic structure. Of the various engineering scaffolds currently under development or in use, the focus here is on microspherical particles to provide 3-D scaffolding matrices for stem cells to anchor to and propagate on, further developing osteogenic processes in concert with the delivery
Conclusions
Calcium-containing silica-based sol–gel-derived microcarriers developed herein demonstrated high mesoporosity with tunable pore size, volume and surface area, bone bioactivity characterizing to rapidly form apatite mineral, self-hardening ability to simply and safely incorporate biological molecules, such as growth factors, and for the long-term sustainable release of loaded molecules, with almost zero-order kinetics over months. Moreover, the microcarriers have the biocompatibility to support
Acknowledgements
This study was supported by a grant from the Priority Research Centers Program (2009-0093829), National Research Foundation, South Korea. Authors thank Drs. Jan JH and Yun YR for their kind help in bFGF study.
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