Acknowledgements
7.2 Multi-Functional Biomaterials for Bone Tissue Engineering
7.2.2 Active Scaffolds
7.2.2.1 Biomaterials for Biomolecular Delivery
to trigger mobilization and phosphorylation of sMad transducing molecules, which regulates the expression of downstream target genes.46
as highlighted recently by several review articles, BMps slowly and con- tinuously released from scaffolds show a better effect for inducing bone formation, compared to free BMps.53,54 these BMp–scaffold systems have enhanced recruitment of osteoprogenitor cells,55 a significant increase in alkaline phosphatase activity,56 as well as greater mineral deposition and higher compressive moduli.57–59 Controlling the release profile of these bio- molecules is a common research theme. Comparing a heparin-conjugated fibrin against a normal fibrin construct, Yang et al. reported that slower BMp-2 release (∼80% after 13 days instead of three days) resulted in ∼10-fold greater calcium concentration and more prominent osteocalcin and osteo- pontin expression, eight weeks after implantation in an ectopic rat model.56 Meanwhile, Wei et al. demonstrated that a nanosphere-immobilized, but not adsorbed rhBMp-7 pLLa scaffold could induce significant ectopic bone for- mation six weeks after rat transplantation, as observed through von kossa staining and radiographic density (Figure 7.2).58
Figure 7.2 Lga nanospheres’ immobilization on a pLLa scaffold enable sustained release of BMp-7 for ectopic bone formation. (a) scanning electron micrograph of pLLa nanofibrous scaffolds after the nanospheres’
incorporation. nanosphere-immobilized scaffolds (iii) showed signifi- cantly greater bone formation than BMp-7 adsorbed scaffolds (ii) and blank scaffolds (i), as observed by radiographic imaging (B) and von kossa staining (C). reproduced from Biomaterials, 28(12), Wei, g., jin, Q., giannobile, W. V., Ma, p. X. the enhancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMp-7 nanospheres, 2087–2096, Copyright (2007) with permission from elsevier.
Published on 03 May 2017 on http://pubs.rsc.org | doi:10.1039/9781788010542-00169
after realizing the benefit of incorporating bioactive molecules, research- ers began to utilize scaffolds for the delivery of two or more growth factors to achieve a synergistic effect.47 one study co-delivered BMp-2 with Wnt1 inducible signaling protein-1 (Wisp-1) using a β-tCp/gelatin scaffold. In vitro, introduction of both factors promoted more than two-fold osteopontin expression on MsCs after seven days, as compared to the singly-delivered molecules. In vivo, there was two-fold greater osteoid formation 10 days after subcutaneous implantation, relative to the BMp2-only group.60 in another study, co-incorporation of BMp-2 and transforming growth factor beta-3 (tgFβ-3; which is involved in the up-regulation of endogenous BMp-2 expression61) in MsCs-containing alginate hydrogel was shown to stimulate significant bone formation from as early as six weeks after transplantation.
Meanwhile, the introduction of individual growth factors resulted in negli- gible bone formation even after 22 weeks.62 to achieve maximal therapeutic effect, the sequence of the gFs’ release may also be manipulated through this co-administration. one such case is the co-delivery of BMp-2 and insu- lin-like growth factor 1 (igF-1). While igF-1 itself can stimulate osteoblast growth and proliferation,63 sequential delivery of BMp-2/igF-1 might poten- tiate these effects, as BMp-2 treatment upregulates the expression of igF-1 receptors.64 indeed, kim et al. showed that sequential release of BMp-2 from chitosan gel followed with igF-1 from a gelatin microsphere provided ∼50%
greater alkaline phosphatase (aLp) activity of W-17-20 cells at day seven, when compared to scaffolds releasing only BMp-2 or simultaneously releas- ing BMp-2 and igF-1 from chitosan gels.65
aside from proteins with osteoinductive benefits, angiogenic ones inclu- ding fibroblast growth factors (FgFs), vascular endothelial growth fac- tors (VegFs) and platelet-derived growth factors (pdgFs) have also been explored, particularly for applications in the treatment of large, critical- ly-sized defects.66–68 in one study involving polyelectrolyte multilayer films with BMp-2 and VegF-165, the co-introduction of both factors resulted in a 33% increase in ectopic bone formation together with a higher trabecu- lar thickness, taking the BMp-2 only treatment as a control.69 in a similar study using a gelatin-microspheres-incorporated poly(propylene fumarate) (ppF) scaffold, co-delivery of BMp-2 and VegF was shown to accelerate defect bridging and healing time in a critical-size cranial defect model, with two- fold bone formation over the BMp2-only group after four weeks of treatment, as evaluated through micro Ct.70
Lastly, to mitigate problems associated with delivering large and full-scale proteins (e.g. high cost, rapid loss of bioactivity),71 researchers have exper- imented as well with truncated versions containing only critical portions.
While its effects may not be as extensive as its complete sequence counter- part, a dose-dependent stimulatory role on bone formation was attained when BMp-2 peptide was delivered using a hap/collagen/pLa composite scaffold.72 in another study, Luo et al. also reported a dose-dependent upreg- ulation on Mg63 osteogenic marker expression, 14 days following treatment with BMp-7-laden mesoporous silica nanoparticles.73
Published on 03 May 2017 on http://pubs.rsc.org | doi:10.1039/9781788010542-00169
7.2.2.1.2 Small Molecules. small molecules are promising alternatives to stimulate bone regeneration, with their ability to minimize the problems or limitations of gFs including limited synthesis scale, high cost and limited stability.74,75 For instance, small molecular compounds can better withstand processing techniques utilized in material preparation (e.g. solvent exposure, thermal heating) as compared to gFs, hence retaining their bioactivity more efficiently after scaffold coupling.76 over the past decade, many osteoinduc- tive small molecules have been identified with the advent of high-through- put screening.77 one example is simvastatin, which helps fracture healing by up-regulating BMp-2 expression in osteoblasts.78 previously, it was shown that simvastatin increased bone formation when injected subcutaneously or orally administered to mice and rats.79 however, statin drugs have limitations with solubility and adverse side effects at high dosage.78 therefore, sustained release from biomaterials is proposed to circumvent this issue. tai et al.
incorporated simvastatin within pLga/hap double emulsion microspheres, for subsequent avascular graft transplantation in a mouse fracture model.
Compared to a graft-only control, they observed improved blood vessel and callus formation around the implant after two and four weeks, respectively.
Cellular ingrowths that facilitated graft substitution were also observed.80 purmorphamine is another example. it induces osteogenesis through the hedgehog signaling pathway.81 When purmorphamine-containing hap beads were injected into defect femurs, the proportion of the trabecular bone area was found to be significantly higher than the empty beads control (∼70% to ∼50% after seven days).82 Last but not least, FtY720 is an analog of sphingosine-1-phosphate (s1p), which promotes recirculation of osteoclast precursors from a bone surface, an effect that ameliorates bone loss. When applied alongside dissolved pLga as a coating for allograft samples, FtY720 promoted greater osteointegration of the implant interface with superior mechanical properties (i.e. elasticity and compressive strength) than both un-coated and pLga-only control allografts six weeks post-implantation.83 7.2.2.1.3 Nucleic Acids. another major area involves delivering nucleic acids, which encompass dna plasmids and micro/small interfering rnas (mirnas/sirnas). in general, the introduction of nucleic acids aims to alter cellular function and phenotype by causing either up- or down-regulation of gene expressions. plasmid transfection is commonly used to trigger gene upregulation, while interfering rna is applied to knock down target gene expression through mrna cleavage.84,85 specifically in bone tissue engineer- ing, various plasmids encoding for osteogenic differentiation genes have been studied, in addition to various interfering rnas targeting osteo-inhibiting genes.86,87
For example, a BMp-2 dna plasmid can be complexed with polyethylene- imine (pei) and encapsulated within pLga microspheres for gelatin sponge transplantation into a rat calvarial defect model. eight weeks later, bone formation at the defect site was visible only in BMp-2-containing sponges, as observed through micro Ct and histological staining.88 BMp-2 plasmid
Published on 03 May 2017 on http://pubs.rsc.org | doi:10.1039/9781788010542-00169
complexation with acetylated pei prior to collagen/pga-fiber scaffold incor- poration has also been shown to promote osteogenesis on seeded MsCs. Fol- lowing scaffold and cell implantation into the backs of rats, homogeneous bone formation was observed with ∼10-fold aLp activity and osteocalcin con- tent, as compared to a blank scaffold control.89
apart from BMp-2, plasmids encoding other genes such as runt-related transcription factor 2 (runX2) and pdgF were also successfully delivered through scaffold incorporation. Monteiro et al. loaded runX2 plasmids, a crucial gene to induce transition towards osteoblast phenotype, onto the surface of electrospun-pCL nanofibers through liposomal encapsulation and immobilization. Following internalization by seeded MsCs, the runX2- loaded liposomes induced continuous overexpression of the runX2 gene, which in turn triggered ∼2.5-fold greater aLp activity at day 21 to unmodified nanofibers, without additional supplements.90 For the purpose of enhancing scaffold vascularization, shea et al. directly combined a pdgF plasmid with pLga matrices for the formation of a plasmid-containing 3d sponge through a gas forming process. here, pdgF was chosen for its angiogenesis-stimu- lating role in mediating endothelial cell proliferation and tube formation.91 Following subcutaneous implantation in Lewis rats, plasmids released from the sponges transfected nearby cells at the adipose and muscle layers, which accelerated blood vessel formation (∼three-fold vessel area to blank sponge at day 14); a useful trait for scaffold infiltration.92
similar to protein delivery, multiple kinds of plasmids can be co-delivered through the same scaffold to achieve synergistic effects. By firstly condens- ing the plasmid with pei for lyophilization, huang et al. incorporated both BMp-4 and VegF encoding plasmids into compressed pLga pellets. When MsC-seeded pLga constructs were subcutaneously implanted, significant bone deposition was observed with micro Ct. importantly, simultaneous loading of both plasmids resulted in three times stiffer bone as compared to when BMp-4 or the VegF plasmid was independently incorporated, 15 weeks post implantation (Figure 7.3).93
Lastly, rna moieties (such as mirna and sirna) have also been incor- porated into biomaterials for bone tissue engineering. in one study, a pos- itively-charged transit-tko/BCL2L2 sirna nanoparticle was proposed for retention within the cavities of naoh-treated pCL films. this local BCL2L2 silencing enhanced early osteogenic differentiation, in terms of collagen type 1 deposition and organization.94 Moreover, prolonged and sustained release of sinoggin (a gene which is reported to inactivate BMp-4 and prevent ossification95) in combination with mir-20a (which is predicted to upregu- late BMp/runx2 signaling by regulating pparγ, Bambi and Crim1 96) from a peg hydrogel was shown to successfully direct MsCs’ osteogenic differentia- tion and induce higher calcium content on day 28 (∼two-fold), as compared to blank hydrogel.87 With various osteogenic-inducing mirnas identified recently (e.g. mirna 26a, mirna 148b, mir-196a),97,98 further exploration to optimize their combination and release profile from scaffold materials is warranted.
Published on 03 May 2017 on http://pubs.rsc.org | doi:10.1039/9781788010542-00169
Figure 7.3 Co-delivery of VegF and BMp-4 encoding plasmid enhances MsC- driven bone regeneration. Von kossa staining revealed calcium depo- sition of MsC-seeded constructs incorporating VegF plasmids (a), BMp-4 plasmids (B) and both plasmids (C). Micro-Ct images of con- structs delivering both plasmids at week three (d), eight (e) and 15 (F).
(g) elastic modulus of engineered bone recovered at week 15. scale bar indicates 20 µm in (a)–(C), and 2 mm in (d)–(F). *represents statistical significance against all other groups (P < 0.05). reproduced with per- mission from huang Y. C. et al., J. Bone Miner. Res., 20, 848, 2005. Copy- right 2005: john Wiley and sons.
Published on 03 May 2017 on http://pubs.rsc.org | doi:10.1039/9781788010542-00169