In situ gelling nanoparticulate systems for tissue engineering using stem cells
thesisposted on 27.02.2017, 06:10 by Carvalho, Edmund
The aim of the current study was to ascertain whether three dimensional scaffold delivery systems could be developed along with nanoparticles to mediate differentiation in situ using the properties of the hydrogel scaffold and nanoparticles. The work was divided into three sections, 1. The development of in situ gelling scaffold systems, 2. Encapsulation and differentiation of ES cells within the scaffold systems, 3. Nanoparticulate approaches to facilitate ES cell differentiation towards the cardiac lineage. Gellan which can be crosslinked with calcium to form scaffolds while HPMC which was thermoresponsive were chosen to form scaffolds. Gellan and gellan HPMC blends were prepared to further employ the thermoresponsiveness of HPMC to improve the properties of the in situ gelling scaffolds prepared. It was found that the average gelation time of the gellan HPMC(GH) crosslinked with 3mM calcium blends 9:1 and 8:2 was 12 min and 16 min respectively and that for 0.5% gellan blended with 3mM calcium, was 26 min which was within the range of ASTM standards for in situ gelation. The volume of pores present in all of the blends were in the range of 80 to 90 % suggesting that the porous volume of the scaffolds was occupied completely with water once hydrated. The calcium crosslinked gels were porous and non-toxic indicating that the gels suitably network and ensure a viable environment. The 0.5GH8:2-3 blend showed promise and indicated that an increase in the concentration coupled with the addition of calcium improves the overall properties of the hydrogel so formed. Temperature dependent rheology suggested that 0.5GH 8:2-3 shared properties similar to 0.5G-3. The extended viability of the cells as well as tan d in vitro, makes 0.5%GH 8:2 with 3mM calcium more similar to 0.5% G-3. We finally considered gellan crosslinked with calcium for encapsulation of embryoid bodies. Scanning electron microscopy indicated EB presence within scaffold matrix. EB, beating efficiency on day 19 from 1.5 mM to 3 mM and 6 mM. showed that there was a significant difference in the beating rate of EBs encapsulated in 0.5G 1.5 compared to 0.5G 3 and 0.5G 6 at p<0.05 and significant difference in beating rates between 0.5G 3 and plated controls at p<0.005. Gene expression of early marker Flk 1 was present in all samples. Late cardiac marker Mlc2v was absent in 1.5mM crosslinked gel on day 19 its expression was faintly present in 3mM calcium encapsulated EBs on both day3 and day 5. Its expression was prominent in 6mM calcium encapsulated gellan and absent in day 3 and day 5 plated EBs. Atrial marker Mlc 2a was expressed uniformly in all of the treatment sets while cardiac TnT too was expressed from day 5 onwards in treated and untreated samples. Tenfold expression of Mlc 2v, four fold Flk 1 and three fold cardiac TnT expression was observed compared to plated controls on day 5 of encapsulation and plating. The gelling time and gene expression studies indicated that a stiffer matrix induced greater expression of cardiac specific genes. Since 3mM calcium crosslinked gellan had a gelling time within ASTM standards for in situ gelling agents; nanoparticle nanoparticle encapsulated molecular mediators could be used to facilitate differentiation along with 3mM crosslinked gellan. Cardiac tissue specific lipids were chosen to prepare liposomes. It was found that the average hydrodynamic diameter for PC liposomes were found to be of 178 nm +/- 13nm before loading and 153nm +/- 15nm after loading with retinoic acid. While average size for PCPE liposomes were found to be of 210nm +/- 7nm and 255nm +/-20nm after retinoic acid loading. The surface charge for PC and PC-RA was - 32 +/-3 mV and - 30 +/- 4 mV respectively, while that of PCPE and PCPE-RA were - 26 +/- 2.5 mV and - 27+/- 2.6 mV respectively. The encapsulation efficiency for the PC-RA liposomes was 57.2% + 0.9 and while that for PCPE-RA liposomes was 54.8% + 0.5. PCPE liposomes resulted in a lower retinoic acid release over 24 hours as compared to PC loaded retinoic acid. PCPE liposomes showed an initial burst release for 2 hours with a sustained release at an average of 30% thereon until 24 hours as compared to 100% release for PC liposomes. Proliferation studies revealed that there was a significant difference between PC-RA at 0.3nM treated embryoid bodies and those treated with free retinoic acid at a concentration of 0.3nM and 0.003nM Moles at p<0.05 and p<0.01 respectively. The number of beating bodies were low in the free 0.3nM retinoic acid and the PCPE 0.3 loaded liposomes, this did not change from day 9 to day 13. There was a greater change in the percentage beating bodies with the untreated EBS , DMSO treated, free 0.003nM, PCPE 0.003nM and PCPE 0.003nM from day 9 to day 13. There was a greater change in the percentage beating bodies with the untreated EBS , DMSO treated, free 0.003nM, SPC 0.003 M and SPC POPE 0.003nM from day 9 to day 13. Early gene Flk 1 expression diminished after day 7. Mlc2a i.e. atrial myocin expression was only observed on day 7 embryoid bodies. Mlc2v was expressed on day 7 EB and at very low levels in samples treated with retinoic acid. Low expression was observed in the presence of RA concentration at 0.003nM in the presence of PC nanoparticles. Cardiac TnT expression was observed at levels slightly lower than levels observed on day7 in all samples. The tunable rheological properties of the gellan can further be used for the differentiation of stem cells in vitro. Liposome entrapped molecular mediators can be used in conjunction with tunable scaffolds to drive in situ differentiation in ES cells.