Impact of Alginate Composition on Neural Stem Cell Behavior and Neurotrophic Factor Releas, Notas de estudo de Engenharia de Produção

Baixe Impact of Alginate Composition on Neural Stem Cell Behavior and Neurotrophic Factor Releas e outras Notas de estudo em PDF para Engenharia de Produção, somente na Docsity! Alginate Composition Effects on a Neural Stem Cell–Seeded Scaffold Erin K. Purcell, Ph.D., Aparna Singh, B.Tech., and Daryl R. Kipke, Ph.D. The purpose of this study was to evaluate the effects of alginate composition on the neurotrophic factor release, viability, and proliferation of encapsulated neural stem cells (NSCs), as well as on the mechanical stability of the scaffold itself. Four compositions were tested: a high guluronic acid (68%) and a high mannuronic acid (54%) content alginate, with or without a poly-L-lysine (PLL) coating layer. Enzyme-linked immunosorbent assay was used to quantify the release of brain-derived neurotrophic factor, glial-derived neurotrophic factor, and nerve growth factor from the encapsulated cells. All three factors were detected from encapsulated cells only when a high L-guluronic acid alginate without PLL was used. Additionally, capsules with this composition remained intact more frequently when exposed to solutions of low osmolarity, potentially indicating superior mechanical stability. Alginate beads with a PLL-coated, high D-mannuronic acid composition were the most prone to breakage in the osmotic pressure test, and were too fragile for histology and proliferation assays after 1 week in vitro. NSCs survived and proliferated in the three remaining alginate compositions similarly over the 21-day study course irrespective of scaffold condition. NSC-seeded alginate beads with a high L-guluronic acid, non-PLL-coated composition may be useful in the repair of injured nervous tissue, where the mechanism is the secretion of neuroprotective factors. We verify the neuroprotective effects of medium conditioned by NSC- seeded alginate beads on the serum withdrawal–mediated death of PC-12 cells here. Introduction Alginate is a biocompatible hydrogel that has beenused to encapsulate many types of cells with the pur- pose of immunoisolation from the host.1–4 Encapsulation protects graft cells from potential damage caused by an im- mune response while allowing the secretion of therapeutic agents from the cells into the surrounding host tissue. Small molecules such as glucose, oxygen, and waste products freely pass through the gel matrix. Recent studies have investigated the use of alginate as a neural stem cell (NSC) scaffold.5–7 However, the effect of alginate composition on NSC function has not been investigated. Administration of stem cells to sites of central nervous system injury may be used as a means of replacing damaged or diseased tissue, where the focus is on directing the dif- ferentiation of implanted cells into neurons and subsequent reinnervation of host tissue. This strategy has been used to intervene in numerous sources of central nervous system injury, including Parkinson’s disease, stroke, ALS, spinal cord injury, traumatic brain injury, and Huntington’s disease.8–10 However, in many studies the degree of functional recov- ery after NSC transplantation is not explained by the quan- tity of differentiated graft cells alone, lending credence to a bystander or supporting role of NSCs.9,11,12 Several studies suggest that NSCs have an innate ability to promote neuro- protection and axonal regeneration of host tissue.11,13–16 Po- tential mechanisms include constitutive secretion of multiple neurotrophic factors,11,14–16 as well as degrading molecules that are inhibitory to axonal growth.13 NSCs derived from various sources have been found to elute nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial- derived neurotrophic factor (GDNF), and matrix metallo- protease-2, which degrades chondroitin sulfate proteoglycan (a molecule inhibitory to axonal growth).6,13–15 Thus, NSCs and progenitor cells may be exploited as a sort of minia- ture drug factory, releasing factors that result in the desired healing response in injured nervous tissue. Encapsulation of these cells in alginate may further enhance their therapeutic utility by localizing the cells to the site of injury and isolating them from a host immune response. Alginate is a biocompatible polysaccharide polymer com- posed of D-mannuronic (M) and L-guluronic acid (G) residues in varying proportions. Cross-linking and gel formation takes place when divalent cations, such as calcium, ionically bind carboxylic acid groups of blocks of guluronic residues be- tween chains. Alginate has been used widely to encapsulate cells after ground-breaking work published by Lim and Sun Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan. TISSUE ENGINEERING: Part C Volume 15, Number 00, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tec.2008.0302 1 in 1980, which demonstrated reversal of diabetes in rats im- planted with alginate-encapsulated pancreatic islets for a pe- riod of 2–3 weeks.3 Several studies have sought to understand the relationship between alginate composition and the func- tion of the graft.4,17–24 Two common themes emerge in the literature regarding alginate composition and graft perfor- mance: the effect of the M=G content of the alginate and the importance of a polycation coating layer. These two variables have been related to gel mechanical stability, viability of en- capsulated cells, in vivo biocompatibility, and diffusion through the alginate gel. In terms of mechanical stability, al- ginates with a high G content are more mechanically stable than those with a high M content.25 However, high G alginate has been shown to initially inhibit the metabolic and secretory activity of cells due to growth inhibition, theoretically because a higher strength gel is more difficult for proliferating cells to displace.17,21 Beads composed of high G alginate are also known to be more porous than high M alginate, thus en- hancing diffusion of molecules into and out of the matrix.26 Poly-L-lysine (PLL) coating is commonly employed as a means of strengthening the alginate bead and providing a barrier to immune system components such as IgG.27,28 However, the PLL coating layer may itself cause an unfa- vorable foreign body response and slight toxicity to encap- sulated cells, and its use remains controversial.19,20,22,23,29–31 In light of the extensive research indicating a relation- ship between alginate composition and encapsulated cell function, as well as the limited amount of data on NSC encapsulation in alginate, the effects of M=G content and PLL coating on entrapped cortical NSCs were investigated. Among the conditions tested, we show that neurotrophic factor release and mechanical stability in response to an os- motic challenge were the most favorable with a high G scaffold without a PLL coating layer. NSCs survived and proliferated in alginate regardless of the compositions tested. Neurotrophic factor release and bioactivity assay data sub- stantiated the use of NSCs encapsulated in alginate to heal injured nervous tissue via a bystander mechanism. These scaffolded cells have therapeutic potential in treating ner- vous system injuries in future studies, and current work in our lab is investigating their ability to repair a cortical lesion in the adult rat brain.32 Materials and Methods Materials Cortical NSCs, nestin antibody, neuronal class III b- tubulin (TUJ-1) antibody, and all NSC cell culture reagents were purchased from StemCell Technologies (Vancouver, BC). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and enzyme- linked immunosorbent assay (ELISA) kits were obtained from Promega Corporation (Madison, WI). Alginate was from No- vaMatrix (Drammen, Norway). Live=Dead Assay and PC-12 medium reagents were from Invitrogen Corporation (Carls- bad, CA). BD Biocoat collagen–coated plates were from BD Biosciences (San Jose, CA). Centricon filters and NG-2 anti- body were from Millipore Corporation (Billerica, MA). Sec- ondary antibodies were from Molecular Probes (Eugene, OR). PC-12 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). All other reagents were from Sigma-Aldrich (St. Louis, MO). Cortical NSC culture and encapsulation in alginate E14 murine cortical NSCs were cultured and expanded with 20 ng=mL epidermal growth factor according to the supplier’s protocol with a penicillin–streptomycin antibiotic supplement. The culture and stem cell characteristics of these cells have been described.33,34 Cell encapsulation (on Day 0) was achieved by mixing a cell slurry with alginate 50:50 and dropping into a 0.1 M calcium chloride solution for 10 min. The encapsulation yielded beads with a final concentration of 500,000 cells=mL in 1% w=v alginate (approximately 650 cells per bead). The weight percentage was chosen based on a recommendation reported in the literature.21 Cells were approximately 90% viable as assessed by trypan blue stain- ing before encapsulation. Bead size was approximately 1 mm in diameter (1.38
0.19 mm, mean
SD, n¼ 158) immedi- ately after production, and size was controlled by parallel air flow through a glass atomizer. Four different conditions were employed to optimize the encapsulation procedure: a high G alginate (68% G content, molecular weight¼ 219,000 g=mol) or a high M alginate (54% M content, molecular weight¼ 222,000 g=mol), with or without a PLL coating layer. The conditions chosen have been shown to have differing mechanical strengths based on M=G content, and molecu- lar weights were closely matched to eliminate this factor as an experimental variable.25,35 These conditions are abbrevi- ated as G, M, G-PLL, and M-PLL. All alginates were highly purified and sterile. PLL coating was achieved as previously described.36 Briefly, beads were rinsed in physiological saline before a 10 min incubation in 0.1% PLL-HCl (15,000–30,000 MW) in saline. After additional saline rinsing, beads were incubated for 10 min in 0.1% alginate in saline. Beads received a final rinse before being returned to medium. Beads were cultured in static transwell dishes containing 500,000 cells (encapsu- lated or unencapsulated) that were allowed to proliferate in the presence of epidermal growth factor over time. ELISA At 1, 4, 7, 14, and 21 day time points, medium samples were collected for quantification of growth factor release with ELISA (n¼ 5 per condition). Untreated medium and supernatant from nonencapsulated cells were used as con- trols. Supernatant was concentrated with YM-3 Centricon filters at 48C. Promega Emax ELISA kits for NGF, BDNF, and GDNF were used according to the manufacturer’s protocol. Average values below the lowest dilution above zero of the standard curve for each kit were considered to be non- detectable. Alginate mechanical stability The mechanical stability of the alginate beads was as- sessed using a semi-quantitative osmotic pressure test.37 Testing was conducted at 1, 7, 14, and 21 day time points. Alginate beads were exposed to solutions of low osmolarity (0, 2.8, and 11.1 mOsm saline with medium used as a control) for a period of 3 h, as previously described.37 Thirty beads per condition were assessed visually for breakage at each time point. The number of intact capsules was compared between conditions by an observer blinded to the experi- mental condition. 2 PURCELL ET AL. Discussion Alginate composition has been shown to affect encapsu- lated cell proliferation and secretion of therapeutic proteins, as well as the mechanical stability of the scaffold.17,21,25 We sought to characterize the effects composition may have on NSCs, as alginate is largely unstudied as a carrier for these cells. NSCs survive and proliferate in alginate irrespective of the scaffold compositions tested in this study. Among the conditions tested, a high G content alginate without a PLL coating layer was the optimal composition based on its me- chanical stability during the osmotic pressure test and sup- port of neurotrophic factor release. Our data reveal that cortical NSCs, even when encapsu- lated in alginate, secrete NGF, BDNF and GDNF into the surrounding medium. Importantly, these neurotrophic fac- tors are known to support neuronal survival and plasticity in various models of axonopathy and neuropathy.42–46 Quan- titative analysis of constitutive neurotrophic factor release from NSCs is relatively rarely reported in the literature, de- spite the popular theory that release of these proteins and their effects on compromised host tissue is a potential mech- anism behind the cells’ restorative capacity.8,9,11,14,16 Time courses of release are generally lacking, as are release profiles from scaffolded cells. Lu et al. reported detection of NGF, BDNF, and GDNF in medium collected from C17.2 NSCs after a 24 h time point, with levels in the 10–70 pg=million cells=day range.15 GDNF and NGF, but not BDNF, secretion was detected from these cells in a separate report.14 GDNF release from alginate encapsulated and unencapsulated hip- pocampal NSCs tended to be approximately 500–1000 pg= million cells after 72 h, as reported by Li et al.6 Therefore, the reported values for neurotrophic factor release from encap- sulated and unencapsulated cells differ from 10 to 1000 pg= million cells, and our results are consistent with this range. This study revealed that the detection of released neuro- trophic factors is influenced by alginate composition and time. BDNF and GDNF are detected at single time points (4 FIG. 1. Neural stem cell (NSC)-seeded alginate microcapsules with a G compo- sition have superior stability in compar- ison to all other conditions. G beads remain intact more frequently than M capsules after exposure to 0 mOsm 7 days after seeding (a) ( p< 0.001, logistic regression). Poly-L-lysine (PLL)-coated capsules break more frequently than uncoated capsules ( p< 0.001, logistic regression). Representative images of G (b), G-PLL (c), M (d), and M-PLL (e) capsules in solution illustrate the stabil- ity of G capsules (intact beads are labeled ‘‘I’’) and breakage (broken beads are denoted ‘‘B’’) of M and G-PLL beads. M-PLL beads are completely dissolved. Insets in (b–d) highlight representative beads (arrows) to improve observation. Error bars are not shown due to the na- ture of the data analysis. Scale¼ 1 mm. ALGINATE COMPOSITION EFFECTS ON NEURAL STEM CELL–SEEDED SCAFFOLD 5 and 14 days, respectively), while NGF release is observed throughout the study by unencapsulated and encapsulated cells. NGF is detected on fewer days (occurring only in the first week postencapsulation) from encapsulated cells in comparison to unencapsulated cells, possibly due to the initial reduction in viability of the cells by encapsulation, degradation of the NGF protein, or interactions of NGF with the alginate matrix. Alternatively, this result may be related to the apparently lower proliferation rate of NSCs in alginate matrix; the proliferation data shown in Figure 2d do not reflect the 2.5 day doubling rate unencapsulated cells typi- cally experience in culture. GDNF and NGF are only detected from encapsulated cells when a high G, non- PLL-coated capsule is used. Meanwhile, BDNF is released from cells in all conditions tested. Why the high G alginate capsules seem more permissive to neurotrophic factor release is unclear, given that previous literature for insulin-secreting cells has suggested reduced proliferation and less secretion at early time points for cells encapsulated in high G alginate.21 No significant effect of alginate composition on NSC prolif- eration was demonstrated here, which may be due to the relatively low alginate weight percentage (1%) used, as well FIG. 2. NSCs survive (a–c) and prolif- erate (d) in alginate regardless of the compositions tested. Viability of the cells is initially reduced after encapsula- tion, but significantly improves for all conditions after 1 week (a). Cells are la- beled with Hoechst (blue), and propi- dium iodide (red) stains nuclei of cells with compromised membranes indica- tive of cell death (b–c). Images of G- encapsulated cells at 21 days illustrate the vulnerability of cells in the center of larger proliferating neurospheres to necrosis, presumably due to reduced nutrient and oxygen diffusion (b–c). NSCs proliferate irrespective of alginate composition, and cell number was significantly increased 21 days postencapsulation over initial 4-day quantities (d). Values were initially nondetectable by the MTS assay 1 day after encapsulation (d). M-PLL capsules were too mechanically unstable to assay after the first week. {Significantly in- creased versus 1 and 4 day values at the 0.05 level (ANOVA). Mean
standard error of the mean (SEM) is shown. Scale¼ 100mm. Color images available online at www.liebertonline.com=ten. 6 PURCELL ET AL. as differences in the cell types studied. Additionally, the in- creased gel porosity of high G alginate (which could be further enhanced by swelling) and the potential for stabili- zation of trophic factors by interaction with the alginate matrix are possible explanations.26,47 Since neurotrophic factors are larger than insulin (approximately 20–30 kDa vs. 5 kDa), the increased porosity of high G alginate may be necessary to allow for adequate release. The release timing of individual neurotrophic factors may be explained by cellu- lar events and their relationship to evolving environmental cues, and the quantities and timing of neurotrophic factor release is likely to vary with culture conditions. BDNF re- lease coincided with low cell viability, while GDNF detection occurred at a time of increased viability and cell number. It is important to note that the initial drop in viability after en- capsulation could affect the types and quantities of trophic factors released from the cells. Further, the traits of the in- dividual neurotrophic factors may have been a factor in their detection. Specifically, the relatively larger size of GDNF may have resulted in its detection at fewer time points in comparison to NGF. While electrostatic interactions with the alginate matrix require consideration, the neurotrophic fac- tors studied here all have similar basic isoelectric points (ranging from 9 to 10), indicating that they each carry a net positive charge at physiological pH. Cellular events, protein characteristics, and their relationships to interactions with the alginate matrices may all play a role in the detection of the neurotrophic factors. However, elucidating the mecha- nisms behind the release profile of neurotrophic factors by the NSCs is beyond the scope of the current work. The mechanical stability of alginate in vivo over time remains an important challenge.29,48 In a cell encapsulation application, alginate localizes graft cells to the transplant site and isolates them from the immune response, and loss of mechanical strength may compromise these functions. Alginates with a high G content are known to be more mechanically stable than those with higher M contents, due to stabilization by addi- tional cross-links.25 That result was verified here, where the stability of alginate capsules of varying compositions was semi- quantitatively compared by an osmotic pressure test. High G capsules remained intact far more frequently when exposed to solutions of low osmolarities compared with M beads and PLL-coated conditions, and no evidence of decreased prolifer- ation and therapeutic factor secretion was observed. A tradeoff between cellular function and scaffold mechanical strength has been reported previously with insulin-secreting cells.21 How- ever, this tradeoff was more pronounced for higher weight percentage (2%) alginates than the weight percentage (1%) used here. Additionally, this phenomenon was reported for a markedly different cell type than the one studied here, with presumably substantial impacts on proliferation and secretion characteristics. It was somewhat surprising that the PLL-coated capsules were more prone to breakage than uncoated beads during the osmotic pressure test, given that PLL has been credited with improving mechanical stability.20,49 This evidence requires careful interpretation, with consideration for the consequences of saline washing, the potential effects of polycation coating on swelling behavior, the impact of alginate concentration, and the definition of mechanical stability. The results may be affected by the multiple saline washes used in the coating protocol. Monovalent sodium ions may compete with divalent calcium ions for binding sites between G residues, and ultimately break the cross-links and weaken the gel.50 We have previously re- ported a study in which uncoated and PLL-coated alginate disks were washed identically in saline, implanted subcuta- neously in rats, and monitored over 3 months for mechanical stability, weight changes related to swelling behavior, and biocompatibility.48 PLL-coating resulted in a slight but signif- icant increase in complex modulus 7 days after implantation in comparison to noncoated gels; no other significant differences in stability between the two conditions were noted. Therefore, PLL coating may confer a slight advantage, or at least have a nondetrimental role, on gel stability when results are not af- fected by saline washing. However, saline washing is a pro- totypic part of PLL coating protocols; thus, if coating is pursued, the current results indicate that the potentially de- stabilizing effects of saline washing should be considered. In the same study, both PLL and noncoated gels initially dropped in weight after implantation, followed by similar trends in increasing weight (interpreted as swelling) for a period of 2 weeks. The weight gains were greater for FIG. 3. Encapsulated NSCs express nestin (green) on the periphery and GFAP (red) in the center of cell masses after entrapment in alginate (a–b). Interestingly, some evidence of limited NG-2 expression (green) was observed in one bead, indicating oligodendrocyte progenitors (c). All nuclei were counterstained with Hoechst (blue). Images are from (a) G, 14 day (b) M-PLL, 14 day, and (c) G-PLL, 4 day, scaffolds and time points. Scale¼ 50mm. Color images available online at www.liebertonline.com=ten. ALGINATE COMPOSITION EFFECTS ON NEURAL STEM CELL–SEEDED SCAFFOLD 7