A pilot study of poly(N-isopropylacrylamide)-g-polyethylene glycol and poly(N-isopropylacrylamide)-g-methylcellulose branched copolymers as injectable scaffolds for local delivery of neurotrophins and cellular transplants into the injured spinal cord

Laboratory investigation

Restricted access

Object

The authors investigated the feasibility of using injectable hydrogels, based on poly(N-isopropylacrylamide) (PNIPAAm), lightly cross-linked with polyethylene glycol (PEG) or methylcellulose (MC), to serve as injectable scaffolds for local delivery of neurotrophins and cellular transplants into the injured spinal cord. The primary aims of this work were to assess the biocompatibility of the scaffolds by evaluating graft cell survival and the host tissue immune response. The scaffolds were also evaluated for their ability to promote axonal growth through the action of released brain-derived neurotrophic factor (BDNF).

Methods

The in vivo performance of PNIPAAm-g-PEG and PNIPAAm-g-MC was evaluated using a rodent model of spinal cord injury (SCI). The hydrogels were injected as viscous liquids into the injury site and formed space-filling hydrogels. The host immune response and biocompatibility of the scaffolds were evaluated at 2 weeks by histological and fluorescent immunohistochemical analysis. Commercially available matrices were used as a control and examined for comparison.

Results

Experiments showed that the scaffolds did not contribute to an injury-related inflammatory response. PNIPAAm-g-PEG was also shown to be an effective vehicle for delivery of cellular transplants and supported graft survival. Additionally, PNIPAAm-g-PEG and PNIPAAm-g-MC are permissive to axonal growth and can serve as injectable scaffolds for local delivery of BDNF.

Conclusions

Based on the results, the authors suggest that these copolymers are feasible injectable scaffolds for cell grafting into the injured spinal cord and for delivery of therapeutic factors.

Abbreviations used in this paper: BDNF = brain-derived neurotrophic factor; CGRP = calcitonin gene-related peptide; CSPG = chondroitin sulfate proteoglycan; LCST = lower critical solution temperature; MA = methacrylic anhydride; MC = methylcellulose; NIPAAm = N-isopropylacrylamide; PBS = phosphate-buffered saline; PEG = polyethylene glycol; PFA = paraformaldehyde; PNIPAAm = poly(N-isopropylacrylamide); RSF = rat skin fibroblast; SCI = spinal cord injury.

Article Information

Address correspondence to: Jennifer Vernengo, Ph.D., Department of Chemical Engineering, Rowan University, 201 Mullica Hill Road, Glassboro, New Jersey 08028. email: vernengo@rowan.edu.

Please include this information when citing this paper: published online September 2, 2011; DOI: 10.3171/2011.7.SPINE11194.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Brightfield images of injury cavities 1 week after grafting. Grafts are outlined by boxes. Left: Brightfield image of PNIPAAm-g-PEG (Group 1a) showing that the polymer gel fills the cavity with minimal gap formation. Right: In comparison, the Gelfoam implant (Group 1b) has many large spaces within the cavity. Bar = 200 μm.

  • View in gallery

    Hydrogels do not cause additional demyelination in adjacent tissue. Myelin staining (blue) highlights the intact white matter (WM) of the spinal cord. A–H: Demyelination is only present at the injury site, not in surrounding tissue. Low- (A) and high- (B) magnification PNIPAAm-g-PEG/RSF cell suspension (Group 2). Low- (C) and high- (D) magnification PNIPAAm-g-PEG/BDNF (Group 3). Low- (E) and high- (F) magnification PNIPAAm-g-MC/BDNF (Group 4). Low- (G) and high- (H) magnification Vitrogen PureCol/BDNF (Group 5). Bar = 500 μm. GM = gray matter.

  • View in gallery

    Hydrogels do not elicit a greater host response than is seen in the control groups. IBA-1 (red) staining was used to assess the presence of reactive macrophages and microglia adjacent to the lesion and in the surrounding host tissue. DAPI labeling (blue) was used to identify cell nuclei. A: Immunohistochemical labeling of macrophages and microglia, rostral to a graft of PNIPAAm-g-PEG (Group 1a). B: IBA-1 labeling rostral to a graft of Gelfoam (Group 1b). C: PNIPAAm-g-PEG/RSF cell suspension (Group 2). D: PNIPAAm-g-PEG/BDNF (Group 3). E: PNIPAAm-g-MC/BDNF (Group 4). F: Vitrogen PureCol/BDNF (Group 5). Bar = 200 μm.

  • View in gallery

    Cellular grafts were successfully delivered to the lesion site by encapsulation within the injectable gels. Upper: Low-magnification Nissl and myelin–stained image to help in evaluating the lesion size and the location of the lesion within the host tissue. DAPI labeling (blue) was used to identify cell nuclei. Lower: The rat fibroblast graft within the lesion (PNIPAAm-g-PEG/RSF cell suspension, Group 2) is identified using the GFP fluorescent marker (green). Bars = 500 μm (upper); 200 μm (lower).

  • View in gallery

    Hydrogels do not contribute to more glial scar formation than the control matrix. Low-magnification Nissl and myelin–stained images obtained in each group to help in evaluating the lesion size and the location of the lesion within the host tissue (left column). GFAP (green, center column) and CS-56 (red, right column) staining of astrocytes and chondroitin sulfate proteoglycans around graft site. DAPI labeling (blue) was used to identify cell nuclei. A–C: PNIPAAm-g-PEG/RSF cell suspension (Group 2). D–F: PNIPAAm-g-PEG/BDNF (Group 3). G–I: PNIPAAm-g-MC/BDNF (Group 4). J and K: Vitrogen PureCol/BDNF (Group 5). L: Vitrogen PureCol/BDNF (Group 5), dorsal to the injury site. Bar = 500 μm (A, D, G, and J); 200 μm (B, C, E, F, H, I, and K); 100 μm (L).

  • View in gallery

    Hydrogels are permissive to axonal growth. Lesion sites were stained for neurofilaments and CGRP. Low-magnification Nissl and myelin–stained images from each group to help in evaluating the lesion size and the location of the lesion within the host tissue (left column). RT-97 (red) stains host axons (center column), and CGRP (red) labels sensory axons. DAPI labeling (blue) was used to identify cell nuclei (right column). In all CGRP images, notice the dorsal root next to the injury site, shown with an arrow. A–C: PNIPAAm-g-PEG/RSF cell suspension (Group 2). D–F: PNIPAAm-g-PEG/BDNF (Group 3). G–I: PNIPAAm-g-MC/BDNF (Group 4). J–L: Vitrogen PureCol/BDNF (Group 5). Bar = 500 μm (A, D, G, and J); 200 μm (B, C, E, F, H, I, K, and L).

References

  • 1

    Aubert-Pouëssel AVenier-Julienne MCClavreul ASergent MJollivet CMontero-Menei CN: In vitro study of GDNF release from biodegradable PLGA microspheres. J Control Release 95:4634752004

  • 2

    Benoit JPFaisant NVenier-Julienne MCMenei P: Development of microspheres for neurological disorders: from basics to clinical applications. J Control Release 65:2852962000

  • 3

    Blight AR: Effects of silica on the outcome from experimental spinal cord injury: implication of macrophages in secondary tissue damage. Neuroscience 60:2632731994

  • 4

    Bryant SJDavis-Arehart KALuo NShoemaker RKArthur JAAnseth KS: Synthesis and characterization of photopolymerized multifunctional hydrogels: water-soluble poly(vinyl alcohol) and chondroitin sulfate macromers for chondrocyte encapsulation. Macromolecules 37:672667332004

  • 5

    Burdick JAWard MLiang EYoung MJLanger R: Stimulation of neurite outgrowth by neurotrophins delivered from degradable hydrogels. Biomaterials 27:4524592006

  • 6

    Chen QZhou LShine HD: Expression of neurotrophin-3 promotes axonal plasticity in the acute but not chronic injured spinal cord. J Neurotrauma 23:125412602006

  • 7

    Comolli NNeuhuber BFischer ILowman A: In vitro analysis of PNIPAAm-PEG, a novel, injectable scaffold for spinal cord repair. Acta Biomater 5:104610552009

  • 8

    Dhar SRadulescu DGharibjanian NAHayes DJEvans GRD: Regulated nerve growth factor delivery on novel polymers by hNGF-EcR-293-TK cells. J Reconstr Microsurg 22:A0282006. (Abstract)

  • 9

    Flanagan LAJu YEMarg BOsterfield MJanmey PA: Neurite branching on deformable substrates. Neuroreport 13:241124152002

  • 10

    Gehrmann JMatsumoto YKreutzberg GW: Microglia: intrinsic immuneffector cell of the brain. Brain Res Brain Res Rev 20:2692871995

  • 11

    Gupta DTator CHShoichet MS: Fast-gelling injectable blend of hyaluronan and methylcellulose for intrathecal, localized delivery to the injured spinal cord. Biomaterials 27:237023792006

  • 12

    Himes BTGoldberger METessler A: Grafts of fetal central nervous system tissue rescue axotomized Clarke's nucleus neurons in adult and neonatal operates. J Comp Neurol 339:1171311994

  • 13

    Himes BTLiu YSolowska JMSnyder EYFischer ITessler A: Transplants of cells genetically modified to express neurotrophin-3 rescue axotomized Clarke's nucleus neurons after spinal cord hemisection in adult rats. J Neurosci Res 65:5495642001

  • 14

    Himes BTNeuhuber BColeman CKushner RSwanger SAKopen GC: Recovery of function following grafting of human bone marrow-derived stromal cells into the injured spinal cord. Neurorehabil Neural Repair 20:2782962006

  • 15

    Hirschberg DLYoles EBelkin MSchwartz M: Inflammation after axonal injury has conflicting consequences for recovery of function: rescue of spared axons is impaired but regeneration is supported. J Neuroimmunol 50:9161994

  • 16

    Horn KPBusch SAHawthorne ALvan Rooijen NSilver J: Another barrier to regeneration in the CNS: activated macrophages induce extensive retraction of dystrophic axons through direct physical interactions. J Neurosci 28:933093412008

  • 17

    Ibarra ACorrea DWillms KMerchant MTGuizar-Sahagún GGrijalva I: Effects of cyclosporin-A on immune response, tissue protection and motor function of rats subjected to spinal cord injury. Brain Res 979:1651782003

  • 18

    Ibarra ADiaz-Ruiz A: Protective effect of cyclosporin-A in spinal cord injury: an overview. Curr Med Chem 13:270327102006

  • 19

    Jain AKim YTMcKeon RJBellamkonda RV: In situ gelling hydrogels for conformal repair of spinal cord defects, and local delivery of BDNF after spinal cord injury. Biomaterials 27:4975042006

  • 20

    Jin YFischer ITessler AHoule JD: Transplants of fibroblasts genetically modified to express BDNF promote axonal regeneration from supraspinal neurons following chronic spinal cord injury. Exp Neurol 177:2652752002

  • 21

    Joosten EA: Corticospinal tract regrowth. Prog Neurobiol 53:1251997

  • 22

    Kawaja MDFagan AMFirestein BLGage FH: Intracerebral grafting of cultured autologous skin fibroblasts into the rat striatum: an assessment of graft size and ultrastructure. J Comp Neurol 307:6957061991

  • 23

    Kim SHealy KE: Synthesis and characterization of injectable poly(N-isopropylacrylamide-co-acrylic acid) hydrogels with proteolytically degradable cross-links. Biomacromolecules 4:121412232003

  • 24

    Lavik ETeng YDSnyder ELanger R: Seeding neural stem cells on scaffolds of PGA, PLA, and their copolymers. Methods Mol Biol 198:89972002

  • 25

    Lepore ACFischer I: Lineage-restricted neural precursors survive, migrate, and differentiate following transplantation into the injured adult spinal cord. Exp Neurol 194:2302422005

  • 26

    Levenberg SBurdick JAKraehenbuehl TLanger R: Neurotrophin-induced differentiation of human embryonic stem cells on three-dimensional polymeric scaffolds. Tissue Eng 11:5065122005

  • 27

    Liu YHimes BTTryon BMoul JChow SYJin H: Intraspinal grafting of fibroblasts genetically modified by recombinant adenoviruses. Neuroreport 9:107510791998

  • 28

    Liu YKim DHimes BTChow SYSchallert TMurray M: Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function. J Neurosci 19:437043871999

  • 29

    Lu JFéron FMackay-Sim AWaite PME: Olfactory ensheathing cells promote locomotor recovery after delayed transplantation into transected spinal cord. Brain 125:14212002

  • 30

    Madigan NNMcMahon SO'Brien TYaszemski MJWindebank AJ: Current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury using polymer scaffolds. Respir Physiol Neurobiol 169:1831992009

  • 31

    Maquet VJerome R: Design of macroporous biodegradable polymer scaffolds for cell transplantation. Mater Sci Forum 250:15421997

  • 32

    McMahon SSAlbermann SRooney GEMoran CHynes JGarcia Y: Effect of cyclosporin A on functional recovery in the spinal cord following contusion injury. J Anat 215:2672792009

  • 33

    Mitsui TShumsky JSLepore ACMurray MFischer I: Transplantation of neuronal and glial restricted precursors into contused spinal cord improves bladder and motor functions, decreases thermal hypersensitivity, and modifies intraspinal circuitry. J Neurosci 25:962496362005

  • 34

    Murray MFischer ISmeraski CTessler AGiszter S: Towards a definition of recovery of function. J Neurotrauma 21:4054132004

  • 35

    Nomura HTator CHShoichet MS: Bioengineered strategies for spinal cord repair. J Neurotrauma 23:4965072006

  • 36

    Novikov LNovikova LKellerth JO: Brain-derived neurotrophic factor promotes axonal regeneration and long-term survival of adult rat spinal motoneurons in vivo. Neuroscience 79:7657741997

  • 37

    Oudega MGautier SEChapon PFragoso MBates MLParel JM: Axonal regeneration into Schwann cell grafts within resorbable poly(alpha-hydroxyacid) guidance channels in the adult rat spinal cord. Biomaterials 22:112511362001

  • 38

    Oudega MHagg T: Neurotrophins promote regeneration of sensory axons in the adult rat spinal cord. Brain Res 818:4314381999

  • 39

    Patist CMMulder MBGautier SEMaquet VJérôme ROudega M: Freeze-dried poly(D,L-lactic acid) macroporous guidance scaffolds impregnated with brain-derived neurotrophic factor in the transected adult rat thoracic spinal cord. Biomaterials 25:156915822004

  • 40

    Prewitt CMNiesman IRKane CJHoulé JD: Activated macrophage/microglial cells can promote the regeneration of sensory axons into the injured spinal cord. Exp Neurol 148:4334431997

  • 41

    Rolls AShechter RLondon ASegev YJacob-Hirsch JAmariglio N: Two faces of chondroitin sulfate proteoglycan in spinal cord repair: a role in microglia/macrophage activation. PLoS Med 5:e1712008

  • 42

    Schwartz MLazarov-Spiegler ORapalino OAgranov IVelan GHadani M: Potential repair of rat spinal cord injuries using stimulated homologous macrophages. Neurosurgery 44:104110461999

  • 43

    Shibayama MHattori SHimes BTMurray MTessler A: Neurotrophin-3 prevents death of axotomized Clarke's nucleus neurons in adult rat. J Comp Neurol 390:1021111998

  • 44

    Stabenfeldt SEGarcía AJLaPlaca MC: Thermoreversible laminin-functionalized hydrogel for neural tissue engineering. J Biomed Mater Res A 77:7187252006

  • 45

    Stichel CCMüller HW: Experimental strategies to promote axonal regeneration after traumatic central nervous system injury. Prog Neurobiol 56:1191481998

  • 46

    Sugar OGerard RW: Spinal cord regeneration in the rat. J Neurophysiol 3:1191940

  • 47

    Taylor LJones LTuszynski MHBlesch A: Neurotrophin-3 gradients established by lentiviral gene delivery promote short-distance axonal bridging beyond cellular grafts in the injured spinal cord. J Neurosci 26:971397212006

  • 48

    Tessler AFischer IGiszter SHimes BTMiya DMori F: Embryonic spinal cord transplants enhance locomotor performance in spinalized newborn rats. Adv Neurol 72:2913031997

  • 49

    Tessler AHimes BTHoule JReier PJ: Regeneration of adult dorsal root axons into transplants of embryonic spinal cord. J Comp Neurol 270:5375481988

  • 50

    Tuszynski MHGrill RJones LLBrant ABlesch ALöw K: NT-3 gene delivery elicits growth of chronically injured corticospinal axons and modestly improves functional deficits after chronic scar resection. Exp Neurol 181:47562003

  • 51

    Vernengo JFussell GWSmith NGLowman AM: Evaluation of novel injectable hydrogels for nucleus pulposus replacement. J Biomed Mater Res B Appl Biomater 84:64692008

  • 52

    Wagner FC JrVan Gilder JCDohrmann GJ: The development of intramedullary cavitation following spinal cord injury: an experimental pathological study. Paraplegia 14:2452501977

TrendMD

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 69 69 4
Full Text Views 46 46 0
PDF Downloads 105 105 0
EPUB Downloads 0 0 0

PubMed

Google Scholar