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

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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).


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.


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.


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.



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    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.

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    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.

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    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.

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    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).

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    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).

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    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).


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