\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{IL-10 lentivirus-laden hydrogel tubes increase spinal progenitor
survival and neuronal differentiation after spinal cord injury}



\author[1]{Andrew Ciciriello}%
\author[2]{Dominique  Smith}%
\author[2]{Mary Munsell}%
\author[1]{Sydney Boyd}%
\author[2]{Lonnie Shea}%
\author[1]{Courtney Dumont}%
\affil[1]{University of Miami}%
\affil[2]{University of Michigan}%


\vspace{-1em}



  
  \date{June 16, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
The pathophysiological response following spinal cord injury (SCI) is
characterized by a complex cellular cascade that limits regeneration.
Biomaterial and stem cell combination therapies have shown synergistic
effects, compared to their interventions independent of each other, and
represent a promising approach towards regaining function after injury.
In this study, we combine our polyethylene glycol (PEG) cell delivery
platform with lentiviral-mediated overexpression of the
anti-inflammatory cytokine interleukin (IL)-10 to improve embryonic day
14 (E14) spinal progenitor transplant survival. PEG tubes loaded with
lentivirus encoding for IL-10 were implanted immediately following
injury into a mouse SCI hemisection model. Two weeks after tube
implantation, mouse E14 spinal progenitors were injected directly into
the integrated tubes, which served as a soft substrate for cell
transplantation. Together, the tubes with the IL-10 encoding lentivirus
improved E14 spinal progenitor survival, assessed at two weeks
post-transplantation (four weeks post-injury). Mice receiving IL-10
lentivirus-laden tubes had on average 8.1\% of E14 spinal progenitors
survive compared to 0.7\% in mice receiving transplants without tubes,
an 11.5-fold difference. Surviving E14 spinal progenitors gave rise to
neurons when injected into tubes. Additionally, axon elongation and
remyelination was observed, in addition to a faster rate of functional
recovery in mice receiving anti-inflammatory tubes with E14 spinal
progenitor delivery. This system affords increased control over the
transplantation microenvironment, offering the potential to improve stem
cell-mediated tissue regeneration.%
\end{abstract}%



\sloppy


\textbf{Title:} IL-10 lentivirus-laden hydrogel tubes increase spinal
progenitor survival and neuronal differentiation after spinal cord
injury

\textbf{Authors and Affiliations:}

Andrew J. Ciciriello\textsuperscript{1,2}, Dominique R.
Smith\textsuperscript{3}, Mary K. Munsell\textsuperscript{3}, Sydney J.
Boyd\textsuperscript{1}, Lonnie D. Shea\textsuperscript{3,4}, and
Courtney M. Dumont\textsuperscript{1,2,*}

\textsuperscript{1}Department of Biomedical Engineering, University of
Miami, Coral Gables, FL, USA

\textsuperscript{2}DJTMF Biomedical Nanotechnology Institute at the
University of Miami (BioNIUM), University of Miami, Miami, FL, USA

\textsuperscript{3}Department of Biomedical Engineering, University of
Michigan, Ann Arbor, MI, USA

\textsuperscript{4}Department of Chemical Engineering, University of
Michigan, Ann Arbor, MI, USA

\textsuperscript{\textbf{*}}\textbf{Corresponding Author}

Courtney M. Dumont

Department of Biomedical Engineering, University of Miami

1251 Memorial Drive

Coral Gables, FL 33156

Email: cdumont@miami.edu

Phone: (305)~243-6142

\textbf{ORCID iD:}

Courtney M. Dumont -- 0000-0001-8455-7188

Lonnie D. Shea -- 0000-0002-9296-9673

\textbf{Funding Information:} This work was supported by the NIH
(R01EB005678).

\textbf{Abstract}

The pathophysiological response following spinal cord injury (SCI) is
characterized by a complex cellular cascade that limits regeneration.
Biomaterial and stem cell combination therapies have shown synergistic
effects, compared to their interventions independent of each other, and
represent a promising approach towards regaining function after injury.
In this study, we combine our polyethylene glycol (PEG) cell delivery
platform with lentiviral-mediated overexpression of the
anti-inflammatory cytokine interleukin (IL)-10 to improve embryonic day
14 (E14) spinal progenitor transplant survival. PEG tubes loaded with
lentivirus encoding for IL-10 were implanted immediately following
injury into a mouse SCI hemisection model. Two weeks after tube
implantation, mouse E14 spinal progenitors were injected directly into
the integrated tubes, which served as a soft substrate for cell
transplantation. Together, the tubes with the IL-10 encoding lentivirus
improved E14 spinal progenitor survival, assessed at two weeks
post-transplantation (four weeks post-injury). Mice receiving IL-10
lentivirus-laden tubes had on average 8.1\% of E14 spinal progenitors
survive compared to 0.7\% in mice receiving transplants without tubes,
an 11.5-fold difference. Surviving E14 spinal progenitors gave rise to
neurons when injected into tubes. Additionally, axon elongation and
remyelination was observed, in addition to a faster rate of functional
recovery in mice receiving anti-inflammatory tubes with E14 spinal
progenitor delivery. This system affords increased control over the
transplantation microenvironment, offering the potential to improve stem
cell-mediated tissue regeneration.

\textbf{Keywords:} spinal cord injury, neural stem cells, biomaterials,
gene delivery, tissue engineering

Introduction

Spinal cord injury (SCI) is an incredibly devastating condition that can
leave patients both physically and financially burdened for the
remainder of their lives. Approximately 12,500 new cases of traumatic
SCI are reported annually in North America alone, with lifetime
healthcare costs averaging about \$2.35 million per patient (Alizadeh,
Dyck, \& Karimi-Abdolrezaee, 2019). Difficulties in treating SCI arise
as both the innate and adaptive immune responses attempting to protect
the spinal cord from further damage. The immune response, while
necessary for removing dead cells and debris, creates an inhospitable
milieu characterized by a cascade of cellular and biochemical signals,
highlighting a delicate balance between neuroplasticity and further
damage. Known as the secondary injury, this induced damage persists
through acute, sub-acute, and chronic stages of injury, spanning hours
to months post-SCI (Dalamagkas, Tsintou, Seifalian, \& Seifalian, 2018;
Donnelly \& Popovich, 2008; Dumont, Margul, \& Shea, 2016) resulting in
glial and fibrotic scarring (Bradbury \& Burnside, 2019; Hackett \& Lee,
2016; Soderblom et al., 2013; Zhu, Soderblom, Trojanowsky, Lee, \& Lee,
2014), axonal dieback and demyelination (Busch, Horn, Silver, \& Silver,
2009; Hill, 2017), apoptosis (X. Z. Liu et al., 1997), and oxidative
stress (Hall, 2011) severely limiting regenerative potential.

Exogenous stem cell transplantation is a promising technique that is
highly dependent on the transplantation microenvironment. Cell
transplantations have shown initial promise both broadly in tissue
engineering (Kwon, Kwon, Lee, Park, \& Kim, 2018) and specifically in
SCI repair models (Dulin \& Lu, 2014). On a cellular level, neural stem
cell (NSC) transplants repopulate lost neural and glial cells in damaged
tissue (Dulin \& Lu, 2014; Mortazavi et al., 2015) in addition to
working on a molecular level through cytokine and chemokine release
post-SCI (P. Lu, Jones, Snyder, \& Tuszynski, 2003). Furthermore,
transplanted NSCs can integrate with endogenous neural circuitry (Ceto,
Sekiguchi, Takashima, Nimmerjahn, \& Tuszynski, 2020; Kumamaru et al.,
2018) and can significantly decrease local expression of
pro-inflammatory cytokines (Cheng et al., 2016). Altogether, NSCs
exhibit numerous pro-regenerative functions that are highly contingent
on cell survival post-transplantation. Transplantation method, medium,
location, and cell age all play pivotal roles in influencing survival
(Chen, Bernreuther, Dihn\selectlanguage{ngerman}é, \& Schachner, 2005; Karimi-Abdolrezaee,
Eftekharpour, Wang, Morshead, \& Fehlings, 2006; Nagoshi et al., 2018),
but perhaps the most important factor to consider with cell therapies is
their transplantation microenvironment. In SCI, NSC delivery is
complicated by the persisting secondary injury. As the immune response
clears damaged cells and cell debris, transplanted cells are also
subjected to inhospitable conditions, leading to poor survival and
limited regenerative effect. SCI cell transplantation strategies
commonly delay delivery to occur after an initial wave of post-injury
inflammation (Tetzlaff et al., 2011). Starting hours after injury,
inflammatory cytokines including interleukin (IL)-1\selectlanguage{greek}β, \selectlanguage{english}tumor necrosis
factor \selectlanguage{greek}α \selectlanguage{english}(TNF\selectlanguage{greek}α\selectlanguage{english}), IL-6, and leukemia inhibitory factor (LIF) are strongly
activated in the wound area, but their concentrations decrease by 1 week
post-injury. Similarly, reactive oxygen species peak in concentration
approximately 12 hours after injury, but by 1 week post-injury, their
concentration drops precipitously (Donnelly \& Popovich, 2008; Dumont et
al., 2016; Lacroix, Chang, Rose-John, \& Tuszynski, 2002; Shamash,
Reichert, \& Rotshenker, 2002). Avoiding these inflammatory mediators by
delaying transplantation has the potential to improve survival, but
further intervention is required for cell transplants to have their
intended regenerative effects.

Biomaterial interventions have proven to be effective following SCI in
shifting the post-injury microenvironment and improving regeneration
(Shuo Liu, Xie, \& Wang, 2019; Straley, Foo, \& Heilshorn, 2010) making
them promising candidates to be used in combination therapies with stem
cell transplants (Shengwen Liu, Schackel, Weidner, \& Puttagunta, 2018;
Shrestha et al., 2014) where the goal is to create a more hospitable
transplantation environment to improve transplant survival. Highly
porous, polymeric scaffold implants can facilitate regeneration in the
spinal cord, where the porosity allows for endogenous progenitor
infiltration, aiding in axonal regrowth and myelination (Pawar et al.,
2015; Shahriari, Koffler, Tuszynski, Campana, \& Sakamoto, 2017; Straley
et al., 2010; Tuinstra et al., 2012; Tuinstra et al., 2013; Y. Yang et
al., 2009). Furthermore, implantable scaffolds provide a defined
architecture that directs axonal elongation. Softer, hydrogel based
implants fabricated from polyethylene glycol (PEG) microspheres have
also proved to support regeneration (Dumont et al., 2019). Macroporous
tubes, molded from PEG microspheres, function similar to rigid,
polymeric scaffolds, but are advantageous in that they better match
native tissue mechanical properties and have the potential to conform to
individual injury anatomy. Biomaterial strategies for SCI can
additionally support stem cell transplantation whether that is
through\emph{ex vivo} culture on a rigid scaffold (Dumont et al., 2018;
Li et al., 2016; Mothe, Tam, Zahir, Tator, \& Shoichet, 2013),
suspension in hydrogel injections (Assun\selectlanguage{ngerman}ção-Silva, Gomes, Sousa, Silva,
\& Salgado, 2015; Cai, Dewi, \& Heilshorn, 2015; Marquardt \& Heilshorn,
2016), or direct injection into an integrated implant (Ciciriello et
al., 2020). Alone, biomaterials passively modify immune cell
infiltration and inflammation, resulting in improved stem cell survival
post-transplantation, but a more active role in immune modulation can be
taken through delivery of an anti-inflammatory factor.

Lentiviral vectors are a highly effective method for active therapeutic
treatment with presently ongoing clinical trials (Milone \& O'Doherty,
2018). Lentivirus-mediated therapies work by integrating within a host
genome, resulting in long-term overexpression of the selected gene of
interest. Implantable biomaterials can serve as a medium for loading and
release of lentivirus, and this has been investigated heavily in both
SCI models (Abdellatif et al., 2006; Boehler et al., 2014; Daniel J.
Margul et al., 2016; Park, Decker, Margul, et al., 2018; Park, Decker,
Smith, et al., 2018; Dominique R. Smith et al., 2020; D. R. Smith et
al., 2019) and for other tissue engineering applications like islet
transplantation (Chou \& Sytwu, 2009; Jimenez-Moreno et al., 2015; J. M.
H. Liu, Zhang, Joe, Luo, \& Shea, 2018), cancer immunotherapies (Arce,
Breckpot, Collins, \& Escors, 2011; Liechtenstein, Perez-Janices, \&
Escors, 2013; Milone \& O'Doherty, 2018), and cardiac tissue repair (Di
Pasquale, Latronico, Jotti, \& Condorelli, 2012; Niwano et al., 2008;
Zhao et al., 2002). Lentiviral-mediated over-expression of cytokines
from the IL family, specifically those targeting macrophages, has shown
promise in altering immune response post-injury. Macrophages are a
well-studied cell population, often viewed as an attractive therapeutic
target. Infiltrating macrophages are traditionally grouped into two
general phenotype classes, pro-inflammatory M1 and pro-regenerative M2,
each with important functions post-injury. In reality, the behavior of
these phenotypes is much more nuanced than the binary options presented
here (Mosser \& Edwards, 2008), but this provides a frame of reference
for distinguishing between pro-inflammatory and pro-regenerative roles.
Furthermore, this described phenotypic difference opens the opportunity
for lentiviral-mediated overexpression of an anti-inflammatory cytokine,
like IL-10, to facilitate the polarization of M1 to M2 macrophages to
create a more pro-regenerative microenvironment.

In this work, we expand the passive microenvironment modulation of our
macroporous PEG tubes to actively modify the transplantation
microenvironment through lentiviral-mediated delivery of IL-10.
Previously, we demonstrated the short-term feasibility of the temporally
flexible transplantation of enhanced green fluorescent protein
(EGFP\textsuperscript{+}) embryonic day 14 (E14) spinal progenitors into
integrated PEG tubes (Ciciriello et al., 2020). Expanding on this prior
work, in our current study we will implant IL-10 encoding
lentivirus-laden tubes into a C5 lateral hemisection, and two weeks
later inject EGFP\textsuperscript{+} E14 spinal progenitors directly
into the tubes at the lesion epicenter. This two week period between
tube implantation and E14 spinal progenitor transplantation affords the
tubes time to integrate and the IL-10 encoding lentivirus to modulate
the immune response, ultimately improving transplantation. Alone, the
tubes served as a privileged cell transplantation site, so we anticipate
that adding lentivirus-mediated IL-10 delivery will further improve
transplantation survival, resulting in long term benefits in
regeneration and functional restoration through both endogenous and
exogenous repair mechanisms, marking an important step forward in the
development of a comprehensive cell delivery platform for SCI repair.

Methods

Fabrication of hydrogel tubes

Hydrogel tubes were generated as previously described (Dumont et al.,
2019). Briefly, 20\% w/v 8-arm polyethylene glycol maleimide (PEG-MAL,
20 kDa; JenKem, Plano, TX) was crosslinked with 5 mM
slow-degrading-plasmin-sensitive YKND cross-linking peptide
(Ac-GCYKNDGCYKNDCG; Genscript, Piscataway, NJ) (Shikanov, Smith, Xu,
Woodruff, \& Shea, 2011) to form microspheres through water-oil emulsion
with diameter ranging between 15 and 150 \selectlanguage{greek}µ\selectlanguage{english}m and an average of 45 \selectlanguage{greek}µ\selectlanguage{english}m. The
PEG-YKND solution was homogenized in silicone oil (Fisher, Hampton, NH)
with 2\% Tween-20 (Sigma, St. Louis, MO) at a speed of 4000 rpm for 1
minute. Microspheres were rinsed by centrifugation three times. Irgacure
2959 photoinitiator (Sigma) dissolved in N-vinylpyrrolidinone (660
mg/mL; Sigma) was added to the microspheres at a final concentration of
1\% w/v. The resulting microspheres were then packed into
polydimethylsiloxane (PDMS, Dow Corning, Midland, MI) molds to generate
PEG tubes (approximate OD: 600 \selectlanguage{greek}μ\selectlanguage{english}m, ID: 250 \selectlanguage{greek}μ\selectlanguage{english}m, porosity 66\%) and
exposed to an ultraviolet lamp for 3 minutes to initiate free radical
polymerization. Tubes were rinsed three times, dehydrated, and stored at
-80 until use. Tubes were cut to length during surgery to ensure fit
within the defect.

Spinal progenitor isolation

All animal work pertaining to progenitor cell isolation was performed
with prior approval and in accordance with the Institutional Animal Care
and Use Committee (IACUC) guidelines at the University of Michigan and
University of Miami. Embryonic spinal progenitors were isolated from the
spinal cords of embryonic day 14 (E14) C57BL/6-Tg(CAG-EGFP)10sb/J mice
(Jackson Laboratory, Bar Harbor, ME), enzymatically dissociated with 10
U/mL papain (Worthington, Lakewood, NY) and 37 \selectlanguage{greek}μ\selectlanguage{english}g/mL DNase (Sigma) into
single cells, and expanded as neurospheres in ultralow attachment flasks
(Corning, Corning, NY), as described previously (Dumont et al., 2018).
Embryonic spinal progenitors were expanded in Dulbecco's Modified Eagle
Medium (DMEM; Gibco, Grand Island, NY) supplemented with 1X B27 (Gibco),
1X N2 (Gibco), N-acetyl cysteine (NAC; Sigma), and 20 ng/mL basic
fibroblast growth factor (FGF2; Peprotech, Rocky Hill, NJ) and leukemia
inhibitory factor (LIF; Peprotech). E14 cell colonies were passaged with
papain as needed and not used beyond the second passage for
transplantation studies.

Production of lentiviral vectors

Lentivirus was produced as described previously (D. R. Smith et al.,
2019). HEK-293FT cells (80-90\% confluent, American Type Culture
Collection, Manassas, VA) were transfected with third generation
lentiviral packaging vectors and pLenti-CMV-Luciferase or
pLenti-CMV-hIL10. Correct insertion was validated via DNA sequencing.
Plasmids were incubated in OptiMEM (Life Technologies, Carlsbad, CA)
with Lipofectamine 2000 (Life Technologies) for 20 minutes prior to
being added to cells. After 48 hours of incubation, supernatant was
collected, centrifuged to remove cellular debris, and then incubated
with PEG-It (System Biosciences, Palo Alto, CA) for 16-24 hours at 4\selectlanguage{ngerman}°C.
Virus was centrifuged at 1500g at 4°C for 30 min, supernatant was
removed, and the pellet was re-suspended in sterile phosphate buffered
saline (PBS; Life Technologies). Viral solution was aliquoted and frozen
at -80°C until use. Viral titers used throughout the study were 4E9
IU/mL as determined by the Lentivirus qPCR Titer Kit (Applied Biological
Materials, Richmond, BC, Canada). Viral vectors used in this study were
from the same batch as described by Smith et. al. (Dominique R. Smith et
al., 2020), thus all \emph{in vivo} protein expression data would be
comparable between these studies. However, it should be noted that
additional vector expression analysis was performed using the In Vivo
Imaging System (IVIS; Perkin Elmer, Waltham, MA) with the
pLenti-CMV-Luciferase vector as described below.

SCI surgeries

All animal work was performed with prior approval and in accordance with
the Institutional Animal Care and Use Committee (IACUC) guidelines at
the University of Michigan. A C5 lateral hemisection SCI was created in
adult C57BL/6J female mice aged 6-8 weeks, as previously described (D.
J. Margul et al., 2016; Thomas et al., 2013; Thomas et al., 2014).
Briefly, mice were anesthetized with 2\% isoflurane and provided
preemptive local pain management (1 mg/kg bupivacaine). After
confirmation of anesthesia via toe pinch, a 2 cm incision was made in
the skin to facilitate the laminectomy performed at C5. A 1.15 mm
lateral hemisection was excised in the left side of the spinal cord.
This ensures any functional deficits are confined to the left forepaw,
providing an internal control with the right paw. PEG tubes were cut to
size, allowed to dehydrate for 30 s, and implanted individually into the
injury site, which accommodated 5 tubes in total. A subset of these mice
received 2 \selectlanguage{greek}μ\selectlanguage{english}L of 4E9 IU/mL pLenti-CMV-hIL10 viral vector injected
directly into the tubes. Gelfoam was used to secure the injury site in
all conditions, after which the muscles were sutured and skin stapled. A
subset of mice (no-treatment control group) did not receive an implant
but did receive gelfoam over the injured spinal cord. Mice were
immediately provided post-operative antibiotics (enrofloxacin 2.5 mg/kg
once a day for 2 weeks), analgesics (0.1 mg/kg buprenorphine twice a day
for 3 days), and supportive hydration (1 mL/20 g lactated ringer
solution once a day for 5 days). Bladders were expressed twice daily
until function recovered and staples were removed after 10 days.
Surgical controls were put in place to limit lesion size variance,
including the order of the incisions made to limit the effects of
swelling to cut lines, measuring the distance between the rostral and
caudal cuts, and verifying the absence of bruising to the contralateral
tissue. Exclusion criteria include any deviations to the surgical
controls, as well as any variance to the recovery timeline, including an
inability to ambulate by post-operative day 3. No mice met these
exclusion criteria for this study.

All mice received a second surgery 14 days after the primary injury.
Protocols for anesthesia and post-operative care were the same as
described with the initial surgery. After anesthesia induction, an
incision in the skin was made, sutures in the muscle were removed, and
any remaining gelfoam was carefully removed from atop the spinal cord.
Two injections of E14 EGFP spinal progenitors (2 \selectlanguage{greek}μ\selectlanguage{english}L of 150,000 cells in
PBS -- 300,000 cells/mouse) or PBS were injected in the rostral-medial
and caudal-lateral regions of the hydrogel implant using a 33G 10\selectlanguage{greek}μ\selectlanguage{english}L
Hamilton syringe at a rate of 1 \selectlanguage{greek}μ\selectlanguage{english}L/minute. Gelfoam was placed back over
the injury, muscles were sutured, and wound clips applied. Mice were
euthanized and spinal cord segments (C4-6) were collected after 4 or 12
weeks (2 or 10 weeks after cell transplantation). For each condition, n
= 4-12 mice at each time point: n = 4 for histology at week 4 (N=24), n
= 6 for bioluminescent imaging (N=12), n = 6 for histology at week 12
(N=36), and n = 12 for ladder beam analysis (N=72).

Bioluminescent imaging

Bioluminescent signal was evaluated with IVIS. Mice used to assess
bioluminescent signal received a single surgery, in which the injury was
made, tubes were implanted, and 2 \selectlanguage{greek}μ\selectlanguage{english}L of 4E9 IU/mL lentivirus with a
luciferase reporter was injected directly into the tubes. A second set
of mice did not receive the viral injection and served as controls.
Gelfoam was placed over the injury, muscles were sutured, and wound
clips applied. After 7 days the wound clips were removed. Mice were
injected with 150 mg/kg D-Luciferin (Promega, Madison, WI)
intraperitoneally 10 minutes prior to imaging. Mice were sedated 5
minutes prior to imaging and transferred to the IVIS chamber under
continuous isoflurane sedation. D-Luciferin injections and IVIS imaging
were repeated at weeks 4, 6, 8, and 12. Living Image (Perkin Elmer)
software was used to evaluate total flux based on bioluminescent signal
within the injury site.

Endogenous progenitor identification

Bromodeoxyuridine (BrdU) was used to identify proliferating cells in the
first 7 days post-SCI, as described previously (Ciciriello et al.,
2020). Intraperitoneal injections of 50 mg/kg BrdU (Roche, Basel,
Switzerland) were pulsed every day for 7 days, allowing 7 additional
days for unbound BrdU to wash out before exogenous spinal progenitors
were implanted, as done by others (Ciciriello et al., 2020; Z. Yang et
al., 2015). Using this protocol, overlap of BrdU and exogenous EGFP cell
transplants was not observed. Any cells that co-localized with BrdU and
neuronal lineage markers described below were considered to arise from
endogenous spinal progenitors giving rise to new cells along the
neuronal lineage. Tissue sections requiring BrdU identification were
first denatured in 2N HCl for 1 hour at 37 \selectlanguage{ngerman}°C followed by neutralization
in 2 five minute rinses of 0.1M borate buffer to facilitate antigen
retrieval. Samples were incubated for 1 hour at room temperature in rat
anti-BrdU (1:200, Abcam, Cambridge, UK) antibody followed by appropriate
fluorophore-conjugated goat anti-rat secondary antibody before
proceeding with additional immunohistochemistry of specific cell
phenotypes described below.

Immunohistochemistry

Isolated spinal cords were flash frozen, and then cryosectioned
transversely (4 or 12 week tissue) in 12 \selectlanguage{greek}μ\selectlanguage{english}m sections.
Immunohistochemistry analysis was performed using nine transverse
sections evenly spaced across the rostral, middle, and caudal regions of
the tubes and average across each animal allowing for comparisons
between animals while considering regional differences (Ciciriello et
al., 2020; Dumont et al., 2019). Samples were fixed, permeabilized
(0.5\% triton-X for 10 minutes) and/or prepared with BrdU staining
(described above) as necessary, and incubated overnight at 4\selectlanguage{ngerman}°C with
primary antibodies. The following antibodies were used for primary
detection: rat anti-F4/80 (1:200, Abcam, Cambridge, United Kingdom),
goat anti-arginase (1:100, Santa Cruz, Dallas, TX, USA), rabbit
anti-neurofilament-200 (1:200, Sigma), goat anti-myelin basic protein
(MBP; 1:500, Santa Cruz), chicken anti-P0 (1:250, Aves Labs, Tigard,
OR), chicken anti-GFP (1:200, Aves Labs), rabbit anti-Tuj1 (1:500,
Sigma), mouse anti-NeuN (1:500, Millipore, Burlington, MA), mouse
anti-Nestin (1:200, Millipore), rabbit anti-GFAP (1:500, Thermo
Scientific, Waltham, MA), and rat anti-IL-10 (1:100, Thermo Scientific).
Species-specific fluorescent secondary antibodies were used for
detection at 1:1000 (Life Technologies, Carlsbad, CA, USA). Hoechst
33342 (Life Technologies) was used as a counterstain in all tissue
sections. Immunostained tissue sections were imaged using an
AxioObserver inverted fluorescent microscope (Zeiss, Oberkochen,
Germany) using a 10X dry objective.

\emph{Progenitor survival, proliferation, and fate:}Immunohistochemistry
was used to verify no overlap between BrdU\textsuperscript{+} and
EGFP\textsuperscript{+} cell types. Subpopulations of exogenous
EGFP\textsuperscript{+} cells and endogenous BrdU\textsuperscript{+}
cells were counted manually by two blinded researchers to evaluate
progenitor-driven neurogenesis. The following cell populations were
quantified: exogenous progenitors
(EGFP\textsuperscript{+}BrdU\textsuperscript{-}Nestin\textsuperscript{+}),
endogenous progenitors
(BrdU\textsuperscript{+}EGFP\textsuperscript{-}Nestin\textsuperscript{+}),
exogenous neuroblasts
(EGFP\textsuperscript{+}BrdU\textsuperscript{-}Nestin\textsuperscript{+}Tuj1\textsuperscript{+}),
endogenous neuroblasts
(BrdU\textsuperscript{+}EGFP\textsuperscript{-}Nestin\textsuperscript{+}Tuj1\textsuperscript{+}),
exogenous early neurons
(EGFP\textsuperscript{+}BrdU\textsuperscript{-}Tuj1\textsuperscript{+}Nestin\textsuperscript{-}),
endogenous early neurons
(BrdU\textsuperscript{+}EGFP\textsuperscript{-}Tuj1\textsuperscript{+}Nestin\textsuperscript{-}),
exogenous neurons
(EGFP\textsuperscript{+}BrdU\textsuperscript{-}NeuN\textsuperscript{+}),
and endogenous neurons
(BrdU\textsuperscript{+}EGFP\textsuperscript{-}NeuN\textsuperscript{+}).
Cells were quantified within the injury and normalized to the implant or
injury (empty control) area. Nine tissues evenly distributed throughout
the rostrocaudal axis of the injury were averaged for each animal.

\emph{Axon Density and Myelination:} Semi-automated counting software,
previously described by McCreedy et al.(McCreedy et al., 2016), was used
to quantify axons and the co-localization of myelin with axons in
transverse sections Briefly, the software was calibrated using manual
NF-200\textsuperscript{+} (axons),
NF-200\textsuperscript{+}MBP\textsuperscript{+} (myelinated axons), and
NF-200\textsuperscript{+}MBP\textsuperscript{+}P0\textsuperscript{+}(Schwann
cell myelinated axons) counts from a subset of transverse 10X images
taken from different animals and regions of the implant. The software
then used a series of Hessian filters and threshold functions within the
bridge region to reduce noise for selected NF-200, MBP, and P0 images
(McCreedy et al., 2016). The software then output total axon counts, as
well as the myelinated axon counts based on the curvilinear MBP
co-localizing with axons with or without P0 co-localization; image
acquisition and analysis was performed by investigators blinded to
treatment condition. ImageJ (NIH, Bethesda, MD, USA) was used to analyze
all other fluorescent images and define the bridge area. Cells were
quantified within the injury and normalized to the implant or injury
(empty control) area. Nine tissues evenly distributed throughout the
rostrocaudal axis of the injury were averaged for each animal.

\emph{Immune Cell Density and Glial Scar Thickness:} Cells positive for
F4/80\textsuperscript{+}(macrophages) and
F4/80\textsuperscript{+}arginase\textsuperscript{+}(M2 macrophages)
containing Hoechst\textsuperscript{+} nuclei were counted manually by
two blinded researchers to quantify macrophage infiltration.
Additionally, researchers were blinded to condition during image
acquisition and quantification to eliminate bias for all histological
analyses. Cells were quantified within the injury and normalized to the
implant or injury (empty control) area. Nine tissues evenly distributed
throughout the rostrocaudal axis of the injury were averaged for each
animal. Glial scar thickness was quantified via GFAP\textsuperscript{+}
staining at the injury as previously reported (Dumont et al., 2019).
Reactive astrocytes strongly express GFAP when forming the glial scar
post-injury. Two manual thickness measurements were averaged within each
tissue, while nine tissues evenly distributed throughout the
rostrocaudal axis of the injury were averaged for each animal.

Locomotor assessment

Ladder beam was used to evaluate mouse locomotor and coordination over a
12 week period post-SCI, as previously described for all conditions
(N=72, 12 per condition) (B. J. Cummings, Engesser-Cesar, Cadena, \&
Anderson, 2007). Each of the 50 rungs were numbered and equally spaced
along the length of the beam with a dark enclosure containing bedding at
the far end of the apparatus. An HD Handycam camcorder (Sony, Tokyo,
Japan) was used to record mouse ambulation across ladderbeam. Mice were
acclimated to the ladder beam over three sessions in the two weeks
preceding the initial surgery. Baseline scores were determined to
separate animals into equal groups (tubes or gelfoam) prior to the
initial surgery. Mice were evaluated on the ladder beam every 2 weeks
over the course of the experiment. The 2 week evaluation occurred prior
to the second surgery in which the mice received spinal progenitors or
vehicle (PBS) injections. This was to allow researchers to separate
animals in equal groups prior to the addition of cell transplants.
Observation and ladder beam scoring were performed by two blinded
observers for 3 trials per animal. Animals were scored by average
forepaw full placement on the ladder beam during the task.

Statistics

Data normality was assessed using a Shapiro-Wilk normality test with an
\selectlanguage{greek}α \selectlanguage{english}value of 0.05, which determined parametric statistical tests were
appropriate for our analyses. Multiple comparison pairs were analyzed
using a one-way or two-way ANOVA with Tukey post-hoc test. All
statistics test significance using an \selectlanguage{greek}α \selectlanguage{english}value of 0.05. For all graphs, *
denotes p \textless{} 0.05, ** denotes p \textless{} 0.01, *** denotes p
\textless{} 0.005, and **** denotes p \textless{} 0.001, unless
otherwise specified in the figure caption. All values are reported as
mean +/- standard error of the mean (SEM). Prism 7 (GraphPad Software,
La Jolla, CA) software was used for all data analysis.

\begin{enumerate}
\tightlist
\item
  Results
\item
  Lentiviral expression is sustained in PEG hydrogel tubes over 12 weeks
  A cohort of mice received a C5 lateral hemisection injury followed by
  immediate implantation of PEG hydrogel tubes with a subsequent
  injection of firefly luciferase (FLuc) encoding lentivirus directly
  into the tube. A subset of these mice did not receive lentiviral
  injections to serve as a background control. Bioluminescent signal
  from the FLuc reporter gene was measured over 12 weeks
  post-implantation to assess lentiviral expression. Lentiviral
  expression of FLuc was observed in the injured area (\textbf{Fig. 1A}
  ). The average bioluminescent signal was assessed across 12 weeks for
  mice receiving lentivirus and compared to those that did not receive
  lentivirus (\textbf{Fig. 1B} ). Increased FLuc expression was observed
  across the 12 weeks in lentivirus-laden PEG hydrogel tubes, with a
  significant increase observed at weeks 4, 6, and 8. These results
  matched lentiviral-mediated IL-10 presence at the injury demonstrated
  with immunohistochemistry (\textbf{Fig. S1} ). IL-10 was strongly
  observed at 4 weeks post-injury in the tubes + IL-10 encoding
  lentivirus condition (\textbf{Fig. S1A} ), but there was a noticeable
  drop by 12 weeks (\textbf{Fig. S1B} ) that coincides with the decrease
  in signal measured via IVIS (\textbf{Fig. 1B} ). Conversely, no IL-10
  was observed at 4 (\textbf{Fig. S1C} ) or 12 weeks (\textbf{Fig. S1D}
  ) in the tubes alone condition.
\item
  Macrophage infiltration is not significantly changed among
  experimental groups This study included six total conditions tested, 3
  without E14 spinal progenitors (SCI, tubes, tubes + IL-10) and 3 with
  E14 spinal progenitors (E14 only, E14 + tubes, E14 + tubes + IL-10).
  Each individual tube has an approximate inner diameter of 250 \selectlanguage{greek}µ\selectlanguage{english}m and
  outer diameter of 600 \selectlanguage{greek}µ\selectlanguage{english}m (\textbf{Fig. S2A} ). Upon implantation, the
  tubes integrate with surrounding tissue for two weeks prior to
  transplanting E14 spinal progenitors (\textbf{Fig. S2B} ). Four weeks
  following implantation tube lumens are visible when evaluating tissue
  sections transversely (\textbf{Fig. S2C} ), and immune cell
  infiltration was quantified through F4/80 staining for macrophages
  (\textbf{Fig. 2A-F} ). Identified F4/80+ macrophages were further
  classified through arginase staining where
  F4/80\textsuperscript{+}arginase\textsuperscript{+} macrophages were a
  pro-regenerative M2-like phenotype. No significant differences were
  observed in total (\textbf{Fig. 2G} ) or M2-specific (\textbf{Fig. 2H}
  ) macrophage density observed across all conditions at 4 weeks. M2
  classified macrophages were then reported as a percent of total
  macrophages, and there were no significant differences observed in M2
  fraction across all conditions (\textbf{Fig. 2I} ).
\item
  Implanted hydrogel tubes attenuate glial scar thickness
\end{enumerate}

Four weeks post-tube implantation, a glial scar was observed in all
conditions. The scar was quantified by measuring the thickness of the
GFAP\textsuperscript{+} astrocyte layer at the injury margin in
transversely cross-section tissue (\textbf{Fig. S3A} ). Formation of the
scar is important for remediating the integrity of the blood-brain
barrier, but it can also act as a physical barrier limiting axon
elongation into the injury site. Attenuation, rather than total
ablation, of the glial scar can improve functional recovery. In all mice
that received hydrogel tube implants (tubes, tubes + IL-10, E14 + tubes,
E14 + tubes + IL-10) a significant decrease in average scar thickness at
the medial interface was observed compared to both SCI only and E14
spinal progenitors only (\textbf{Fig. S3B} ).

E14 progenitor survival increases when combined with IL-10 encoding
lentivirus loaded hydrogel tubes

Following hemisection injury, five tubes either loaded with IL-10
encoding lentivirus or empty, were implanted into the lesion.
Additionally, an injury only condition with no tube implantation served
as a control. These mice recovered for two weeks, at which point a
second surgery was performed where mice were injected with two doses of
150,000 EGFP\textsuperscript{+} E14 spinal progenitors or PBS as a
control, delivered into the integrated tubes (\textbf{Fig. 3A} ). A
subset of mice (n = 4) were sacrificed after another two weeks (4 weeks
post-injury) to assess the short-term survival of transplanted E14
spinal progenitors. EGFP\textsuperscript{+} cells were observed in all
conditions receiving transplants (\textbf{Fig. 3B-D} ). Survival was
assessed as both a density normalized to the injury area and as a
percent of transplanted cells. E14 spinal progenitor transplantation
alone resulted in the lowest density (20.3 \selectlanguage{ngerman}± 7.5
cells/mm\textsuperscript{2}) compared to E14 spinal progenitor
transplants with tubes (44.1 ± 3.7 cells/mm\textsuperscript{2}) and
tubes with IL-10 encoding lentivirus (67.2 ± 19
cells/mm\textsuperscript{2}) (\textbf{Fig. 3E} ). Similarly, mice
receiving E14 spinal progenitor transplants with no additional
treatments resulted in 0.7\% of cells surviving, and those with
transplants directly into tubes had 4.3\% survival. When the E14 spinal
progenitors were transplanted into IL-10 lentivirus loaded tubes, cell
survival increased to 8.1\%, an 11.5-fold increase over the E14 spinal
progenitor alone condition (\textbf{Fig. 3F} ).

Implanted tubes support exogenous-sourced mature neuron formation by 12
weeks

Surviving transplanted E14 spinal progenitor commitment to a neuronal
lineage was assessed at both 4 and 12 weeks post-injury. At 4 weeks
post-injury (\textbf{Fig. S4A-D} ), no significant differences were
observed in exogenous progenitor
(EGFP\textsuperscript{+}Nestin\textsuperscript{+}) or neuroblast
(EGFP\textsuperscript{+}Nestin\textsuperscript{+}Tuj1\textsuperscript{+})
densities (\textbf{Fig. S4E-F} ), but there was a significant increase
in exogenous-sourced immature neurons
(EGFP\textsuperscript{+}Nestin\textsuperscript{-}Tuj1\textsuperscript{+})
in mice receiving E14 spinal progenitor transplants into tubes
(\textbf{Fig. S4G} ). No significant differences were observed in mature
neuron formation (EGFP\textsuperscript{+}NeuN\textsuperscript{+}) at
this early time point (\textbf{Fig. S4H} ). Similarly, endogenous
progenitors were quantified at 4 weeks post-injury. To label endogenous
progenitors, BrdU was injected for 7 days post-injury, followed by 7
days with no injections to allow sufficient clearance of BrdU prior to
exogenous E14 spinal progenitor transplantation. Endogenous progenitor
(BrdU\textsuperscript{+}Nestin\textsuperscript{+}) (\textbf{Fig. S5A} ),
neuroblast
(BrdU\textsuperscript{+}Nestin\textsuperscript{+}Tuj1\textsuperscript{+})
(\textbf{Fig. S5B} ), immature neuron
(BrdU\textsuperscript{+}Nestin\textsuperscript{-}Tuj1\textsuperscript{+})
(\textbf{Fig. S5C} ), and mature neuron
(BrdU\textsuperscript{+}NeuN\textsuperscript{+}) (\textbf{Fig. S5D} )
densities had no significant differences across all conditions
(\textbf{Fig. S5E-H} ).

Neuron formation at 12 weeks post-injury was assessed from both
exogenous and endogenous progenitor cells (\textbf{Fig. 4A} ). For the
conditions receiving transplanted EGFP\textsuperscript{+} E14 spinal
progenitors, neurons were identified as
EGFP\textsuperscript{+}NeuN\textsuperscript{+} cells and quantified as a
density normalized to the injury cross section and as a percent of total
EGFP\textsuperscript{+} positive cells. No significant differences were
observed in EGFP\textsuperscript{+}NeuN\textsuperscript{+} densities
between E14 spinal progenitors only (0.12 ± 0.1
cells/mm\textsuperscript{2}), tubes with E14 spinal progenitors (1.7 ±
0.4 cells/mm\textsuperscript{2}), and IL-10 lentivirus loaded tubes with
E14 spinal progenitors (1.9 ± 0.8 cells/mm\textsuperscript{2})
(\textbf{Fig. 4B} ). When expressed as a percent of the total
EGFP\textsuperscript{+} cells mice receiving transplanted E14 spinal
progenitors only had on average 12.8 ± 10\% of their transplants form
neurons. This was significantly lower than the average percentages for
mice receiving E14 spinal progenitors with blank tubes (38.9 ± 5.1\%)
and with IL-10 lentivirus loaded tubes (44.3 ± 1.7\%) (\textbf{Fig. 4C}
). Endogenous-derived neurons were assessed as a density, and there were
no significant differences across any of the conditions tested
(\textbf{Fig. 4D} ).

Axon elongation increases with IL-10 lentivirus-laden hydrogel tubes and
E14 spinal progenitors

Transverse cross sections of isolated spinal cords were evaluated to
assess axon elongation in the lesion space at 12 weeks post-injury.
Facilitating and encouraging axon elongation through the injury is
crucial for any SCI treatment. Axon counts quantified using NF-200
staining were normalized to the injury area (\textbf{Fig. S6} ), shown
as the solid white border (\textbf{Fig. 5A-F} ) to give an axon density
for each condition. Assessed samples were evenly spaced across rostral,
middle, and caudal regions of the injuries and averaged as no
significant differences in axon density were observed between individual
regions within each condition. All conditions exhibited axon elongation
into the injury (\textbf{Fig. 5A-F} ) at 12 weeks. Infiltrating axons
were typically observed growing in fascicle-like bundles (\textbf{Fig.
5G} ) or as individual, elongated structures (\textbf{Fig. 5H} ). Mice
receiving IL-10 lentivirus loaded tubes with E14 spinal progenitors had
significantly greater axon formation (490 ± 41
axons/mm\textsuperscript{2}) compared to all other conditions. No other
significant differences were observed between conditions at this time
point (\textbf{Fig. 5I} ).

Axon remyelination increases with E14 spinal progenitor delivery

Elongated axons were further characterized 12 weeks post-injury by the
extent and source of their myelination to indicate functional axonal
regrowth. Myelination was quantified as a density of
NF-200\textsuperscript{+}MBP\textsuperscript{+} axons normalized to the
injury area of a transverse cross section (\textbf{Fig. S6} ). Mice not
receiving tubes (SCI only and E14 only) exhibited a decreased
cross-sectional area compared to the other conditions, most likely due
to tissue collapse with no support from the implanted biomaterial. All
conditions had myelinated axons present in the injury (\textbf{Fig.
6A-F} ). Mice receiving IL-10 lentivirus loaded tubes with E14 spinal
progenitors had 83 ± 13 myelinated axons/mm\textsuperscript{2} which was
a significantly greater density compared to the injury only control (24
± 6 myelinated axons/mm\textsuperscript{2}), but they were not different
compared to any of the other experimental conditions. Similarly, the E14
spinal progenitor only condition had a significant increase in myelin
density (105 ± 12 myelinated axons/mm\textsuperscript{2}) compared to
the injury only but not any of the other conditions (\textbf{Fig. 6G} ).
It is possible that the density calculations for the E14 spinal
progenitor only condition are artificially inflated as a result of lower
ipsilateral tissue area to normalize to, which might be attributable to
tissue collapse with no biomaterial. Ipsilateral tissue in the E14
spinal progenitor only condition was approximately 67\%, 70\%, and 39\%
lower by comparison to tubes + IL-10, E14 + tubes, and E14 + tubes +
IL-10, respectively (\textbf{Fig. S6} ). The percent of regenerated
axons was determined by normalizing the number of
NF-200\textsuperscript{+}MBP\textsuperscript{+} axons to the total
number of NF-200\textsuperscript{+} axons and reported as a percent.
Mice receiving E14 spinal progenitors had a significant increase in
myelination percent compared to all other conditions tested, with 42 ±
3\% of their axons having myelin. No other significant differences were
observed for myelination percent (\textbf{Fig. 6H} ).

Myelination source was determined where axons expressing
MBP\textsuperscript{+}P0\textsuperscript{+} were identified as Schwann
cell-derived myelinated axons and
MBP\textsuperscript{+}P0\textsuperscript{-} were assumed to be
oligodendrocyte-derived. Both longitudinally and transversely myelinated
axons were observed in the tissue (\textbf{Fig. S7A} ). Mice receiving
E14 spinal progenitors alone and delivered into tubes had significant
increases in Schwann cell-derived myelinated axon densities compared to
the injury only control (\textbf{Fig. S7B} ). The percent of Schwann
cell-derived myelinated axons was determined by normalizing the
NF-200\textsuperscript{+}MBP\textsuperscript{+}P0\textsuperscript{+}axons
to all NF-200\textsuperscript{+}MBP\textsuperscript{+} axons and
reported as a percent. The E14 spinal progenitor only condition had a
significant increase in Schwann-cell derived myelin compared to all
other conditions with 31 ± 4\% of axons coming from Schwann cells
(\textbf{Fig. S7C} ). No other significant differences were observed.

IL-10 lentivirus loaded tubes with E14 spinal progenitors increase rate
of functional recovery

The horizontal ladder beam test assessed the functional recovery of the
left forelimb of mice at 2, 4, 8, and 12 weeks post-injury. An average
left paw placement score out of 50 possible rungs was assessed at each
time point for each condition (\textbf{Fig. 7} ). At 4 weeks post-injury
the mice receiving IL-10 lentivirus loaded tubes and E14 spinal
progenitors exhibited increased function compared to the injury only
control. By 8 weeks, any combination of condition where the mice had
tubes implanted demonstrated increased function, and at 12 weeks, all
conditions had significantly more successful placements compared to the
injury only control.

Discussion

Biomaterial-based strategies are increasingly relevant in SCI tissue
engineering with numerous clinical trials investigating scaffold-based
therapies (Tsintou, Dalamagkas, \& Seifalian, 2015). As a singular
therapy, biomaterial implants can promote endogenous repair mechanisms
and modulate immune responses, but combination therapies involving
biomaterials, stem cells, and therapeutic factors are more effective as
they can have therapeutic synergism, improving the potential to
facilitate repair (Ashammakhi et al., 2019). In this work, we modified
our PEG hydrogel tube platform to incorporate lentiviral-mediated
anti-inflammatory overexpression while also serving as a soft substrate
for delayed E14 spinal progenitor transplantation. Alone, the PEG tubes
are soft enough to conform to injury anatomy while maintaining an
aligned structure for axonal elongation guidance in an acute SCI (Dumont
et al., 2019). Once implanted, the tubes create a more pro-regenerative
environment, serving as a privileged injection site for E14 spinal
progenitors 2 weeks after injury. Our previous work showed that
transplanted cell survival is enhanced when injected into the tubes,
compared to direct transplantation into the injury, demonstrating the
feasibility of using this as a cell-biomaterial system for SCI
(Ciciriello et al., 2020). While survival did improve with the
integrated tubes, the stem cell-influenced results of that study were
minimized as functional and histological assessments occurred only 2
weeks after cell transplantation (4 weeks after injury). Axon elongation
and myelination were comparable between the tubes only and tubes with
E14 spinal progenitor conditions indicating the tubes played a bigger
role at this early time point. For the work presented here, we
hypothesized that adding lentivirus encoding for the anti-inflammatory
cytokine IL-10 to our implanted tubes would allow for active immune
modulation to complement the immunomodulatory properties of the
integrated tubes, further remediating the cell transplantation site.
Herein we developed a combinatorial therapy that both improved cell
transplantation survival while also exhibiting synergistic regenerative
gains at 12 weeks post-injury in an acute cervical SCI.

Lentiviral-mediated therapies present an attractive option for local
therapeutic delivery as the virus genome integrates into the host genome
and gene-of-interest expression is sustained over long periods of time
(Sakuma, Barry, \& Ikeda, 2012). Moreover, lentivirus can easily be
loaded onto a biomaterial scaffold that can be implanted directly at an
injury site (Avilés \& Shea, 2011; Shin, Salvay, \& Shea, 2010). For
this work, we added lentivirus encoding the gene for the
anti-inflammatory cytokine IL-10 to our hydrogel PEG tubes once
implanted in an acute cervical SCI model. Previous reports have
similarly used lentiviral delivery of anti-inflammatory cytokines from
multi-channel poly(lactide-\emph{co} -glycolide) (PLG) bridges in SCI
models (Park, Decker, Margul, et al., 2018; Park, Decker, Smith, et al.,
2018; Dominique R. Smith et al., 2020; D. R. Smith et al., 2019). In
these studies, rigid PLG bridges were loaded with multiple rounds of
virus \emph{ex vivo} prior to implantation. Bridges loaded with IL-10
lentivirus significantly increased M2 macrophage phenotype density in
the injured region compared to control conditions demonstrating the
efficacy of lentiviral-mediated IL-10 overexpression in facilitating a
shift towards a more pro-regenerative microenvironment. For our study,
we injected IL-10 encoding lentivirus directly into our tubes
immediately after implantation to modulate the immune response after
injury and create a more hospitable microenvironment for transplanting
E14 spinal progenitors two weeks later. Macrophage phenotype was
assessed four weeks post-injury, and no significant differences across
all conditions were observed for total macrophage density or M2
macrophage density and fraction. It should be noted that mice receiving
IL-10 lentivirus injections did have a trending increase in M2 density
and fraction. Other work with IL-10 lentivirus biomaterials has shown
that the elevated M2 concentrations begin to resolve by 4 weeks
post-injury, indicating an earlier assessment point for our work would
likely show a significant increase in M2 density (Park, Decker, Margul,
et al., 2018). Additionally, our viral load was lower by comparison to
the dose used with rigid PLG bridges. Park et al. loaded their bridges
with 2 \selectlanguage{greek}µ\selectlanguage{english}L of 2E9 IU/mL of virus and incubating for 2 minutes to allow it
to absorb. This process was repeated three additional times for four
total doses (Park, Decker, Margul, et al., 2018; Park, Decker, Smith, et
al., 2018). By comparison, our viral loading conditions were somewhat
restricted, as the tubes are dried prior to implantation for handling
ease, and upon implantation, they swell to fit an injury. As a result,
we could not load the tubes with virus \emph{ex vivo} prior to
implanting, potentially limiting the total viral load absorbed by the
bridge. Loading concentrations were restricted to a single 2 \selectlanguage{greek}µ\selectlanguage{english}L dose of
4E9 IU/mL, half of the total dose used by Park et al. Moving forward,
viral loading concentrations and conditions will need to be optimized in
order to observe a greater anti-inflammatory effect when used in
conjunction with stem cell transplants. Furthermore, affinity peptides
for the lentivirus can be incorporated into the hydrogel tubes to
improve their retention and localization at the injury (Skoumal,
Seidlits, Shin, \& Shea, 2016).

Stem cell transplantation into the injured spinal cord is a treatment
option with high potential but has clinically been met with difficulty
in cell sourcing and scale-up (Anderson, Piltti, Hooshmand, Nishi, \&
Cummings, 2017; Levi et al., 2018; Temple \& Studer, 2017). In research,
another notable barrier is low survival post-transplantation, which can
largely be attributed to a cytotoxic transplantation site at the injury.
As the post-injury immune response clears out dead cells and debris, it
also targets transplanted cells, limiting their durability and potential
to promote repair (Pereira, Marote, Salgado, \& Silva, 2019). Improving
exogenous cell transplant survival would allow spinal progenitors to
integrate with endogenous circuitry (Lien, Tuszynski, \& Lu, 2019; Paul
Lu et al., 2017; Paul Lu et al., 2019) and remediate extracellular space
(Hawryluk et al., 2012; P. Lu et al., 2003). Biomaterial strategies have
long proven to be effective vehicles for transplanting stem cells to
central nervous system (CNS) injuries as they can be functionalized for
cell adhesion and offer a protective role against transplantation
conditions (Higuchi et al., 2019; Katoh, Yokota, \& Fehlings, 2019).
Bulk biomaterial implants, like porous PLG bridges (Dumont et al., 2018)
or 3D printed scaffolds (Koffler et al., 2019), can be cultured \emph{ex
vivo} with stem cells and implanted into the injury. Rigid implants with
a defined architecture also provide directional cues for axon elongation
once in the injury. Alternatively, cells can be suspended in synthetic
(Marquardt et al., 2020) or natural (Cerqueira et al., 2018; Mothe et
al., 2013) hydrogels that are soft enough to be injected and form a
biomimetic extracellular matrix once in the injury. Soft hydrogel
implants are advantageous as they better mimic the mechanical properties
of the surrounding native tissue and can swell to fit unique injury
shapes, advancing their translatability.

Unfortunately, both \emph{ex vivo} culturing and hydrogel suspension
techniques for cell transplantation offer little control over delivery
time relative to biomaterial implantation as cell transplants are
dependent on the biomaterial as a carrier. The temporal dependence on
the biomaterial limits the opportunity for addressing regenerative
barriers, such as inflammation, prior to cell transplantation.
Alternatively, stem cells can be directly injected into a previously
implanted biomaterial that has had time to integrate and modulate the
immune response. In our previous report we saw approximately 4.3\% of
transplanted E14 spinal progenitors survive two weeks after
transplantation when injected into integrated PEG tubes (Ciciriello et
al., 2020). For the present study, we similarly delayed E14 spinal
progenitor transplantation for 2 weeks, but we additionally incorporated
IL-10 encoding lentivirus to our PEG tubes at implantation. A buffer
period between 1 and 2 weeks is common in SCI-cell transplant strategies
(Brian J. Cummings et al., 2005; F\selectlanguage{ngerman}ührmann et al., 2016; Mothe et al.,
2013), and we chose 2 weeks for delaying E14 spinal progenitor delivery
to coincide with IL-10 lentivirus-attributable M2 polarization shown by
others (Park, Decker, Margul, et al., 2018). Transplant survival was
assessed after another 2 weeks, 4 weeks after tube implantation, and
8.1\% of E14 spinal progenitors transplanted into IL-10 lentivirus
loaded tubes survived, an 11.5-fold difference compared to the
transplantation only control in this study. The 8.1\% survival for the
E14 spinal progenitors presented here is a significant increase in
transplant engraftment compared to what has previously been demonstrated
in immunocompetent models (Tejeda, Ciciriello, \& Dumont, 2021).
Combining the independent immunomodulatory properties of the tubes with
lentiviral-mediated overexpression of IL-10 resulted in synergistic
gains for the transplants, significantly improving their survival.

Exogenous NSC transplants can facilitate \emph{de novo} neurogenesis,
improving regenerative potential post-SCI (Ogawa et al., 2002). We
assessed neurogenesis from surviving exogenous E14 spinal progenitors at
both 4 and 12 weeks post-injury (2 and 10 weeks post-transplantation,
respectively) using NeuN staining, a marker for mature neuron formation.
At 4 weeks post-injury, very little staining was observed for
EGFP\textsuperscript{+}NeuN\textsuperscript{+} cells, indicating low
levels of neurogenesis from exogenous cells, however an observable
increase in neuronal lineage commitment was observed with Tuj staining
in EGFP\textsuperscript{+} cells transplanted with tubes or IL-10
lentivirus-laden tubes. Ogawa et al., transplanted E14.5 spinal
progenitors 9 days after a contusive injury, and they observed
neurogenesis beginning at 5 weeks post-transplantation, albeit in a rat
model (Ogawa et al., 2002), so it is unsurprising that we saw few mature
neurons at our earlier time point. By 12 weeks post-injury, we did
observe an increase in NeuN staining densities across conditions with a
significant increase in the percentage of EGFP\textsuperscript{+} cells
co-staining for NeuN\textsuperscript{+} cells observed in conditions
that received tube implants.

In a hemisection injury model, as used in the present study, all tissue
on the ipsilateral side of the injury is completely removed, thus any
axons identified histologically are attributable to post-injury
regrowth. Increasing axon density and elongation is an important factor
when designing a treatment for SCI as this will bridge the healthy
tissue rostral and caudal to the injury. Additionally, remyelination of
these new axons is important for signal propagation along axons in the
CNS, a process that is largely attributed to the myelin sheath. We
observed a significantly higher axon density at the injury in mice
receiving IL-10 lentivirus loaded tubes with E14 spinal progenitors
compared to all other experimental conditions. With that in mind, it
should be noted that sparing, plasticity, axonal growth, and
remyelination likely occurred early after implantation and
transplantation. It is suspected that by 12 weeks post-injury the
anti-inflammatory effects of the tubes and E14 spinal progenitors have
resolved. Benefits from the tubes and E14 spinal progenitors are known
to have an early impact as we previously saw in mice with E14 spinal
progenitors alone, tubes alone, and tubes with E14 spinal progenitors
had increased axonal densities and myelinated axon densities at 4 weeks
post-injury (Ciciriello et al., 2020). In this study, mice receiving E14
spinal progenitors with IL-10 lentivirus-laden tubes possibly had a
longer therapeutic regime compared to all other conditions, resulting in
the significant increase in axon density. Directly comparing densities
between conditions can be misleading if the overall context is not
considered, and this is more apparent with the myelin data. The mice
that received E14 spinal progenitors only had an average injury area of
1.10 ± 0.5 mm\textsuperscript{2} compared to the expected area of 1.75
mm\textsuperscript{2} based on the cross-sectional area of resected
tissue. These mice may have experienced some tissue collapse as there
was no biomaterial to fill the lesion, effectively lowering the area to
which cell counts are normalized and artificially inflating densities.
Axonal remyelination results in the E14 spinal progenitor only condition
indicate they had significantly improved remyelination compared to other
conditions, but this might be attributable to a lower cross-sectional
area resulting in a higher apparent density. This discrepancy is also
highlighted when comparing axon myelination to functional data. The E14
only condition had the most myelin of all the conditions, but it was the
last treatment group to reach significantly more successful placements
compared to the SCI only group. This disparity between histological and
functional data raises a point of disconnect that could possibly be
attributable to improper synapse formation or poor myelin quality, which
could be investigated to better understand this relationship.

Conclusion Stem cell transplantations are a promising therapy across
tissue engineering, especially in CNS damage and disease. Unfortunately,
therapeutic effect is minimized by poor survival resulting from poor
flexibility in transplantation strategies, subjecting the cells to
inhospitable conditions upon injection. Biomaterials and therapeutic
factors can facilitate improved conditions, but they are often delivered
simultaneously with cell transplants, limiting the perceived
immunomodulatory effect on the cells. In this study, our hydrogel tube
system affords temporal independence between cell transplantation and
biomaterial implantation, an advantage absent in many other
cell-biomaterial strategies. In addition to temporal independence, we
enhanced the passive immunomodulatory properties of our hydrogel tube
system with active immunomodulation via IL-10 encoding lentivirus
release from the tubes. In a 2 week course of combined passive and
active immunomodulation prior to transplantation, the IL-10 lentivirus
loaded tubes effectively created a privileged cell transplantation site
for E14 spinal progenitors. To that end, we observed a significant
increase in transplant survival and axon elongation in addition to a
more rapid functional recovery. Altogether, this therapy synergizes the
therapeutic benefits of biomaterials, stem cells, and therapeutic factor
delivery, representing a combinatorial approach capable of addressing
several of the challenges that limit recovery post-SCI.

\textbf{Supporting Information:} Additional supporting information may
be found in the online version of this article.

\textbf{Figure S1:} IL-10 expression at 4 and 12 weeks

\textbf{Figure S2:} PEG implants facilitate tissue ingrowth

\textbf{Figure S3:} Glial scarring is decreased in mice receiving tubes

\textbf{Figure S4:} Exogenous progenitor characterization at 4 weeks

\textbf{Figure S5:} Endogenous progenitor characterization at 4 weeks

\textbf{Figure S6:} Ipsilateral tissue area is lower in conditions not
receiving PEG implants

\textbf{Figure S7:} Remyelinated axons have Schwann cell derived myelin

\textbf{Data Availability:} The datasets generated during and/or
analyzed during the present study are available from the corresponding
author upon reasonable request.

\textbf{Conflict of Interest:} The authors declare no conflicts of
interest.

\textbf{Author Contributions:} Study design -- AJC, DRS, LDS, CMD; Data
collection -- AJC, DRS, MKM, CMD; Data analysis -- AJC, DRS, MKM, SJB,
CMD; Manuscript drafting -- AJC, CMD; Revising and approving final
manuscript -- AJC, DRS, MKM, SJB, LDS, CMD

\textbf{References}

Abdellatif, A. A., Pelt, J. L., Benton, R. L., Howard, R. M., Tsoulfas,
P., Ping, P., . . . Whittemore, S. R. (2006). Gene delivery to the
spinal cord: comparison between lentiviral, adenoviral, and retroviral
vector delivery systems. \emph{Journal of Neuroscience Research, 84}
(3), 553-567. doi:10.1002/jnr.20968

Alizadeh, A., Dyck, S. M., \& Karimi-Abdolrezaee, S. (2019). Traumatic
Spinal Cord Injury: An Overview of Pathophysiology, Models and Acute
Injury Mechanisms. \emph{Frontiers in Neurology, 10} (282).
doi:10.3389/fneur.2019.00282

Anderson, A. J., Piltti, K. M., Hooshmand, M. J., Nishi, R. A., \&
Cummings, B. J. (2017). Preclinical Efficacy Failure of Human
CNS-Derived Stem Cells for Use in the Pathway Study of Cervical Spinal
Cord Injury. \emph{Stem Cell Reports, 8} (2), 249-263.
doi:10.1016/j.stemcr.2016.12.018

Arce, F., Breckpot, K., Collins, M., \& Escors, D. (2011). Targeting
lentiviral vectors for cancer immunotherapy. \emph{Current cancer
therapy reviews, 7} (4), 248-260. doi:10.2174/157339411797642605

Ashammakhi, N., Kim, H.-J., Ehsanipour, A., Bierman, R. D., Kaarela, O.,
Xue, C., . . . Seidlits, S. K. (2019). Regenerative Therapies for Spinal
Cord Injury. \emph{Tissue Engineering Part B: Reviews, 25} (6), 471-491.
doi:10.1089/ten.teb.2019.0182

Assunção-Silva, R. C., Gomes, E. D., Sousa, N., Silva, N. A., \&
Salgado, A. J. (2015). Hydrogels and Cell Based Therapies in Spinal Cord
Injury Regeneration. \emph{Stem cells international, 2015} ,
948040-948040. doi:10.1155/2015/948040

Avilés, M. O., \& Shea, L. D. (2011). Hydrogels to modulate lentivirus
delivery in vivo from microporous tissue engineering
scaffolds.\emph{Drug delivery and translational research, 1} (1),
91-101. doi:10.1007/s13346-010-0011-1

Boehler, R. M., Kuo, R., Shin, S., Goodman, A. G., Pilecki, M. A.,
Leonard, J. N., \& Shea, L. D. (2014). Lentivirus delivery of IL-10 to
promote and sustain macrophage polarization towards an anti-inflammatory
phenotype. \emph{Biotechnology and Bioengineering, 111} (6), 1210-1221.
doi:10.1002/bit.25175

Bradbury, E. J., \& Burnside, E. R. (2019). Moving beyond the glial scar
for spinal cord repair. \emph{Nature Communications, 10} (1), 3879.
doi:10.1038/s41467-019-11707-7

Busch, S. A., Horn, K. P., Silver, D. J., \& Silver, J. (2009).
Overcoming macrophage-mediated axonal dieback following CNS
injury.\emph{Journal of Neuroscience, 29} (32), 9967-9976.
doi:10.1523/JNEUROSCI.1151-09.2009

Cai, L., Dewi, R. E., \& Heilshorn, S. C. (2015). Injectable Hydrogels
with In Situ Double Network Formation Enhance Retention of Transplanted
Stem Cells. \emph{Advanced functional materials, 25} (9), 1344-1351.
doi:10.1002/adfm.201403631

Cerqueira, S. R., Lee, Y.-S., Cornelison, R. C., Mertz, M. W., Wachs, R.
A., Schmidt, C. E., \& Bunge, M. B. (2018). Decellularized peripheral
nerve supports Schwann cell transplants and axon growth following spinal
cord injury. \emph{Biomaterials, 177} , 176-185.
doi:10.1016/j.biomaterials.2018.05.049

Ceto, S., Sekiguchi, K. J., Takashima, Y., Nimmerjahn, A., \& Tuszynski,
M. H. (2020). Neural Stem Cell Grafts Form Extensive Synaptic Networks
that Integrate with Host Circuits after Spinal Cord Injury. \emph{Cell
stem cell} . doi:10.1016/j.stem.2020.07.007

Chen, J., Bernreuther, C., Dihné, M., \& Schachner, M. (2005). Cell
Adhesion Molecule L1--Transfected Embryonic Stem Cells with Enhanced
Survival Support Regrowth of Corticospinal Tract Axons in Mice after
Spinal Cord Injury. \emph{Journal of neurotrauma, 22} (8), 896-906.
doi:10.1089/neu.2005.22.896

Cheng, Z., Zhu, W., Cao, K., Wu, F., Li, J., Wang, G., . . . He, X.
(2016). Anti-Inflammatory Mechanism of Neural Stem Cell Transplantation
in Spinal Cord Injury. \emph{International journal of molecular
sciences, 17} (9), 1380. doi:10.3390/ijms17091380

Chou, F.-C., \& Sytwu, H.-K. (2009). Overexpression of thioredoxin in
islets transduced by a lentiviral vector prolongs graft survival in
autoimmune diabetic NOD mice. \emph{Journal of Biomedical Science, 16}
(1), 71-71. doi:10.1186/1423-0127-16-71

Ciciriello, A. J., Smith, D. R., Munsell, M. K., Boyd, S. J., Shea, L.
D., \& Dumont, C. M. (2020). Acute Implantation of Aligned Hydrogel
Tubes Supports Delayed Spinal Progenitor Implantation. \emph{ACS
Biomaterials Science \& Engineering, 6} (10), 5771-5784.
doi:10.1021/acsbiomaterials.0c00844

Cummings, B. J., Engesser-Cesar, C., Cadena, G., \& Anderson, A. J.
(2007). Adaptation of a ladder beam walking task to assess locomotor
recovery in mice following spinal cord injury. \emph{Behav Brain Res,
177} (2), 232-241. doi:10.1016/j.bbr.2006.11.042

Cummings, B. J., Uchida, N., Tamaki, S. J., Salazar, D. L., Hooshmand,
M., Summers, R., . . . Anderson, A. J. (2005). Human neural stem cells
differentiate and promote locomotor recovery in spinal cord-injured
mice. \emph{Proceedings of the National Academy of Sciences of the
United States of America, 102} (39), 14069. doi:10.1073/pnas.0507063102

Dalamagkas, K., Tsintou, M., Seifalian, A., \& Seifalian, A. M. (2018).
Translational Regenerative Therapies for Chronic Spinal Cord
Injury.\emph{International journal of molecular sciences, 19} (6), 1776.
doi:10.3390/ijms19061776

Di Pasquale, E., Latronico, M. V. G., Jotti, G. S., \& Condorelli, G.
(2012). Lentiviral vectors and cardiovascular diseases: a genetic tool
for manipulating cardiomyocyte differentiation and function. \emph{Gene
Therapy, 19} (6), 642-648. doi:10.1038/gt.2012.19

Donnelly, D. J., \& Popovich, P. G. (2008). Inflammation and its role in
neuroprotection, axonal regeneration and functional recovery after
spinal cord injury. \emph{Experimental Neurology, 209} (2), 378-388.
doi:10.1016/j.expneurol.2007.06.009

Dulin, J. N., \& Lu, P. (2014). Bridging the injured spinal cord with
neural stem cells. \emph{Neural regeneration research, 9} (3), 229-231.
doi:10.4103/1673-5374.128212

Dumont, C. M., Carlson, M. A., Munsell, M. K., Ciciriello, A. J.,
Strnadova, K., Park, J., . . . Shea, L. D. (2019). Aligned hydrogel
tubes guide regeneration following spinal cord injury. \emph{Acta
Biomater, 86} , 312-322. doi:10.1016/j.actbio.2018.12.052

Dumont, C. M., Margul, D. J., \& Shea, L. D. (2016). Tissue Engineering
Approaches to Modulate the Inflammatory Milieu following Spinal Cord
Injury. \emph{Cells Tissues Organs, 202} (1-2), 52-66.
doi:10.1159/000446646

Dumont, C. M., Munsell, M. K., Carlson, M. A., Cummings, B. J.,
Anderson, A. J., \& Shea, L. D. (2018). Spinal Progenitor-Laden Bridges
Support Earlier Axon Regeneration Following Spinal Cord
Injury.\emph{Tissue Eng Part A, 24} (21-22), 1588-1602.
doi:10.1089/ten.TEA.2018.0053

Führmann, T., Tam, R. Y., Ballarin, B., Coles, B., Elliott Donaghue, I.,
van der Kooy, D., . . . Shoichet, M. S. (2016). Injectable hydrogel
promotes early survival of induced pluripotent stem cell-derived
oligodendrocytes and attenuates longterm teratoma formation in a spinal
cord injury model. \emph{Biomaterials, 83} , 23-36.
doi:10.1016/j.biomaterials.2015.12.032

Hackett, A. R., \& Lee, J. K. (2016). Understanding the NG2 Glial Scar
after Spinal Cord Injury. \emph{Frontiers in Neurology, 7} (199).
doi:10.3389/fneur.2016.00199

Hall, E. D. (2011). Antioxidant therapies for acute spinal cord
injury.\emph{Neurotherapeutics : the journal of the American Society for
Experimental NeuroTherapeutics, 8} (2), 152-167.
doi:10.1007/s13311-011-0026-4

Hawryluk, G. W. J., Mothe, A., Wang, J., Wang, S., Tator, C., \&
Fehlings, M. G. (2012). An in vivo characterization of trophic factor
production following neural precursor cell or bone marrow stromal cell
transplantation for spinal cord injury. \emph{Stem Cells and
Development, 21} (12), 2222-2238. doi:10.1089/scd.2011.0596

Higuchi, A., Suresh Kumar, S., Benelli, G., Ling, Q.-D., Li, H.-F.,
Alarfaj, A. A., . . . Murugan, K. (2019). Biomaterials used in stem cell
therapy for spinal cord injury. \emph{Progress in Materials Science,
103} , 374-424. doi:10.1016/j.pmatsci.2019.02.002

Hill, C. E. (2017). A view from the ending: Axonal dieback and
regeneration following SCI. \emph{Neuroscience Letters, 652} , 11-24.
doi:10.1016/j.neulet.2016.11.002

Jimenez-Moreno, C. M., Herrera-Gomez, I. d. G., Lopez-Noriega, L.,
Lorenzo, P. I., Cobo-Vuilleumier, N., Fuente-Martin, E., . . .
Martin-Montalvo, A. (2015). A Simple High Efficiency Intra-Islet
Transduction Protocol Using Lentiviral Vectors. \emph{Current gene
therapy, 15} (4), 436-446. doi:10.2174/1566523215666150630121557

Karimi-Abdolrezaee, S., Eftekharpour, E., Wang, J., Morshead, C. M., \&
Fehlings, M. G. (2006). Delayed Transplantation of Adult Neural
Precursor Cells Promotes Remyelination and Functional Neurological
Recovery after Spinal Cord Injury. \emph{The Journal of Neuroscience,
26} (13), 3377. doi:10.1523/JNEUROSCI.4184-05.2006

Katoh, H., Yokota, K., \& Fehlings, M. G. (2019). Regeneration of Spinal
Cord Connectivity Through Stem Cell Transplantation and Biomaterial
Scaffolds. \emph{Frontiers in cellular neuroscience, 13} , 248-248.
doi:10.3389/fncel.2019.00248

Koffler, J., Zhu, W., Qu, X., Platoshyn, O., Dulin, J. N., Brock, J., .
. . Tuszynski, M. H. (2019). Biomimetic 3D-printed scaffolds for spinal
cord injury repair. \emph{Nature medicine, 25} (2), 263-269.
doi:10.1038/s41591-018-0296-z

Kumamaru, H., Kadoya, K., Adler, A. F., Takashima, Y., Graham, L.,
Coppola, G., \& Tuszynski, M. H. (2018). Generation and post-injury
integration of human spinal cord neural stem cells. \emph{Nature
Methods, 15} (9), 723-731. doi:10.1038/s41592-018-0074-3

Kwon, S. G., Kwon, Y. W., Lee, T. W., Park, G. T., \& Kim, J. H. (2018).
Recent advances in stem cell therapeutics and tissue engineering
strategies. \emph{Biomaterials research, 22} , 36-36.
doi:10.1186/s40824-018-0148-4

Lacroix, S., Chang, L., Rose-John, S., \& Tuszynski, M. H. (2002).
Delivery of hyper-interleukin-6 to the injured spinal cord increases
neutrophil and macrophage infiltration and inhibits axonal
growth.\emph{J Comp Neurol, 454} (3), 213-228. doi:10.1002/cne.10407

Levi, A. D., Anderson, K. D., Okonkwo, D. O., Park, P., Bryce, T. N.,
Kurpad, S. N., . . . Gant, K. (2018). Clinical Outcomes from a
Multi-Center Study of Human Neural Stem Cell Transplantation in Chronic
Cervical Spinal Cord Injury. \emph{Journal of neurotrauma, 36} (6),
891-902. doi:10.1089/neu.2018.5843

Li, X., Liu, S., Zhao, Y., Li, J., Ding, W., Han, S., . . . Dai, J.
(2016). Training Neural Stem Cells on Functional Collagen Scaffolds for
Severe Spinal Cord Injury Repair. \emph{Advanced functional materials,
26} (32), 5835-5847. doi:10.1002/adfm.201601521

Liechtenstein, T., Perez-Janices, N., \& Escors, D. (2013). Lentiviral
vectors for cancer immunotherapy and clinical
applications.\emph{Cancers, 5} (3), 815-837. doi:10.3390/cancers5030815

Lien, B. V., Tuszynski, M. H., \& Lu, P. (2019). Astrocytes migrate from
human neural stem cell grafts and functionally integrate into the
injured rat spinal cord. \emph{Experimental Neurology, 314} , 46-57.
doi:10.1016/j.expneurol.2019.01.006

Liu, J. M. H., Zhang, X., Joe, S., Luo, X., \& Shea, L. D. (2018).
Evaluation of biomaterial scaffold delivery of IL-33 as a localized
immunomodulatory agent to support cell transplantation in adipose
tissue. \emph{Journal of Immunology and Regenerative Medicine, 1} ,
1-12. doi:10.1016/j.regen.2018.01.003

Liu, S., Schackel, T., Weidner, N., \& Puttagunta, R. (2018).
Biomaterial-Supported Cell Transplantation Treatments for Spinal Cord
Injury: Challenges and Perspectives. \emph{Frontiers in cellular
neuroscience, 11} (430). doi:10.3389/fncel.2017.00430

Liu, S., Xie, Y.-Y., \& Wang, B. (2019). Role and prospects of
regenerative biomaterials in the repair of spinal cord
injury.\emph{Neural regeneration research, 14} (8), 1352-1363.
doi:10.4103/1673-5374.253512

Liu, X. Z., Xu, X. M., Hu, R., Du, C., Zhang, S. X., McDonald, J. W., .
. . Choi, D. W. (1997). Neuronal and glial apoptosis after traumatic
spinal cord injury. \emph{The Journal of neuroscience : the official
journal of the Society for Neuroscience, 17} (14), 5395-5406.
doi:10.1523/JNEUROSCI.17-14-05395.1997

Lu, P., Ceto, S., Wang, Y., Graham, L., Wu, D., Kumamaru, H., . . .
Tuszynski, M. H. (2017). Prolonged human neural stem cell maturation
supports recovery in injured rodent CNS. \emph{The Journal of clinical
investigation, 127} (9), 3287-3299. doi:10.1172/JCI92955

Lu, P., Gomes-Leal, W., Anil, S., Dobkins, G., Huie, J. R., Ferguson, A.
R., . . . Tuszynski, M. (2019). Origins of Neural Progenitor
Cell-Derived Axons Projecting Caudally after Spinal Cord
Injury.\emph{Stem Cell Reports, 13} (1), 105-114.
doi:10.1016/j.stemcr.2019.05.011

Lu, P., Jones, L. L., Snyder, E. Y., \& Tuszynski, M. H. (2003). Neural
stem cells constitutively secrete neurotrophic factors and promote
extensive host axonal growth after spinal cord injury.\emph{Experimental
Neurology, 181} (2), 115-129. doi:10.1016/S0014-4886(03)00037-2

Margul, D. J., Park, J., Boehler, R. M., Smith, D. R., Johnson, M. A.,
McCreedy, D. A., . . . Seidlits, S. K. (2016). Reducing
neuroinflammation by delivery of IL-10 encoding lentivirus from
multiple-channel bridges. \emph{Bioeng Transl Med, 1} (2), 136-148.
doi:10.1002/btm2.10018

Margul, D. J., Park, J., Boehler, R. M., Smith, D. R., Johnson, M. A.,
McCreedy, D. A., . . . Seidlits, S. K. (2016). Reducing
neuroinflammation by delivery of IL-10 encoding lentivirus from
multiple-channel bridges. \emph{Bioengineering \& Translational
Medicine, 1} (2), 136-148. doi:10.1002/btm2.10018

Marquardt, L. M., Doulames, V. M., Wang, A. T., Dubbin, K., Suhar, R.
A., Kratochvil, M. J., . . . Heilshorn, S. C. (2020). Designer,
injectable gels to prevent transplanted Schwann cell loss during spinal
cord injury therapy. \emph{Science Advances, 6} (14), eaaz1039.
doi:10.1126/sciadv.aaz1039

Marquardt, L. M., \& Heilshorn, S. C. (2016). Design of Injectable
Materials to Improve Stem Cell Transplantation. \emph{Current stem cell
reports, 2} (3), 207-220. doi:10.1007/s40778-016-0058-0

McCreedy, D. A., Margul, D. J., Seidlits, S. K., Antane, J. T., Thomas,
R. J., Sissman, G. M., . . . Shea, L. D. (2016). Semi-automated counting
of axon regeneration in poly(lactide co-glycolide) spinal cord
bridges.\emph{J Neurosci Methods, 263} , 15-22.
doi:10.1016/j.jneumeth.2016.01.021

Milone, M. C., \& O'Doherty, U. (2018). Clinical use of lentiviral
vectors. \emph{Leukemia, 32} (7), 1529-1541.
doi:10.1038/s41375-018-0106-0

Mortazavi, M. M., Jaber, M., Adeeb, N., Deep, A., Hose, N., Rezaei, M.,
. . . Tubbs, R. S. (2015). Engraftment of neural stem cells in the
treatment of spinal cord injury. \emph{Translational Research in
Anatomy, 1} , 11-16. doi:10.1016/j.tria.2015.10.002

Mosser, D. M., \& Edwards, J. P. (2008). Exploring the full spectrum of
macrophage activation. \emph{Nature reviews. Immunology, 8} (12),
958-969. doi:10.1038/nri2448

Mothe, A. J., Tam, R. Y., Zahir, T., Tator, C. H., \& Shoichet, M. S.
(2013). Repair of the injured spinal cord by transplantation of neural
stem cells in a hyaluronan-based hydrogel. \emph{Biomaterials, 34} (15),
3775-3783. doi:10.1016/j.biomaterials.2013.02.002

Nagoshi, N., Khazaei, M., Ahlfors, J.-E., Ahuja, C. S., Nori, S., Wang,
J., . . . Fehlings, M. G. (2018). Human Spinal Oligodendrogenic Neural
Progenitor Cells Promote Functional Recovery After Spinal Cord Injury by
Axonal Remyelination and Tissue Sparing. \emph{Stem cells translational
medicine, 7} (11), 806-818. doi:10.1002/sctm.17-0269

Niwano, K., Arai, M., Koitabashi, N., Watanabe, A., Ikeda, Y., Miyoshi,
H., \& Kurabayashi, M. (2008). Lentiviral Vector\&\#x2013;mediated
\textless{}em\textgreater{}SERCA2\textless{}/em\textgreater{} Gene
Transfer Protects Against Heart Failure and Left Ventricular Remodeling
After Myocardial Infarction in Rats. \emph{Molecular Therapy, 16} (6),
1026-1032. doi:10.1038/mt.2008.61

Ogawa, Y., Sawamoto, K., Miyata, T., Miyao, S., Watanabe, M., Nakamura,
M., . . . Okano, H. (2002). Transplantation of in vitro-expanded fetal
neural progenitor cells results in neurogenesis and functional recovery
after spinal cord contusion injury in adult rats. \emph{Journal of
Neuroscience Research, 69} (6), 925-933. doi:10.1002/jnr.10341

Park, J., Decker, J. T., Margul, D. J., Smith, D. R., Cummings, B. J.,
Anderson, A. J., \& Shea, L. D. (2018). Local Immunomodulation with
Anti-inflammatory Cytokine-Encoding Lentivirus Enhances Functional
Recovery after Spinal Cord Injury. \emph{Molecular Therapy, 26} (7),
1756-1770. doi:10.1016/j.ymthe.2018.04.022

Park, J., Decker, J. T., Smith, D. R., Cummings, B. J., Anderson, A. J.,
\& Shea, L. D. (2018). Reducing inflammation through delivery of
lentivirus encoding for anti-inflammatory cytokines attenuates
neuropathic pain after spinal cord injury. \emph{Journal of Controlled
Release, 290} , 88-101. doi:10.1016/j.jconrel.2018.10.003

Pawar, K., Cummings, B. J., Thomas, A., Shea, L. D., Levine, A., Pfaff,
S., \& Anderson, A. J. (2015). Biomaterial bridges enable regeneration
and re-entry of corticospinal tract axons into the caudal spinal cord
after SCI: Association with recovery of forelimb
function.\emph{Biomaterials, 65} , 1-12.
doi:10.1016/j.biomaterials.2015.05.032

Pereira, I. M., Marote, A., Salgado, A. J., \& Silva, N. A. (2019).
Filling the Gap: Neural Stem Cells as A Promising Therapy for Spinal
Cord Injury. \emph{Pharmaceuticals (Basel, Switzerland), 12} (2), 65.
doi:10.3390/ph12020065

Sakuma, T., Barry, M. A., \& Ikeda, Y. (2012). Lentiviral vectors: basic
to translational. \emph{Biochem J, 443} (3), 603-618.
doi:10.1042/bj20120146

Shahriari, D., Koffler, J. Y., Tuszynski, M. H., Campana, W. M., \&
Sakamoto, J. S. (2017). Hierarchically Ordered Porous and High-Volume
Polycaprolactone Microchannel Scaffolds Enhanced Axon Growth in
Transected Spinal Cords. \emph{Tissue Eng Part A, 23} (9-10), 415-425.
doi:10.1089/ten.TEA.2016.0378

Shamash, S., Reichert, F., \& Rotshenker, S. (2002). The cytokine
network of Wallerian degeneration: tumor necrosis factor-alpha,
interleukin-1alpha, and interleukin-1beta. \emph{The Journal of
neuroscience : the official journal of the Society for Neuroscience, 22}
(8), 3052-3060. doi:10.1523/JNEUROSCI.22-08-03052.2002

Shikanov, A., Smith, R. M., Xu, M., Woodruff, T. K., \& Shea, L. D.
(2011). Hydrogel network design using multifunctional macromers to
coordinate tissue maturation in ovarian follicle
culture.\emph{Biomaterials, 32} (10), 2524-2531.
doi:10.1016/j.biomaterials.2010.12.027

Shin, S., Salvay, D. M., \& Shea, L. D. (2010). Lentivirus delivery by
adsorption to tissue engineering scaffolds. \emph{Journal of biomedical
materials research. Part A, 93} (4), 1252-1259. doi:10.1002/jbm.a.32619

Shrestha, B., Coykendall, K., Li, Y., Moon, A., Priyadarshani, P., \&
Yao, L. (2014). Repair of injured spinal cord using biomaterial
scaffolds and stem cells. \emph{Stem Cell Research \& Therapy, 5} (4),
91. doi:10.1186/scrt480

Skoumal, M., Seidlits, S., Shin, S., \& Shea, L. (2016). Localized
lentivirus delivery via peptide interactions. \emph{Biotechnology and
Bioengineering, 113} (9), 2033-2040.
doi:https://doi.org/10.1002/bit.25961

Smith, D. R., Dumont, C. M., Park, J., Ciciriello, A. J., Guo, A.,
Tatineni, R., . . . Shea, L. D. (2020). Polycistronic Delivery of IL-10
and NT-3 Promotes Oligodendrocyte Myelination and Functional Recovery in
a Mouse Spinal Cord Injury Model. \emph{Tissue Engineering Part A} .
doi:10.1089/ten.tea.2019.0321

Smith, D. R., Margul, D. J., Dumont, C. M., Carlson, M. A., Munsell, M.
K., Johnson, M., . . . Shea, L. D. (2019). Combinatorial lentiviral gene
delivery of pro-oligodendrogenic factors for improving myelination of
regenerating axons after spinal cord injury. \emph{Biotechnol Bioeng,
116} (1), 155-167. doi:10.1002/bit.26838

Soderblom, C., Luo, X., Blumenthal, E., Bray, E., Lyapichev, K., Ramos,
J., . . . Lee, J. K. (2013). Perivascular Fibroblasts Form the Fibrotic
Scar after Contusive Spinal Cord Injury. \emph{The Journal of
Neuroscience, 33} (34), 13882. doi:10.1523/JNEUROSCI.2524-13.2013

Straley, K. S., Foo, C. W. P., \& Heilshorn, S. C. (2010). Biomaterial
design strategies for the treatment of spinal cord
injuries.\emph{Journal of neurotrauma, 27} (1), 1-19.
doi:10.1089/neu.2009.0948

Tejeda, G., Ciciriello, A. J., \& Dumont, C. M. (2021). Biomaterial
Strategies to Bolster Neural Stem Cell-Mediated Repair of the Central
Nervous System. \emph{Cells Tissues Organs} .

Temple, S., \& Studer, L. (2017). Lessons Learned from Pioneering Neural
Stem Cell Studies. \emph{Stem Cell Reports, 8} (2), 191-193.
doi:10.1016/j.stemcr.2017.01.024

Tetzlaff, W., Okon, E. B., Karimi-Abdolrezaee, S., Hill, C. E.,
Sparling, J. S., Plemel, J. R., . . . Kwon, B. K. (2011). A systematic
review of cellular transplantation therapies for spinal cord
injury.\emph{Journal of neurotrauma, 28} (8), 1611-1682.
doi:10.1089/neu.2009.1177

Thomas, A. M., Kubilius, M. B., Holland, S. J., Seidlits, S. K.,
Boehler, R. M., Anderson, A. J., . . . Shea, L. D. (2013). Channel
density and porosity of degradable bridging scaffolds on axon growth
after spinal injury. \emph{Biomaterials, 34} (9), 2213-2220.
doi:10.1016/j.biomaterials.2012.12.002

Thomas, A. M., Seidlits, S. K., Goodman, A. G., Kukushliev, T. V.,
Hassani, D. M., Cummings, B. J., . . . Shea, L. D. (2014). Sonic
hedgehog and neurotrophin-3 increase oligodendrocyte numbers and
myelination after spinal cord injury. \emph{Integr Biol (Camb), 6} (7),
694-705. doi:10.1039/c4ib00009a

Tsintou, M., Dalamagkas, K., \& Seifalian, A. M. (2015). Advances in
regenerative therapies for spinal cord injury: a biomaterials
approach.\emph{Neural regeneration research, 10} (5), 726-742.
doi:10.4103/1673-5374.156966

Tuinstra, H. M., Aviles, M. O., Shin, S., Holland, S. J., Zelivyanskaya,
M. L., Fast, A. G., . . . Shea, L. D. (2012). Multifunctional,
multichannel bridges that deliver neurotrophin encoding lentivirus for
regeneration following spinal cord injury. \emph{Biomaterials, 33} (5),
1618-1626. doi:10.1016/j.biomaterials.2011.11.002

Tuinstra, H. M., Margul, D. J., Goodman, A. G., Boehler, R. M., Holland,
S. J., Zelivyanskaya, M. L., . . . Shea, L. D. (2013). Long-Term
Characterization of Axon Regeneration and Matrix Changes Using Multiple
Channel Bridges for Spinal Cord Regeneration. \emph{Tissue Engineering
Part A, 20} (5-6), 127-1037. doi:10.1089/ten.tea.2013.0111

Yang, Y., Laporte, L. D., Zelivyanskaya, M. L., Whittlesey, K. J.,
Anderson, A. J., Cummings, B. J., \& Shea, L. D. (2009). Multiple
Channel Bridges for Spinal Cord Injury: Cellular Characterization of
Host Response. \emph{Tissue Engineering Part A, 15} (11), 3283-3295.
doi:10.1089/ten.tea.2009.0081

Yang, Z., Zhang, A., Duan, H., Zhang, S., Hao, P., Ye, K., . . . Li, X.
(2015). NT3-chitosan elicits robust endogenous neurogenesis to enable
functional recovery after spinal cord injury. \emph{Proc Natl Acad Sci U
S A, 112} (43), 13354-13359. doi:10.1073/pnas.1510194112

Zhao, J., Pettigrew, G. J., Thomas, J., Vandenberg, J. I., Delriviere,
L., Bolton, E. M., . . . Lever, A. M. (2002). Lentiviral vectors for
delivery of genes into neonatal and adult ventricular cardiac myocytes
in vitro and in vivo. \emph{Basic Res Cardiol, 97} (5), 348-358.
doi:10.1007/s00395-002-0360-0

Zhu, Y., Soderblom, C., Trojanowsky, M., Lee, D.-H., \& Lee, J. K.
(2014). Fibronectin Matrix Assembly after Spinal Cord
Injury.\emph{Journal of neurotrauma, 32} (15), 1158-1167.
doi:10.1089/neu.2014.3703

\textbf{Figure 1:} FLuc loaded PEG tubes sustained expression over a
course of 12 weeks. Qualitatively, (A) bioluminescence was detected in
highest concentrations at the injured spinal cord. (B) Bioluminescent
signal of lentivirus-laden hydrogel tubes was assessed over a course of
12 weeks with higher expression compared to background across all times
evaluated. n=6 per condition, * p\textless{}0.05, ** p\textless{}0.01,
**** p\textless{}0.001.

\textbf{Figure 2:} Macrophage infiltration is not exacerbated by tube
implantation or E14 spinal progenitor transplantation. Macrophage
densities in (A) SCI only, (B) Tubes only, (C)Tubes+IL-10, (D) E14
spinal progenitors only, (E) Tubes+E14, and (F) Tubes+E14+IL-10 in
transverse tissue cross sections exhibit both M1 macrophages
(F4/80\textsuperscript{+}, red) denoted by \^{} and M2 macrophages
(F4/80\textsuperscript{+}arginase\textsuperscript{+}, red and green)
denoted by \textsubscript{*}. At 4 weeks after implantation, there were
no differences in (G) total macrophage density, (H) M2 macrophage
density, and (I) M2 percent across all conditions. Data are presented as
mean ± SEM. n=4 per condition, 50 \selectlanguage{greek}µ\selectlanguage{english}m scale bars.

\textbf{Figure 3:} (A) Experimental timeline detailed with major
interventions. Delayed EGFP\textsuperscript{+} spinal progenitor
transplantation into IL-10 encoding lentivirus loaded tubes 2 weeks
post-injury improves survival. Five hydrogel tubes, either blank or
IL-10 lentivirus loaded, were implanted directly after lateral C5
hemisection injury, and EGFP\textsuperscript{+} spinal progenitors were
transplanted 2 weeks post-injury into (B) SCI only, (C) blank tubes, or
(D) IL-10 lentivirus loaded tubes. Quantitatively, survival was assessed
4 weeks post-injury (2 weeks post-transplantation), and IL-10 lentivirus
loaded tubes exhibited an increase in (E) cell density and (F) percent
survival over the other two conditions. Data are presented as mean \selectlanguage{ngerman}±
SEM. n=4 per condition, * p\textless{}0.05, ** p\textless{}0.01, 50 \selectlanguage{greek}µ\selectlanguage{english}m
scale bars.

\textbf{Figure 4:} Neuron formation at 12 weeks post-injury is increased
when hydrogel tubes are implanted. (A) Formed neurons were observed at
12 weeks post-injury arising from both exogenous
(EGFP\textsuperscript{+}NeuN\textsuperscript{+}\textbar{}green\textsuperscript{+}gray\textsuperscript{+},
green arrow) and endogenous
(BrdU\textsuperscript{+}NeuN\textsuperscript{+}\textbar{}red\textsuperscript{+}gray\textsuperscript{+},
pink arrow) sources. (B) No statistically significant differences was
observed in exogenous-sourced neuron density. (C) A significant increase
in NeuN\textsuperscript{+} cells as a percent of total
EGFP\textsuperscript{+} cells was observed when E14 spinal progenitors
were transplanted into either blank tubes or IL-10 lentivirus-loaded
tubes. (D) There were no statistically significant differences observed
in endogenous-sourced neuron formation. Data are represented as mean \selectlanguage{ngerman}±
SEM, n = 4-7 animals per conditions, * p\textless{}0.05, **
p\textless{}0.01, 50 \selectlanguage{greek}µ\selectlanguage{english}m scale bars.

\textbf{Figure 5:} E14 spinal progenitor transplants into IL-10
lentivirus-laden tubes improve new axon formation at 12 weeks
post-injury. Axon expression (NF-200\textsuperscript{+}) in (A) SCI
only, (B) Tubes only, (C) Tubes+IL-10, (D) E14 spinal progenitors only,
(E) Tubes+E14, and (F) Tubes+E14+IL-10. Examples of (G) bundles and (H)
elongated axons observed in the ipsilateral tissue. Quantitatively,
animals receiving Tubes+E14+IL-10 exhibited a higher elongated axon
density (I) in the ipsilateral tissue. Data are presented as mean \selectlanguage{ngerman}± SEM.
n=6 animals per condition, * p\textless{}0.05, ** p\textless{}0.01, ***
p\textless{}0.005, 200 \selectlanguage{greek}µ\selectlanguage{english}m scale bars (A-F), 50 \selectlanguage{greek}µ\selectlanguage{english}m scale bars (G-H).

\textbf{Figure 6:} E14 spinal progenitor delivery increases axon
remyelination 12 weeks post-injury. Axon (NF-200\textsuperscript{+},
red) co-localization with myelin (MBP\textsuperscript{+}, green) was
assessed in (A) SCI only, (B) Tubes only, (C) Tubes+IL-10, (D) E14
spinal progenitors only, (E) Tubes+E14, and (F) Tubes+E14+IL-10. (G) E14
only transplants and Tubes+E14+IL-10 exhibited an increase in overall
myelinated axon density compared to the SCI only condition. (H) As a
percent, myelination was significantly increased in the E14 only
condition compared to all other conditions. Data are represented as mean
\selectlanguage{ngerman}± SEM, n = 4-7 animals per condition, * p\textless{}0.05, **
p\textless{}0.01, *** p\textless{}0.005, **** p\textless{}0.001, 20 \selectlanguage{greek}µ\selectlanguage{english}m
scale bars.

\textbf{Figure 7:} Animals receiving E14 spinal progenitors transplanted
into IL-10 lentivirus-laden tubes exhibited a quicker return of forelimb
motor function. To assess mobility, a horizontal ladder beam test was
used. Mice were trained on walking across the ladder prior to injury.
Mobility was assessed through successful placements as a score out of 50
possible rungs. By 4 weeks post-injury Tubes+E14+IL-10 (a,
p\textless{}0.005) had improved stepping compared to SCI only. At 8
weeks all conditions with tubes had improved stepping compared to
(Tubes+E14, b, p\textless{}0.005; Tubes+IL-10, c, p\textless{}0.05;
Tubes, d, p\textless{}0.05). At 12 weeks all conditions exhibited
improved stepping (E14 only, e, p\textless{}0.01).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-7/Figure-7}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

