Cell-Laden Hydrogel Constructs of Hyaluronic Acid,Collagen,and Laminin for Neural Tissue Engineering

Abstract

Various neural tissue engineering approaches that are under development for applications ranging from guidance conduits to cell-based therapies rely on the ability to encapsulate cells in three-dimensional (3D) scaffolds. Schwann cells play a key role in peripheral nerve regeneration by forming oriented paths for regrowing axons. We have engineered collagen and hyaluronic acid interpenetrating polymer network (IPN) hydrogels with and without laminin as a 3D culture system for Schwann cells in an attempt to devise novel neural regeneration therapies. Encapsulation of Schwann cells in 3D hydrogel constructs did not affect cell viability and cells were viable for 2 weeks in all hydrogel samples. Moreover, in hydrogels with high cell density, cells underwent spreading and proliferation, and the cell numbers increased by day 14 as assessed qualitatively using a Live/dead^® assay and scanning electron microscopy (SEM), and quantitatively using a CellTiter^® 96 AQueous non-radioactive cell proliferation assay. In some cases, the cells aligned parallel to each other and formed structures reminiscent of Bands of Büngner. Schwann cells in cell–hydrogel constructs with high cell density were not only viable but also actively secreting nerve growth factor and brain-derived neurotrophic factor. Of particular importance was the observation that addition of laminin in these hydrogels increased the overall production of nerve growth factor and brain-derived neurotrophic factor from the cells. Immunostaining revealed that S100 expression and cell spreading were differentially affected by cell density. Interestingly, in the co-culture of dissociated neurons with Schwann cells, neurons were able to extend neurites and some neurites were observed to follow Schwann cells. Therefore, we conclude that Schwann cells encapsulated in the 3D extracellular matrix–mimicking hydrogel may hold promise in nerve regeneration therapies and may form the basis for understanding the underlying mechanisms of Schwann cell interactions with neurons and various extracellular matrix components.

Introduction

Hydrogels represent an attractive class of biomaterial scaffolds and have been utilized in a number of biomedical and tissue engineering applications. Hydrogels are three-dimensional (3D) crosslinked polymer networks with high water content similar to tissues.¹ Hydrogels have been used to encapsulate cells in a 3D environment to grow different types of tissues.^2–5 Recently, studies have been focused on fabricating hydrogels using native extracellular matrix (ECM) components, including proteins and glycosaminoglycans, in an attempt to recapitulate ECM's structural, biological, and mechanical properties.^6,7 In the native environment, ECM regulates vital cell processes, including cell migration, adhesion, differentiation, and proliferation by providing physical, chemical, and biological cues to the cells.⁸ Hydrogels fabricated from ECM biomolecules are promising since they are biocompatible and biodegradable and have functional domains for cell signaling.⁹

Schwann cells are glial cells that play a fundamental role in peripheral nerve tissue repair by promoting axonal regeneration postinjury.^10,11 After a nerve injury, the key requirement during regeneration is to guide the axons across the injury site, leading to functional reinnervation. Schwann cells play a major role in axonal guidance to establish functional synapses by forming oriented paths termed “Bands of Büngner.” Schwann cells express CD44, which is a major hyaluronan-binding protein that mediates various cellular processes, including cell growth, migration, and cell–matrix interactions.¹² Studies have shown that CD44 plays a significant role in mediating Schwann cell response to neuregulins, which is important for Schwann cell–axon interactions.¹³ Schwann cells are surrounded by endoneurium, the ECM of neural tissue, which is enriched in hyaluronic acid (HA), collagen IV, and laminin. Surrounding the nerve cables is epineurium, which is enriched in collagens I, II, and III.¹⁴ Schwann cells assist with the regeneration process by performing major roles after injury, including (1) secretion of neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF); (2) presenting a permissive environment for regenerating axons by secreting ECM molecules such as collagen, laminin, and proteoglycans; (3) expression of cell adhesion molecules such as neural-cell adhesion molecule (N-CAM), N-cadherin, and L1, which enhance glia–neuron interactions; and (4) remyelinating the injured axons.^10,15,16

Our choice of natural biomaterials such as HA, collagen, and laminin is marked by the need to mimic natural ECM composition so that the cells interact with the scaffold in a similar way as they interact in native tissues. HA is a nonsulfated, unbranched, high-molecular-weight glycosaminoglycan composed of repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine linked by alternating β(1→4) and β(1→3) linkages. The glucuronic acid residues of HA contain a carboxyl group, which is negatively charged at physiological pH and ionic strength, giving rise to the polyanionic character of HA. Because of this polyanionic behavior, HA does not favor protein and cell adhesion. HA is ubiquitously present in ECM and is a major component of vitreous humor of the eye, synovial fluid of joints, cartilage, and umbilical cord.¹⁷ HA is highly conserved among species and thus nonimmunogenic.¹⁸ It has been approved by Food and Drug Administration and has been employed for the treatment of osteoarthritis,^19,20 as a vitreous humor substitute in eye,²¹ for the prevention of postsurgical adhesions,²² drug delivery applications,²³ and wound healing.²⁴

Collagen and laminin are the major proteins found in neural tissue ECM. Several studies have been performed using collagen tubes and gels for neural tissue regeneration.²⁵ Collagen is the most abundant protein of the ECM and also plays an important role in cell–cell and cell–matrix interactions.^26,27 Laminin is a three-chain protein and is present at a high concentration on the inner face of the endoneurium where it is in close contact with neurons and Schwann cells.²⁸ Laminin enhances neurite extension and is vital for Schwann cell migration. Patterned laminin microgrooves, for example, have been shown to provide directional cues to regenerating axons experimentally.^29,30

Previous research by our group showed that interpenetrating polymer networks (IPNs) of collagen and HA do not interfere with the viability and proliferation of Schwann cells.³¹ In the present study, we synthesized 3D ECM-mimicking IPN hydrogels of HA and collagen in the presence of Schwann cells to yield cell-laden hydrogels. In some gels, laminin was also added, since it is a vital component of neural ECM and regulates neuron–glial interactions. For controls, Schwann cells were cultured in collagen hydrogels, which have been used previously for their encapsulation.³² Encapsulated cells were cultured in 3D hydrogels for 2 weeks and their viability, metabolic activity, growth factor release, and morphology were monitored as a function of time. The effects of low cell density and high cell density on cell function and behavior in collagen–HA IPNs were also examined. Expression of S100, a Schwann cell–specific marker, was studied via immunohistochemistry to analyze expression in 3D.

Materials and Methods

Materials

Sodium hyaluronate from Streptococcus equi of molecular weight 1.6 × 10⁶ Da was obtained from Sigma-Aldrich (St. Louis, MO). Photoinitiator Irgacure 2959 (I2959) was obtained from Ciba Specialty Chemicals (Basel, Switzerland). A longwave UV lamp filtered around 365 nm and with an intensity of 5 mW/cm² was used for photopolymerization of hydrogels (Blak-Ray B-100A; UVP, Upland, CA). Silicone molds with 2 mm thickness and 4 mm diameter were purchased from Grace Biolabs (Bend, OR). NGF and BDNF ELISA kits were obtained from Promega (Madison, WI). All other chemicals were acquired from Sigma-Aldrich unless otherwise specified.

Synthesis of glycidyl methacrylate–modified HA

Photopolymerizable methacrylate groups were attached to HA by a method described previously.³³ Briefly, a 1% w/v solution of HA was prepared in water by stirring overnight. Triethylamine and glycidyl methacrylate were added to the HA solution in 20-fold molar excess and stirred overnight at room temperature. Glycidyl methacrylate–modified HA (GMHA) was purified by precipitating the solution twice in a 20-fold volume of acetone. The precipitate was redissolved in deionized water and lyophilized. The lyophilized GMHA was dessicated and stored at −20°C in the dark until use.

Schwann cell isolation and culture

Schwann cells were harvested following established protocols.³⁴ Neonatal Sprague-Dawley rats were euthanized with CO₂, and sciatic nerves were aseptically harvested under a dissecting microscope and rinsed with phosphate-buffered saline (PBS). The extraneous tissue was carefully removed, and nerves were cut into 1 mm pieces. Sheared nerve pieces were enzymatically digested for 30 min at 37°C using a solution containing 250 U/mL collagenase in serum-free Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich). This was followed by another digestion step using 0.1 mg/mL collagenase and 0.25% trypsin in serum-free DMEM for 30 min at 37°C. The tissue was triturated through a 5 mL pipette, then a Pasteur pipette, and then a flame polished Pasteur pipette. The resulting solution was transferred to a centrifuge tube containing 10 mL of DMEM with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and was centrifuged at 1000 rpm for 5 min. The cells were counted and plated on uncoated tissue culture dishes with DMEM and 10% FBS, and incubated at 37°C with 95% humidity and 5% CO₂. After 24 h, the culture medium was replaced with a fresh medium containing 10⁻⁵ M cytosine arabinoside, an antimicotic drug, to minimize the rapidly dividing fibroblast population. After 3 days, cytosine arabinoside was removed, and remaining fibroblasts were killed by complement-mediated lysis using mouse anti-rat CD90/Thy1.1 (Accurate, Westbury, NY). Subsequently, Schwann cell culture was maintained in DMEM containing 10% FBS, 30 μg/mL bovine pituitary extract, and 2 μM forskolin. Schwann cells obtained from this method were >99% pure as confirmed by immunostaining using anti-S100 antibody (Dako, Carpinteria, CA). Cells were passaged when they were 70% confluent and were discarded after eight passages.

Cell encapsulation

Cell encapsulation was performed at a cell concentration of 8 × 10⁶ cells/mL in three different groups: collagen–GMHA IPNs (ColHA), collagen–GMHA IPNs with 100 μg/mL laminin (ColHAlam), and collagen gel alone (Col; control) (Table 1). To study the effect of low cell density on the IPNs, collagen–GMHA IPNs were also prepared with a cell concentration of 2 × 10⁶ cells/mL (ColHA2). The encapsulation was performed as follows. Collagen type I solution (BD Biosciences, Sparks, MD) was diluted by adding cold sterile-filtered 0.2% acetic acid. GMHA solution was prepared by dissolving 15 mg/mL GMHA in PBS (pH 7.4) containing 0.3% Irgacure 2959 (w/v) overnight. HEPES solution was prepared by adding 2.2 g sodium bicarbonate and 4.77 g HEPES in 100 mL of 0.5 N sodium hydroxide.

Table 1.

Nomenclature of Various Samples Used in the Study

Sample nomenclature	Collagen concentration (mg/mL)	HA concentration (mg/mL)	Laminin concentration (μg/mL)	Cell concentration (cells/mL)
Col	3	0	0	8
ColHA	3	5	0	8
ColHAlam	3	5	100	8
ColHA2	3	5	0	2

To prepare the collagen gels, collagen solution, 10 × DMEM, and HEPES solution were mixed at the ratio of 8:1:1. For collagen–GMHA IPNs, ice-cold GMHA and collagen solutions were mixed together to yield a collagen–GMHA solution, which was then mixed with 10 × DMEM and HEPES solution in the ratio of 8:1:1. Concentrated cell solution was added to the prepolymer solution to achieve the desired cell concentration (8 × 10⁶ cells/mL or 2 × 10⁶ cells/mL) in the final solution. A volume of 30 μL of the pregel solution containing cells was transferred to sterile silicone molds (4.5 × 1.5 mm; Grace Biolabs) and placed in an incubator at 37°C for 60 min to allow the fibrillogenesis of collagen. To create collagen–GMHA IPNs, collagen was first allowed to form a gel at 37°C, followed by UV exposure for 5 min to allow free radical polymerization of GMHA chains by photocrosslinking. All IPNs had final Irgacure concentration of 0.1%. After polymerization, the gels were suspended in the Schwann cell maintenance medium and were maintained in an incubator at 37°C with 95% humidity and 5% CO₂. The medium was changed after 3 h to remove unreacted components and every 3 days afterward. A schematic of cell encapsulation in IPN hydrogels is provided in Figure 1. The cell-encapsulated hydrogels were prepared with a collagen final concentration of 3 mg/mL (for collagen gels), and with a final collagen concentration of 3 mg/mL and GMHA concentration of 5 mg/mL (for IPNs).

FIG. 1.

The overall methodology for Schwann cell encapsulation in 3D interpenetrating polymer network (IPN) hydrogels. The cell pellet was dissolved in a photocrosslinkable form of hyaluronan (GMHA) and collagen solution, and the final cell-containing hydrogel precursor solution was suspended in the silicone mold. Collagen was permitted to undergo fibrillogenesis at 37°C. This results in the formation of a semi-IPN (SIPN), which was then exposed to UV light for GMHA crosslinking to yield a Schwann cell–encapsulated 3D collagen–HA IPN. 3D, three-dimensional; GMHA, glycidyl methacrylate-modified HA. Color images available online at www.liebertonline.com/ten.

Cell viability

The viability of the encapsulated cells was evaluated at days 4, 7, and 14 using a Live/Dead^® assay (Molecular Probes, Eugene, OR). Briefly, the medium was removed and cell-encapsulated gels were incubated with 4 μM ethidium and 2 μM calcein AM in PBS for 15 min at 37°C in the dark. Live cells were stained green because of the cytoplasmic esterase activity, which results in reduction of calcein AM into fluorescent calcein, and dead cells were stained red by ethidium, which enters the cells via damaged cell membranes and becomes integrated into the DNA strands. Cells were imaged using confocal microscopy and z-stacks were acquired for a total of 200 μm thickness. The z-stacks were projected onto a single plane image to show the entire thickness; 3D distribution of the cells inside the hydrogels was viewed by 3D reconstruction of z-stacks using IMARIS software (Bitplane, Zurich, Switzerland). Quantitative image analysis was performed on a series of live/dead images using Image J to determine the percentage of viable cells present at each time point. We also observed the long-term viability of these cells in hydrogels where the cells remained viable and continued to grow for 1 month in the gels (Supplemental Fig. S1, available online at www.liebertonline.com/ten).

Apoptosis assay

Apoptosis in the encapsulated cells was assessed using two methods: Caspase-Glo^® 3/7 assay and annexin-V staining. Caspase activity was measured at days 0, 4, 7, and 14 as reported previously by Lin et al.³⁵ Briefly, at each time point, the cell-laden hydrogels were incubated in 200 μL of the fresh medium containing 40 μL CellTiter-Blue reagent (Promega) for 3 h in an incubator at 37°C and 5% CO₂. The solution was transferred to a 96-well plate and the fluorescence was read (Ex. 560 nm; Em. 590 nm) using a fluorescence plate reader (Biotek FLx800) to determine the relative cell number in each gel. The gels were then transferred to a 50:50 solution of fresh medium and Caspase 3/7 Glo reagent (Promega); the gels were kept on an orbital shaker for 1 h at room temperature. The solution was transferred to a white-walled 96-well plate and the luminescence signal was measured. The luminescence signal was then normalized to the previously obtained CellTiter-Blue signal for the respective cell-laden hydrogel and was plotted as relative caspase 3/7 activity.

Apoptotic cells were identified and imaged within hydrogel samples using annexin V/propidium iodide–based Vybrant^® apoptosis assay (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. This assay detects the externalization of phosphatidylserine in apoptotic cells. Propidium iodide stains the dead cells with red fluorescence, whereas annexin V stains the cells undergoing apoptosis with green fluorescence. Viable cells display very little or no fluorescence.

Cell metabolism in hydrogels

The metabolic activity of cells in the hydrogels was assessed at days 0, 4, 7, and 14 using the CellTiter^® 96 AQueous nonradioactive cell proliferation assay (MTS assay; Promega) following the manufacturer's protocol. Briefly, at each time point, the old culture medium was aspirated and 200 μL of fresh culture medium and 40 μL of MTS reagent was added to each well containing a single gel in a 48-well plate. The gels with the reagent were incubated for 4 h in an incubator at 37°C and 5% CO₂ on an orbital shaker.³⁶ The absorbance of the resulting solution was read at 490 nm using a microplate reader (Thermo-labsystems, Franklin, MA). The background absorbance was subtracted from all the samples. The background absorbance was measured from a sample containing hydrogel (without cells), medium, and MTS reagent. The absorbance of samples for all days was normalized to day 0 values to illustrate fold change in cell metabolism.

Immunostaining

For immunohistochemistry, cell–gel constructs were washed in PBS and fixed with 4% paraformaldehyde (Sigma, St. Louis, MO) in PBS for 40 min, followed by three washes in PBS for 40 min each. The blocking step was performed with 0.3% Triton X-100 (Fisher, Pittsburgh, PA) and 5% goat serum (Sigma) in PBS for 45 min. The cells were incubated in primary antibodies (rabbit-anti S100, 1:200 [Dako]; mouse anti N-CAM, 1:250 [Abcam, Cambridge, MA]) in blocking buffer (0.3% Triton X-100 and 5% goat serum in PBS) and incubated overnight at 4°C. Hydrogels were extensively washed in PBS to eliminate any unbound primary antibody. Secondary antibodies conjugated to Alexa Fluor dyes were purchased from Invitrogen and were used at a dilution of 1:500 in PBS for 6 h at room temperature. This incubation time was found to be sufficient for the antibody to diffuse inside the hydrogel. Hydrogel constructs were washed several times in PBS (1 h each wash) and stained with 4′,6-diamidino-2-phenylindole (DAPI) at a dilution of 1:1000 in PBS.

Fluorescence microscopy was performed using either a wide-field fluorescence (IX70; Olympus, Center Valley, PA) or confocal fluorescence microscope (SP2 AOBS; Leica, Wetzlar, Germany). Images were acquired using Olympus Magnafire or Leica confocal software. All image analysis was performed using NIH Image J software.

Scanning electron microscopy

Cell morphology and cell interaction with the matrix components were observed using SEM. The medium was aspirated from the gels and the cell–gel constructs were fixed by adding 3% glutaraldehyde solution in 0.1 M cacodylate buffer. The gels were cut to expose their inner structure. Postfixation, gels were washed with PBS three times for 30 min each. The gels were dehydrated by incubating with graded ethanol solutions (30%, 50%, 70%, 85%, 90%, 95%, and 100% for 1 h each). The final dehydration step was performed by adding hexamethyl disilazine and drying in a fumehood overnight. Dried hydrogels were coated with gold using a sputter coater (Cressington 208 HR, Watford, United Kingdom) and imaged using SEM (Zeiss SUPRA 40 VP, Thornwood, NY). We understand that SEM images are not the accurate reflection of the microstructural details of hydrogels because of possible drying artifacts; however, the technique has successfully been used by researchers to provide insights into general properties and relative differences among cell-seeded biomaterial scaffolds.^37,38

NGF and BDNF production and release

Supernatant media (500 μL) were collected from all the hydrogel groups on days 1, 4, 7, and 14, and were stored at −20°C until analyzed. Growth factor release from the encapsulated cells was quantified using a commercially available sandwich-ELISA kit (NGF Emax kit, BDNF Emax kit; Promega) following the manufacturer's protocol. Briefly, for NGF quantification, a 96-well ELISA plate was coated with anti-NGF polyclonal antibody overnight at 4°C followed by incubation with blocking buffer for 1 h. Samples and standards were added to the wells and incubated for 6 h with shaking at room temperature followed by overnight incubation of the secondary antibody at 4°C. Anti-rat IgG conjugated to horseradish peroxidase was added to the plate and incubated for 2.5 h, and color was developed by addition of 3,3′,5,5′-tetramethylbenzidine substrate for 10 min.

For BDNF quantification, a 96-well ELISA plate was coated with anti-BDNF monoclonal antibody overnight at 4°C followed by incubation with blocking buffer for 1 h. Samples and standards were added and were incubated for 2 h with shaking at room temperature followed by 2 h incubation with anti-human BDNF polyclonal antibody at room temperature. Anti-IgY-conjugated horseradish peroxidase was added to the plate and incubated for 1 h and color was developed by addition of tetramethylbenzidine substrate for 10 min.

The reaction was terminated by adding 1 M hydrochloric acid and absorbance was measured at 450 nm using an ELISA plate reader (Biotek Instruments, Winooski, VT). Growth factor release was normalized to cell number and was presented as NGF or BDNF released per million cells to compare multiple groups.

Schwann cell–dorsal root ganglia coculture

All animal work was performed in accordance with the Institutional Animal Care and Use Committee at the University of Texas at Austin. Dorsal root ganglia (DRG) neurons were isolated from neonatal Sprague-Dawley rats (P0-P3) as described previously.³⁹ Cells were dissociated by incubation with collagenase (0.25%; Sigma-Aldrich) for 30 min, followed by incubation with trypsin (0.25%; Sigma-Aldrich) for 15 min. Finally, DRGs were dissociated with a series of flame-polished Pasteur pipettes. Dissociated neurons were mixed with Schwann cells (1:1, total cell concentration 8 × 10⁶ cells/mL and 2 × 10⁶ cells/mL for high and low cell density hydrogels, respectively) and were encapsulated in hydrogels as described in the Cell viability section. Cell-laden hydrogels were cultured in DMEM/F12-Ham's (Sigma-Aldrich), 10% FBS, 50 ng/mL NGF, and 1% antibiotic–antimicotic solution for 2 weeks. Cells in the hydrogels were stained with Live/Dead assay as described in the Cell viability section. Cells were fixed in 4% paraformaldehyde, blocked, and immunostained for S100 protein, βIII tubulin (1:1000; Abcam), and SYTOX^® green (1:5000; Invitrogen) for nuclei as described in the Immunostaining section.

Statistical analysis

All data are presented as mean ± standard deviation. For multiple comparisons, one-way analysis of variance was performed followed by Bonferroni correction using OriginPro 8. In all tests, p < 0.05 was regarded as statistically significant.

Results

Cell viability

Schwann cells were encapsulated in the IPNs of collagen and GMHA, with and without laminin, and were cultured for 2 weeks. Qualitative images are presented in Figure 2 and quantitative analysis is shown in Figure 3. The results from this study demonstrate that the cells remained viable in 3D hydrogels for 2 weeks and that the encapsulation process was not cytotoxic to the cells, which was further confirmed by quantitative results. There was no difference in the viability of all gel types on day 4. The figure also shows that, on day 4, some cells were round, whereas many cells had started to spread and form extensions in Col, ColHA, and ColHAlam hydrogels. Some cells were also observed to make cell–cell contacts, which were increased by day 7. By day 14, the cell numbers in the hydrogels visually started to appear higher and there were a larger number of physical cell–cell interactions. The cells in ColHA2 were sparse and most of the cells maintained a round or undifferentiated morphology. Because of low cell density, there were few cell–cell contacts observed among the cells in the ColHA2 gels.

FIG. 2.

Viability of Schwann cells encapsulated in different extracellular matrix hydrogels. Schwann cells were encapsulated at a cell density of 8 million cells/mL in collagen (Col), Col-HA (ColHA), and Col-HA with laminin (ColHAlam) hydrogels, and at a cell density of 2 million cells/mL in Col-HA hydrogels (ColHA2). Encapsulated cells were cultured for 2 weeks, and viability was assessed at days 4, 7, and 14 using the Live/dead^® assay. Cells were imaged using confocal microscopy and z-stacks were acquired at every 10 μm interval for a total of 200 μm thickness. The z-stacks were projected onto a single plane image to show the entire thickness. Scale bar is 150 μm. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Quantitative analysis of Schwann cells' viability in hydrogel samples on days 4, 7, and 14. A series of live/dead images for each time point were analyzed using NIH Image J software and the results were plotted against time.

The organization of the cells in the hydrogel constructs is depicted in Figure 4A and B, which was obtained by reconstructing the confocal z-stacks using IMARIS software. This figure clearly illustrates that the cells were organized in 3D, and were extending processes in all directions and appeared to be making contacts with other cells.

FIG. 4.

Three-dimensional image showing distribution of Schwann cells in ColHAlam hydrogel on day 4. (A) and (B) were acquired at low and high magnifications, respectively. The confocal z-stacks of live/dead images were reconstructed using Imaris software to observe the 3D distribution of cells in the hydrogel. Scale bar is 150 μm and 75 μm, respectively. Color images available online at www.liebertonline.com/ten.

Cell apoptosis

Apoptosis of Schwann cells in the hydrogel samples was assessed using caspase 3/7 activity and annexin-V staining. Figure 5 illustrates the apoptosis of cells in various hydrogel samples. There were very few apoptotic cells in the hydrogel samples on day 0 as well as on day 14, indicating that the 3D environment of the hydrogel was not detrimental for cell survival and growth (Fig. 5A). This was further confirmed by the caspase 3/7 activity (Fig. 5B). There was no significant increase in the relative apoptotic index for cells in hydrogel samples for various time points compared to that of day 0, further supporting the fact that the hydrogels were permissive for cell proliferation and growth.

FIG. 5.

(A) Annexin V/propidium iodide staining of cells in hydrogel samples on days 0 and 14 for apoptotic activity. Cells in early stage of apoptosis were stained green by annexin V, whereas necrotic cells were stained red by propidium iodide. (B) Apoptotic activity of cells in hydrogel samples. Apoptotic activity was obtained by normalizing the luminescent signal (Caspase-Glo 3/7 activity) to the fluorescent signal (CellTiter Blue) from the respective cell–hydrogel constructs. *p < 0.05 of a sample across days. Color images available online at www.liebertonline.com/ten.

Cell metabolic activity in hydrogel constructs

A cell titer proliferation assay (MTS) was performed to quantify the metabolic activity of the encapsulated Schwann cells. Figure 6 depicts the results of the MTS assay for days 4, 7, and 14 for all hydrogel groups. The metabolic activity was normalized against values for day 0 to depict fold change. As indicated by the graph, the metabolic activity of the cells initially decreased on day 4 compared to day 0, except for the ColHAlam group. This decrease can be attributed to the quiescent state of the cells. The normalized absorbance for ColHAlam gels was significantly higher than the other groups for all time points, indicating higher metabolic activity and thus higher proliferation. Further, within the ColHAlam gel, there was a significant increase in cell metabolic activity with time (2.5-fold on day 14 compared to day 0), suggesting that the laminin likely increased the proliferation rate of Schwann cells in 3D culture. Previous studies have also indicated that laminin supports Schwann cell proliferation and that disruption of laminin expression in Schwann cells prevents their proliferation.⁴⁰ For other groups, there was no significant difference in the numbers of cells at day 4 and 7, indicating that the cells had just started to proliferate in these gels. After 14 days of culture, there was a significant increase in absorbance values for Col and ColHA (∼1.5-fold for Col and ColHA gels compared to day 0). In ColHA2 gels, there was no significant change in the cell number on day 14 compared with day 4 or 7. This suggests that the low cell density does not allow a sufficient number of cell–cell contacts, which is vital for cell spreading and proliferation.⁴¹ These MTS results, combined with cell viability and apoptosis results, indicate that the encapsulation process was not detrimental to the cells and did not impede their proliferation. Further, there appears to be a sufficient number of cells in the 3D matrix to allow for cell–cell communication and contacts, which are essential for proliferation and normal functioning of the cells.

FIG. 6.

Metabolic activity of encapsulated Schwann cells in various hydrogel samples as assessed by a cell proliferation assay (MTS). The MTS results for a particular time point were normalized to day 0 for all hydrogel samples. There was a significant increase (p < 0.05) in metabolic activity of cells in the hydrogels with high cell density on day 14 compared to day 0. ^#p < 0.05 of sample versus Col, *p < 0.05 of sample versus ColHAlam, ^$p < 0.05 of sample versus ColHA, ^&p < 0.05 of sample versus ColHA2 for respective day.

S100 protein expression

The morphologies of Schwann cells in the hydrogel were observed on day 7 by immunostaining (Fig. 7). Since the hydrogels were optically transparent, the entire cell-laden hydrogel constructs were immunostained for S100 protein. The presence of Schwann cells in the hydrogels was revealed by S100 marker immunolocalization. At high cell density, for which the cells were able to make sufficient cell–cell contacts and were likely able to signal to adjacent cells, the Schwann cells were elongated and exhibited a spread, polymorphic morphology with multiple extensions or processes (Fig. 7A–C). In contrast, ColHA2 hydrogels contained cells with mostly rounded morphologies and without processes, even after 7 days (Fig. 7D). To compare among different hydrogels, quantitative analysis of S100 protein expression was also performed as demonstrated in Figure 7E. ColHAlam hydrogels expressed significantly higher levels of S100 protein compared to other groups. Lower expression of S100 protein by ColHA2 hydrogels as indicated by Figure 7D was further confirmed by quantitative image analysis (p < 0.05).

FIG. 7.

Immunohistochemistry after 7 days of Schwann cell culture in 3D hydrogels. S100 (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) staining in (A) Col, (B) ColHA, (C) ColHAlam, and (D) ColHA2 hydrogels. The encapsulated Schwann cells extended processes in the hydrogels (arrows) and expressed high levels of S100 marker protein as observed by confocal. In ColHA2 hydrogel samples, cells were round and expressed low levels of S100 protein (arrows). Scale bars are 40 μm. (E) Quantitative analysis of S100 fluorescence intensity was performed using NIH Image J software and the results were plotted. ^#p < 0.05 of sample versus Col, *p < 0.05 of sample versus ColHAlam, ^$p < 0.05 of sample versus ColHA, ^&p < 0.05 of sample versus ColHA2. Color images available online at www.liebertonline.com/ten.

Morphological analyses

SEM ultrastructural analysis demonstrated that the hydrogel components appeared to form a 3D matrix around the cells. As observed by SEM images, cells were successfully able to spread in the porous hydrogel and were extended processes in all directions. Figure 8 shows the cells in the hydrogel matrix on days 4 and 14. SEM micrographs also demonstrated increased number of cells in the hydrogel indicative of increased cell–cell interactions on day 14. The day 14 micrographs for high-cell-density hydrogels also displayed altered hydrogel morphology with what appeared to be deposited matter in the hydrogel matrix, in comparison to day 4. We speculate that this deposited matter might be the ECM secreted by the cells. Cells in ColHAlam hydrogels were also found to align parallel to each other in columns and to form structures similar to Bands of Büngner, which was intriguing since axons normally migrate on these patterned paths created by Schwann cells during regeneration.¹⁰ In ColHA2 hydrogels, there were very few cells. Those cells exhibited either a rounded morphology or extended processes but were unable to establish cell–cell contacts. Further, the SEMs suggest that the hydrogel morphology of ColHA2 gels was not significantly altered overtime, indicating minimal secretion and deposition of ECM matrix by the low number of encapsulated cells.

FIG. 8.

Pseudocolor scanning electron micrographs of Schwann cells encapsulated in hydrogels after 4 and 14 days of culture. Note that Schwann cells proliferated and exhibited numerous cell–cell contacts on day 14 in hydrogels with high cell density. Schwann cells were also found to align parallel to each other (arrows), similar to Bands of Büngner, in ColHAlam hydrogels. In ColHA2 gels, there were very few cells with minimal cell–cell contacts. Scale bars are 2 μm. Color images available online at www.liebertonline.com/ten.

NGF and BDNF production

To evaluate the functional activity of encapsulated Schwann cells in various hydrogel matrices, NGF and BDNF production was monitored on days 4, 7, and 14 using an ELISA (Fig. 9A, B). The release was normalized for the number of cells and was expressed as NGF released per million cells. After 2 weeks, NGF production by cells in ColHAlam hydrogels increased ∼2.5-fold compared to the Col and ColHA hydrogels. This increase in NGF can likely be attributed to increased proliferation rate in these gels, therefore resulting in increased growth factor production. Cells in the ColHAlam hydrogels continued to secrete NGF throughout the culture period. There was no significant difference in NGF released form ColHA gels and Col gels for the entire culture period. In ColHA2 hydrogels, there was a very low level of NGF secreted by the cells and a constant level of NGF was maintained throughout the culture period. The negligible levels of NGF released from cells in ColHA2 hydrogels could be attributed to the low cell density, which results in fewer cell–cell interactions and signaling. This can result in the failure of the cells to spread and proliferate, thus leading to loss of cell functionality. The cells were alive but acquired an undifferentiated or rounded morphology and remained in a quiescent state throughout the culture period.

FIG. 9.

(A) Nerve growth factor (NGF) and (B) brain-derived neurotrophic factor (BDNF) release from encapsulated Schwann cells in various hydrogel samples as a function of time. The release was normalized against the number of cells and was plotted as NGF released per million cells. Reported values are the average ± standard deviation of at least three independent measurements (p < 0.05, indicated by *).

The production of BDNF from encapsulated Schwann cells in various hydrogel matrices was also monitored on days 1, 4, 7, and 14 using an ELISA (Fig. 9B). BDNF production by cells in ColHAlam hydrogels increased 1.5-fold compared to the Col hydrogels or ColHA hydrogels on day 14. Cells in the ColHAlam hydrogels continued to secrete BDNF throughout the culture period. There was no significant difference in BDNF released from cells in ColHA gels and Col gels for the entire culture period. In the hydrogels with low cell density (ColHA2), there was a very low level of BDNF secreted by the cells compared to other hydrogel samples, again potentially attributed to negligible cell–cell interactions.

Schwann cell–dissociated neurons coculture

Since Schwann cells play a vital role in peripheral nerve regeneration and form a platform for the regrowing axons, cocultures of Schwann cells and dissociated neurons were evaluated in hydrogel samples. As indicated by Figure 10, the cells in the coculture that contained neurons and Schwann cells demonstrated excellent cell viability and dissociated neurons were able to extend neurites in the hydrogel constructs. Figure 11 shows the 3D organization of a Schwann cell–DRG coculture obtained by reconstructing confocal z-stacks. As shown in the figure, Schwann cells were observed to form oriented pathways for growing neurites (white arrows in figure), which is a crucial step for guiding neuron regeneration after injury. Very few cells were observed to spread and extend neurites in the ColHA2 hydrogels (Fig. 11D).

FIG. 10.

Live/dead image showing viable Schwann cells and dissociated neurons in ColHAlam hydrogel on day 7. The neurons were extending thread like neurites in all directions. Scale bar is 150 μm. Color images available online at www.liebertonline.com/ten.

FIG. 11.

Three-dimensional image showing immunostained Schwann cells and neurons in (A) Col, (B) ColHAlam, (C) ColHA, and (D) ColHA2 hydrogels. Schwann cells (green), neurons (red), and nuclei (blue). Neurites were observed to follow the path of Schwann cells (white arrows). The confocal z-stacks of immunostained images were reconstructed using Imaris software to observe the 3D distribution of cells in the hydrogel. Scale bar is 75 μm. Color images available online at www.liebertonline.com/ten.

Discussion

Current development of nerve tissue engineering scaffolds is aimed at mimicking the native nerve environment in an attempt to improve regeneration. ECM-mimicking scaffolds for neural tissue repair are of great interest because of their potential to mimic the native connective tissue and interact with glia cells and neurons. Schwann cells are effective in supporting nerve regeneration in the peripheral nervous system as well as in the central nervous system. Here, we present a novel paradigm to study Schwann cell viability, proliferation, and function in 3D ECM-mimicking scaffolds containing major neural ECM components present together in a single hydrogel system.

Previous studies on Schwann cells on two-dimensional matrices and inside 3D hydrogels^15,42–45 have been performed to better understand nerve repair mechanisms and in an attempt to develop nerve regeneration scaffolds. Recently, Dadsetan et al. studied the culture of Schwann cells and DRGs on positively charged hydrogels of oligo (polyethylene glycol) fumarate and demonstrated the dependence of cell adhesion and neurite extension on the density of positive charges.⁴⁵ Recently, porous two-dimensional and 3D collagen scaffolds were prepared with varying crosslinking density using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), and Schwann cell migration was monitored through the porous hydrogels. Cells were able to infiltrate and migrate through the porous gels.³⁷ Although these results were encouraging, the use of EDC prohibits mixing cells in the hydrogel before crosslinking, which may ultimately limit cell loading and uniformity of cell distribution.

Other studies on Schwann cell–encapsulated collagen gels revealed that the cells were not able to spread inside the scaffold and maintained a spherical or undifferentiated morphology as opposed to their normal spindle- and star-shaped morphology.¹⁵ Schwann cells with a spherical morphology have altered expression of cell adhesion molecules (e.g., N-CAM) and thus fail to provide guidance cues to regenerating axons.¹⁵ Further, the cells did not secrete physiological levels of growth factors such as NGF.¹⁵

Therefore, we attempted to recapitulate the physical, chemical, and biological properties of neural tissue by synthesizing cell-laden hydrogels of HA, collagen, and laminin. The process did not involve the use of any toxic crosslinkers such as EDC or glutaraldehyde. The current system demonstrates a synergistic approach providing various physiochemical, biological, and chemical cues in the form of hydrogel matrix components, encapsulated Schwann cells, and the released growth factors, respectively, to enhance the neural regeneration process. The cells in the hydrogels exhibited a spread morphology and were successfully able to express cell adhesion molecules such as N-CAM (Supplemental Fig. S2; available online at www.liebertonline.com/ten). Collagen gels were utilized as controls since collagen gels have previously been used for Schwann cell encapsulation and neural regeneration.^15,42

Our results clearly demonstrate that the hydrogels developed here support cell viability for 2 weeks. The encapsulated Schwann cells were not only viable in the hydrogels but also underwent spreading and proliferation over the culture period. As clearly evident from the results, Schwann cells proliferated and increased in number on day 14 in hydrogel groups containing high cell density. The cells in these gels exhibited close cell–cell contacts, which is necessary for cell proliferation and tissue formation. SEM results suggest that the cells in the 3D environment may be remodeling the matrix by secreting ECM molecules (Fig. 8). In contrast, samples with low cell density resulted in minimal cell proliferation. This is likely attributed to negligible cell–cell communication. In this case, the cells maintained a quiescent morphology and expressed very low levels of the S100 Schwann cell marker (Fig. 7). Among samples containing high cell density, no significant difference was observed in the viability and proliferation rates of Schwann cells.

The functional activity of the encapsulated Schwann cells was further explored in terms of growth factor release from these samples—in particular, by NGF and BDNF quantification. Laminin-containing hydrogels induced significantly higher NGF and BDNF secretion overall compared to other samples. Previous studies have established that laminin is a permissive molecule for Schwann cells, which enhances Schwann cell adhesion, proliferation, and growth factor secretion. Armstrong et al. demonstrated that Schwann cells grown on laminin, when compared to other ECM molecules, secrete higher levels of growth factors leading to enhanced neurite length.⁴⁶ These growth factors play important roles in neural regeneration by participating in various neuronal cell signaling events. During nerve regeneration, for example, Schwann cells in the distal stump release growth factors, which provide chemical cues to the regenerating axons.⁴⁷ Griffin et al. studied the effect of NGF concentrations on neurite extensions. The study demonstrated that increasing the NGF concentration resulted in altered growth cone and neurite morphology.⁴⁸ Not only do these neurotrophic factors promote neurite outgrowth, but they also promote the survival of injured neurons indirectly via glial cells. Anton et al. demonstrated that NGF and low-affinity NGF receptors promote the migration of Schwann cells. Schwann cells migrated more rapidly on regenerating sciatic nerves and the addition of NGF antibodies inhibited their migration.⁴⁹ Verderio et al.⁵⁰ found that mutual interaction between nerves and Schwann cells in normal and regenerating neurons results in elevated BDNF secretion from Schwann cells and the growth factor in turn enhanced growth and survival of neurons.

Schwann cell alignment is a crucial step for axonal regrowth. The current study was aimed at devising an ECM-mimicking scaffold composed of native ECM components that can support neural cell survival and growth, which was successfully demonstrated with our results. SEM images also revealed an interesting observation in which Schwann cells appeared to align in parallel and form columnar structures reminiscent of Bands of Büngner, which are formed during Wallerian degeneration after nerve injury. Future refinement of these hydrogels should be directed toward obtaining directional cellular guidance either via magnetic alignment of hydrogels as demonstrated earlier for pure collagen gels.¹⁵ It would also be interesting to introduce this hydrogel matrix in a multi-lumen tube mimicking nerve fascicles to guide neurons.

Conclusions

Schwann cells were encapsulated in novel collagen–HA IPN scaffolds with and without laminin, and were analyzed for viability, proliferation, and growth factor release. The ECM-mimicking hydrogel and the mild fabrication conditions supported cell viability in all hydrogels groups. Schwann cells underwent spreading and proliferation and secreted NGF and BDNF in hydrogels with high cell density. In hydrogels with low cell density, Schwann cells mostly maintained a rounded morphology, expressed very low levels of S100 protein, and secreted minimal levels of growth factors. Schwann cells in laminin-containing hydrogels secreted the highest levels of growth factors compared to other groups.

To our knowledge, no other study has demonstrated synergistic effects of multiple ECM components on Schwann cell viability, proliferation, and growth factor release in 3D in a biomimetic scaffold. Further, the hydrogel matrix also supported the co-culture of Schwann cells and neurons. We hypothesize that biomimetic scaffolds employing native ECM components and support cells will contribute to the general understanding of how biomaterials such as hyaluronan, collagen, and laminin participate and augment the nerve regeneration process.

Footnotes

Acknowledgments

The authors would like to thank Angela M. Bardo and Dwight Romanovicz for technical assistance. The authors would like to acknowledge the financial support from National Science Foundation (CBET 0500969).

Disclosure Statement

No competing financial interests exist.

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