Spinal cord injuries (SCIs) are characterized by the inability of injured neurons to self-repair or self-regenerate, leaving patients with SCIs permanently paralyzed below the site of injury. Several therapeutic interventions have been tested with intentions of reversing the devastating consequences of SCIs. Recently, emerging evidence from the literature demonstrates that cell transplantation is one of the most promising approaches that is expected to yield great outcomes. Schwann cells (SCs),1 olfactory ensheathing cells (OECs),2,3 nerve stem cells4 and bone marrow mesenchymocytes5 have been transplanted to overcome SCI in animal models in vivo. Among these cells, OECs are thought to be most promising for transplantation, since once transplanted they are shown to provide a permissive local micro-environment for nerve regeneration in a generally inhibitory mature central nerve system.6 Promising as they may be, the therapeutic efficacy of the cell-transplantation approach is limited because most of the transplanted cells are currently delivered to hosts in suspensions. This may have negatively affected the expected outcomes of the approach because of the absence of extracellular matrix (ECM) proteins in suspensions, which are critical for survival, adhesion, and proliferation of transplanted cells.
Various researches are being directed at the search for bioengineered scaffolds suitable for cell transplantation for SCIs. Recently, fibrinogen (FG), fibronectin (FN) and laminin (LN) have been applied as base materials or modifying motifs to repair injured spinal cords.7 Fibrin gel has enjoyed widespread applications in neuronal tissue repair to encourage neurite outgrowth in vitro, and nerve regeneration across spinal cord lesion gaps in vivo.8–10 Recent studies confirmed that salmon-derived fibrin gels indeed promoted extensive neurite outgrowth, and suggested that a fibrin-based tissue-engineered scaffold could be conducive to nerve regeneration following a SCI.11,12 FN-based materials prepared from human plasma have been used in various forms as substrates for tissue engineering. A shear-aggregated FN gel could support in vitro growth of fibroblasts, SCs and astrocytes, and implantation of this FN gel into a rat model of SCI provided a permissive environment for axonal growth with promoted orientation and straightness.13 Likewise, many studies have indicated that LN sustained cell survival and promoted nerve fiber extension in both peripheral and central nervous systems.14 Therefore, we believe that in cell transplantation strategies, it is advantageous to prepare a scaffold that serves initially as a provisional ECM for the transplanted cells to survive, proliferate and differentiate, and gradually degrades over time while the transplanted cells migrate within and remodel the ECM scaffold to achieve tissue repair.
In this study, we described a new ECM-based scaffold (ECM-S) that can be easily prepared by gelling FG, FN and LN using fresh rat plasma. We also explore the structural properties of the novel scaffold and its biological effects on seeded OECs in vitro. The findings of these studies provide insights into design strategies of a novel implantable bioengineered hybrid scaffold to promote axonal regeneration following SCIs.
Primary rat OECs were isolated and cultured as previously described.15 OECs were derived from the olfactory mucosa, and then cultured in DMEM-F12 medium (Invitrogen, USA) with 10% fetal bovine serum (FBS, Invitrogen, USA). In order to obtain more homogenous OEC cultures, protocols by Nash et al16 were used. Following the differential adhesion hypothesis, cell suspensions were collected after the first 24-hour culture. After washing, the collected cells were plated onto a culture flask pre-coated with LN. Using this process, we consistently achieved an OEC culture of 93% purity; for even better homogenous OEC cultures, the purification procedure was repeated three times. Purified OECs were used to seed the ECM-S after a parallel culture was characterized and confirmed by immunostaining (described below) using various markers, such as glial fibrillary acidic protein (GFAP), low-affinity nerve growth factor receptor (NGFRp75), S-100, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), matrix metallo-proteinase-3 (MMP-3), MMP-9 and laminin-R (Sigma, USA).
Fabrication and stability test of the ECM-S
To prepare the ECM-S, FN and LN (Sigma) were first mixed in serum-free culture medium (pH 7.2) to achieve a concentration of 10 μg/ml each. This was followed by dissolving FG (Sigma) in the FN/LN mixture to reach a concentration of 100 mg/ml. This resulted in a clear, viscous and homogeneous solution which gelled in approximately 30 minutes after mixing with an equal volume of fresh rat plasma. Applying this technique, different shapes of scaffolds were prepared using various molds; mat-like scaffolds using round coverslips in a 24-well plate or cylindrical scaffolds using hollow tubular molds. For macroscopic observations, the mat-like scaffold was lifted off the coverslip mold and photographed with a digital camera. To show the in vitro stability of the scaffold, the cylindrical scaffold was first stained with 0.2% trypan blue, immersed in sterile tris-buffered saline (TBS) in a 1.5 ml-Eppendorf tube and photographed with a digital camera. The incorporation of the trypan blue was used to test for any significant short-term scaffold dissolution in an aqueous solution. As described below, the surface ultra-structure of the mat-like scaffold and the ultra-structure inside of the cylindrical scaffold were recorded using a scanning electron microscope (SEM) and a transmission electron microscope (TEM), respectively.
Seeding of OECs on the ECM-S
Purified OECs were removed from the culture flasks to seed the scaffolds immersed in a trypsin-EDTA solution. To study the effects of the scaffold on cell adhesion and growth, 1 ml of the purified OEC suspension was used to seed the cells on top of surfaces of mat-like scaffolds (experimental groups), or poly-lysine coated coverslips (control groups, hereinafter referred to as coverslip) in 24-well plates or 35 mm-dishes (for immunoblotting analysis) at a plating density of 1×105 cells/cm2. In parallel, in order to study OEC growth within scaffolds, cells were first suspended in the FN/LN solution at a concentration of 1×106 cells/ml. To the OECs containing FN/LN mixture, FG was added to make up a final concentration of 100 mg/ml. The mixture was then mixed with an equal volume of fresh rat plasma and molded into OEC-laden cylindrical scaffolds. Finally, the OEC-laden cylindrical scaffolds were cultured in DMEM-F12 culture medium containing 10% FBS. The culture medium was changed every three days. The surface ultra-structure of the OEC-seeded mat-like scaffolds and the ultra-structure inside of the seeded cylindrical scaffolds were recorded using a SEM and a TEM, respectively.
At pre-determined time intervals of the 1st, 3rd and 5th hours, the scaffolds were carefully rinsed with the culture medium to remove the non-adherent cells, and the cultured cells were labeled with Hoechst 33342 (0.5%, w/v). The labeled OECs were counted under five random non-overlapping fields (count-box, 0.2 square mm in dimension) using a Leica DM LB2 florescence microscope (Leica Microsystems, Germany). Treated under the same conditions, OECs cultured directly on coverslips were used as controls.
Assay for cellular proliferation and process extension
At pre-determined time intervals, i.e. days in vitro (DIV) 1, 4, 7, 14, 21, 28 and 35, the scaffolds and cultured cells were treated as previously described in the cell adhesion experiment, and cell numbers were recorded. In parallel, separate cultures were fixed with 4% paraformaldehyde at the same time points, and the OECs were immunostained with GFAP so that the OEC processes could be visualized under the florescence microscope. The lengths of the longest processes from 50 random (and different) OECs were measured and the average lengths from these cells were calculated. OECs cultured directly on coverslips were used as controls.
Capacity of producing NGF, MMP-3 and MMP-9
At pre-determined time intervals of DIV 4, 7 and 14, the cultured OECs on the scaffolds were studied by immunoblotting (as described below) using antibodies against representative neurotrophin (NGF) and two antibodies against MMPs (MMP-3 and MMP-9) with the β-actin as the standard.
Samples to be analyzed were fixed overnight with 4% paraformaldehyde at 4°C. After being washed twice with TBS (0.5 mol/L, pH 7.6), the cells were first permeabilized with 0.3% Triton X-100 in TBS and then immuno-blocked with 20% (v/v) normal goat serum and 1% (w/v) BSA in TBS. Next, the samples were incubated separately overnight with respective primary antibodies in TBS containing 1% (w/v) BSA and 0.3% (v/v) Triton X-100 at 4°C. The samples were subsequently rinsed in TBS and incubated with Cy3-conjugated secondary antibodies at 37°C for 1 hour. Parallel negative controls were subjected to the same procedures without using either primary or secondary antibodies. Finally, the preparations were washed with TBS, stained with nuclear fluorescent dye Hoechst 33342 (0.5%, w/v) and coverslips were placed with neutral buffered glycerol. Observations and photographs were documented using a Leica DM LB2 fluorescence microscope (Leica Microsystems) coupled with a digital camera, and the results were analyzed with a Leica QWin image analysis software (Leica Microsystems).
Scanning electron microscopy
Samples for SEM were fixed in 2.5% glutaraldehyde (Sigma) for 24 hours, dehydrated with a graded ethanol series, and at the critical-point dried in CO2. The dried samples were mounted on aluminum and sputter-coated with gold.
Transmission electron microscopy
Samples for TEM were fixed in 2.5% glutaraldehyde (Sigma) for 24 hours, embedded and sectioned into ultra-thin sections and mounted on copper grids. The mounted samples were post-stained with 1% uranyl acetate followed by lead citrate.
Briefly, OEC lysate was extracted using a RIPA lysis buffer, solubilized in SDS-PAGE sample buffer, electrophoresed in a 10% SDS-PAGE gel then blotted onto a PVDF membrane. The PVDF membrane was incubated with the antibodies against NGF or MMPs and HRP-conjugated goat anti-rabbit immunoglobulin, visualized using the ECL Plus Western blotting detection system PRN2132 (Amersham Biosciences, UK) and finally scanned with a Typhoon 9400 variable mode imager (Amersham Biosciences).
One-way analysis of variance (ANOVA) or paired t test was used to compare the average number of the adherent cells per count-box and the average length of the longest process of the OECs on both scaffold and poly-lysine coated coverslip surfaces at different post-seeding hours or DIV. Fisher's protected least significant difference (PLSD) was used as a post-hoc test with the SPSS 12.0. A P value less than 0.05 was considered statistically significant.
Labeling of the cultured OECs
In order to obtain a purified OEC culture, OECs from primary cultures were purified from heterogeneous cell populations based on the preferential adherence of different cell types (e.g. fibroblasts) to culture substrates. As shown in Figure 1, after the purification process, nearly all of the cultured cells were bound by antibodies against specific OEC markers including GFAP, NGFRp75 and S-100 (Figure 1A, 1B and 1C, respectively). The results indicated that the purified cells were OECs, suggesting that a relatively pure OEC culture could be established in vitro by following the protocols described in this study. Expression of NGF, BDNF and NT-3 (Figure 1D, 1E and 1F, respectively) in OECs confirmed their neurotrophic roles. Furthermore, OECs expressed MMP-3 (Figure 1G) and MMP-9 (Figure 1H) which possessed the ability to cleave most ECM proteins. They also expressed the cell adhesion receptor LN-R (Figure 1I), confirming the presence of LN receptors on their cell surface. These data suggested that a highly purified OEC culture could easily be obtained and maintained in vitro using the cell purification and culture protocols used in this study and that GFAP was as a reliable OEC marker.
Structural properties of ECM-S without seeded OECs
In this study, two ECM-S with different shapes were created as follows:
The mat-like ECM-S
On the coverslip per well in a 24-well culture plate, 20 μl of a mixture containing FG, FN and LN with an equal volume of fresh rat plasma (total 40 μl of gel per well) could form a transparent, mat-like scaffold with a 12-mm diameter and a thickness of 0.35 mm (Figure 2A). SEM observations showed that the mat-like scaffold was a three-dimensional (3-D) porous network with 1.5 μm diameter pores (Figure 2B).
The cylindrical ECM-S
Using a tuber mold, the same 40 μl gel used for the mat-like ECM-S could be molded into a cylindrical scaffold with a 2.8-mm diameter and a length of 7.0 mm. To demonstrate the structural integrity of the resulting structure, a cylindrical scaffold stained with trypan blue dye was soaked in sterile saline. As shown in Figure 3A, the structure was well maintained even after 4 weeks in sterile saline, demonstrating its stability in an aqueous environment. Our TEM observations showed that the scaffold had a sponge-like porous structure which could potentially facilitate cell growth and migration within the scaffold (Figure 3B). Interestingly, the hydrogel network of the scaffold was composed of many fibers of ECM molecules, which were randomly interwoven to form porous structures 1 to 2 μm in size.
Effects of the scaffold on the adhesion and growth of the OECs
In order to study the effect of the scaffold on OEC adhesion and growth, OECs were seeded on mat-like flat ECM-S surfaces, and cell numbers as well as the length of the longest cell processes from 50 random cells were characterized. The expression of some beneficial proteins such as NGF and MMP-3, MMP-9 were also studied.
Effect of the ECM-S on OEC adhesion
As shown in Figure 4, the ECM-S promoted OEC adhesions in comparison with coverslips (P <0.01). The results from the cell number measurements showed that at the 1st, 3rd, and 5th hours post-seeding, the percentage of the cell adhesions were 26.8%, 68.4% and 96.5% in the scaffold group, and 8.5%, 29.3% and 66.0% in the coverslip group, respectively. One-way ANOVA indicated that there was a significant difference in cell adhesion at the 1st, 3rd and 5th hours for both the scaffold and coverslip groups (P <0.05).
Effect of the ECM-S on OEC proliferation
As shown in Figure 5, the ECM-S promoted OEC proliferation in comparison with coverslips. There were more cells on the scaffolds than on the coverslips during the course of the experiment except for DIV 1 (P >0.05) and DIV 14 (P <0.01). The number of the OECs on the scaffold peaked on DIV 7, seven days earlier than in the control (peaked on DIV 14), although there was no significant difference in the peak cell numbers between the scaffold group and the control.
Effect of the ECM-S on OEC process extension
As shown in Figure 6A, the length of the longest processes of 50 random OECs both on the scaffold and the coverslip was characterized. In comparison with those on the coverslip, OECs on the scaffold had significantly longer processes (P <0.05 for DIV 1 and P <0.001 for other six pre-determined time intervals). In addition, GFAP immuno-labeling showed that after 14 days in culture, OECs cultured on the ECM-S (Figure 6C) showed dramatically longer and more aligned growth processes as well as more fasciculations of these processes than those on the coverslip (Figure 6B). Furthermore, the SEM micrographs of the OECs on the scaffold further confirmed that, after 4 days in culture, OECs arranged loosely and extended short, few processes (Figure 6D), whereas on DIV 14, the cells tightly adhered to the surface of the scaffold, extended longer processes in more aligment, which nearly covered the surface of the mat-like scaffold (Figure 6E).
Expression of NGF and two MMPs in OECs on the ECM-S
To explore whether OECs on the ECM-scaffold maintained the capacity of producing the neurotrophin NGF, as well as MMP-3 and MMP-9, the OECs on the scaffolds and the coverslips cultured for 4, 7 and 14 days were studied using immunoblots with antibodies against these proteins. As shown in Figure 7A, the OECs on the scaffolds expressed more NGF, MMP-3 and MMP-9 than those on the coverslips on different culturing days. Additionally, NGF and MMP-3 immuno-labeling revealed that their granules were distributed in the cell body as well as in the long processes of the spindle-shaped OECs on the scaffold cultured for 14 days (Figure 7B and 7C).
Growth of the OECs in the cylindrical ECM-S
In order to study the cell distribution of the OECs within the scaffold after seeding, Hoechst 33342 was used to stain the nuclei of live cells after 7 days of seeding the scaffold. As shown in Figure 8A, a relatively high seeding density and multilayer distribution of OECs in the scaffold for 7 days were achieved; it was evident that the OECs were well maintained within the scaffold in vitro for that time duration. Figure 8B shows OECs stained for GFAP 7 days after seeding. It is interesting to see that the OECs seeded inside of the scaffold had differentiated and assumed different cell morphology (Figure 1A and 1B). It should also be noted that there seemed to be an OEC alignment after 7 days in culture (Figure 8B). Interestingly, as shown in Figure 8C, the spindle-shaped OECs arranged in parallel and extended long processes in the scaffold with some tissue space (marked by asterisks) around the OECs, which might be due to the cellular degradation of the scaffold by enzymes (such as MMPs) secreted by OECs.
We applied a simple method for harvesting and purifying olfactory mucosa-derived OECs which were used in this study to evaluate the performance of our ECM-S in vitro. After identification with antibodies against three common OEC marker proteins (GFAP, NGFR-p75 and S10017–19), the purified OECs were seeded onto scaffolds. The expression of NGF, BDNF and NT-3 in the OECs showed that this glial cell type from the olfactory mucosa also had a neurotrophic role.20 OECs also expressed LN-R, the receptors responsible for cell adhesion and migration. Interestingly, in addition to expressing MMP-2 (as reported by Pastrana et al21), we found that OECs could express MMP-3 and MMP-9. This finding, which indicated that the OECs could degrade ECM proteins of the scaffold, will be discussed in detail.
In this study, an ECM-S was prepared by initially dissolving three major soluble ECM components (FG, FN and LN), which were subsequently solidified by introducing fresh rat plasma to the aqueous mixture. We showed that in a fibrinogen environment (a concentration of 50 mg/ml and pH 7.2), the ECM-S could be molded into two different shapes which were transparent and stable in buffered solution for 4 weeks. These data were well in agreement with results obtained from other researchers,22–24 who had reported that, with increasing fibrinogen concentration (range, 10–100 mg/ml), the fibrin-formed scaffold increased in tensile strength and stability, had smaller pore sizes, and became transparent in a pH range of 6.8 and 9.0. It is well known that plasma can provide activated thrombin, which in turn polymerizes water soluble fibrinogen into insoluble and relatively stable fibrin gel.25–27 We showed that the resulting ECM-S exhibited a porous, 3-D network. This porous structure could allow the diffusion of nutrients and other soluble molecules in the cell culture medium, thus acting as a conducive tissue-engineered scaffold. The formation of porous structures may be the result of interactions between the three ECM proteins. Besides the apparent crosslinking of fibrinogen to form fibrin in the presence of externally introduced thrombin, it is also reported that the addition of LN, as well as the binding between FN and fibrin at the fibrin-binding domain of the FN peptide sequence could contribute to the gel formation in the mixture.28
For a bioengineered scaffold to be successful, the scaffold must promote cell adhesion, proliferation, migration and differentiation. Our scaffold showed significantly increased OEC adhesion in comparison with the coverslip control group. The time expended by most cells on adhering to the scaffold was about 5 hours, when there was lower than 70% cell attachment in the coverslip group. This enhanced cell adhesion can be attributed to cell adhesions to all three components of the scaffold, i.e., FG, FN, and LN, which have been reported to play key roles in promoting cell adhesion.29,30 All three components have a common cell adhesive peptide sequence RGD (Arg-Gly-Asp) to bind specifically with integrin receptors, such as LN-R, on the cell surface. It is worthwhile to note that our results clearly showed that the OECs expressed LN-R when seeded within the scaffold (Figure 1I). This likely explains why the ECM-scaffold exhibited enhanced cell adhesion and promoted OEC growth and process extensions when compared with coverslip controls.31,32 For example, in comparison with the control group, there were significantly higher adherent cell counts in the scaffold group; these cell counts peaked earlier than in the control group. The result suggests that the scaffold could promote cell proliferation especially during the early stage of the OEC culture. It also should be noted that at the later stage of the OEC culture, despite of the fact that the cell numbers in both groups appeared to decrease, the cell number in the scaffold group remained constantly at a higher level, suggesting that ECM-S was a better substrate for cell growth. Well in agreement with the data obtained from Eyrich et al22 which showed that the growth of bovine chondrocytes was well maintained for about 5 weeks in fibrin gels in a final fibrinogen concentration of 50 mg/ml, our study showed that OECs plated on or in the scaffold exhibited significant migration by forming alignment after 7 days in culture and extended long processes (Figures 6A and 8B). This finding, however, has conflicts with data published by Bensaïd et al.24 According to their results, the fibrin scaffold was too dense to allow cell growth or migration at fibrinogen concentrations of 50 mg/ml. However, we believe that the preferred directional migration and cell alignment might be due to the cell type seeded but not the orientation of ECM-fibers in the scaffold, since the OECs in the scaffold are capable of secreting a series of MMPs (an enzyme family including earlier mentioned MMP-2, MMP-3 and MMP-9) that could digest the cross-linked fibrin to facilitate cell migration and process extension. The existence of tissue space around the cell body especially at the headend of process extension may be the best proof of scaffold degradation. Recent studies have confirmed that MMP-3 carries out the degradation of cross-linked fibrin through specific hydrolysis of the gamma Gly 404-Ala 405 peptide bond, which is different from plasmin's mechanism of action.33,34 In addition, MMP-9 has been reported to participate in fibrin degradation by the plasmin-independent mechanism of fibrinolysis.35
The ultimate goal of this study was to prepare implantable OECs seeded on a biosynthetic hybrid scaffold to promote nerve regeneration after a SCI. We believe that early proliferation and subsequent cell process extensions of OECs on or within the scaffold are of extreme importance for the transplant to be effective when the OEC-seeded ECM-S is transplanted into the spinal cord lesion. In our study, OECs on the scaffold were shown to consistently express neurotrophic factors such as NGF. Therefore, an increasing OEC population within the scaffold at the early stage of the implantation would eventually increase the expression of neurotrophic factors, which would be made available for nerve regeneration.36 At a later stage, OEC migrations, alignments and the process extensions will facilitate elongations and assist in pathway alignment of regenerating nerve fibers — a task that OECs have long been shown to carry out in developing and regenerating olfactory systems that guide the olfactory nerve fibers to the olfactory bulb and help form synapses with mitral cells.37 Tisay et al18 reported that this outgrowth and guidance of olfactory axons were promoted by OECs during the development, based on the foundation of an ECM region rich in LN and heparan sulfate proteoglycans secreted by OECs. In addition, recent studies suggested that the presence of MMPs was beneficial for cell migration and neurite extension during development or after a stroke or brain injury.38,39 In adults, tangential migration along the subventricular zone, olfactory bulb pathway (termed the rostral migratory stream, RMS) takes place rapidly in “chains” of cells contained within glial tubes. In neonates, the RMS (which contains MMPs but lacks defined glial tubes and chains) allows newborn neuroblasts to migrate instead as individual cells. Therefore, MMPs play an important role in the migration of newborn neuroblasts. We believe that the MMPs secreted by OECs in our study could have opened up channels for migration and process extensions within the scaffold matrix. When implanted in vivo, this might result in the formation of glial tubes that could guide axonal regeneration. Therefore, an OEC differentiation phase is desired during the late stage of the implantation. It is important to note that if the OECs proliferate excessively during the late stage, a new glial scar tissue could form as a result of excessive OEC growth, which could eventually prevent nerve regeneration. Our results showed that the scaffold did promote OEC proliferation in the first 7 days of seeding and maintained cell numbers at a relatively high level for up to 28 days in the culture after seeding, suggesting that the ECM-S is the ideal scaffold for this approach. However, the length of processes of OECs on the scaffold during the late stage of the culture gradually decreased (Figure 6A), and eventually reached the 28-day control level. This decrease in both the length of processes and cell counts during the late stage of the culture is highly likely due to degradation of the scaffold material mediated by MMPs.
As has been discussed already, the ECM-S is an ideal scaffold for various tissue engineering applications. This is because ECM molecules have been shown to possess cell adhesive peptide sequences (such as RGD) for enhanced cell adhesion.40,41 In addition, LN has a domain full of EGF-like sequences that have been shown to promote cell growth. Furthermore, ECMs have also been shown to be involved in cell proliferation and differentiation. For example, previous investigations indicated that LN was over-expressed in neuroepithelial cells at both protein and mRNA levels after stimulation with soluble fibroblast growth factors (FGF); and after a long nerve growth factor stimulation, PC12 cells were shown to extend their processes on an uncoated plastic culture dish; ECMs molecules — supposedly produced by the cultured PC12 cells — were detected in the culture medium.42 Combined, these evidence suggest that the ECMs also serve as soluble signals that promote cell proliferation and differentiation, especially during the early stage of proliferation. This conclusion is consistent with our observations demonstrating that when compared with the controls, the ECM-S showed significant OEC proliferation and differentiation during the DIV 4-DIV 21 time intervals.
From this study, we conclude that our tissue engineered ECM-S can promote adhesion, proliferation, differentiation, alignment of the OECs, thus making it a promising candidate for preparation of OEC-populated, tissue engineered, biosynthetic hybrids. These hybrids could probably be used as implants to help overcome SCIs.
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