Implantation of 3D Constructs Embedded with Oral Mucosa-Derived Cells Induces Functional Recovery in Rats with Complete Spinal Cord Transection

Abstract

Spinal cord injury (SCI), involving damaged axons and glial scar tissue, often culminates in irreversible impairments. Achieving substantial recovery following complete spinal cord transection remains an unmet challenge. Here, we report of implantation of an engineered 3D construct embedded with human oral mucosa stem cells (hOMSC) induced to secrete neuroprotective, immunomodulatory, and axonal elongation-associated factors, in a complete spinal cord transection rat model. Rats implanted with induced tissue engineering constructs regained fine motor control, coordination and walking pattern in sharp contrast to the untreated group that remained paralyzed (42 vs. 0%). Immunofluorescence, CLARITY, MRI, and electrophysiological assessments demonstrated a reconnection bridging the injured area, as well as presence of increased number of myelinated axons, neural precursors, and reduced glial scar tissue in recovered animals treated with the induced cell-embedded constructs. Finally, this construct is made of bio-compatible, clinically approved materials and utilizes a safe and easily extractable cell population. The results warrant further research with regards to the effectiveness of this treatment in addressing spinal cord injury.

Keywords: oral mucosa; regenerative medicine; spinal cord injury; stem cells; tissue engineering.

Figure 1

Characterization of hOMSC constructs. (A) Preparation scheme of naïve and induced hOMSC constructs. (B) Viability of hOMSC after on-scaffold induction (green indicates viable cells and red indicates dead cells), scale bar = 500 μm. (C) GFP signals showing cellular projections in hOMSC-GFP engineered cells after induction, scale bar = 10 μm. (D) RT-PCR-based comparison between 3D on-scaffold-induced hOMSCs (dotted bars) and hOMSCs induced in culture plates (solid bars): pluripotency and neural crest markers (green and magenta), neuronal markers (yellow), astrocytic markers (red), and neurotrophic factors (blue). Bars represent fold-increase compared to naïve hOMSCs (mean ± SEM). Statistical differences between 2D and 3D induced hOMSC (n = 3/group) were assessed by T-test (^*p < 0.05). Astrocyte markers GFAP (green) and EAAT1 (red) in naïve hOMSC constructs (E) and induced constructs (F) (scale bar = 100 μm, n = 5/group).

Figure 2

In-vivo analysis of therapeutic effects of implanted induced constructs. (A) Implantation scheme. Following complete transection at T10, cell-embedded, or acellular scaffolds are implanted in the transection site and sealed with an acellular PLLA/PLGA scaffold. (B) Representative images of rat posture 43-days following implantation of an induced-construct (bottom) vs. transection only (top). (C) BBB scores over time of rats treated with induced constructs (n = 12, blue), naïve constructs (n = 5, green), acellular scaffolds (n = 10, red), or left untreated (none, n = 16, cyan). The dotted line indicates the results of a long-term efficacy study for the last group treated with induced constructs (n = 3). Data are presented as mean ± SEM, and two-way ANOVA with Bonferroni post-hoc test evaluated statistical differences between the four groups over time (^*p < 0.05, ^**p ≤ 0.01, ^***p < 0.001, ^****p ≤ 0,0001 between induced and acellular constructs: blue-red indication; between induced constructs and none: blue-cyan indication). (D) Maximum BBB score per experimental group histogram (E) Coordinated gait analysis of intact (upper left), acellular (upper right), and induced construct groups, on days 11 (lower left) and 35 (lower right) post-implantation. Gait pattern legend- hind-right (HR), front right (FR), hind left (HL), front left (FL). (F) Results of the nociceptive perception test of the hind limbs and tail in rats treated with induced constructs vs. acellular scaffolds (+: responsive, −: unresponsive to the stimulus, n = 4/group).

Figure 3

In vivo imaging and electrophysiology. (A) FA analysis obtained from MRI data of intact rats, rats treated with induced constructs (n = 3, mean ± SEM) and rats treated with acellular scaffolds (n = 3/group, mean ± SEM). Data are presented as mean ± SEM, one-way ANOVA with Bonferroni post-hoc test evaluated statistical differences between the groups at each respective position (^*p < 0.05). (B) Electrophysiology scheme. The rat motor cortex was stimulated by single spikes. The contralateral sciatic nerve was exposed and MEPs were recorded. Following recording, the spinal cord was retransected at C5 and stimulation and recording were performed again to verify signal propagation through the spinal cord. (C) Representative recordings of the sciatic nerve in intact rats (blue), rats treated with induced constructs (brown) or with acellular scaffolds (black), and re-transected rats treated with induced constructs (green). (D) Quantification of MEP amplitudes in rats treated with induced constructs (n = 3), acellular scaffold (n = 3) and retransected induced constructs (n = 3). Data are presented as mean ± SEM, one-way ANOVA with Bonferroni post-hoc test evaluated statistical differences between the groups (^*p < 0.05, ^**p < 0.01).

Figure 4

Spinal cord immunofluorescence demonstrating neuronal integrity and axonal elongation-associated markers. DAPI is marked in blue in all immunohistochemistry images. (A) Images of spinal cord immunofluorescence staining 8 weeks after implantation of induced constructs and acellular constructs (in descending order): Human nuclear staining, TUJ1 and NF200, GAP43, MBP, nestin and spinal cord beta III tubulin. Scale bar = 200 μm, magnification insets scale bar = 40 μm, n = 5/group. (B) Computer-based quantification of staining (n = 5/group). Top—axonal and neuronal axonal elongation markers, middle—MBP-positive elongated processes, bottom—nestin-positive cells. (C) Immunofluorescence staining of longitudinally sectioned spinal cord tissue in rats with acellular PLLA/PLGA scaffolds (left) or induced constructs (right) 8-weeks after lesion. Dotted line indicates scaffold area. Axonal elongation marker GAP43 (green) and DAPI (blue), scale bar = 200 μm, zoom in inset = 50 μm. Data are presented as mean ± SEM, one-way ANOVA with Bonferroni post-hoc test evaluated statistical differences between the groups (^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 5

Spinal cord expression of glial scar and inflammation-associated markers. (A) Spinal cord immunofluorescence staining 8 weeks after implantation of different treatment groups (in descending order): CSPGs and GAP43, and GFAP and CD11b, DAPI is marked in blue in all immunohistochemistry images (scale bar = 200 μm, magnification insets scale bar = 40 μm, n = 5/group). (B) Computer-based quantification of staining (n = 5/group). Data are presented as mean ± SEM, one-way ANOVA with Bonferroni post-hoc test evaluated statistical differences between the groups (^*p < 0.05, ^**p < 0.01).