Adult spinal cord tissue transplantation combined with local tacrolimus sustained‐release collagen hydrogel promotes complete spinal cord injury repair

Abstract The strategy of replacing a completely damaged spinal cord with allogenic adult spinal cord tissues (aSCs) can potentially repair complete spinal cord injury (SCI) in combination with immunosuppressive drugs, such as tacrolimus (Tac), which suppress transplant rejection and improve graft survival. However, daily systemic administration of immunosuppressive agents may cause harsh side effects. Herein, a localized, sustained Tac‐release collagen hydrogel (Col/Tac) was developed to maximize the immune regulatory efficacy but minimize the side effects of Tac after aSC transplantation in complete SCI recipients. Thoracic aSCs of rat donors were transplanted into the complete thoracic spinal cord transection rat recipients, after which Col/Tac hydrogel was implanted. The Tac‐encapsulated collagen hydrogel exhibited suitable mechanical properties and long‐term sustained Tac release behaviour. After Col/Tac hydrogel implantation in SCI rats with aSC transplantation, the recipients' survival rate significantly improved and the side effects on tissues were reduced compared with those with conventional Tac medication. Moreover, treatment with the Col/Tac hydrogel exhibited similarly reduced immune rejection levels by regulating immune responses and promoted neurogenesis compared to daily Tac injections, and thus improved functional restoration. Localized delivery of immunosuppressive agents by the Col/Tac hydrogel may be a promising strategy for overcoming immune rejection of transplants, with significant potential for clinical application in the future.

however, can cause spinal cord injury (SCI), which leads to cell and/or tissue death, interrupting the connections below the injured area, resulting in loss of sensation, voluntary motor dysfunction and even paralysis. Currently, SCI is still incurable. 2 Despite the existence of strategies involving stem-cell transplantation with or without biomaterials that could partially replace damaged cells and tissues, thereby recovering some function in SCI animals, limitations remain. [3][4][5] For instance, extracellular matrix with complex structures and components, and various types of neural and nonneural cells of the spinal cord tissue are difficult to replace or restore.
In previous studies, transplantation of allogenic adult spinal cord tissues (aSCs) promoted locomotor function recovery in complete SCI rats and canines. 6,7 aSC has a natural tissue architecture and microenvironment that may be a potential substrate for replacing damaged neurons and glia and supporting axonal regrowth. 8,9 During aSC transplantation, graft survival at the lesion site is critical for successful SCI repair. Thus, systemic administration of immunosuppressive drugs has been clinically applied to avoid graft rejection-induced apoptosis.
Tacrolimus (Tac, FK506) is a clinical immunosuppressive drug that efficiently inhibits T-lymphocyte proliferation and activation by binding to FK506-binding protein 12 (FKBP12) to block calcineurin activation within lymphocytes. 10,11 However, severe complications, including infection, chronic allograft nephropathy, neurotoxicity, liver damage and even tumour malignancy, may occur with continuous systemic Tac administration. [12][13][14] Compared with systemic administration, local drug delivery systems offer therapeutic levels of Tac at the transplantation area via sustained release for a prolonged period, which reduces toxic effects by decreasing the total drug dosage. [15][16][17] Deng et al. developed poly (lactide-co-glycolide) nanoparticles for Tac delivery and release, which exhibited longer residence time and elimination half-life than free Tac after injection. Hence, with this treatment, acute rejection of heterotopic transplanted hearts was reduced and allograft survival time was increased in comparison with treatment with free FK506. 15 Further, Wu et al. designed a peptide-based hydrogel for Tac release with immune-responsive behaviour. The supramolecule could form a hydrogel, encapsulate the drug through π-π stacking and hydrogen bonding, and release the drug by responding to T-cell activation.
Smearing this responsive hydrogel on graft surfaces after liver transplantation significantly prolonged the hosts' survival time compared to the gavage therapeutic dose of Tac. 16 Hence, effective Tac delivery may be a useful technique for suppressing organ transplantation rejection with fewer side effects. Furthermore, Tac reportedly enhances neural regeneration after peripheral nerve injuries. 18,19 In vitro experiments using human neural stem cells (hNSCs) co-cultured with Tac for 7 days showed that more β-tubulin positive neural cells were observed compared with the control, indicating the potential role of Tac in neuronal differentiation. 20 Collagen is the main structural protein in organs and is widely used in regenerative medicine because of its excellent properties, including low immunogenicity, good biocompatibility and superior biodegradability. [21][22][23] Previous studies have demonstrated that collagen hydrogel can facilitate neural cell migration, growth and differentiation, and also act as a platform to support cell and functional factor (small molecules, genes and proteins) encapsulation and delivery. 24 Clinical studies have also indicated the potential of using collagen scaffolds developed by our laboratory in various tissue regeneration. [25][26][27] Additionally, in aSC transplantation rats, adding a collagen hydrogel-containing growth factor cocktail significantly improved graft survival levels. 6 In this study, we developed a collagen/Tac (Col/Tac) hydrogel to locally deliver immunosuppressive agents to improve the repair effects of the transplanted aSC by immune regulation and reduction of Tac side effects. Tac was loaded onto the collagen hydrogel (Col), and then the sustained release property was measured. The neurogenesis effects of Col/Tac were evaluated using hNSCs. Thoracic aSCs of rat donors were transplanted into the complete thoracic spinal cord transection rat recipients, after which the Col/Tac hydrogel was applied to the transplants and donor-host interfaces to 'glue' the tissues together ( Figure 1A). We hypothesized that the Col/Tac hydrogel could construct an immunosuppressive microenvironment after aSC transplantation to SCI recipients, consequently improving graft survival and reducing transplant rejection, thereby improving functional recovery. The effects of the Col/Tac hydrogels on regulating immune responses and promoting neural regeneration and SCI repair were studied by immunohistology analysis and transcriptomics.
The side effects of the Col/Tac hydrogels were also investigated.

| Animals
Eighty adult female Sprague-Dawley rats (6-8 weeks old) were used as recipients, and 60 adult green fluorescent protein (GFP)-expressing female transgenic Sprague-Dawley rats (6-8 weeks old) were used as donors. All rats were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Rats were housed at a standard temperature and humidity in a 12 h dark/light cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Sciences.

| Surgical procedure
The surgical procedure for the T9 complete transection SCI rats was in accordance with a previous report. 28,29 All rats were anaesthetised via intraperitoneal injection with sodium pentobarbital (50 mg/kg) under aseptic conditions. A laminectomy was performed at the T9 vertebral level with an iridectomy scissor, a complete transection and removal of a 2-mm-long spinal cord was performed for generating hosts, and whole T8-T10 spinal cord tissues were collected from GFPtransgenic rats. aSCs were incubated with phosphate-buffered saline (PBS) containing 500 IU/mL heparin solution (Solarbio, China). All SCI rats were randomly divided into four groups: (1) Group aSC: aSC transplantation into lesion sites in a physiological orientation; (2) Group Col/Tac 5mg : transplantation of 25 μL Col/Tac hydrogel with Tac concentration of 5 mg/mL at defects; (3) Group aSC + Col/Tac 5mg : combination treatment with aSC transplantation and Col/Tac hydrogel.
Briefly, the Col/Tac hydrogel was symmetrically paved at the bottom of the lesion sites and injected at the host-graft interface, following which the spinal cord tissue surface was covered using a microsyringe  Figure S1); and (4) Group aSC + iv.Tac d : daily intraperitoneal Tac injection (1.5 mg/kg) after aSC transplantation. The bladders were manually emptied twice daily after surgery.

| Preparation and characterization of the Col/Tac hydrogel
Collagen hydrogels were prepared from bovine collagen as previously described. 23 Fresh bovine aponeurosis was treated with 1% tri(n-butyl) phosphate for 48 h and 1% trypsin for 1 h successively, then washed with deionized water to remove cells and other proteins.
The treated collagen was then dissolved in 0.5 mol L À1 acetic acid for 24 h, dialyzed in deionized water for 10 days, and then lyophilized.

| Pharmacokinetic study
To test blood concentrations of Tac, blood samples were collected from rats in all four SCI groups at 10, 30 and 60 days post-surgery.
Serum samples were collected after centrifuging the blood samples at 3000 rpm for 15 min, after which HPLC-MS/MS analysis was performed.

| In vivo biocompatibility test
Blood biochemistry indicators for liver, heart and kidney function were measured to assess the systemic toxicity of the Col/Tac hydrogel. The blood samples, hearts, livers, spleens, lungs and kidneys were collected. The organs were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm. The sections were stained with Masson and haematoxylin and eosin (H&E) and observed using an optical microscope.

| NSC culture and differentiation
The derivation, culture and characterization of hNSCs has been described previously. 29 Table S1.

| Electrophysiology
Eight weeks post-surgery, the cortical motor-evoked potentials (MEPs) were measured using the Keypoint bichannel-evoked potential/electro-myography system (9033A07, Dantec Company, Copenhagen, Denmark) as previously described. 31 All rats received the same intraperitoneal administration of anaesthesia.

| Behavioural assessment
The Basso-Beattie-Bresnahan (BBB) locomotor rating scale 28 was recorded weekly by two independent observers who were unaware of the experimental conditions. The main characteristics observed were ankle movement of the hind limbs, state of feet during walking and body stability.

| Histological analysis and immunohistochemistry
After treatment for 10 and 60 days, the animals were anaesthetised and perfused with 4% paraformaldehyde. Subsequently, 4 cm-long spinal cord samples were collected, immersed in 30% sucrose, and embedded in a Tissue-Tek ® O.C.T compound (Sakura Finetek, Torrance, CA, USA). Further, 15 μm sections were made using a Leica CM1950 cryostat (Leica Microsystems, Wetzlar, Germany). Immunohistochemistry was performed as described previously. 28,29 Images were obtained using a Leica SP8 confocal microscope (Leica Microsystems).
The following primary antibodies were incubated overnight at

| RNA-sequencing
RNA-Sequencing (RNA-Seq) was performed to identify the differential genes and their related pathways between the aSC + Col/Tac 5mg and aSC groups, and between the aSC + Col/Tac 5mg and aSC + iv.
Tac d groups, respectively. Three different batches of spinal cord samples were collected at 10 days post-surgery and extracted their total RNA. RNA library sequencing was performed on the Illumina nova-seq6000 by CapitalBio Technology Co., Ltd (Beijing, China). Bioinformatic analysis was performed using R/R-studio and KOBAS.

| Statistical analyses
All data were expressed as mean ± standard deviation. Statistical analyses were performed using analysis of variance test, and p < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS (26.0, IBM, Armonk, NY, USA) and GraphPad Prism

| Effects of the Col/Tac hydrogel on hNSC differentiation
The in vitro toxicity of the Col/Tac hydrogels with different Tac concentrations (0, 1, 2, 5 and 10 mg/mL) was investigated using live/dead cell staining after culturing with hscNSCs ( Figure S3A The effects of the Col/Tac hydrogel on human spinal cord-derived neural stem cell (hscNSC) differentiation were measured by the marker gene expression levels after a 20-day co-cultivation of hscNSCs and Col/Tac hydrogels (containing 0, 2, 5 and 10 mg/mL Tac) in neural differential medium. Nestin, a widely used marker for NSCs, was used to identify the stemness. 32,33 Nestin expression levels were higher in the Col/Tac 5mg group than in the Col group (Figure 2A). The expression level of Tuj-1, a neuron-specific marker, was increased in both Col/-Tac 5mg and Col/Tac 10mg groups ( Figure 2B). Islet1, a motor neuronrelated gene, showed significantly high expression in the Col/Tac 2mg , Col/Tac 5mg and Col/Tac 10mg groups. Map2, a neuronal dendritic marker, was overexpressed in the Col/Tac 2mg group and showed slightly high expression levels in the Col/Tac 5mg group ( Figure 2D). Moreover, the marker for astrocyte GFAP showed its highest expression level in the Col group ( Figure 2E). Finally, PDGF expression was enhanced in the Col/Tac 5mg group, which is related to vessel regeneration. The gene expression behaviors in the Col/Tac 10mg group may have also been influenced by cell quantity as higher Tac concentrations led to cell death. These results indicated that the Col/Tac hydrogels containing appropriate Tac concentrations maintained hscNSC properties and promoted neural differentiation in vitro.

| Pharmacokinetic analysis of Tac after Col/Tac hydrogel implantation
Tac pharmacokinetics after Col/Tac 5mg hydrogel implantation is crucial for immunoregulation. We evaluated Tac concentrations in blood using an HPLC/MS system at specific time points ( Figure 3A). Serum Tac concentration was maintained at >1.5 ng/mL in the aSC + iv.Tac d group at all time points. The Col/Tac 5mg and aSC + Col/Tac 5mg groups maintained relatively sustained and effective Tac concentrations of 1.11 ± 0.25 and 1.12 ± 0.39 ng/mL, respectively, at 10-day post-surgery, which decreased to 0.18 ± 0.07 and 0.25 ± 0.05 ng/mL, respectively, at 30 days. There were relatively few serum Tac residues at 60 days in the Col/Tac hydrogel implantation groups.

| Biocompatibility evaluation of the Col/Tac hydrogels
Long-term persistence with Tac injections is an important causal factor for tissue damage and mortality following organ transplantation. 34 There were significant differences in the survival rates among the aSC, Col/Tac 5mg , aSC + Col/Tac 5mg and aSC + iv.Tac d groups during the first 30-day post-surgery (80%, 75%, 72% and 46.7%, respectively) and 8 weeks post-surgery (80%, 75%, 64% and 40%, respectively). The Col/Tac hydrogel treatment enhanced the survival of aSC-transplanted SCI rats compared to the conventional Tac treatment ( Figure 3B).
Then, we performed histological tests of major organs and blood biochemistry analyses to assess the in vivo toxicity of the implantation of aSCs and Col/Tac hydrogels (containing 5 mg/mL Tac) in SCI rats at 60-day post-surgery. H&E staining results showed that glomerulonephritis pathomorphism was observed in the glomeruli in the aSC + iv.
Tac d group as they had narrow glomerular capsule gaps and glomerular basement membrane incrassation, which did not occur in the other three groups ( Figure S4). This was consistent with the Masson staining results of the kidney samples in the aSC + iv.Tac d group ( Figure 3C).
There were also large areas of abnormal fibrous connective tissues in the Masson staining results of the heart and spleen in the aSC + iv.
Tac d group, which may be associated with hypofunction. Furthermore, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were chosen as biochemical indicators for assessing liver toxicity, creatine kinase (CK) for cardiac toxicity, and uric acid (UA) and serum creatinine (CRE) for kidney toxicity ( Figure 3D-H). Significantly higher serum levels of the above metabolites were detected in the aSC + iv.
Tac d group compared with the others. The results showed that the Col/Tac localized delivery and sustained release hydrogel, when combined with aSC transplantation, did not result in significant toxicity 60-day post-surgery, while the aSC + iv.Tac d treatment affected the heart, liver and kidney function. The local Tac sustained-release hydrogel caused lower toxicity levels than the daily Tac injection method.
F I G U R E 3 In vivo toxicity evaluation of the Col/Tac hydrogel-treated SCI rats after aSC implantation. (A) Tac concentrations in serum in the aSC, Col/Tac 5mg , aSC + Col/ Tac 5mg and aSC + iv.Tac d groups at different time points. Data are presented as mean ± SD (n = 4). (B) Survival curves of SCI rats with aSC transplantation. (C) Masson staining of the major organs. The abnormal morphologies in kidney, heart and spleen are shown with red arrows. The levels of (D) ALT, (E) AST, (F) CK, (G) CRE and (H) UA in the serum. There were no significant abnormal changes in the aSC, Col/Tac 5mg and aSC + Col/Tac 5mg groups, while noteworthy changes were observed in the aSC + iv.Tac d group for kidney, heart, spleen and liver biochemistry results. Bar = 50 μm. *p < 0.05; **p < 0.01; ***p < 0.001, n = 3. ALT, alanine aminotransferase; aSC, adult spinal cord tissue; AST, aspartate aminotransferase; CK, creatine kinase; CRE, creatinine; Col/Tac, collagen/tacrolimus; SCI, spinal cord injury; UA, uric acid.
Tac administration and localized Col/Tac hydrogel implantation, peripheral immune cell counts were reduced during the acute rejection period (10-day post-transplantation; Figure 4A-D). In particular, LY, MO and total WBC counts were significantly reduced when compared with those in the aSC group (p < 0.001), and no significant difference was found between the aSC + Col/Tac 5mg and aSC + iv.Tac d groups at subacute stage. For 60-day post-transplantation, LY and WBC counts were persistently lower in the aSC + iv.Tac d group than in the aSC, Col/Tac 5mg and aSC + Col/Tac 5mg groups ( Figure 4E-H). The local Tac sustained-release hydrogel showed similar effects with daily Tac administration on peripheral immune cells regulation at acute rejection periods, but could not maintain in long-term.

| Col/Tac hydrogel attenuated immune rejection and inflammation at lesion site
Severe and rapid reactive immune rejection is the main cause for cell and organ transplantation failure. 35,36 Cellular immunity mediated by T cells, including regulatory T cells (CD4+) and cytotoxic T cells (CD8+), plays important roles in this process. 37 Regulatory T cells can recognize and present antigens and secrete cytokines, including IL-2, IFN-γ and TNF. 15 Cytotoxic T cells can directly induce cell apoptosis and death. 38 To investigate immune rejection after aSC transplantation, immunofluorescence staining of CD4 and CD8 was performed for T cells at 10-day post-transplantation. Fewer CD4+ and CD8+ T cells were observed in the Col/Tac 5mg , aSC + Col/Tac 5mg and aSC + iv.Tac d groups compared to the aSC group ( Figure 5A,C). The number of CD4+ and CD8+ T cells was not visibly different among the Col/Tac 5mg , aSC + Col/Tac 5mg and aSC + iv.Tac d groups ( Figure 5B,D). These data indicated that localized Tac delivery via Col/Tac hydrogel exhibited similar immunoregulatory effects to the daily Tac administration during subacute phase, which reduced local immune rejection.
The activated macrophage-and microglia-mediated inflammatory microenvironment impairs neurological recovery after SCI. 39 Immunofluorescence staining of CD68, a marker of reactive microglia/macrophages, was used to determine the inflammatory response in the transplant area 10-day post-surgery ( Figure 5E). The number of CD68-positive cells was decreased in the Col/Tac 5mg , aSC + Col/ Tac 5mg and aSC + iv.Tac d groups when compared with those in the aSC group ( Figure 5F). The staining results of CD68, CD4 and CD8 indicated that an immunosuppressive microenvironment at the lesion site was constructed by Col/Tac hydrogel implantation, which might be beneficial for graft survival and neural restoration.
RNA-Seq analysis at day 10 of aSC transplantation showed that 711 genes exhibited significant differential expression levels with neuroactive-ligand receptor interaction and axon guidance categories, rather than immune response-related categories (Figures S5 and S6).
The results of the GO enrichment analysis showed that DEGs between the aSC + Col/Tac 5mg and aSC + iv.Tac d groups were primarily involved in chemokine-related pathways and extracellular region ( Figure S7).

| Implantation of Col/Tac hydrogel improves the transplanted aSC survival
A previous report evidenced that aSC transplantation could improve locomotor function recovery in SCI rats. 6  We further evaluated neuron survival in the transplanted site by immunostaining of neuron-specific Tuj-1 and GFP ( Figure 7A).
Many GFP-and Tuj-1-co-labelled neurons were detected in the aSC + Col/Tac 5mg and aSC + iv.Tac d groups 10-and 60-day postsurgery. Moreover, we found that the Col/Tac hydrogel implantation could only increase neuron cell numbers at defects compared to aSC transplantation ( Figure 7B). This phenomenon is consistent with the above in vitro experiments. Additionally, the Col/Tac hydrogel implantation could prevent myelin degeneration of transplanted aSCs ( Figure S9).

| aSC + Col/Tac implantation improved function recovery
MEPs and BBB scores were used to evaluate motor function recovery levels after treating SCI rats with aSC and Col/Tac hydrogel. After treatment for 8 weeks, aSC and Col/Tac 5mg rats showed slight sweeping hind limbs, while aSC + iv.Tac d and aSC + Col/Tac 5mg rats could frequently swing their hind limbs and take occasional weight- supported plantar steps ( Figure 7C). Mean BBB scores of the aSCtransplanted SCI rats with Col/Tac treatment were restored to approximately 6, which were similar to those with daily Tac administration, indicating that two joints were involved in substantial movement and one in light movement; moreover, SCI rats in the aSC group had less than 4 mean BBB scores ( Figure 7F). SCI rats in the aSC + Col/Tac 5mg and aSC + iv.Tac d groups showed notable electrophysiological improvements compared to those in the aSC group ( Figure 7D). The mean MEP amplitude in the aSC + Col/Tac 5mg and aSC + iv.Tac d groups was restored to 0.18 and 0.19, respectively, which was higher than that in the aSC and Col/Tac 5mg groups ( Figure 7E).
These results suggested that the co-implantation of aSC and Col/Tac hydrogel facilitated motor function recovery in SCI rats.

| DISCUSSION
Transplantation of central neural tissues was reported as an effective strategy for replacing damaged spinal cord and reconstructing neural circuits for SCI therapy. 6 However, immune rejection and inflammatory responses after organ transplantation result in reduced graft survival and function loss, leading to poor therapeutic efficacy.
Tac is currently the drug of choice for immunosuppression, which has been commercialized in over 70 countries to date. 35,40 Tac is also the first choice for transplanting allogeneic or xenogeneic grafts for SCI therapy. 41,42 However, the toxicity and side effects caused by immunosuppressants are largely ignored. 43  New strategies for Tac administration were designed to reduce side effects and improve therapeutic efficiency. 14,16,45 An immuneresponsive hydrogel was applied in a local controlled release system, which prolonged liver transplant survival. 16  showing great potential for cell and organ transplantations. However, the complex synthetic process and uncertain biosecurity of the high molecular synthetic material mean that it is not yet ready for clinical application. 46 Collagen is a natural protein that occurs mainly in the extracellular matrix, with reliable biocompatibility. 47 Our laboratory developed a series of clinical grade collagen scaffolds for various growth factor-localized delivery and sustainedrelease, and have been used to treat patients of SCI, intrauterine adhesion, so forth. 21 Tac binds FKBP12 to form a complex that inhibits calcineurin and leads to the inhibition of T-lymphocyte proliferation. 11 Notably, FKBP and calcineurin are co-expressed at high levels in most brain and spinal cord regions, indicating that they have key regulatory functions. 51 The Tac-FKBP12 complex increased neuronal levels of GAP-43 protein, which is involved in neural injury response and regeneration. 52 In contrast to cyclosporin A and rapamycin, Tac did not inhibit hNSC proliferation. 20 When released in a locally injured peripheral nerve area, Tac enhanced axon regeneration and repaired sciatic and peroneus nervous injury. 53 The neuroprotective effects and neurotrophy of Tac were evidenced in peripheral nerve injury. 18,45 Although the functional restoration effects of Tac administration in SCI animals were disputed, 54,55 Tac was found to drive hNSCs toward a neuronal fate in vitro, likely owing to Notch blockade. 20 We also observed that the presence of Col/Tac enhanced neural differentiation of hNSCs.
The RNA-seq analysis also confirmed that treatment of Col/Tac in the aSC transplanted SCI rats exhibited positive effects on axon growth compared to conventional Tac daily injection. Thus, Tac may not only be an immunosuppressive agent but also a potential contributor to SCI therapy. For instance, combined treatment with Tac and nerve growth factor exhibited synergistic effects in both peripheral nerve injury and SCI. 56 In addition, transcriptional analysis also revealed that Col/Tac implantation in the aSC transplanted SCI rats might regulate cellular development and axon compared to conventional daily Tac administration, which might exhibit positive effects on SCI repair.
Cell and tissue transplantation reportedly produced potential benefits in SCI treatments in the previous two decades; however, a lack of standardization between studies, including immunosuppression regimes, has limited development. 43