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Combination of ultra-purified stem cells with an in situ-forming bioresorbable gel enhances intervertebral disc regeneration

Summary Background Lumbar intervertebral disc (IVD) herniations are associated with significant disability. Discectomy is the conventional treatment option for IVD herniations but causes a defect in the IVD, which has low self-repair ability, thereby representing a risk of further IVD degeneration. An acellular, bioresorbable, and good manufacturing practice (GMP)-compliant in situ-forming gel, which corrects discectomy-associated IVD defects and prevents further IVD degeneration had been developed. However, this acellular matrix-based strategy has certain limitations, particularly in elderly patients, whose tissues have low self-repair ability. The aim of this study was to investigate the therapeutic efficacy of using a combination of newly-developed, ultra-purified, GMP-compliant, human bone marrow mesenchymal stem cells (rapidly expanding clones; RECs) and the gel for IVD regeneration after discectomy in a sheep model of severe IVD degeneration. Methods RECs and nucleus pulposus cells (NPCs) were co-cultured in the gel. In addition, RECs combined with the gel were implanted into IVDs following discectomy in sheep with degenerated IVDs. Findings Gene expression of NPC markers, growth factors, and extracellular matrix increased significantly in the co-culture compared to that in each mono-culture. The REC and gel combination enhanced IVD regeneration after discectomy (up to 24 weeks) in the severe IVD degeneration sheep model. Interpretation These findings demonstrate the translational potential of the combination of RECs with an in situ-forming gel for the treatment of herniations in degenerative human IVDs. Funding Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Agency for Medical Research and Development, and the Mochida Pharmaceutical Co., Ltd.


Introduction
Lumbar intervertebral disc (IVD) herniations, which can cause sciatica, are associated with significant disability and a decreased quality of life. 1 Discectomy is the conventional surgical procedure involving the removal of IVD materials compressing the nerve root to relieve symptoms of IVD herniations. 1À4 However, discectomy causes a large defect in IVDs, which have low self-repair ability, posing a substantial risk of further progression and degeneration of the IVD. 2À4 The prevalence of reherniation has been reported to be between 5 and 26% of approximately 500,000 lumbar discectomies per year. 4À6 Consequently, patients are likely to experience disabling discogenic lower back pain as well as reherniation that may require additional surgery, such as repeated discectomy or intervertebral fusion. 5,7 Integration of a next-generation surgical procedure that could prevent further IVD degeneration following the discectomy procedure might improve long-term surgical outcomes. Recent studies have demonstrated that cell transplantation and biomaterials could potentially be used to treat IVD degeneration. 2À4,8À16 The transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) into IVDs has shown promise for the treatment of chronic lower back pain. 10 However, BMSC transplantation has several limitations. Traditionally, the isolation of BMSCs from the unfractionated, whole bone marrow has been retrospectively confirmed based on their adherence to plastic dishes as fibroblast-like colony-forming cells. 17,18 Consequently, it has been difficult to maintain cell quality, because the cell population harvested using this method always includes non-differentiating contaminant cells. 18,19 Extended cell culture is often needed to remove contaminant cells and isolate a moderately pure BMSC population. 18 Moreover, the proliferation, differentiation, and migration abilities of BMSCs gradually decrease as the cells start exhibiting a more mature phenotype during culture. 20,21 Consequently, BMSCs used for clinical studies often lead to variable or even contradictory findings. 22,23 To overcome these limitations, we developed a single cell isolation technique for directly obtaining BMSCs from the bone marrow by sorting cells based on the expression of surface markers. 18,24À27 Human BMSCs were isolated from the bone marrow based on the expression of two cell-surface markers (CD271 and CD90) as according to the findings of previous studies, the use of these two markers serves as the best combination for the efficient collection of BMSCs. 18,26,27 After cell isolation and expansion, we obtained genetically stable, ultra-purified, good manufacturing practice (GMP)compliant, clonogenic BMSCs (known as rapidly expanding clones; RECs) that exhibited all the functions of BMSCs and did not show lot-related differences in clinical applications (Figure 1). 18 A carrier biomaterial is necessary to prevent cell leakage during discectomy. 2,15 To date, no biomaterials have been clinically approved for this purpose because each biomaterial did not meet criteria for both biologic and mechanical safety. We had previously developed an original, acellular, bioresorbable, ultra-purified alginate (UPAL) gel to repair IVD degeneration in small 2,3,15,16 and large animal models in vivo. 3 Alginate is a polysaccharide derived from brown seaweed, Phaeophyceae, and exhibits low-level endotoxicity (1/10,000 th that of commercially available laboratory alginate), and thus precludes immunologic reactions. 3 This material gelates within 5 min in situ via calcium ion-mediated crosslinking and exhibits appropriate biomechanical characteristics, without resulting in material protrusion after discectomy. 3,15 In addition, the UPAL gel showed a lack of immunogenicity during in vivo biological safety testing, performed according to International Organization for Standardization and Good Laboratory Practice (GLP)

Research in context
Evidence before this study Discectomy is the conventional surgical procedure involving the removal of intervertebral disc (IVD) materials compressing the nerve root. However, discectomy causes a defect in IVDs, which have low self-repair ability, posing a substantial risk of further degeneration of the IVD. Previously, we demonstrated that combination of allogenic bone-derived mesenchymal stem cells (BMSCs) and an in-situ forming gel was effective at inducing IVD regeneration. However, BMSCs used for clinical studies often lead to variable or even contradictory findings because these BMSCs always include nondifferentiating contaminant cells. To overcome these limitations, we developed genetically stable, ultra-purified, clonogenic BMSCs (known as rapidly expanding clones; RECs) that exhibited all the functions of BMSCs and did not show lot-related differences in clinical applications.

Added value of this study
The present study demonstrates that the expression levels of nucleus pulposus cell (NPC) markers, growth factors, and extracellular matrix components were significantly increased in the co-culture of human NPCs and RECs compared with those observed in the co-culture of NPCs with commercially available BMSCs. The efficacy of the combination of RECs and the in-situ forming gel was observed at the site of IVD degeneration in a sheep lumbar spine model.

Implications of all the available evidence
The findings reveal that the combination of ultra-purified BMSCs in the form of RECs and in situ-forming gel enhances IVD regeneration after discectomy in a sheep model exhibiting severe IVD degeneration, without resulting in neoplastic changes, and enable the maintenance of mechanical functions after implantation. These findings and strategy need to be validated in clinical settings in future studies, especially in an elderly cohort such as individuals with combined lumbar canal stenosis. Articles standards. 3 Consequently, we are currently conducting a first-in-human clinical trial using an acellular matrixbased strategy and implanting this GMP-compliant gel into the defect after discectomy in young patients (age, 20À40 y). 28 We have previously demonstrated that the intrinsic nucleus pulposus (NP) and NP progenitor cells transmigrate into the UPAL matrix for endogenous IVD repair. 3 Because residual IVD tissues observed after discectomy act as a possible source of reparative cells and the grade of IVD degeneration affects repair capabilities, the strategy of using UPAL gel alone may have limitations in an elderly population, as it could result in the development of conditions such as combined lumbar canal stenosis (e.g., IVD herniation complicated by lumbar canal stenosis). 2,3 Notably, most animal models of IVD degeneration were created using healthy IVDs and therefore might exhibit natural healing capabilities. 3,4,8,11À14,16 To identify the course of post-surgical IVD degeneration from the onset, we generated a rabbit model of IVD degeneration. 2,15 We demonstrated that following discectomy, the use of UPAL gel alone suppressed IVD degeneration compared to the extent of degeneration without UPAL gel treatment. Additionally, the combination of allogenic BMSCs and UPAL gel was more effective at inducing IVD regeneration compared to UPAL alone. 2,15 To further advance the clinical translation to enhance IVD regeneration post-discectomy for degenerated IVDs, we developed a large animal ovine model of IVD degeneration and successfully implanted RECs encapsulated in the UPAL gel into the IVDs-as a medical product-in a GLP-adapted laboratory. We hypothesized that the implantation of RECs encapsulated in the UPAL gel would promote spontaneous IVD regeneration after discectomy. Here, we aimed to demonstrate that the combination of these GMP-compliant, ultrapurified, BMSCs and in situ-forming gel enhances IVD regeneration after discectomy in a sheep model exhibiting severe IVD degeneration, without resulting in neoplastic changes, and enable the maintenance of mechanical functions after implantation.

Study design
The primarily explored parameters in this study were: (i) potential mechanisms of IVD regeneration via the 3dimensional (3D) co-culture of human NPCs and RECs in vitro; (ii) comparison of the effects of RECs and commercial human BMSCs on NPCs via the 3D co-culture of these cells with NPCs; (iii) measurement of the mechanical properties of UPAL and REC + UPAL gels; (iv) evaluation of regenerative capacity of severely degenerated IVDs after discectomy in vivo; and (v) evaluation of tumorigenesis in the implanted IVDs.
Healthy human NPCs and RECs were co-cultured in a 3D system in UPAL gel to evaluate the mechanism underlying IVD regeneration. At 0 and 7 days after culture, we analyzed the expression of NPC markers, including HIF-1a, GLUT-1, and brachyury; growth factors, including CDMP-1, TGF-b, and IGF-1; and extracellular matrix (ECM) components, including Type II collagen and aggrecan (Table 1), in all cell types, using quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 4). 2,9,29,30 In addition, NPCs and commercial BMSCs were co-cultured to compare the effects of RECs and commercial BMSCs on NPCs. Then, we measured the mechanical properties of UPAL and REC + UPAL gels using both unconfined and confined compression tests (see 2.12. Elasticity analysis of the gels using both unconfined and confined compression tests). Disc-shaped gels were compressed at a constant speed of 0¢5 mm/min, and Young's moduli were calculated (n = 4). 3,15,31,32 In animal procedures, 14 sheep (56 IVDs in total) were divided into the following four groups: Intact control (4 weeks, n = 6; 24 weeks, n = 6), Discectomy (4 weeks, n = 6; 24 weeks, n = 6), Gel (4 weeks, n = 8; 24 weeks, n = 8), and REC + gel (4 weeks, n = 8; 24 weeks, n = 8) groups. In a preliminary study, we found that IVD degeneration occurred after 4 weeks, with the removal of 20 mg or more of NP tissue in a sheep model (Supplementary Figure 1). Consequently, to create severely degenerated IVDs, we initially removed 20 mg of NP tissue from the treated IVDs and further removed 70 mg of the gel-like NP tissue separate from the AF from the degenerated IVDs, four weeks after the initial surgery. For the second removal procedure, the amount of 70 mg was determined based on the ratio of the removed amounts of normal and degenerated IVDs in previous studies. 2,3 In a previous study, the total amount of NP tissue in a rabbit normal IVD was 15-18 mg, whereas the amount in a rabbit degenerated IVD, created by needle puncture was 10-12 mg, corresponding to approximately 70% of the amount in the normal IVD. 2 Since the amount of NP tissue removed from a sheep normal IVD was 100 mg in our previous study, 3 the amount of NP tissue removed from a degenerated IVD in this study was set to 70 mg, which was equivalent to 70% of the amount of NP tissue in the normal IVD. After the second discectomy, UPAL or a solution containing a combination of RECs with UPAL was implanted into the IVD cavity. Sheep were euthanized 4 and 24 weeks after implantation. IVDs were qualitatively analyzed to assess IVD degeneration using a 3¢0-T magnetic resonance imaging (MRI). 2,3,15,33,34 Subsequently, IVDs were stained with hematoxylin & eosin (H&E) and safranin-O for histological analysis, and immunohistochemistry (IHC) was performed to evaluate the levels of Type II and I collagen for analyzing the ECM components. Finally, tumorigenesis analysis was performed using histological specimens. 2,3,15

Ethics statement
The ethics committee of the Hokkaido University Graduate School of Medicine approved the use of healthy human IVDs. Animal procedures were approved by the Institutional Animal Care and Use Committee at Hokkaido University and Hamri Co., Ltd. (Ibaraki, Japan), and were performed according to the guidelines recommended by these committees.

Preparation of RECs
Frozen GMP-compliant RECs provided by Japan Tissue Engineering, Inc. were used in this study. In the comparative analysis of RECs and commercial human BMSCs, we prepared three clones of RECs, namely, REC-02 prototype #003-P6-191220, REC-02 HLWF-1-P6-210114, and REC-02 QRKF-1-P6-210210. They were prepared by the company and received in passage 6. The cells were thawed in a warm bath at 37 C prior to 3D culture and were directly used after thawing.

Articles
Thermo Fisher Scientific), and 0¢1% bFGF (Kaken Pharmaceutical Co., Ltd., Tokyo, Japan) in a humidified atmosphere (20% O 2, 5% CO 2 , 37°C) according to the manufacturer's instructions. The medium was replaced twice per week, and BMSCs from the fourth passage process were used (six passages in total similar to that with RECs).

Preparation of UPAL gel and 3D culture
The UPAL gel (Mochida Pharmaceutical Co. Ltd., Tokyo, Japan) was used as an alginate scaffold for 3D culture, as described previously. 2, 3 We prepared a 2% (w/v) UPAL solution dissolved in phosphate-buffered saline (PBS; FUJIFILM Wako Pure Chemical Industries) and used CaCl 2 solution (102 mM) for gelation. RECs or commercial BMSCs were mixed with the UPAL solution at a final cell concentration of 1 £ 10 6 cells/mL. 2,30,35 The cell-UPAL mixed solution, total 1 mL in each group, was pipetted into a 102 mM CaCl 2 solution (100mL) using a 22-gauge needle for gelation. The two types of the gel obtained in the shape of beads were cultured with the medium for 7 days in a humidified atmosphere (20% O 2, 5% CO 2 , 37°C). The medium was changed every 3 days. To retrieve cells after 7 days of 3D culture, we collected the cells from gel beads after dissolution using 55 mM sodium citrate and centrifugation (110 £ g for 10 min at 4°C), as described previously. 2,3 Additionally, we used the RECs and commercial BMSCs prepared as plane cultures (2D culture) for 7 days under normal condition as the control group, as mentioned previously (i.e., the following four groups were set up as the experimental groups: 1) 2D-cultured RECs, 2) 3D-cultured RECs, 3) 2D-cultured BMSCs, and 4) 3D-cultured BMSCs).

Analysis of cell proliferating capacity
After 7 days of 2D or 3D culture, cells obtained in the 4 experimental groups (see 2.5. Preparation of UPAL gel and 3D culture) were seeded again as a secondary culture. We collected the cells at confluency and evaluated their cell proliferative capacity based on the doubling time (Td) defined as the time required for the cell number to double. Td was calculated using the following formula: where N 2 and N 1 were the cell number at time t 2 and t 1 .

Preparation of healthy human NPCs
Human NP samples were obtained from nine patients (mean age § standard deviation (SD), 15¢3 § 3¢3 years) who had undergone anterior spinal fusion for adolescent idiopathic scoliosis at Hokkaido University Hospital. We obtained written informed consent from all participants of this study. The samples were obtained during the surgical procedure. All IVDs were analyzed before surgery via MRI and graded for degenerative changes using the Pfirrmann classification system. 36 MRI measurement was performed within 3 months before surgical removal. All IVDs were classified as grade 1 IVDs, suggesting that all the samples were nondegenerated IVDs. NP cells were isolated within 1 hr after the harvesting of human IVDs during the surgery and cultured as described previously. 2,3 Briefly, each gel-like NP was separated from the annulus fibrosus (AF) under a dissecting microscope. The tissue specimens were placed in the medium as described above (section 2.4). The preparations were washed twice via centrifugation (200 £ g, 3 min) and resuspended in the medium supplemented with 0¢25% collagenase. For cell isolation, the preparations were incubated in a shaking incubator (37°C, 4 h) and then centrifuged twice (200 £ g, 3 min). Cells that were separated from the matrix were placed in 10 cm tissue culture dishes and incubated under the conditions indicated in section 2.4. The medium was changed twice a week, and NPCs from the fourth passage were used.

Preparation of RECs and commercial human BMSCs
The RECs (REC-02_prototype #003-P6-191220) and commercial human BMSCs as above were also used for 3D co-culture. Two types of the cells were cultured according to the manufacturer's instructions in the same way as the 2D culture above. The medium was replaced twice a week. Both of cells from the second passage (RECs, 8 passages in total; BMSCs, 4 passages in total) were used.

3D co-culture and mono-culture
We prepared a 2% UPAL solution and used CaCl 2 solution (102 mM) for gelation, as described previously. 2,3 In advance of the 3D culture, the RECs and BMSCs were labeled with 20 mM 5,6-caboxyfluorescein diactetate succinimidyl ester (CFDA-SE) (CFDA-SE Cell Proliferation Assay Kit; BIO RAD, Hercules, CA, USA) fluorescently, referring to the manufacturer's manuals. 2,15,37 Afterward, the labeled cells (BMSCs or RECs) and unlabeled NPCs were mixed (i.e. a combination of two cell types, NPCs + BMSCs or NPCs + RECs) with the UPAL solution at the same ratio (each cell 1 £ 10 6 cells/mL respectively), 2,30,35 with a final cell concentration of 2 £ 10 6 cells/mL. The cell-UPAL mixed solution was pipetted into CaCl 2 solution of 102 mM using a 22-gauge needle to gelate. The obtained gels were cultured with the medium for 7 days under hypoxic conditions (5% O 2 and 5% CO 2 ). 2,35 Additionally, NPCs or RECs were mixed in the UPAL solution separately at a concentration of 1 £ 10 6 cells/mL. The cell concentration was selected on the basis of the results of a preceding research. 2 After gelation, the gel beads were cultured in the same manner, under hypoxic conditions. At 0 and 7 days after culture, all gel beads were dissolved in 55 mM sodium citrate until the gel was separated from the cells as above. In the co-cultured groups, collected cells were sorted using a BD FACSAria III High speed cell sorter using Diva software version 7¢0 (BD Biosciences, San Jose, CA, USA). 2,30 Fluorescent cell with 530 nm were selected as RECs and BMSCs, and non-fluorescing cells were selected as NPCs after the excluding debris and dead cells (Figure 2a and b). Subsequently, we had 7 experimental groups ( Table 2).

RNA extraction and qRT-PCR
The collected cells were lysed in 1 mL of TRIzol Ò (Invitrogen, Thermo Fisher Scientific), and total RNA was extracted from the samples using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). Real-time qRT-PCR was performed using TaqMan Ò Gene Expression Assays (Applied Biosystems, Thermo Fisher Scientific). Assay IDs used for the assay are presented in Supplementary  Table 1. We obtained a cycle threshold (Ct) value for each sample. Moreover, the relative mRNA expression levels of each target gene, NPC markers, growth factors, and ECM components were calculated via the 2 ÀDCt method; 2 the levels were normalized to those of the housekeeping gene GAPDH. 2,30 Elasticity analysis of the gels using both unconfined and confined compression tests The mechanical properties of the UPAL and REC-UPAL gels were evaluated using both unconfined and confined compression tests. We prepared two types of discshaped gels with a diameter of 4¢5 mm and thickness of 2 mm (Figure 3a). In the confined compression test, the disc-shaped gels were fitted within the confining chamber made of acrylic resin with a cylindrical hole (diameter, 4¢5 mm; depth, 3 mm), and pressed with a brass hammer. The samples were placed in a tensilecompressive mechanical tester (Autograph AG-X; Shimadzu Corporation, Kyoto, Japan) and compressed at a constant rate of 0¢5 mm/min using a 100 N load cell, until the gels collapsed (Figure 3b and c). 3,15 Young's moduli were calculated based on the obtained stressstrain curve (Figure 3d) using an approximate straight line between compression values of 10À20% (n = 4 gels per group) (Figure 3e). 3,15,31,32 These tests did not mimic the IVD; however, Young's modulus was used to assess the mechanical properties of alginate gels with and without cells. 3,15,32 or NP of the human IVD. 31 In vivo study in a sheep model of degenerated IVD In this study, all procedures involving the sheep model were performed in a medical product GLP-adapted laboratory (Hamri Co., Ltd.). 3 Fourteen male, Suffolk sheep (2-year old, weighing 40À60 kg) were used for the qualitative analysis of IVD degeneration. 3 A total of 56 IVDs were randomly allocated to the Intact control (4 weeks, n = 6; 24 weeks, n = 6), Discectomy (4 weeks, n = 6; 24 weeks, n = 6), Gel (4 weeks, n = 8; 24 weeks, n = 8), and REC + gel (4 weeks, n = 8; 24 weeks, n = 8) groups. First, the NP tissue removal surgery was performed to create a severely degenerated IVD model. Anesthesia induction was performed via the intramuscular injection of a 4:1 mixture of ketamine (0¢2 mg/kg) and xylazine (20 mg/kg), at a rate of 0¢5 mL/kg, and maintenance of anesthesia was achieved via inhalation anesthesia (isoflurane). The operation was performed through a right lateral retroperitoneal approach, and vertebral bodies and IVDs from L1 to L5 were exposed. A solid cancellous screw (ZIMMER BIOMET, Warsaw, IN, USA) was inserted into the L2 vertebral body as a vertebral landmark. In the Discectomy, Gel, and REC + gel groups, 20 mg of NP tissue was removed after AF incision (5 £ 3 mm), and IVD degeneration was induced (Figure 4a and b). 3,8 Removal of degenerated NP tissue and implantation Four weeks after the initial surgery, the vertebral bodies and IVDs were exposed using the same approach. We further extracted 70 mg of NP tissues from the degenerated IVDs obtained from the three treatment groups to create IVD cavities after an AF incision, as described above (Figure 4c). After discectomy, IVD cavities were implanted with 110À120 µL of 2% UPAL solution in the Gel groups, and 110À120 µL of a mixture of RECs and UPAL solution (final concentration, 1£10 6 cells/ mL) was implanted into the REC + gel group (Figure 4d). 2 Immediately, 102 mM CaCl 2 solution was injected onto the surface of the solution, and gelation was confirmed after 5 min. After 4 and 24 weeks of implantation, the sheep were euthanized using pentobarbital and their lumbar spines were removed. 3

MRI analysis
T2-weighted midsagittal section images were obtained using a 3¢0-T MR scanner (MAGNETOM Prisma; Siemens, Munich, Germany). To evaluate the change in the signals of treated IVDs, we scored the degree of IVD degeneration using the Pfirrmann classification method, 36 which involves five grades (1: normal to 5: highly degenerated). In addition, using Analyze 14¢0 software (AnalyzeDirect, Overland Park, KS, USA), the MRI index values (the product of the mean signal intensity of the NP and the NP area) were measured to quantify the radiance of the NP tissue. We evaluated the relative MRI index, which is the ratio of the MRI index in the Intact control group, in all three treatment groups. 2,3,15,33,34 The disc height index (DHI), which is the ratio of the disc height to the height of the adjacent vertebral body on the cranial side, was also measured. 4,38 We determined the relative DHI, which is a percentage value of the DHI of the Intact control group (Supplementary Figure 2).

Histological analysis
After MRI imaging, samples were fixed in 10% formaldehyde, demineralized with 10% EDTA (pH 7¢5), and embedded in paraffin. Sagittal 5-micrometer-thick paraffin sections were deparaffinized with xylene for 20 min, treated with alcohol for 15 min, washed with water for 5 min, rinsed with distilled water for 30 s, and then stained with H&E and safranin-O. The degrees of IVD degeneration were scored from 0 (normal) to 36 (highly degenerative), using the modified Boos' classification system. 3,39,40 IHC analysis IHC was performed to determine the expression of Type II and I collagen in the IVDs. Sections were deparaffinized in xylene and treated with 0¢1% trypsin for 30 min for antigenic activation. Then, sections were treated with 3% H 2 O 2 in methanol for 10 min, followed by protein blocking for 30 min using the Protein Block Serum-Free solution (DAKO, Agilent, Santa Clara, CA, USA). Goat anti-Type I Collagen (1:40; Southern Biotech, Birmingham, AL, USA; Cat# 1310-01, RRID: AB_2753206) was used with anti-Type I collagen antibodies and anti-hCL (II) and purified IgGs (1:400; Kyowa Pharma Chemical Co., Ltd., Toyama, Japan; Cat # F-57) were used with anti-Type II collagen antibodies as the primary antibodies. The sections were incubated at room temperature for 1 h for the primary antibody response. After washing cells with PBS, the EnVision+ System-HRP Labeled polymer anti-mouse (DAKO; Cat # K4001) for Type II collagen and Histofine Ò Simple Stain Max PO (G) (Nichirei Biosciences, Tokyo, Japan; Cat # 414162F) for Type I collagen were used as the secondary antibodies. The sections were incubated at room temperature for 30 min for the secondary antibody response. Finally, sections were stained with DAB (DAKO) and hematoxylin. The number of Type II and I collagen-positive cells was determined in five randomly selected fields, and the positive cell percentages in total cells were calculated. 2,3,15

Tumorigenesis analysis
To evaluate the tumorigenesis of implanted and existing cells in implanted IVDs, the following aspects were evaluated: (i) invasive growth, (ii) karyomitosis, (iii) binucleate cells, and (iv) nucleoli. Using H&E-stained specimens in the Intact control and REC + gel groups during the 24-week evaluation period, the total number of positive cells was determined in 15 randomly selected visualization fields (15 visualization fields at a magnification of 400£; 26¢5 visualization fields, 5 mm 2 in total), and we calculated the numbers per square millimeter.

Statistical analysis
Sample sizes for quantitative data were determined by power analysis, using the Tukey-Kramer test, with an a level of 0¢05 and power of 0¢8. Statistical analyses were performed using the JMP Pro version 14¢0 software (SAS Institute, Cary, NC, USA), and values were considered significant if p < 0¢05. All data are presented as mean § SD values. One-way analysis of variance (ANOVA) and the Tukey-Kramer post hoc test were conducted for multigroup comparisons. Student's t-test or Mann-Whitney U test and Welch's test were used for two-group comparisons. We performed sample randomization and were blinded to the samples being tested.

Role of the funding source
The funders had no role in study design, data collection, data analysis, data integration, or report preparation.

Comparison of RECs and commercially available human BMSCs
Details of the results of the stability checks for RECs and commercially available human BMSCs prior to and after their incorporation into the gel are shown in Tables 3  and 4.
The ratio of PI-unstained cells to the total number of cells was defined as the cell viability. There was no significant difference in the ratios between RECs and BMSCs (p = 0¢6087, Student's t-test). The coefficient of variation (CV) of the forward scattered light proportional to the cell diameter was calculated to evaluate the cell size uniformity. The %CV value was significantly lower in RECs than in BMSCs (p = 0¢0366, Student's ttest). Cell proliferative capacity was evaluated by calculating the Td. The Td were significantly shorter in RECs than in BMSCs (p = 0¢0074, Student's t-test). Cell surface antigen confirmation test showed that the percentage of CD90-positive cells was significantly higher in RECs than in BMSCs (p = 0¢0004, Student's t-test). There was no significant difference in CD45-positive cells between the groups (p = 0¢0907, Student's t-test) ( Table 3).
We further compared RECs and commercially available BMSCs after 1 week of culture. There was no significant difference between RECs and BMSCs in terms of the Td after both 2D and 3D cultures (p = 0¢3339, p = 0¢6996, Student's t-test). However, the percentages of CD90-positive cells were significantly higher among RECs than among BMSCs after both 2D and 3D cultures (p = 0¢0039, p = 0¢0007, Student's t-test). Regarding the CD45-positive cells, the percentage was significantly lower in RECs than in BMSCs after 2D culture (p = 0¢0038, Student's t-test); however, there was no significant difference between RECs and BMSCs after 3D culture (p = 0¢3221, Student's t-test) ( Table 4).

Co-culture of RECs and human NPCs
We investigated the effects of co-culture of RECs with human NPCs and compared them with co-culture of commercially available human BMSCs and human NPCs of different cell types. We measured the expression of NPC markers, including HIF-1a, GLUT-1, and brachyury; growth factors, including CDMP-1, TGF-b, and IGF-1; and ECM components, including Type II collagen and aggrecan, in these cells via real-time qRT-PCR. 2,9,29,30 The expression levels of three NPC markers, HIF-1a, GLUT-1, and brachyury, were significantly higher in NPCs co-cultured with RECs than those in control NPCs, mono-cultured NPCs, and NPCs co-cultured with commercial BMSCs. GLUT-1 expression levels were significantly higher in NPCs co-cultured with commercial BMSCs than those in control NPCs (Figure 2cÀe). The expression levels of CDMP-1 and IGF-1 were significantly increased in NPCs co-cultured with RECs compared with those in control NPCs, mono-cultured NPCs, and NPCs co-cultured with commercial BMSCs. The TGF-b expression levels were significantly increased in NPCs co-cultured with RECs compared with those in control and mono-cultured NPCs. NPCs co-cultured with commercial BMSCs showed a significant increase in CDMP-1 and IGF-1 expression compared with control NPCs and mono-cultured NPCs (Figure 2fÀh). The expression levels of Type II collagen and aggrecan were significantly higher in NPCs co-cultured with RECs than those in control and mono-cultured NPCs; their levels in NPCs co-cultured with commercial BMSCs were also significantly higher than those in control and mono-cultured NPCs (Figure 2i and j).
The expression levels of all three NPC markers in cocultured RECs were significantly higher than those in control and mono-cultured RECs (Figure 2cÀe). The expression levels of CDMP-1, TGF-b, and IGF-1 were also significantly increased in co-cultured RECs compared with those in control and mono-cultured RECs (Figure 2fÀh). Similarly, the expression levels of Type II collagen and aggrecan were significantly higher in cocultured RECs than those in control and mono-cultured RECs (Figure 2i and j).

Effects of RECs embedded in the UPAL gel
Both unconfined and confined compression tests were performed to evaluate and compare the mechanical properties of the REC-UPAL and UPAL gels, as described previously. 3,15,31,32 We prepared two types of cylindrical gel samples (diameter: 4¢5 mm, thickness: 2 mm) and calculated the ratio of longitudinal compressive stress to strain (Figure 3aÀc). At compression levels of 10À20%, the Young's moduli were 18¢0 § 3¢5 kPa and 18¢8 § 2¢1 kPa for the UPAL and REC-UPAL gels in the unconfined test, respectively, and 24¢3 § 4¢2 kPa and 26¢5 § 8¢5 kPa for the UPAL and REC-UPAL gels in the confined test, respectively; no significant differences in mechanical properties were observed between the two groups (p = 0¢6942 in unconfined test and p = 0¢6623 in the confined test; p values measured with

RECs
BMSCs Student's t-test) (Figure 3d and e). Additionally, these two values were comparable to those of healthy human NP tissues. 3,15,31 Combination of RECs and UPAL gel facilitates IVD regeneration The degenerative changes in treated IVDs were evaluated using T2-weighted midsagittal section images obtained via MRI (Figure 5a). Based on the Pfirrmann scores, the grades of the Gel group were significantly lower than those of the Discectomy group at 24 weeks (p = 0¢0123, TukeyÀKramer test), and the grades of the REC + gel group were significantly lower than those of the Discectomy and Gel groups at both 4 and 24 weeks (4 weeks; p < 0¢0001 (vs Discectomy), 24 weeks; p < 0¢0001, p = 0¢0208, TukeyÀKramer test) (Figure 5b). The MRI index was significantly higher in the REC + gel group than in the Discectomy and Gel groups at 4 weeks (p = 0¢0032, p = 0¢0032, TukeyÀKramer test). At 24 weeks, the MRI index of the Gel group was significantly higher than that of the Discectomy group (p = 0¢0158, TukeyÀKramer test) and that of the REC + gel group was significantly higher than that of the Discectomy and Gel groups (p < 0¢0001, p = 0¢0064, TukeyÀKramer test) (Figure 5c).
Additionally, the disc heights of treated IVDs were measured using MRI images. The DHI values of the three treated groups were significantly lower than those of the Intact control group at both 4 and 24 weeks. Although there was no significant difference among the three groups 4 weeks after implantation, the DHI of the Gel group was significantly higher than that of the Discectomy group (p < 0¢0001, TukeyÀKramer test) and that of the REC + gel group was significantly higher than that of the Gel and Discectomy groups 24 weeks after implantation (p = 0¢0123, p < 0¢0001, TukeyÀKramer test) (Figure 5d).
Histological analysis was performed using H&E and safranin-O staining. At both 4 and 24 weeks in the Intact control group, the IVDs exhibited a spindle shape, no fibrotic changes in the NP tissue, concentric AF, and uniform staining with safranin-O. Although IVD tissue structures were preserved effectively in the REC + gel group, the tissues were mildly crushed and exhibited many fibrotic changes; a partial loss of safranin-O staining was observed in the Gel group. Considerable amount of scar tissue, several fibrotic changes, tissue loss, and endplate collapse and destruction were observed in the Discectomy group (Figure 6a and b). Histological grades determined based on the modified Boos' classification 3,39,40 in the Gel group were significantly lower than those in the Discectomy group at both 4 and 24 weeks (p < 0¢0001, p = 0¢0006, TukeyÀKramer test) and were significantly lower in the REC + gel group than those in the Discectomy and Gel groups at both 4 and 24 weeks (4 weeks; p < 0¢0001, p = 0¢0001, 24 weeks; p < 0¢0001, p < 0¢0001, TukeyÀKramer test) (Figure 6c).
The expression levels of Type II and I collagen in treated IVDs were assessed by IHC (Figure 7a and b). The percentages of Type II and I collagen-positive cells-in the total number of cells-in five randomly selected visualization fields were determined in NP tissues. 2,3,15 Type II collagen is an essential ECM component in NP tissues and is replaced by Type I collagen with the progression of degeneration. Analysis of Type II collagen showed that NP tissues were uniformly stained in the Intact control group, but a slight decrease in intensity was observed in the REC + gel groups. Scattered non-stained areas were observed in the Gel groups and broadly non-stained areas were observed in the Discectomy group. The percentage of Type II collagen-positive cells was significantly higher in the REC + gel group than that in the Discectomy group at the 4-week evaluation period (p < 0¢0001, TukeyÀKramer test) and was significantly higher than that in the Discectomy and Gel groups at the 24-week evaluation period (p < 0¢0001, p < 0¢0001, TukeyÀKramer test). The percentage of these cells were significantly higher in the Gel group than those in the Discectomy group at 24 weeks (p = 0¢0388, TukeyÀKramer test) (Figure 7c). However, the percentages of Type I-collagen-positive cells were significantly lower in the REC + gel group than those in the Discectomy and Gel groups at both 4 and 24 weeks (4 weeks; p < 0¢0001, p = 0¢0379, 24 weeks; p < 0¢0001, p = 0¢0001, TukeyÀKramer test). In addition, the percentage of these cells was significantly lower in the Gel group than in the Discectomy group at 24 weeks (p = 0¢0014, TukeyÀKramer test) (Figure 7d).

Effects of REC + gel implantation on IVDs
Tumorigenesis was analyzed in histological specimens in the Intact control and REC+ gel groups during the 24-week evaluation period. Invasive growth, along with karyomitosis, was assessed in all specimens of both groups, and the number of binucleate cells and nucleoli per square millimeter was determined. Invasive growth, karyomitosis, and nucleoli were not observed in both groups. The number of binucleate cells in the Intact control and REC + gel groups was 0¢03 § 0¢07 and 0¢23 § 0¢25, respectively, i.e., the values were < 1 /mm 2 and no significant differences were observed between the groups (p = 0¢3311, Mann-Whitney U test) (Supplementary Table 2).

Discussion
Although several studies have reported the regenerative ability of various BMSCs in IVDs and the effect of BMSCs incorporated in various gel-types and model systems has been shown earlier, 2,15,43À45 further biomedical advancement is needed to achieve better clinical Disc height index for IVD treatment. Numerical numbers are shown as percentages relative to those of intact control IVDs. Data represent mean § SD values (Intact control, n = 6; Discectomy, n = 6; Gel, n = 8; REC + gel, n =8). Significant differences were assessed by one-way ANOVA with post hoc analysis using the Tukey-Kramer test. safety and efficacy in applied regenerative medicine. BMSCs with consistently high cell quality are required for clinical applications. The use of RECs, characterized according to the International Society for Cellular Therapy (ISCT) criteria for human BMSCs, 46,47 represents a notable advantage of this study, as the cell source yields high-quality BMSCs with minimal batch-to-batch variance. 18 In this study, excellent characteristics of RECs were shown in terms of cell proliferative capacity, cell size uniformity, and the expression of cell surface antigens compared to those of commercially available human BMSCs. In addition, the present study demonstrates that the expression levels of NPC markers, growth factors, and ECM components were significantly increased in the 3D co-culture of human NPCs and RECs compared with those observed in the 3D co-culture of NPCs with commercially available BMSCs. The efficacy of the combination of RECs and UPAL gel was observed at the site of IVD degeneration in a sheep lumbar spine model. Though the UPAL gel alone delayed IVD degeneration (compared to that following discectomy) the combination of RECs and gel enhanced IVD regeneration more effectively.
In this study, the expression levels of all three NPC markers in co-cultured RECs were significantly higher than those in the control and mono-cultured RECs, indicating that the co-culture of RECs with NPCs results in the differentiation of RECs into NPCs, thereby improving ECM component production in both cell types. This is consistent with the findings of our previous-and other-in vitro studies, which revealed the crosstalk between NPCs and BMSCs through growth factor production 2,30,48,49 and enhanced ECM synthesis. 2,9,30,35,50 Similar results were observed in our previous study in rabbits, which revealed increased expression of NPC markers after the implantation of BMSCs encapsulated in the UPAL gel and increased  production of ECM compared with that observed following discectomy without the implantation of BMSCs encapsulated in the UPAL gel. 2 Based on the results of present and previous studies, 2,30,48,49,50À52 the following are the possible mechanisms underlying IVD regeneration in response to the implantation of RECs encapsulated in the UPAL gel: (1) RECs can be implanted in IVD defects through gel encapsulation (gel encapsulation is not a mechanism of IVD regeneration and just the technique used in the study); (2) RECs produce growth factors and ECM components, thereby activating the existing NPCs (paracrine mechanism); (3) NPCs produce more ECM and growth factors; (4) consequently, RECs directly differentiate into NPCs; (5) finally, RECs and NPCs have positive intercellular feedback loop resulting in IVD regeneration. 2 The biomaterials/hydrogels used for IVD restoration should be biologically and mechanically suitable. Hence, comprehensive biomechanical investigations need to focus on the clinically relevant loads in the context of daily human activity, which are thought to be essential for the regulation of IVD repair. 3 Presently, there are no commercially available soft biomaterials used in routine clinical practice to fix discectomy-associated defects, likely because of the risk that high intradiscal pressures might result in their ejection. 3 Food and Drug Administration (FDA) guidelines or American Society for Testing and Materials (ASTM) International standards are not currently available for evaluating the adhesion of hydrogel to IVD tissues. 53,54 However, an ex vivo experiment could be used to investigate the mechanical properties of a hydrogel. Here, we selected a sheep lumbar spine model for implanting the candidate hydrogel into a preclinical animal model, because the biomechanics and geometry of lumbar IVDs are comparable to those of humans. 3,4,41 We had previously shown that the UPAL gel implanted into cadaveric sheep lumbar IVDs after discectomy exhibited appropriate biomechanical characteristics, and did not result in material protrusion and require suturing of the AF after discectomy in sheep spines. 3 In addition to six-axis static loading tests, sinusoidal dynamic axial loading ( § 300 N) tests were applied for 1000 cycles, in the context of daily human activity, mimicking how an individual stoops down 10 times per day, and the approximate period after which the gel disappears (100 days) was determined. 3 Although there have been some studies on hydrogels, wherein dynamic mechanical evaluations were performed immediately after the injection of the gel into the IVD using a human or large animal cadaveric model, 55,56 to our knowledge, none of the previous studies investigating GMP-compliant gels have addressed this issue. 3 In this study, the results of both the unconfined and confined compression tests revealed that there were no significant differences in the Young's moduli between the UPAL and REC-UPAL groups, indicating that the RECs embedded in the UPAL gel did not alter the mechanical characteristics of the gel. 15 This could be expected given the low concentration of BMSCs, which essentially are non-adherent and therefore non-contracting. Also, cells generally do not adhere to alginate. It should, however, be considered that the REC concentration utilised in this study (1 £ 10 6 cells/ ml) was based on the results of a previous study that used a canine IVD degeneration model to determine the optimal BMSC number for transplantation; 57 the results showed that both the structural microenvironment and ECM were maintained following the transplantation of 1 £ 10 6 BMSCs to the canine IVDs compared to that of 1 £ 10 7 BMSCs. Accordingly, we did not attempt to apply higher concentrations of RECs to IVDs, greatly beyond the current concentration, and did not test the effect of the cells on the mechanical properties of the gel at a higher concentration. Thus, owing to its crucial ability to bring about rapid healing, the combination of the UPAL gel with RECs at the current concentration offers clinical advantages in terms of preventing cell leakage without suturing the AF. 15 Many studies have reported on the discectomy of non-degenerated IVDs in a large animal model. 3,4,8,12À14,42,45 However, to the best of our knowledge, this is the first study to report on a discectomy for degenerated IVDs in a large animal model. MRI and histological evaluation in animals implanted (and not implanted) with UPAL gel indicates that IVD degeneration was delayed following the implantation of UPAL gel alone. Moreover, the combination of RECs and UPAL gel prevented degeneration even more effectively. There was no significant difference in the number of binucleate cells between the Intact and REC + gel groups, suggesting that the combination of RECs with the gel did not induce tumorigenesis. In addition, IHC suggests that the combined use of RECs and gel enhances ECM synthesis in the degenerated NP tissue, a phenomenon that is essential for the functioning of native IVD, and prevents the progression of IVD degeneration by downregulating the production of Type I collagen. These results are consistent with our previous resultsobtained using a rabbit model of IVD degenerationand demonstrate that BMSCs encapsulated in the UPAL gel prevented degeneration more effectively. 2,15 The previous results also showed that the histological grading score for degeneration was lower after BMSC + UPAL gel implantation, compared to that in animals not subjected to discectomy, in which degenerated IVDs were created via AF needle puncture. This suggests that BMSC implantation resulted in IVD regeneration. 2,15 Taken together, our present and previous results indicate that the implantation of RECs encapsulated in the UPAL gel enhances IVD regeneration in vivo. 2,15 The present study provided valuable insights into the translational approach of combining GMP-compliant BMSCs with soft biomaterials to enhance IVD regeneration post-discectomy; however, a few limitations need to be addressed. First, since unconfined compression test is the representative mechanical testing method for measuring the mechanical properties of cylindrical hydrogel samples 3,15,32 and as we previously reported the mechanical properties of a UPAL gel with and without cells using this test, 3,15 in the present study, we conducted the same test. However, the characterization of the hydrogel with Young's modulus based on results of the simple compression test is insufficient because hydrogels and soft tissues are poro-visco-elastic tissues with evident time-dependent properties. To investigate the complexity of the material, aggregate modulus and permeability based on confined compression tests should be further assessed using human NP. 58 Second, the animal models of IVD degeneration used in the present study were generated using sheep IVDs; therefore, the surgical procedure of discectomy was not strictly replicated in clinical settings. Although discectomy was performed in the sheep model through the antero-lateral approach, clinical discectomy is typically performed through the posterior approach. 2À4 However, sheep models subjected to discectomy performed through the antero-lateral approach with subsequent UPAL gel implantation exhibited IVD defect repair. These results were similar to those obtained in the present model. 3 Additionally, discectomy of cadaveric sheep lumbar IVDs through the posterior approach demonstrated sufficient biomechanical advantages, and did not result in material protrusion 3 . No material protrusion was observed in a clinical trial, in which the gel was implanted into the defect after discectomy in 40 young patients (data not shown). 28 Therefore, the current results might be applicable to humans. Third, although the AF shows a material with a highly complex structure and has a pivotal role in the containment of the NP and/or its substitutes, a detailed evaluation of AF regeneration was not performed. It appears that the UPAL gel is capable of containing the alginate within, also under in vivo conditions, however, this requires an attachment of the gel to the adjacent vertebrae and the remainder of the AF. Because IVD degeneration is characterized by the degradation of the NP ECM and our previous study showed no UPAL gel protrusion from the cadaveric sheep IVDs, 3 the current study concentrated principally on NP preservation/regeneration. 2,3,15 However, the present study evaluated the morphological changes in the AF structure based on a histological scale. 2,3,39 Last, the intervention has been carried out in sheep in acute and sub-acute phases of IVD disruption, and therefore the efficacies shown here may be different from those in chronic cases, which were not the subject of this model. However, as most patients will present with chronic stages of IVD, the efficacy of the implants in such cases will require further evaluation. In addition, though the pre-degeneration of the IVD carried out in this study could be considered an improvement over the investigation of non-degenerated IVDs undertaken in previous studies, this improvement of the model was not quantified. Furthermore, it is not completely clear whether a four-week degeneration period is sufficient for obtaining a representative model of the human IVD after herniation. It is known that the AF of the human IVD is damaged over the entire posterior part, while there is only a protrusion on one posterior-lateral part. The posterior-contralateral region of the AF in human IVD is also damaged, unlike the AF in the degenerated sheep IVD.

Conclusions
We demonstrate that the combination of GMP-compliant, ultra-purified, BMSCs in the form of RECs and in situ-forming gel enhances IVD regeneration after discectomy in a sheep model exhibiting severe IVD degeneration, without resulting in neoplastic changes, and enable the maintenance of mechanical functions after implantation. Our findings and strategy need to be validated in clinical settings in future studies, especially in an elderly cohort such as individuals with combined lumbar canal stenosis.

Declaration of interests
T. S. and Y. M. are employees of PuREC Co., Ltd. M. W. is an employee of Japan Tissue Engineering Co., Ltd. They do not hold any other financial interests in these companies. These companies had no role in the study design, data collection, analysis, writing of the manuscript, the decision to submit the manuscript for publication or any aspect pertinent to the study. D. U., T. S., Y. M., and H. S. are listed as inventors on a patent application related to this work. The other authors declare that they have no competing interests. / Cellular Medicine and Gene Therapies" at the Japan Agency for Medical Research and Development, AMED (JP20lm0203098h0001 and JP21bm0404068h0002), and Mochida Pharmaceutical Co., Ltd for H.S.. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or report preparation.

Data sharing statement
All experimental data obtained in this study are available upon request to the corresponding author. All the data supporting the conclusions of this study are included within the article and supplementary data. All the other data and materials will be available upon request to the corresponding author.

Supplementary materials
Supplementary material associated with this article can be found in the online version at doi:10.1016/j. ebiom.2022.103845.