Tissue‑engineered bone used in a rabbit model of lumbar intertransverse process fusion: A comparison of osteogenic capacity between two different stem cells
- Authors:
- Published online on: February 11, 2020 https://doi.org/10.3892/etm.2020.8523
- Pages: 2570-2578
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Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
From the pathological standpoint, in several diseases, including spondylosis, deformity, tumor, infection, fracture and instability, it is necessary to reconstruct a stable structure and correct an abnormal relationship between adjacent vertebral structures (1). Spinal fusion may enhance the mechanical stability of the spine via reconstruction and stabilization of the vertebral column; therefore, spinal fusion is currently one of the main treatment options for the aforementioned diseases (2,3). Until now, autografts have been the gold standard for use as spinal fusion materials; however, limited bone graft sources and donor-site morbidity hinder their extensive use, especially for those cases in which large amounts of bone graft material are required. By contrast, allograft and xenogenic bone, and other potential options for bone graft substitution or supplementation, including ceramics, calcium phosphate compounds, collagen gel and demineralized bone matrix, have shown significant variability in osteoinductive properties and clinical efficacy (4-6). Certain biological factors, such as bone morphogenetic protein have shown similar or improved fusion rates compared with autografts; however, potential safety concerns require further clarification (5). With the development of tissue engineering as an alternative approach for spinal fusion, bone tissue engineering has become a topic of particular interest (7,8).
The elements of tissue engineering comprise seed cells, biological scaffolding and growth factors. Seed cells serve an important role in the effects of tissue engineering technology (9). As classical seed cells, bone marrow mesenchymal stem cells (BM-MSCs) have frequently been employed in bone tissue engineering due to their multi-lineage differentiation potential and rapid in vitro amplification (10,11). MSCs have been reported to exist in many types of mesenchymal tissue, and different tissue-derived MSCs differ from each other in properties including proliferation and differentiation potential, and tissue regeneration capacity (12,13). Therefore, specific types of MSCs should be chosen as appropriate for the intended tissue engineering application.
The intervertebral disc (IVD) is composed of annulus fibrosus (AF), nucleus pulposus (NP) and cartilage endplate (CEP). Previous studies reported that different types of MSCs exist in AF and NP regions (14,15). The present research team identified MSCs in CEP, which they designated as CEP-derived stem cells (CESCs), and found that CESCs share similar morphology, proliferation rate, cell cycle, immunophenotype and stem cell gene expression with BM-MSCs (16). Furthermore, CESCs have exhibited superior chondrogenic and osteogenic potentials compared with BM-MSCs in vitro (16,17). In an in vivo study, CESCs showed more powerful NP regeneration potential compared with AF-derived stem cells, NP-derived stem cells and BM-MSCs derived from the same patient following transplantation into the rabbit IVD, and displayed no obvious immune rejection as heterografts (18). However, the osteogenic characteristics of CESCs in vivo are unclear. Large quantities of CEP samples that are usually discarded as clinical waste in spinal fusion surgeries could be collected and reused for the extraction of CESCs, and may serve as an adequate seed cell source for experimental or clinical studies. Therefore, it is necessary to investigate the in vivo bone formation capacity of CESCs, and explore whether they have the potential to serve as seed cells for bone tissue engineering.
In the present study, CESCs and BM-MSCs were harvested from the same donors who received a lumbar spinal fusion procedure. After culturing and expanding, the cells were each seeded into porous hydroxyapatite (PHA). After 14 days in vitro induction, the cell/PHA composites were tested to determine the difference in osteogenic mRNA expression between the two types of seed cells. In addition, cell/PHA composites were implanted into a rabbit lumbar intertransverse process fusion model after 3 days in vitro induction. Eight weeks later, those grafts were gross observed, palpated and inspected with three-dimensional (3D) computed tomography (CT) reconstruction, micro-CT and quantitative histology to obtain bone formation indices for the comparison of in vivo osteogenic capacity.
Materials and methods
Ethics statement
All procedures were approved by the Institutional Review Board of Xinqiao Hospital and the patients provided written informed consent in the study before surgery. All animal experiments were also approved by the Xinqiao Hospital Committee on Ethics for the Care and Use of Laboratory Animals.
Isolation and culture of CESCs
The procedures for the isolation and culture of CESCs were performed as previously described (17,18). CEP samples were derived from 11 patients (age range: 37.9-61.2 years) who received lumbar fusion surgery at Xinqiao Hospital (Chongqing, China) between June 2015 and August 2016. The severity of CEP damage was determined as described by Rajasekaran et al (19). The characteristics of the patients and the tests in which their CESCs were used are shown in Table I.
Isolation and culture of BM-MSCs
Bone marrow samples were obtained from the aforementioned patients. Isolation and culture procedures for BM-MSCs were performed as previously described (20,21). In brief, 6 ml bone marrow was aspirated from the iliac crest and centrifuged at 900 x g for 25 min at 20˚C with an equal volume of 1.073 g/ml Percoll solution (Sigma-Aldrich; Merck KGaA). Mononuclear cells were carefully extracted and rinsed twice with PBS. Finally, the cells were suspended with DMEM/F12 (Hyclone) supplemented with 10% fetal calf serum (FCS; Hyclone) and 100 U/ml penicillin-streptomycin (Hyclone), then cultured in 25-cm2 cell culture flasks (Costar; Corning, Inc.) with an atmosphere of 5% CO2 at 37˚C. Thereafter, the culture medium was refreshed every 3 days. When 90% confluence was reached, the cells were passaged.
Determination of the cell surface antigen profile
BM-MSCs and CESCs from 3 patients were analyzed to determine their respective surface immunophenotypes by flow cytometry. Cells were washed with PBS twice and fixed with 4% paraformaldehyde at 4˚C for 10 min, then were incubated in the dark for 20 min with fluorescein isothiocyanate (FITC)-coupled monoclonal antibodies: CD11b-FITC, CD34-FITC, CD45-FITC, CD90-FITC, and CD105-FITC. The cells were then washed with PBS twice and re-suspended in 200 µl PBS. Finally, the cell suspension was passed through a Flow Cytometer, and the antigen phenotype was analyzed using Flow Jo software (version 7.5, Flow Jo LLC). Mouse isotype antibodies were used as controls.
Stem cell seeding in the PHA graft
BM-MSCs and CESCs derived from 8 patients were trypsinized, rinsed and re-suspended in fresh medium. After microscopic counting, 3x106 cells were dropped into PHA (1.0x1.0x3.0 cm; porosity 42.2±1.8%; average pore diameter 180±60 µm) and centrifuged at 80 x g for 1 min at 20˚C. Cells/PHA grafts were incubated in an incubator with 5% CO2 at 37˚C for 24 h. Then, they were induced for 2 weeks with basal medium supplemented with 100 nM dexamethasone, 0.2 mM ascorbate and 10 mM β-glycerophosphate (Sigma-Aldrich; Merck KGaA). During the induction period, the osteogenic medium was changed every 3 days.
Quantitative assay of alkaline phosphatase (ALP) activity
To quantify the ALP activity of the in vitro cultured grafts, a modified procedure was used (22). After induction for 1 or 2 weeks in the osteogenic medium, the grafts were washed with PBS, and then incubated in 1.0 ml lysis solution comprising 10 mM Tris-HCl, 1 mM MgCl2 and 1% Triton X-100 at 4˚C. The supernatant was transferred to a 96-well plate (50 µl/well), and incubated with 100 µl substrate (p-nitrophenyl phosphate; 6.7 mM/l) at room temperature for 10 min. Then, 100 µl NaOH (0.1 M) was added to stop the reaction. The optical density (OD) at 405 nm (OD405) was measured using a spectrophotometer. The OD405 value of a PHA graft containing no cells served as control, and each sample was tested in triplicate.
Reverse transcription-quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assay
To evaluate the expression of osteogenic-specific genes in cells/PHA grafts in vitro, RT-qPCR was used (23). Stem cells harvested from 3 patients were each assigned to a PHA graft. After 2 weeks of induction, total RNA was extracted from each cell/PHA graft using a Total RNA Extraction kit (Qiagen GmbH) (24). RNA concentration and quality were evaluated on the basis of the OD 260/280 ratio. The mRNA (1.0 µl) was reversely transcribed to cDNA using a First Strand cDNA kit (Qiagen GmbH) according to the manufacturer's instructions. A total reaction volume of 25 µl containing SYBR-Green Master Mix reagent (Applied Biosystems; Thermo Fisher Scientific, Inc.) was amplified via qPCR (ABI Prism 7000; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 30 sec; 40 cycles of 95˚C for 5 sec and 60˚C for 32 sec; and a final dissociation stage at 95˚C for 15 sec, 60˚C for 60 sec and 95˚C for 15 sec. The osteogenic-specific genes and reaction conditions are shown in Table II, and β-actin served as an internal control to normalize the expression of the mRNA of these genes in different cell types. The quantitative determination of target mRNA expression was conducted according to the 2-ΔΔCq method as previously described (24,25).
Animal model
An animal model was produced using previously reported methods (26,27). A total of 24 New Zealand white rabbits (The Third Military Medical University; age range 8-12 weeks), of mixed sexes (13 mala and 11 female) weighing 2.0-2.5 kg were used in the study. Rabbits were fed with rabbit pellets and drinking water ad libitum and reared in a constant temperature room at 20˚C with 50±5% humidity, 0.03% CO2 and 12-h light/dark cycles. The rabbits were randomly divided into 3 groups (each n=8) as follows: BM-MSCs/PHA grafts; CESCs/PHA grafts; and PHA only grafts containing no cells to serve as a control. The rabbits were anesthetized with sodium pentobarbital (30 mg/kg) via intravenous injection. Following removal of all the soft tissues, decortication of transverse process L4-L5 was performed to provide the fusion bed. Then, grafts that had undergone 3 days in vitro induction were implanted into bilateral sides of the intertransverse process interval, in parallel with the spine. Finally, the surgical incision was closed layer by layer.
Spiral CT scanning
To evaluate bone formation and fusion conditions, spiral CT scanning was conducted at 8 weeks after implantation surgery. The lumbar spine segment L3-L6 was scanned at 1-mm slice thickness and reconstructed into 3D images (SOMATOM Emotion; Siemens Healthineers). To observe the fusion conditions, 5 axial slices were scanned at positions containing the L4 and L5 transverse process attachments to the graft, and three intermediate regions, respectively.
Manual palpation
At 8 weeks after implantation, animals were sacrificed with an overdose of sodium pentobarbital. The objective lumbar spine (L4-L5 processes) was exposed after the removal of soft tissues, then manually palpated as previously described (28,29). Only if no movement was detected in the L4-L5 segment, and was confirmed by two checkers in a blinded manner, was the implanted graft considered as fused.
Micro-CT analysis
To assess the quality of the newly formed bone in the grafts, micro-CT was used (29,30). At 8 weeks after implantation, all the extraneous vertebrae and soft tissues were dissected, and the implants were scanned using micro-CT (GE Healthcare, Canada) using the following parameters: 60 kV; 0.6 mm aluminum filter; 800 µA; number of players=150. More than 1,000 axial images were obtained from each graft at the threshold of 1,200 HU. The region of interest was chosen symmetrically in the left and right grafts as a cylinder (0.5x0.5x0.5 cm3) at rdifferent coronary positions. The grafts were equally portioned into five segments by 4 cross-sections. To evaluate osteogenesis, six morphometric indices were measured as follows: i) bone mineral density (BMD); ii) bone mineral content (BMC); iii) tissue mineral density (TMD); iv) tissue mineral content (TMC); v) bone volume fraction (BVF); vi) bone volume (BV) (30,31). PHA containing no stem cells served as control. Two photographers analyzed the data in a blinded manner.
Histological analysis
Animals were sacrificed with an overdose of sodium pentobarbital at 8 weeks after implantation. The graft specimen was harvested, fixed in 10% neutral buffered formalin for 24 h at 20˚C and sequentially dehydrated in ethanol solutions. Then, the graft was embedded in polymethylmethacrylate solution for 1 week. Grafts were sectioned to 50 µm using a diamond saw (Leica Microsystems GmbH, Germany). The slices were stained with Villanueva-Goldner's trichrome (VG) at 20˚C for 30 min and observed with a light microscope (Olympus Corporation) to evaluate osteogenesis by two pathologists in a blinded manner. A total of 9 sections from 3 grafts, with 3 random sections obtained from each graft, were quantitatively analyzed for newly formed bone and collagen I in VG staining using Image-Pro Plus software 6.0 (Media Cybernetics, Inc.). Osteogenesis was quantified on the basis of the area volume of light blue and red staining, which represented collagen I and newly formed trabecular bone, respectively (32).
Statistical analysis
SPSS version 13.0 software (IBM Corp.) was used for statistical analysis. All data are presented as the mean ± standard deviation. The two-tailed Student's t-test was used when comparing only two groups, and one-way ANOVA followed by Fisher's Least Significant Difference or Bonferroni's correction post-hoc tests were used to analyze differences among three groups. P<0.05 was considered to indicate a statistically significant difference.
Results
Cell morphology and antigenic phenotype
The BM-MSCs and CESCs exhibited a similar spindle-shaped appearance while in culture (passage 2; Fig. 1A). Flow cytometric analysis indicated that CESCs and BM-MSCs shared an analogous antigenic phenotype (Fig. 1B and C). Both cell types were negative for CD34, CD11b and CD45 (<2%), positive for CD90, and moderately positive for CD105. No marked differences were detected in the expression levels of CD11b, CD90, CD34 and CD45 between the two cell types.
ALP activity
The ALP activity results indicated that the OD405 values of both two cell types contained in the grafts increased from 1 to 2 weeks of induction (Fig. 2A). At the 1-week culture time point, the OD405 value of the CESCs/PHA group was significantly higher compared with that of the BM-MSCs/PHA group (1.80±0.26 vs. 1.47±0.24; P<0.01; Fig. 2A). A significant difference was also detected between the two groups at 2 weeks (2.36±0.28 vs. 1.92±0.25; P<0.01; Fig. 2A).
Osteogenic capacity in 3D culture
According to the RT-qPCR assay results, after 2 weeks of induction, the CESCs/PHA group exhibited a significantly higher expression level of ALP mRNA compared with the BM-MSCs/PHA group (1.45±0.20 vs. 1.0-fold; P<0.01; Fig. 2B). For Runx2 and osteocalcin (OC) mRNA, significantly higher expression was also observed in the CESCs/PHA group compared with the BM-MSCs/PHA group (1.19±0.18 vs. 1.0-fold for Runx2; 1.24±0.20 vs. 1.0 for OC; P<0.01; Fig. 2B). However, both groups exhibited comparable expression levels for osteopontin and bone sialoprotein mRNA (P>0.05; Fig. 2B).
3D CT evaluation and fusion status
Although all grafts broke into several parts during surgery, at 8 weeks after implantation, reconstructed 3D CT demonstrated bony healing of the fractured segments and definite fusion existing between the transverse processes (L4 and L5) and the graft in the majority of the cell-containing grafts (Fig. 3A and B).
Analysis using manual palpation revealed that 7/8 animals (87.50%) in the CESCs/PHA group and 6/8 animals (75.0%) in the BM-MSCs/PHA group achieved fusion; however, fusion was obtained in only 3/8 samples (37.5%) for the graft comprising only PHA. The fusion rate in the control group was lower compared with the CESCs/PHA and BM-MSCs/PHA groups (P<0.05). Furthermore, no significant difference in fusion rate was detected between the CESCs/PHA and BM-MSCs/PHA groups (P>0.05; Fig. 3C).
Bone formation analysis by micro-CT
According to the micro-CT data, all the osteogenesis indices in the CESCs/PHA group had higher values compared with those of the BM-MSCs/PHA group (Fig. 4). Significant differences were observed between the two stem cell-containing groups for BV, BVF, BMC, BMD and TMD (P<0.01; Fig. 4B).
Histological assessment
In VG stained slices, collagen I, newly formed trabecular bone and PHA were stained as blue, red and black, respectively (Fig. 5A-F). The CESCs/PHA grafts exhibited more newly formed collagen I and trabecular bone than the BM-MSCs/PHA group. For the only PHA graft, the content of collagen I and trabecular bone was clearly the lowest. Quantitative data indicated that the CESCs/PHA group had 740±62 µm2 newly formed trabecular bone and 863±84 µm2 of collagen I, whereas the respective values in the BM-MSCs/PHA group were 381±36 and 740±54 µm2, respectively (Fig. 5G). Significant differences were detected between each pair of the three groups for collagen I and trabecular bone (P<0.01).
Discussion
MSCs are an attractive cell population for use in the regeneration of various tissues due to their multilineage differentiation potential (10). Studies have indicated the extensive use of MSCs, especially BM-MSCs, in bone tissue engineering (8). The MSCs used in the present study were obtained using previously described methods (10,18). In addition, the cell surface antigen profiles were also basically consistent with those in previous studies, and indicate that the cells used in the present study possess the basic characteristics of MSCs described by the International Society for Cellular Therapy (18,33).
Generally, the degenerative status of NP and CEP is hemi-quantitatively judged by magnetic resonance imaging (34,35). In the present study, CESCs were obtained from human degenerated CEP of types V and VI according to the previously described classification (19). Whether the bone formation ability correlates with the degeneration level of the extracted sample remains to be elucidated. The possible discrepant biological characteristics and bone formation potential of the CESCs derived from clinical CEP samples with different degenerative degrees merits investigation in future studies.
Unlike 2D culturing, the 3D culture environment closely resembles the in vivo environment. Different growing conditions may lead to differences in biological characteristics. ALP mRNA expression and ALP activity were significantly upregulated in CESCs as compared with BM-MSCs in the present study, and were accordant in 2D and 3D culturing environments (16). OC as a marker for the late stage of osteoblast differentiation was expressed at a significantly highly level in CESCs/PHA compared with BM-MSCs/PHA, which was consistent with previous 2D culture data (16,36). In addition, significantly higher expression of Runx2, another specific matrix protein expression marker for bone maturation, was observed in CESCs/PHA compared with BM-MSCs/PHA in the 3D environment, whereas no significant difference was detected between CESCs and BM-MSCs in 2D culture (16,37). It is hypothesized that the aforementioned differences may be partially attributed to the favorable cell-cell and cell-extracellular matrix (ECM) interactions in a 3D multilayered-cell environment.
PHA is a classical scaffold material with good biocompatibility, bone induction and bone conduction properties, and is often used in bone tissue engineering research (38). The PHA used in the present study had a porosity of 42.2±1.8%, an average pore diameter of 180±60 µm, and a 3D framework in which cellular proliferation and differentiation, and ECM deposition may occur. However, the weak fracture resistance of PHA predisposes it to break under torsion or shear force (39). In the present study, nearly all PHA grafts broke into two to four parts following transplantation into the intertransverse process. However, the broken grafts were restored to integrity by the newly formed bone that gradually bridged the defects of the broken material. Furthermore, the grafts with CESCs or BM-MSCs demonstrated significantly higher fusion rates compared with the PHA only control. Generally, in the present study, PHA breakage during the experimental process did not affect the evaluation of bone formation capacity. Instead, it further certified that the implanted stem cells, especially CESCs, provided a stronger osteogenic and repair capacity, even under the challenging environment caused by PHA breakage.
Micro-CT is a reliable in vivo method for the quantitative and qualitive evaluation of newly formed bone without physical disruption of the sample. Indices such as BV, BVF, BMC, BMD, TMC and TMD indirectly reflect fusion quality (40). In the CESCs/PHA group, BV, BVF, BMC, BMD and TMD values were significantly higher compared with those of the BM-MSCs/PHA group; only the TMC values exhibited no significant differences between these two groups. These results indicate that the CESCs/PHA complex was able to induce bone regeneration more efficiently. In addition, quantitative histology complemented the micro-CT data by revealing that the volume of collagen I, the main organic component of bone (41), and newly regenerated trabecular bone in the CESCs/PHA group were significantly higher than those in the BM-MSCs/PHA group. Therefore, the results of micro-CT and histological analysis confirmed that the CESCs/PHA composite enhanced the quantity and quality of bone formation.
However, the present study has certain limitations. Firstly, rabbits are relatively small in size, and the mechanical stress that the implanted grafts endured in rabbits are likely to differ from those in the human spine. Different mechanical factors might have a profound effect on the biological characteristics of seed cells, including their osteogenic capacities. Therefore, in subsequent studies, larger animals such as goats or nonhuman primates might be used to provide a more restrictive biomechanical environment that is more analogous to that of the human spine. Secondly, autologous bone grafting was not set as the gold standard in this study due to the small number of experimental animals; therefore, the final fusion efficacy that CESCs could achieve relative to the gold standard remains unknown. Thirdly, no biomechanical tests were performed to further assess the quality of the newly formed bones due to the small size of the rabbit spine. To address this issue, larger animals should be included in future studies.
To the best of our knowledge, the present study was the first to use stem cells derived from human degenerated CEP as seed cells for tissue-engineered bone products, and compare their osteogenesis with the traditional seed cells, BM-MSCs, in the 3D in vitro environment and in vivo rabbit spinal fusion model. Although the present study yielded encouraging results, the definite osteogenic efficacy relative to the gold standard and the long-term safety for in vivo implantation require further investigation prior to clinical application. In addition, PHA should be improved to enhance its fracture resistance, or an alternative scaffold with fine biocompatibility and mechanical properties should be considered for further study.
In conclusion, the present study preliminarily compared the osteogenic capacity between CESCs and BM-MSCs derived from the same donors in the rabbit lumbar intertransverse process fusion model. CESCs exhibited superior bone formation ability than BM-MSCs when used with PHA under a 3D environment in vitro and in vivo. The results indicate that CESCs have potential as an efficient and sufficient seed cell source for bone tissue engineering, and CESC-based products show promise as superior candidates for future clinical application in spinal fusion or other bone regeneration and repair issues.
Acknowledgements
Not applicable.
Funding
This study was supported by the National Natural Science Foundation of China (grant nos. 81472076 and 81560369).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
HW performed the experiments, wrote the manuscript and collected, analyzed and interpreted data. YZ contributed to study design and conception, wrote the manuscript and analysed and interpreted data. CQL, TWC and JW performed the experiments and obtained clinical samples. BH contributed to study design and conception, analysed and interpreted data, and wrote and gave final approval of the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All procedures were approved by the Institutional Review Board of Xinqiao Hospital. All patients provided written informed consent for participation in the study. All animal experiments were approved by the Xinqiao Hospital Committee on Ethics for the Care and Use of Laboratory Animals.
Patient consent for publication
Not applicable.
Competing interests
All the authors declare that they have no competing interests.
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