2.1 Selection criteria and baseline data
From June 2019 to December 2020, patients who were diagnosed with degenerative lumbar diseases and were ready for the transforaminal lumbar interbody fusion(TLIF) procedure were recruited into this study. The inclusion criteria for patients were as follows: (1) aged from 18–65 years, regardless of whether patients were male or female; (2) diagnosed with degenerative lumbar diseases; (3) did not participate in another clinical study within three months; and (4) voluntarily participated and provided signed informed consent. The exclusion criteria were as follows: (1) fracture; (2) tumour; (3) infection; (4) severe osteoporosis; (5) revision surgery; (6) mental illness; (7) metabolic or immune dysfunction; (8) drug abuse; (9) severe primary diseases of vital organs; (10) allergy; (11) pregnancy and breastfeeding; or (12) poor compliance. After the patients signed the informed consent form, they were randomly divided into two groups using a previously established randomized number table. One group using decompressed laminal bone graft (LBG) for intervertebral fusion and another group received MSCs/β-TCP composites prepared by the SECCS for intervertebral fusion. Eventually 37 patients were enrolled into the study, of which 19 received LBG treatment and 18 received SECCS treatment (Figure 1). The detailed data of patients included into the two groups were demonstrated at table 1.
2.2 MSCs/β-TCP composite preparation
After general anaesthesia, the patient was placed in a supine position, and a 16-gauge bevel medullo-puncture needle was used to puncture the anterior superior iliac spine. Approximately 80 mL bone marrow was collected before surgery (Figure 2A). The SECCS system mainly consists of the following parts: (1) a detachable columnar double-layer filter box, which used for loading the porous biomaterial and haemofiltration; (2) a sealed pipeline for fluid circulation; and (3) a peristaltic pump that exerts a force to drive the continuous circulation of the bone marrow (Figure 2B–2D). When the procedure was started, 3~6 g β-TCP particles (Bio-Lu, Shanghai, China) with mechanical strength > 2 Mpa, porosity > 40%, a diameter of 1–3.5 mm, and a mean pore size of 500 ± 200 μm were placed in the inner box. The inner and outer filter boxes were then screwed together. Approximately 60 mL bone marrow was injected into the pipeline, and the power pump was started to circulate the bone marrow in the pipeline and filter it through the porous β-TCP (Figure 2E and F). After 6 min (60 r/min,50HZ), the MSC-enriched β-TCP (MSCs/β-TCP composites) particles were manufactured and ready for implantation. In addition, 5 mL bone marrow was collected before and after enrichment for routine blood tests.
2.3 Surgical procedure
After general anaesthesia, the patient was placed in a prone position, and sterile drapes were placed on the surgical field after disinfection. TLIF was performed as previously reported(14). Specifically, for patients allocated to the LBG group, the bone graft filled in the intervertebral fusion cage was decompressed laminal bone, while MSC/β-TCP composites were used for fusion in patients in the SECCS group. In addition, bilateral pedicle screws and rods were used for fixation.
2.4 Enrichment efficiency evaluation
The bone marrow was collected before and after enrichment for MSC counting and maintained in Alpha Minimum Essential Medium (Sigma, USA) with 10% foetal bovine serum (FBS; HyClone). Then, the suspension was added to a 6-well plate for adherent cell culture in an incubator with 5% CO2 at 37 °C. The medium was refreshed every two days, and 10–14 days later, the culture medium was replaced with osteogenic induction solution (complete medium + 50 µM sodium ascorbate [Sigma] + 10 mM glycerophosphate [Sigma, USA] + 100 nM Mdexamethasone [Sigma, USA]) and cultured for another 10–14 days. Next, the cell colonies were observed under a microscope and then stained with alkaline phosphatase (ALP; Beyotime, Shanghai, China). The number of ALP-positive colony-forming units (CFUs/ALP+) with a diameter of more than 2 mm were counted. The average number of CFUs/ALP+ colonies before and after enrichment represents the difference between the numbers pre- and post-SECCS for each patient. Enrichment efficiency was formulated as (PreCFUs/ALP+ – PostCFUs/ALP+)/PreCFUs/ALP+ × 100%. For erythrocyte, leukocyte, and haemoglobin counts, the bone marrow pre- and post-SECCS was detected using a blood cell detector.
2.5 Cell count and cell viability test
Erythrocytes were eliminated using erythrocyte lysate (SCIGE, Shanghai, China) according to the manufacturer’s instructions. The retained cells were resuspended and 10 µL of the resuspended solution was mixed with trypan blue and added to a cell counting plate (Invitrogen, USA). Cell number and cell viability were detected using a cell counting machine (Beckman Coulter, Brea, CA, USA).
2.6 Flow cytometry
The MSC/β-TCP composite granules that remained after surgery were collected, and the MSCs were eluted by pancreatic enzymes. The cells were resuspended and cultured in complete medium for 14 days. The cells were collected and labelled with CD44, CD73, CD90, CD105, CD34, CD11b, CD19, CD45, and HLA-DR antibodies (BD Biosciences, USA) to identify MSC markers by flow cytometry (Becton, Dickinson and Company, USA).
2.7 Osteogenic, adipogenic, and chondrogenic differentiation
As per the abovementioned procedure, the cells adhering to the wall were obtained and passaged, and the first-generation MSCs were collected. For osteogenic differentiation, the cells were cultured in osteo-inductive medium (Stemcell, Canada) for 21 days. They were then fixed with 95% absolute ethanol for 30 min and stained with Alizarin Red (Solarbio, USA) for 30 min at 37 C. For adipogenic differentiation, cells were cultured in adipogenic induction medium (Stemcell) for two days. The medium was then changed to the adipogenic maintenance medium containing 10 μg/mL insulin and cells were incubated for one day. After 14 days, the cells were stained with Oil Red O (Sigma). For chondrogenic differentiation, aliquots of 250,000 cells were added to serum-free medium, resuspended in a 15 mL tube, and centrifuged at 600 × g for 5 min. Then, 0.5 mL chondrogenic medium (Stemcell) was added into the tube. After 48 h incubation at 37 °C with 5% CO2, the cell pellets were flipped into the tube and cultured for 28 d. Then, the pellets were fixed with 4% paraformaldehyde (Servicebio, Shanghai, China) for 15 min, followed by Alcian blue staining (Solarbio).
2.8 Scanning electron microscope (SEM) observation
Some MSC/β-TCP composite granules that remained after surgery were collected and divided into two parts, which were cultured in complete medium for 2 h and 2 weeks, respectively. Particles were fixed in 2% glutaraldehyde (Servicebio) for 2 h and washed three times with 0.1 M PBS (pH 7.4) for 10 min each time. Then, 1% osmic acid (Best-Reagent, Shanghai, China) was added to fix the particles again for 1 h, and particles were post-rinsed in 0.1 mol/L phosphate buffer (pH 7.2) for 1 h. Ethanol was added for stepwise gradient dehydration. Isoamyl acetate (Sigma) was added and mixed with ethanol at a ratio of 1:1 for replacement. After 10 min, the replacement solution was discarded and pure isoamyl acetate was added to soak for 15 min. Finally, the critical point drying method was used to dry the samples. After sputter-coating with a layer of gold, the specimens were placed under an SEM for observation.
2.9 Patient follow-up
All patients were routinely followed up within one week, three months (± 7 days), six months (± 15 days) and 1 year after surgery. The interbody fusion rate was evaluated using X-ray and computed tomography (CT) scans. According to dynamic radiographs (lateral flexion and extension), with the angle between the vertebral bodies less than 5°, and continuous trabecular bone formation in the segment analysed by CT is recognized as fusion. Fusion failure is determined to have occurred when the cage is loosened and shifted, there is a translucent band > 2 mm on the cage surface, and the angle between the vertebral bodies is > 5° on the dynamic radiographs . Intervertebral height collapse can also reflect the fusion quality; as such, we observed the change in intervertebral height, which was calculated as follows: (anterior disc height + posterior disc height)/2. Functional improvement was evaluated using the Japanese Orthopaedic Association (JOA) and Oswestry Disability Index (ODI) scores. As the accuracy of pedicle screw insertion affects function improvement, we also evaluated this factor by CT scan after surgery. The screw position was classified into four grades, as follows: grade 0, no branch; grade 1, only the threads outside the pedicle are less than 2 mm; grade 2, core screw diameter outside the pedicle or breach 2–4 mm; and grade 3, complete screw outside the pedicle(15). Two senior orthopaedic surgeons and one radiologist assessed the images, and the unanimously judged result of two or three doctors was confirmed to be the approved result.
2.10 Statistical analysis
SPSS 22.0 statistical software (IBM Corp., Armonk, NY, USA) was used for data management and statistical analysis. The measurement data are expressed as`X ± S, and the count data are expressed as rate (%). The data were tested for normality by the Shapiro–Wilk test. Two sample t-test or the paired t-test were used to compare normally distributed variables. Chi-squared test were used to compare the proportion. P < 0.05 was considered to indicate a statistically significant difference.