1. Patient inclusion criteria and exclusion criteria
Inclusion criteria: 1. Benign bone tumors occurring in the long bones of the limbs with local medullary cavity involvement associated with large bone defects after surgery and with bone grafting indications suitable for rapid stem cell screening and enrichment technology; 2. No hematopoietic systemic disease and 3. A follow-up time of no less than 12 months and with complete follow-up data available. Exclusion criteria: 1. Defects involving all of the medullary cavity requiring a large segment osteotomy; 2. Pathological findings showing that the tumor had abundant cells with a high atypia degree, indicating a high possibility of recurrence and 3. A loss of visitor.
2. Case data
A total of 22 patients were included in this study and divided into two groups: an experimental and control group. In the experimental group, the 11 patients using the enriched stem cell technique for bone defect repair included 8 males and 3 female, with an average age of 40.6 years old. The pathological diagnoses were as follows: 7 cases of giant cell tumors, 2 cases of fatty sclerosing mucinous fibrous tumor and 2 cases of fibrous dysplasia. In the control group, conventional methods were carried out to repair the bone defect. There were three cases in total, including 7 males and 4 females. The average age was 30 years old. Their pathological diagnoses were 6 cases of giant cell tumor, 4 cases of non-ossifying fibroma and 1 case of atypical cartilaginous tumor, respectively. This study was carried out after the approval of the Shanghai Ninth People’s Hospital Ethics Committee. We have obtained the consent for publication from the patient.
3. Preparation of the beta-TCP enriched stem cells
After general anesthesia, the anterior superior iliac spine was routinely disinfected and a towel was laid. A total of 75–80 ml of bone marrow blood was extracted. Following which, porous beta-TCP particles with around a 3–5 mm diameter and 75%±10% average porosity were put into a filter box, and the stem cell rapid screening–enrichment–composite system was then assembled. A total of 65–70 ml of bone marrow blood was injected into the system and filtered through the porous beta-TCP particles at a frequency of 70 Hz for 10 minutes. Finally, the bioactive beta-TCP particles were prepared.
4. Nucleated cell count and cell viability
For each sample, 1 mL of pre- and post-enrichment bone marrow was treated with a red blood cell lysis buffer (BioTime, Shanghai, China). Then, the nucleated cells (NCs) were counted using a hemocytometer (Beckman Coulter, Brea, California, USA). Cell viability was assessed by the trypan blue exclusion rate (Vi-CELL XR Cell Viability Analyzer Software, Beckman Coulter). The difference between the pre- and post-enrichment bone marrow was estimated for each patient.
5. Osteogenesis evaluation of porous beta-TCP loaded with BMSCs
A small amount of porous beta-TCP particles were implanted subcutaneously into 3-month-old nude mice in order to evaluate in vivo osteogenesis effects. The control group was treated with equal-quality pure TCP particles. In detail, a transverse incision of about 0.5 cm long was made on both sides of the spine. Porous beta-TCP particles enriched with BMSCs were placed subcutaneously on the left, while the control groups were placed on the right. Three weeks later, the nudes were sacrificed, and the materials were taken out and stained with picric acid and magenta to observe the inside osteogenesis under microscope.
6. Intraoperative operating
After general anesthesia, we firstly spent 10 minutes collecting the bone marrow blood. Then, bone tumor resection combined with bone grafting was performed after the diseased limbs had been disinfected. During the operation, all bone tumor focus areas were completely removed and the tumor boundaries were inactivated with anhydrous ethanol. In the test group, about 25–47 ml of porous beta-TCP particles enriched with BMSCs were implanted into the defect area, while in the control group, about 18–30 ml of pure beta-TCP particles were implanted into the defect area.
7. Evaluation of postoperative efficacy
X-ray films at 1 week after surgery were used as the baseline. Bone formation at 3 months after surgery was taken as the main observation index to evaluate the degree of bone healing in the bone defect site for both groups. At the same time, Musculoskeletal Tumor Society (MSTS) scores for the two groups were recorded and the functional recoveries of the affected limbs were compared. The degree of bone defect healing was evaluated by two orthopedic surgeons and one radiologist. Simultaneously, the MSTS score was assessed and recorded through a “single blind” method by two orthopedic surgeons who did not participate in this project.
8. Statistical method
SPSS Statistics 20.0 (IBM, America) was used for the statistical analysis. The mean, standard error of the mean and p values based on two-tailed t tests were calculated. Differences were considered significant at P < 0.05.