Healing capacity of bone marrow mesenchymal stem cells versus platelet-rich fibrin in tibial bone defects of albino rats: an in vivo study

Ethical statement

The study protocol was approved by the Research ethics committee of Faculty of Dentistry, Cairo University (151031).

Experimental procedure

A total of 36 male Wistar rats weighing 175–200 g, aged 12–14 weeks-old were used in this study. The animals were obtained from and housed in the Animal house, Faculty of Medicine, Cairo University. The animals were randomly placed in separate cages under controlled room temperature 25±2°C with 12/12 h light/dark cycle and were fed food and water ad libitum.

Isolation, culture and identification of BM-MSCs

BM-MSCs were isolated from the femurs of 6 Wistar donor (6-weeks-old male) rats (100±20 g), BM-MSCs isolation and propagation occur in 14 days before experimental procedures under aseptic conditions as previously described¹⁷. Briefly, bone marrow was harvested by flushing the femurs with Dulbecco’s modified Eagle’s medium (DMEM, GIBCO/BRL) supplemented with 10% fetal bovine medium (GIBCO/BRL). Cells were isolated with a density gradient [Ficoll/Paque (Pharmacia)] and cultured in culture medium supplemented with 1% penicillin-streptomycin (GIBCO/BRL) at 37°C in a humidified 5% CO₂ incubator. When large colonies developed (80–90% confluence), cultures were washed twice with phosphate buffer saline (PBS) and cells were trypsinized with 0.25% trypsin in 1 mM EDTA (GIBCO/BRL) for 5 minutes at 37°C. After centrifugation (at 2400 rpm for 20 minutes), cells were re-suspended with serum-supplemented medium and incubated in 50 cm² culture flask Falcon. On day 14, the adherent colonies of cells were trypsinized, and counted.¹⁷. Cultures confluence was monitored by inverted light microscope (Olympus, USA) with a digital camera (Nikon, Japan).

Surface antigens CD90 and CD34 were detected by flow cytometry to allow identification of BM-MSCs as follows. Following blocking in 0.5% BSA and 2% FBS in PBS, 100,000 cells were incubated in the dark at 4°C for 20 min with the following monoclonal antibodies; FITC CD 90 (PN IM1839U; Beckman Coulter), PE CD 34 (PN IM1871U; Beckman Coulter, USA). Mouse isotype PE antibody (Beckman Coulter, USA) were used as controls (dilution of all antibodies, 1:1500). Cells were washed and suspended in 500 µl fluorescence activated cell sorting (FACS) buffer and analyzed using a Cytomics FC 500 flow cytometer (Beckman Coulter, USA) using CPX software version 2.2. BM-MSCs osteogenic differentiation was induced by StemPro osteogenic induction medium; incubation for 7 days at the third passage, 1 × 10³ cells/each well by osteocyte StemPro osteogenesis differentiation kit (Gibco, Life Technology) at concentration 300 µl of osteogenic medium (stempro medium) and identified by alizarin red staining (Sigma-Aldrich) for 30 min at room temprature, the mineralized nodules were stained and monitored using inverted light microscope (Olympus, USA) with a digital camera (Nikon, Japan). The results were presented by descriptive analysis.

Establishment of bone defects

The surgical approach was under general anaesthesia via intramuscular injection of 50-75 mg/kg ketamine chlorohydrate (Amoun CO) and 20 mg/ Kg body weight xylazine HCL (Xyla-Ject®, Phoenix^TM, Pharmaceutical Inc.) in the proximal–medial area of each tibia. While blood samples were being prepared, a 3-mm diameter bone defect was created using a round surgical bur³ under constant irrigation with saline solution in both tibiae of the same animal (split-body design) to avoid selection bias and neutralize any confounders that may affect the outcomes of both treatments. Experimental groups were standardized among all the animals: group A, left tibia defect received PRF clot immediately placed by sterile tweezers in the defect; Group B, right tibia of the same animal received BM-MSCs seeded on chitosan scaffold then implanted in the tibial bone defect using sterile spatula. Both groups were randomly sub-divided according to time of euthanasia into three sub-groups (1, 2 and 3); at 3, 10 days and 3 weeks, respectively (n = 12) (Table 1). Postoperatively, periosteum flaps and skin were sutured. Anti-inflammatories and antibiotics were applied on the skin and injected for 3 days. Each animal received IM 10 mg/kg flumox (Eipico, Egypt) to avoid secondary bacterial infection, 10 mg/kg cataflam (Novartis, Egypt) to relieve postoperative pain and topical antibiotic spray; Bivatracin (Egyptian Company For Advanced Pharma, Egypt) to avoid local infection. The animals were euthanized by intra-cardiac overdose of sodium thiopental (80 mg/kg).

Scaffold fabrication

To obtain a porous chitosan scaffold to deliver BM-MSCs into the defect, 1 g chitosan (Merck Germany) was dissolved in 200 µl 0.2% M acetic acid , stored for 1 day at room temperature, poured into a 3-mm diameter stainless steel circular mould, stored in deep freezer at −70°C for 5 days, then lyophilized for 3 days as follows. In the lyophiliser (Thermo Fisher Scientific), there were three phases of preparation. The first phase was freezing phase, where the sample was exposed to −40°C in a vacuum for 10 min. The second phase was warm up vacuum pump phase, where sample was exposed to −15°C in a vacuum for 20 min. The third phase was main drying phase, where sample was exposed to 30°C in a vacuum for 3 days; after the 3 days, a blank porous chitosan scaffold was prepared^18,19.

Preparation of BM-MSCs

Prior to cell seeding, the lyophilized scaffolds were immersed in absolute ethanol for sterilization. Hydration was accomplished by sequential immersion in serially diluted ethanol solutions of 70, 50, and 25%, during intervals of 30 min each. Scaffolds were finally equilibrated in PBS followed by standard culture medium (30 min; 3 times), and then placed in tissue culture plates ready to be seeded. BM-MSCs were seeded at a density of 2.5x10⁶ cells/scaffold under static conditions, by means of a cell suspension. The seeded scaffold was then placed in the defect to deliver the stem cells to the defect, which was then sutured closed.

Preparation of PRF

A total of 2 ml venous blood was drawn from the caudal vein of rats used in the experiment into a plain tube and immediately centrifuged at room temperature with a lab centrifuge (Electronic centrifuge 800, China) for 10 min at 3000 rpm²⁰. In the middle of the tube, a fibrin clot was formed between the supernatant acellular plasma and the lower red corpuscles. The PRF clot was detached using sterile tweezers applied to the bone defect.

Scanning electron microscopy/energy-dispersive X-ray (SEM/EDX) analysis

Tibiae were carefully dissected free from soft tissue; bone specimens of each group were sectioned using a disc in low speed hand piece under constant irrigation to include the entire defect sites. Specimens were placed in 2.5% buffered glutaraldehyde solution (pH 4.7) for 6 hours. Then, dehydrated in increasing concentrations of ethanol (50, 70, 85, 95 and 100%) for 10 minutes at each concentration. Finally, they were mounted on EM stubs and examined using SEM (Model Quanta 250 FEG FEI Thermo Fisher Scientific, USA, with accelerating voltage 30 K.V., magnification14–1,000,000× and resolution for Gun.1n). The EDX analysis system works as an integrated feature of SEM Quanta FEG 250 attached with EDX unit, (FEI Company, Netherlands). EDX analysis of the bone surfaces was performed, the elemental distribution of phosphorus and calcium (expressed as weight percentage) were determined. Composition scans were collected at randomly selected points in the bone surfaces of the defect using the backscattered electron mode. Data were obtained by calculating the mean of ten independent determinations²¹.

Statistical analysis

One-way analysis of variance (ANOVA) was used to compare different observation times within the same group. This was followed by Tukey’s post hoc test when the difference was found to be significant. A t-test was used to compare between both groups using IBM SPSS 18.0 version 21 for windows (SPSS Inc., Chicago, IL, USA). The significance level was set at p ≤ 0.05.

In vitro evaluation of BM-MSCs

Cells were rounded non-adhesive isolated bone marrow cells (Figure 1a). Adhesive cells started to get fusiform spindle shaped fibroblast like cells. Cell confluence was 30–40% (Figure 1b). Cells became adhesive fusiform spindle shaped fibroblast like cells. Cell confluence was 50–70% (Figure 1c). Cells were long and spindle-shaped, reaching the highest confluence (90%) at 14 days (Figure 1d). BM-MSCs were negative for CD34 and positive for CD90 (>98.7%) (Figure 2). BM-MSCs were positively stained with alizarin red (Figure 3).

Figure 1. BM-MSCs in culture at different durations.

(a) Cells cultured for 3 days (x100). (b) Cells cultured for 7 days (x100). (c) Cells cultured for 10 days (x100). (d) Cells cultured for 14 days (x100).

Figure 2. FACS analysis of bone marrow mesenchymal stem cells.

(a) Positive for CD90 (98.7%). (b) Negative for CD34 (0.04%).

Figure 3. Bone marrow mesenchymal stem cells (arrows) differentiated into osteoblasts and stained with Alzarin red (x200).

SEM/EDX analysis

Fibro-cellular tissue and traces of PRF material were seen in sub-group A1 (Figure 4). In sub-group A2, along the margins, bone was actively forming; blood vessels were seen to be emerging and inserted close to the newly formed bone. PRF remnants were observed in the defect centre (Figure 5). Sub-group A3 revealed spongy-like pattern with abundant non-remodelled vascular spaces containing fibro-cellular tissue. In addition, bone formation extended beyond the perimeter of the original defect site compared to sub-group A2 (Figure 6). In sub-group B1, numerous vessels appeared, along with dis-organized architecture of newly formed bone (Figure 7). The bone in sub-group B2 was nearly restored, with new bone extending partly beyond the perimeter of the defect (Figure 8). Sub-group B3 revealed more organized bone architecture; well-oriented thick and smooth interconnecting bone trabeculae with large-centred vascular space. Many blood vessels were noticed. The borders between the newly formed bone and the pre-existing old bone were almost remodeled; no longer detectable in most areas and cannot be distinguished from the cortical surroundings (Figure 9). Raw EDX data are shown in Dataset 1²².

Figure 4. Scanning electron microscopy/energy-dispersive X-ray analysis sub-group A1 showing: PRF traces (red arrows), fibro-cellular tissue (yellow arrow) and old bone (OB).

(A) x100; (B) x500.

Figure 5. Scanning electron microscopy/energy-dispersive X-ray analysis sub-group A2 showing: new bone (NB), blood vessels (BV), PRF traces (red arrow) and old bone (OB).

(A) x100; (B) x500.

Figure 6. Scanning electron microscopy/energy-dispersive X-ray analysis sub-group A3 showing bone trabeculae (BT), and blood vessels (BV).

Note the borders between the newly formed bone and the cortical surroundings were no longer detectable (white arrow). (A) x100; (B) x500.

Figure 7. Scanning electron microscopy/energy-dispersive X-ray analysis sub-group B1 showing new bone (NB), blood vessels (BV) and old bone (OB).

(A) x100; (B) x500.

Figure 8. Scanning electron microscopy/energy-dispersive X-ray analysis sub-group B2 showing new bone (NB), blood vessel (BV) and old bone (OB).

Note the interface between new and old bone (white arrows). (A) x100; (B) x500.

Figure 9. Scanning electron microscopy/energy-dispersive X-ray analysis sub-group B3 showing bone trabeculae (BT), blood vessels (BV), vessel-space (VS) and old bone (OB).

Note the more integrated interface between new and old bone. (A) x100; (B) x500.

Statistical results

There was significant increase in the mean calcium and phosphorous weight percentage of group B relative to group A at all-time intervals. Moreover, calcium and phosphorus weight percent mean value increased by time in both groups (Figure 10 and Table 2–Table 5).

Figure 10. Calcium and phosphorous weight percentage between groups at 3, 10 days and 3 weeks postoperatively.