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

Background: Various techniques for tissue engineering have been introduced to aid the regeneration of defective or lost bone tissue. The aim of this study was to compare the in vivo bone-forming potential of bone marrow mesenchymal stem cells (BM-MSCs) and platelet-rich fibrin (PRF) on induced bone defects in rats’ tibiae. Methods: In total, one defect of 3-mm diameter was created in each tibia of 36 Wistar male rats. There were two groups: group A, left tibia bone defects that received PRF; and group B, right tibia bone defects of the same animal that received BM-MSCs loaded on a chitosan scaffold. Subsequently, Scanning electron microscope/energy-dispersive X-ray (SEM/EDX) analyses was performed at 3 and 10 days, and 3 weeks post‑implantation and following euthanasia; (n=12). Results: The EDX analysis performed for each group and time point revealed a significant increase in the mean calcium and phosphorous weight percentage in the BM-MSC-treated group relative to the PRF-treated group at all-time intervals (P < 0.05). Moreover, the mean calcium and phosphorus weight percentage increased as time progressed since the surgical intervention in the PRF-treated and BM-MSCs groups (P < 0.05). Conclusions: In the present study, both BM-MSCs and PRF were capable of healing osseous defects induced in a rat tibial model. Yet, BM-MSCs promoted more adequate healing, with higher mean calcium and phosphorous weight percentages than PRF at all-time points, and showed greater integration into the surrounding tissues than PRF.


Introduction
Several biomaterials are used to treat bone deficiencies 1 . Autologous bone graft limitations are related to harvesting process including the quality and quantity of grafted bone and complications at the second surgical site, while allogenic bone grafts carry the risk of disease transmission and immunological rejection. Hence, there are considerable motivations for developing alternative solutions for bone regeneration 2 . The use of tissue engineering approaches has proven to be effective in inducing bone formation by applying mesenchymal stem cells (MSCs) 3 or platelet-rich fibrin (PRF) 4 . The capacity of bone marrow mesenchymal stem cells (BM-MSCs) for bone repair has been well reported in vivo with promising results; BM-MSCs remain the most widely used source of osteogenic cells in bone tissue engineering studies 5-7 MSCs are undifferentiated cells capable of replication 8 that have the potential to differentiate along multiple cell lineages, giving rise to cells that form mesenchymal tissues, including bone, cartilage and muscle 9 . PRF is a second-generation platelet-rich biomaterial 10 . PRF is derived from a natural and slowly progressive polymerization process occurring during centrifugation, which increases incorporation of the circulating cytokines and growth factors in the fibrin mesh and prevents them from undergoing proteolysis 11 . In addition, the PRF fibrin matrix provides an optimal support for MSCs which constitute the determining elements responsible for real therapeutic potential 12,13 . Platelets are active growth factor-secreting cells that initiate woundhealing, connective tissue healing and cell proliferation 14 . Therefore, PRF is considered as an inexpensive autologous fibrin scaffold prepared in approximately one minute and hence no cost for membrane and bone graft 15 . In the present research, rats were used as they are easy to handle and less expensive. In addition, breeding cycles are substantially shorter, providing enough animals in a reasonable amount of time 16 . Research on bone tissue engineering is focused on the development of alternatives to autologous bone grafts for bone reconstruction. Although multiple stem cell-based products and biomaterials are currently being examined, comparative studies are rarely achieved to evaluate the most appropriate approach in this context. The purpose of this study was to compare the regenerative capacity of bone marrow (BM)-MSCs and PRF implanted in surgically induced bone defects in rats' tibiae.

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 described 17 . 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% penicillinstreptomycin (GIBCO/BRL) at 37°C in a humidified 5% CO 2 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 cm 2 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 × 10 3 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 proximalmedial area of each tibia. While blood samples were being prepared, a 3-mm diameter bone defect was created using a round surgical bur 3 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 2.5x10 6 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 rpm 20 . 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 determinations 21 .

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.

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 nonremodelled 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 disorganized 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 largecentred 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 1 22 .

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). Also included are raw SEM images

Discussion
Bone regeneration using BMSCs was very well reported and standardized in many protocols, Donzelli et al., 2007 showed that the adult rat bone marrow was a suitable source for MSCs that can be easily induced to differentiate into an osteogenic lineage, so they are thought to be a promising candidate, supporting cells for bone reconstruction 23 . Most in vitro and many in vivo studies have proposed that MSCs possess the ability to increase osteoinduction and osteogenesis 24-27 .
Through the current study, BM-MSCs and PRF promoted bone regeneration; where the newly formed bone was almost    remodelled and integrated into the surrounding old bone with well-vascularized fibro-cellular tissue. In addition, evidence of osteogenesis was reflected by the presence of blood vessels. However, there was an improved bone regenerative capacity in defects treated with BM-MSCs compared to those treated with PRF. SEM-EDX analysis revealed a significant increase in the mean calcium and phosphorous weight percentage in the BM-MSCs group relative to the PRF group at all-time intervals.
Considering PRF, growth factors released are postulated to be promoters of tissue regeneration, tissue vascularity, mitosis of MSCs and osteoblasts and rate of collagen formation, playing a key role in the rate and extent of bone formation 28 . This would help explain the significant increase in calcium and phosphorus weight percentage in the PRF group through the experiment.
Accordingly, there was a marked drop in elemental analysis of calcium and phosphorous in sub-group A1, that increased gradually in sub-groups A2 and A3. This can be explained by the findings of a previous study on the pattern of growth factor release where PRF sustained long-term release of TGF-1 and PDGF-AB that peaked at day 14, which led to increase mineralization then decreased mildly but had a delayed peak of release 11 .
The BM-MSC-treated group exhibited more organized bone architecture than the PRF-treated group; sub-group B3 exhibited well-oriented thick and smooth interconnecting bone trabeculae filling the defect compared to sub-group A3; which revealed spongy-like pattern with abundant non-remodelled vascular spaces containing fibro-cellular tissue. The proposed mechanism through which BM-MSCs contribute to bone regeneration enhancement is via maturation into osteoblasts in vivo, or via an indirect pathway by paracrine effects on host stem or progenitor cells 29 . Notably, a significant increase in the mean calcium and phosphorus weight percentage was observed in BM-MSCtreated group at all time intervals throughout the experiment when compared with the corresponding PRF group. In accordance with the findings of a previous study, SEM/EDX analysis of osteogenic differentiated MSCs seeded on collagen scaffold demonstrated that calcium co-localized with phosphorous, with a gradual increase of both chemical elements observed from day 7 up to very high levels at day 28 23 . In conclusion, we confirmed that PRF yielded inferior bone formation to that by BM-MSCs after implantation in rat tibiae.

Grant information
The author(s) declared that no grants were involved in supporting this work.

2.
3. Overall the study was well performed and showed some interesting results. After looking at referees' reports which summarized some revisions required for this paper, I would like to add the following:

Open Peer Review
A statement needs to be added in the methods outlining the power analysis performed to determine the sample size in each group.
Despite the localized effect of BM-MSCs and PRF being applied on a scaffold, the authors should add in their discussion the potential systemic effect of both treatments especially that the choice was made to have both defects created in the same animal (Right and left tibis with different treatments). I am guessing that this was done to reduce the number of animals used in the experiment. However, the systemic effect of treatments should be addressed as a limitation of the study and/or a discussion should made to clarify how the systemic effect could not have contributed to the significant results. Statistical analysis: A two ANOVA analysis should be more approriate to use in order to investigate the single main effects of time and treatment as well as the possible interaction between treatment and time. This will add more strength to the results rather than using post hoc results and t-tests.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
No competing interests were disclosed. Overall, the paper is clear, substantially easy to read and well constructed but still, there are suggested minor comments the authors could deal with, or at least discuss for additional impact.

Methods
It is worthy mentioning the number of rats per cage Further details for clarifying the methodology and aseptic conditions of BM-MSCs isolation from the femurs of donor rats will be a valuable add. It is recommended to mention how the depth of bone defect was controlled to be standard in all experiment induced defects . It is an add to mention the place of the lab where the BM-MSCs isolation procedures were done. You need to give further details about the size and methodology of BM-MScs and PRF pellet loading in the bone defect Results Fig. 3 need show monitoring of the positively stained calcified nodules as mentioned in the methodology Recommendations would be worth mentioning depending on the paper results

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results?

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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