Mesenchymal stem cells surpass the capacity of bone marrow aspirate concentrate for periodontal regeneration

Abstract Regenerative approaches using mesenchymal stem cells (MSCs) have been evaluated to promote the complete formation of all missing periodontal tissues, e.g., new cementum, bone, and functional periodontal ligaments. MSCs derived from bone marrow have been applied to bone and periodontal defects in several forms, including bone marrow aspirate concentrate (BMAC) and cultured and isolated bone marrow mesenchymal stem cells (BM-MSCs). This study aimed to evaluate the periodontal regeneration capacity of BMAC and cultured BM-MSCs in the wound healing of fenestration defects in rats. Methodology: BM-MSCs were obtained after bone marrow aspiration of the isogenic iliac crests of rats, followed by cultivation and isolation. Autogenous BMAC was collected and centrifuged immediately before surgery. In 36 rats, fenestration defects were created and treated with suspended BM-MSCs, BMAC or left to spontaneously heal (control) (N=6). Their regenerative potential was assessed by microcomputed tomography (µCT) and histomorphometry, as well as their cell phenotype and functionality by the Luminex assay at 15 and 30 postoperative days. Results: BMAC achieved higher bone volume in 30 days than spontaneous healing (p<0.0001) by enhancing osteoblastic lineage commitment maturation, with higher levels of osteopontin (p=0.0013). Defects filled with cultured BM-MSCs achieved higher mature bone formation in early stages than spontaneous healing and BMAC (p=0.0241 and p=0.0143, respectively). Moreover, significantly more cementum-like tissue formation (p<0.0001) was observed with new insertion of fibers in specimens treated with BM-MSCs within 30 days. Conclusion: Both forms of cell transport, BMAC and BM-MSCs, promoted bone formation. However, early bone formation and maturation were achieved when cultured BM-MSCs were used. Likewise, only cultured BM-MSCs were capable of achieving complete periodontal regeneration with inserted fibers in the new cementum-like tissue.


Introduction
Restoring periodontal structures functionally lost by periodontal disease remains one of the major challenges in Periodontology due to the limited capacity of the periodontium tissues for selfregeneration and the complexity of their architecture and microenvironment. 1 Regenerative approaches, including the use of bone replacement grafts, guided tissue regeneration (GTR), and various growth factors and/or host modulating agents, such as enamel protein derivatives, have shown some efficacy in achieving only partial reconstruction of bone defects, especially in advanced cases. 1 New techniques and treatment options involving tissue engineering and cell-based therapies to promote regeneration of periodontal and bone defects have been evaluated. 1 The literature shows that mesenchymal stem cells (MSCs) can be advantageous for periodontal regeneration and promising for tissue regeneration in clinical applications. 2,3 MSCs are multipotent cells that can adhere to plastic, differentiate into many tissues, and show a specific surface antigen expression, including CD90+ and CD45-. 4 These cells can be obtained from different sources in the adult organism, 2 including bone marrow. 5 MSCs derived from bone marrow can be used in several ways, such as bone marrow aspirates (BMA), 6 bone marrow aspirate concentrates (BMAC) 7-9 or cultured and isolated bone marrow mesenchymal stem cells (BM-MSCs). [9][10][11][12][13][14][15][16] BM-MSCs used in periodontal defects to achieve regeneration have shown promising but conflicting results. 2 Clinically, a recent systematic review only showed clinical reports, case series, and phase I/II trials using BM-MSCs in intrabony or furcation defects, with substantial variation in terms of reduction and gain of clinical attachment. 17 Therefore, most of the literature on the use of BM-MSCs is focused on preclinical studies to gain a greater understanding of the action and effect of their use to further promote their application in clinical practice. Thus, further preclinical research on cell therapy is encouraged. 1 Preclinical studies have investigated the effects of BM-MSCs on bone, cementum, and periodontal ligament regeneration. [9][10][11][12][13][14][15][16] The use of BM-MSCs has achieved reliable results in terms of bone regeneration, such as the increase of local bone density in the subperiosteal buccal alveolar bone surface at the tooth extraction site. 18 Few studies have shown the induction of cementum formation in fenestration defects. [9][10][11] However, the implementation of these deliveryisolated BM-MSCs in periodontal therapy is hampered by inbuilt difficulties in the isolation, characterization, and identification of the appropriate ex vivo culture before reimplantation. 1 Moreover, this process remains expensive, difficult, and requires specialized labor and highly sophisticated laboratories. 19 BMACs are another source of bone marrow-derived mesenchymal stem cells. Although the percentage of MSC content in BMAC represents a very limited fraction of the implanted cells, 19 its manipulation and implantation are simplified and less expensive than isolated BM-MSCs. To produce BMAC, after bone marrow collection, the tissue only needs to be centrifuged twice: first to remove red cells, and second, to separate the platelets and nucleate cells from the platelet-poor plasma. 7 Therefore, BMAC represents a minimally manipulated whole tissue fraction, which preserves the physiological microenvironment of multiple cell types, such as platelets that are rich in growth factors in their natural proportions. 7,19 Promising results have been shown when BMAC is used to regenerate different bone sites. 8,9,[20][21][22]  In total, 36 old male isogenic Wistar Kyoto rats weighing 250-300 g were used, and all procedures were done under intramuscular anesthesia (ketamine, 7 mg/100 g of body weight; xylazine, 0.6 mg/100 g of body weight). Animals were randomly assigned to one of three experimental groups: i) spontaneous healing -Control, ii) BM-MSCs, and iii) BMAC. Each group was further divided into two randomized subgroups according to two healing time points: 15 and 30 days.
The number of animals per group was six (N=6) at each time point, and each animal was an experimental unit. All analyses were performed on animals from all groups (N=6).

BM-MSCs collection and isolation
From every iliac crest from two isogenic rats, 0.5 mL of bone marrow was aspirated using a 10-mL sterile syringe, containing 0.1 mL of heparin. 7 Cells were isolated with Ficoll-Paque PREMIUM (Gibco-  To evaluate the expression of peroxisome proliferatoractivated receptor gamma-2 (PPARγ2, Rn00440945_ m1, Thermo Fisher Scientific), cells were seeded in 6-well plates (1 × 10 5 cells/well) in an adipogenic medium, and the total RNA was collected after seven, 10, and 14 days to carry out the RT-PCR, as described above.

BMAC collection
BMACs were collected and prepared for each rat at the time of surgery. After anesthesia, 0.5 mL of bone marrow was aspirated from the iliac crest of each rat using a 10-mL sterile syringe containing 0.1 mL of heparin (to prevent clotting) and stored in sterile microtubes. The concentration of the bone marrow aspirate (BMA) was determined using a previously described protocol. 7 Briefly, microtubes were centrifuged at 160 × g for 20 min at 22°C to separate the plasma, which contained platelets and mononuclear cells (upper layer), from the red cells.

In vivo experimental surgery
All surgical procedures were performed under a 10-40× magnifying stereomicroscope (Nikon SMZ800, Nikon Instruments Inc., Tokyo, Japan) to identify anatomic landmarks. The same trained operator performed the procedures to reduce risk of bias. Both sides of the mandible underwent the same treatment.
After intramuscular anesthesia, surgical sites were shaved off and disinfected. A superficial extraoral incision was made at the base of the mandible to expose the bones. Periodontal fenestration defects of 2 mm, 4 mm, and 1 mm in height, length, and depth, respectively, were created to denude the distal and buccal (mesial length limit) roots of the first molar and the mesial root of the second molar (distal length limit) using a round bur with high-speed instrumentation under irrigation. 29 The height limit of the defect was 1 mm above and below the center of the defect.
Defects were filled with one of the treatments or The non-bone tissue in the defect represents the newly formed connective tissue, characterized by µCT as a radiolucent area, e.g., empty.    improve osteogenesis, 20,24 has been shown in previous studies, 8,19,20,22,24 In our study, BMAC bone regeneration capacity could be observed, although it was unable to induce cementum formation. This concentrated form of bone marrow aspirate could reach bone maturation in 30 days, compared to spontaneous healing. Besides We can speculate that these distinctive results, in  10-13,15,16 Paknejad, et al. 15 (2015) found that 80% of the height of three-wall intrabony defects was filled by new cementum after the transplantation of cultured BM-MSCs in an organic bovine bone mineral in a canine model with induced periodontitis.

Descriptive histological and histomorphometry analyses
Some conflicting results can be found in the literature regarding the use of BM-MSCs for periodontal regeneration, hampered by inherent difficulties in the stage of differentiation, lineage, and heterogeneity, as well as the number of cells transferred to the defects. 2 We can consider that the protocol used in this study was efficient. Isolated and cultured BM-MSCs, when applied to fenestration defects at a density of 1.2 x 10 6 cells, could promote complete periodontal regeneration. This cell density was selected based on previous studies that showed the regeneration capacity of BM-MSCs. [13][14][15][16] These studies used a larger number of cells than those used in this study (varying from 2 × 10 6 to 2 × 10 7 ). However, reduced cell survival and, consequently, reduced regeneration capacity can occur when high concentrations of MSCs are used due to greater difficulty in inserting nutrients into the deepest areas of the periodontal defects. 37 Therefore, after a pilot study, 1.2 x 10 6 cells were considered sufficient to achieve periodontal regeneration without hindering cell capacities in this type of defect. Many studies have used carriers and biomaterial scaffolds to hold and support MSCs. However, these carriers can help or interfere with new tissue formation, favoring the abundant presence of multinucleated giant cells. 14 Therefore, because this pre-clinical study is a proof of principle, BM-MSCs were applied alone, in suspended form, to avoid any influence of carriers on the periodontal tissue formation capacity and, therefore, facilitate the real influence of BM-MSCs on the morphological and molecular aspects of periodontal regeneration. While promising results were found in the use of BM-MSCs, including their potential to communicate with dental tissue and lead to periodontal regeneration, future translational studies using other vehicles can verify the best way to support these cells in large periodontal defects.
To the best of our knowledge, this is the first study and maturation. Therefore, our animal model is recommended to investigate early healing processes that occur after blood coagulation as a consequence of material implantation. Furthermore, it benefits the extraoral approach of surgery by isolating the defect from the oral cavity, which prevents any contamination or infection by saliva and the resident oral flora. 38