It is estimated that a small amount of bone fractures gives alterations in the consolidation process and lead to nonunion. This still happens even with good understanding of recent advancements in the biotechnology of bone healing Bastos Filho et al., 2012Giannotti et al., 2013aLiebergall et al., 2013. The surgical approach is still the most important tool in the management of nonunion: revitalizing, opening of the intramedullary canal, synthesizing, and filling the gap of the bone defect with autologous cancellous bone Giannotti et al., 2013aTressler et al., 2011. Autologous cancellous bone graft, which is the most often removed from the iliac crest, is currently used as the gold standard option. This is due to its high potentials of osteogenesis, osteoinduction and osteoconduction Bastos Filho et al., 2012Jäger et al., 2011. However, there is a limit to the amount of autologous cancellous bone that can be harvested in the reconstruction of extensive bone defects Yoshikawa et al., 2011. In addition, the donor site morbidity presents a major disadvantage to autologous bone grafting Jäger et al., 2011.

Various alternative treatment modalities to stimulate bone healing, including tissue engineering, have been investigated all with the aim of minimizing patient morbidity Dimitriou et al., 2011Fox and Genever, 2014Gómez-Barrena et al., 2015Kim and Cho, 2013. Embedding viable cells within the biological scaffolds appears to be extremely promising since it allows osteocompetent cells to generate new bone tissue and contribute to the improved tissue healing Connolly et al., 1989Gamie et al., 2012Giannotti et al., 2013a. Mesenchymal stem cells (MSCs) represent a good candidate cell source because of their biological characteristics and potential role for clinical bone regeneration Chanda et al., 2010Jäger et al., 2011Kuroda et al., 2014.

The purpose of this clinical study was to assess the outcome of autologous bone marrow mesenchymal stem cells combined with a cancellous bone allograft as compared to an autologous bone graft in the treatment of bone nonunion.

No significant differences were found between the two groups in demographic characteristics or fracture parameters following previous procedures at the baseline time point. But there was a statistically significant difference in the mean time to treatment between both groups. For the group of BMAC-ACB graft, it was 11.8 months (range, 4 - 24 months) and the autograft group it was 5.4 months (range, 3 - 8 months) (p = 0.0034). The baseline characteristics of each group are shown in Table 1 . None of the patients presented perioperative complications in both groups. Mean followup time was 15.5 ± 7.7 months.

In the group of autologous iliac bone graft, intramedullary nailing was performed in four patients, and revision by locking plating was done in four patients. No revision of the existing external fixation was performed in one patient. There were no postoperative complications. There was one nonunion after 2-year follow-up in the case of nailing at the femur that needed revision of autologous cancellous bone graft. Bone healing was obtained in eight (88.9%) patients. The mean interval between surgery and union was 4.6 ± 1.5 months (range 3 - 7 months).

In the group of BMAC-ACB graft, revision by locking plating was done in nine patients. Nailing was performed in two patients. External fixation was carried out in one patient. No revision of the existing fixation was performed in six patients. There were two cases of postoperative wound infections. Antibiotic treatment was used during 6 weeks following the culture from the wound. Two patients were performed debridement, and one needed a local rotated flap to cover the wound (2 x 2 cm). These infections were controlled after two months of aggressive treatment. There was one patient that had a treatment failure due to nonunion and a broken nail after 12 months. Revision nailing and bone grafting were indicated for this patient. Bone healing after BMAC-ACB grafting was obtained in seventeen (94.4%) patients, where the interval between surgery and union was 3.3 ± 0.90 months (range, 2 - 5 months) in radiology. However, two patients showed delayed union of the fracture, manifested by no radiographic progress in any of the four cortices after 6 months. The first patient that had ongoing pain at the femoral fracture site was successfully treated by removal of the distal locking screws of the intramedullary nail and healing was evident at the 12- month follow-up ({fig3$A 22-year-old woman had a poly-trauma with splenic rupture associated IIIA Gustilo open fracture of thee left femur 7 months ago$Patient was needed a second debridement due to develop wound infection. Antibiotic treatment was used in 6 weeks following the culture from the wound. The infection was controlled after three months off aggressive treatment. (A) Clinical and laboratory examination were done to rule out infection. χ-ray was taken after external device removal and showed non-healing fracture of femur at 77 months after surgery. (B) χ-ray at one month after bone stability using interlocking intramedullary nail and a composite BMAC-ACB grafting. (C) Small new callus bridging the nonunion site on χ-ray at 3-month follow-up. (D) Bone consolidation was obtained by χ-ray at 6-month follow-up. (E) χ-ray showed mature remodeled callus in the area of the graft placement at 12-month follow-up.

}). The second patient had no specific complaints and wanted a delay of a further intervention ( Figure 4 ). Patient started to have new small callus at the 8-monthfollow-up. No difference in bone healing rate was identified between the treatment groups (p = 0.4168), but there was a significant difference in the mean interval between surgery and union (p = 0.038). Survival analysis showed no significant difference in the time to bone healing between the two treatment groups during postoperative follow-up (logrank test) ( Figure 2 ).

The present study shows that that application of BMAC-ACB in 17 out of 18 patients (94.4%) resulted in bone union during the 24-month follow-up period. The mean interval between surgery and union for BMAC-ACB implantation is 3.3 ± 0.90 months (range, 2 - 5 months), and lead to faster healing when compared to the autologous cancellous bone graft.

As is widely accepted, the "diamond concept" of bone fracture healing is applied to treating nonunion Fayaz et al., 2011Giannoudis et al., 2007Gómez-Barrena et al.,2015. A triangular-shaped complex of interactions between the potent osteogenic cell populations, the osteoinductive stimulus and the osteoconductive matrix scaffolds are extensively studied and applied in search for the optimal grafting material Giannoudis et al., 2007Khan et al., 2011Virk and Lieberman, 2012. Mechanical stability is added to the diamond model as a crucial factor for bone healing, and essential for the formation of a callus that bridges the fracture site allowing loads to be transmitted across the fracture line Fayaz et al., 2011Hernigou et al., 2005b.

Historically, autologous cancellous bone grafts, usually from the iliac crest, is the gold standard and represents the most common therapeutic approach in orthopaedics. It is the only graft capable of providing all 3 elements of bone regeneration including osteogenic and osteoinductive as well as osteoconductive properties Giannotti et al.,2013aHatzokos et al., 2011Tressler et al., 2011. However, a bone autograft is limited in quantity and its harvesting represents an additional surgical intervention and donor site morbidity Gómez-Barrena et al., 2015. Therefore, investigations are needed to provide a safe and effective alternative. Tissue engineering has appeared to be a very promising technique in several regenerative applications in different anatomic regions Fayaz et al., 2011Hernigou et al., 2006Jäger et al., 2011Pacini, 2014. Indeed, the combination of cells and scaffolds could represent the optimal solution for the management of many bone defects, accelerating fracture healing, and reducing complications Giannotti et al., 2013bIsmail et al., 2013Ohgushi, 2014. According to the above mentioned "diamond concept", BM-MSCs provide the most appropriate cells for inducing bone repair, as they have a strong osteogenic potential and are easily obtained by culturing iliac crest aspirates Akram et al., 2014Gómez-Barrena et al., 2015. But the graft of BM-MSCs should be seen as the catalyst for bone healing and not the only determining factor for the resolution of the disease. A surgical approach still remains the most important factor Giannotti et al., 2013b. BM-MSCs aspiration from the iliac crest contains osteoprogenitor cells and has osteogenic and osteoinductive but not osteoconductive properties Hatzokos et al., 2011. For the management of bone defects, autologous BM-MSCs have been applied together with scaffolds at the lesion site, exploiting their trophic and differentiative properties Hernigou et al., 2005aKuroda et al., 2014. The need for cell attachment, proliferation, and differentiation in tissue engineering requires the design of functional scaffolds, preferably bioabsorbable. Cancellous bone allograft offers a scaffold providing advantages in terms of biocompatibility and good osteoconductive and osteoinductive properties Giannotti et al., 2013b. There was no significant difference in the mean rate of union between the treatment groups, but there was the relatively short time to achieve union in the group of composite BMAC-ACB grafting as compared to the allograft group. The high rate of union and the relatively short time to union in the clinical trial group represent a good outcome of BM-MSCs implantation.

Previous studies have confirmed that it was important to increase the number of progenitor cells in the graft after implantation Connolly, 1998Hernigou et al., 2005a. Bone marrow aspirations combined with cell concentration techniques have been proposed to increase the density of progenitor cell populations Hatzokos et al., 2011Jacobsen et al., 2008. The importance of the concentration of cells may be related to the survival of progenitors. The amount of available oxygen is probably one of the most limiting factors after transplantation. Because the transplanted progenitor cells compete with other cells for oxygen, the limitation of the transplanted cells is the best way to optimize progenitor cell survival that contribute to the bone formation Hernigou et al.,2005bPountos et al., 2010. Hernigou et al. reported that autologous bone marrow must contain at least 1500 progenitor cells per milliliter in order to be effective for the treatment of nonunion Hernigou et al., 2005a. The mean ratio of CD34⁺ cells in bone marrow grafting prior injection in the study of Gangji et al. achieved 1.0 ± 0.2(%) of CD34⁺ cells, which are precursors of hematopoietic cells Gangji, 2004. According to the results of our studies, a mean CD34⁺ cells ratio was 1.46 ± 0.74(%), a mean concentration of autologous progenitor cells was 2.43 ± 1.03 x10⁶ CD34⁺ cells/ml, and a mean viability of CD34⁺ cells was 97.97 ± 1.47(%). Bacterial cultures of BMAC were completely negative, and all the cultures of the CFU and CFU-F displayed positive staining in vitro. We believe that the application of BMAC is a safe procedure and that we can harvest a relevant amount of potent mesenchymal stem cells to let them differentiate into osteoblasts in vitro. Furthermore, the excellent bone regeneration by BMAC combined with ACB reduced the amount of autologous bone grafting procedures in our patient population and expedited the bone healing Jäger et al., 2011.

The disadvantages of these series are the limited number of patients, the diversity of indications, the various locations of nonunion, defect sizes, as well as a measurement of the number of CFU-F was not performed. Therefore, it is very difficult to allow conclusive judgments.

The result from this study, the implantation of BMAC has presented the efficiency in the treatment of bone nonunion in the initial outcome and may provide an available alternative to autologous cancellous bone graft. As the surgical procedure remains the treatment of choice for bone nonunion, tissue engineering using BM-MSCs could provide a useful method to accelerate and complete the bone union process. The use of BMAC-scaffold composites may contribute to improved outcome in patients. But large clinical application of BM-MSCs requiresmore appropriate and profound scientific investigations.

Mesenchymal stem cells (MSCs); allograft cancellous bone (ACB); bone marrow mesenchymal stem cells (BM-MSCs); bone marrow aspiration concentrate (BMAC); and fibroblast colony-forming unit (CFU-F).