inadequate bone quantity in the posterior maxilla secondary to pneumatization of the maxillary sinus and/or postextraction alveolar ridge resorption can compromise dental implant placement , therefore requiring site grafting prior to implant placement using techniques such as maxillary sinus floor elevation . the ideal material for bone grafting should be biocompatible ,
induce no host rejection , present no risk of disease transmission , promote support for bone regeneration , and have mechanical stability from the outset , which should be maintained throughout the healing period . the maxillary sinus floor elevation technique described by tatum and published by boyne and james in 1980   described autologous bone as the filling material for the sinus cavity , which is still regarded as the gold standard in bone reconstruction . if , on the one hand , autologous grafts present osteoconductive , osteoinductive , and osteogenic potential , on the other , they present some risks .
this has translated into a constant search for the development of biomaterials to substitute autologous bone grafts , for instance , xenografts [ 5 , 6 ] , homologous grafts [ 7 , 8 ] , and synthetic grafts [ 9 , 10 ] .
bone substitute biomaterials lack cellularity , which has encouraged much research into the field of tissue engineering in order to combine autologous osteogenic cells with osteoconductive materials . consequently
, the bone marrow is currently the most explored source of autologous cells ,   since it contains a number of bone regenerating cells , such as undifferentiated mesenchymal cells that can differentiate into osteoblasts [ 12 , 13 ] .
they also present an angiogenic potential due to the production and release of vascular endothelium growth factor , which is highly desirable for graft integration .
osteoconductive graft enrichment in reconstructive surgery for maxillary sinus floor elevation can be performed using cells from a bone marrow aspirate concentrate obtained by centrifugation [ 1619 ] .
this method is regarded as simple and safe because it is performed using autologous material immediately before surgery .
therefore , the aim of this study was to histomorphometrically evaluate the use of a bone xenograft enriched with autologous bone marrow aspirate concentrate ( bmac ) for maxillary sinus floor lifting .
this study was conducted in the outpatient clinic of the department of implant dentistry of the so leopoldo mandic dental school ( campinas , sp , brazil ) , upon approval by the research ethics committee ( 694.065/2014 ) and free and informed consent forms for all the patients .
the inclusion criteria involved completely edentulous patients needing implants in the posterior maxillary region with no more than 4 mm of remaining alveolar ridge , with need for maxillary sinus floor augmentation .
the patients also committed to returning for follow - up appointments and to maintaining adequate oral hygiene .
patients were excluded if they had a history of neoplastic disease treated with radiotherapy or chemotherapy , if they were pregnant or breastfeeding , if they were receiving treatment or were affected by an illness that could have an effect on bone homeostasis , allergy to any of the materials used , and sinus pathologies , or if they were smokers .
cone - beam computed tomography scans of the posterior maxillary region were obtained to measure the height of the posterior maxillary bone and the size of the maxillary sinus .
the ct scans were also used to evaluate possible sinus pathologies ( figure 1 ) .
eight patients with a mean age of 55.4  9.2 years were included in this study .
the patients were randomly divided using online - based software available at http://www.randomization.com/ into two groups according to the material used , control group ( cg ) ( n = 8) with bio - oss only and test group ( tg ) ( n = 8) with bio - oss combined with bone marrow concentrate obtained by the bmac method , and each patient had the same graft material placed in each sinus . following the principles of the guided bone regeneration technique , collagen membranes were placed over the bone window for all sinus floor augmentation procedures in both groups .
all patients were dentally rehabilitated using osseointegrated implants and fixed prostheses at the end of the study . according to the instructions by the manufacturer ,
bone marrow was harvested and processed directly in the operating room using the bmac system ( bone marrow procedure pack , harvest technologies , plymouth , ma , usa ) .
briefly , in an outpatient setting and using local anesthesia ( 2% xylocaine without a vasoconstrictor ) , 30  ml of bone marrow was collected from all the patients by aspiration through a puncture 2  cm laterocaudally from the superior posterior iliac crest , using a bone marrow needle ( included in the pack ) , with 30  ml syringes previously heparinized ( 1  ml of 5.000  u / ml heparin ) ( figure 2 ) .
the syringe containing 30  ml of bone marrow was connected to a filter bag , to which 8  ml of acd - a anticoagulant was added .
after appropriate homogenization , new syringe and needle were connected and the filtered 30  ml was removed .
the bone marrow aspirate was then transferred into specific process disposables , which were placed in a smartprep2 centrifuge .
after centrifugation for 14 minutes , two phases were obtained within the container , that is , the supernatant plasma and the precipitated bone marrow cells concentrate ( figure 3 ) .
the plasma was removed using specific syringes provided in the kit and the cell concentrate was resuspended and approximately 4  ml was aspirated .
a lateral window was prepared using number 3  pm spherical diamond bur ( medical burs ind . e com .
the schneiderian membrane was carefully released ( figure 4 ) using specific curettes ( neodent , curitiba , pr , brazil ) and the maxillary sinuses were grafted with xenogeneic bone from bovine hydroxyapatite , ( 1 - 2  mm bio - oss , geistlich biomaterials , wolhusen , switzerland ) ( figure 5 ) , either alone or combined with the bone marrow concentrate ( figure 6 ) .
a collagen membrane of porcine origin ( bio - gide , geistlich biomaterials , wolhusen , switzerland ) was used to cover the graft and the osteotomy of the lateral maxillary sinus wall , thus impeding migration of soft tissue to the graft region .
after 6 months , 16 bone biopsies ( one per sinus ) were taken using a trephine bur ( 2.0  mm in diameter and 18  mm in length ) ( figure 7 ) .
immediately after that , the implants ( blackfix , as technology , so jos dos campos , brazil ) were placed ( figure 8) .
the bone biopsies were fixed in 4% formaldehyde solution ( merck , darmstadt , germany ) .
the suture was made with ethilon - nylon 4 - 0 ( ethicon , ma , usa ) .
after the surgery , each patient was prescribed amoxicillin 500  mg ( 12/12 hours for 5 days ) .
all patients were rehabilitated with metal - ceramic prosthesis over the implants six months after its installation .
the histological analysis was performed at the histopathology laboratory of the so leopoldo mandic dental school ( campinas , sp , brazil ) .
the biopsies underwent decalcification in 10% edta for 36 hours and then processed following a conventional histological method for hard tissue .
the entire area of the trephine biopsy above the native bone of the sinus was defined as region of interest and histomorphometrically evaluated .
the fragments were masson 's trichrome - stained and four different areas of each fragment were evaluated in the histology slides ( upper left , lower left , upper right , and lower right ) , which were then averaged out .
, diagnostic instruments , sterling heights , mi , usa ) coupled with an optical microscope ( 1.25x magnification ) .
the digital images were merged to create a single image for each histological cut , using adobe photoshop elements 2.0 software ( adobe systems , san jose , ca , usa ) ( figures 9(a ) and 9(b ) ) .
the examiners traced new bone formation on all the images using imagej pro plus 4.5 for windows software ( national institute of health , nih , usa ) .
the following parameters were considered for histomorphometry : ( 1 ) nonvital mineralized tissue ( nvmt ) , ( 2 ) vital mineralized tissue ( vmt ) , and ( 3 ) nonmineralized tissue ( nmt ) .
all the results were noted in square micrometers and , subsequently , stated as a percentage of the total area . for analysis of the nonvital mineralized tissue ( nvmt ) , vital mineralized tissue ( vmt ) , and nonmineralized tissue ( nmt ) parameters ,
the nonparametric mann - whitney test was applied with correction using the sidak - bonferroni test for the statistical analysis .
at least two implants were placed in each previously grafted sinus and all of them were osseointegrated .
cg and the tg showed percentages of vital mineralized tissue ( vmt ) area of 27.30  5.55% and 55.15  20.91% , respectively .
the same groups showed percentages of nonvital mineralized tissue ( nvmt ) area ( represented by remaining bio - oss particles ) of 22.79  9.60% and 6.32  12.03% , respectively .
finally , the percentages of nonmineralized tissue ( nmt ) area were 49.90  7.64% and 38.53  13.08% , respectively ( table 1 ) .
surgery to augment the maxillary sinus is a well - documented method to generate adequate amount of bone for implant installation in the posterior maxilla [ 2125 ] , for which various types of bone grafts have been tested .
the autologous bone graft technique is considered the gold standard because of its osteoinductive , osteoconductive , and osteogenic characteristics .
nevertheless , it presents some drawbacks , especially regarding operative morbidity , namely , the need for two or more surgical sites in cases of greater amount of donor tissue , including extraoral sources .
this raises the operative risk and surgical costs and generates postoperative discomfort , causing fewer patients to opt for this approach .
consequently , a search for bone biomaterials that could replace the autologous bone has taken place . nonetheless , these are not always graced with the advantages of osteogenesis and osteoinduction inherent of the autologous grafts .
biomaterials that feature the physical , chemical , and mechanical characteristics of autologous bone have become increasingly desired , given the need for further use of the grafted area for the installation of dental implants .
the literature reports on a xenogeneic bovine bone substitute , bio - oss , as a biomaterial with very similar characteristics to those of human bone , including osteoconduction [ 2629 ] .
lyophilized xenogeneic bone tissue , or other bone substitute graft materials , lacks factors that promote osteogenesis and osteoinduction . in turn , this increases healing time compared to autologous bone , reaching a waiting period for implant placement ranging from six to eight months .
this is greater than the time required for autologous grafts , which feature live cells and growth factors , therefore fulfilling their osteogenic and osteoinductive potentials .
such properties reflect positively on the time required for bone healing , which may vary from four to six months .
based on the aforementioned properties , methods to enhance bone substitutes combined with bone marrow cells have been investigated .
studies have described techniques for harvesting and applying fresh bone marrow [ 3133 ] and isolating and expanding mesenchymal stem cells from the bone marrow [ 3335 ] as well as concentrating the bone marrow cells [ 18 , 33 , 36 , 37 ] in combination with a mineralized carrier . despite these various methods , however , there is no consensus on the best alternative for bone remodeling in humans . regarding the use of cell culture ,
mesenchymal stem cells must be carefully considered , because they usually require a waiting period of several weeks between harvesting , culture , and transplantation , thus risking contamination .
therefore , in this study , preference was given to test a clinically plausible cell concentration method from a bone marrow aspirate concentrated by centrifugation within a fully closed system .
the main reason for this choice was the versatility of the technique and the unlikelihood of contamination due to the fact that the system is closed .
,  to standardize the bone marrow transplantation for bone regeneration , a simple , safe , clean and cost - effective system is needed .  in the present study , the waiting period of six months between grafting and reopening for implant placement was adopted , as it stands within the waiting period for autologous ( 46 months ) and xenogeneic grafting ( 68 months ) . however , on the grounds that bone neoformation levels ( vital mineralized tissue ) in the test group were significantly higher ( p = 0.002 ) than in the control group , 55.15  20.91% and 27.30  5.55% , respectively , one can speculate the possibility of early reopening when using the bmac method .
( 2010 ) achieved 19.9% of new bone after lifting maxillary sinuses with bio - oss associated with the bmac method .
the discrepancy between the results from both studies could probably be justified on the time delay between grafting and reopening surgery . in the study by sauerbier et al .
, a healing period of approximately 4.1 months was chosen , but in some cases , reopening occurred after just 3 months .
the lack of time standardization for reopening combined with a precocious second intervention may have resulted in lower levels of bone tissue as stated by sauerbier et al .   when compared to the present study , which standardized the reopening procedures at 6 months .
rickert et al .   compared the combination of bio - oss / bmac method with bio - oss ( 70% ) associated with autologous bone ( 30% ) and detected no significant difference between the groups , further suggesting that the bmac method improved the reconstructive standard .
nevertheless , the authors also opted for an early reopening time of approximately 3 months after grafting , which also hindered further comparisons between their data and those of the present study .
regarding the levels of residual particles of bio - oss ( nvmt ) , a significantly lower percentage ( p = 0.006 ) was observed in the test group compared to the control , 6.32  12.03% and 22.79  9.60% , respectively .
this can be hypothesized as an acceleration of the healing process in the test group , which corroborates the vmt results , since a higher rate of bone formation should translate into a higher biomaterial resorption . regarding the levels of nmt , despite the lack of significant difference between the groups ( p = 0.09 ) , a decreasing trend in the amount of nonmineralized tissue in the test group was observed , which was , on average , 10% lower than in the control group .
there have been very few reports on the use of the biomaterials and techniques investigated in the present preliminary study .
this study indicates that the clinical use of bone marrow aspirate concentrate obtained by the bmac method associated with a xenograft for maxillary sinus elevation resulted in more adequate bone repair than the xenograft alone .
 
 purpose . to investigate the regenerative results obtained with the association of bone marrow aspirate concentrate using the bone marrow aspirate concentrate ( bmac ) method to a xenogeneic bone graft ( bio - oss ) in sinus floor elevation . 
 materials and methods . using a randomized controlled study design in eight consecutive patients ( age of 55.4  9.2 years ) , 
 16 sinus floor lift procedures were performed with bio - oss alone ( control group , cg , n = 8) or combined with bone marrow aspirate concentrate obtained via the bmac method ( test group , tg , n = 8) . 
 six months after the grafting procedures , bone biopsies were harvested during implant placement and were analyzed by histomorphometry . 
 results . 
 histomorphometric analysis revealed a significantly higher amount ( p < 0.05 ) of vital mineralized tissue in tg when compared to the cg ( 55.15  20.91% and 27.30  5.55% , resp . ) . 
 for nonvital mineralized tissue , tg presented a statistically higher level of bio - oss resorption ( p < 0.05 ) when compared with the cg ( 6.32  12.03% and 22.79  9.60% , resp . ) . 
 both groups ( tg and cg ) showed no significantly different levels ( p > 0.05 ) of nonmineralized tissue ( 38.53  13.08% and 49.90  7.64% , resp . ) . 
 conclusion . 
 the use of bone marrow concentrate obtained by bmac method increased bone formation in sinus lift procedures . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion