Table 4.

Studies evaluating concentrated bone marrow aspirate in bone healing

Ref.	Tissue	BMAC preparation	Concen-tration	Study design/methods/follow up	Outcomes measured	Results	LOE
Bastos Filho et al[25]	Tibia/femur nonunion	11G × 10 cm bone marrow aspiration needle into posterior iliac crest to obtain a total of 100 to 110 mL for each patient - concentrated to 20 mL with Sepax system	NS	n = 6 patients with nonunion of tibia or femur. Four received percutaneous infusion of autologous bone marrow aspirated without Sepax processing. Two received with processing. Follow up to 6 mo	Clinical examination and radiographic evaluation at 2, 4, 6 mo. Clinical criteria included full weight bearing tolerance and absence of pain upon palpation at the fracture site. Radiographic healing checked with AP, lateral and oblique films to look for bone callus. Patient satisfaction questionnaire scale from 0-10	Bone consolidation obtained in all the patients. Bone callus observed in the radiographic between 3 and 24 wk, average 13.8 wk in group without processing. Mean satisfaction increased in all patients	II
Desai et al[26]	Nonunion/delayed union of tibia	Total of 60 cc bone marrow aspirated from iliac crest with 16 gauge Jamshidi needle (Harvest system). Concentrated to 10 cc for injection	101.48 ± 64.13/cc	n = 49 patients with tibial nonunion had BMAC injection with DBM and/or rhBMP-2. Follow up until radiographic union or another procedure was performed	Radiographic healing (bridging of 3 out of 4 cortices on AP and lateral films)	No difference in healing rate between patients with fracture gaps less than and greater than 5 mm	III
Garnavos et al[27]	Humeral shaft delayed union	With the use of a 10 cm long and 3 mm wide biopsy needle, 60 mL of bone marrow was aspirated from each patient’s iliac wing and was centrifuged to provide 10 mL of concentrated mesenchymal stem cells. The concentrated bone marrow mixed with 10 cc of DBM putty	NS	n = 5. Intramedullary nailing with antegrade/unreamed technique was performed for 4 patients. One patient was treated previously with retrograde/unreamed nailing left in situ. The concentrated mixture was infused percutaneously in the area of nonunion with a biopsy needle under fluoroscopy. Patients were followed up every 4-6 wk for 12 mo	Patients were assessed for union process, discomfort, level of activities and functional improvement	There were no peri-or postoperative complications. Sound union was obtained in all cases from 12 to 20 wk after the operation. At final followup, all patients had regained a satisfactory range of shoulder and elbow motion. They had also returned to pre-injury level of activities and were happy with their treatment and outcome	IV
Guimaraes et al[28]	Femoral shaft nonunion	11G × 10 cm needle used for aspiration from iliac crest. The marrow samples were harvested in small amount (2 mL) and the contents of each syringe were pooled in the container of the bone-marrow-collection kit containing anticoagulant solution. The final volume of bone marrow aspirate (200 mL) was then filtered through a sequence of successively smaller-diameter mesh filters. The cells were finally collected in a blood transfer pack unit. The aspirated material was reduced to a final volume of 40 mL by removing most of the RBC the plasma by centrifugation	9.8 ± 4.3 × 106 vs 20.2 ± 8.6 × 106	n = 16 patients with aseptic nonunion of femur were treated with injection of BM-MSCs who had locked IMN. Follow up: 3-8 mo	Radiographic RUST scores	Bone union occurred in 8 of 16 patients according to RUST. The grafts used in patients whom treatment failed contained significantly lower number of total nucleated cells (9.8 ± 4.3 × 106 vs 20.2 ± 8.6 × 106)	IV
Hernigou et al[29]	Ankle nonunion	150 mL of bone marrow aspirate obtained from anterior portion of the ipsilateral iliac crest then treated with a cell separator	27.3 ± 14.6 × 106	n = 86 ankle nonunion in diabetic patients treated with BM-MSCs vs n = 86 diabetic matched nonunion treated with a standard bone iliac crest autograft	Time of union, callus volume, complication, morbidity of graft harvesting vs bone marrow aspiration in diabetic patients	70 out of 86 patients (82.1%) treated with BMC achieved healing with a low number of complications; 53 (62.3%) of patients treated with iliac bone graft had healing and major complications were observed: Amputations, osteonecrosis of fracture wound edge, infections	III
Hernigou et al[30]	Tibial shaft nonunion	Bone marrow aspirated from anterior iliac crest total of 300 mL then concentrated to 50 mL	18 ± 7 million	BMAC injected into 60 noninfected atrophic nonunion of tibia. Follow up until union	Radiographic union; healing time; volume of callus	Patients who did not achieve union had significantly lower number of progenitor cells comparing to the 53 patients who achieved union. There was positive correlation between the volume of mineralized callus at 4 mo and the number and concentration of fibroblast colony-forming units in the graft; there was a negative correlation between the time needed to obtain union and the concentration of CFU in the graft	IV
Ismail et al[31]	Long bone nonunion	40 mL of bone marrow was aspirated from posterior iliac crest and transferred into a container prefilled with 5000 U/mL of heparin. Aspirate was diluted with phosphate-buffered saline at a ratio of 1:1 and centrifuged at room temperature at 3000 rpm for 30 min. The collected buffy coat was washed and transferred into a culture flask containing Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. Cells were incubated at 37 °C at 5% CO2 with a routine culture medium change every two to three days. Subculture was performed between days 7 and 10. Mixed with 5 g/cm3 defect of HA granules	14-18 million BMSCs	n (total) = 10. n = 5, treated with combination of 15 million BM-MSCs, 5 g/cm3 (HA) granules and internal fixation. n = 5, control subjects were treated with iliac crest autograft, 5 g/cm3 HA granules with internal fixation. Follow up = 12 mo	VAS, LEFS, DASH score. Radiological assessments for union were conducted by a blinded radiologist using two radiological scoring systems: The Lane-Sandhu and Tiedeman radiological scores	No significant differences in post-op pain between the two groups. The treatment group demonstrated initial radiographic and functional improvements. Statistically significant differences in functional scores were present during the first (P = 0.002), second (P = 0.005) and third (P = 0.01) month. Both groups achieved similar outcomes by the end of one year follow up	III
Le Nail et al[32]	Open tibia fracture	Hernigou’s technique. Bone marrow from posterior iliac crest by needle aspiration. Around 500 mL concentrated by centrifugation to obtain 50 mL	171 ± 107 × 106 vs 118 ± 28 × 106	n = 43 cases of open tibial fractures with initial surgical treatment that developed nonunion or delayed union, subsequently treated with injection of BMAC	Clinical success (consolidation without any subsequent procedure): Non painful callus palpation and a full weight bearing without any contention system. Radiographic bone healing 3 out of 4 cortices	23 successes (53.5%) within 17 wk after BMAC	IV
Thua et al[33]	Long bone nonunion	BMA (300-350 mL) were obtained by Jamshidi vacuum. Both posterior iliac crests of patients were harvested under loco-regional anaesthesia. BMAC was produced via density gradient centrifugation using the Sorvall centrifuge at 3670 rpm for 7 min. Afterwards, a total volume of 8 mL BMAC was mixed with freeze-dried allograft cancellous bone chips. BMAC was incubated for 15 min with bone chips as a composite of BMAC-ACB prior to transplantation	2.43 ± 1.03 (× 106) CD34 cells/mL (staining)	n (total) = 27. n = 9 control treated with autologous cancellous bone graft from iliac crest. n = 18 clinical trial group treated with BMSCs and allograft cancellous bone chips. Correction and optimization of fixation device were done for previously failed procedures. Patients were followed up in outpatient clinic for 1, 3, 6, 9 12, 18, 24 mo	Functional outcomes, radiographic outcomes based on modified Lane and Sandhu radiological scoring system	Bone consolidation was obtained in 88.9% and mean interval between cell transplantation and union was 4.6 ± 1.5 months in autograft group. Bone union rate was 94.4% in group of composite BMAC-ACB implantation. The time to union in BMAC-ACB grafting group was 3.3 ± 0.9 mo, and led to faster healing when compared to the autograft	III

NS: Not significant; BM-MSC: Bone marrow-derived mesenchymal stem cell; BMA: Bone marrow aspirate; RBC: Red blood cell; CFU: Colony-forming units; BMSC: Bone marrow derived stroma cell.