title: Cell-based therapy by implanted human bone marrow-derived mononuclear cells improved bone healing of large bone defects in rats.
abstract: QUESTION/AIM: Cell-based therapy by cultivated stem cells (mesenchymal stem cells [MSC] and endothelial progenitor cells [EPC]) in a large-sized bone defect has already shown improved vascularization and new bone formation. However, these methods are clinically afflicted with disadvantages. Another heterogeneous bone marrow cell population, the so-called human bone marrow-derived mononuclear cells (BMC), has nevertheless been used clinically and showed improved vascularization in ischemic limbs or in the myocardium. For clinical use, a certified process has been established; thus, BMC were isolated from bone marrow aspirate by density gradient centrifugation, washed, cleaned, and given back to patients within several hours. This investigation tested the ability of human BMC seeded on beta-tricalcium phosphate (β-TCP) and placed into a large bone defect in rats to improve the bone healing process in vivo.
  
  
  METHODS
  Human EPC were isolated from buffy coat, and MSC or BMC, respectively, were isolated from bone marrow aspirate by density gradient centrifugation. 1.0×10(6) cells were loaded onto 750 μL β-TCP (0.7-1.4 mm). Large femoral defects (6 mm) in athymic rats were created surgically and stabilized with an internal fixateur. The remaining defects were filled with β-TCP granules alone (group 1), β-TCP+EPC/MSC (group 2), or β-TCP+BMC (group 3). After 8 weeks, histomorphometric analysis (new bone formation), radiological microcomputer tomography analysis (bony bridging), and biomechanical testing (three-point bending) were achieved. Moreover, a tumorigenicity study was performed to evaluate the safety of BMC implantation after 26 weeks. For statistical analysis, the Kruskal-Wallis test was used.
  
  
  RESULTS
  Eight weeks after implantation of EPC/MSC or BMC, respectively, we detected a more significant new bone formation compared to control. In group 2 and 3, bony bridging of the defect was seen. In the control group, more chondrocytes and osteoid were detected. In the BMC and EPC/MSC group, respectively, less chondrocytes and a significantly more advanced bone formation were observed. The biomechanical stability of the bone regenerate was significantly enhanced if BMC and EPC/MSC, respectively, were implanted compared to control. Moreover, no tumor formation was detected either macroscopically or histologically after 26 weeks of BMC implantation.
  
  
  CONCLUSIONS
  Implanted BMC suggest that a heterogeneous cell population may provide a powerful cellular therapeutic strategy for bone healing in a large bone defect in humans.
authors.affiliation: 1 Department of Trauma Surgery, Johann-Wolfgang-Goethe University , Frankfurt/Main, Germany .
authors.forename: Caroline
authors.initials: C
authors.lastname: Seebach
authors.forename: Dirk
authors.initials: D
authors.lastname: Henrich
authors.forename: Alexander
authors.initials: A
authors.lastname: Schaible
authors.forename: Borna
authors.initials: B
authors.lastname: Relja
authors.forename: Manfred
authors.initials: M
authors.lastname: Jugold
authors.forename: Halvard
authors.initials: H
authors.lastname: Bönig
authors.forename: Ingo
authors.initials: I
authors.lastname: Marzi
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001696:Biomechanical Phenomena; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D063646:Carcinogenesis / Q000473:pathology; D002454:Cell Differentiation / Q000235:genetics; D066026:Endothelial Progenitor Cells / Q000166:cytology; D005269:Femur / Q000000981:diagnostic imaging / Q000473:pathology* / Q000503:physiopathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D020007:Implants, Experimental; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010012:Osteogenesis / Q000235:genetics; D011923:Rats, Nude; D060888:Real-Time Polymerase Chain Reaction; D013194:Staining and Labeling; D033581:Stem Cell Transplantation; D014945:Wound Healing; D055114:X-Ray Microtomography
chemical_list: D015415:Biomarkers