The translation of tissue engineering approaches to the clinic has been hampered by the inability to find suitable multipotent cell sources requiring minimal in vitro expansion. Enhanced bone marrow (eBM), which is obtained by reaming long bone medullary canals and isolating the solid marrow putty, has large quantities of stem cells and demonstrates significant potential to regenerate bone tissues. eBM, however, cannot impart immediate load-bearing mechanical integrity or maintain the gross anatomical structure to guide bone healing. Yet, its putty-like consistency creates a challenge for obtaining the uniform seeding necessary to effectively combine it with porous scaffolds. In this study, we examined the potential for combining eBM with mechanically strong, osteoinductive trabecular bone scaffolds for bone regeneration by creating channels into scaffolds for seeding the eBM. eBM was extracted from the femurs of adult Yorkshire pigs using a Synthes reamer-irrigator-aspirator device, analyzed histologically, and digested to extract cells and characterize their differentiation potential. To evaluate bone tissue formation, eBM was seeded into the channels in collagen-coated or noncoated scaffolds, cultured in osteogenic conditions for 4 weeks, harvested and assessed for tissue distribution and bone formation. Our data demonstrates that eBM is a heterogenous tissue containing multipotent cell populations. Furthermore, coating scaffolds with a collagen hydrogel significantly enhanced cellular migration, promoted uniform tissue development and increased bone mineral deposition. These findings suggest the potential for generating customized autologous bone grafts for treating critical-sized bone defects by combining a readily available eBM cell source with decellularized trabecular bone scaffolds.

Keywords:
Enhanced bone marrow, Bone scaffold, Bone regeneration, Mesenchymal stem cells
1.
Clarke, K.I., S.E. Graves, A.T.C. Wong, J.T. Triffitt, M.J.O. Francis, J.T. Czernuszka (1993) Investigation into the formation and mechanical-properties of a bioactive material based on collagen and calcium-phosphate. J Mater Sci 4: 107-110.
2.
Correia, C., W.L. Grayson, M. Park, D. Hutton, B. Zhou, X.E. Guo, L. Niklason, R.A. Sousa, R.L. Reis, G. Vunjak-Novakovic (2011) In vitro model of vascularized bone: synergizing vascular development and osteogenesis. PLoS One 6: e28352.
3.
Cox, G., D. McGonagle, S.A. Boxall, C.T. Buckley, E. Jones, P.V. Giannoudis (2011) The use of the reamer-irrigator-aspirator to harvest mesenchymal stem cells. J Bone Joint Surg Br 93B: 517-524.
4.
Frohlich, M., W.L. Grayson, D. Marolt, J.M. Gimble, N. Kregar-Velikonja, G. Vunjak-Novakovic (2010) Bone grafts engineered from human adipose-derived stem cells in perfusion bioreactor culture. Tissue Eng 16: 179-189.
5.
Frölke, J.P., H. Van de Krol, F.C. Bakker, P. Patka, H.J. Haarman (2000) Destination of debris during intramedullary reaming: an experimental study on sheep femurs. Acta Orthop Belg 66: 337-340.
6.
Grayson, W.L., S. Bhumiratana, P.H. Grace Chao, C.T. Hung, G. Vunjak-Novakovic (2010a) Spatial regulation of human mesenchymal stem cell differentiation in engineered osteochondral constructs: effects of pre-differentiation, soluble factors and medium perfusion. Osteoarthritis Cartilage 18: 714-723.
7.
Grayson, W.L., M. Frohlich, K. Yeager, S. Bhumiratana, M.E. Chan, C. Cannizzaro, L.Q. Wan, X.S. Liu, X.E. Guo, G. Vunjak-Novakovic (2010b) Engineering anatomically shaped human bone grafts. Proc Natl Acad Sci USA 107: 3299-3304.
8.
Grayson, W.L., F. Zhao, R. Izadpanah, B. Bunnell, T. Ma (2006) Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol 207: 331-339.
9.
Hammer, T.O., R. Wieling, J.M. Green, N.P. Sudkamp, E. Schneider, C.A. Muller (2007) Effect of re-implanted particles from intramedullary reaming on mechanical properties and callus formation: a laboratory study. J Bone Joint Surg Br 89: 1534-1538.
10.
Harakas, N.K. (1984) Demineralized bone-matrix-induced osteogenesis. Clin Orthop 188: 239-251.
11.
Hutton, D.L., E.A. Logsdon, E.M. Moore, F. Mac Gabhann, J. Gimble, W.L. Grayson (2012) Vascular morphogenesis of adipose-derived stem cells is mediated by heterotypic cell-cell interactions. Tissue Eng 18: 1729-1740.
12.
Jones, E.A., A. English, K. Henshaw, S.E. Kinsey, A.F. Markham, P. Emery, D. McGonagle (2004) Enumeration and phenotypic characterization of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis. Arthritis Rheum 50: 817-827.
13.
Kobbe, P., I.S. Tarkin, H.C. Pape (2008) Use of the ‘reamer irrigator aspirator' system for non-infected tibial non-union after failed iliac crest grafting. Injury 39: 796-800.
14.
Lima, E.G., P.H.G. Chao, G.A. Ateshian, B.S. Bal, J.L. Cook, G. Vunjak-Novakovic, C.T. Hung (2008) The effect of devitalized trabecular bone on the formation of osteochondral tissue-engineered constructs. Biomaterials 29: 4292-4299.
15.
Lima, E.G., R.L. Mauck, S.H. Han, S. Park, K.W. Ng, G.A. Ateshian, C.T. Hung (2004) Functional tissue engineering of chondral and osteochondral constructs. Biorheology 41: 577-590.
16.
Mauney, J.R., J. Blumberg, M. Pirun, V. Volloch, G. Vunjak-Novakovic, D.L. Kaplan (2004) Osteogenic differentiation of human bone marrow stromal cells on partially demineralized bone scaffolds in vitro. Tissue Eng 10: 81-92.
17.
Mauney, J.R., C. Jaquiery, V. Volloch, M. Herberer, I. Martin, D.L. Kaplan (2005) In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering. Biomaterials 26: 3173-3185.
18.
O'Keefe, R.J., J. Mao (2011) Bone tissue engineering and regeneration: from discovery to the clinic - an overview. Tissue Eng 17: 389-392.
19.
Peyton, S.R., Z.I. Kalcioglu, J.C. Cohen, A.P. Runkle, K.J. Van Vliet, D.A. Lauffenburger, L.G. Griffith (2010) Marrow-derived stem cell motility in 3D synthetic scaffold is governed by geometry along with adhesivity and stiffness. Biotechnol Bioeng 108: 1181-1193.
20.
Pittenger, M.F., A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284: 143-147.
21.
Porter, R.M., F. Liu, C. Pilapil, O.B. Betz, M.S. Vrahas, M.B. Harris, C.H. Evans (2009) Osteogenic potential of reamer irrigator aspirator (RIA) aspirate collected from patients undergoing hip arthroplasty. J Orthop Res 27: 42-49.
22.
Salter, E.K., B.C. Goh, B.P. Hung, D.L. Hutton, N.V. Ghone, W.L. Grayson (2012) Bone tissue engineering bioreactors: a role in the clinic? Tissue Eng 18: 62-75.
23.
Serre, C.M., M. Papillard, P. Chavassieux, G. Boivin (1993) In vitro induction of a calcifying matrix by biomaterials constituted of collagen and/or hydroxyapatite: an ultrastructural comparison of three types of biomaterials. Biomaterials 14: 97-106.
24.
Stolzing, A., E. Jones, D. McGonagle, A. Scutt (2008) Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev 129: 163-173.
25.
Tydings, J.D., L.J. Martino, M. Kircher, R. Alfred, J. Lozman (1986) The osteoinductive potential of intramedullary canal bone reamings. Curr Surg 43: 121-124.
26.
Urist, M.R. (1965) Bone: formation by autoinduction. Science 150: 893-899.
27.
Urist, M.R., A. Mikulski, A. Lietze (1979) Solubilized and insolubilized bone morphogenetic protein. Proc Natl Acad Sci USA 76: 1828-1832.
28.
Wahl, D.A., J.T. Czernuszka (2006) Collagen-hydroxyapatite composites for hard tissue repair. Eur Cell Mater 11: 43-56.
29.
Wenisch, S., K. Trinkaus, A. Hild, D. Hose, K. Herde, C. Heiss, O. Kilian, V. Alt, R. Schnettler (2005) Human reaming debris: a source of multipotent stem cells. Bone 36: 74-83.
30.
Yoneno, K., S. Ohno, K. Tanimoto, K. Honda, N. Tanaka, T. Doi, T. Kawata, E. Tanaka, S. Kapila, K. Tanne (2005) Multidifferentiation potential of mesenchymal stem cells in three-dimensional collagen gel cultures. J Biomed Mater Res 75: 733-741.
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2013
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