Three-Dimensional Perfusion Culture of Human Bone Marrow Cells and Generation of Osteoinductive Grafts
http://www.100md.com
《干细胞学杂志》
a Departments of Surgery and of Research, University Hospital Basel, Basel, Switzerland;
b Department of Oncology, Biology and Genetics, University of Genova, Genova, Italy
Key Words. Bone marrow stromal cells ? Bone marrow cells ? Colony formation ? Expansion ? Hematopoiesis ? Mesenchymal stem cells ? Osteoprogenitor ? Tissue regeneration
Correspondence: Ivan Martin, Ph.D., Institute for Surgical Research and Hospital Management, University Hospital Basel, Hebelstrasse 20, ZLF, Room 405, 4031 Basel, Switzerland. Telephone: 41-61-265-2384; Fax: 41-61-265-3990; e-mail: imartin@uhbs.ch
ABSTRACT
Bone marrow stromal cells (BMSCs) have received increasing experimental and clinical interest, owing to their surprising degree of plasticity and their potential use for treatment of genetic or immunologic pathologies. In the field of regenerative medicine, BMSCs have been most extensively used for bone repair because their default pathway seems to be osteogenic . This has led to encouraging findings in heterotopic models , in orthotopic implants , and in a few clinical cases . Given their low frequency among bone marrow–nucleated cells (approximately 0.01%), BMSCs are typically selected and expanded by sequential passages in monolayer (two-dimensional ) cultures. However, 2D-expanded BMSCs have a dramatically reduced differentiation capacity compared with those found in fresh bone marrow , which limits their potential use for therapeutic purposes .
Reasoning that a three-dimensional (3D) culture system may represent a more physiological environment than a Petri dish for a variety of cells and that fluid flow is an important component for seeding and culturing BMSCs in 3D environments , we aimed in this work at developing an innovative procedure to seed and expand BMSCs directly into porous 3D scaffolds under perfusion. We demonstrated that perfusion of bone marrow–nucleated cells through the pores of 3D ceramic scaffolds resulted in the efficient expansion of clonogenic BMSCs and in the generation of highly osteoinductive grafts. Moreover, the developed system allowed us to coculture BMSCs with hematopoietic cells and to support hematopoiesis.
MATERIALS AND METHODS
BMSC Expansion Under 3D Perfusion
Using a bioreactor system recently developed for efficient and uniform seeding of anchorage-dependent cells into 3D scaffolds , we perfused the nucleated cells of human bone marrow aspirates in alternate directions through the pores of disk-shaped ceramic scaffolds, and we hypothesized that BMSCs would attach to the ceramic substrate and proliferate. The number of BMSCs perfused through each scaffold, estimated by CFU-F assays, averaged 4.8 ± 2.6 x 103 cells. Medium was first changed after 5 days (cell seeding phase), which resulted in the elimination of the non-attached cell population, containing negligible numbers of CFU-F (<1% of those seeded in the scaffolds). Fresh medium was further perfused for an additional 14 days (cell expansion phase), during which time the total number of cells, monitored by Alamar blue, was found to increase at a nearly exponential rate (Fig. 1). At 19 days, the number of BMSCs found within the ceramic pores, calculated as the CD105+ fraction of the extracted cells, averaged 9 ± 3 x 105 cells for each scaffold. These data demonstrate that BMSCs can be seeded and extensively expanded (average of 8.2 ± 0.9 doublings in 19 days) by perfusion of bone marrow cell suspensions through 3D porous scaffolds, thereby avoiding typical 2D expansion.
Figure 1. Total number of cells per construct detected in the three-dimensional (3D) system by Alamar blue assays. At day 0, the number of cells corresponds to the total number of cells added to the 3D system. At day 5, after removing the non-adherent cells with the first medium change, the total number of cells corresponds to the cells attached to the scaffold.
Bone Formation by Expanded BMSCs
The osteoinductivity of the constructs resulting from BMSC seeding and expansion in the porous ceramic under perfusion (total of 19 days culture) was verified by ectopic implantation in nude mice. Reproducible, extensive, and markedly uniform bone formation was found in implanted constructs from four out of four independent experiments, performed using aspirates from different donors. Mature lamellar bone, organized in typical bone/marrow ossicles , filled an average of 52.1% ± 7.7% of the total available pore space and was distributed throughout the scaffold volume with high uniformity (Fig. 2). In contrast, when 2D-expanded BMSCs from the same donors were loaded into ceramic scaffolds at the same density as measured in the corresponding 3D cultured constructs, bone tissue was formed in only one of the four experiments. Moreover, in those constructs positive for bone formation, bone tissue filled only 9.6% ± 2.7% of the total available pore space and was localized to scattered peripheral regions (Fig. 2). The increased osteoinductivity of constructs generated using the developed system may have been supported by the ceramic substrate used for BMSC expansion , the 3D cell–cell interactions during culture , the regimen of fluid flow applied , or combinations of these variables that remain to be further elucidated. Interestingly, constructs implanted immediately after the cell seeding phase, in which BMSCs were attached to the ceramic but had not significantly expanded, were never osteoinductive. This suggests that a critical density of osteoprogenitor cells is necessary to initiate bone formation and points out the limit of approaches based on direct implantation of scaffolds mixed with bone marrow aspirates, especially considering the known variability in the number of BMSCs per aspirate volume .
Figure 2. Bone tissue formation by bone marrow stromal cells (BMSCs) expanded in monolayers (two-dimensional ) or under three-dimensional (3D) perfusion. (A, B): Representative hematoxilin/eosin-stained cross-sections of BMSC-ceramic constructs implanted ectopically in nude mice and harvested after 8 weeks. BMSCs expanded directly in the ceramic scaffolds in the 3D system yielded massive and uniformly distributed bone tissue (A), in contrast to BMSCs loaded in the ceramic after traditional 2D culture (B). White spaces correspond to the decalcified ceramic (c), whereas scaffold pores are filled with fibrous (f), adipose (a), or bone (b) tissue. Bar = 400 μm. (C, D): Quantitative image analysis of constructs generated using bone marrow aspirates from four independent donors further highlighted the increased reproducibility, amount (C), and uniformity (D) of bone tissue formation after BMSC expansion under 3D compared with 2D. Values are presented as mean and SE of the percentages calculated for each cross-section. The crosses indicate no bone formation in any of the implanted constructs.
BMSC Characterization
We then preliminarily characterized the morphology, phenotype, and clonogenicity of cells seeded and expanded within the developed 3D system. Scanning electron microscopy indicated the formation of a stromal-like tissue within the ceramic pores, consisting of a 3D network of spheroidal cells in contact with heterogeneously shaped fibroblastic cells (Fig. 3A). The mRNA expression levels of genes encoding for the osteoblast-related proteins BSP, CI, and OP averaged, respectively, 3.6%, 35.3%, and 48.0% of those previously quantified in human osteoblast cultures (Fig. 3B). Levels were similar to those measured in 2D-expanded BMSCs and lower than those measured in BMSCs after osteogenic differentiation . Fluorescence-activated cell sorting analyses indicated that 68% ± 18% of the cells extracted from the ceramic scaffolds were positive for CD105, a surface marker typically expressed by cells of the mesenchymal lineage (Fig. 3C). These CD105+ cells expressed low levels of STRO-1 (proposed as a marker of early mesenchymal progenitors ) and NGFR (proposed as a marker of multipotent BMSCs ) and high levels of BSP, OP, and CI (Figs. 3D–3H). The percentage of CD105+ cells capable of forming a fibroblastic colony (CFU-F) was markedly higher after expansion in the 3D than in typical 2D cultures (29.4% vs. 10.7%, respectively). Taken together, these data suggest that BMSCs generated in the developed 3D system were neither early undifferentiated mesenchymal precursors nor fully differentiated osteoblast-like cells but comprised a large population of clonogenic osteoprogenitor cells. Future studies should address whether changes in the substrate used (e.g., scaffold composition or architecture), flow rate, and culture medium composition will regulate the phenotype, proliferation, and multilineage differentiation capacity of the expanded BMSCs.
Figure 3. Morphology and phenotype of cells expanded under three-dimensional (3D) perfusion. (A): Scanning electron microscopy images of the constructs generated by perfusion of bone marrow–nucleated cells through the pores of ceramic scaffolds for 19 days. The ceramic pores were filled with a stromal-like tissue, consisting of a 3D network of heterogeneously shaped cells and extracellular matrix. Bar = 10 μm. (B): mRNA expression levels of bone sialoprotein (BSP), collagen type I (CI), and osteopontin (OP) in the cells. Values are presented as mean and SE of three independent experiments. (C–H): Surface markers expressed by cells extracted from the ceramic scaffolds after 19 days culture. Cells positive for (C) CD105 expressed low levels of (D) nerve growth factor receptor (NGFR) and (E) STRO-I and high levels of (F) BSP, (G) CI, and (H) OP. Light line, isotype control; dark line, specific antibody.
Hematopoietic Cell Characterization
The finding that a substantial fraction of the cells cultured in the developed 3D system was not of the mesenchymal lineage, as suggested by the rounded morphology and demonstrated by the lack of expression of CD105, induced us to investigate whether both hematopoietic and mesenchymal cells were cocultured within the ceramic pores. Indeed, in the engineered constructs we found cells positive for CD45, a surface marker of hematopoietic cells, at percentages (30% ± 15%) equivalent to those of cells negative for CD105 (Figs. 4A–4I). It is likely that cocultured hematopoietic cells, possibly including CD14-positive adherent macrophages, regulated the phenotype of BMSCs and played a critical role in determining the osteoinductivity of the constructs, possibly by maintaining a higher fraction of clonogenic BMSCs. It has been described that upon transplantation into a host animal, BMSCs form an ectopic ossicle in which bone cells, myelo supportive stroma, and adipocytes are of donor origin where as hematopoiesis and the vasculature are of recipient origin . Considering that in our 3D system human hematopoietic cells were coimplanted with BMSCs, future studies should aim at determining whether human cells contributed to hematopoiesis in this model.
Figure 4. Fraction and clonogenicity of hematopoietic cells. (A–H): Representative profiles of cells labeled for CD105 or CD45 after two-dimensional (2D) or three-dimensional (3D) culture in standard or hematopoietic medium (HM). Light line, isotype control; dark line, specific antibody. (I): Percentages of CD105+ and CD45+ cells in the above conditions. Values are presented as mean and SE of four independent experiments. (J): Quantification of the following types of hematopoietic colony-forming units present within the populations generated in the above conditions: neutrophils (CFU-N), macrophages (CFU-M), burst-forming-unit-erythroid (BFU-E), and granulocyte-erythroblast-macrophage-megakariocyte (CFU-GEMM).
We next hypothesized that, through the addition of specific medium supplements, the developed 3D culture model allows the regulation of the relative proportions of hematopoietic and mesenchymal cells. Using supplements typically used for culture of hematopoietic cells (i.e., interleukin-3, stem cell factor, and platelet-derived growth factor-bb, hematopoietic medium) , the fraction of CD45+ cells found after 19 days of 3D culture was increased to more than 90% (Fig. 4I) whereas BMSC proliferation capacity was still sustained (average of 4.5 ± 0.7 doublings in 19 days). Interestingly, the use of this culture medium further increased the percentage of CFU-F within CD105+ cells from 29.4%–38.8% and generated relevant fractions of hematopoietic CFUs, including those with a mixed phenotype, indicative of early multilineage progenitor populations (Fig. 4J). Remarkably, the use of the same medium supplements in 2D cultures was not able to modulate the fractions of hematopoietic/mesenchymal cells nor their clonogenicity, possibly due to the fact that most of the non-adherent cells were not entrapped within the 3D niches of the ceramic or newly formed stromal-like tissue and were thus discarded during medium changes. This evidence further highlights the potential of the developed culture system, in which the 3D configuration under perfusion flow provides an extension of the concept of stromal feeder layer for the support and development of hematopoietic cells and thus modifies standard paradigms for culture of bone marrow cells.
CONCLUSIONS
We would like to thank Raffaella Arbicò, Andrea Barbero, Marcel Dueggelin, Anna Marsano, Anca Reschner, and Silvia Scaglione for assistance and cooperation in conducting this research and Walter Dick, Oliver Frank, and Stefan Sch?ren for providing human bone marrow aspirates. We are grateful to Roberta Martinetti (Fin-Ceramica Faenza) for the generous supply of Engipore ceramic scaffolds.
REFERENCES
Bianco P, Gehron RP. Marrow stromal stem cells. J Clin Invest 2000;105:1663–1668.
Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation of rapidlyself-renewingandmultipotentialadultstemcellsincoloniesofhuman marrow stromal cells. Proc Natl Acad Sci U S A 2001;98:7841–7845.
Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–313.
Le Blanc K, Rasmusson I, Sundberg B et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004;363:1439–1441.
Javazon EH, Beggs KJ, Flake AW. Mesenchymal stem cells: paradoxes of passaging. Exp Hematol 2004;32:414–425.
Haynesworth SE, Goshima J, Goldberg VM et al. Characterization of cells with osteogenic potential from human marrow. Bone 1992;13:81–88.
Muraglia A, Martin I, Cancedda R et al. A nude mouse model for human bone formation in unloaded conditions. Bone 1998;22:131S–134S.
Kon E, Muraglia A, Corsi A et al. Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res 2000;49:328–337.
Petite H, Viateau V, Bensaid W et al. Tissue-engineered bone regeneration. Nat Biotechnol 2000;18:959–963.
Quarto R, Mastrogiacomo M, Cancedda R et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 2001;344:385–386.
Banfi A, Muraglia A, Dozin B et al. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 2000;28:707–715.
Mendes SC, Tibbe JM, Veenhof M et al. Bone tissue-engineered implants using human bone marrow stromal cells: effect of culture conditions and donor age. Tissue Eng 2002;8:911–920.
Bianchi G, Banfi A, Mastrogiacomo M et al. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp Cell Res 2003;287:98–105.
Abbott A. Cell culture: biology’s new dimension. Nature 2003;424:870–872.
Jacks T, Weinberg RA. Taking the study of cancer cell survival to a new dimension. Cell 2002;111:923–925.
Bancroft GN, Sikavitsas VI, van den DJ et al. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner. Proc Natl Acad Sci U S A 2002;99:12600–12605.
Wendt D, Marsano A, Jakob M et al. Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity. Biotechnol Bioeng 2003;84:205–214.
Frank O, Heim M, Jakob M et al. Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro. J Cell Biochem 2002;85:737–746.
Baksh D, Davies JE, Zandstra PW. Adult human bone marrow-derived mesenchymal progenitor cells are capable of adhesion-independent survival and expansion. Exp Hematol 2003;31:723–732.
Martin I, Mastrogiacomo M, De Leo G et al. Fluorescence microscopy imaging of bone for automated histomorphometry. Tissue Eng 2002;8:847–852.
Wodnar-Filipowicz A, Tichelli A, Zsebo KM et al. Stem cell factor stimulates the in vitro growth of bone marrow cells from aplastic anemia patients. Blood 1992;79:3196–3202.
Bianco P, Riminucci M, Gronthos S et al. Bone marrow stromal stem cells: nature, biology, and potential applications. STEM CELLS 2001;19:180–192.
Heng BC, Cao T, Stanton LW et al. Strategies for directing the differentiation of stem cells into the osteogenic lineage in vitro. J Bone Miner Res 2004;19:1379–1394.
Kale S, Biermann S, Edwards C et al. Three-dimensional cellular development is essential for ex vivo formation of human bone. Nat Biotechnol 2000;18:954–958.
Kapur S, Baylink DJ, Lau KH. Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways. Bone 2003;32:241–251.
Muschler GF, Boehm C, Easley K. Aspiration to obtain osteoblast progenitor cells from human bone marrow: the influence of aspiration volume. J Bone Joint Surg Am 1997;79:1699–1709.
Gronthos S, Zannettino AC, Graves SE et al. Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells. J Bone Miner Res 1999;14:47–56.
Gronthos S, Simmons PJ. The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro. Blood 1995;85:929–940.
Quirici N, Soligo D, Bossolasco P et al. Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 2002;30:783–791.
Friedenstein AJ, Latzinik NV, Gorskaya Y et al. Bone marrow stromal colony formation requires stimulation by haemopoietic cells. Bone Miner 1992;18:199–213.
Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 1977;91:335–344.
Wang Y, Uemura T, Dong J et al. Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials. Tissue Eng 2003;9:1205–1214.(Alessandra Braccinia, Dav)
b Department of Oncology, Biology and Genetics, University of Genova, Genova, Italy
Key Words. Bone marrow stromal cells ? Bone marrow cells ? Colony formation ? Expansion ? Hematopoiesis ? Mesenchymal stem cells ? Osteoprogenitor ? Tissue regeneration
Correspondence: Ivan Martin, Ph.D., Institute for Surgical Research and Hospital Management, University Hospital Basel, Hebelstrasse 20, ZLF, Room 405, 4031 Basel, Switzerland. Telephone: 41-61-265-2384; Fax: 41-61-265-3990; e-mail: imartin@uhbs.ch
ABSTRACT
Bone marrow stromal cells (BMSCs) have received increasing experimental and clinical interest, owing to their surprising degree of plasticity and their potential use for treatment of genetic or immunologic pathologies. In the field of regenerative medicine, BMSCs have been most extensively used for bone repair because their default pathway seems to be osteogenic . This has led to encouraging findings in heterotopic models , in orthotopic implants , and in a few clinical cases . Given their low frequency among bone marrow–nucleated cells (approximately 0.01%), BMSCs are typically selected and expanded by sequential passages in monolayer (two-dimensional ) cultures. However, 2D-expanded BMSCs have a dramatically reduced differentiation capacity compared with those found in fresh bone marrow , which limits their potential use for therapeutic purposes .
Reasoning that a three-dimensional (3D) culture system may represent a more physiological environment than a Petri dish for a variety of cells and that fluid flow is an important component for seeding and culturing BMSCs in 3D environments , we aimed in this work at developing an innovative procedure to seed and expand BMSCs directly into porous 3D scaffolds under perfusion. We demonstrated that perfusion of bone marrow–nucleated cells through the pores of 3D ceramic scaffolds resulted in the efficient expansion of clonogenic BMSCs and in the generation of highly osteoinductive grafts. Moreover, the developed system allowed us to coculture BMSCs with hematopoietic cells and to support hematopoiesis.
MATERIALS AND METHODS
BMSC Expansion Under 3D Perfusion
Using a bioreactor system recently developed for efficient and uniform seeding of anchorage-dependent cells into 3D scaffolds , we perfused the nucleated cells of human bone marrow aspirates in alternate directions through the pores of disk-shaped ceramic scaffolds, and we hypothesized that BMSCs would attach to the ceramic substrate and proliferate. The number of BMSCs perfused through each scaffold, estimated by CFU-F assays, averaged 4.8 ± 2.6 x 103 cells. Medium was first changed after 5 days (cell seeding phase), which resulted in the elimination of the non-attached cell population, containing negligible numbers of CFU-F (<1% of those seeded in the scaffolds). Fresh medium was further perfused for an additional 14 days (cell expansion phase), during which time the total number of cells, monitored by Alamar blue, was found to increase at a nearly exponential rate (Fig. 1). At 19 days, the number of BMSCs found within the ceramic pores, calculated as the CD105+ fraction of the extracted cells, averaged 9 ± 3 x 105 cells for each scaffold. These data demonstrate that BMSCs can be seeded and extensively expanded (average of 8.2 ± 0.9 doublings in 19 days) by perfusion of bone marrow cell suspensions through 3D porous scaffolds, thereby avoiding typical 2D expansion.
Figure 1. Total number of cells per construct detected in the three-dimensional (3D) system by Alamar blue assays. At day 0, the number of cells corresponds to the total number of cells added to the 3D system. At day 5, after removing the non-adherent cells with the first medium change, the total number of cells corresponds to the cells attached to the scaffold.
Bone Formation by Expanded BMSCs
The osteoinductivity of the constructs resulting from BMSC seeding and expansion in the porous ceramic under perfusion (total of 19 days culture) was verified by ectopic implantation in nude mice. Reproducible, extensive, and markedly uniform bone formation was found in implanted constructs from four out of four independent experiments, performed using aspirates from different donors. Mature lamellar bone, organized in typical bone/marrow ossicles , filled an average of 52.1% ± 7.7% of the total available pore space and was distributed throughout the scaffold volume with high uniformity (Fig. 2). In contrast, when 2D-expanded BMSCs from the same donors were loaded into ceramic scaffolds at the same density as measured in the corresponding 3D cultured constructs, bone tissue was formed in only one of the four experiments. Moreover, in those constructs positive for bone formation, bone tissue filled only 9.6% ± 2.7% of the total available pore space and was localized to scattered peripheral regions (Fig. 2). The increased osteoinductivity of constructs generated using the developed system may have been supported by the ceramic substrate used for BMSC expansion , the 3D cell–cell interactions during culture , the regimen of fluid flow applied , or combinations of these variables that remain to be further elucidated. Interestingly, constructs implanted immediately after the cell seeding phase, in which BMSCs were attached to the ceramic but had not significantly expanded, were never osteoinductive. This suggests that a critical density of osteoprogenitor cells is necessary to initiate bone formation and points out the limit of approaches based on direct implantation of scaffolds mixed with bone marrow aspirates, especially considering the known variability in the number of BMSCs per aspirate volume .
Figure 2. Bone tissue formation by bone marrow stromal cells (BMSCs) expanded in monolayers (two-dimensional ) or under three-dimensional (3D) perfusion. (A, B): Representative hematoxilin/eosin-stained cross-sections of BMSC-ceramic constructs implanted ectopically in nude mice and harvested after 8 weeks. BMSCs expanded directly in the ceramic scaffolds in the 3D system yielded massive and uniformly distributed bone tissue (A), in contrast to BMSCs loaded in the ceramic after traditional 2D culture (B). White spaces correspond to the decalcified ceramic (c), whereas scaffold pores are filled with fibrous (f), adipose (a), or bone (b) tissue. Bar = 400 μm. (C, D): Quantitative image analysis of constructs generated using bone marrow aspirates from four independent donors further highlighted the increased reproducibility, amount (C), and uniformity (D) of bone tissue formation after BMSC expansion under 3D compared with 2D. Values are presented as mean and SE of the percentages calculated for each cross-section. The crosses indicate no bone formation in any of the implanted constructs.
BMSC Characterization
We then preliminarily characterized the morphology, phenotype, and clonogenicity of cells seeded and expanded within the developed 3D system. Scanning electron microscopy indicated the formation of a stromal-like tissue within the ceramic pores, consisting of a 3D network of spheroidal cells in contact with heterogeneously shaped fibroblastic cells (Fig. 3A). The mRNA expression levels of genes encoding for the osteoblast-related proteins BSP, CI, and OP averaged, respectively, 3.6%, 35.3%, and 48.0% of those previously quantified in human osteoblast cultures (Fig. 3B). Levels were similar to those measured in 2D-expanded BMSCs and lower than those measured in BMSCs after osteogenic differentiation . Fluorescence-activated cell sorting analyses indicated that 68% ± 18% of the cells extracted from the ceramic scaffolds were positive for CD105, a surface marker typically expressed by cells of the mesenchymal lineage (Fig. 3C). These CD105+ cells expressed low levels of STRO-1 (proposed as a marker of early mesenchymal progenitors ) and NGFR (proposed as a marker of multipotent BMSCs ) and high levels of BSP, OP, and CI (Figs. 3D–3H). The percentage of CD105+ cells capable of forming a fibroblastic colony (CFU-F) was markedly higher after expansion in the 3D than in typical 2D cultures (29.4% vs. 10.7%, respectively). Taken together, these data suggest that BMSCs generated in the developed 3D system were neither early undifferentiated mesenchymal precursors nor fully differentiated osteoblast-like cells but comprised a large population of clonogenic osteoprogenitor cells. Future studies should address whether changes in the substrate used (e.g., scaffold composition or architecture), flow rate, and culture medium composition will regulate the phenotype, proliferation, and multilineage differentiation capacity of the expanded BMSCs.
Figure 3. Morphology and phenotype of cells expanded under three-dimensional (3D) perfusion. (A): Scanning electron microscopy images of the constructs generated by perfusion of bone marrow–nucleated cells through the pores of ceramic scaffolds for 19 days. The ceramic pores were filled with a stromal-like tissue, consisting of a 3D network of heterogeneously shaped cells and extracellular matrix. Bar = 10 μm. (B): mRNA expression levels of bone sialoprotein (BSP), collagen type I (CI), and osteopontin (OP) in the cells. Values are presented as mean and SE of three independent experiments. (C–H): Surface markers expressed by cells extracted from the ceramic scaffolds after 19 days culture. Cells positive for (C) CD105 expressed low levels of (D) nerve growth factor receptor (NGFR) and (E) STRO-I and high levels of (F) BSP, (G) CI, and (H) OP. Light line, isotype control; dark line, specific antibody.
Hematopoietic Cell Characterization
The finding that a substantial fraction of the cells cultured in the developed 3D system was not of the mesenchymal lineage, as suggested by the rounded morphology and demonstrated by the lack of expression of CD105, induced us to investigate whether both hematopoietic and mesenchymal cells were cocultured within the ceramic pores. Indeed, in the engineered constructs we found cells positive for CD45, a surface marker of hematopoietic cells, at percentages (30% ± 15%) equivalent to those of cells negative for CD105 (Figs. 4A–4I). It is likely that cocultured hematopoietic cells, possibly including CD14-positive adherent macrophages, regulated the phenotype of BMSCs and played a critical role in determining the osteoinductivity of the constructs, possibly by maintaining a higher fraction of clonogenic BMSCs. It has been described that upon transplantation into a host animal, BMSCs form an ectopic ossicle in which bone cells, myelo supportive stroma, and adipocytes are of donor origin where as hematopoiesis and the vasculature are of recipient origin . Considering that in our 3D system human hematopoietic cells were coimplanted with BMSCs, future studies should aim at determining whether human cells contributed to hematopoiesis in this model.
Figure 4. Fraction and clonogenicity of hematopoietic cells. (A–H): Representative profiles of cells labeled for CD105 or CD45 after two-dimensional (2D) or three-dimensional (3D) culture in standard or hematopoietic medium (HM). Light line, isotype control; dark line, specific antibody. (I): Percentages of CD105+ and CD45+ cells in the above conditions. Values are presented as mean and SE of four independent experiments. (J): Quantification of the following types of hematopoietic colony-forming units present within the populations generated in the above conditions: neutrophils (CFU-N), macrophages (CFU-M), burst-forming-unit-erythroid (BFU-E), and granulocyte-erythroblast-macrophage-megakariocyte (CFU-GEMM).
We next hypothesized that, through the addition of specific medium supplements, the developed 3D culture model allows the regulation of the relative proportions of hematopoietic and mesenchymal cells. Using supplements typically used for culture of hematopoietic cells (i.e., interleukin-3, stem cell factor, and platelet-derived growth factor-bb, hematopoietic medium) , the fraction of CD45+ cells found after 19 days of 3D culture was increased to more than 90% (Fig. 4I) whereas BMSC proliferation capacity was still sustained (average of 4.5 ± 0.7 doublings in 19 days). Interestingly, the use of this culture medium further increased the percentage of CFU-F within CD105+ cells from 29.4%–38.8% and generated relevant fractions of hematopoietic CFUs, including those with a mixed phenotype, indicative of early multilineage progenitor populations (Fig. 4J). Remarkably, the use of the same medium supplements in 2D cultures was not able to modulate the fractions of hematopoietic/mesenchymal cells nor their clonogenicity, possibly due to the fact that most of the non-adherent cells were not entrapped within the 3D niches of the ceramic or newly formed stromal-like tissue and were thus discarded during medium changes. This evidence further highlights the potential of the developed culture system, in which the 3D configuration under perfusion flow provides an extension of the concept of stromal feeder layer for the support and development of hematopoietic cells and thus modifies standard paradigms for culture of bone marrow cells.
CONCLUSIONS
We would like to thank Raffaella Arbicò, Andrea Barbero, Marcel Dueggelin, Anna Marsano, Anca Reschner, and Silvia Scaglione for assistance and cooperation in conducting this research and Walter Dick, Oliver Frank, and Stefan Sch?ren for providing human bone marrow aspirates. We are grateful to Roberta Martinetti (Fin-Ceramica Faenza) for the generous supply of Engipore ceramic scaffolds.
REFERENCES
Bianco P, Gehron RP. Marrow stromal stem cells. J Clin Invest 2000;105:1663–1668.
Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation of rapidlyself-renewingandmultipotentialadultstemcellsincoloniesofhuman marrow stromal cells. Proc Natl Acad Sci U S A 2001;98:7841–7845.
Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–313.
Le Blanc K, Rasmusson I, Sundberg B et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004;363:1439–1441.
Javazon EH, Beggs KJ, Flake AW. Mesenchymal stem cells: paradoxes of passaging. Exp Hematol 2004;32:414–425.
Haynesworth SE, Goshima J, Goldberg VM et al. Characterization of cells with osteogenic potential from human marrow. Bone 1992;13:81–88.
Muraglia A, Martin I, Cancedda R et al. A nude mouse model for human bone formation in unloaded conditions. Bone 1998;22:131S–134S.
Kon E, Muraglia A, Corsi A et al. Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res 2000;49:328–337.
Petite H, Viateau V, Bensaid W et al. Tissue-engineered bone regeneration. Nat Biotechnol 2000;18:959–963.
Quarto R, Mastrogiacomo M, Cancedda R et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 2001;344:385–386.
Banfi A, Muraglia A, Dozin B et al. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 2000;28:707–715.
Mendes SC, Tibbe JM, Veenhof M et al. Bone tissue-engineered implants using human bone marrow stromal cells: effect of culture conditions and donor age. Tissue Eng 2002;8:911–920.
Bianchi G, Banfi A, Mastrogiacomo M et al. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp Cell Res 2003;287:98–105.
Abbott A. Cell culture: biology’s new dimension. Nature 2003;424:870–872.
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