- Email: [email protected]
The osteogenic capacity of bone narrow cells
742
Cell Biology
international
Reports, Vol. 4, No. 8, August
1980
THE OSTEOGENIC CAPACITY OF BONE NARROW CELLS C.C. Eaglesomx, B.A. Ashtor?, T.O. Allen*, and M.E. Ower?. ‘NRC Bone Research Laboratory, Nuffield Orthopaedic Centre, Oxford OX3 7L0. *Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 98X. The stromal cells of bone marrow have been shown to have osteogenic potential in diffusion chambers. These same cells in monolayer culture give rise to colonies of fibroblastic cells, each colony being derived from a single cell. Osteogenic potential is also displayed by these fibroblasts. It has been proposed that the colony forming cells can be regarded as stem cells for marrow stromal tissue(l). In this study we have investigated the bone forming and colony forming ability of cells from different regions of the marrow. Three populations of cells were taken from the femurs of male New Zealand White rabbits (l-l.5 kg). The first (M21, a mid marrow population, was obtained by passing a needle ci.d.1.21 mm, o.d.1.59 mm) down the centre of the marrow. Cells displaced were drawn into the attached syringe. Passage of a piece of tubing (i.d.1.9 mm1 just large enough to mm, o.d.2.76 pass easily down the marrow cavity withdrew the next population [M3). The remaining cells CM41 were removed by flushing the endosteal surface with medium. The cells of each population were suspended and dispersed in a small volume of MEN by repeated passage through needles of decreasing size The cells were innocu(19-23 gauge]. lated into either flasks (Z-2.5 x IO7 cells/flask1 or into diffusion chambers (1.5-3.9 x IO7 cells/chamber]. The diffusion chambers used were of approximate volume 0.1 ml bounded by 0.45 urn membrane filters [Millipore Corp.). After innoculation with cells they were implanted intraperitoneally to NZW rabbits (1.5-2.5 kg). The chambers were harvested after four some fixed for histology, weeks, others taken for estimation of alkaline phosphatase activity, ONA, calcium and phosphorus content. Chambers for light microscopy were fixed in 95% ethanol, embedded in glycolmethacrylate (2) at 4oC and sectioned (I-4 vml. The sections were stained with 1% toluidine blue or by the Von Kossa method or for alkaline phosphatase activity using an azo dye method (Sigma kit 65). Chambers for electron micmscopy were fixed in 2.5% glutaraldehyde, postfixed in osmium tetroxide embedded in Epon-Araldite and stained with uranyl acetate and lead acetate. Cells were also innoculated into 25
030!+1651/60/060742~1/$02.00/0
cm2 flasks with 5ml modified Minimal Essential Medium [MEN), 20 mfl HEPES, supplemented with 10% Foetal Calf Serum. The flasks were incubated for IO-14 days at 37’C, fixed with 95% ethanol and stained with Ray Griinwald-Giesma stain. The number of colonies with more than 50 cells are counted under a microscope. Colonies are divided into two categories according to how tightly the cells were packed together. Both types of colony were fibroblastic and together accounted for over 98% of the colonies formed. The table below shows an increase in the Colony Forming Efficiency [CFE, the number of colonies counted/the number of cells innoculatedl from mid to peripheral marrow populations. -6 Population n CFE x 10 median range 64 0- 2.78 r12 0.63 r13 60 1.09 0.056.79 M4 62 2.40 0.1 -11.71 The differences between the populations have been shown to be significant LpCO.011 by the Mann Whitney test. The results for “tight” and “loose” colonies considered separately show that at the mid marrow the predominant colony type is “tight” (61%1 whereas at the periphery the predominent colony type is “loose” (73%1. Morphological studies of the tissue formed in diffusion chambers show that bone, cartilage, and fibrous tissue are formed by all populations. The first sign of bone formation is the appearance of alkaline phosphatase activity on cytoplasmic membranes. As calcification proceeds the enzyme activity decreases and is confined to a layer adjacent to, but separate from, the calcified area. Examination of the tissue by electron microscopy confirmed that the bone and cartilage formed in diffusion chambers are morphologically identical to their skeletal counterparts. Analyses of diffusion chamber contents demonstrated no difference in DNA content of alkaline phosphatase activity between the different populations. However the higher Ca and P content found in the M4 and M3 population compared to M2 is an indication of more bone formation by these populations since all calcification observed histologically was associated with bone tissue. 1.
Friedenstein, A.J.: 47, 327,(1976). 2. Ems EI.:Microscopy
Int.Rev.Cytol. 101,223
01960 Academic
[1974).
Press Inc. (London) Ltd.
Cell Biology
international
Reports, Vol. 4, No. 8, August
1980
THE OSTEOGENIC CAPACITY OF BONE NARROW CELLS C.C. Eaglesomx, B.A. Ashtor?, T.O. Allen*, and M.E. Ower?. ‘NRC Bone Research Laboratory, Nuffield Orthopaedic Centre, Oxford OX3 7L0. *Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 98X. The stromal cells of bone marrow have been shown to have osteogenic potential in diffusion chambers. These same cells in monolayer culture give rise to colonies of fibroblastic cells, each colony being derived from a single cell. Osteogenic potential is also displayed by these fibroblasts. It has been proposed that the colony forming cells can be regarded as stem cells for marrow stromal tissue(l). In this study we have investigated the bone forming and colony forming ability of cells from different regions of the marrow. Three populations of cells were taken from the femurs of male New Zealand White rabbits (l-l.5 kg). The first (M21, a mid marrow population, was obtained by passing a needle ci.d.1.21 mm, o.d.1.59 mm) down the centre of the marrow. Cells displaced were drawn into the attached syringe. Passage of a piece of tubing (i.d.1.9 mm1 just large enough to mm, o.d.2.76 pass easily down the marrow cavity withdrew the next population [M3). The remaining cells CM41 were removed by flushing the endosteal surface with medium. The cells of each population were suspended and dispersed in a small volume of MEN by repeated passage through needles of decreasing size The cells were innocu(19-23 gauge]. lated into either flasks (Z-2.5 x IO7 cells/flask1 or into diffusion chambers (1.5-3.9 x IO7 cells/chamber]. The diffusion chambers used were of approximate volume 0.1 ml bounded by 0.45 urn membrane filters [Millipore Corp.). After innoculation with cells they were implanted intraperitoneally to NZW rabbits (1.5-2.5 kg). The chambers were harvested after four some fixed for histology, weeks, others taken for estimation of alkaline phosphatase activity, ONA, calcium and phosphorus content. Chambers for light microscopy were fixed in 95% ethanol, embedded in glycolmethacrylate (2) at 4oC and sectioned (I-4 vml. The sections were stained with 1% toluidine blue or by the Von Kossa method or for alkaline phosphatase activity using an azo dye method (Sigma kit 65). Chambers for electron micmscopy were fixed in 2.5% glutaraldehyde, postfixed in osmium tetroxide embedded in Epon-Araldite and stained with uranyl acetate and lead acetate. Cells were also innoculated into 25
030!+1651/60/060742~1/$02.00/0
cm2 flasks with 5ml modified Minimal Essential Medium [MEN), 20 mfl HEPES, supplemented with 10% Foetal Calf Serum. The flasks were incubated for IO-14 days at 37’C, fixed with 95% ethanol and stained with Ray Griinwald-Giesma stain. The number of colonies with more than 50 cells are counted under a microscope. Colonies are divided into two categories according to how tightly the cells were packed together. Both types of colony were fibroblastic and together accounted for over 98% of the colonies formed. The table below shows an increase in the Colony Forming Efficiency [CFE, the number of colonies counted/the number of cells innoculatedl from mid to peripheral marrow populations. -6 Population n CFE x 10 median range 64 0- 2.78 r12 0.63 r13 60 1.09 0.056.79 M4 62 2.40 0.1 -11.71 The differences between the populations have been shown to be significant LpCO.011 by the Mann Whitney test. The results for “tight” and “loose” colonies considered separately show that at the mid marrow the predominant colony type is “tight” (61%1 whereas at the periphery the predominent colony type is “loose” (73%1. Morphological studies of the tissue formed in diffusion chambers show that bone, cartilage, and fibrous tissue are formed by all populations. The first sign of bone formation is the appearance of alkaline phosphatase activity on cytoplasmic membranes. As calcification proceeds the enzyme activity decreases and is confined to a layer adjacent to, but separate from, the calcified area. Examination of the tissue by electron microscopy confirmed that the bone and cartilage formed in diffusion chambers are morphologically identical to their skeletal counterparts. Analyses of diffusion chamber contents demonstrated no difference in DNA content of alkaline phosphatase activity between the different populations. However the higher Ca and P content found in the M4 and M3 population compared to M2 is an indication of more bone formation by these populations since all calcification observed histologically was associated with bone tissue. 1.
Friedenstein, A.J.: 47, 327,(1976). 2. Ems EI.:Microscopy
Int.Rev.Cytol. 101,223
01960 Academic
[1974).
Press Inc. (London) Ltd.