Rabbit marrow cells inoculated into diffusion chambers (107 cells/chamber) were implanted intraperitoneally into athymic mouse hosts and cultured in vivo for 20 days. A connective tissue consisting of bone, cartilage and fibrous tissues is formed by the stromal fibroblastic cells of marrow within the chambers. Cell kinetics and tissue differentiation have been studied using histo-morphometric and biochemical analyses. Haemopoietic cell numbers decrease to less than 0·05% of the initial inoculum during the 20-day period. At 3 days an average of 15 stromal fibroblastic cells only are identifiable within the chambers. After 3 days there is a high rate of stromal cell proliferation with a doubling time of 14·5 h during the period from 3 to 8 days and an increase in the total stromal cell population by more than six orders of magnitude from 3 to 20 days. Thirteen to fourteen population doublings occur before expression of the first observable differentiation parameter, alkaline phosphatase activity. The data demonstrate that the mixture of connective tissues formed within the chamber is generated by a small number of cells with high capacity for proliferation and differentiation. This is consistent with the current hypothesis that stromal stem cells are present in bone marrow.

Stromal cell lines of marrow include cells of the fibroblastic-reticular network, adipocytic and osteogenic cells. The hierarchy of these cell lines has not yet been elucidated and they are often encompassed by the term ‘fibroblastic’ (Owen, 1985). Studies of the potential for differentiation of marrow stromal cells were pioneered by Friedenstein, who demonstrated that either marrow cell suspensions or fibroblastic cells grown from them in vitro are able to form a calcified tissue in diffusion chambers (DC) implanted in vivo (Friedenstein et al. 1970; Friedenstein, 1973, 1976). Recent investigations have shown that the bone, cartilage and fibrous tissues formed by marrow stromal cells within DC are morphologically similar to their counterparts in the skeleton (Ashton et al. 1980; Bab et al. 1984b) and that there is an increase in alkaline phosphatase activity and accumulation of mineral, parameters that are indicative of osteogenic differentiation (Bab et al. 1984a; Ashton et al. 1984).

Current evidence for the existence of pluripotent stromal stem cells in the bone marrow has recently been reviewed (Owen, 1985). Circumstantial evidence supports the hypothesis that a single stromal stem cell is able to generate several cell lines (Friedenstein, 1980). Results from experiments in our laboratory have led us to suggest that the mixture of fibrous and osteogenic tissues formed by marrow cells within DC is likely to arise from a small number of cells with stem cell characteristics (Owen, 1982, 1985; Bab et al. 1984a).

Formation of osteogenic tissue within DC has many similarities to other bone developmental systems: namely, rapid cell proliferation followed by production of a fibrous anlage prior to osteogenesis. In the present experiments stromal cell kinetics have been studied in relationship to the expression of parameters of differentiation following implantation of rabbit marrow cell suspensions in DC in athymic mouse hosts. The results obtained add support to the hypothesis that a small population of cells with the characteristics of stem cells generate the tissues formed within the chambers.

Preparation of cell suspensions

Suspensions of marrow or spleen cells for inoculation into DC were prepared under sterile conditions as described previously (Ashton et al. 1980, 1984). Male New Zealand White rabbits weighing 570–630 g were used as cell donors in all experiments. Single cell suspensions of femoral marrow cells were prepared in Minimal Essential Medium (MEM) by drawing the cells several times through graded needles. Cell density was determined by counting in a haemocytometer. Approximately 2 ×108 total marrow cells were obtained from each femur and suspended in 2 ml of culture medium. Similarly dispersed spleen cells were used as non-osteogenic controls.

In vivo culture in diffusion chambers

DC were assembled from commercially available components (Millipore Corporation). Chamber dimensions were: 13mm external diameter, 9mm internal diameter, 2mm thick, approximately 127 mm3 capacity. The pore size of membrane filters was 0 ·45 μm. Pairs of DC were assembled by cementing their lucite rings edge to edge (Fig. 1) and sterilized with ethylene oxide. Approximately 107 marrow or spleen cells in 100 μl samples were inoculated into the chambers through a hole in the ring, which was then sealed with a tapered plastic plug coated with glue (Fig. 1A). One donor rabbit and four host athymic mice (25 g male MFl/nu nu/Ola mice, Olac 1976 Ltd, Oxon., England) were used in each of three experiments with marrow cells and in one control experiment with spleen cells. A single experiment consisted of 18 DC inoculated with samples from the same suspension. Sixteen of the DC were implanted into the peritoneal cavity of hosts, two pairs per mouse (Fig. 1A). The remaining pair of chambers was not implanted and served as the 0-day controls. DC were harvested at 0, 3, 8, 12 and 20 days after implantation; the contents of one chamber from each pair was prepared for histology and that of the other for biochemical analysis (Fig. IB); four chambers were examined by each method at each time point.

Fig. 1.

Diagram illustrating: A, experimental procedure for inoculating and implanting DC. DC-containing marrow cells (marrow DC) were cemented together in pairs, plugged and implanted intraperitoneally into athymic mice, each pair on either side of the abdominal mid-line; B, subsequent analysis of paired DC. One DC from each pair is either processed for histology or the total contents prepared for biochemical analysis. * * Numbering is arbitrary.

Fig. 1.

Diagram illustrating: A, experimental procedure for inoculating and implanting DC. DC-containing marrow cells (marrow DC) were cemented together in pairs, plugged and implanted intraperitoneally into athymic mice, each pair on either side of the abdominal mid-line; B, subsequent analysis of paired DC. One DC from each pair is either processed for histology or the total contents prepared for biochemical analysis. * * Numbering is arbitrary.

Biochemical analysis of DC

The analysis was carried out as described previously (Ashton et ql. 1984). The contents of one DC from each pair (Fig. IB) was homogenized in 10 ml of triple-distilled water. Samples of the homogenate were assayed for alkaline phosphatase activity and the results expressed as μmolpNPh−1DC−1. The remainder of each homogenate was freeze-dried and resuspended in 0’4M-perchloric acid (500 μl), and the samples were kept at 4°C for 2 h and centrifuged for 15 min at 1800 g. The amounts of calcium and phosphorus present in the supernatant were determined by atomic absorption spectrophotometry and the colorimetric method of Chen et al. (1956), respectively. The results were expressed as μgDC−1.

Histological processing of DC

Immediately after removal from the host animal, one DC from each pair (Fig. IB) was immersed in cold 95% (v/v) ethanol. Dehydration with three changes of absolute ethanol and embedding in glycol methacrylate were carried out in the cold to preserve alkaline phosphatase activity (Ashton et al. 1980). Sections, 5 μm thick, were cut with a glass-knife microtome (Auto-Cut, Reichert-Jung) at three parallel, equally spaced planes perpendicular to the surface of the membrane filters (Fig. IB). At each plane, two sections were taken for each of the following stains: (1) haematoxylin and eosin, (2) Von Kossa and Toluidine Blue, and (3) for alkaline phosphatase activity using an azo dye method (Sigma kit no. 85), counterstained with Von Kossa and Meyer’s haematoxylin.

His tomorphome try

The inner DC in a section is the area bounded by the membrane filters and lucite ring. Measurements of the proportion of this area occupied by tissue and of differential counts of intact cells and of alkaline phosphatase positive cells (see Results for criteria on cell typing) were carried out using a computerized digitizing system (Bab et al. 1985). The light point tracer of the digitizer was superimposed on the microscopical image via a drawing apparatus and quantification was performed at ×250 magnification. An average of 280 microscopical fields (34 mm2) was examined for each DC and the following parameters were calculated according to Weibel (1963): volume of tissue per DC and number of each cell type per DC.

The specific activity of alkaline phosphatase was expressed as the ratio between the total activity per DC estimated biochemically, and the number of alkaline phosphatase positive cells in the paired DC.

Histology

At day 0 the cell population in DC inoculated with marrow cells (marrow DC) contains an abundance of rounded haemopoietic cells, 7–25 μm in diameter with a regular perimeter, together with occasional adipocytes distinguishable by the large lipid vacuole in their cytoplasm. The haemopoietic and fat cells occur singly or in small clusters (Fig. 2B).

Fig. 2.

Changes with time after implantation in the cellular composition in marrow DC inoculated with 107 marrow cells. A. Stromal (• —•), haemopoietic (▵ — —A),adipose (○ — —), alkaline phosphatase positive cells (▴ — —). Each point is the mean ± S.E.M. of four DC. B-F. Photomicrographs of sections from DC harvested at 0, 3, 8, 12 and 20 days, respectively, after implantation. B,C,E. Stained with haematoxylin and eosin (H & E); × 187. D. Stained with H & E; ×120. F. Stained with Toluidine Blue and Von Kossa; × 187. Membrane filter (mf), adipocyte (ad), phagocytic giant cell (ph), osteoblast (ob), cartilage (cr), fibrous tissue (ft), mineral deposits (mn), haemopoietic cells (double arrows), stromal cells (single arrows), degenerating haemopoietic cells (arrowheads).

Fig. 2.

Changes with time after implantation in the cellular composition in marrow DC inoculated with 107 marrow cells. A. Stromal (• —•), haemopoietic (▵ — —A),adipose (○ — —), alkaline phosphatase positive cells (▴ — —). Each point is the mean ± S.E.M. of four DC. B-F. Photomicrographs of sections from DC harvested at 0, 3, 8, 12 and 20 days, respectively, after implantation. B,C,E. Stained with haematoxylin and eosin (H & E); × 187. D. Stained with H & E; ×120. F. Stained with Toluidine Blue and Von Kossa; × 187. Membrane filter (mf), adipocyte (ad), phagocytic giant cell (ph), osteoblast (ob), cartilage (cr), fibrous tissue (ft), mineral deposits (mn), haemopoietic cells (double arrows), stromal cells (single arrows), degenerating haemopoietic cells (arrowheads).

At 3 days haemopoietic and fat cells still predominate, distributed mainly close to the Millipore membrane filters. Many of the haemopoietic cells are in different stages of degeneration. Giant cells with phagocytosed nuclear elements within their cytoplasm are also present (Fig. 2C). The central portion of the DC is occupied by a loose fibrin clot.

At 8 days the occurrence of stromal cells other than adipocytes is first noted mainly in proximity to the membrane filters, admixed with many intact or degenerating haemopoietic cells (Fig. 2D). These stromal cells can be distinguished from haemopoietic cells by their shape and nuclear morphology. They have either elongated, often fusiform fibroblastic morphology or are polygonal with at least one angle along their perimeter that enables them to be distinguished from rounded haemopoietic cells of a similar size (Fig. 2D). A large regular ovoid nucleus with prominent nucleoli is also a consistent feature of these cells.

The majority of cells in DC at 12 days are stromal with many polygonal forms (Fig. 2E, top) and appear slightly larger than those seen at 8 days (Fig. 2D). In addition, at 12 days many stromal cells reside within large rounded lacunae of slightly irregular shape in a fibrous matrix (Fig. 2E, bottom). Staining the tissue in DC at 12 days for alkaline phosphatase activity reveals scattered foci of these lacunar cells with the reaction product localized at their plasma membrane (Fig. 3B).

Fig. 3.

A. Changes with time after implantation in alkaline phosphatase activity in marrow DC (• — •) and control DC (○ — ○). Each point is the mean ± S.E.M. of four DC. B,C. Photomicrographs of sections from marrow DC. B, 12 days; ×80; C, 20 days; ×190. Stained for alkaline phosphatase activity and with Von Kossa. Membrane filter (mf), mineral deposits (mn), alkaline phosphatase positive cells (arrows). P< 0 ·014; **,P< 0 ·001.

Fig. 3.

A. Changes with time after implantation in alkaline phosphatase activity in marrow DC (• — •) and control DC (○ — ○). Each point is the mean ± S.E.M. of four DC. B,C. Photomicrographs of sections from marrow DC. B, 12 days; ×80; C, 20 days; ×190. Stained for alkaline phosphatase activity and with Von Kossa. Membrane filter (mf), mineral deposits (mn), alkaline phosphatase positive cells (arrows). P< 0 ·014; **,P< 0 ·001.

Fig. 4.

Photomicrograph of section from marrow DC at 20 days. Stained with Toluidine Blue and Von Kossa; ×160. Membrane filter (mf), fibrous tissue (ft), mineral deposits (mn), cartilage (cr), adipocytes (ad).

Fig. 4.

Photomicrograph of section from marrow DC at 20 days. Stained with Toluidine Blue and Von Kossa; ×160. Membrane filter (mf), fibrous tissue (ft), mineral deposits (mn), cartilage (cr), adipocytes (ad).

At 20 days DC contain a mixture of bone, cartilage and fibrous tissues as has been described in detail (Ashton et al. 1980; Bab et al. 1984b). Initially, fibrous tissue is formed adjacent to the membrane filters and then extends into the interior of the chamber. Bone and cartilage develop within this fibrous anlage, cartilage mainly towards the centre of the chamber and bone nearer to the filter (Figs 2F, 3C, 4). With Toluidine Blue fibrous tissue stains weakly and cartilage strongly metachromatically (Fig. 2F). Mineral (Von Kossa positive deposits) is seen at 20 days in regions of developing bone in proximity to the cartilage (Figs 2F, 4). An osteoblast-preosteoblast layer of cells with high alkaline phosphatase activity is consistently found at the mineralizing front organized in short palisades; in some areas it is close to the membrane filter (Fig. 3C) and in others fibrous tissue intervenes (Fig. 2F). Small clusters of adipocytes can be seen in all chambers examined (Figs 2B,D, 4).

In DC inoculated with spleen cells (control DC) from 8 days onwards a fibrous tissue with some haemopoietic cells included in it is found (Fig. 5). This tissue fails to stain positively for either alkaline phosphatase activity or with Von Kossa.

Fig. 5.

Photomicrograph of section from control DC at 20 days. Stained with H & E; X 160. Membrane filter (mf), fibrous tissue (ft), haemopoietic cell (arrow).

Fig. 5.

Photomicrograph of section from control DC at 20 days. Stained with H & E; X 160. Membrane filter (mf), fibrous tissue (ft), haemopoietic cell (arrow).

Morphometry and cells counts

In marrow DC the volume of tissue, consisting of cells and extracellular substance, shows a highly significant linear increase (r = 0 ·77; P< 0 ·01) throughout the 20-day experiment (Fig. 6). During this period there is a marked change in the cellular composition of the tissue (Fig. 2A). Counts of stromal cells are for stromal cells excluding mature adipocytes; the latter are counted separately. Adipocytic precursors, which are indistinguishable from other stromal precursors by the present techniques, are included in the stromal cell count. During the first 3 days the total number of cells decreases to about 0 ·2% of the inoculated levels. Approximately 90% of those surviving are haemopoietic cells, with somewhat less than 10% being adipocytic. The average number of stromal cells identified at day 3 in the four chambers examined is 15 per DC (Fig. 2A), ranging from zero to 58.

Fig. 6.

Tissue volume (cells + extracellular substance) in marrow DC plotted against time after implantation. Each point represents the mean±S.E.M. of 4 DC. r = 0 ·77; P<0 ·01.

Fig. 6.

Tissue volume (cells + extracellular substance) in marrow DC plotted against time after implantation. Each point represents the mean±S.E.M. of 4 DC. r = 0 ·77; P<0 ·01.

From 3 days onwards the stromal cells show exponential growth with a 6 log increase between day 3 and day 20. Over the same period there is a 1 log decrease in the number of haemopoietic cells and a 1 log increase in the number of adipocytes (Fig. 2A). The rate of stromal cell proliferation is very high between 3 and 8 days with a doubling time of 14 ·5 h. Then the doubling time increases to 63 ·6 and 112 ·7 h for the 8-to 12- and 12-to 20-day periods, respectively (Table 1). The number of cells added per day to the stromal cell population in the DC increases throughout the 20-day period (Table 1).

Table 1.

Doubling time and growth rate of stromal cells in marrow DC during different periods of implantation

Doubling time and growth rate of stromal cells in marrow DC during different periods of implantation
Doubling time and growth rate of stromal cells in marrow DC during different periods of implantation

In control DC stromal cells grow rapidly and have reached 90% of their maximum number by 8 days of incubation.

Alkaline phosphatase activity

The total alkaline phosphatase activity measured in marrow DC on days 0, 3 and 8 is similar to that recorded in control DC (Fig. 3A). There are no alkaline phosphatase positive cells in the paired DC taken for histological examination. The number of alkaline phosphatase positive cells and the total enzyme activity rises from day 8 onwards (Figs 2A, 3A). The two parameters approximately parallel each other up to 20 days (Figs 2A, 3A) so the specific activity of the enzyme does not change from 12 to 20 days (Table 2). The percentage of stromal cells positive for the enzyme also increases rapidly during this period and constitutes more than 20% of the stromal cell population by 20 days (Table 2).

Table 2.

Alkaline phosphatase activity (AlPase act.) in marrow DC after different periods of implantation

Alkaline phosphatase activity (AlPase act.) in marrow DC after different periods of implantation
Alkaline phosphatase activity (AlPase act.) in marrow DC after different periods of implantation

Calcium content

The total calcium content of marrow DC shows a significant increase over control DC only at the 20-day time point (Fig. 7). At this time Von Kossa positive areas are first seen in the histological preparations. There is a wide variability in the calcium content of the four DC measured, which is not unexpected at this early stage of mineralization.

Fig. 7.

Ca content in marrow DC (• — •) and control DC (○ — ○) plotted against time after implantation. Each point represents the mean ±S.E.M. of 4 DC. *, P< 0 ·014; * *.PC0 ·001.

Fig. 7.

Ca content in marrow DC (• — •) and control DC (○ — ○) plotted against time after implantation. Each point represents the mean ±S.E.M. of 4 DC. *, P< 0 ·014; * *.PC0 ·001.

The formation of bone, cartilage and fibrous tissue, the increase in alkaline phosphatase activity and accumulation of calcium within diffusion chambers inoculated with rabbit marrow cells are in agreement with previous results where rabbit hosts were used (Ashton et al. 1980, 1984; Budenz & Bernard, 1980; Bab et al. 1984a). The use of athymic mice as hosts was based on preliminary experiments that indicated better chamber-to-chamber reproducibility in these animals compared to rabbits. This facilitated the present morphometric study where cellular changes prior to and during osteogenesis are reported. Direct evidence has been obtained that the fibrous—osteogenic tissues formed within the chambers are generated by a small number of stromal cells. Implantation of diffusion chambers containing marrow cells therefore provides a unique system for studying the differentiation of these tissues in the adult from early cellular precursors.

There has been no previous study of the kinetics of stromal cell differentiation in marrow DC culture, whereas there have been many studies (Steinberg & Robinson, 1985) of the kinetics of haemopoietic cells. The latter cannot be compared directly with the present work for several reasons. Haemopoietic cells recovered from the chambers were counted and identified morphologically after they had been brought into suspension by limited proteolysis. Under these conditions it is unlikely that stromal cells would be identified. The morphometric methods used here enable necessary additional factors such as cell shape and extracellular matrix characteristics to be invoked in the recognition of different cell populations and tissue differentiation. In many studies of haemopoiesis (Steinberg & Robinson, 1985) the host animals were subjected to irradiation or other conditions that promote survival of haemopoietic stem cells. Furthermore, the cell inoculum used here (107 cells DC−1) is at least 10-fold higher than in studies of haemopoiesis. This may be important since, in a previous study in rabbits (Willemze et al. 1978), the number of haemopoietic cells harvested decreased as the inoculum was increased up to 106 cells per DC.

In the present work there is a marked decrease in haemopoietic cell number between implantation and day 3 as was also generally found in studies of haemopoietic differentiation in DC. In rats and mice, however, there was then exponential growth of the surviving haemopoietic cells (Benestad, 1972; Steinberg & Robinson, 1985), whereas in rabbits (the present study; and Willemze et al. 1978) the initial decrease is followed first by a modest increase in haemopoietic numbers to day 8, and then a steady decrease to day 20. Over the same period, from 3 to 20 days, there is a tremendous growth of the stromal population in our experiments. This is reminiscent of what happened following transplantation of intact marrow tissue under the renal capsule, where death of haemopoietic cells occurred in conjunction with the survival and differentiation of stromal cells into bone and marrow stroma (Tavassoli & Crosby, 1968; Friedenstein et al. 1978). The diminution of the haemopoietic component may account for the absence of osteoclastic resorption of the osteogenic tissue in the DC. It is widely accepted that osteoclasts are a differentiated form of a haemopoietic cell line (Chambers, 1985; Marks, 1983) and their absence has been considered a major advantage of the DC method as a system for studying osteogenesis per se (Ashton et al. 1980; Budenz & Bernard, 1980).

Marrow adipocytes are a cell line of the stromal system and are, apparently, a specialized form of fat cells with different hormonal responses to adipocytes from extramedullary tissues (Green & Meuth, 1974). The majority of mature adipocytes are probably injected with the initial inoculum and their number underwent only slight changes in the DC compared to other stromal cells. A close morphological relationship between mature adipocytes and the mineralized tissue was often seen and deserves further study.

The data obtained herein demonstrate the presence of a population of stromal cells in post-natal marrow with a high potential for proliferation and differentiation. The number of cells with stromal-fibroblastic morphology identified at day 3 in DC ranged from zero to 58, and suggests that a small number of cells with stem cell characteristics generates the mixture of fibrous and osteogenic tissues in the chambers. The doubling time, initially 14 ·5 h between days 3 and 8, increased during the subsequent weeks in culture in vivo, probably as a consequence of increasing cellular differentiation. Given stromal numbers of approximately 50 cells at day 3, some 13–14 population doublings occur between the onset of stromal cell proliferation and the initial appearance of alkaline phosphatase positive cells between days 8 and 12.

It is not possible to decide from the available evidence whether the cells must go through a set number of divisions before alkaline phosphatase is acquired as a differentiated cell product, or whether the expression of this cell surface marker is secondary to other factors such as the local cell density. In vitro, the alkaline phosphatase activity of osteoblast-like cells (MC 3T3-E1; Sudo et al. 1983; ROS 18/2.8; Majeska & Rodan, 1982) increases once the cells have reached confluence,although a lower level of enzyme activity was expressed in the proliferative phase (Rodan & Rodan, 1984). In vivo thymidine-labelling studies have shown that the alkaline phosphatase-rich preosteoblast population in bone is a proliferating population (Owen, 1971), and epiphyseal chondrocytes in the proliferative zone of the growth plate display the enzyme on the cell membrane. There is no evidence on the proliferative capacity of the alkaline phosphatase positive cells in the marrow diffusion chamber system. However, the continued increase in the stromal cell population in the second and third weeks in DC culture indicates the persistence of a proliferating cell pool during the differentiation phase.

When rabbit marrow cell suspensions are cultured in vitro the number of fibroblastic colony-forming cells (FCFC or CFU-F) per number of cells inoculated (colony-forming efficiency) is about 30 per 107 marrow cells (Eaglesom et al. 1980; Ashton et al. 1984). This is of the same order of magnitude as the number of stromal cells found in DC at 3 days. Although the methods used can give only a crude estimate of the number of early stromal precursors, this result suggests that colonyforming efficiencies for CFU-F cells in vitro and in DC in vivo are similar.

Circumstantial evidence supports the current hypothesis of a stromal cell system with stromal stem cells able to generate several cell lines present in bone marrow (Friedenstein, 1980; Owen, 1980, 1985). The data in this paper are consistent with the hypothesis, but conclusive proof of it must await the results of further experiments.

Support from The Wellcome Trust, The Royal Society and The British Council for visits to the MRC Bone Research Laboratory by I. Bab in the course of this study, is gratefully acknowledged.

Part of this study was presented as partial fulfilment of the requirement for a DMD degree at the Hebrew University of Jerusalem.

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