Cell Suspension Comparisons of Bone Production in Bone Marrow Stem Cells of Young and Adult Aged Rats as Measured In Vitro by Bone Colony Assays

Stephanie A. Dodson

1. Abstract
2. Introduction
3. Chapter One
4. Chapter Two
5. Chapter Three
6. Chapter Four
7. References

CHAPTER TWO

Introduction

Materials and Methods

Adult male aged and young Sprague-Dawley rats (Simonsen, Zivec Miller) weighing 230-250g and 500-550g respectively, were used as the source of bone marrow cells in this study. The young group of animals consisted of 55 day old males, which are considered to be sexually mature at this time point. The aged animals were 18 to 22 months old. Figure 3 and Figure 4 The life expectancy of the Sprague-Dawley rat is an average of 24 months. During the bone marrow harvesting surgery, the animals were kept alive under general anesthesia until all bone marrow cells had been harvested.

During bone marrow harvesting surgery, each rat was anesthetized with 0.2 ml of sodium nembutal injection solution (Abbott Laboratories, North Chicago, IL 60064, USA) intraperiotoneally. The animals were shaved form the forelegs down to the tail with a surgical #40 Oster clipper blade. After shaving, each animal was cutaneously prepped with an iodophore scrub (Betadine). A skin incision was made over each rear leg and hip, and the entire skin covering the lower half of the animal was removed. After the removal of the skin, a second set of sterile surgical instruments was used to separate the muscles of the thigh and leg and to expose the whole length of the femur and tibia. Then the instruments are used to dissect the femur and tibia/fibula complex of each rear leg. The periosteum was scraped from the bones, and both femurs and tibiae were excised. Figure 5 Bone marrow was then flushed gently from these tubular bones, with a 21 gauge needle containing 3-5 ml BGJb medium (Gibco Laboratories, Life Technologies, Inc., Grand Island, New York 14073, USA). Figure 6 The bone marrow cells were collected into a 100 x 15 mm glass Petri dish (Fisher Scientific, 2170 Martin Avenue, Santa Clara, CA 95050, USA) with 10 ml BGJb medium which contained 0.4 ml heparin, supplemented with 15% fetal calf serum, 0.1% (v/v) of a 29.2 mg/ml solution of L-glutamine, 0.1% (v/v) of a 10,000 u/ml solution of penicillin-streptomycin and 0.1% (v/v) of a 250 ug/ml solution of Fungizone (Gibco Laboratories, Grand Island, NY 14072).

The harvested whole marrow cells were gently pipetted to break up cell clumps, then centrifuged at 2375 rpm for 30 minutes. Following centrifugation, the fat and serum layers were discarded. The buffy coat, which represents all the white cells of the marrow, along with the upper portion of the red cell layer, was carefully pipetted into a test tube containing Ficoll-Paque. The remainder of the red cell layer and the bone spicules were discarded. The resulting cell suspension was composed of virtually all the original marrow white cells and about 10% bone marrow red cells. Figure 7

The whole bone marrow suspension was divided into two sub-populations by density gradient separation using a mixture of Ficoll and Hypaque. A tube with Ficoll-Paque (bottom layer), buffy coat white cells (middle layer), and tissue culture medium (top layer) was centrifuged at 2375 rpm for 30 minutes. The serum/medium layers were discarded and the band of light density (LD) white cells are collected. The layer of fat was carefully pipetted and collected into an additional test tube to be added to light cell gradient prior to counting and plating. These cells, which consist of monocytes, lymphocytes, blast cells and stem cells, comprised about 15% of the white cells of the whole marrow. The collected LD white cells were washed with Hanks¹ balanced salt solution, centrifuged at 2375 rpm for 10 minutes twice and resuspended in BGJb medium. The remaining layer of Ficoll-Paque was discarded, leaving a pellet of granulocytes, macrophages, and fibroblastic cells on the top of the remaining red blood cells. The top one-third of this pellet (all the remaining white cells and some of the red cells) was washed and resuspended in BGJb medium and becomes the pellet suspension of heavy density (HD) cells. Figure 8

The pellet or HD suspension, which consisted of granulocytes, macrophages and fibroblastic cells, was used to develop the feeder layer of fibroblasts. Approximately 5 x 10 6 heavy density pooled white cells obtained from rats are plated into 60 x 15 mm Petri dishes for development of the feeder layer. The number of white cells placed in each dish was counted by mixing a small aliquot of the cell suspension with an equal volume of a 0.5% trypan blue stain, and counting the non-stained viable cells in a hemocytometer. The fibroblast feeder layer grew to semiconfluency in 10 to 14 days. At the time when the feeder layer of fibroblasts were well developed, light density pooled white cells were obtained, pipetted and plated into each 60 mm Petri dish (3 x 10 6 LD white cells/Petri dish). The cells in Petri dishes were cultured in a 95% humidified atmosphere of 95% air and 5% CO2 at 37 C in a Forma 3028 incubator.

The medium was not changed for 2-4 days after adding LD white cells to the feeder layer of fibroblasts in order that the cells could attach to the feeder layer. Two to four days after adding LD white cells, 5ml of BGJb medium was added to the Petri dishes. The light density cells containing the bone marrow stem cells were allowed to grow for 14 days. On the fourteenth day of culture, the cells were fixed and prepared for assessment. Figure 10

Monolayer Fixation, Staining and Embedment

On the fourteenth day of culture, the BGJb medium was drained from the dishes for the last time. Each dish was fixed for 30 minutes with 2% gluteraldehyde in 0.1 M sodium cacodylate buffer solution. Then some of the dishes, in preparation for transmission electron microscopy were post fixed for one hour in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 5 minutes. Now the dishes were washed twice in 0.1 M Sodium cacodylate buffer for five minutes.

In Situ Von Kossa Staining consisted of placing 5% silver nitrate solution in each dish and setting the dishes under a UV lamp for ten minutes. The dishes were then rinsed copiously with phosphate buffer three times for two minutes each. Now a 5% solution of sodium thiosulfate was placed in each dish for 10 minutes. Dishes were rinsed with phosphate buffer and exposed to direct sunlight for 10 minutes in 0.1 M sodium cacodylate buffer.

Dehydration consisted of two changes, five minutes each of 70% alcohol, followed by two changes of five minutes each of 95% alcohol. Next came embedment, which consisted of two changes, 10 minutes each of one part absolute alcohol and 4 parts Glycolmethacrylate. this was followed by two changes each of pure GMA of 10 minutes each; two parts GMA to one part EPON (Pelco) for 30 minutes; 12 hours in the refrigerator; then one part GMA to two parts EPON for 30 minutes; one change of pure EPON for 30 minutes and embedment by adding a 5mm height of EPON to the Petri dish. The EPON was polymerized in an oven for 24 hours at 60C.

With low power light microscopy, the osteogenic colonies can be differentiated from hemopoietic colonies, because the former give a positive reaction to Von Kossa silver staining in areas of calcification as they form microscopic to macroscopic nodules of calcified bone within their centers. Sections taken for light microscopy were stained with Toluidine blue. The unsectioned dishes were examined and photographed with a Zeiss phase contrast inverted microscope while the slides with stained histologic sections were examined with an Olympic AH-2 microscope.

The dishes to by processed for TEM were post fixed in osmium tetroxide as described previously. The dishes were then broken away form the hardened EPON, and blocks were prepared so that sections could be cut either parallel or perpendicular to the bottom of the dish. The blocks were cut with glass knives on a Porter-Blum MT-1 ultramicrotome. Semi-thin sections (One micron) were cut and stained with aqueous toluidine blue for light microscopy, while thin sections, 600 Å - 800 Å were stained with uranyl acetate and lead citrate for electron microscopy. A Siemens 1A Elmskop Electron Microscope was used, operating at 80 kV.

To confirm that a positive Von Kossa reaction did in fact signify bone formation, positive areas were located using the phase contrast microscope. These positive colonies were sectioned and examined by light and TEM microscopy. Quantitative results were tabulated for both bone and hemopoietic colonies for the test and control dishes. The area for each colony, bone and hemopoietic was measured utilizing and IBM digital analyzer. Again, the numbers and size of colonies were statistically analyzed by the student's t test.

Results

The pellet suspension, which contained fibroblasts, macrophages and granulocytes, was placed into a 60mm Petri dish and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37C. After 5 days in culture, fibroblasts were attached to the bottom of the Petri dish. At days 12-14, an adherent layer which consisted of several cell types formed. The major component of this adherent feeder layer was fibroblasts. At day 14, light density bone marrow white cells were added onto the prepared feeder layer of fibroblasts.

Between 2 and 5 days in culture, stem cells which appeared to resemble medium to large lymphocytes, Yoffey, (1971); Dicke et al., (1973); Rosse, (1973); Budenz and Bernard, (1980) having a smooth nuclear profile,. a high nucleo-cytoplasmic ratio, a poorly differentiated cytoplasm and a smooth cell membrane, Murphy et al., (1971) and Allen, (1978) began to attach to the feeder layer and formed either hemopoietic or osteogenic colonies. After Von Kossa staining, the osteogenic colony was identified either by visual examination or under a Zeiss phase contrast light microscope.

The purpose of this trial was to investigate the difference in osteogenic colony forming potential between aged and young animals by single cell suspensions from red bone marrow with the addition of fatty marrow. In every trial, cell suspensions of marrow produced bone forming colonies, as well as hemopoietic colonies, on adherent layers of cells. These adherent layers and the two types of colonies were characterized using light microscopy, phase contrast light microscopy and transmission electron microscopy.

Morphologically identical adherent layers were formed using whole marrow suspensions of cells (see Materials and Methods for details). These adherent layers consisted of a complex multilayer of several cell types. The major component of the adherent layer was a fibroblastic cell. The fibroblastic cells formed multiple layers and showed the parallel orientation of fibroblasts grown in culture. Figure 9

A second component of the adherent layer was a large flattened cell labeled an epitheliod cell by Allen (1978), which has been described by Tibone (1981) to be another morphological expression of the fibroblastic cell. A third major cell type was the macrophage. This mononuclear cell has a well demarcated leading edge, a characteristic of motile cells.These three major adherent cell types are strikingly similar to those described by Dexter (1979) and Tibone (1981).

In all of the micrographs, many rounded smooth cells can be seen on or around the adherent cells. These cells represent granulocytes, lymphocytes, monocytes and stem cells in different stages of differentiation. Because no pure population of stem cells has been isolated, there are no absolute morphological criteria for their identification. However several groups of researchers report that stem cells appear to resemble medium to large lymphocytes [Dicke et al (1973), Rosse (1973) and Yoffey (1973)]. Stem cells are also reported as having a smooth nuclear profile, a thin rim of marginal condensed chromatin, a high nucleocytoplasmic ratio, undifferentiated cytoplasm and a smooth cell membrane [Murphy et al (1971) Allen (1978)].

Colonies could easily be identified in this In Vitro system, because they formed on top of the adherent layer. These young but as yet uncharacteristic colonies contained numerous cells closely associated with each other and piled up in a three-dimensional orientation.

Both granulocytic and erythrocytic colonies were identified in our bone marrow cultures. These colonies appeared very round under the phase contrast microscope. There was not a large size difference between the two age groups, however, with the addition of fatty marrow, there was a statistically significant increase in the number of hemopoietic colonies derived from the aged animals. Figure 15, Figure 17, and Figure 18

Osteogenic colonies, like hemopoietic colonies grew on top of the adherent layer. In a von Kossa stained In Situ colony the bone is represented by the intensely dark stained areas. Figure 11, Figure 12, Figure 13, and Figure 14 Many cells were included within the colony. Among these identifiable cells are fibroblastic cells on the surface, and giant fat cells on the interior. Cross sections were cut through plastic-embedded osteogenic colonies, both parallel to the growing surface and perpendicular to it. These sections were stained with toluidine blue, which stains bone purple. The cells, primarily osteoblasts are closely apposed to the bone surface and completely surround it in these small colonies. Figure 19 and Figure 23

~ P < 0.002
* P< 0.000:
Statistically Significant increase from red marrow only number of hemopoietic colonies for aged animals, P < 0.000. Increase of P < 0.002 over the number of colonies formed for young animals with the addition of fatty marrow.