As the treatment of patients with periodontitis becomes more and more sophisticated, the goals of clinicians consistently reach higher for the ideal outcome, including regeneration of the periodontium and successful placement of osseointegrated endosseous implants to replace missing teeth. Numerous factors may play a role in the healing potential of the type of periodontal wounds, including microbial, immune reactions, systemic disease and possibly patient age. For this reason, this study was initiated to examine the most basic common denominator of bone wound healing; the presence of stem cells, and to quantitate the presence of stem cells and their potential in contributing to osteogenesis. It is of course difficult to correlate this In Vitro animal study to human patients, but the basic tenets of the cellular aspects of wound healing and aging are certainly the same in human subjects.
The same number of light density cells, or cells containing osteogenic and hemopoietic stem cells were plated for each age group in this study. In young bone marrow, one stem cell gives rise to each colony, and there are approximately 1.6 osteogenic stem cells per 1 x 10 6 light density cells plated [Tibone (1981) and Shih (1991)]. In the current study, we found 1.2 stem cells per one million cells plated, not a statistically significant difference. Each stem cell then proliferates to give rise to osteoprogenitors and osteoblasts.
During the course of laboratory procedures it was dually noted that less stem cells, or light density gradient could be obtained from aged animals. Additionally, it was noted during the data collection for this study that the volume of fatty marrow present after primary centrifugation in the aged animals was markedly increased as compared to the young animals. Increase in the fatty component of tissue is associated with aging, and may be due to entrapment of cholesterol esters in the extracellular compartments of connective tissues [Shock (1962)]. It has been reported that cells of the fatty marrow can be converted to stem cells under certain circumstances. For this reason it was decided to combine the fatty marrow with the red marrow of both age groups to investigate whether there were stem cells present in the fatty marrow, particularly of the aged animals; which would produce an increase in the number of osteogenic colonies or hemopoietic colonies for that matter. It was considered that there is less cellularity in aged individuals that parallels an increase in fats and a decrease in hydration, however, when the body is steeled, such as in bone fracture or inflammatory states, there must be a source of stem cells to provide healing capacity. For this reason, there might have been a possibility that the fatty marrow, which is usually discarded in the density gradient separation, might have harbored stem cells which would express osteogenic capability in tissue culture. The following is a discussion of the various segments of this investigation.
II. Osteogenic and Hemopoietic Colonies Produced Between Aged and Young Animals from Red Bone Marrow
Young animals produced 3.6 +/- 2.3523 osteogenic colonies as compared to 0.45 +/- 0.6863 osteogenic colonies formed from aged animals per 3 million light density cells plated and grown in tissue culture for 14 days. These figures are statistically significant as calculated by the Student's t Test P< 0.000. This finding is consistent with what has previously been reported in the literature regarding the osteogenic stem cell potential of young animals. Tonna in 1959, reported on a study investigating the mean number of osteoblasts per unit area estimated from midfemoral regions of 60 male and female rats of different ages. A ten fold decrease was noted in both male and female rats between the ages of 8 and 104 weeks. These findings, as far as size and number of osteogenic colonies are congruent with the findings of Shih (1990) and Shayegsteh (1992), who utilized the same tissue culture protocol to test the effects of various growth peptides on the osteogenic stem cells of 55 day old male Sprague-Dawley rats. Also Tsuji (1990) reported on an In Vitro study which compared the difference of stem cells harvested from immature and aged male rats, that there was a three fold difference in the number of osteogenic colonies grown in culture from the immature animals as compared to the aged animals.
In addition it was noted that colonies did not differ significantly in size as measured In Situ parallel to the bottom of the Petri dish between age groups. Colonies from young animals measured 0.1276 +/- 0.0518 mm 2 as opposed to aged colonies which measured 0.1250 +/- 0.1033 mm 2 . These findings infer that once a committed stem cell begins its journey of differentiation, there is no difference in the rate at which it proliferates. Tsuji (1990) examined the doubling times of aged and immature stem cell cultures and did not report any statistically significant difference between doubling times and age groups.
The osteogenic colonies were observed In Situ after staining with Von Kossa silver stain for complexed calcium, and it was noted that the colonies derived from the young animals consistently stained much darker and denser than those from the aged animals, indicating the elevated presence of calcified matrix. Figure 11, Figure 12, Figure 13, and Figure 14 These findings can be explained due to the mechanisms by which cells from different age groups divide and behave, as well as the activity of protein making machinery. Tonna (1959), quantitated the mean number of mitochondria of the periosteal osteoblasts of femurs of rats of different ages, and found a decline of 37.1 +/- 2.4 mitochondria per osteoblast at 5 weeks to 16.8 +/-0.9 at 8 weeks to 3.3 +/- 0.5 at 104 weeks. In addition, he noted a decline in the surface volume of mitochondria with age in periosteal osteoblasts. Adenosine triphosphatase activity also followed a declining pattern in these cells, Tonna (1971). Tsuji (1990) reported a decrease in the alkaline phosphatase activity of aged rat stem cells in tissue culture. These reports help to underscore the findings of decreased calcified tissue matrix in the osteogenic colonies derived from our aged animals. There is an apparent decrease in the protein making ability of the stem cells derived form aged animals.
The effects of time and aging can be best appreciated in response to stress, such as fractures. With H3-thymidine labeling studies, Tonna (1961) showed reactivation of cell proliferation about 10 hours after fracture. A peak labeling index was reached at about 24 hours in a young mouse. Away from the fracture site, the labeling indices returned to normal by about 4 to 5 days. At the fracture site, labeling indices decreased significantly until an elevated plateau was reached, also by about 5 days. This level was maintained until repair was complete. In aged animals, cell proliferation was also initiated at about 10 hours post trauma, but from a somewhat lower baseline. A peak was reached by about day 2, after which time the labeling indices of the areas away from the fracture site returned to normal by day 4. At the fracture site, activation of cell proliferation continued to a new peak at 4 days. The author postulated that decreased cell numbers and increased bone surface size, together with osteocyte formation depleted periosteal cells with time. Consequently, a suitable population size adequate for repair was not achieved until later in aged animals. This peak was however, followed by a decrease in labeling indices to an elevated plateau which was gradually reduced as repair was completed, in a similar manner as occurred in young animals. The author concluded that aging did not change the biological qualitative response of bone cells; only the quantitative response was effected. The animal was therefore assured of effective skeletal repair throughout its lifespan via an emergency mechanism wherein the cell proliferation potential could be reactivated. The proliferative potential is not lost with aging; only the proliferative activity is considerably diminished. Respiratory enzyme studies in rats, however, show progressively diminished levels of enzyme activity when examined 1 to 2 weeks post trauma. This is not the case with acid and alkaline phosphatases, in that enzyme activity is increased following trauma at all ages. Furthermore, the depleted cell population is replenished and is responsible for fracture repair, not the original old population. H3-proline autoradiographic studies reveal that in old mice, the metabolic activity of newly formed cells is delayed one week. However, in time they reveal the capability of contributing to repair in measure equal to or exceeding that of younger animal cells [Tonna (1959)].
The hemopoietic colonies in this system differed statistically between age groups in numbers, but not in size. Young animals produced 8.23 +/- 3.3601 colonies per dish and aged animals produced 5.25 +/- 2.2449 colonies per dish which was statistically significant by computation with the Student's t Test P < 0.002. In the past, there have been numerous reports to indicate that there is a decline in erythropoiesis as mammals age [Fand et al (1957) and Reigle et al (1966)]. There are also reports that indicate an increase in granulocytes with age [Fand (1957)]. Our results can be explained with the increase in hemopoietic colonies for the red marrow only, of the young adults over the aged animals in that there were apparently more erythropoietic colonies present than with the aged animals. These results become more meaningful, and the dynamic aspect of the cells harvested when one examines the results of adding the fatty marrow to the light density layer of both age groups.
The marrow harvested from young animals produced hemopoietic colonies sized 0.06010 +/- 0.0172 mm 2 and bone marrow harvested from aged animals produced colonies sized 0.07591 +/- 0.0514, which was not statistically significant. The lack of size difference between age groups can be similarly explained as the lack of size difference between osteogenic colonies. Once the stem cell becomes committed to differentiation, there is no difference in the rate at which proliferation takes place. These colonies are slightly smaller than the osteogenic colonies and that is a reasonable observation, as there is little extracellular matrix in hemopoietic colonies, only a cellular aspect.
III. Osteogenic and Hemopoietic Colonies Derived From Young and Aged Animals from Stem Cells Harvested From Red Marrow with the Addition of Fatty Marrow
The addition of the fatty marrow did not statistically effect the numbers or sizes of osteogenic colonies formed between those formed from red marrow only. However, there was still a statistically significant increase in the number of colonies formed between marrow harvested from young and aged animals. Young animals produced 3.8333 +/- 1.4720 colonies which was a statistically significant increase by the Student's t Test, P < 0.002 over colonies produced from marrow harvested from aged animals. Aged animals produced 1.125+/-1.1260 colonies per Petri dish. This was not statistically different from the number of colonies produced from the red marrow only of aged animals. Osteogenic colonies produced from young animals measured 0.1285 +/- 0.0285 mm2 and osteogenic colonies produced from aged animals measured 0.08803 +/- 0.0694 mm2, which was not statistically different from the size of young colonies without fatty marrow added. The addition of the fatty marrow therefore had no statistically significant effect, either in size or number of osteogenic colonies formed between the two age groups. In the aged animals, we conclude, there were no stem cells harbored in the fatty marrow which expressed their osteogenic potential when plated in tissue culture.
There was an increase in the number of hemopoietic colonies produced between aged red marrow and aged red marrow with fatty marrow added. This difference was also statistically significant when compared to the number of Hemopoietic colonies formed from young animals, with or without fatty marrow added. Colonies derived from aged animals with fatty marrow added numbered 19.62 +/- 6.7387, whereas colonies derived from young animals numbered 6.5 +/-1.8708. This was a statistically significant increase between aged and young in the number of hemopoietic colonies formed as tabulated by the Student's t Test of P < 0.002. The difference for hemopoietic colonies formed between aged red marrow and aged red marrow with fatty marrow added was statistically significant as calculated by the Student's t Test of P < 0.000.
Previous studies have quantitated the number of colony forming units of hemopoietic cells from the bone marrow of mice. In Guinea Pigs, it has been shown that there is a decrease in erythrocytes with age, and an increase in granulocytes [Fand (1957)] These studies used whole bone marrow and not red marrow only, this explains why our results of red marrow only in the aged animals yielded fewer hemopoietic colonies. As has been mentioned previously, there is an increase in the fat content, particularly of connective tissue extracellular spaces. Hemopoietic cells are found in the light density band after Ficoll-Paque gradient separation and most likely remain in the fatty marrow after the primary centrifugation. When the fatty marrow is added to the light density gradient cells for inclusion in the tissue culture, the hemopoietic stem cells differentiate and form an increased number of colonies as compared to the red marrow only. After the initial centrifugation, the marrow harvested from the young animals normally has very little fatty marrow present, therefore there is little impact upon adding the fatty marrow of the young animals to the tissue culture.
In summary then, this study reports upon the behavior of red bone marrow and red bone marrow with the addition of fatty marrow stem cells harvested from young adult and aged Sprague-Dawley rats In Vitro.
The results indicate that there is a statistically significant increase in the number of osteogenic colonies, and therefore stem cells in young adult male rats, regardless of the addition of fatty marrow, which might possibly harbor osteogenic stem cells. This study therefore concludes that no statistically significant population of osteogenic stem cells are present in the fatty marrow portion In addition, the results indicate that at fourteen days in tissue culture, there is less calcified matrix present in the osteogenic colonies formed from the stem cells harvested from the aged animals. These results are in concert with previous reports of decreased cellular machinery in aged animals.
The hemopoietic colonies examined in our study indicated a statistically significant increase in the number of colonies formed between young and aged with red marrow only, no statistically significant difference between the numbers of hemopoietic colonies formed for young animals with the addition of fatty marrow and no significant difference in colony size. With the addition of fatty marrow to the aged animal light density cells there was a significant increase of hemopoietic colonies over both young groups and red marrow only aged colonies. This finding is in congruence with other reports.
Some final considerations include: Aged humans may show various effects upon their bones as a result of their age, including as this study indicates, a decrease in the number and metabolic activity of osteogenic stem cells. Elderly humans, especially postmenopausal females lose bone density, have an increase in bone brittleness, have delayed bone wound healing and increased inflammatory reactions. Fractures due to successful implant placement in highly resorbed, poorly vascularized bone may end in non-healing fractures with disastrous results. Not enough information has been accrued besides case reports and highly biased industry-driven studies of short duration to assess how important age may play a role in the placement of these fixtures. It is important for the future study of the role of long term success and healing expectations, both in young and elderly human patients to have a reproducible model with which to study the effects of various cellular healing aspects as they relate to clinical subjects such as the relationship of cellular activity to various substrates and the addition of growth factors. By utilizing a reproducible, In Vitro animal system, basic biologic tenets can be enumerated before moving on to clinical animal models and finally human studies.