Historical Perspective of Periodontal Progenitor Cells: Early Studies That Clarified Identity and Function
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Pioneering studies using cell kinetics, transplantation and wound healing models that were conducted in the 1960s–1980s provided critical insights into the proliferative and differentiation capacity of cell populations in the periodontium. These experiments showed that the periodontium, in general, and the periodontal ligament, in particular, comprise progenitor cell populations that can give rise to cells that synthesize bone, cementum and fibrous connective tissue. These insights ultimately led to the discovery of multipotent progenitors that are just now being studied to define their contributions to periodontal regeneration. Despite the limitations that were inherent in these earlier approaches, data from these experiments enabled major strides in exploiting the transformative potential of stem cell biology to provide improved outcomes in periodontal therapy.
KeywordsCell kinetics Stem cells Periodontal ligament Cell proliferation 3H-Tdr
Introduction: Why Are We Interested in Progenitor Cells of the Periodontium?
The PL and Its Central Role in Homeostasis and Wound Healing
It has been known for many years that the functions of the PL include proprioception, tooth support and attachment ; these latter functions are evidently provided by the principal collagen fibres of the PL, which undergo rapid turnover in health . Homeostasis of the PL and contiguous periodontal tissues is maintained by cells that include formative and resorptive cells for cementum, PL and bone. In contrast to the rapid collagen turnover in the PL , the turnover of cells is very slow . Under normal conditions of function, these cells are actively engaged in protein metabolism for PL homeostasis rather than in proliferation, but their exact role in regeneration was not known. Expert clinicians have provided evidence to show that under favourable circumstances, periodontal regeneration was possible , but the determinants of successful treatment at the cellular level were not defined. The recognition that cells of the PL play critical roles in periodontal regeneration  and may contribute to the formation of new cementum, bone and PL generated considerable interest in the proliferative and differentiation capacity of these cell populations.
In the 1960s, 1970s and 1980s, most of our understanding of the contribution of periodontal cell populations to regeneration came from morphological, wound healing and transplantation studies, as well as the clever use of cell kinetic methods to examine cell origins and fate. Insights from these experiments were instrumental in advancing our current understanding of progenitor cells in the periodontium and their contributions to periodontal regeneration. In the next section, research approaches used in the 1960s–1980s will be discussed in order to place the notion of progenitor and stem cells in a larger perspective.
Experimental Approaches for Investigating Progenitor Cells and Cell Turnover
The rate of turnover of cells varies considerably between different tissues: while no new cell production occurs in the neurons of the central nervous system, turnover of the epithelial cells in the small intestine can be as fast as 3 days . Cell population kinetic studies involve the clarification and quantification of proliferation, differentiation, migration and death of cells within a definable cell population . An essential requirement of cell proliferation is that each daughter cell should have exactly the same genome as the parent cell. For this reason, the mother cell’s DNA must be replicated prior to cell division. Howard and Pelc  discovered, by monitoring the uptake of radioactively labelled nucleotide precursors, that DNA is synthesized during a limited part of interphase and not during cell division. They proposed the concept of a cell cycle, during which the cell goes through recognizable phases between one mitosis and the next. The period of DNA synthesis was designated the S phase; the periods preceding and following DNA synthesis were denoted G1 and G2, respectively, and represent gaps in the proliferative cycle. G2 was followed by mitosis and, it in its turn, by G1 (or G0).
Charles LeBlond of McGill University in Montreal  described cell populations as “being groups of cells with a common origin and the same morphological and functional potential,” and he and his colleagues used the techniques of cell kinetics to group different mammalian tissues on the basis of their proliferative behaviour. Leblond and his colleagues  used two parameters to characterize cell populations: the rate at which cells incorporate 3H-Tdr into DNA and the rate at which labelled cells are lost from the tissue. Two tests were used to measure these parameters, a cell formation test and a cell retention test. In the cell formation test, rats and mice were injected with 3H-Tdr (ages of either 3 days or 6 months old) and killed within a few hours after injection. The fraction of labelled cells in a number of different tissues was determined by radioautography. In the cell retention test, animals were injected with 3H-Tdr when they were 3 days old and killed when they were 6 months old, and the fraction of labelled cells was determined as stated above. Tissues such as smooth muscle, cardiac muscle and neurons of the cerebrum exhibited no labelling in the cell formation test and minimal labelling in the cell retention test: these were termed static cell populations. They could not divide, but their precursors could divide and differentiate into end cells which were incapable of further mitoses. In the proximal convoluted tubules of the kidney, parenchymal cells of the liver and the epithelial cells of the thyroid gland, a small percentage of cells, were labelled in both the cell formation and cell retention tests so that there was both limited cell division and cell retention throughout the adult life of the animal. In addition, the daily increase in cell number was about equal to the daily mitotic rate , and therefore, new cells were retained in situ, thereby expanding the size of the cell population with age. These were expanding cell populations.
In the epithelial cells of the small intestine, the cortical cells of the thymus, bone marrow cells and the epithelial cells of the tongue, many cells were labelled in the cell formation test at 3 days and 6 months, but none was labelled in the cell retention test. These tissues contain cells that divide frequently and thereby dilute their label through successive cell divisions until it is not detectable. The high mitotic activity is balanced by cell loss as a result of migration or death, and the size of the cell population is maintained in a steady state. These cells comprise renewal populations.
However, there were three tissues that did not fit into these classifications. Dermal connective tissue, lung alveolar tissue and cells of the PL of continuously erupting incisors exhibited high labelling indices in the cell formation test and low labelling indices in the cell retention test. It was not expected that labelled cells would be renewed or lost from these tissues. Leblond  therefore concluded that “connective tissue cells may constitute a mixed cell population with very different proliferative potencies, or else local conditions determine whether or not connective tissue cells are to divide.” Cameron  subdivided renewal cell populations into rapidly and slowly renewing. On the basis of data from a continuous labelling experiment, he classified the PL as a slowly renewing tissue. Later data obtained from continuous 3H-Tdr labelling experiments showed that there is indeed a renewal cell population in PL, but there is also a static, non-dividing cell population that may be formed during PL development and that does not undergo renewal or turnover thereafter .
Concepts of Stem Cell, Transit Cell and Static Cell Populations
In certain renewal tissues such as the epithelium of the seminal vesicles, cells throughout the tissue have the capacity to divide . However, in most tissues with renewing cell populations, such as the stratified squamous epithelium of the oesophagus, cell division is restricted to the relatively undifferentiated cells of the basal cell layers . In the oesophagus, the migration of cells into the spinous and granular layers is accompanied by increasing differentiation and loss of proliferative potential. On the basis of these and other data, Leblond proposed that proliferation is restricted to relatively undifferentiated cells, which he denoted as stem cells while differentiation occurred in post-mitotic cells. By contrast, in haematopoiesis and spermatogenesis, cell replacement by mitosis was observed in differentiating cell compartments in which further differentiation and maturation may occur . In this model, the proliferative compartment is double (or multitiered) because it contains self-renewing cells and cells committed to terminal maturation that can undergo further rounds of cell division. These latter cells were denoted as transit cells. It was thought that if the burden of proliferation rests solely on self-renewing (stem) cells, large genetic pressures would compromise the integrity of the stem cell DNA because of the large number of replications that occur during the life of the organism.
In a multitier proliferative model, stem cells cycle slowly, but their transit cell progenies are responsible for the bulk of proliferation and do so by repeated cell divisions . Transit cells were thought to be derived from stem cells and would have a finite life span governed by loss of proliferative capacity and expulsion from the tissue . Using the criteria of proliferation alone, transit cells cannot be distinguished from stem cells, although it had been suggested that the proliferative rate of stem cells is low in blood-forming tissues  and epidermis . It was known that the turnover rates of these tissues cannot be accounted for solely on the basis of stem cell proliferation . Repeated division in the transit cell populations would account for the bulk of cell division in a multitier model of proliferation . The essential functional difference between stem cell and transit cell populations is the proliferative lifetime of the population . Stem cell function relies on self-renewal during the entire lifetime of the animal; otherwise, the tissue would die. Transit cell populations have a limited lifetime although, while proliferating, they have a tremendous amplification potential. Ten cell divisions in a population of transit cells could result in a 1000-fold increase in cell number .
Static cell populations are built up during an early stage of development of the organism, lose the ability to divide  and are usually present in slowly decaying numbers . The neurons of the central nervous system, muscle cells and oocytes are examples of such cells. They will not take up 3H-Tdr after a pulse injection because they do not proliferate. The important difference between static cells and cells of expanding cell populations is the ability to divide. Liver cells (which belong to expanding cell populations) do not divide rapidly under normal conditions but can be stimulated to divide after partial liver resection . Static cell populations are not capable of such a response to wounding, and this explains the irreversible damage to cells of the central nervous system after injury.
What Are Stem Cells?
Criteria for stem cells from the early 1980s
Be precursors for specialized cells
Be capable of self-renewal
Demonstrate regenerative or clonogenic capacity
Exhibit sparse distribution within the proliferative compartment
Respond to control mechanisms,
Have a long cell cycle time and hence a long G0 period,
Possess a mechanism for conservation of the integrity of DNA
Demonstrate a characteristic thymidine metabolism
Searching for Stem Cells
One of the early studies to examine the stem cell concept was the work of James Till and Ernest McCulloch at the Ontario Cancer Institute . They injected bone marrow suspensions into lethally irradiated mice and found that the mice could survive because of the following: (1) the repopulation of the haematopoietic system, (2) the clonogenic capacity of the hematopoietic stem cells in the suspension and (3) their extensive capacity for self-renewal. They extended this experimental approach with 3H-Tdr suicide experiments combined with a spleen colony assay system to show that the proliferative rates of colony-forming unit-stem cells (CFU-S) were considerably higher in foetal liver and spleen than adult marrow or spleen . These data suggested that the CFU-S were regulated by the rate of production of blood cells and that in the adult, when the steady state is obtained, few CFU-S are traversing the cell cycle. They remain in a resting or G0 part of the cell cycle. The same group  showed that a single, cytogenetically marked hematopoietic pluripotent stem cell can produce colonies of heterogeneous differentiated cells. These observations taken together suggest that the CFU-S fulfils most of the criteria for a putative stem cell as outlined earlier .
In epidermis, transplanted epidermal stem cells exhibit considerable capacity for regeneration and self-renewal and they can generate differentiated cells in irradiated sites deprived of epidermal stem cells . Epidermal stem cells have a slow turnover rate, a prolonged G0 and a rate of proliferation that is sensitive to external factors such as irradiation . It has been suggested that the nascent DNA of epithelial stem cells is not semi-conserved upon cell division. Instead, the newly synthesized DNA, which is more subject to errors of replication, is sequestered in one of the daughter cells . Indirect experimental evidence to substantiate the selective segregation of DNA in epithelial stem cells came from examination of radioautographs of 3H-Tdr-labelled mouse skin . Cells which occupied the position of stem cells were initially labelled and then passed their nascent labelled DNA to one of the daughter, migratory cells. The stem cells thereafter were unlabelled. These data are not consistent with other work showing that putative stem cells in the skin are labelled and cycle very slowly .
Cell Populations in the PL
Despite the many characteristics attributed to stem cells described above, these cells remain difficult to identify and separate in most mammalian tissues; the PL is no exception. Work conducted by Melcher and colleagues at the University of Toronto showed that (1) in both steady-state and wounding conditions, the regions around blood vessels in the PL (paravascular sites) were enriched with progenitor cells [33, 34]; (2) these progenitors could give rise to fibroblasts, cementoblasts and osteoblasts; (3) the PL was not a closed cell system, but instead, cells from adjacent endosteal spaces migrated into the PL and there contribute to the progenitor cell populations that could synthesize bone, cementum and PL .
Experimental Challenges of Identifying Progenitor Cells
It is difficult to discriminate fibroblasts from macrophages in the light microscope . Thin plastic sections have been used to improve the accuracy of cell identification and so partially overcome this difficulty . In another approach to improve cell identification , animals were irradiated to lower the number of circulating monocytes and, correspondingly, the number of labelled macrophages that could be identified as fibroblasts and, second, identified labelled cells by their ultrastructure. Further, when animal models employing sacrifice times greater than 1 day are used, it is uncertain whether all 3H-Tdr-labelled cells are derived from cells initially resident in the PL.
Another general problem with application of cell kinetic methods in the PL is the low level of cell proliferation under physiological conditions . To derive data of 3H-Tdr labelling index that can be analyzed statistically, large numbers of cells must be counted. This can be accomplished by either using many sections from a small number of animals or vice versa. In this context, analysis of variance  of osteoclast histogenesis in the PL indicated that designs in which the numbers of animals are increased are preferable to designs with more sections per animal. Stimulations of the PL by wounding , orthodontic tooth movement  or heat  have also been used to increase the rate of cell proliferation and so address in part these statistical problems. However, because there is massive cell death in these experimental systems, steady-state conditions do not exist and those factors that regulate proliferation under normal function can only be surmised.
Origin and Kinetics of Cells Resident in the PL: Fibroblasts
Debate over the origins of fibroblasts began with Virchow (1871)  who favoured local division of undifferentiated mesenchymal cells while Maximow (1926)  suggested a hematogenous origin after observing a rapid accumulation of cells in tissue culture without obvious increase of local mitotic activity. Experiments by Russell Ross and colleagues  clarified this issue with a parabiotic model in which the identity of labelled cells was established using electron microscopy. These data demonstrated that fibroblasts taking up 3H-Tdr after wounding are not blood-borne and that proliferation is associated with local division of cells closely associated with blood vessels. A follow-up in vitro study of human bone marrow fibroblasts  added further support to the belief in separate origins for fibroblasts and macrophages.
PL fibroblasts may originate embryologically from the dental follicle , and an electron microscope examination of developing mouse tooth germs  demonstrated that cell division was confined to paravascular cells. The cells of the investing layer around the developing PL exhibited the ultrastructural characteristics of “undifferentiated fibroblasts” . Gould et al. [34, 40] used stathmokinetic agents and 3H-Tdr labelling with radioautography to identify proliferating cells in wounded PL of adult mouse molars. At 30 h after wounding, uptake of 3H-Tdr by cells was maximal and the labelling index curve closely resembled that of liver after partial hepatectomy , indicating that PL cells could be stimulated to entry into the cell cycle by wounding. Labelled cells were initially located paravascularly, but after 3 days of healing, labelled cells were seen at a distance of up to 100 μm from blood vessels. This indicated a migration of proliferating cells away from the paravascular zone. Both undifferentiated and cytodifferentiated paravascular cells took up the 3H-Tdr label as demonstrated by the electron microscope radioautographs. Some of the labelled cells contained collagen in phagosomes, which suggested a more differentiated state. This last observation has received further substantiation from data  showing that in the PL of the rat molar stimulated by orthodontic tooth movement, dividing fibroblasts exhibited collagen-containing phagosomes.
A functional analysis of stem cells as demonstrated in the blood-forming system  has not yet been conducted for PL fibroblasts. However, phenotypic and functional differences of PL cells compared with those of other connective tissues have been examined in combined cell culture/transplantation experiments. In one report, the roots of dog teeth were incubated with connective tissue cells from either PL, skin or gingiva; the roots were then implanted in bone . Only fibroblasts from PL formed structures that resembled the PL. A phenotypic difference between PL and skin fibroblasts derived from Macaca irus has also been demonstrated in culture .
The kinetics of PL fibroblasts have been investigated in detail using mitotic counts and labelling with injected 3H-Tdr. The large inter-animal variation, diurnal periodicity and difficulty in localizing mitotic cells accurately in thick paraffin sections caused early investigators  to report mitotic indices with large variations in normally functioning rat molar PL. Later workers  examined rat molar PL and found that 3H-Tdr-labelled cells were located preferentially in the bone half of the PL. Further, labelled cells were most abundant in the bifurcation zones of the PL and higher at the apical and coronal levels of the root compared to the middle of the root. The authors suggested that the tooth rotates very slightly about an axis located at the level of the middle of the root and that the increased function at the coronal and apical portions of the root accounts for the higher labelling indices. No evidence was presented which detected or measured “function,” and indeed, later studies  found no gradient of 3H-proline uptake in the PL of rat molars at different levels of the root. Because most of the protein metabolism in the PL involves collagen , it is reasonable to assume that 3H-proline uptake in PL measures collagen synthesis.
When PL cell proliferation was examined in rats, the labelling indices were ∼9-fold higher in 30-day rats than 400-day rats , data that are consistent with observations of aging fibroblasts in culture [57, 58]. Shorter life spans and progressively fewer cell population doublings were observed in cultures originating from adult human tissues compared with cultures prepared from foetal tissue. Studies of PL cell proliferation were also conducted in aging mice , and these data were interpreted as providing evidence for a pluripotent cell labelled with 3H-Tdr that could produce differentiated progeny for the synthesis of cementum, PL and bone.
The turnover time of PL cells was studied with the use of continuous labelling of 3H-Tdr in the drinking water of mice to label all proliferating cells . After 30 days of labelling, 50 % of the PL cells were labelled, suggesting that the PL is a renewal tissue and that its turnover time is ∼60 days. In contrast, if the turnover time is calculated from the birth rate and the mean labelling index derived from a 3H-Tdr pulse label experiment, then the turnover time is about 20 days . The reason for this difference is the assumption in the calculated turnover time that all cells in the PL are cycling, which may not be correct based on later data showing that after prolonged labelling with 3H-Tdr in the drinking water of mice, many of the PL cells were not labelled . The identity and function of these non-proliferating cells in the PL have not been resolved.
When rat molar teeth are subjected to orthodontic forces, there are very large increases of the PL labelling index [61, 62], which may be attributable to entry of cells into the cell cycle and expansion of the proliferating fraction of cells. Another report suggested that cells in the normally functioning PL are released from G2 block by orthodontic force [63, 64]. Collectively, these data indicate the recruitment of non-proliferating cells into the cell cycle for generating repopulation responses in the PL. Further, measurements of nuclear volume density of proliferating PL cells indicate that there are separate progenitor populations for osteoblasts and fibroblasts in the PL .
Three experimental systems were used extensively in the 1960s–1970s to evaluate osteoblast histogenesis: (1) regeneration of marrow after depletion of the marrow cavity, (2) transplantation of marrow to an ectopic site and (3) culture of marrow stromal cells. The common finding from the first two methods is that marrow stroma and osteogenic soft connective tissue are both capable of producing bone tissue and marrow stroma [66, 67]. The production of marrow stroma relies upon the prior formation of bone tissue and that haematopoiesis is initiated at sites of bone resorption . The third method utilized an in vivo/in vitro system pioneered by Friedenstein . Fibroblast colonies were formed from a single-cell suspension derived from marrow tissue and grown in vitro. When these cells were placed in a diffusion chamber with thymus cells and implanted subcutaneously, viable mineralized tissue was formed which, upon light and electron microscopic examination, closely resembled bone.
Friedenstein  described two classes of bone progenitors, the determined osteogenic precursor cell (DOPC), which generates colony-forming units in culture and which exhibits extensive self-renewal without heterogenic inductive stimuli, and the inducible osteogenic precursor cell (IOPC) which requires stimulation for bone formation. Pre-osteoblasts in vivo are likely to be the DOPCs because they are capable of self-renewal throughout the life of the organism. The existence and the location of the stem cell for osteoblasts were not definitively demonstrated although they were more abundant near bone surfaces .
Little work has been done on the local origin of osteoblasts in the PL. In one experiment, 3H-Tdr-labelled ectomesenchymal cells of dental follicle were transplanted into adult mice , but grain counts over bone cells were so low that the possibility of labelling error was very high. These experiments were repeated  using the electron microscope to examine mononuclear cells as a marker for a rejection mechanism. The presence of lymphocytes around bone was interpreted to indicate a donor origin of the bone cells, but the data do not provide insight on whether periodontal osteoblasts arise from a stem cell common to fibroblasts, cementoblasts and osteoblasts. In experiments using recombination of mouse dental papilla and dental sac in ocular grafts, there was formation of fibrous connective tissue resembling PL and bone, which appeared similar to alveolar bone .
The number of cells labelled with 3H-Tdr (designated as osteoprogenitors)  in the orthodontically stimulated PL peaks several hours after the number of labelled fibroblasts when multiple injections of 3H-Tdr are given. In a pulse-label experiment, osteoblasts were never observed to be labelled 1 h after injection, and after multiple labelling, labelled osteoblasts were never observed up to 34 h after stimulation. From these data, pre-osteoblasts were considered to be either G1 or G2 blocked, in which case they either could become two functional osteoblasts or go through only one mitosis prior to becoming functional osteoblasts. There is an apparently separate proliferative response in mouse PL adjacent to bone after wounding , indicating that a separate population of bone progenitors may exist. After orthodontic stimulation and multiple labelling by 3H-Tdr to detect proliferating cells, PL fibroblasts evolved into local osteoblasts on the alveolar bone surface . This report also indicated similarities of the rates of increase of labelling index between PL fibroblasts and osteoblasts, which was cited as indirect evidence that PL fibroblasts are the source of the osteoblasts.
As yet, there is no definitive evidence for the origin of osteoblasts in the PL. It is known that after extirpating PL from teeth, subsequent wound healing will result in the formation of a bony union between cementum and alveolar bone. If the ligament is allowed to remain on the tooth root after removal of the overlying bone, it will become necrotic but will prevent osteogenesis and ankyloses . These findings indicate that some factor(s) in the PL inhibits osteogenesis and preserves the fibrous domain of the ligament, which was shown subsequently to involve prostaglandin E2 . This is a curious finding because the PL may then, under different circumstances, serve both to provide osteoprogenitor cells and to inhibit their differentiation and expression as osteogenic cells. This phenomenon suggests that the cells of the PL regulate and coordinate all osteogenic, cementogenic and fibrogenic activities occurring within its domain. The nature of these regulatory mechanisms is unknown.
The origin of cementoblasts has been examined in transplantation and kinetic experiments : cementoblasts were derived from the cells of the dental follicle, and 3H-Tdr labelling of donor cementoblasts was heavy. A wound healing study in cats  suggested that cementoblasts differentiate from the fibroblasts adjacent to the denuded cementum surface. However, it was difficult to assign cell lineage with certainty because of a lack of distinct markers for cementoblasts and their precursor cells. After injection of 3H-Tdr into adult rats and sacrifice 1 h later, cementoblast labelling was ∼25 % of the value of osteoblast labelling indices . The highest labelling indices were observed in the bifurcation of the root where there is virtually no cementum deposition , and there were somewhat lower labelling indices measured at the middle and apical levels of the root. As cementogenesis is greatest around the apical part of the root , it is difficult to see how one can relate this pattern of cell labelling with cementogenesis.
Mice exhibit much lower rates of cell proliferation in periodontal tissues than rats, and with aging, both cementoblast and osteoblast proliferation decreases rapidly, presumably a response to reduced growth and metabolic function . What is interesting is that the amount of cementum deposited on the apical portion of the root increases with age while the labelling index of cementoblasts decreases in this area. These data further support the belief that the proliferation of cells and the synthesis of mineralized extracellular matrix are unrelated events in the PL.
The PL is a very narrow, soft fibrous ligament in which cells expressing three distinct phenotypes (bone, cementum and fibrous connective tissue) enable homeostasis and regenerative processes in the periodontium. The progenitor cells of the PL stimulated by wounding [34, 40] and orthodontic tooth movement  were characterized in terms of their kinetics, spatial distribution and cytology. Wounding induces repopulation of the damaged PL by PL cells; in contrast, orthodontic forces cause a complex response which involves remodelling of alveolar bone and PL to accommodate the new position of the tooth . The pioneering studies described above were not able to resolve whether there were three different progenitor cells for each of the cementoblast, fibroblast and osteoblast populations or whether a single progenitor cell gives rise to all three synthetic cells. In spite of the experimental limitations, data obtained from cell kinetic, wounding and transplantation experiments conducted more than 35 years ago have been instrumental in paving the way for a measured and logical approach to harnessing the potential power of stem cell biology to produce improved outcomes for periodontal therapy. While this promise has not yet been realized in the clinic, current trends reviewed in other papers in this issue point to a bright future.
Compliance with Ethics Guidelines
Conflict of Interest
Dr. McCulloch has no conflict of interest.
Human and Animal Rights and Informed Consent
This article does not contain any studies with human or animal subjects performed by any of the authors.
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