This study was completed to investigate the osteogenic potential of bone marrow stem cells harvested from aged and young animals. In recent years, the goals of periodontal therapy have focused more closely on the regeneration of the periodontium in previously diseased sites, also there has been significant interest in the formation of bone around dental implants, in situations where there is minimal host bone at the time of fixture placement or where there was bone loss due to periimplant diseases. The key to guided tissue regeneration, of the periodontium is dependent on histodifferentiation of primitive mesenchymal stem cells migrating into the surgical wound which have the potential for osteogenesis, cementogenesis and formation of the periodontal ligament. Successful implant therapy is also dependent on the differentiation of osteoprogenitor cells arising from the surrounding bone marrow.
The objectives of this study were to investigate and compare the differences, if any between the number and size of osteogenic and hemopoietic colonies produced from the stem cells derived from the bone marrow of young and aged animals. In addition, to investigate the effect of addition of the fatty marrow to the red marrow of these two age group cohorts and to compare the results. This study seeks to quantify the potential for differentiation and histomorphogenesis of bone marrow stem cells in an In Vitro system. The purpose of this In Vitro study was to determine whether there were differences in formation of osteogenic colonies of male Sprague-Dawley rats, with bone marrow stem cells harvested from sexually mature 55 day old rats and those harvested from 18 to 22 month old, aged adult rats. Single cell suspensions from the red marrow of the long bones were cultured In Vitro and assessed In Situ for number and size, utilizing a computerized histomorphometric linear measuring system to assess colony area in square millimeters. The results clearly showed that young animals have a markedly increased potential for bone formation. Aged animals showed the formation of 0.45 +/- 0.6863 osteogenic colonies per Petri dish, while younger animals had 3.6 +/- 2.3523 colonies per dish (P ¾ 0.000). The second part of the study quantified the formation of bone and hemopoietic colonies after the addition of the fatty marrow, which may also harbor stem cells, to the red marrow. The addition of the fatty marrow, which is much more voluminous in the aged animals did not effect the number of bone colonies formed. These results may have implications for the healing aspects of older patients undergoing regenerative and implant therapy in periodontics.
1). To compare the number of bone and hemopoietic colonies formed from red bone marrow cells derived from aged and young animals, and to compare the number of bone and hemopoietic colonies formed from aged and young animals with the addition of fatty marrow to red marrow.
2). To compare the size of bone and hemopoietic colonies formed from aged and young animals, and to compare the size of bone and hemopoietic colonies formed from aged and young animals with the addition of fatty marrow to red marrow.
REVIEW OF THE LITERATUREThe ultimate goal of treatment of destructive periodontitis is the regeneration of the periodontium on the surface of tooth roots that have been deprived both of their periodontal attachment apparatus, and have been exposed to plaque pathogens, as well as the oral environment. The formation of new attachment has been studied for many years, however, the keys to predictable success have not yet been acquired. Melcher (1976) described four tissues which could contribute cells to the repopulation of the root surface during wound healing; Cementum, periodontal ligament (PDL), epithelium and bone. It has been widely recognized that epithelium is the fastest migrating of these four tissues, and will grow quickly down the root surface to form an epithelial attachment. The cellular rate limiter to new connective tissue attachment would seem to be the cementum, for without cementum there can be no functional arrangement of the connective tissue fibers, and without bone, no periodontal ligament fibers. Various clinicians have performed successful therapy which limited the epithelial downgrowth by excluding it from the wound and root surface for extended periods. Although positive clinical results have been acquired, very little histologic evidence exists to show predictable new attachment. Nonetheless, it has been shown histologically in humans that the formation of new attachment is possible [Frank (1972 and 1974), Cole (1980), Caton and Nyman (1980) Karring (1980)]. Histologic proof of new attachment following regenerative procedures was particularly tenuous until Cole (1980), utilized the most apical extent of clinically visible calculus as a marker.
The currently accepted definitions of periodontal wound healing as listed in the World Workshop in Periodontics (1989):
Early workers felt that scaling and root planing and curettage were clinical endpoints for new attachment. It was generally accepted that requirements for new attachment were: The removal of the sulcular epithelium, stabilization of the blood clot between the wound and the root surface, and complete removal of calculus and diseased cementum. There were numerous animal and human studies which showed positive results, particularly with a reduction in pocket depth; this was thought to be due to shrinkage, as well as new connective tissue attachment at the bottom of the pocket [Ratcliff (1966), Leonard (1935), Linghorne (1954), Waerhaug (1952) and Ramfjord (1951)]. Not until the study by Caton and Zander (1979), was it conclusively shown that periodic scaling and root planing does not achieve a new attachment, rather a long junctional epithelium with connective tissue windows; which is otherwise defined as repair.
Flap techniques for new attachment have been popular since the 1950s. The technique has basically consisted of raising full thickness mucoperiosteal flaps, degranulating the soft tissue, scaling and root planing under direct vision and then suturing the flaps closed. Some clinicians also made small perforations into surrounding alveolar bone to allow repopulation of the surgical wound with stem cells from the bone marrow spaces. With the advent of the description of the walls of osseous defects by Cohen and Goldman (1958), clinicians began to notice, and accurately communicate to others several phenomena which could be correlated at the time of surgery, and post surgically by radiographs. It was recognized that the more osseous walls remaining, generally the better bone fill was seen on post operative X-rays. However, there was scant histologic evidence to show that the soft tissue to root interface was not more than a long junctional epithelium [Patur and Glickman (1962), Ramfjord and Nissle (1974), Rossling (1976) and Polson and Heijl (1978)].
Early in the attempts at surgical endpoints for new attachment, it was recognized that the gingival epithelium proliferated very quickly and that proliferating epithelial cells reach the pre-surgical level of pocket epithelium by 1 week postoperatively [Moskow (1964) and Kon et. al. (1969)]. An early contact between the gingival connective tissue and the root came to be regarded as the prime prerequisite for the formation of a new connective tissue attachment. In other words, it was recognized that the epithelium had to be excluded or delayed from repopulating the wound, [Bjorn (1961), Bjorn et al (1965) and Hiatt et al (1968)].
In order to exclude the epithelium from the wound, Ellegard et al (1974) used free gingival grafts to cover osseous defects and essentially exclude the epithelium from the site. Prichard (1967) and Becker, Becker and Berg (1986) showed radiographic evidence of new attachment by completely excluding tissue from periodontal defect sites. Nilveus et al (1978) reported the technique for coronally positioned flaps in an animal study. This was also a technique for epithelial exclusion. Again, histologic studies were lacking to show the result of healing at the root surface.
With the renewed interest in achieving a true, new attachment, research was again pursued in the area of root treatment. The idea was to expose collagen fibrils within the root surface with which the connective tissue fibrils could bond, opening the way for functionally oriented, tooth-bonded periodontal ligament fibers. Many substances were tried, and those with the most promise were citric acid and tetracycline [Register and Burdick (1975 and 1976) and Terranova (1986)]. More recent controlled histologic studies, particularly using Cole's method of marking the apical extent of the calculus, have yielded equivocal results for root conditioning in the technique for new attachment [Moore (1987)]. However there is no known detrimental effect to the use of root conditioners, and they may empirically aid in actually cleaning the root surface free of bacterial plaque.
Guided Tissue Regeneration:Historically, the concept of guided tissue regeneration was begun with the idea of excluding the epithelial cells and allowing tissues from the other three compartments described by Melcher (1976) to repopulate the root surface. An additional element to success in this technique is the long term elimination of inflammation. Periodontal wound healing studies during the 1970s and 1980s supported Melcher's hypothesis. Caton et al (1980) studied four different types of treatment modalities in an animal model. The treatments included; scaling and root planing, Modified Widman surgical flaps with debridement alone or used in conjunction with autogenous or synthetic grafts. The healing in each case was characterized by a long junctional epithelium to the base of the defect indicating that conventional periodontal therapy does not usually result in new attachment.
A series of studies were now embarked upon by the researchers at the University of Gothenburg in Sweden as an investigation of the behavior of the four cellular compartments to the tooth surface. Nyman et al (1980) examined the effects of exclusion of epithelium from the periodontal wound. In this study, periodontitis was induced around selected teeth in monkeys and dogs using the experimental periodontitis protocol described by Caton and Zander (1975). The affected roots were then planed and sectioned from their crowns. Each root was extracted and embedded longitudinally in a trough made in alveolar bone, so that half its circumference was in contact with bone, and the other with gingival connective tissue from the overlying flap. In this way, four zones for histologic study were created: Planed root surface, and periodontal ligament covered surface in contact with either bone or connective tissue. After 3 months of healing, histologic examinations showed that the planed root surfaces in contact with connective tissue were affected by resorption, and that the planed root surfaces in contact with bone showed both root resorption and ankylosis. The areas which were still covered by PDL demonstrated fibrous reattachment, supporting Melcher's concept that the periodontal ligament may have a regenerative and protective role as well. These findings demonstrated that more than the mere exclusion of the epithelium was required for new attachment. In 1982, Nyman et al performed another classic study. In this uncontrolled monkey study, fenestration defects were created on the facial aspect of canines and covered with Millipore filters to exclude the connective tissue in the flap. In this way the PDL was allowed to repopulate the wound from four sides. The histologic sections showed new cementum and inserting connective tissue fibers as well as alveolar bone, this was seen primarily in the apical portion of the 2-5mm defects. Conclusions from this paper point to cells from the PDL having the potential to initiate regeneration when both epithelium and gingival connective tissue are excluded from the wound. Nyman et al (1982) tested this hypothesis on a human mandibular incisor which was severely affected by periodontitis. The tooth was treated by flap surgery, thoroughly scaled and root planed to the base of the shallow three walled defect, then a Millipore filter was trimmed and sutured tightly around the tooth, covering the defect like an apron. After 3 months of healing, the tooth was removed in a block biopsy. Histologic sections showed an unexpectedly large gain in new attachment, 5mm from the the apical extent of the defect. The authors concluded that new attachment could be formed on previously diseased root surfaces. Gottlow (1986) published case reports from 10 patients successfully employing the technique of guided tissue regeneration around periodontally involved teeth utilizing the e-PTFE membranes manufactured by Gore-Tex. These investigations formed the scientific basis for the principles of guided tissue regeneration (GTR). In GTR, a physical barrier is placed between the gingival flap and the instrumented root surface. Placement of this barrier excludes the gingival epithelium and connective tissue from the wound and root surface, creating a space for progenitor cells from the periodontal ligament and bone to migrate into the defect. Although it is generally believed that periodontal ligament cells have the greatest potential to promote new attachment, recent investigations have suggested that bone cells may also have an important role [Melcher et al (1970) and McCulloch et al (1987)].
Guided Bone RegenerationNew techniques have arisen very recently which entail guided bone regeneration around dental implants. This consists of bone growth as opposed to periodontal ligament and cementum regeneration around teeth with periodontal defects. Dahlin (1988) showed that circular defects in the jaws of rats healed predictably with bone fill in three weeks, whereas the same defects when not isolated by membranes did not fill with bone by 22 weeks. This study spurred other leaders in the field of endosseous oral implantology to apply the principles of guided tissue regeneration to the sophisticated management of implants. New techniques are currently developing for the augmentation of insufficient alveolar ridges prior to, as well as post implant placement, as well as in the treatment of ailing fixtures. The principles of guided tissue regeneration, or guided bone regeneration, seems to be even more appropriate for use where only one cell type from Melcher¹s four cellular compartments is being addressed. It seems that an artificial extraction socket can be somewhat reproduced, and the architectural benefits harnessed for surgeon directed wound healing. The clinician must bear in mind however, that the biologic tenets must be adhered to just as in guided tissue regeneration around teeth. There must be a space created underneath the membrane as well as sufficient surface area for containment of the clot and sufficient walls or surface area for bone formation. Studies have been carried out to show the potential gains of this technique. Becker and others (1991) reported a pilot animal test which gave encouraging results in the use of GBR for immediate implant placement and ridge augmentation. Jovanovic and others (1992) published a recent human study on the treatment of dehisced fixtures also giving positive results. Buser (1990) used small surgical steel screws to provide space under PTFE membranes for ridge augmentation prior to fixture placement. Generally it appears that GBR may be a very useful step in giving patients and clinician more options in endosseous implant therapy.
Extensive work has been carried out for both GTR and GBR utilizing animal models. Not much conclusive histologic evidence has been presented for humans however; and particular concerns arise as to how newly regenerated tissue around implants will fare over time with loading. Some facts seem to hold true for the possibility of GTR and GBR to be successful. These include the understanding that regeneration of lost tissues will be provided for by the stem cells of the tissues sought after, and the strict control of inflammation. In periodontal lesions, the greatest success seems to be found in deep narrow lesions, providing they can be successfully debrided of plaque and calculus. The reason seems to be that these three walled lesions provide a large surface area for periodontal ligament stem cells to migrate into, to close the defect. Shallower, wide lesions seem to be the most difficult to treat by GTR. These are often treated with bone grafting procedures.
Bone Grafting:
Definitions of bone grafting procedures are as follows:, Lindhe (1989)
In periodontics, the most common grafts currently in use are autografts, alloplasts and allografts. Many studies have been carried out in humans and animals to examine the result of grafting procedures upon periodontal defects. The ultimate goal of periodontal therapy is to achieve regeneration of the periodontium. A secondary, but clinically important, and possibly more achievable goal is to reduce the depth of periodontal pockets, acquire firm, pink gingival tissue which does not show signs and symptoms of inflammation, and to gain clinical attachment. Studies in wound healing with autografts from both intra-oral and iliac crest sites which include bone marrow have shown that regeneration is possible [Froum et al (1975) and Hiatt et al 1978]. Extensive studies utilizing decalcified, freeze-dried bone allografts have also shown that regeneration is a possible achievement with this therapy [Bowers et al (1989)]. Clinical parameters such as gain in clinical attachment level have been shown to be predictable outcomes utilizing alloplasts, particularly porous hydroxyapatite [Kenney et al (1988) and Oreomuno (1990)].
Orthopedists and hematologists have been using red marrow grafts for many years, these are primarily taken as cores from the sternum and ileum. Bone marrow or trabecular bone transplants have the greatest osteogenic potential because osteogenic stem cells are present in the hemopoietic marrow. These stem cells will divide and differentiate into bone producing osteoblasts. Friedenstein (1976) and Owen (1980) called this entity a stromal cell, and Tibone and Bernard (1981) named it a colony forming unit - osteogenic (CFUo). Whether these are all variations of the same cell or are two different cells types remain to be determined. All of these cells will produce osteogenic colonies In Vivo and In Vitro and therefore potentially can be the most potent moiety for bone defect filling. To understand the underlying factors of success for healing of periodontal defects, dehisced implants and bony ridge augmentations, one must first understand the formation of bone.
II. Bone Wound Healing:Healing of a fracture or extraction site recapitulates embryogenesis. Stem cells from the bone marrow, which are morphologically similar to medium sized lymphocytes, but genetically different, become stimulated to form osteoprogenitor cells, the osteoprogenitors divide again to form one osteoprogenitor and one osteoblast. The osteoblast is an end differentiated bone matrix producing machine and therefore does not divide again, relying on the more primitive stem cells for this purpose. Extra cellular matrix is essential. Cells become entrapped as osteocytes, more osteoblasts are differentiated and bone eventually fills the defect [Tibone and Bernard, (1981)].
Bone tissue is divided into two categories, depending on the original model from which it was derived during embryogenesis [Rubin et al (1988)]. Most of the skeleton is derived from endochondral bone formation, the facial bones are derived from intramembranous ossification where bone develops within a fibrous tissue template [Ten Cate (1989)]. The original type of ossification generally dictates the type of vasculature for each bone [Rubin et al (1989)]. Generally, bones formed by endochondral ossification have a central artery, and intramembranous formed bones rely on the vasculature of the periosteum for their blood supply. The mandible is architecturally similar to, but not the same as, bones formed by endochondral ossification in that it has a central artery, the inferior alveolar artery. This is important from a wound healing standpoint, because fracture healing relies on vascularity as a key factor, [Rowe et al (1985) and Wlodarski (1989)].
There are three phases of wound healing in bone which are generally recognized [Kent and Zide (1984) and Manoli (1984)]. The first phase occurs during the first week after fracture and is often called the inflammatory phase. This phase consists of organization of the blood clot and phagositization of soft tissue necrosis and damage by the polymorphonucleocytes. Within 7 days, blood vessels invade the clot and early osteoid is formed at the periphery of the wound, where the blood vessels first invaded. The peripheral vascularization is provided by soft tissue and surrounding periosteum, which also provides stem cells to repopulate the defect, with osteoid forming osteoblasts [Wlodarski (1989)]. One can see that even at this early stage, the integrity of the periosteum and blood supply are very important for wound healing.
The second, or reparative phase begins after one week, and may last several weeks to months. During this time the clot is resorbed, while blood vessel ingrowth and osteoid deposition continue. This phase, more than the others depends on the nutritional status of the patient, as various tissues, including collagen and osteoid are being synthesized and organized. Adequate supplies of cofactors such as vitamins C and D, and a positive nitrogen balance must be ensured. These nutritional concerns may be even more important in the elderly individual and should be addressed, [Friedman and Costantino (1990)].
The third phase of fracture healing, or the remodeling phase begins with ingrowth of bone spicules into the osteoid from the edges of the fracture, these are known as filling cones, [Ten Cate (1989)]. The remodeling phase is similar to the embryonic phase of remodeling woven bone to lamellar bone, and can last from several weeks to months.
In the case of a healing extraction socket, this process begins as the socket fills with blood which becomes a clot, the clot seals off the bony walls from invading microorganisms and provides a matrix for replacement by granulation tissue [Bertolami (1990) and Shetty and Bertolami (1990)]. Within the first 24 hours, organization of the clot begins by means of leukocytic migration, fibroblastic proliferation and capillary budding. Polymorphonuclear leukocytes, lymphocytes and plasma cells densely populate the fibrin matrix at the coronal surface but diminish in number near the apex. Epithelial proliferation in the gingival wound margins and fibroblastic invasion into the clot begin at this time. Although periodontal ligament remnants can be recognized within the extraction site, they degenerate and do not contribute significantly to the healing process. During early repair, the alveolar crest lacunae appear to be empty and thereby set the stage for active crestal resorption. By the end of the first week, the granulation tissue is more highly organized and fibroblasts are distributed throughout the alveolus. The gingival epithelium establishes contact with the surface of the granulation tissue and should completely cover the wound within two weeks. Previously, appositional bone deposition was thought to begin along the lateral walls and fundus of the socket during the second week after extraction. In fact, bone formation starts at the end of the first week in the surrounding marrow spaces, not the socket itself [Bertolami (1990)]. By 14 days there is no radiographic evidence of new bone, but histologic examination reveals numerous foci of osteogenesis, particularly along the wall and at the fundus. At the end of the third week, bone in the socket has assumed a characteristic architecture consisting of distinct trabeculae that are oriented parallel to the long axis. The inflammatory infiltrate persists within the remaining granulation tissue but is greatly diminished, and the sub-epithelial connective tissue becomes arranged into distinct fiber bundles that extend between the cementum of the teeth adjacent to the extraction site. Bone fills the socket by the end of the fourth week and may extend slightly beyond the alveolar crest. Although the lamina dura may be visible radiographically at 6 weeks, the concurrent processes of bone apposition and bone resorption cause the parallel trabecular pattern to be lost. By 8 weeks, resorption of the external plate of the alveolus uncovers the new bone within the socket. Newly deposited bone is also subject to resorption, so a rounded alveolar contour is established while maintaining the general shape of the alveolar ridge. After 2 months, the socket outlines are no longer distinct because of deposition of new bone and resorption of the lamina dura. By 10 to 12 weeks, the extraction site is virtually indistinguishable from surrounding bone and the overlying epithelium cannot be distinguished from the normal mucosa. [Bertolami (1990)].
Primitive stem cells are contained within the bone marrow. Some of these stem cells are osteogenic and have the potential to become osteoblasts Tibone and Bernard, (1981) found 1.6 stem cells per one million cells derived from the red bone marrow of New Zealand White rabbits to be osteogenic. The osteogenic stem cells divide to become one stem cell, which can divide again, and one osteoprogenitor cell. The osteoprogenitor again divides to become an osteoblast and an osteoprogenitor cell. From this cascade of division, one can see that the most potential for osteogenesis is provided when the primitive stem cells are recruited during wound healing. The osteoblast now secretes collagen, glycosaminoglycans and the trilaminar matrix vesicles which are the primary locus of calcification.
III. The Effects of Aging:Various studies have shown a decrease in the healing abilities of aged and young humans as well as animal models. Holm-Pedersen (1971) completed a study on experimental gingivitis between healthy young and aged human adult groups and found an earlier onset of inflammatory changes, as well as significantly delayed return to baseline in the aged group. Grant (1970) has performed autopsy studies which showed artherosclerotic changes in the lamina propria of aged individuals. This leads to lingering inflammation in the aged, as there is a decreased vascular capability in dealing with the inflammatory infiltrate. Gingival biopsy studies point to delayed healing in the aged, as well as a lack of true regeneration of the lamina propria. This is understandable, because with age the regenerative capacities of the body begin to wane significantly [Hall (1976)]. However, few studies have been completed to quantify the potential of bone marrow to form bone between aged and young individuals. Recently, in 1990, Tsuji and co-workers published a study investigating the effect of donor age on the production of bone-like tissue and expression of cellular alkaline phosphatase in cultures of cells obtained from rat bone marrow. These researchers found a three fold increase in the numbers of bone nodules produced by immature young rats as compared to aged adult rats. They also reported a decrease in the number of alkaline phosphatase positive cells from the cultures derived from the aged animals. There was no difference in the size of colonies. As many of the patients who are treated for periodontal disease and implant therapy are not in the very young age groups, it is important to consider the potential of bony regeneration and healing in the aging population. To study this, it would also be appropriate to establish an animal model which more closely imitates this age group. Perhaps with this information, further research can be completed in the area of growth factors which would enhance the potential of stem cells in older populations.
Research in aging is relatively new, however it has been a subject discussed and thought about for centuries. Aristotle observed and commented on the definite life spans of various animals and plants, and described his own hypothesis on aging. ³The incapacity of old age is due to an affection not of the soul but of its vehicle, as occurs in drunkenness or disease.² Quoted in Griffin (1950).
During the Renaissance, when the sciences were beginning to bud, careful observation of the outside world was in vogue. Leonardo da Vinci dissected 30 bodies, and was particularly interested in anatomic changes with age, [Belt (1952)]. da Vinci paid particularly close attention to connective tissues and blood vessels, and concluded that the cause of aging was ³Veins, which by thickening of their tunics in the old restrict the passage of the blood, and by this lack of nourishment destroy the life of the aged without any fever, the old coming to fail little by little in slow death.²
Research in earnest, directed at the aging process took off in the 1950s when a number of long term studies were launched at various centers in Europe and the United States [Arking (1991)].
Senescence can be described as a loss of adaptability and a progressive loss of the capacity of the individual to maintain homeostasis [Buetow (1985)]. Long term studies of age changes in the human show that on average, a variety of physiological functions begin to decline by 30 to 40 years of age [Shock (1960 and 1977)]. This can be characterized as a complex interaction between growth processes and degenerative processes, including changes in the cellular content and proliferative capacity of tissues. An interesting and important concept of aging is the chronologic and biologic age of an individual [Hall (1976)]. As of yet, there is no organ or tissue that sets the gold standard for the measure of chronologic age, and age from the date of birth is accepted as the primary parameter. However, individuals and tissues within individuals age differently, due in part to genetics, systemic disease and environmental influences, [Hall (1976)].
In general, there can be seen a general increase in the fat content of the body and a decrease in the water content, [Buetow (1985)]. The decrease in water content may be due to replacement of well hydrated extracellular spaces with collagen. For example, a dramatic demonstration of change in water content with age can be seen in bone. Developing bone is 60ater, whereas aged cortical bone is only made up of 10ater, [Arking (1991)]. This may be primarily due to replacement of water by collagen and inorganic crystals. Hydration is important for cellular homeostasis and regulatory functions, therefore the reduction of water content in age is an important change. The increase in lipid content of the body is probably due to the increase in cholesterol esters and sphingomyelin primarily found in collagen rich extracellular tissues of decreased cellularity. Lipids are probably not synthesized locally, but bound as they diffuse through the extracellular compartment [Kohn (1978)].
Collagen makes up 25-30111f total body protein and has been studied extensively, not only because physiologically it is important in organ function and pathologic processes, but also because it has been important historically in industry as leather, gelatin and glue, [Kohn (1978)]. Accumulated information describe collagen as existing in at least two metabolic pools. The collagen that is most recently synthesized has an appreciable turnover rate that varies from site to site as a function of physiologic alterations, and tissue stresses [Heikken (1972)]. Most of the body collagen comprising the second pool is mature and insoluble and either non-renewable or has a very low rate of turnover. It appears that a significant fraction of collagen remains present for the life of an animal, and is an excellent candidate for observing age related changes, [Kohn (1978)]. In general aging changes apparent in collagen consist of decreased solubility and increased cross linking; in humans, the most rapid and extensive cross linking occurs between ages 30 to 50 years. It has been noted that aged collagen is less susceptible to denaturization by collagenase, Hamlin (1972), this is probably due to increased cross linking. Collagen is synthesized as soluble pro-collagen molecules. These are converted to tropocollagen molecules that aggregate to form insoluble collagen fibrils. During growth, tissues contain large amounts of neutral salt-soluble collagen. With maturation, soluble collagen drops to very low levels and is incorporated into mature collagen in the form of insoluble fibrils. In the absence of severe tissue stresses or inflammation, the amount of collagen remains stable and demonstrates very little turnover, it is still subjected to increased cross linking however, [Kohn (1978)]. According to Sobel (1967), the amount of collagen present in some organs is due to processes that occur at the borderline between normal aging and pathological change. Collagen fibers are synthesized in response to severe or prolonged tissue stresses and are oriented in tissues parallel to lines of stress. Collagen is also formed as part of the healing phase of inflammation. Thus wherever prolonged inflammation or stresses occur, a scarring resulting from collagen synthesis will be observed. Analysis of fibrosis at sites such as the arteries and arterioles have led to the concept that tissue fibrosis is a generalized aspect of aging, however, examination of other organs and tendons have shown that collagen concentrations remain constant with age [Kohn (1978)]. It is possible that collagen production is regulated by feedback mechanisms that insure that when sufficient collagen is synthesized to maintain form and provide strength in an organ, further production is inhibited, and the amount remains constant, when more strength is needed in damaged or stressed tissue, collagen synthesis is initiated [Tomanek (1972)]. In summary, it seems that the most significant age related changes in collagen are in its properties and not its amount.
As da Vinci correctly noted, some of the most important and far reaching changes in aging are due to changes in the blood vessels. Atherosclerosis is a finding in virtually all humans with increasing age, and the plaques formed are directly and indirectly a primary cause of death in humans. A variety of factors including nutritional, metabolic, genetic and the presence of other systemic diseases influence the rate of progression of atherosclerosis. The essential changes consist of a chronic inflammatory reaction in the walls of arteries characterized by the proliferation of small vessels, by the accumulation of minerals, lipids and glycoproteins and by scarring associated with the formation of large amounts of collagen. The older artery is dilated, the wall is thickened and stiff and the inner surface shows many atherosclerotic plaques in conjunction with ulceration's and adherent blood clots. Based on extensive autopsy observations, Stary (1987) described the progression of atherosclerosis as a diffuse thickening of the intima as the hallmark of the disease. The lesions are composed of assemblages of macrophages and smooth muscles cells embedded in an extracellular matrix containing elastic fibers. These four components of the plaque are unevenly distributed into two distinct layers. In the early stages, the innermost or luminal layer is rich in matrix and poor in elastic fibers. It contains a loose and irregular arrangement of smooth muscle and macrophages. Conversely, the underlying musculoelastic layer is poor in matrix and rich in elastic fibers. In advanced lesions, it also contains dense and orderly arrangements of lipid-rich smooth muscle cells and macrophage foam cells layered above a thick extracellular lipid core. This lipid or necrotic core is composed of the partially degraded lipid droplets and dead macrophages. Other secondary processes also take place, such as deposition of calcium into elastin in the cores of the vessels and the slow formation of a collagenous cap over the intima region above the core. This leads to a massive thickening of the intima and the narrowing of the arterial channel to critical limits, [Arking (1991)].
Bone is a dynamic tissue, constantly remodeling in response to the metabolic needs of the body, and is dictated by hormonal regulation. The changes that are observed in the aging of bone and the skeleton are therefore changes in anabolic and catabolic imbalances.
Osteoporosis is an entity which has drawn more attention by researchers in recent years. It is thought that the rate of osteogenesis by osteoblasts during remodeling in older individuals does not change, however bone resorption by osteoclasis outstrips regeneration, resulting in a total deficit of bone density, leading to reduced strength and concurrent fractures. Osteoporotic changes occur in all bony structures of the body, in both the cancellous and trabecular region of bone, but are normally confined to the spine, pelvis and more rarely the skull and extremities, [Hall (1976)]. Research has shown that osteoporosis, defined by Schlenker (1984) as a decrease in bone mass with no change in the chemical ratio of mineral to protein matrix, is not a disease entity separate from aging, but rather a more extreme version of the normal processes of bone loss. The role of gender in osteoporosis and loss of bone mass and density with age cannot be argued. At any given age, bone mass is greater in men than in women, but not the rate of bone loss. A male with a 4,000gm skeleton will lose approximately 450gm or 12111ver a 12 year period. Over an equivalent period, a female with a 3,000gm skeleton will lose approximately 750gm or 250f her bone mass, much of it during the post menopausal years [Arking (1991)]. Estrogen's tend to protect bone from the stimulating effect of parathyroid hormone on the osteoclasts. After menopause, there is a rise in bone resorption due to the increased sensitivity of the osteoclasts to parathyroid hormone. Quantitiative measurements of the amount of bone resorption in postmenopausal women has shown it to be about equal to 425 mg/day of calcium, while bone formation equals only 387 mg/day, this results in a net daily loss of 38 mg., at this rate an average female loses 1.5 percent of her bone per year, [Schlenker (1984)]. Bone loss can be measured with a variety of different methods, each of which yields different numbers, but all yield the same patterns of age dependent loss [Exton-Smith (1985)]. It is known that estrogen also effects the levels of calcium absorption and excretion. The effect of estrogen is the dual one of an indirect suppression of remodeling, and improved efficiency in the utilization of dietary calcium. The loss of bone strength with age has been attributed to at least two different processes [Whitbourne (1985)]. Increased porosity arising from the continuous bone remodeling reduces the structural strength of the bone and the remaining structure becomes more brittle with age, which is a result of increased mineralization of the remaining bone structure [Matrovic (1979)]. As a result, the bone of an elderly individual when subjected to pressure is more likely to snap and cause a clean fracture. Such fractures are more likely to result in non-union and less likely to heal. The bone of younger persons is more flexible, due to higher organic content of the bone matrix, therefore when it is subjected to fracture producing stresses, it is more likely to bend and crack [Whitbourne (1985)]. Bone resorption occurs primarily on the surfaces of existing bone. As trabecular bone accounts for 907431064566f the total surface area of the skeleton, it is not surprising that most bone remodeling takes place on the trabecular surfaces of spongy bone. The pattern of loss in spongy bone differs in several ways form that in compact bone. In both sexes the onset of bone loss occurs at least a decade earlier in spongy bone [Arking (1991)].
Age related changes in the facial skeleton and fracture wound healing has become a greater clinical concern for various reasons, including the inreased population of upper age groups, and the large number of facial fractures incurred by automobile and other accidents, as well as sophisticated surgical procedures involving the placement of endosseous implants in edentulous jaws. The importance of vascular supply to wound healing is paramount. There are no papers documenting an age related decrease in vascular supply of the soft tissues of the face or the periosteum of the jaws [Friedman and Costantino (1990)]. Assuming that a decline in reparative capacity due to cellular senescence comes with age, it is also reasonable to assume that maximizing the blood supply to the region of the facial fracture takes on an added importance in the elderly. With the exception of the mandible, the vascular supply to the facial skeleton is almost entirely by the subperiosteal plexus and the surrounding soft tissues, therefore, preservation of these tissues is imperative [Friedman and Costantino (1990)]. A fragment or region of intramembranous facial bone that has been stripped of its investing periosteum is nothing more than a free bone graft. In this setting, fracture healing would be impaired and partial or complete resorption of the devascularized bone could result. Therefore, even in severely comminuted fractures resulting in an egg shell appearance, the membranous attachment to the fragments should be preserved when feasible. When considering the mandibular blood supply, the input of the intermedullary artery must be considered as well. The inferior alveolar artery augments the periosteal blood supply of the mandible to the region of the mandibular body, and the mandibular angle below the level of the lingula and the parasymphysis. Therefore, a good collateral circulation exists in these areas in contrast to the regions of the mandibular ramus. The subperiosteal vascular plexus or the regional muscular attachments supply blood for the coronoid process, the neck of the condylar process and to the fibrocartilaginous joint [Rowe (1985)]. Prior to age 40, approximately 50111f the mandiblular fractures involve those areas of the mandibular body and angle with a collateral blood supply [Friedman and Costantino (1990)]. With increasing age, atherosclerosis of the inferior alveolar artery usually occurs, resulting in cessation of blood flow in approximately on third of cases studied angiographically [Bradley (1975)]. In fact, the inferior alveolar artery begins to show signs of atherosclerosis approximately 15 years before it is detected within the carotid system. By the time an individual reaches the age of 40 years, atherosclerotic changes can begin to be seen clinically. After 65, however, it can be assumed that a significant number of patients no longer have a functioning inferior alveolar artery, and this collateral supply to the mandibular body is absent [Friedman and Costantino (1990)].
Fractures located in regions where the blood supply is derived solely from muscular attachments, such as the symphysis, require rigorous preservation of the muscular attachment. Osteonecrosis with spontaneous fracture of the symphysis has been reported in an elderly individual who underwent surgery of the genial region, which resulted in disruption of the primary source of perforating vessels to the symphysis. Although many factors such as physical stress and adequacy of stabilization, certainly contribute to the eventful outcome of treatment, it is notable that the area of the mandible with the most tenuous blood supply, the symphysis, has the highest proportionate rate of fracture non-union. Approximately 14111f all mandibular fractures are of the symphysis, yet over 250f all nonhealing fractures are located there [Mathog (1983)].
Changes also occur in the growth structure of the facial skeleton, these changes primarily involve resorption of both upper and lower alveolar ridges with increasing age. Alveolar resorption usually occurs secondary to a loss of dentition, although periodontal resorption occurs while the teeth are still present, the alveolar ridges are usually resorbed down to or below the level where the root tips were located [Friedman and Costantino (1990)]. This can result in a loss of over 50111f the total mandibular height, and therefore its vertical cross-sectional area [Friedman and Costantino (1990)]. With respect to facial trauma, alveolar resorption poses two primary problems. First, lower alveolar ridge resorption significantly weakens the mandible to the point where spontaneous fractures can occur, although they are extremely rare [Cope (1982)] Secondly the edentulous mandible, coupled with an alveolar height inadequate for stable support of a denture, can make the establishment of occlusion and maxillo-mandibular fixation difficult.
In summary, this discussion has focused on three aspects of aging upon bone. First, that there are changes in the blood supply, due to atherosclerotic changes in the vasculature. Second that there are changes in the hormonal milieu with age, especially in women, that effect the catabolic and anabolic processes of the skeleton. And third, that there may be differences in the number of stem cells available in aged individuals and a reduction in their metabolic activity. This research focuses on the third aspect of aging in bone in the preceding discussion.
As clinicians, we have treated patients at both ends of the spectrum and noticed that the biologic age of individuals makes a large degree of difference in their ability to heal. This research may stimulate further studies in the elucidation of growth factors which could even out the plane of healing potential and make GTR as well as GBR techniques more predictable.
IV. Description of the Tissue Culture System:The formation of bone has been replicated In Vitro utilizing a rabbit system first described by Tibone and Bernard (1981). This system was replicated for a rat model by Shih and Bernard (1991) and other systems have been successfully executed by McCullough and Melcher (1989) and Bellows and Aubin (1986). It has been well established that the cells of adult bone marrow possess both hemopoietic and osteogenic potential. In 1961, the existence of pluripotent hemopoietic stem cells in bone marrow was demonstrated In Vivo by Till and McCullough. In 1966, Bradley and Metcalf reported that these stem cells had the ability to differentiate and to form discrete hemopoietic colonies In Vitro. They also demonstrated a linear relationship between the number of cells plated and the number of hemopoietic colonies formed. From this, they concluded that each colony was the result of the proliferation of a single stem cell. The growth of colonies from a single stem cell coined the use of the term colony forming units of CFU, for the bone marrow stem cells responsible for the colony growth. The osteogenic potential of bone marrow In Vivo was reported as early as 1881 by Bruns, but there was much debate over whether the bone from bone marrow grafts was produced by cells from the host or by the bone marrow cells of the donor. It was not until 1968 that Friedenstein demonstrated conclusively that the bone marrow itself contains a cell with the potential to form bone. Since Friedenstein¹s work in 1968 many In Vivo experiments have been performed in order to learn more about the osteogenic stem cell of adult bone marrow. More recently, Budenz and Bernard (1980) separated bone marrow cells into 7 subpopulations based on density gradients, and found that one of these subpopulations produced significantly more bone than the others when implanted in chambers In Vivo. Lindholm and Urist (1980) demonstrated that the osteogenic cells of bone marrow can be influenced In Vivo by the presence of an ³Osteoinductive agent² in the microenvironment. The ability of cultured bone marrow ³fibroblasts² to form bone In Vivo has been demonstrated [Owen (1970), Friedenstein et al (1970), Miskarova et al. (1970), Friedenstein (1976), Ashton et al. (1980) and Hirano and Urist (1981)]. In all of these experiments, adult whole bone marrow cells were cultured, and the adherent fibroblastic cells were harvested. These cells were then placed in chambers and implanted In Vivo. In all cases, bone formed within the chambers, even after 17 passages of the fibroblastic cells in culture [Miskarova (1970)]. These bone marrow fibroblasts have been labeled ³stromal cells² by Owen (1980), and have been characterized as a mesenchymal cell, unrelated to hemopoietic stem cells [Golde et al (1980), and Owen (1980)]. The osteogenic precursor cells found in adults have been classified into two categories: determined osteogenic precursor cells (DOPC) and inducible osteogenic precursor cells (IOPC) [Friedenstein et al., (1968); Owen, (1970); Friedenstein, (1973)]. Originally, DOPC were defined as a self-perpetuating population of cells which could be isolated from the bone marrow, the stromal or fibroblastic cells of bone marrow cultures, and which would form bone in transplantation In Vivo without any stimuli required. IOPC were cells found in connective tissue or in the lymphoid cells populations of the spleen, the thymus or the peripheral blood, which could be induced to form bone in chambers In Vivo by osteogenic inducers (Transitional epithelium or decalcified bone matrix). This original definition has since been modified, because evidence now exists that both DOPC and IOPC can be found in the cells of adult bone marrow [Budenz and Bernard, (1980) and Owen, (1980)]. Thus far, DOPC have been shown to be present only in bone marrow and on bone surfaces, whereas IOPC are found throughout the body in lymphoid tissues and connective tissues, Urist (1965), as well as in bone marrow. Tibone and Bernard (1982), were the first to show that osteogenic stem cells of adult bone marrow were capable of forming bone In Vitro.
In order to demonstrate that true osteogenesis has occurred In Vitro, a number of criteria must be met. These criteria, from initial calcification to intramembranous osteogenesis, were developed by Bernard and Pease (1969), Marvaso and Bernard (1977) and Tibone and Bernard (1981).
Criteria for In Vitro Osteogenesis:Tibone and Bernard (1981) and Shih and Bernard (1990), showed that bone could be produced In Vitro by bone marrow cells. It was shown that osteogenic stem cells present in bone marrow could differentiate, proliferate and express their ability to make bone in a tissue culture.
This system proved to be reliable, and has been shown to be reliable in quantititating the number of stem cells and bone colonies responsible for bone formation from the marrow of long bones, it has been chosen to study the questions proposed by the current study.