BONE IS D E A D A N D THERE A R E N O REAL OSTEOCYTES
JAMES C STINSON. Department of Anatomical Pathology, Scott and White Clinic, Temple, Texas 76501, USA. SUMMARY Fresh bone fixed in formalin and subsequently impregnated with silver nitrate before decalcification reveals certain microscopic features that are not apparent with the usual techniques. Microscopic studies of prepared bone and physical experiments on fresh bone suggest that the "osteocytes" may represent only spaces and canals containing electrically conductive material. Living cells are not present. Explanations for some physiologic processes in bone are suggested by the observations.
Few heretics are popular but fortunately there is now little inclination for physical roasting. Since the penance of today is neither permanent nor especially painful, the heresy that "bone is a nonliving structure" is proposed and supported. Bone is dead, or depending on one's definition of life, it is as viable as a toenail. Of the basic components of bone, collagen is a product of living cells, but collagen is not considered to be alive. The hydroxyapatite crystals of bone are piezoelectric, are poor conductors, and are not alive; yet, "living" bone is a conductor of electricity. .Another basic component o f bone is said to be the "osteocyte" which is alledged to be derived fron an osteoblast. In the subperiosteal layer of bone and in bone adjacent to cartilage, cells may be observed in lacunar spaces; however, identifiable cells or their processes are rare in subcortical or cancellous bone. If osteocytes and their processes are present in such bone, why don't they appear as other cells appear in decalcified bone? Is bone analgous to a building in which there are electrical conduits? In this comparison, the bricks (crystal structure) and mortar (collagen) are not particularly controversial; but the electrical circuitry represents a potent area for disagreement. The "osteocyte" with its processes may represent the unit of electrical conduction; and its development, structure, and physiology are therefore of special interest. When calcified bone is impregnated with silver, various microscopic structures become apparent. These are never seen in decalcified bone .stained with the usual stains or silver, are consistent, and do not have the appearance or staining characteristics of cells. They may be reservoirs and conduits for a material which is similar to phosphatase in its chemical reaction with silver and which may be electrically conductive (Fig. 1). Some of these structures have a graceful, spider-like a p p e a r a n c e , have staining characteristics similar to some nerve tissue stained with silver, and there are interconnecting processes (Fig. 2). There appears to be some association with haversian canals, and they appear in prominent colonies in some abnormal conditions (osteoid osteoma, some osteochon-
dromas, and amputation stumps). Could this structure have a sensory function in bone? Extensions from the reservoirs appear by electron microscopy to have valve-like constrictions (Fig. 3). Several layers of these pools may extend beneath the periosteum and the processes probably terminate as canaliculi opening into spaces. The silver-stained material either obscures the cell or only a remnant persists in the space (Fig. 4). The reservoirs, canaliculi, or "osteocytes" are not evident in the deeper bone that is decalcified prior to staining. Is s o m e t h i n g r e m o v e d froffl t h e s e s p a c e s d u r i n g decalcification? It seems unlikely that cells in the lacunae and their processes in the canaliculi would selectively be unstained while other cells in the same section would stain normally. If this hypothetical material is a fluid or gel, there should be some suggestion as to its origin. It would be convenient to assign this responsibility to the "osteoblast," but what is an ostcoblast? Is it a modified fibroblast that is capable of secreting this material, such as the modified fibroblast which produces argyrophilic reticulum? In new bone, the " o s t e o b l a s t " has a strong reaction for alkaline phosphatase; and silver stains reveal dark granules in the cytoplasm of cells in the periosteum as well as in fibroblasts in the area. This granularity in the fibroblasts may be especially noticeable in callus formation, in some exostoses, and in ectopic bone formation where identifiable periosteum or adjacent living cells are not associated with osteoid or bone. As mineralization progresses, adjacent fibroblasts may be incorporated in the matrix of the bone and continue to function for some time. Truly viable appearing cells are rarely seen in the deeper layers or in cancellous bone. The cells in the lacunae of cartilage also contain silver stained cytoplasmic granules and, in addition, there is often a halo of these granules in the cartilage matrix adjacent to the space. So far, this hypothesis of bone origin and development would not be too inconsistent with accepted theories. However, Flow does one explain bone formation in callus and infant bone'where ~ceUular pools of black material are 33
o
present and where interconnecting canals appear (Fig. 5)2 How can the myriads of canaliculi or canalicuiar segments seen in some specimens of exostose, ectopic, fetal, and avian bone be explained when cells are absent and few lacunae are present? To be consistent in this fall from grace, electrical conduction that does not involve living cells should be considered. Fresh bone can conduct an electrical current and it acts as a condenser by discharging over a relatively long period of time. It will conduct a current better in one direction than in the other and has less resistence when cooled, but greater resistence to current flow when frozen. From very preliminary experimentation, fresh bone inadvertently left in a refrigerator for several days was found to retain its original electrical characteristics. This abuse of tissue would suggest that electrical conduction is not dependent on viable cells. Do the canalicufi and lacunae represent electrical circuits and junction boxes in the bone? If cellular extensions are not in the deeper canals, is the black material in direct contact with the wall of its space7 Electron micrographs from deeper bone do not reveal a definite membrane (Fig. 6). Could it be assumed, then, that a difference in the polarity or amperage of the current would result in formation or dissolution of the crystal structure? Are osteoclasts actually only macrophages clearing up the collagen and crysalline debris? In another preliminary expermiment, fresh long bone s s centrifuged prior to silver impregnation. After treating tlk~ bone with silver and decalcifying it, the silver positive material was seen concentrated in the subperiosteal region, in the "reservoirs" and in the distal end of the centrifuged bone (Fig. 7). Cells could not be identified in the reservoirs or lacunae, and canaliculi were not visible in other areas. And what is osteoid?* The explanation is not difficult if one is satisfied with the idea that osteoid is merely nonmineralized bone. In normal and abnormal bone, (1) osteoid may or may not be on the bone surface (2) cells may or may not be on the surface of the osteoid (3) cells may or may not be incorporated in the osteoid, and (4) mineralization may be partial or absent in the osteoid. If one is of the opinion that there are no osteocytes and what is seen with the silver stains of bone represent electrical channels, however, how is osteoid produced? Is it possible that an electrical charge in bone causes electrophoretic migration and precipitation of protein and other materials onto the exposed surface? Are there chemotaxic inflt. ,Ices that attract the modified fibroblasts to the osteoia where they produce the additional conductive material that is required for growth and mineralization? What are osteoid osteomas7 Perhaps it is unsporting to mention the practice of bovine bone transplantation. This bone apparently disregards the laws of foreign tissue rejection and has served its purpose quite well in some instances. In one femur, the foreign bone
(in place for I0 years) probably would still be functional had the patient not fractured another area. The silver stains indicated that a "weld" had taken place between the host and the guest, and there was no inflammation. Although silver-stained material was absent from the central portion, fluid or gel in the host bone had begun to permeate the canaliculi and lacunae in the periphery of the implant. While not especially germalne to this discussion, what may have been a remote ancestor of the cow in question surfaced as part of a petrified skeleton in a Central Texas gully. This petrified bone would have pleased a rock hound as it could be polished and the marrow spaces were prominent. Some fragments were treated with hydrochloric acid, the undissolved crystals removed manually and the remaining debris examined microscopically (Fig. 8). There is no doubt that this bone was dead but the skeletons of the prehistoric "osteocyte" were well preserved. Bases of the heresy that bone is not a living tissue and that true functional osteocytes are mythical include hypotheses and a few facts from observations and experiments: 1. Modified fibroblasts produce some material that can be stained with silver, much as reticulum formed by altered fibroblasts. (These cells may be incorporated into the bony matrix.) 2. This substance is delivered into subperiosteal reservoirs and subsequently transmitted into preformed canals and lacunae that possibly remain from previous cell inclusions. 3. In subcortical bone, protoplasmic extensions rarely are visible and the content of a canaliculus appears to be in direct contact with its wall. 4. The material in the canaliculi and lacunar spaces is conductive, and an electrical current will effect crystal formarion and dissolution. This substance can be physicafiy displaced by centrifugation. (Could this physical displacement be a factor in the bone alterations observed in bedfast patients, astronauts, and deep sea divers...) 5. Osteoid may be the result of an electrophroetic phenomenon rather than the direct product of cell secretion. 6. Bovine bone is dead too. Because manipulation of bone for microscopic examination is difficult, some deduced "classical facts" probably are hypothetical. Some nonconforming theories and hypotheses have evolved from staining calcified bone with silver as a spare-time project, and these dissenting views are based on experiments and observations of normal and pathologic bone. "Cause and effect" relationships are suggested by light and electron microscopic examinations. (There has been a moderate amount of intuitive guessing.) The absence of extensive "solid" experimental proof is conspicuous, but the experimental portion will he perfected "mafiana."
* Osteoid does not stain with silver unless partially mineralized. Eosin is a good counterstain to show this substance.
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JAMES C STINSON. Department of Anatomical Pathology, Scott and White Clinic, Temple, Texas 76501, USA. SUMMARY Fresh bone fixed in formalin and subsequently impregnated with silver nitrate before decalcification reveals certain microscopic features that are not apparent with the usual techniques. Microscopic studies of prepared bone and physical experiments on fresh bone suggest that the "osteocytes" may represent only spaces and canals containing electrically conductive material. Living cells are not present. Explanations for some physiologic processes in bone are suggested by the observations.
Few heretics are popular but fortunately there is now little inclination for physical roasting. Since the penance of today is neither permanent nor especially painful, the heresy that "bone is a nonliving structure" is proposed and supported. Bone is dead, or depending on one's definition of life, it is as viable as a toenail. Of the basic components of bone, collagen is a product of living cells, but collagen is not considered to be alive. The hydroxyapatite crystals of bone are piezoelectric, are poor conductors, and are not alive; yet, "living" bone is a conductor of electricity. .Another basic component o f bone is said to be the "osteocyte" which is alledged to be derived fron an osteoblast. In the subperiosteal layer of bone and in bone adjacent to cartilage, cells may be observed in lacunar spaces; however, identifiable cells or their processes are rare in subcortical or cancellous bone. If osteocytes and their processes are present in such bone, why don't they appear as other cells appear in decalcified bone? Is bone analgous to a building in which there are electrical conduits? In this comparison, the bricks (crystal structure) and mortar (collagen) are not particularly controversial; but the electrical circuitry represents a potent area for disagreement. The "osteocyte" with its processes may represent the unit of electrical conduction; and its development, structure, and physiology are therefore of special interest. When calcified bone is impregnated with silver, various microscopic structures become apparent. These are never seen in decalcified bone .stained with the usual stains or silver, are consistent, and do not have the appearance or staining characteristics of cells. They may be reservoirs and conduits for a material which is similar to phosphatase in its chemical reaction with silver and which may be electrically conductive (Fig. 1). Some of these structures have a graceful, spider-like a p p e a r a n c e , have staining characteristics similar to some nerve tissue stained with silver, and there are interconnecting processes (Fig. 2). There appears to be some association with haversian canals, and they appear in prominent colonies in some abnormal conditions (osteoid osteoma, some osteochon-
dromas, and amputation stumps). Could this structure have a sensory function in bone? Extensions from the reservoirs appear by electron microscopy to have valve-like constrictions (Fig. 3). Several layers of these pools may extend beneath the periosteum and the processes probably terminate as canaliculi opening into spaces. The silver-stained material either obscures the cell or only a remnant persists in the space (Fig. 4). The reservoirs, canaliculi, or "osteocytes" are not evident in the deeper bone that is decalcified prior to staining. Is s o m e t h i n g r e m o v e d froffl t h e s e s p a c e s d u r i n g decalcification? It seems unlikely that cells in the lacunae and their processes in the canaliculi would selectively be unstained while other cells in the same section would stain normally. If this hypothetical material is a fluid or gel, there should be some suggestion as to its origin. It would be convenient to assign this responsibility to the "osteoblast," but what is an ostcoblast? Is it a modified fibroblast that is capable of secreting this material, such as the modified fibroblast which produces argyrophilic reticulum? In new bone, the " o s t e o b l a s t " has a strong reaction for alkaline phosphatase; and silver stains reveal dark granules in the cytoplasm of cells in the periosteum as well as in fibroblasts in the area. This granularity in the fibroblasts may be especially noticeable in callus formation, in some exostoses, and in ectopic bone formation where identifiable periosteum or adjacent living cells are not associated with osteoid or bone. As mineralization progresses, adjacent fibroblasts may be incorporated in the matrix of the bone and continue to function for some time. Truly viable appearing cells are rarely seen in the deeper layers or in cancellous bone. The cells in the lacunae of cartilage also contain silver stained cytoplasmic granules and, in addition, there is often a halo of these granules in the cartilage matrix adjacent to the space. So far, this hypothesis of bone origin and development would not be too inconsistent with accepted theories. However, Flow does one explain bone formation in callus and infant bone'where ~ceUular pools of black material are 33
o
present and where interconnecting canals appear (Fig. 5)2 How can the myriads of canaliculi or canalicuiar segments seen in some specimens of exostose, ectopic, fetal, and avian bone be explained when cells are absent and few lacunae are present? To be consistent in this fall from grace, electrical conduction that does not involve living cells should be considered. Fresh bone can conduct an electrical current and it acts as a condenser by discharging over a relatively long period of time. It will conduct a current better in one direction than in the other and has less resistence when cooled, but greater resistence to current flow when frozen. From very preliminary experimentation, fresh bone inadvertently left in a refrigerator for several days was found to retain its original electrical characteristics. This abuse of tissue would suggest that electrical conduction is not dependent on viable cells. Do the canalicufi and lacunae represent electrical circuits and junction boxes in the bone? If cellular extensions are not in the deeper canals, is the black material in direct contact with the wall of its space7 Electron micrographs from deeper bone do not reveal a definite membrane (Fig. 6). Could it be assumed, then, that a difference in the polarity or amperage of the current would result in formation or dissolution of the crystal structure? Are osteoclasts actually only macrophages clearing up the collagen and crysalline debris? In another preliminary expermiment, fresh long bone s s centrifuged prior to silver impregnation. After treating tlk~ bone with silver and decalcifying it, the silver positive material was seen concentrated in the subperiosteal region, in the "reservoirs" and in the distal end of the centrifuged bone (Fig. 7). Cells could not be identified in the reservoirs or lacunae, and canaliculi were not visible in other areas. And what is osteoid?* The explanation is not difficult if one is satisfied with the idea that osteoid is merely nonmineralized bone. In normal and abnormal bone, (1) osteoid may or may not be on the bone surface (2) cells may or may not be on the surface of the osteoid (3) cells may or may not be incorporated in the osteoid, and (4) mineralization may be partial or absent in the osteoid. If one is of the opinion that there are no osteocytes and what is seen with the silver stains of bone represent electrical channels, however, how is osteoid produced? Is it possible that an electrical charge in bone causes electrophoretic migration and precipitation of protein and other materials onto the exposed surface? Are there chemotaxic inflt. ,Ices that attract the modified fibroblasts to the osteoia where they produce the additional conductive material that is required for growth and mineralization? What are osteoid osteomas7 Perhaps it is unsporting to mention the practice of bovine bone transplantation. This bone apparently disregards the laws of foreign tissue rejection and has served its purpose quite well in some instances. In one femur, the foreign bone
(in place for I0 years) probably would still be functional had the patient not fractured another area. The silver stains indicated that a "weld" had taken place between the host and the guest, and there was no inflammation. Although silver-stained material was absent from the central portion, fluid or gel in the host bone had begun to permeate the canaliculi and lacunae in the periphery of the implant. While not especially germalne to this discussion, what may have been a remote ancestor of the cow in question surfaced as part of a petrified skeleton in a Central Texas gully. This petrified bone would have pleased a rock hound as it could be polished and the marrow spaces were prominent. Some fragments were treated with hydrochloric acid, the undissolved crystals removed manually and the remaining debris examined microscopically (Fig. 8). There is no doubt that this bone was dead but the skeletons of the prehistoric "osteocyte" were well preserved. Bases of the heresy that bone is not a living tissue and that true functional osteocytes are mythical include hypotheses and a few facts from observations and experiments: 1. Modified fibroblasts produce some material that can be stained with silver, much as reticulum formed by altered fibroblasts. (These cells may be incorporated into the bony matrix.) 2. This substance is delivered into subperiosteal reservoirs and subsequently transmitted into preformed canals and lacunae that possibly remain from previous cell inclusions. 3. In subcortical bone, protoplasmic extensions rarely are visible and the content of a canaliculus appears to be in direct contact with its wall. 4. The material in the canaliculi and lacunar spaces is conductive, and an electrical current will effect crystal formarion and dissolution. This substance can be physicafiy displaced by centrifugation. (Could this physical displacement be a factor in the bone alterations observed in bedfast patients, astronauts, and deep sea divers...) 5. Osteoid may be the result of an electrophroetic phenomenon rather than the direct product of cell secretion. 6. Bovine bone is dead too. Because manipulation of bone for microscopic examination is difficult, some deduced "classical facts" probably are hypothetical. Some nonconforming theories and hypotheses have evolved from staining calcified bone with silver as a spare-time project, and these dissenting views are based on experiments and observations of normal and pathologic bone. "Cause and effect" relationships are suggested by light and electron microscopic examinations. (There has been a moderate amount of intuitive guessing.) The absence of extensive "solid" experimental proof is conspicuous, but the experimental portion will he perfected "mafiana."
* Osteoid does not stain with silver unless partially mineralized. Eosin is a good counterstain to show this substance.
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