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Autogenous bone marrow transplants in the rat
Research
Autogenous bone marrow transplants in the rat Surindar N. Bhaskar, Colonel, DC, UXA,” Duane E. Cutright, Lieutenant Colonel, DC, USA,“+ and Robert E. Boyers, Lieutenant Colonel, DC, USA”“’ UNITED
STATES
WALTER
REED
ARMY ARMY
INSTITUTE MEDICAL
OF DENTAL CENTER,
RESEARCH,
WASHINGTON,
D. C.
T
he possibility that bone marrow transplants can be a source of osteogenic tissue has been of interest to biologists for many decades. The feasibility of this technique in periodontal therapy was recently demonstrated by Schallhorn,’ who successfully transplanted hip bone marrow from the posterior superior iliac crest to the intrabony periodontal defects. The osteogenetic potential of the hematopoietic marrow has also been demonstrated in the healing of maxillary and mandibular wounds in dogs.?, 3 Although bone marrow transplants have been shown to stimulate bone formation, the mechanism of this process is unknown. It has been suggested that the necrosis in the transplant is necessary for the differentiation of osteoblasts4 On the other hand, such necrosis was not observed in marrow transplants in dogs which were examined 4 to 10 weeks postoperatively,3 Because of the wide clinical applications of this procedure, the present serial study was undertaken to determine the sequential changes which occur in small bone marrow transplants. METHODS
AND
MATERIALS
Thirty-six albino rats, weighing about 200 grams each, were anesthetized with 0.2 cc. of Pentosol. The cranial vault and the hind leg area of each animal were then shaved. Incisions were made along the anterior crest of the right tibia, and the proximal end of the bone was exposed. A trephine in a conventional dental handpiece was then used to remove a. plug of bone marrow 2 mm. in *Chief, Division of Oral Pathology. **Division of Oral Pathology. ***Assistant Chief, Division of Oral
472
Pathology.
Volume 29 Number 3
Fig. 1. Transplanted bone trabecula.
bone marrow graft at 0 days showing hematopoietic elements :~u11
diameter and from 2 to 3 mm. in length from the area of the proximal metaphysis. An incision was made on the cranial vault, and a subdermal “pocket“ was created between the skin and the periosteum. The bone marrow transplant was then inserted into the supraperiosteal “pocket,” and the incision line was closed with silk sutures. Four animals were killed, 0, 1, 3, 5, 7, 10, 14, 21, 30 days postoperatively and the cranial vaults were recovered, fixed in 10 per cent formalin, decalcified, cut at 6 microns, and stained with hematoxylin and cosin. FINDINGS Zero days
The transplant which was recovered at 0 days contained dense sheets of hematopoietic marrow and its usual elements (Fig. 1). Areas of hematopoiesis were interspersed by a moderate number of fat cells. In addition to these elements, the transplanted material also contained bone t,rabeculae, islands of bone dust, and occasionally a few fragments of the striated muscle. The bone tra.beculae included cores of the cartilage matrix, which implied that they represented the primary spongiosa of the metaphysral region; this feature was one of many which made it possible to identify them in later stages. One
to three
days
At 1 and 3 days after transplantation the autogenous graft showed both the degenerating and viable cells of the hematopoietic marrow (Figs. 2 and 3). The fat and the osseous components of the graft were unchanged. The bone trabeculae contained cartilaginous cores (Fig. 3). While some of the Iacunae were empty, others still contained osteocytes. The surface of the bone trabecnlae revealed neither bone apposition nor resorption (Fig. 3). Five
to seven
days
Five days after transplantation most of the hematopoietic elements in the graft had degenerated. However, this period was marked by a. proliferation of
474
Bhaskar,
Cutright,
Oral Surg. March, 1970
and Boyers
Fig. d. Transplant in situ 1 day postoperatively, Note presence of a number of bone trabeculae.
Fig. 3. Transplant 3 days postoperatively. necrosis (H). Bone trabecula shows cartilaginous
lying
betxeen
Most hematopoietic core (C).
cranial
elements
vault
and dermis.
have undergone
fusiform cells with distinct cytoplasm and vesicular nuclei (Figs. 4 and 5). They packed the spaces between the fat and appeared to represent the reticulum cells. The bone trabeculae showed no change from the earlier stage. Connective tissue cells around the bone “dust” particles showed differentiation of osteoclasts and beginning resorption. The 7-day specimens were essentially similar to those seen at 5 days, except that the reticulum-cell proliferation was more marked and numerous areas of the graft showed the formation of eosinophilic homogeneous material (Figs. 6
Volume Number
Autogenous bone marrow
29 3
tmnsplmts
Figs. 4 and 5. Low- and high-power photomicrographs of area of tra.nsplant Note a few bone fragments and dense mass of reticulum-cell proliferation.
475
Fig.
4
Fig.
5
seen at 7 days.
and 7). This material was seen on bone trabeculae as well a.s between the reticulum cells of the bone marrow. Ten duys
At 10 days new bone formation was seen in the graft. This was evidenced by the presence of osteophytic bone on and near the bone trabeculae which were transpIanted with the graft (Figs. 8 and 9). These trabeculae either contained cartilaginous cores or were lamellated and could be easily distinguished from the newly formed bone. The new bone trabeculae were surrounded by prominent osteoblasts (Figs. 9 to 13). In some areas differentiation of osteoblasts from the reticulum cells could be seen. Fourteen
days
Although individual variations were seen in the 14day specimens, all of them showed evidence of new bone formation. Upon examination of the specimen
476
Bhaskar,
Fig.
6
Fig.
7
Cutright,
Oral Surg. March, 1970
and Boyers
Figs. 6 and 7. Hcven days after transplantation, be seen between reticulum cells and on bone trabeculae.
eosinolhilic
homogeneous
material
can
with maximum bone activity as compared to those with minimal new bone, it appeared that the osteogenetic potential of the graft depended on its size and configuration. Grafts which had more bone marrow elements and those with minimal amounts of bone “dust” had the greatest osteogenetic potential. Twenty-one
to thirty
days
Between 21 and 30 days the rate of bone apposition diminished, and at 30 days new bone formation occurred primarily as surface apposition. Osteoblasts were smaller in size and almost fusiform in shape, and they did not show the crowding that was observed earlier. Particles of bone dust and debris which mere included with the graft had undergone almost complete osteoclastic resorption. In these areas fibroblasts and giant cells remained. The bone tissue seen at the host site consisted of new bone as well as that which was transplanted with the graft. These could be distinguished from each other by their different staining reactions and by the presence of the reversal lines. As evidenced by their osteocytes, both types of bone were viable. There was no evidence of sequestration.
Volume Number
21) :I
E
Fig.
9
Figs. 8 rind 9. Two IO-da,y specimens showing rapid apposition of bane ou and l~etncwu transplanted bone trabeculae. Old and nex bone can 1~ easily distinguiahrd hp thclir sfaiuiug and lamellar features and 1)~ their ostcocyte populations.
DISCUSSION
The transplanted tissue in this experiment was not “protected” from the surrounding connective tissue by a mieropore filter, the experimental animals were not given any type of drugs during the postoperative period, and the transplanted tissue was no more than 2 by 2 mm. in size. In spite of t,hese conditions, the transplant survived and produced new bone. The bone trabeculae which were included with the graft could be recognized by their deeper staining, their lamellar pattern, and the presence of cartilaginous cores. During the course of the experiment none of these trabeculae underwent sequestration or exfoliation. This bone tissue did not undergo resorption, and
478 Bhaskar, Gutright, and Boyers
Oral Surg. March, 1970
10
Fig.
11
Figs. 10 and 11. Low(lamellated),
new bone (light
and high-power views of area of transplant staining), and dense osteoblast aggregation.
showing
old bone
osteoeytes could be identified in it at 30 days. Fragments of bone chips and bone dust which were included in the transplant were seen on its periphery and consistently showed evidence of osteoclastic resorption. It may be concluded from the observation of this diversified fate of the two types of bone tissue that, when bone trabeculae are transplanted with the bone marrow or with the mesenchyme which surrounds them, they are able to survive while bone dust and bone chips undergo osteoclastic resorption. This observation should once again cast some doubt on the therapeutic use of bone chips in the management of osseous defects. A study of the bone marrow cells in the transplanted tissue reveals that the first 3 days are characterized by necrosis of the hematopoietic elements. Although at 5 days some of the hematopoietic cells survive, most of them degenerate. At this time, however, there is a rapid proliferation of young mesenchymal cells of the bone marrow. At ‘7 and 10 days after transplantation many of these mesen-
Volume
29
Number 3
Autogenous
bone marrow
transplants
479
chymal
(reticulum) cells differentiate into osteohlasts, and new hone fomatiorl It may he suggested from this observation that the undifferentiated mesenchymal (reticulum) cell component of these transplants is the main reason for their survival. The necrosis of the hematopoietic elements observed in the first few days does not, in our opinion, prove or disprove the concept proposed by Burwell” that marrow necrosis provides an inductive mechanism for the differentiation of marrow cells into osteoblasts. The formation of new bone started about 10 days after transplantation, and at about 20 days it became markedly slowed until at 30 days only surface apposition could be seen. This observation would seem to indicate that any bone marrow graft has a limited osteogenetic potential. Whether this is determined by the size of the original transplant or is inherent in some other factor is open to speculation. OCCUIS.
SUMMARY This histologic study concerns autogenous bone marrow grafts in thirty-six rats in which the proximal tibia1 metaphysis and the cranial subcutaneous tissues were the donor and recipient sites, respectively. The transplanted tissue measured about 2 by 2 mm. This study revealed that: 1. Small autogenous bone marrow transplants can survive and produce bone. 2. In the rat, at least, the use of antibiotics and mechanical protective devices is not necessary for osteogenesis. 3. The first 3 days after transplantation are characterized by necrosis of hematopoietic cells. At 5 days the undifferentiated mesenchymal cells of the bone marro’w undergo rapid proliferation, and at 10 days bone formation is a prominent feature. 4. Osteogenetic potential of the marrow transplants is limited. In the present series the maximum osteogenesis was observed at between 10 and 20 postoperative days. The mechanism governing the duration of osteogenesis in a given transplant is not known. The size of the transplant, however, may have a bearing on its productive life. 5. Whereas bone chips and bone dust undergo resorption on transplantation, bone trabeculae which are surrounded by marrow elements do not. This would seem to indicate that the clinical use of bone chips or bone dust in the management of bone defects is of questionable value. It is suggested that, for the survival of a bone trabecula, it should be transplanted as a “hemato-osseous” unit. REFERENCES
1. Sehallhorn, R. G.: The Use of Autogenous Hip Marrow Biopsy Implants for Bony Crater Defects, J. Periodont. 39: 145, 1968. 2. Lyons, H. W.: Effect of Hemopoietic Bone Marrow Transplants on Healing of Maxillofacial Osseous Defects, J. Dent. Res. 43: 827, 1964. of Osteogenesis in the 3. Richter, H. E., Sugg, W. E., Jr., and Boyne, P. J.: Stimulation Dog Mandible by Autogenous Bone Marrow Transplants, ORAL SURG. 26: 396, 1968. 4. Burwell, R. G.: Studies in the Transplantation of Bone, J. Bone Joint Surg. 46-B: 110, 1964.
Autogenous bone marrow transplants in the rat Surindar N. Bhaskar, Colonel, DC, UXA,” Duane E. Cutright, Lieutenant Colonel, DC, USA,“+ and Robert E. Boyers, Lieutenant Colonel, DC, USA”“’ UNITED
STATES
WALTER
REED
ARMY ARMY
INSTITUTE MEDICAL
OF DENTAL CENTER,
RESEARCH,
WASHINGTON,
D. C.
T
he possibility that bone marrow transplants can be a source of osteogenic tissue has been of interest to biologists for many decades. The feasibility of this technique in periodontal therapy was recently demonstrated by Schallhorn,’ who successfully transplanted hip bone marrow from the posterior superior iliac crest to the intrabony periodontal defects. The osteogenetic potential of the hematopoietic marrow has also been demonstrated in the healing of maxillary and mandibular wounds in dogs.?, 3 Although bone marrow transplants have been shown to stimulate bone formation, the mechanism of this process is unknown. It has been suggested that the necrosis in the transplant is necessary for the differentiation of osteoblasts4 On the other hand, such necrosis was not observed in marrow transplants in dogs which were examined 4 to 10 weeks postoperatively,3 Because of the wide clinical applications of this procedure, the present serial study was undertaken to determine the sequential changes which occur in small bone marrow transplants. METHODS
AND
MATERIALS
Thirty-six albino rats, weighing about 200 grams each, were anesthetized with 0.2 cc. of Pentosol. The cranial vault and the hind leg area of each animal were then shaved. Incisions were made along the anterior crest of the right tibia, and the proximal end of the bone was exposed. A trephine in a conventional dental handpiece was then used to remove a. plug of bone marrow 2 mm. in *Chief, Division of Oral Pathology. **Division of Oral Pathology. ***Assistant Chief, Division of Oral
472
Pathology.
Volume 29 Number 3
Fig. 1. Transplanted bone trabecula.
bone marrow graft at 0 days showing hematopoietic elements :~u11
diameter and from 2 to 3 mm. in length from the area of the proximal metaphysis. An incision was made on the cranial vault, and a subdermal “pocket“ was created between the skin and the periosteum. The bone marrow transplant was then inserted into the supraperiosteal “pocket,” and the incision line was closed with silk sutures. Four animals were killed, 0, 1, 3, 5, 7, 10, 14, 21, 30 days postoperatively and the cranial vaults were recovered, fixed in 10 per cent formalin, decalcified, cut at 6 microns, and stained with hematoxylin and cosin. FINDINGS Zero days
The transplant which was recovered at 0 days contained dense sheets of hematopoietic marrow and its usual elements (Fig. 1). Areas of hematopoiesis were interspersed by a moderate number of fat cells. In addition to these elements, the transplanted material also contained bone t,rabeculae, islands of bone dust, and occasionally a few fragments of the striated muscle. The bone tra.beculae included cores of the cartilage matrix, which implied that they represented the primary spongiosa of the metaphysral region; this feature was one of many which made it possible to identify them in later stages. One
to three
days
At 1 and 3 days after transplantation the autogenous graft showed both the degenerating and viable cells of the hematopoietic marrow (Figs. 2 and 3). The fat and the osseous components of the graft were unchanged. The bone trabeculae contained cartilaginous cores (Fig. 3). While some of the Iacunae were empty, others still contained osteocytes. The surface of the bone trabecnlae revealed neither bone apposition nor resorption (Fig. 3). Five
to seven
days
Five days after transplantation most of the hematopoietic elements in the graft had degenerated. However, this period was marked by a. proliferation of
474
Bhaskar,
Cutright,
Oral Surg. March, 1970
and Boyers
Fig. d. Transplant in situ 1 day postoperatively, Note presence of a number of bone trabeculae.
Fig. 3. Transplant 3 days postoperatively. necrosis (H). Bone trabecula shows cartilaginous
lying
betxeen
Most hematopoietic core (C).
cranial
elements
vault
and dermis.
have undergone
fusiform cells with distinct cytoplasm and vesicular nuclei (Figs. 4 and 5). They packed the spaces between the fat and appeared to represent the reticulum cells. The bone trabeculae showed no change from the earlier stage. Connective tissue cells around the bone “dust” particles showed differentiation of osteoclasts and beginning resorption. The 7-day specimens were essentially similar to those seen at 5 days, except that the reticulum-cell proliferation was more marked and numerous areas of the graft showed the formation of eosinophilic homogeneous material (Figs. 6
Volume Number
Autogenous bone marrow
29 3
tmnsplmts
Figs. 4 and 5. Low- and high-power photomicrographs of area of tra.nsplant Note a few bone fragments and dense mass of reticulum-cell proliferation.
475
Fig.
4
Fig.
5
seen at 7 days.
and 7). This material was seen on bone trabeculae as well a.s between the reticulum cells of the bone marrow. Ten duys
At 10 days new bone formation was seen in the graft. This was evidenced by the presence of osteophytic bone on and near the bone trabeculae which were transpIanted with the graft (Figs. 8 and 9). These trabeculae either contained cartilaginous cores or were lamellated and could be easily distinguished from the newly formed bone. The new bone trabeculae were surrounded by prominent osteoblasts (Figs. 9 to 13). In some areas differentiation of osteoblasts from the reticulum cells could be seen. Fourteen
days
Although individual variations were seen in the 14day specimens, all of them showed evidence of new bone formation. Upon examination of the specimen
476
Bhaskar,
Fig.
6
Fig.
7
Cutright,
Oral Surg. March, 1970
and Boyers
Figs. 6 and 7. Hcven days after transplantation, be seen between reticulum cells and on bone trabeculae.
eosinolhilic
homogeneous
material
can
with maximum bone activity as compared to those with minimal new bone, it appeared that the osteogenetic potential of the graft depended on its size and configuration. Grafts which had more bone marrow elements and those with minimal amounts of bone “dust” had the greatest osteogenetic potential. Twenty-one
to thirty
days
Between 21 and 30 days the rate of bone apposition diminished, and at 30 days new bone formation occurred primarily as surface apposition. Osteoblasts were smaller in size and almost fusiform in shape, and they did not show the crowding that was observed earlier. Particles of bone dust and debris which mere included with the graft had undergone almost complete osteoclastic resorption. In these areas fibroblasts and giant cells remained. The bone tissue seen at the host site consisted of new bone as well as that which was transplanted with the graft. These could be distinguished from each other by their different staining reactions and by the presence of the reversal lines. As evidenced by their osteocytes, both types of bone were viable. There was no evidence of sequestration.
Volume Number
21) :I
E
Fig.
9
Figs. 8 rind 9. Two IO-da,y specimens showing rapid apposition of bane ou and l~etncwu transplanted bone trabeculae. Old and nex bone can 1~ easily distinguiahrd hp thclir sfaiuiug and lamellar features and 1)~ their ostcocyte populations.
DISCUSSION
The transplanted tissue in this experiment was not “protected” from the surrounding connective tissue by a mieropore filter, the experimental animals were not given any type of drugs during the postoperative period, and the transplanted tissue was no more than 2 by 2 mm. in size. In spite of t,hese conditions, the transplant survived and produced new bone. The bone trabeculae which were included with the graft could be recognized by their deeper staining, their lamellar pattern, and the presence of cartilaginous cores. During the course of the experiment none of these trabeculae underwent sequestration or exfoliation. This bone tissue did not undergo resorption, and
478 Bhaskar, Gutright, and Boyers
Oral Surg. March, 1970
10
Fig.
11
Figs. 10 and 11. Low(lamellated),
new bone (light
and high-power views of area of transplant staining), and dense osteoblast aggregation.
showing
old bone
osteoeytes could be identified in it at 30 days. Fragments of bone chips and bone dust which were included in the transplant were seen on its periphery and consistently showed evidence of osteoclastic resorption. It may be concluded from the observation of this diversified fate of the two types of bone tissue that, when bone trabeculae are transplanted with the bone marrow or with the mesenchyme which surrounds them, they are able to survive while bone dust and bone chips undergo osteoclastic resorption. This observation should once again cast some doubt on the therapeutic use of bone chips in the management of osseous defects. A study of the bone marrow cells in the transplanted tissue reveals that the first 3 days are characterized by necrosis of the hematopoietic elements. Although at 5 days some of the hematopoietic cells survive, most of them degenerate. At this time, however, there is a rapid proliferation of young mesenchymal cells of the bone marrow. At ‘7 and 10 days after transplantation many of these mesen-
Volume
29
Number 3
Autogenous
bone marrow
transplants
479
chymal
(reticulum) cells differentiate into osteohlasts, and new hone fomatiorl It may he suggested from this observation that the undifferentiated mesenchymal (reticulum) cell component of these transplants is the main reason for their survival. The necrosis of the hematopoietic elements observed in the first few days does not, in our opinion, prove or disprove the concept proposed by Burwell” that marrow necrosis provides an inductive mechanism for the differentiation of marrow cells into osteoblasts. The formation of new bone started about 10 days after transplantation, and at about 20 days it became markedly slowed until at 30 days only surface apposition could be seen. This observation would seem to indicate that any bone marrow graft has a limited osteogenetic potential. Whether this is determined by the size of the original transplant or is inherent in some other factor is open to speculation. OCCUIS.
SUMMARY This histologic study concerns autogenous bone marrow grafts in thirty-six rats in which the proximal tibia1 metaphysis and the cranial subcutaneous tissues were the donor and recipient sites, respectively. The transplanted tissue measured about 2 by 2 mm. This study revealed that: 1. Small autogenous bone marrow transplants can survive and produce bone. 2. In the rat, at least, the use of antibiotics and mechanical protective devices is not necessary for osteogenesis. 3. The first 3 days after transplantation are characterized by necrosis of hematopoietic cells. At 5 days the undifferentiated mesenchymal cells of the bone marro’w undergo rapid proliferation, and at 10 days bone formation is a prominent feature. 4. Osteogenetic potential of the marrow transplants is limited. In the present series the maximum osteogenesis was observed at between 10 and 20 postoperative days. The mechanism governing the duration of osteogenesis in a given transplant is not known. The size of the transplant, however, may have a bearing on its productive life. 5. Whereas bone chips and bone dust undergo resorption on transplantation, bone trabeculae which are surrounded by marrow elements do not. This would seem to indicate that the clinical use of bone chips or bone dust in the management of bone defects is of questionable value. It is suggested that, for the survival of a bone trabecula, it should be transplanted as a “hemato-osseous” unit. REFERENCES
1. Sehallhorn, R. G.: The Use of Autogenous Hip Marrow Biopsy Implants for Bony Crater Defects, J. Periodont. 39: 145, 1968. 2. Lyons, H. W.: Effect of Hemopoietic Bone Marrow Transplants on Healing of Maxillofacial Osseous Defects, J. Dent. Res. 43: 827, 1964. of Osteogenesis in the 3. Richter, H. E., Sugg, W. E., Jr., and Boyne, P. J.: Stimulation Dog Mandible by Autogenous Bone Marrow Transplants, ORAL SURG. 26: 396, 1968. 4. Burwell, R. G.: Studies in the Transplantation of Bone, J. Bone Joint Surg. 46-B: 110, 1964.