Calcif. Tissue Int. 29, 225-237 (1979)
Calcified Tissue International 9~, 1979 b} Springer-Verlag
Early Osteogenesis in Compact Bone lsografts: A Quantitative Study of the Contributions of the Different Graft Cells J. Craig Gray and M.W. Elves* Professorial Research Unit. Institute of Orthopaedics, Royal National Orthopaedic Hospital, Stanmore. Middlesex, HA7 4LP. England
Summary. Isografts of cortical bone were transplanted subcutaneously in the rat and the rate of osteogenesis 12 to 14 days later was assessed by measurement of 8-'Sr uptake and by histology. Some grafts were implanted complete whereas others had had one or more of their cellular components (viz. periosteum, endosteum, osteocytes, marrow) removed by mechanical or enzymatic pretreatment. From an analysis of the differences in osteogenesis between grafts devoid of different combinations of cellular components, the contribution of each component to osteogenesis was determined. The results indicate that the endosteal lining cells and marrow stroma together produce more than half of the new bone, the periosteal cells contribute about 30%, the osteocytes possibly make a small (10%) contribution, and the free, hemopoietic cells of the marrow make no significant contribution. Evidence about the relative contributions to osteogenesis of graft and host cells is reviewed and the possible osteogenetic role of bone marrow is discussed. Key words: Osteogenesis -- Experimental bone grafts.
Introduction The osteogenesis that occurs within and around a successful bone graft usually depends on both the graft and the host. For example, a graft that would produce abundant new bone within 2 weeks if implanted as a fresh autograft [1-4] fails to do so if implanted as a fresh ailograft [5-9] or as an X-irradiated (850 rad) isograft [10]. However, 4 to 8 weeks *Present adr Department of Immt, nobiology, GlaxoAIlenburys Research (Greenford) Ltd., Greenford Road. Middlesex. Send qffprint requests to J. Craig Gray at the above address.
after grafting, devitalized isografts such as X-irradiated or freeze-dried, decalcified matrix [1 l] and, under some circumstances, bone allografts [6, 12, 13] can produce new bone. Such evidence favors the two-phase theory of osteogenesis, first put forward by Axhausen [14, 15] to the effect that the main contribution to osteogenesis within the first 4 weeks after implantation of a bone isograft or autograft comes from cells of the graft, whereas after 4 to 8 weeks, cells from the host begin to contribute significantly to new bone formation. There is now strong evidence for this theory, which has been sumarized by Elves [16] and Craig Gray [17]. The studies to be described here deal directly only with the early phase ofosteogenesis, although behavior during this phase may have a profound effect on later, host-derived osteogenesis and hence on the ultimate fate of the graft, as will be discussed later. The objective was to seek evidence for the particular cells within the graft that give rise to osteogenesis. The four main cellular components of a bone graft that are possible sources of osteogenetic cells include the bone marrow, the endosteal lining cells, the periosteum, and the osteocytes. The first three of these have all been suggested at various times to be the main source of bone-forming cells: the purpose of the present study was to ascertain, quantitatively, the relative contributions to first-phase osteogenesis of each of these groups of cells.
Materials and Methods lsografts of rat femur were prepared and implanted subcutaneously, some of the grafts being complete with marrow, endosteum, and periosteum and others with one or more of these components removed, as described below. From the measured differences in osteogenesis 2 weeks after implantation of grafts
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J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in C o m p a c t Bone Isografts
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Table 1. Effect of marrow and e n d o s t e u m
Mean RI SE No.
FEMUR GRAFT Fig. 1. Schematic drawing of a complete c o m p a c t bone graft. P, periosteal cells: O, osteocytes; E, endosteal cells; M, marrow
devoid of different cellular c o m p o n e n t s and implanted into the same recipients, the osteogenetic contribution of each component was determined. M e a s u r e m e n t s of the a m o u n t of new bone formation were made using 85Sr retention histiometry [18] and histology.
Animals The animals used as both donors and recipients of the bone isografts were male rats of the AS2 inbred strain. T h e y were 2-4 m o n t h s of age (average weight 260 g) at the time of grafting.
Grafts Nine different types of cortical grafts were prepared under aseptic conditions from the femora of freshly killed rats. After removal of the femoral diaphysis, excess adherent muscle was trimmed o f f a n d the bone was split longitudinally to produce two hemicylinders of c o m p a c t cortical bone with its constituent marrow, e n d o s t e u m , and periosteum all intact. Such a graft is s h o w n schematically in Figure 1 and is referred to as F.poem. The letters indicate the type of matrix and the cellular constituents of the graft at the time of implantation: F, femur; p, periosteum; o, osteocytes; e, e n d o s t e u m ; m, marrow. From this basic graft, eight other types were prepared as follows: I. F.poe(m). Most of the marrow was r e m o v e d from the bone hemicylinders by gently agitating the graft in sterile saline after which sterile saline was dropped very gently into the concavity from a 19SWG hypodermic needle. T h e s e grafts were still pink in parts of the medulla: the parentheses around the "'m" indicate the presence of a little marrow. 2. F.poe. The marrow was r e m o v e d from the bone hemicylinders by m e a n s of a gentle jet of sterile saline from a 23SWG hypodermic needle fitted to a 20 ml syringe, leaving the grafts almost uniformly white. 3. F.po. The marrow was r e m o v e d by a forcible jet of saline and then the endosteal lining cells were removed by scraping with a small scalpel. For the five remaining types of cortical grafts, the adherent muscle and p e r i o s t e u m were scraped off with a scalpel.
F.oem
F.oe(m)
F.oe
F.o
1 -23
0.906 0.074 19
0.672 0.068 10
0.52 0.035 23
4. F.oem. This is a graft devoid of periosteum but with the marrow and e n d o s t e u m intact. 5. F.oe(m). Most of the marrow was r e m o v e d from the periosteum-free bone as in (1) above. 6. F.oe. The marrow was r e m o v e d from the grafts as in (2) above. 7. F.o. The marrow and e n d o s t e u m were removed as in (3) above. 8. F.(dead). T h e s e grafts were prepared from F.o grafts by immersing them in boiling water for 5 min followed by two cycles of freezing in liquid nitrogen and thawing.
Graft Incubations Some of the grafts were incubated in tissue culture medium or in solutions of e n z y m e s before implantation. The medium used was TC199 (Wellcome) buffered to pH 7.4 with 10,e~ Hepes. The two e n z y m e solutions used were 0.25% (w/v) trypsin (Wellcome) and 750 units/ml collagenase (C-0130, Sigma), both in TC 199. In addition. TC199 with 20% inactivated fetal calf serum [CS] (Flow Labs.) was u s e d as a rinse after each e n z y m e treatment to remove e n z y m e activity. The incubations were carried out at 37~ in a shaking water bath as follows: collagenase, 1 h; TC199 + CS rinse: trypsin, 1 h: TC199 + CS rinse. Control grafts were incubated in TC199 m e d i u m without e n z y m e s but with the two rinses in TC199 + CS.
Implantation Grafts were inserted, using aseptic procedures, into pockets created b e t w e e n the dermis and the panniculus carnosus muscle of the back of the recipient rats. There were normally 6-8 different grafts per rat, each in a separate pocket. The skin incisions were closed by m e a n s of Michel skin clips and sprayed with 'Hibispray" plastic dressing (ICI, Ltd.).
Assessment of Osteogenesis Osteogenesis in the grafts was m e a s u r e d by the s~Sr retention technique [18]. Twelve days after grafting, the rats received 8 /zCi of 8'~SrCI~ per 100 g body weight by the i.p. route. Four days later, the rats were killed and the grafts r e m o v e d together with the hosts' femora; these were all weighed, put into 10% formal saline, and their 8~Sr activities were m e a s u r e d . From the specific activities (counts/min/mg) of the grafts and of the host femur, an osteogenic index (OI) for each graft was calculated thus: 01=
sp act of graft sp act of host femur
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in C o m p a c t Bone Isografts
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Fig. 2. F,oe graft 16 days after implantation. Living bone fib) lining a small cavity in the dead bone (db) of the graft
A further index o f osteogenesis, the relative index (RI), was devised to reduce the effect on the results of any variations in host physiology. The value o f RI for each test graft was calculated by taking the ratio o f its specific activity to that o f a control F . o e m graft which had been in the same recipient. T h u s RI =
sp act o f test graft sp act of control graft
The m e a n (_+ SE o f mean) o f the RI values for each group o f grafts was used to calculate the differences in osteogenesis attributable to the different cellular c o m p o n e n t s of the grafts. However, significance testing, using S t u d e n t ' s two-tailed paired t test [20] was performed on the individual OI values as these, although subject to more variability than RI, should be normally distributed. Values of P < 0.05 were taken to indicate a significant difference in the m e a n s of the populations from which the samples were drawn.
Histology After g a m m a counting, the grafts were put into fresh fixative and were then decalcified and taken to paraffin wax. Representative sections, cut at 5 tzm, were stained with hematoxylin, eosin, and alcian blue [21] and were examined by light microscopy.
Results
Contribution of Marrow The osteogenetic contribution of the marrow in cortical grafts was assessed by comparing F.oem grafts with F.oe(m) and F.oe grafts (Table 1). A comparison o f the mean RI values suggests that the F.oe(m) grafts may have slightly lower osteo-
genesis than the F.oem grafts. However, comparing OI for the two graft types indicates that the difference does not attain significance (P = 0.09). When the mean RI of the marrow-free grafts (F.oe) is examined, it is found to be considerably lower (0.672), and comparison of OI o f the marrow-free grafts against that of the F.oem grafts shows the difference to be highly significant (P = 0.008). To interpret these results, the effect on the graft cells of the procedures used to produce the F.oe(m) and F.oe grafts must be considered. During preparation of the F.oe(m) grafts, about 80% of the marrow was removed, as could be seen in histological sections of nonimplanted F.oe(m) grafts. In the F.oem grafts the medulla was full of marrow, and endosteal lining cells were quite apparent along much of the medullary surface and in the small marrow cavities, whereas the amount o f marrow present in the main cavity of the F.oe(m) grafts varied from about 30% of that in the marrow-intact grafts to almost none. However, the smaller marrow cavities were all full of marrow and the endosteal surfaces were generally lined with cells much as in the marrow-intact grafts, although one graft seemed rather deficient in lining cells. During the slightly more vigorous procedure used to produce an F.oe graft, over 90% of the marrow was removed: histologically, these grafts appeared devoid of marrow in the main cavity although a few of the smaller cavities contained a little: considerably fewer endosteal lining cells were seen in the main recess of these preparations than in the F.oem and F.oe(m) grafts, but the smaller marrow spaces were lined with them.
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J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts
From the results in Table 1, it appears that removal of the final 10-15% of marrow reduces osteogenesis by more than twice as much as the removal of the first 80%: c o m p a r e d with the F . o e m grafts, RI is reduced by (9.4 + 7.4)% in the F.oe(m) grafts and by (32.8 -+ 6.8)% in the F.oe grafts. The difference between these two values (23.4 -+ 10.0)% is significantly different from zero (P < 0.05). So it would appear that the last 10-15% of cells r e m o v e d are qualitatively different from the first 80% of cells and, as the last 10-15% cells are closest to the endosteal surface of the bone, it is likely that they also include endosteal osteoblasts, which will be shown later to be highly osteogenetic. This interpretation is also consistent with the histological findings described a b o v e and below. It appears, therefore, that the bulk of the marrow contributes little or nothing to early osteogenesis: no more than (9.4 + 7.4)% of the F . o e m activity. Histologically, none of the grafts examined after 16 days revealed any periosteal new bone, but luxuriant endosteal w o v e n new bone was seen spanning the medullary cavity in all the F . o e m grafts and in all but one of the F.oe(m) grafts. In that graft, there was not so much endosteal new bone although there was still new bone around most of the haversian canals and lining the smaller marrow recesses. The F.oe grafts had a lot of woven endosteal bone lining the inner surface of the graft, but in only one case did it span the marrow cavity. Osteogenesis had also occurred within the smaller cavities and haversian canals (Fig. 2). A relatively acellular fibrous tissue filled the center of the grafts, but a richer granulation tissue seemed to be entering through the open end of the bone.
Contribution of Endosteum ( Mechanic'al Removal) The main comparisons were of F.o with F.oe(m) and with F . o e m , but a c o m p a r i s o n was also made with F.oe. The histological appearance of the F.oem, F.oe(m), and F.oe grafts has already been described; the F.o grafts were also examined immediately after preparation and after 16 days' implantation. In the fresh grafts, little or no marrow was seen. No endosteal lining cells were observed in the main cavity, but there were a few in some of the smaller recesses. After 16 days in the recipient, an occasional graft showed small amounts of new bone in recesses of the marrow cavity or in haversian canals; however, little or no new bone was o b s e r v e d in most grafts. There was no marrow present and not much granulation tissue in any of the grafts, although there appeared to be some fibrous invasion from outside. The other predominant cell types
seen were osteoclasts or giant cells and there was a fair n u m b e r of healthy looking osteocytes in the grafted bone. The strontium results from which the effect of the e n d o s t e u m was assessed are s u m m a r i z e d in Table 1. The value of RI for the F.o grafts was 0.52 compared with 1.0 for the F . o e m grafts and 0.906 for the F.oe(m) grafts. Thus removing m a r r o w and endosteum together reduced osteogenetic activity by 48.0 -+ 3.5%, a highly significant result (P < 0.001); whereas removing most of the marrow alone (and possibly disrupting the endosteum) was shown above to reduce osteogenesis by only 9.4 -+ 7.4%, which does not attain significance (P = 0.09). Similarly, c o m p a r i s o n of the F.oe(m) graft with the F.o graft (Table 1) shows that RI in the latter is reduced by a further 38.6 -+ 8.2% of the baseline activity, again a highly significant result (P < 0.001). The c o m b i n e d contribution to osteogenesis of the endosteal cells and the last 10-20% of marrow cells (stroma?) is thus great: at least 38.6 +_ 8.2% and probably 48.0 -+ 3.5%, if the free marrow cells' contribution is taken to be nil. The histological results, discussed later, suggest that most of this osteogenetic activity is due to endosteal cells rather than to m a r r o w stroma.
Contribution of Endosteum (Enzymatic Removal) In addition to the experiment involving mechanical removal of the endosteum described above, the osteogenetic activities were measured for F.oe(m) incubated either in TC 199 for 2 h or in collagenase and trypsin, as described above. Measurements were also made using fresh F.oe(m), fresh F.oem, and TC199-incubated F.oem grafts. These experiments were performed partly as a guide to the interpretation of results from a study of osteogenesis in cancellous grafts (Craig Gray and Elves, in preparation), but they also confirm the results obtained above by mechanical removal of endosteum. Histological examination of the enzyme-treated F.oe(m) grafts both before and after transplantation gave results similar to those o b s e r v e d with the F.o (mechanical) grafts. A small or very small amount of endosteal new w o v e n bone was observed in half the grafts examined; none was seen in the others. There seemed to be a high rate of survival of graft osteocytes. The TC199-incubated F.oe(m) grafts a p p e a r e d very similar to their nonincubated counterparts already described. In fact, the incubated grafts, as well as having luxuriant endosteal new bone, possibly had rather more new bone in and around the haversian canals and other recesses of the graft. There also seemed to be greater survival
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Table 2. Effect of e n d o s t e u m (enzyme treatment)
Mean RI SE No.
F.oem
F.oe(m) enzymes
F.oe(m) TC199
F.oe(m)
F.oem TCI99
1 -23
0.552 0.041 16
1.028 0.081 8
0.835 0.091 8
0.910 0.082 15
of superficial osteocytes of the grafted bone than was seen with the unincubated grafts. Comparison of the s'~Sr results from the enzyme incubated F.oe(m) grafts with those of the fresh control F.oem grafts (Table 2) shows a highly significant drop (P < 0.00l) for the enzyme-treated grafts. The reduction in RI is 44.8 +- 4.1% of the control activity, which is close to the value 48.0 -+ 3.5% found previously for mechanical removal of the endosteum and marrow. Such a close correspondence (P = 0.55) suggests that the effect of the enzymes is similar to that of mechanical removal o f endosteum. A surprising result shown in Table 2 is that, although F.oe(m) grafts incubated in TC 199 alone for 2 h gave an SSSr index not significantly different from that of fresh F.oem control grafts (P = 0.75) they did significantly better than fresh F.oe(m) grafts (P < 0.005), whereas 2 h incubation of F.oem grafts in TC199 did not raise the eventual level o f osteogenesis (P = 0.12). Taking these results with the previous finding that removing 80% of the marrow possibly reduced osteogenesis in a fresh graft by 9.4 +_ 7.4%, it appears that, whatever the mechanism by which this possible reduction occurred, it can be reversed by 2 h incubation of the marrow-depleted graft in TC199. However, when the incubation was performed with the marrow intact, a rise did not occur, presumably because it was due to the recovery or stimulation of osteogenic endosteal or stroma cells damaged during the depletion procedure: the release of enzymes from dying marrow cells may be harmful to those cells. These findings, of course. add further weight to the argument that the free marrow cells make no positive contribution whatsoever to early osteogenesis.
Contribution of Periosteum Histological examination of F.poem and F.oem grafts revealed the presence of periosteal new bone in the former (Fig. 3A) but not in the latter, the periosteal surface of which generally had a shredded appearance (Fig. 3B). The periosteal new bone of the
Fig. 3A. The periosteal surface of an F . p o e m graft 16 days after implantation. M a n y living osteocytes are seen in the lacunae of the periosteal new bone (nb) at the top of the micrograph but most of the lacunae of the graft bone (gb) below are empty. Compare this with Fig. 3B
F.poem grafts was lamellar in form whereas the luxuriant endosteal new bone present in both sets of grafts, and bridging the medullary space, was of woven appearance: its trabeculae were lined by plump osteoblasts in many places (Fig. 4). Within the old bone of the F . p o e m grafts, some of the superficial osteocytes, both periosteal and endosteal, appeared normal although the deeper lacunae were all empty. In the F.oem grafts, no surviving periosteal osteocytes were seen but there were some close to the endosteal surface. In both sets of grafts, osteoclasts were seen occasionally, usually (but not always) against the surface of the graft bone. There were blood vessels entering cavities near the surfaces of the old bone (Fig. 5) and in the endosteal and periosteal new bone (Fig. 3A), and larger ones were seen in the inflammatory tissue close to the endosteal new bone (Fig. 6). For the s~'Sr assessment of the osteogenetic contribution of the periosteum, eight different types of grafts were used. The sets of grafts varied in the presence or absence of marrow, endosteum, and periosteum, but they could be arranged in four pairs
230
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in C o m p a c t Bone lsografts
Fig. 4. The endosteal tissue in an F.poem graft 16 days after implantation. T h e s e trabeculae of woven new bone appear quite healthy, with o s t e o c y t e s in the lacunae and osteoblasts lining m a n y of the surfaces
Fig. 3B. The periosteal surface of an F.oem graft 16 days after implantation. No new bone is seen at the periosteal surface (top) and the outer layers of the graft bone (gb) have a shredded appearance. C o m p a r e this with Fig. 3A
Table 3. Effect of periosteum
Mean RI SE No.
F.oem
F.poem
F.oe
F.poe
F.oelm)
F.poelm)
F.o
F.po
1 -23
1.367 0.212 8
0.688 0.066 4
1.006 0.147 4
0.982 0.088 4
1.112 0.085 4
0.560 0.053 7
0.717 0.082 7
where one of each pair was periosteum-free and the other had the periosteum intact but was otherwise identical. It will be seen in Table 3 that the value for the mean RI is in each case greater for the periosteumintact grafts than for the corresponding periosteumfree grafts, although the differences did not generally attain significance as measured by the paired t test. H o w e v e r , if all the periosteum-free grafts are pooled and all the periosteum-intact grafts are pooled, the difference between these two groups is significant (P < 0.01). This suggests that the periosteum makes a small but significant contribution to osteogenesis. The proportion of the total graft osteogenesis due to the periosteum was estimated by taking the difference in mean RI of the perioste-
um-intact and the corresponding periosteum-free grafts of each type and calculating the weighted mean of these. Using the relevant data in Table 3, the increase in osteogenesis due to periosteum, expressed as a fraction of the osteogenesis in F.oem graft, is 0.253 _+ 0.087. So the periosteum would have contributed approximately a further 25.3 -+ 8.7% of osteogenesis to the F . o e m graft, which was the base line for RI calculations.
Contribution (~['Osteocytes The importance of the graft osteocytes for osteogenesis in cortical bone was examined by comparing the osteogenesis in F(dead) grafts with that
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts
231
Table 4. Effect of osteocytes and of matrix
Mean SE No.
F.oem
F.o
F(dead)
F.o ~
F(dead) b
1 -7
0.515 0.049 12
0.368 0.028 12
0.437 0.031 9
0.338 0.032 9
a F.o a as F.o, but omitting grafts with perihaversian new bone (see text) F(dead) h as F(dead), but omitting the grafts in same animals as those omitted in F.o a
Fig. 5. The endosteal surface of an F.poem graft 16 days after implantation. A blood vessel can be seen penetrating from the medulla, through a lining of living new bone (nb) into a cavity in the dead graft bone (gb)
in f r e s h F . o g r a f t s ( T a b l e 4). T h e d i f f e r e n c e in m e a n R I o f t h e d e a d a n d l i v i n g g r a f t s w a s f o u n d to b e 14.7 -+ 5 . 6 % o f t h e a c t i v i t y o f t h e c o n t r o l F . o e m g r a f t s . T h e p a i r e d t t e s t s h o w s this d i f f e r e n c e to be signifi c a n t (P < 0.02). It s e e m s l i k e l y , h o w e v e r , t h a t t h i s d i f f e r e n c e in osteogenesis between the dead and living grafts was n o t e n t i r e l y d u e to t h e o s t e o c y t e s . T h e r e w o u l d have been an osteogenetic contribution from any endosteal osteoblasts that had not been removed from the F.o grafts by the scraping procedure. Evid e n c e f o r this is the h i s t o l o g i c a l f i n d i n g o f e n d o s t e a l lining c e l l s in s o m e o f t h e s m a l l e r m a r r o w c a v i t i e s and haversian canals of the freshly prepared F.o g r a f t s a n d t h e a p p e a r a n c e , 16 d a y s l a t e r , o f s m a l l a m o u n t s o f n e w b o n e in t h e s e a r e a s o f t h r e e o f t h e grafts. The F(dead) grafts, on the other hand, showed no new bone. If the osteocyte contribution
Fig. 6. The endosteal surface and medullary cavity of an F.oem graft 16 days after implantation. Several large blood vessels (by) may be seen close to the trabeculae of woven new bone filling the medullary cavity, and there are 3 resorption cavities in the dead graft bone (gb) below
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J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in C o m p a c t Bone Isografts
to osteogenesis is recomputed, omitting both the F.o grafts with new bone in the areas mentioned and the corresponding F(dead) grafts, it is reduced to 9.9 • 4.5% and the paired t test shows the difference between the grafts to be on the borderline of significance (P = 0.08). The value 9.9 • 4.5% may thus be ascribed to activity of the osteocytes.
o ~ 70
0
040
Contrib.tion gf Matrix
30 (b
The contribution of the matrix to the strontium uptake is given in Table 4 by the value of RI for F(dead). This value (0.368 • 0.028) is significantly different from zero (P < 0.001). However, histological examination of the grafts did not reveal new bone in any of the dead matrix grafts, so we conclude that the matrix contribution to osteogenesis is negligible. Therefore most, if not all, of the strontium uptake must have been due to passive deep exchange between the mineral ions of the matrix and the ~sSr in the blood. Such activity will be present, as an approximately constant background, in all the bone grafts. Thus the values found for the relative contributions of the different cell populations will not be affected by the deep exchange, since those values were found by subtraction. However, in expressing each cell population's contribution as a fraction of total graft osteogenesis, one must allow for the deep exchange, as shown below.
Summary gf Results The contribution of each component of the grafts to strontium uptake has been calculated above with respect to the F.oem graft taken as 100%. On this basis, the following percentages have been found: Periosteum Free marrow cells Endosteum + marrow stroma Osteocytes Matrix (passive exchange)
25.3 _~ 8.8 9.4 _+ 7.4 or 0 38.6 + 8.2 or 48.0 • 3.5 9.9 • 4.5 36.8 : 2.8 120.0 + 15.1 or 120.0 _~ 10.9
The total is well over 100% because it includes a contribution of approximately 25% from the periosteum which is not included in the "'baseline" graft: the remaining discrepancy is adequately covered by the standard error. In order to normalize these results to take account of (a) the passive exchange contribution from the matrix and (b/ the fact that the baseline graft
,~
,-7 " - i ~ "1 ; I
: i I
P
O(E~S) M
Fig. 7. Histogram of the percentage contributions to osteogenesis of the periosteum (P), osteocytes (Ot. endosteal lining cells and marrow s t r o m a (E + S/, and free marrowy cells tM). Bars indicate SEM
does not include periosteum, the following changes are made: 1. The matrix contribution of 36.8 + 2.8 is omitted in adding up the cellular osteogenetic activity, giving 83.2 • 14.8 or 83.2 • 10.5 for the totals. 2. The totals are multiplied by the normalizing factor, 100/83.2 = 1.2, and the contribution of each graft element is multiplied by the same factor to express it as a fraction of the F.poem osteogenetic activity. This gives: Periosteum Free marrow cells Endosteum + marrow stroma Osteocytes
30.4 • 10.6 !1.3 • 8.9 or 0 46.3 _+ 9.8 or 57.6 • 4.2 11.9 • 5.4
These values are expressed as percentages of the activity of the complete F . p o e m graft, assuming from the histological evidence that all the activity in the dead matrix is due to passive uptake or deep exchange and not to osteogenesis. However, the relative importance of each of the above cell populations for osteogenesis is independent of this last assumption. The above results are shown graphically in the histogram (Fig. 7).
Discussion
The above plants with sSSr figures tween days
results are all based on 16-day transs'~Sr administered on day 12. Thus the represent the osteogenetic activity be12 and 16 after grafting, and, because of
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts the relatively rapid clearance of strontium from the blood, most of the label will be in new bone mineral laid down on days 12 to 14 after grafting. It is quite probable, however, that labeling of new bone continues during days 15 and 16 at a higher rate than the blood-strontium level would suggest. There is likely to be local reusage of strontium that has been passively exchanged into. then out of, bone mineral. The histological results, on the other hand, represent the integrated activity of new bone formation and reformation over the whole 16-day period. Both the "~'Sr and the histological data and their interpretation apply directly only to early new bone growth in grafts: that is, to first-phase, graft-derived osteogenesis, in terms of the two-phase theory of Axhausen [14, 15] and Chalmers [6]. There is, however. some evidence that the second phase of osteogenesis, although probably produced primarily by host-derived cells, is also dependent to some extent on the cells of the graft. In fact, a good first phase of osteogenesis appears to be a stimulus to a good second phase, as suggested by Vainio [22] and by Axhausen [15]. This is d e m o n s t r a t e d in a comparison of the amount of late-phase osteogenesis in devitalized and living autografts where the new bone formation during the period 4 to 8 weeks after implantation is much less prolific in the former than in the latter grafts [10]. A similar difference is also observed in the second-phase bone growth in allografts transplanted to normal and immunosuppressed recipients, respectively [23]: once again, a good first phase, in the i m m u n o s u p p r e s s e d recipients, leads to a good second phase: a poor first phase in the normal recipients results in a poorer second phase. It is also found that allografts containing many minor antigen disparities give a much poorer first phase than allografts having only a few minor disparities with one H-I (Ag-B) disparity and that no second-phase osteogenesis is o b s e r v e d in the former group whereas the latter group show a significant late phase in about 30% of recipients [24]. In sum, it appears that a good early phase of osteogenesis usually leads to a good late phase, either by supplying graft-derived cells to supplement the osteogenic cells coming from the host or else by producing some osteogenic stimulus that ensures a good supply of host-derived osteoblasts. Thus, although the results recorded in this work apply directly only to early osteogenesis, they may well bear indirectly on the long-term success or failure of bone grafts.
Perioste,m The periosteum in the young mammal is a two-layered structure, the inner (cambiuml layer contain-
233
ing closely spaced, interdigitating osteogenic cells and functional osteoblasts, whereas the outer, fibrous layer contains more widely spaced fibroblasts [25]. It is now generally accepted that the cambium layer, certainly in a young animal, contributes to graft and fracture osteogenesis [26-343. The results presented here confirm and quantify these observations for cortical grafts prepared from young bone.
Osteocytes The small possible osteogenetic contribution attributed to the osteocytes is in broad agreement with results for the osteocytes of cancellous grafts [17; Craig G r a y and Elves, in preparation]. The histological findings for the o s t e o c y t e s were similar to those described in most recent reports [3, 9, 35], including the quantitative studies of Heslop et al. [36], who reported a steep reduction in the number of surviving osteocytes in the first few days after subcutaneous autografting of cylinders of c o m p a c t bone. The decrease continued at a reduced rate over the following 2 weeks and then stabilized with about 40% survival. In these older grafts, the surviving osteocytes were found in a band less than 300 p,m wide around the cortical surface. This was contrary to the findings of Nishimura et al. [37], who noted best survival in the mid-layers of the cortex. A t e n d e n c y for superficial osteocytes to survive was also noticed in the present experiments, but this applied to o s t e o c y t e s near the medullary as well as the cortical surface. This difference from the findings of Heslop and her colleagues may be because the grafts in the present experiment were hemicylinders which allowed direct contact of tissue and fluids from the host bed with both the periosteal and the endosteal surfaces. Another observation, which corroborates this interpretation, was made during histological examination of grafts incubated in TC199 before implantation. Such grafts seemed to contain a higher proportion of living osteocytes in the old bone than did similar grafts implanted fresh. This impression was not subjected to any quantitative m e a s u r e m e n t s , but it may give an explanation of an enhancing effect on osteogenesis of short incubation in culture medium before grafting [17: Craig Gray and Elves, in preparation]. It seems likely that while the grafts are being incubated in the shaking water bath, the moving culture fluid penetrates much more readily along the haversian canals and small marrow spaces to the deeper osteocytes than does the relatively static tissue fluid in the early graft bed. The culture
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J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts
medium that reaches many osteocytes may be sufficient to keep them alive for the first few days after grafting until the ingrowing capillaries have been established. If this additional osteocyte survival is the explanation of the enhanced osteogenesis in some incubated grafts, it suggests that the osteocytes play some part in osteogenesis. However, it seems more likely that other osteogenic elements of the graft, such as osteoblasts lining the haversian canals, also have improved survival after incubation and that they are responsible for the increased osteogenesis. Heslop and her colleagues [36] came to no firm conclusion as to the possible role of the osteocytes in new bone formation. Although they found no quantitative relationship between osteogenesis and osteocyte survival, they suggested, on the basis of the results of de Bruyn and Kabisch [27] where dead and living grafts were compared, that living osteocytes may stimulate osteogenesis in their vicinity. However, it appears that such a stimulus, if it exists, has a relatively small effect compared with other factors: Heslop and her colleagues found much more abundant new bone in the hollow center of the grafts where no osteocytes survived than they did around the outer margin of the grafts where osteocyte survival was good. Although not all workers accept that some osteocytes survive in an autograft [34, 38, 39], there appears to be general agreement that any direct osteogenetic contribution by them is small.
Endosteum
The endosteal lining cells and the marrow have been shown to be responsible for well over half the new bone formed, and evidence has been presented suggesting that there may be an additional small contribution from intrahaversian osteoblasts. The free marrow cells, on the other hand, have been shown to make a negligible contribution to osteogenesis, suggesting that the endosteal cells alone make the major contribution. However, it might be argued that the "'easily removed ~" marrow is made up largely of free hemopoietic cells only and that there may remain within the F.oe(m) grafts elements of marrow stroma, fibrous or vascular tissue. It might be further argued that the stroma may have an osteogenetic role and that subjecting the graft to a jet of saline to produce the F.oe graft may disrupt or remove the stroma as well as part of the endosteum. The consequent reduction in osteogenesis would then possibly be due in part to the removal of the marrow stroma. This hypothesis is compatible with the tracer experi-
ments of the present studies, but the histological evidence does not favor it. The new bone in the F.oe grafts lay mainly around the inner rim of the graft in most cases, although it did bridge the marrow cavity in one graft. In the F . o e m and all but one of the F.oe(m) grafts, on the other hand, the marrow cavity was always bridged by new bone. There are three possible explanations of this: (a) The remaining endosteal cells in the F.oe grafts are too few to effect a bridge in 16 days. (b) The stroma of the marrow is partly responsible for producing the new bone that forms the bridge. (c) The marrow stroma acts by osteoconduction [39] of endosteal bone-forming cells into the marrow space, where they lay down trabeculae of woven bone. Point (c) fails to explain why osteogenesis in the F.oe(m) grafts was almost as great as in the F.oem grafts and why new bone spanned the medullary cavity of both sets of grafts. If the marrow stroma was disrupted, as it certainly was in the F.oe(m) grafts, its osteoconductive role would fail. If point (b) were true the F.oe(m) grafts would be expected to have almost as much stroma as (although much less marrow than) the F.oem grafts. In other words, the remaining marrow would be particularly rich in stroma in the freshly prepared grafts. No such effect was observed, although it is impossible to be definite about such a negative observation. Thus it appears that point (a) is the most likely explanation and that the drop in strontium uptake observed in marrow-free grafts was due largely, if not entirely, to disruption and loss of endosteal cells. However, there remains the possibility that some of the drop was due to the removal of stromal elements of the marrow. The free marrow cells, however, certainly did not contribute to osteogenesis. The important role of the endosteum for early osteogenesis in bone grafts does not appear to have been demonstrated quantitatively before. Probably the nearest approach was made by Bassett and his colleagues [26] who concluded, on the basis of a histological study of the repair of osseous defects with and without endosteal curettage, that ~'endosteal cells play a critical role in osteogenesis'" and that "'osteogenic cells resident in Haversian canal systems . . . seem to participate in the formation of new b o n e . " Other authors have, of course, attributed an osteogenetic role to endosteal cells but usually on the basis of rather circumstantial evidence and without any quantitative measure of the endosteal contribution: see, however, Marotti et al. [40] and Zallone [41]. Tonna and Cronkite [42], on the basis of :~H-
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts thymidine uptake by femur fractures in mice, concluded that the proliferative activity of the osteogenic cells of the periosteum and within the intertrabecular spaces and lining the trabeculae was much greater than that of the endosteal cells lining the medullary canal of the diaphyseal cortex. H a m and Harris [9], however, in discussing the endosteal contribution to the formation of the internal callus during fracture repair, stressed the c o m m o n osteogenic lineage of the cells of the cambium layer of the periosteum, of the endosteal cells of the main medullary cavity and trabeculae, and of the osteoblasts that line the haversian canals. They attributed an osteogenetic role to all these cells. In considering the clinical relevance of these results, it must be rem e m b e r e d that the rat is a small mammal c o m p a r e d with man and the proportions of the different osteogenic cells in the bones of the two species are different. In particular, the relative number of haversian. c o m p a r e d with medullary, endostea[ cells increases as the thickness of the bone increases [9]. Thus the proportional osteogenetic contribution of the osteogenic cells of the haversian canals, compared with those of the m a r r o w cavity and the periosteum, is probably much greater in man than in small laboratory mammals.
Marrow
Perhaps the major surprise in the present results is the unimportance of the free marrow cells for early osteogenesis. On the face of it, there appears to be a definite contradiction between the present findings and those of the many workers who have ascribed an important osteogenetic role to cells within the marrow [43-51]. Most of these other experiments have involved the grafting of marrow that has been r e m o v e d from the shaft of a long bone (usually the femur) by washing out, aspiration, or curettage. Many of those employing these techniques admit the possibility or likelihood that endosteal cells are disturbed during removal of the marrow and may be included in the marrow specimen obtained. For example, Burwell [52] states that in his experiments, "'it cannot be denied that endosteal osteoblasts may have contributed to the formation of bone by at least some of the marrow a u t o g r a f t s " ; see also papers by Petrakova et al. [53], Friedenstein et al, [46], and Boyne [54]. H o w e v e r , several authors mention that care was taken to exclude m a c r o s c o p ic bone spicules from the marrow, although most do not mention any particular precautions, and it is also pointed out that occasional bone spicules observed in grafts do not appear to act as foci of osteogenesis.
235
The results of the present work with F.oe grafts, produced by subjecting the m a r r o w in the cavity to a gentle jet of saline, show how easily the endosteum may be disrupted and r e m o v e d along with the marrow and how. without dislodging any bone spicules, one may inadvertently include endosteal osteoblasts with the marrow. Some other evidence for this view is the inconsistency of results obtained by m a n y authors using marrow autografts. The usual method of scoring has been on the basis of the proportion of grafts yielding bone: generally no account has been taken of the quantity of new bone produced which may vary from a "'solitary small bone spicule" to "'very a b u n d a n t " [31]. Danis [55] and Burwell [52] present lengthy tables summarizing the incidence of bone formation within heterotopic m a r r o w autografts, obtained by m a n y workers. The success rate varies from nil to 100%, most authors achieving about 30% to 60%. It seems quite likely that a similar proportion of grafts may have been contaminated by a significant number of endosteal cells, especially as the m a r r o w has, in e v e r y case but one, been r e m o v e d from the shaft of a long bone (tibia, femur, radius, or ulna) where the endosteum is easily dislodged. A n o t h e r criticism of some o f these e x p e r i m e n t s is that the method o f assessing osteogenesis, usually by histological examination with an arbitrary scoring system, is subjective and is statistically suspect. Often the n u m b e r of grafts examined at each time interval is very small, sometimes grafts implanted for different intervals are pooled unjustifiably and inappropriate statistical tests are used. H o w e v e r , although the present experiments show that the free marrow cells do not contribute to early osteogenesis, there are two further possibilities for reconciling the present findings with those mentioned above. 1. In the present experiments, a possible osteogenetic role for the marrow s t r o m a is not excluded. The lack o f osteogenetic capacity d e m o n s t r a t e d for the free m a r r o w cells, however, is consistent with the findings of Friedenstein and his colleagues [46, 53, 56-58] and Amsel and Dell [19] that the hemopoietic stem cell line of the m a r r o w is independent o f the osteogenic cell line. 2. Most e x p e r i m e n t s supporting the idea o f an osteogenetic role for marrow use grafts implanted for more than 2 weeks. It is possible that, if the present work comparing osteogenesis in marrowdepleted and marrow-intact grafts had been performed o v e r 28 days instead of 16 days, there would have been a significant difference between the two types of grafts. In that case, it could be asserted either that the osteogenic stem cells in the m a r r o w take more than 2 weeks to b e c o m e functional osteo-
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J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone lsografts
b l a s t s o r t h a t , as L e v a n d e r [31] m a i n t a i n e d , t h e m a r r o w h a s a r o l e in i n d u c i n g o s t e o g e n e t i c a c t i v i t y in o t h e r g r a f t o r h o s t cells.
General Conclusions E v i d e n c e h a s b e e n c i t e d s u g g e s t i n g t h a t t h e ultim a t e f a t e o f a b o n e g r a f t is l a r g e l y d e t e r m i n e d b y its o s t e o g e n e t i c p e r f o r m a n c e in t h e first f e w w e e k s aft e r g r a f t i n g . T h e r e is r e a s o n to b e l i e v e , t h e r e f o r e , that the results presented here, although based on t h e s t u d y o f 16-day h e t e r o t o p i c c o m p a c t b o n e isog r a f t s in y o u n g a d u l t rats, h a v e a m o r e g e n e r a l r e l e v a n c e to t h e b e h a v i o r o f b o n e g r a f t s . T h e f o l l o w i n g observations are based on the experimental evidence that has been presented. The living cells of a bone graft play an important p a r t in p r o d u c i n g t h e e a r l y n e w b o n e in a n d a r o u n d the graft. The endosteal and intrahaversian osteob l a s t s p r o d u c e a b o u t 60%. o f t h e n e w b o n e , i n c l u d ing a p o s s i b l e c o n t r i b u t i o n b y s t r o m a l c e l l s o f t h e marrow, and the periosteal cells are responsible for a p p r o x i m a t e l y 3 0 % o f t h e o s t e o g e n e s i s . T h e r e is little o r n o c o n t r i b u t i o n to o s t e o g e n e s i s b y g r a f t osteocytes or by the free hemopoietic cells of the marrow. The endosteal osteoblasts of cortical bone a r e e a s i l y r e m o v e d w h e n m a r r o w is g e n t l y w a s h e d out of the medullary cavity.
Acknowledgments. This study was performed in partial fulfillment of the requirements for the degree of Master of Philosophy of the University of London. The authors express their thanks to Mr. D. Sayers for technical assistance and to Mrs. V. King and Mrs. A. Bradley for typing the manuscript.
References 1. Burwell, R.G.: The fate of bone grafts. In A.G. Apley (ed.): Recent Advances in Orthopaedics. Churchill, London, 1969 2. Lance, E.M.: Bone and cartilage. In J.S. Najarian, R.L. Simmons (eds.): Transplantation. Lea & Febiger, Philadelphia, 1972 3. Thorogood, P.V., Craig Gray, J.: The cellular changes during osteogenesis in bone and bone marrow composite autografts, J. Anat. 120:27-47, 1975 4. Urist, M.R.: The substratum for bone morphogenesis, Dev. Biol. [Suppl.]4:125-163, 1974 5. Burwell, R.G.: Studies in the transplantation of bone. V. The capacity of fresh and treated homografts of bone to evoke transplantation immunity, J. Bone Joint Surg. 45:386401. 1963 6. Chalmers, J.: Transplantation immunity in bone homografting, J. Bone Joint Surg. 41:160-179, 1959 7. Dubuc, F.L., Urist, M.R.: The accessibility of the bone induction principle in surface-decalcified bone implants. Clin. Orthop. 55:217-223, 1967
8. Elves, M.W., Craig Gray, J., Thorogood, P.V.: The cellular changes occurring with allografts of marrow-containing cortical bone, J. Anat. 122:253-269, 1976 9. Ham, A.W., Harris, W.R.: Repair and transplantation of bone. In G.H. Bourne (ed.): The Biochemistry and Physiology of Bone, 2rid Ed., Vol. III, Chap. 10. Academic Press, New York, 1971 10. Elves, M.W., Pratt, L.M.: The pattern of new bone formation in isografts of bone. Acta Orthop. Scand. 46:549-560. 1975 11. Urist, M.R.: Bone formation by autoinduction, Science 150:893-899, 1965 12. Anderson, K.J.: The behaviour of autogenous and homogenous bone transplants in the anterior chamber of the rat's eye, J. Bone Joint Surg. 43:980-995, 1961 13. Enneking, W.F.: Histological investigations of bone transplants in immunologically prepared animals, J. Bone Joint Surg. 39:597-615, 1957 14. Axhausen, W.: Die Knochenregeneration--ein zweiphasisches Geschehen!, Zbl. Chir. 11:435-442, 1952 15. Axhausen. W.: The osteogenetic phases of regeneration of bone, J. Bone Joint Surg. 38:593-600, 1956 16. Elves, M.W.: Newer knowledge of immunology of bone and cartilage, Clin. Orthop. 120:232-259. 1976 17. Craig Gray, J.: Factors affecting osteogenesis of bone isografts in the rat, M. Phil. Thesis, University of London, 1977 18. Elves, M.W.: An evaluation of the use of strontium-85 for the assessment of experimental bone grafts, Acta Orthop. Scand. 45:641-651, 1974 19. Amsel, S.. Dell, E.S.: The radiosensitivity of the bone-forming process of heterotopically-grafted rat bone-marrow, Int. J. Radiat. Biol. 20:119-127, 1971 20. Ostle, B.: Statistics in Research. Iowa State University Press, 1963 21. Sayers, D.C.J.: A general staining technique for the demonstration of new bone. J. Sci. Technol. 17:14, 1973 22. Vainio, S.: Observation on the regeneration of an autogenous transplant of the bone. An experimental investigation, Acta Chir. Scand. 100:86-109, 1950 23. Goldberg, V.M., Lance, E.M.: Revascularization and accretion in transplantation, J. Bone Joint Surg. 54:807-816, 1972 24. Elves, M.W.: Studies of the behaviour of allogenic cancellous bone grafts in inbred rats, Transplantation 19:416423, 1975 25. Tonna, E.A.: Electron microscopy of aging skeletal cells. III. The periosteum, Lab. Invest. 31:609-632, 1974 26. Bassett, C.A.L., Creighton. D.K., Stinchfield, F.E.: Contributions of endosteum, cortex, and soft tissue to osteogenesis, Surg. Gynecol. Obstet. 112:145-152, 1961 27. de Bruyn, P.P.H., Kabisch, W.T.: Bone formation by fresh and frozen, autogenous and homogenous transplants of bone, bone marrow and periosteum. Am. J. Anat. 96:375417, 1955 28. Burman, M.S.. Umansky, M.: An experimental study of free periosteal transplants, wrapped around tendon, J. Bone Joint Surg. 12:579-594, 1930 29. Kahnberg, K.-E.: Influence of periosteum on healing of tibial defects in the rat, Odontol. Rev. 25:157-164, 1974 30. Leadbetter, G.W.: Periosteum, a living bone suture, Arch. Surg. 22:754-785, 1931 31. Levander, G.: An experimental study of the role of the bonemarrow in bone regeneration, Acta Chit. Scand. 83:545-560, 1940
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts
32. Narang, R., Laskin, D.M.: Experimental osteogenesis at fracture sites and gaps, J. Oral Surg. 34:225-231, 1976 33. Ritsil/i, V., Alhopuro, S. Rintala, A.: Bone formation with free periosteal grafts in reconstruction of congenital maxillary clefts, Ann. Chir. Gynaecol. 65:342-344, 1976 34. Urist, M.R.. McLean, F.G.: Osteogenetic potency and newbone formation by induction in transplants to the anterior chamber of the eye, J. Bone Joint Surg. 34:443-476, 1952 35. Hutchison, J.: The fate of experimental bone autografts and homografts. Br. J. Surg. 39:552-56l, 1952 36. Heslop, B.F., Zeiss, I.M., Nisbet, N.W.: Studies on transference of bone. I. A comparison of autologous and homologous bone implants with reference to osteocyte survival, osteogenesis and host reaction, Br. J. Exp. Pathol. 41:269287, 1960 37. Nishimura, K.K.. Yaeger, J.A., Sabet, T.Y.: Fate ofosteocytes in adult mouse whole bone isografts and homografts, Anat. Rec. 144:85-96, 1962 38. Reynolds, F.C., Oliver, D.R.: Experimental evaluation of homogenous bone grafts, J. Bone Joint Surg. 32:283-297, 1950 39. Urist, M.R.: Practical applications of basic research on bone graft physiology. In B. Evans (ed.): Instructional Course Lectures, Am. Acad. Orthop. Surgeons, 25:1-26. C.V. Mosby Co., St. Louis, 1976 40. Marotti, G., Zallone, A.Z., Ledda, M.: Number, size and arrangement of osteoblasts in osteons at different stages of formation, Calcif. Tissue Res. [Suppl.]21:96-101, 1976 41. Zallone, A.Z.: Dimensioni e forma degli osteoblasti su superfici di osso spugnoso a diversa attivita osteogenica. Boll. Soc. Ital. Biol. Sper. 52:528-531, 1976 42. Tonna, E.A., Cronkite, E.P.: Cellular response to fracture studied with tritiated thymidine, J. Bone Joint Surg. 43:352362. 1961 43. Burwell. R.G.: Biological mechanisms in foreign bone transplantation. In J.M.P. Clarke (ed.): Modern Trends in Orthopaedics. Vol. 4, Chap. 8. Butterworth, London, 1964 44. Cummine, J., Nade, S.: Osteogenesis after bone and bonemarrow transplantation, Acta Orthop. Scand. 48:15-24, 1977 45. Danis, A.: Le r61e de la moelle osseuse dans l'ost6ogen~se r6paratrice, Bull. Acad. Med. Belg. 129:173-198. 1974 46. Friedenstein. A.J., Piatetzky-Shapiro, I.I.. Petrakova, K.V.: Osteogenesis in transplants of bone marrow cells, J. Embryol. Exp. Morphol. 16:381-390, 1966
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47. Nade, S., Burwell, R.G.B.: Decalcified bone as a substrate for osteogenesis. An appraisal of the interrelation of bone and marrow in combined grafts, J. Bone Joint Surg. 59:189196. 1977 48. Plenk, H., Hollmann, K., Wilfert, K.-H.: Ekperimental bridging of osseous defects in rats by implantation of Kiel bone containing fresh autologous marrow. J. Bone Joint Surg. 54:735-743, 1972 49. Simmons, D.J., Lester, P.A., Ellsasser, J.C.: Survival of osteocompetent marrow cells in vitro and the effect of PHAstimulation on osteoinduction in composite bone grafts, Proc. Soc. Exp. Biol. Med. 148:986-990, 1975 50. Tavassoli, M., Maniatis, A., Binder, R.A., Crosby, W.H.: Studies on marrow histogenesis. II. Growth characteristics of extramedullary marrow autotransplants, Proc. Soc. Exp. Biol. Med. 138:868-870, 1971 51. Yeager, J.E., Boyne, P.J.: Use of bone homografts and autogenous marrow in restoration of edentulous alveolar ridges, J. Oral Surg. 17:185-189, 1969 52. Burwell, R.G.: Studies in the transplantation of bone. VII. The fresh composite homograft-autograft of cancellous bone. An analysis of factors leading to osteogenesis in marrow transplants and in marrow-containing bone grafts, J. Bone Joint Surg. 46:110-140, 1964 53. Petrakova, K.V., Tolmacheva, A.A., Friedenstein, A.J.: Bone formation occurring in bone marrow transplantations in diffusion chambers, Bull. Exp. Biol. Med. 12:87-98, 1963 54. Boyne, P.J.: Autogenous cancellous bone and marrow transplants, Clin. Orthop. 73:199-209, 1970 55. Danis, A.: Etude de l'ossification darts les greffes de moelle osseuse, Acta Chit. Belg. [Suppl.]Ill:l-120. 1957 56. Friedenstein, A.J., Chailakhjan, R.K., Lalykina, K.S.: The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow' and spleen cells, Cell Tissue Kinet. 3:393-403, 1970 57. Friedenstein, A., Kuralesova, A.I.: Osteogenic precursor cells of bone marrow in radiation chimeras, Transplantation 12:99-108, 1971 58. Friedenstein. A.J.. Petrakova, K.V., Kurolesova, A.I., FroIova, G.P.: Heterotopic transplants of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues, Transplantation 6:230-247, 1968 Received October 26, 1978 / Accepted July II, 1979
Calcified Tissue International 9~, 1979 b} Springer-Verlag
Early Osteogenesis in Compact Bone lsografts: A Quantitative Study of the Contributions of the Different Graft Cells J. Craig Gray and M.W. Elves* Professorial Research Unit. Institute of Orthopaedics, Royal National Orthopaedic Hospital, Stanmore. Middlesex, HA7 4LP. England
Summary. Isografts of cortical bone were transplanted subcutaneously in the rat and the rate of osteogenesis 12 to 14 days later was assessed by measurement of 8-'Sr uptake and by histology. Some grafts were implanted complete whereas others had had one or more of their cellular components (viz. periosteum, endosteum, osteocytes, marrow) removed by mechanical or enzymatic pretreatment. From an analysis of the differences in osteogenesis between grafts devoid of different combinations of cellular components, the contribution of each component to osteogenesis was determined. The results indicate that the endosteal lining cells and marrow stroma together produce more than half of the new bone, the periosteal cells contribute about 30%, the osteocytes possibly make a small (10%) contribution, and the free, hemopoietic cells of the marrow make no significant contribution. Evidence about the relative contributions to osteogenesis of graft and host cells is reviewed and the possible osteogenetic role of bone marrow is discussed. Key words: Osteogenesis -- Experimental bone grafts.
Introduction The osteogenesis that occurs within and around a successful bone graft usually depends on both the graft and the host. For example, a graft that would produce abundant new bone within 2 weeks if implanted as a fresh autograft [1-4] fails to do so if implanted as a fresh ailograft [5-9] or as an X-irradiated (850 rad) isograft [10]. However, 4 to 8 weeks *Present adr Department of Immt, nobiology, GlaxoAIlenburys Research (Greenford) Ltd., Greenford Road. Middlesex. Send qffprint requests to J. Craig Gray at the above address.
after grafting, devitalized isografts such as X-irradiated or freeze-dried, decalcified matrix [1 l] and, under some circumstances, bone allografts [6, 12, 13] can produce new bone. Such evidence favors the two-phase theory of osteogenesis, first put forward by Axhausen [14, 15] to the effect that the main contribution to osteogenesis within the first 4 weeks after implantation of a bone isograft or autograft comes from cells of the graft, whereas after 4 to 8 weeks, cells from the host begin to contribute significantly to new bone formation. There is now strong evidence for this theory, which has been sumarized by Elves [16] and Craig Gray [17]. The studies to be described here deal directly only with the early phase ofosteogenesis, although behavior during this phase may have a profound effect on later, host-derived osteogenesis and hence on the ultimate fate of the graft, as will be discussed later. The objective was to seek evidence for the particular cells within the graft that give rise to osteogenesis. The four main cellular components of a bone graft that are possible sources of osteogenetic cells include the bone marrow, the endosteal lining cells, the periosteum, and the osteocytes. The first three of these have all been suggested at various times to be the main source of bone-forming cells: the purpose of the present study was to ascertain, quantitatively, the relative contributions to first-phase osteogenesis of each of these groups of cells.
Materials and Methods lsografts of rat femur were prepared and implanted subcutaneously, some of the grafts being complete with marrow, endosteum, and periosteum and others with one or more of these components removed, as described below. From the measured differences in osteogenesis 2 weeks after implantation of grafts
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O
E
M
Table 1. Effect of marrow and e n d o s t e u m
Mean RI SE No.
FEMUR GRAFT Fig. 1. Schematic drawing of a complete c o m p a c t bone graft. P, periosteal cells: O, osteocytes; E, endosteal cells; M, marrow
devoid of different cellular c o m p o n e n t s and implanted into the same recipients, the osteogenetic contribution of each component was determined. M e a s u r e m e n t s of the a m o u n t of new bone formation were made using 85Sr retention histiometry [18] and histology.
Animals The animals used as both donors and recipients of the bone isografts were male rats of the AS2 inbred strain. T h e y were 2-4 m o n t h s of age (average weight 260 g) at the time of grafting.
Grafts Nine different types of cortical grafts were prepared under aseptic conditions from the femora of freshly killed rats. After removal of the femoral diaphysis, excess adherent muscle was trimmed o f f a n d the bone was split longitudinally to produce two hemicylinders of c o m p a c t cortical bone with its constituent marrow, e n d o s t e u m , and periosteum all intact. Such a graft is s h o w n schematically in Figure 1 and is referred to as F.poem. The letters indicate the type of matrix and the cellular constituents of the graft at the time of implantation: F, femur; p, periosteum; o, osteocytes; e, e n d o s t e u m ; m, marrow. From this basic graft, eight other types were prepared as follows: I. F.poe(m). Most of the marrow was r e m o v e d from the bone hemicylinders by gently agitating the graft in sterile saline after which sterile saline was dropped very gently into the concavity from a 19SWG hypodermic needle. T h e s e grafts were still pink in parts of the medulla: the parentheses around the "'m" indicate the presence of a little marrow. 2. F.poe. The marrow was r e m o v e d from the bone hemicylinders by m e a n s of a gentle jet of sterile saline from a 23SWG hypodermic needle fitted to a 20 ml syringe, leaving the grafts almost uniformly white. 3. F.po. The marrow was r e m o v e d by a forcible jet of saline and then the endosteal lining cells were removed by scraping with a small scalpel. For the five remaining types of cortical grafts, the adherent muscle and p e r i o s t e u m were scraped off with a scalpel.
F.oem
F.oe(m)
F.oe
F.o
1 -23
0.906 0.074 19
0.672 0.068 10
0.52 0.035 23
4. F.oem. This is a graft devoid of periosteum but with the marrow and e n d o s t e u m intact. 5. F.oe(m). Most of the marrow was r e m o v e d from the periosteum-free bone as in (1) above. 6. F.oe. The marrow was r e m o v e d from the grafts as in (2) above. 7. F.o. The marrow and e n d o s t e u m were removed as in (3) above. 8. F.(dead). T h e s e grafts were prepared from F.o grafts by immersing them in boiling water for 5 min followed by two cycles of freezing in liquid nitrogen and thawing.
Graft Incubations Some of the grafts were incubated in tissue culture medium or in solutions of e n z y m e s before implantation. The medium used was TC199 (Wellcome) buffered to pH 7.4 with 10,e~ Hepes. The two e n z y m e solutions used were 0.25% (w/v) trypsin (Wellcome) and 750 units/ml collagenase (C-0130, Sigma), both in TC 199. In addition. TC199 with 20% inactivated fetal calf serum [CS] (Flow Labs.) was u s e d as a rinse after each e n z y m e treatment to remove e n z y m e activity. The incubations were carried out at 37~ in a shaking water bath as follows: collagenase, 1 h; TC199 + CS rinse: trypsin, 1 h: TC199 + CS rinse. Control grafts were incubated in TC199 m e d i u m without e n z y m e s but with the two rinses in TC199 + CS.
Implantation Grafts were inserted, using aseptic procedures, into pockets created b e t w e e n the dermis and the panniculus carnosus muscle of the back of the recipient rats. There were normally 6-8 different grafts per rat, each in a separate pocket. The skin incisions were closed by m e a n s of Michel skin clips and sprayed with 'Hibispray" plastic dressing (ICI, Ltd.).
Assessment of Osteogenesis Osteogenesis in the grafts was m e a s u r e d by the s~Sr retention technique [18]. Twelve days after grafting, the rats received 8 /zCi of 8'~SrCI~ per 100 g body weight by the i.p. route. Four days later, the rats were killed and the grafts r e m o v e d together with the hosts' femora; these were all weighed, put into 10% formal saline, and their 8~Sr activities were m e a s u r e d . From the specific activities (counts/min/mg) of the grafts and of the host femur, an osteogenic index (OI) for each graft was calculated thus: 01=
sp act of graft sp act of host femur
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in C o m p a c t Bone Isografts
227
Fig. 2. F,oe graft 16 days after implantation. Living bone fib) lining a small cavity in the dead bone (db) of the graft
A further index o f osteogenesis, the relative index (RI), was devised to reduce the effect on the results of any variations in host physiology. The value o f RI for each test graft was calculated by taking the ratio o f its specific activity to that o f a control F . o e m graft which had been in the same recipient. T h u s RI =
sp act o f test graft sp act of control graft
The m e a n (_+ SE o f mean) o f the RI values for each group o f grafts was used to calculate the differences in osteogenesis attributable to the different cellular c o m p o n e n t s of the grafts. However, significance testing, using S t u d e n t ' s two-tailed paired t test [20] was performed on the individual OI values as these, although subject to more variability than RI, should be normally distributed. Values of P < 0.05 were taken to indicate a significant difference in the m e a n s of the populations from which the samples were drawn.
Histology After g a m m a counting, the grafts were put into fresh fixative and were then decalcified and taken to paraffin wax. Representative sections, cut at 5 tzm, were stained with hematoxylin, eosin, and alcian blue [21] and were examined by light microscopy.
Results
Contribution of Marrow The osteogenetic contribution of the marrow in cortical grafts was assessed by comparing F.oem grafts with F.oe(m) and F.oe grafts (Table 1). A comparison o f the mean RI values suggests that the F.oe(m) grafts may have slightly lower osteo-
genesis than the F.oem grafts. However, comparing OI for the two graft types indicates that the difference does not attain significance (P = 0.09). When the mean RI of the marrow-free grafts (F.oe) is examined, it is found to be considerably lower (0.672), and comparison of OI o f the marrow-free grafts against that of the F.oem grafts shows the difference to be highly significant (P = 0.008). To interpret these results, the effect on the graft cells of the procedures used to produce the F.oe(m) and F.oe grafts must be considered. During preparation of the F.oe(m) grafts, about 80% of the marrow was removed, as could be seen in histological sections of nonimplanted F.oe(m) grafts. In the F.oem grafts the medulla was full of marrow, and endosteal lining cells were quite apparent along much of the medullary surface and in the small marrow cavities, whereas the amount o f marrow present in the main cavity of the F.oe(m) grafts varied from about 30% of that in the marrow-intact grafts to almost none. However, the smaller marrow cavities were all full of marrow and the endosteal surfaces were generally lined with cells much as in the marrow-intact grafts, although one graft seemed rather deficient in lining cells. During the slightly more vigorous procedure used to produce an F.oe graft, over 90% of the marrow was removed: histologically, these grafts appeared devoid of marrow in the main cavity although a few of the smaller cavities contained a little: considerably fewer endosteal lining cells were seen in the main recess of these preparations than in the F.oem and F.oe(m) grafts, but the smaller marrow spaces were lined with them.
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From the results in Table 1, it appears that removal of the final 10-15% of marrow reduces osteogenesis by more than twice as much as the removal of the first 80%: c o m p a r e d with the F . o e m grafts, RI is reduced by (9.4 + 7.4)% in the F.oe(m) grafts and by (32.8 -+ 6.8)% in the F.oe grafts. The difference between these two values (23.4 -+ 10.0)% is significantly different from zero (P < 0.05). So it would appear that the last 10-15% of cells r e m o v e d are qualitatively different from the first 80% of cells and, as the last 10-15% cells are closest to the endosteal surface of the bone, it is likely that they also include endosteal osteoblasts, which will be shown later to be highly osteogenetic. This interpretation is also consistent with the histological findings described a b o v e and below. It appears, therefore, that the bulk of the marrow contributes little or nothing to early osteogenesis: no more than (9.4 + 7.4)% of the F . o e m activity. Histologically, none of the grafts examined after 16 days revealed any periosteal new bone, but luxuriant endosteal w o v e n new bone was seen spanning the medullary cavity in all the F . o e m grafts and in all but one of the F.oe(m) grafts. In that graft, there was not so much endosteal new bone although there was still new bone around most of the haversian canals and lining the smaller marrow recesses. The F.oe grafts had a lot of woven endosteal bone lining the inner surface of the graft, but in only one case did it span the marrow cavity. Osteogenesis had also occurred within the smaller cavities and haversian canals (Fig. 2). A relatively acellular fibrous tissue filled the center of the grafts, but a richer granulation tissue seemed to be entering through the open end of the bone.
Contribution of Endosteum ( Mechanic'al Removal) The main comparisons were of F.o with F.oe(m) and with F . o e m , but a c o m p a r i s o n was also made with F.oe. The histological appearance of the F.oem, F.oe(m), and F.oe grafts has already been described; the F.o grafts were also examined immediately after preparation and after 16 days' implantation. In the fresh grafts, little or no marrow was seen. No endosteal lining cells were observed in the main cavity, but there were a few in some of the smaller recesses. After 16 days in the recipient, an occasional graft showed small amounts of new bone in recesses of the marrow cavity or in haversian canals; however, little or no new bone was o b s e r v e d in most grafts. There was no marrow present and not much granulation tissue in any of the grafts, although there appeared to be some fibrous invasion from outside. The other predominant cell types
seen were osteoclasts or giant cells and there was a fair n u m b e r of healthy looking osteocytes in the grafted bone. The strontium results from which the effect of the e n d o s t e u m was assessed are s u m m a r i z e d in Table 1. The value of RI for the F.o grafts was 0.52 compared with 1.0 for the F . o e m grafts and 0.906 for the F.oe(m) grafts. Thus removing m a r r o w and endosteum together reduced osteogenetic activity by 48.0 -+ 3.5%, a highly significant result (P < 0.001); whereas removing most of the marrow alone (and possibly disrupting the endosteum) was shown above to reduce osteogenesis by only 9.4 -+ 7.4%, which does not attain significance (P = 0.09). Similarly, c o m p a r i s o n of the F.oe(m) graft with the F.o graft (Table 1) shows that RI in the latter is reduced by a further 38.6 -+ 8.2% of the baseline activity, again a highly significant result (P < 0.001). The c o m b i n e d contribution to osteogenesis of the endosteal cells and the last 10-20% of marrow cells (stroma?) is thus great: at least 38.6 +_ 8.2% and probably 48.0 -+ 3.5%, if the free marrow cells' contribution is taken to be nil. The histological results, discussed later, suggest that most of this osteogenetic activity is due to endosteal cells rather than to m a r r o w stroma.
Contribution of Endosteum (Enzymatic Removal) In addition to the experiment involving mechanical removal of the endosteum described above, the osteogenetic activities were measured for F.oe(m) incubated either in TC 199 for 2 h or in collagenase and trypsin, as described above. Measurements were also made using fresh F.oe(m), fresh F.oem, and TC199-incubated F.oem grafts. These experiments were performed partly as a guide to the interpretation of results from a study of osteogenesis in cancellous grafts (Craig Gray and Elves, in preparation), but they also confirm the results obtained above by mechanical removal of endosteum. Histological examination of the enzyme-treated F.oe(m) grafts both before and after transplantation gave results similar to those o b s e r v e d with the F.o (mechanical) grafts. A small or very small amount of endosteal new w o v e n bone was observed in half the grafts examined; none was seen in the others. There seemed to be a high rate of survival of graft osteocytes. The TC199-incubated F.oe(m) grafts a p p e a r e d very similar to their nonincubated counterparts already described. In fact, the incubated grafts, as well as having luxuriant endosteal new bone, possibly had rather more new bone in and around the haversian canals and other recesses of the graft. There also seemed to be greater survival
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in C o m p a c t Bone Isografts
229
Table 2. Effect of e n d o s t e u m (enzyme treatment)
Mean RI SE No.
F.oem
F.oe(m) enzymes
F.oe(m) TC199
F.oe(m)
F.oem TCI99
1 -23
0.552 0.041 16
1.028 0.081 8
0.835 0.091 8
0.910 0.082 15
of superficial osteocytes of the grafted bone than was seen with the unincubated grafts. Comparison of the s'~Sr results from the enzyme incubated F.oe(m) grafts with those of the fresh control F.oem grafts (Table 2) shows a highly significant drop (P < 0.00l) for the enzyme-treated grafts. The reduction in RI is 44.8 +- 4.1% of the control activity, which is close to the value 48.0 -+ 3.5% found previously for mechanical removal of the endosteum and marrow. Such a close correspondence (P = 0.55) suggests that the effect of the enzymes is similar to that of mechanical removal o f endosteum. A surprising result shown in Table 2 is that, although F.oe(m) grafts incubated in TC 199 alone for 2 h gave an SSSr index not significantly different from that of fresh F.oem control grafts (P = 0.75) they did significantly better than fresh F.oe(m) grafts (P < 0.005), whereas 2 h incubation of F.oem grafts in TC199 did not raise the eventual level o f osteogenesis (P = 0.12). Taking these results with the previous finding that removing 80% of the marrow possibly reduced osteogenesis in a fresh graft by 9.4 +_ 7.4%, it appears that, whatever the mechanism by which this possible reduction occurred, it can be reversed by 2 h incubation of the marrow-depleted graft in TC199. However, when the incubation was performed with the marrow intact, a rise did not occur, presumably because it was due to the recovery or stimulation of osteogenic endosteal or stroma cells damaged during the depletion procedure: the release of enzymes from dying marrow cells may be harmful to those cells. These findings, of course. add further weight to the argument that the free marrow cells make no positive contribution whatsoever to early osteogenesis.
Contribution of Periosteum Histological examination of F.poem and F.oem grafts revealed the presence of periosteal new bone in the former (Fig. 3A) but not in the latter, the periosteal surface of which generally had a shredded appearance (Fig. 3B). The periosteal new bone of the
Fig. 3A. The periosteal surface of an F . p o e m graft 16 days after implantation. M a n y living osteocytes are seen in the lacunae of the periosteal new bone (nb) at the top of the micrograph but most of the lacunae of the graft bone (gb) below are empty. Compare this with Fig. 3B
F.poem grafts was lamellar in form whereas the luxuriant endosteal new bone present in both sets of grafts, and bridging the medullary space, was of woven appearance: its trabeculae were lined by plump osteoblasts in many places (Fig. 4). Within the old bone of the F . p o e m grafts, some of the superficial osteocytes, both periosteal and endosteal, appeared normal although the deeper lacunae were all empty. In the F.oem grafts, no surviving periosteal osteocytes were seen but there were some close to the endosteal surface. In both sets of grafts, osteoclasts were seen occasionally, usually (but not always) against the surface of the graft bone. There were blood vessels entering cavities near the surfaces of the old bone (Fig. 5) and in the endosteal and periosteal new bone (Fig. 3A), and larger ones were seen in the inflammatory tissue close to the endosteal new bone (Fig. 6). For the s~'Sr assessment of the osteogenetic contribution of the periosteum, eight different types of grafts were used. The sets of grafts varied in the presence or absence of marrow, endosteum, and periosteum, but they could be arranged in four pairs
230
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in C o m p a c t Bone lsografts
Fig. 4. The endosteal tissue in an F.poem graft 16 days after implantation. T h e s e trabeculae of woven new bone appear quite healthy, with o s t e o c y t e s in the lacunae and osteoblasts lining m a n y of the surfaces
Fig. 3B. The periosteal surface of an F.oem graft 16 days after implantation. No new bone is seen at the periosteal surface (top) and the outer layers of the graft bone (gb) have a shredded appearance. C o m p a r e this with Fig. 3A
Table 3. Effect of periosteum
Mean RI SE No.
F.oem
F.poem
F.oe
F.poe
F.oelm)
F.poelm)
F.o
F.po
1 -23
1.367 0.212 8
0.688 0.066 4
1.006 0.147 4
0.982 0.088 4
1.112 0.085 4
0.560 0.053 7
0.717 0.082 7
where one of each pair was periosteum-free and the other had the periosteum intact but was otherwise identical. It will be seen in Table 3 that the value for the mean RI is in each case greater for the periosteumintact grafts than for the corresponding periosteumfree grafts, although the differences did not generally attain significance as measured by the paired t test. H o w e v e r , if all the periosteum-free grafts are pooled and all the periosteum-intact grafts are pooled, the difference between these two groups is significant (P < 0.01). This suggests that the periosteum makes a small but significant contribution to osteogenesis. The proportion of the total graft osteogenesis due to the periosteum was estimated by taking the difference in mean RI of the perioste-
um-intact and the corresponding periosteum-free grafts of each type and calculating the weighted mean of these. Using the relevant data in Table 3, the increase in osteogenesis due to periosteum, expressed as a fraction of the osteogenesis in F.oem graft, is 0.253 _+ 0.087. So the periosteum would have contributed approximately a further 25.3 -+ 8.7% of osteogenesis to the F . o e m graft, which was the base line for RI calculations.
Contribution (~['Osteocytes The importance of the graft osteocytes for osteogenesis in cortical bone was examined by comparing the osteogenesis in F(dead) grafts with that
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts
231
Table 4. Effect of osteocytes and of matrix
Mean SE No.
F.oem
F.o
F(dead)
F.o ~
F(dead) b
1 -7
0.515 0.049 12
0.368 0.028 12
0.437 0.031 9
0.338 0.032 9
a F.o a as F.o, but omitting grafts with perihaversian new bone (see text) F(dead) h as F(dead), but omitting the grafts in same animals as those omitted in F.o a
Fig. 5. The endosteal surface of an F.poem graft 16 days after implantation. A blood vessel can be seen penetrating from the medulla, through a lining of living new bone (nb) into a cavity in the dead graft bone (gb)
in f r e s h F . o g r a f t s ( T a b l e 4). T h e d i f f e r e n c e in m e a n R I o f t h e d e a d a n d l i v i n g g r a f t s w a s f o u n d to b e 14.7 -+ 5 . 6 % o f t h e a c t i v i t y o f t h e c o n t r o l F . o e m g r a f t s . T h e p a i r e d t t e s t s h o w s this d i f f e r e n c e to be signifi c a n t (P < 0.02). It s e e m s l i k e l y , h o w e v e r , t h a t t h i s d i f f e r e n c e in osteogenesis between the dead and living grafts was n o t e n t i r e l y d u e to t h e o s t e o c y t e s . T h e r e w o u l d have been an osteogenetic contribution from any endosteal osteoblasts that had not been removed from the F.o grafts by the scraping procedure. Evid e n c e f o r this is the h i s t o l o g i c a l f i n d i n g o f e n d o s t e a l lining c e l l s in s o m e o f t h e s m a l l e r m a r r o w c a v i t i e s and haversian canals of the freshly prepared F.o g r a f t s a n d t h e a p p e a r a n c e , 16 d a y s l a t e r , o f s m a l l a m o u n t s o f n e w b o n e in t h e s e a r e a s o f t h r e e o f t h e grafts. The F(dead) grafts, on the other hand, showed no new bone. If the osteocyte contribution
Fig. 6. The endosteal surface and medullary cavity of an F.oem graft 16 days after implantation. Several large blood vessels (by) may be seen close to the trabeculae of woven new bone filling the medullary cavity, and there are 3 resorption cavities in the dead graft bone (gb) below
232
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in C o m p a c t Bone Isografts
to osteogenesis is recomputed, omitting both the F.o grafts with new bone in the areas mentioned and the corresponding F(dead) grafts, it is reduced to 9.9 • 4.5% and the paired t test shows the difference between the grafts to be on the borderline of significance (P = 0.08). The value 9.9 • 4.5% may thus be ascribed to activity of the osteocytes.
o ~ 70
0
040
Contrib.tion gf Matrix
30 (b
The contribution of the matrix to the strontium uptake is given in Table 4 by the value of RI for F(dead). This value (0.368 • 0.028) is significantly different from zero (P < 0.001). However, histological examination of the grafts did not reveal new bone in any of the dead matrix grafts, so we conclude that the matrix contribution to osteogenesis is negligible. Therefore most, if not all, of the strontium uptake must have been due to passive deep exchange between the mineral ions of the matrix and the ~sSr in the blood. Such activity will be present, as an approximately constant background, in all the bone grafts. Thus the values found for the relative contributions of the different cell populations will not be affected by the deep exchange, since those values were found by subtraction. However, in expressing each cell population's contribution as a fraction of total graft osteogenesis, one must allow for the deep exchange, as shown below.
Summary gf Results The contribution of each component of the grafts to strontium uptake has been calculated above with respect to the F.oem graft taken as 100%. On this basis, the following percentages have been found: Periosteum Free marrow cells Endosteum + marrow stroma Osteocytes Matrix (passive exchange)
25.3 _~ 8.8 9.4 _+ 7.4 or 0 38.6 + 8.2 or 48.0 • 3.5 9.9 • 4.5 36.8 : 2.8 120.0 + 15.1 or 120.0 _~ 10.9
The total is well over 100% because it includes a contribution of approximately 25% from the periosteum which is not included in the "'baseline" graft: the remaining discrepancy is adequately covered by the standard error. In order to normalize these results to take account of (a) the passive exchange contribution from the matrix and (b/ the fact that the baseline graft
,~
,-7 " - i ~ "1 ; I
: i I
P
O(E~S) M
Fig. 7. Histogram of the percentage contributions to osteogenesis of the periosteum (P), osteocytes (Ot. endosteal lining cells and marrow s t r o m a (E + S/, and free marrowy cells tM). Bars indicate SEM
does not include periosteum, the following changes are made: 1. The matrix contribution of 36.8 + 2.8 is omitted in adding up the cellular osteogenetic activity, giving 83.2 • 14.8 or 83.2 • 10.5 for the totals. 2. The totals are multiplied by the normalizing factor, 100/83.2 = 1.2, and the contribution of each graft element is multiplied by the same factor to express it as a fraction of the F.poem osteogenetic activity. This gives: Periosteum Free marrow cells Endosteum + marrow stroma Osteocytes
30.4 • 10.6 !1.3 • 8.9 or 0 46.3 _+ 9.8 or 57.6 • 4.2 11.9 • 5.4
These values are expressed as percentages of the activity of the complete F . p o e m graft, assuming from the histological evidence that all the activity in the dead matrix is due to passive uptake or deep exchange and not to osteogenesis. However, the relative importance of each of the above cell populations for osteogenesis is independent of this last assumption. The above results are shown graphically in the histogram (Fig. 7).
Discussion
The above plants with sSSr figures tween days
results are all based on 16-day transs'~Sr administered on day 12. Thus the represent the osteogenetic activity be12 and 16 after grafting, and, because of
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts the relatively rapid clearance of strontium from the blood, most of the label will be in new bone mineral laid down on days 12 to 14 after grafting. It is quite probable, however, that labeling of new bone continues during days 15 and 16 at a higher rate than the blood-strontium level would suggest. There is likely to be local reusage of strontium that has been passively exchanged into. then out of, bone mineral. The histological results, on the other hand, represent the integrated activity of new bone formation and reformation over the whole 16-day period. Both the "~'Sr and the histological data and their interpretation apply directly only to early new bone growth in grafts: that is, to first-phase, graft-derived osteogenesis, in terms of the two-phase theory of Axhausen [14, 15] and Chalmers [6]. There is, however. some evidence that the second phase of osteogenesis, although probably produced primarily by host-derived cells, is also dependent to some extent on the cells of the graft. In fact, a good first phase of osteogenesis appears to be a stimulus to a good second phase, as suggested by Vainio [22] and by Axhausen [15]. This is d e m o n s t r a t e d in a comparison of the amount of late-phase osteogenesis in devitalized and living autografts where the new bone formation during the period 4 to 8 weeks after implantation is much less prolific in the former than in the latter grafts [10]. A similar difference is also observed in the second-phase bone growth in allografts transplanted to normal and immunosuppressed recipients, respectively [23]: once again, a good first phase, in the i m m u n o s u p p r e s s e d recipients, leads to a good second phase: a poor first phase in the normal recipients results in a poorer second phase. It is also found that allografts containing many minor antigen disparities give a much poorer first phase than allografts having only a few minor disparities with one H-I (Ag-B) disparity and that no second-phase osteogenesis is o b s e r v e d in the former group whereas the latter group show a significant late phase in about 30% of recipients [24]. In sum, it appears that a good early phase of osteogenesis usually leads to a good late phase, either by supplying graft-derived cells to supplement the osteogenic cells coming from the host or else by producing some osteogenic stimulus that ensures a good supply of host-derived osteoblasts. Thus, although the results recorded in this work apply directly only to early osteogenesis, they may well bear indirectly on the long-term success or failure of bone grafts.
Perioste,m The periosteum in the young mammal is a two-layered structure, the inner (cambiuml layer contain-
233
ing closely spaced, interdigitating osteogenic cells and functional osteoblasts, whereas the outer, fibrous layer contains more widely spaced fibroblasts [25]. It is now generally accepted that the cambium layer, certainly in a young animal, contributes to graft and fracture osteogenesis [26-343. The results presented here confirm and quantify these observations for cortical grafts prepared from young bone.
Osteocytes The small possible osteogenetic contribution attributed to the osteocytes is in broad agreement with results for the osteocytes of cancellous grafts [17; Craig G r a y and Elves, in preparation]. The histological findings for the o s t e o c y t e s were similar to those described in most recent reports [3, 9, 35], including the quantitative studies of Heslop et al. [36], who reported a steep reduction in the number of surviving osteocytes in the first few days after subcutaneous autografting of cylinders of c o m p a c t bone. The decrease continued at a reduced rate over the following 2 weeks and then stabilized with about 40% survival. In these older grafts, the surviving osteocytes were found in a band less than 300 p,m wide around the cortical surface. This was contrary to the findings of Nishimura et al. [37], who noted best survival in the mid-layers of the cortex. A t e n d e n c y for superficial osteocytes to survive was also noticed in the present experiments, but this applied to o s t e o c y t e s near the medullary as well as the cortical surface. This difference from the findings of Heslop and her colleagues may be because the grafts in the present experiment were hemicylinders which allowed direct contact of tissue and fluids from the host bed with both the periosteal and the endosteal surfaces. Another observation, which corroborates this interpretation, was made during histological examination of grafts incubated in TC199 before implantation. Such grafts seemed to contain a higher proportion of living osteocytes in the old bone than did similar grafts implanted fresh. This impression was not subjected to any quantitative m e a s u r e m e n t s , but it may give an explanation of an enhancing effect on osteogenesis of short incubation in culture medium before grafting [17: Craig Gray and Elves, in preparation]. It seems likely that while the grafts are being incubated in the shaking water bath, the moving culture fluid penetrates much more readily along the haversian canals and small marrow spaces to the deeper osteocytes than does the relatively static tissue fluid in the early graft bed. The culture
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J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts
medium that reaches many osteocytes may be sufficient to keep them alive for the first few days after grafting until the ingrowing capillaries have been established. If this additional osteocyte survival is the explanation of the enhanced osteogenesis in some incubated grafts, it suggests that the osteocytes play some part in osteogenesis. However, it seems more likely that other osteogenic elements of the graft, such as osteoblasts lining the haversian canals, also have improved survival after incubation and that they are responsible for the increased osteogenesis. Heslop and her colleagues [36] came to no firm conclusion as to the possible role of the osteocytes in new bone formation. Although they found no quantitative relationship between osteogenesis and osteocyte survival, they suggested, on the basis of the results of de Bruyn and Kabisch [27] where dead and living grafts were compared, that living osteocytes may stimulate osteogenesis in their vicinity. However, it appears that such a stimulus, if it exists, has a relatively small effect compared with other factors: Heslop and her colleagues found much more abundant new bone in the hollow center of the grafts where no osteocytes survived than they did around the outer margin of the grafts where osteocyte survival was good. Although not all workers accept that some osteocytes survive in an autograft [34, 38, 39], there appears to be general agreement that any direct osteogenetic contribution by them is small.
Endosteum
The endosteal lining cells and the marrow have been shown to be responsible for well over half the new bone formed, and evidence has been presented suggesting that there may be an additional small contribution from intrahaversian osteoblasts. The free marrow cells, on the other hand, have been shown to make a negligible contribution to osteogenesis, suggesting that the endosteal cells alone make the major contribution. However, it might be argued that the "'easily removed ~" marrow is made up largely of free hemopoietic cells only and that there may remain within the F.oe(m) grafts elements of marrow stroma, fibrous or vascular tissue. It might be further argued that the stroma may have an osteogenetic role and that subjecting the graft to a jet of saline to produce the F.oe graft may disrupt or remove the stroma as well as part of the endosteum. The consequent reduction in osteogenesis would then possibly be due in part to the removal of the marrow stroma. This hypothesis is compatible with the tracer experi-
ments of the present studies, but the histological evidence does not favor it. The new bone in the F.oe grafts lay mainly around the inner rim of the graft in most cases, although it did bridge the marrow cavity in one graft. In the F . o e m and all but one of the F.oe(m) grafts, on the other hand, the marrow cavity was always bridged by new bone. There are three possible explanations of this: (a) The remaining endosteal cells in the F.oe grafts are too few to effect a bridge in 16 days. (b) The stroma of the marrow is partly responsible for producing the new bone that forms the bridge. (c) The marrow stroma acts by osteoconduction [39] of endosteal bone-forming cells into the marrow space, where they lay down trabeculae of woven bone. Point (c) fails to explain why osteogenesis in the F.oe(m) grafts was almost as great as in the F.oem grafts and why new bone spanned the medullary cavity of both sets of grafts. If the marrow stroma was disrupted, as it certainly was in the F.oe(m) grafts, its osteoconductive role would fail. If point (b) were true the F.oe(m) grafts would be expected to have almost as much stroma as (although much less marrow than) the F.oem grafts. In other words, the remaining marrow would be particularly rich in stroma in the freshly prepared grafts. No such effect was observed, although it is impossible to be definite about such a negative observation. Thus it appears that point (a) is the most likely explanation and that the drop in strontium uptake observed in marrow-free grafts was due largely, if not entirely, to disruption and loss of endosteal cells. However, there remains the possibility that some of the drop was due to the removal of stromal elements of the marrow. The free marrow cells, however, certainly did not contribute to osteogenesis. The important role of the endosteum for early osteogenesis in bone grafts does not appear to have been demonstrated quantitatively before. Probably the nearest approach was made by Bassett and his colleagues [26] who concluded, on the basis of a histological study of the repair of osseous defects with and without endosteal curettage, that ~'endosteal cells play a critical role in osteogenesis'" and that "'osteogenic cells resident in Haversian canal systems . . . seem to participate in the formation of new b o n e . " Other authors have, of course, attributed an osteogenetic role to endosteal cells but usually on the basis of rather circumstantial evidence and without any quantitative measure of the endosteal contribution: see, however, Marotti et al. [40] and Zallone [41]. Tonna and Cronkite [42], on the basis of :~H-
J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts thymidine uptake by femur fractures in mice, concluded that the proliferative activity of the osteogenic cells of the periosteum and within the intertrabecular spaces and lining the trabeculae was much greater than that of the endosteal cells lining the medullary canal of the diaphyseal cortex. H a m and Harris [9], however, in discussing the endosteal contribution to the formation of the internal callus during fracture repair, stressed the c o m m o n osteogenic lineage of the cells of the cambium layer of the periosteum, of the endosteal cells of the main medullary cavity and trabeculae, and of the osteoblasts that line the haversian canals. They attributed an osteogenetic role to all these cells. In considering the clinical relevance of these results, it must be rem e m b e r e d that the rat is a small mammal c o m p a r e d with man and the proportions of the different osteogenic cells in the bones of the two species are different. In particular, the relative number of haversian. c o m p a r e d with medullary, endostea[ cells increases as the thickness of the bone increases [9]. Thus the proportional osteogenetic contribution of the osteogenic cells of the haversian canals, compared with those of the m a r r o w cavity and the periosteum, is probably much greater in man than in small laboratory mammals.
Marrow
Perhaps the major surprise in the present results is the unimportance of the free marrow cells for early osteogenesis. On the face of it, there appears to be a definite contradiction between the present findings and those of the many workers who have ascribed an important osteogenetic role to cells within the marrow [43-51]. Most of these other experiments have involved the grafting of marrow that has been r e m o v e d from the shaft of a long bone (usually the femur) by washing out, aspiration, or curettage. Many of those employing these techniques admit the possibility or likelihood that endosteal cells are disturbed during removal of the marrow and may be included in the marrow specimen obtained. For example, Burwell [52] states that in his experiments, "'it cannot be denied that endosteal osteoblasts may have contributed to the formation of bone by at least some of the marrow a u t o g r a f t s " ; see also papers by Petrakova et al. [53], Friedenstein et al, [46], and Boyne [54]. H o w e v e r , several authors mention that care was taken to exclude m a c r o s c o p ic bone spicules from the marrow, although most do not mention any particular precautions, and it is also pointed out that occasional bone spicules observed in grafts do not appear to act as foci of osteogenesis.
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The results of the present work with F.oe grafts, produced by subjecting the m a r r o w in the cavity to a gentle jet of saline, show how easily the endosteum may be disrupted and r e m o v e d along with the marrow and how. without dislodging any bone spicules, one may inadvertently include endosteal osteoblasts with the marrow. Some other evidence for this view is the inconsistency of results obtained by m a n y authors using marrow autografts. The usual method of scoring has been on the basis of the proportion of grafts yielding bone: generally no account has been taken of the quantity of new bone produced which may vary from a "'solitary small bone spicule" to "'very a b u n d a n t " [31]. Danis [55] and Burwell [52] present lengthy tables summarizing the incidence of bone formation within heterotopic m a r r o w autografts, obtained by m a n y workers. The success rate varies from nil to 100%, most authors achieving about 30% to 60%. It seems quite likely that a similar proportion of grafts may have been contaminated by a significant number of endosteal cells, especially as the m a r r o w has, in e v e r y case but one, been r e m o v e d from the shaft of a long bone (tibia, femur, radius, or ulna) where the endosteum is easily dislodged. A n o t h e r criticism of some o f these e x p e r i m e n t s is that the method o f assessing osteogenesis, usually by histological examination with an arbitrary scoring system, is subjective and is statistically suspect. Often the n u m b e r of grafts examined at each time interval is very small, sometimes grafts implanted for different intervals are pooled unjustifiably and inappropriate statistical tests are used. H o w e v e r , although the present experiments show that the free marrow cells do not contribute to early osteogenesis, there are two further possibilities for reconciling the present findings with those mentioned above. 1. In the present experiments, a possible osteogenetic role for the marrow s t r o m a is not excluded. The lack o f osteogenetic capacity d e m o n s t r a t e d for the free m a r r o w cells, however, is consistent with the findings of Friedenstein and his colleagues [46, 53, 56-58] and Amsel and Dell [19] that the hemopoietic stem cell line of the m a r r o w is independent o f the osteogenic cell line. 2. Most e x p e r i m e n t s supporting the idea o f an osteogenetic role for marrow use grafts implanted for more than 2 weeks. It is possible that, if the present work comparing osteogenesis in marrowdepleted and marrow-intact grafts had been performed o v e r 28 days instead of 16 days, there would have been a significant difference between the two types of grafts. In that case, it could be asserted either that the osteogenic stem cells in the m a r r o w take more than 2 weeks to b e c o m e functional osteo-
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J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone lsografts
b l a s t s o r t h a t , as L e v a n d e r [31] m a i n t a i n e d , t h e m a r r o w h a s a r o l e in i n d u c i n g o s t e o g e n e t i c a c t i v i t y in o t h e r g r a f t o r h o s t cells.
General Conclusions E v i d e n c e h a s b e e n c i t e d s u g g e s t i n g t h a t t h e ultim a t e f a t e o f a b o n e g r a f t is l a r g e l y d e t e r m i n e d b y its o s t e o g e n e t i c p e r f o r m a n c e in t h e first f e w w e e k s aft e r g r a f t i n g . T h e r e is r e a s o n to b e l i e v e , t h e r e f o r e , that the results presented here, although based on t h e s t u d y o f 16-day h e t e r o t o p i c c o m p a c t b o n e isog r a f t s in y o u n g a d u l t rats, h a v e a m o r e g e n e r a l r e l e v a n c e to t h e b e h a v i o r o f b o n e g r a f t s . T h e f o l l o w i n g observations are based on the experimental evidence that has been presented. The living cells of a bone graft play an important p a r t in p r o d u c i n g t h e e a r l y n e w b o n e in a n d a r o u n d the graft. The endosteal and intrahaversian osteob l a s t s p r o d u c e a b o u t 60%. o f t h e n e w b o n e , i n c l u d ing a p o s s i b l e c o n t r i b u t i o n b y s t r o m a l c e l l s o f t h e marrow, and the periosteal cells are responsible for a p p r o x i m a t e l y 3 0 % o f t h e o s t e o g e n e s i s . T h e r e is little o r n o c o n t r i b u t i o n to o s t e o g e n e s i s b y g r a f t osteocytes or by the free hemopoietic cells of the marrow. The endosteal osteoblasts of cortical bone a r e e a s i l y r e m o v e d w h e n m a r r o w is g e n t l y w a s h e d out of the medullary cavity.
Acknowledgments. This study was performed in partial fulfillment of the requirements for the degree of Master of Philosophy of the University of London. The authors express their thanks to Mr. D. Sayers for technical assistance and to Mrs. V. King and Mrs. A. Bradley for typing the manuscript.
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J. Craig Gray and M.W. Elves: Quantitative Osteogenesis in Compact Bone Isografts
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47. Nade, S., Burwell, R.G.B.: Decalcified bone as a substrate for osteogenesis. An appraisal of the interrelation of bone and marrow in combined grafts, J. Bone Joint Surg. 59:189196. 1977 48. Plenk, H., Hollmann, K., Wilfert, K.-H.: Ekperimental bridging of osseous defects in rats by implantation of Kiel bone containing fresh autologous marrow. J. Bone Joint Surg. 54:735-743, 1972 49. Simmons, D.J., Lester, P.A., Ellsasser, J.C.: Survival of osteocompetent marrow cells in vitro and the effect of PHAstimulation on osteoinduction in composite bone grafts, Proc. Soc. Exp. Biol. Med. 148:986-990, 1975 50. Tavassoli, M., Maniatis, A., Binder, R.A., Crosby, W.H.: Studies on marrow histogenesis. II. Growth characteristics of extramedullary marrow autotransplants, Proc. Soc. Exp. Biol. Med. 138:868-870, 1971 51. Yeager, J.E., Boyne, P.J.: Use of bone homografts and autogenous marrow in restoration of edentulous alveolar ridges, J. Oral Surg. 17:185-189, 1969 52. Burwell, R.G.: Studies in the transplantation of bone. VII. The fresh composite homograft-autograft of cancellous bone. An analysis of factors leading to osteogenesis in marrow transplants and in marrow-containing bone grafts, J. Bone Joint Surg. 46:110-140, 1964 53. Petrakova, K.V., Tolmacheva, A.A., Friedenstein, A.J.: Bone formation occurring in bone marrow transplantations in diffusion chambers, Bull. Exp. Biol. Med. 12:87-98, 1963 54. Boyne, P.J.: Autogenous cancellous bone and marrow transplants, Clin. Orthop. 73:199-209, 1970 55. Danis, A.: Etude de l'ossification darts les greffes de moelle osseuse, Acta Chit. Belg. [Suppl.]Ill:l-120. 1957 56. Friedenstein, A.J., Chailakhjan, R.K., Lalykina, K.S.: The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow' and spleen cells, Cell Tissue Kinet. 3:393-403, 1970 57. Friedenstein, A., Kuralesova, A.I.: Osteogenic precursor cells of bone marrow in radiation chimeras, Transplantation 12:99-108, 1971 58. Friedenstein. A.J.. Petrakova, K.V., Kurolesova, A.I., FroIova, G.P.: Heterotopic transplants of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues, Transplantation 6:230-247, 1968 Received October 26, 1978 / Accepted July II, 1979
