Evolution of marrow regeneration as revealed by transplantation studies

Experimental Cell Research 71 (1972) 307-312

EVOLUTION OF MARROW REGENERATION AS REVEALED BY TRANSPLANTATION STUDIES H. M. PATT and MARY A. MALONEY Laboratory of Radiobiology, University of California, San Francisco, CaIif. 94122, USA

SUMMARY This study is concerned with the regeneration of bone marrow as an organized tissue. It is addressed specifically to the time in the regenerative program when the emerging tissue acquires the potential to reconstitute marrow. The evolution of this potential was investigated in rabbits by making autologous subcutaneous implants of tissue obtained at intervals after evacuation of a femur shaft. Analysis of 140 implant sites (89 of regenerating tissue and 51 of normal marrow) reveals a striking similarity in development of implants of a 2 to 4 day regenerating tissue and of normal marrow. New marrow encapsulated by bone can be seen in each instance 5 weeks after implantation. This property of regenerating tissue is uncovered before there are any obvious hemic elements. Significantly, the likelihood of a take is greatly increased when the implant contains a connective tissue. Marrow was always found in the implantation site when an ossicle was forming. It never occurred in the absence of bone. We conclude from this study that the appearance of modulated mesenchymal elements in the early regenerating tissue imparts the quality of normal marrow.

When marrow reforms in an evacuated medullary cavity, its ontogeny is revealed in the regenerative program [l-3]. We have found that the regeneration is basically a local phenomenon, which apparently depends upon an induction system involving mesenchymal cells derived from the adjacent bone [4, 51.The objective of the present study was to determine the time at which the emerging tissue in a mechanically depopulated medullary cavity acquires the potential of normal marrow. This was evaluated by examining the fate of autologous subcutaneous implants of tissue taken at various times of regeneration. It is well known that extramedullary implants of normal marrow fragments undergo a characteristic sequencein which hemic cells disappear and the remaining reticular cells contribute to the formation of an ossicle containing active marrow [6, 7J

Although 35 to 40 days are required for complete marrow restoration in an evacuated rabbit femur [8], the basic cellular transformations occur within a few days. Tissue obtained at this time has essentially all of the attributes of a normal marrow implant in a subcutaneous site. Yet, such tissue is devoid of any obvious hemic or osseousprogenitors. Rather it consists of granulation tissue and undifferentiated connective tissue elements. We have reason to think that it is the latter which represent the regenerative blastema and imparts the quality of normal marrow upon heterotopic transplantation. MATERIALS AND METHODS New Zealand white female rabbits 2.54 kg were used as the experimental animal. Bone marrow was removed from a 3 cm segment of the right femur shaft by in vivo perfusion with a dextran solution in a Exptl Cell Res 71

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H. hf. Putt & Mary A. Maloney

manner described previously [9]. At frequent intervals during the ensuing 6 weeks, samples of the regenerating medullary tissue were transplanted autologously into the subcutaneous tissue of the groin. Normal marrow was taken from the contralateral femur and similarly transplanted. Tissue for transplantation was obtained under sterile conditions in an anesthetized (i.v. sodium pentobarbital) rabbit. The femur was exposed and a bone punch, 3 mm in diameter, was made with a trephine attached to a low-speed drill. The medullary tissue was lifted out with a curette and placed in normal rabbit serum while the implantation site was prepared. Bone wax was inserted in the evacuated marrow cavity and the connective tissue and skin were sutured. For imnlantation of the regenerating tissue or normal bone-marrow, a 1 cm incision was made in the groin. The tissue (25-150 mm*) was then ulaced into a subcutaneous pocket made ‘with a hemostat and the skin was sutured. A representative piece of regenerating tissue not used for transplantation was fixed in neutral formalin in preparation for histological examination. The rabbits were usually sacrificed between 35 to 40 days after transplantation, although in a few cases this occurred after intervals of up to 70 days. Upon sacrifice. the imnlantation site was insnected for growth..If growth had occurred, the tissue was removed and fixed in neutral formalin. Paraffin sections were prepared and treated with either Harris’ hematoxylin and eosin, or Mallory’s aniline blue collagen stain. Histological examination of the regenerating tissue prior to implantation included estimates of the relative distribution of components (i.e., granulation tissue, connective tissue, bone, hemic cellularity, and blood clot). Microscopic examination of the implanted tissue was generally confined to noting the presence of bone and marrow and their extent.

RESULTS Immediately after femur evacuation, the medullary cavity is filled with a leucocyteinfiltrated blood clot. Macrophages and undifferentiated cells soon appear and lead to the formation of a granulation tissue as the clot retracts from the endosteal surface. The granulation tissue adjacent to bone is the initial site of active regeneration, and all samples selected for transplantation represented such regenerative foci. Relative changes in the composition of these foci with time after femur depopulation are depicted in fig. 1. The presumptive transplant consists mainly of granulation tissue during the first few days. There is, however, a sharp increase in Exptl Cell Res 71

IOOr

60 40 20 0

5

IO

15

20

25

30

35

40

Fig. 1. Abscissa: days after marrow removal; ordinate: tissue type as % of total. 0, granulation tissue; A, connective tissue; 0, bone; W, marrow. Relative change in composition of regenerating medullary tissue with time after femur evacuation.

regenerating connective tissue from the first to the sixth day. As shown in fig. 1, scattered osseous and hemic progenitors are seen occasionally by the end of the first week. This is followed by extensive trabeculization of the regenerating area during the second week along with the emergenceof hematopoietic islands. The subsequent picture is characterized by a progressively increasing marrow component coincidental with bone resorption and the disappearance of residual granulation tissue and connective tissue unassociated with hematopoiesis.

.

01

I 5

I IO

I 15

I 20

I 25

I 30

I 35

I 40

Fig. 2. Abscissa: age of regenerating tissue in days; ordinate: frequency. Frequency of implant growth as a function of age of regenerating medullary tissue.

Marrow regeneration

309

Table 1. Frequency of regenerating medullary tissue components in relation to transplantation potential No. of implants with Age of regenerating tissue, days

: 4 ii 12 17 40 Normal marrow

No. of takes with No. of implants

marrow tabsa

new connective tissue

7 25 27 10

2

0

0

0

1

23 2

1’: :

0 I

0 1

:z 10

z 3 3

8 -

9 3 0

92 3 0

i 3 3

51

-

-

-

-

osseous repopulating growth hemic ceils

No. of takes

bone

marrow

1 11 16

1 5 13 3

47 3 3

: 6 3 3

2 3 3

51

47

43

a Refers to residual marrow after evacuation of the medullary cavity.

The incidence of successfultransplantation (growth) of the regenerating tissue increases dramatically during the first week (fig. 2). In this respect, an implant of tissue taken 6 days after femur depopulation is identical with that of normal marrow. The basic data pertaining to the frequency of various medullary tissue components during regeneration in relation to transplantation potential are summarized in table 1. It will be seen that implant growth does not require the presence of osseous or hemic elements. Apropos of this, it is noteworthy that the rather high transplantation efficiency during the first few days cannot be attributed to the occasional small tabs of residual marrow which may remain after evacuation of the medullary cavity. Growth of subcutaneous implants of the early regenerating tissue seems to coincide with the appearance of new connective tissue (table 1). The probability of a take increases by a factor of nearly 3 when the implant contains a clearly recognizable connective tissue. Thus, as shown in table 2, there were 91% takes with a 1 to 4 day implant containing connective tissue in contrast to 35 Y0 takes in the absence of such tissue.

Successfulimplants of a young regenerating tissue tend to show a somewhat lower incidence of marrow than of bone in comparison to the findings with implants of older regenerating tissue or normal marrow. However, this difference can be related to differences in the quantity of bone which was formed. When all regenerating tissue implants were considered irrespective of age, marrow was seenin only 6 of 22 takes with 1 + bone (small island of osseoustissue) and in 24 of 24 takes with 2 to 4+ bone (beginning to complete ossicle formation). Over 50% takes with 1 to 6 day implants showed 1 + bone in contrast to only 10 % takes with older implants. In all of the implant growths, marrow eleTable 2. Transplanation efficiency of a l-4 day regenerating medullary tissue (RMT) with and without connective tissue (CT) % takes with Implant

No. of implants

RMTwithCT 22 RMT without CT 37 Normal marrow 51

% takes bone marrow 91

85

60

35

85

54

100

92

84

Exptl Cell Res 71

Fig. 3. Photomicrographs of regenerating medullary tissue and implant growth. (a) 4 day regenerating tissue in situ. H & E, x 190. Note granulation tissue and emerging connective tissue adjacent to bone. (b, c) Implant sites 35 days after transplantation of a 4 day regenerating tissue (b) and normal marrow from the contralateral femur (c). Mallory’s aniline blue collagen stain, x 30. Note ossicles containing marrow and residual trabecular bone.

culature, where the availability of a mere handful of hematopoietic stem cells is sufficient to reestablish normal function. The reconstitution of bone marrow in a vacant medullary cavity consists of several well defined transitions. These begin with the formation of granulation tissue and involve successively the emergence of a connective tissue and vasculature, and the transient invasion by trabecular bone as a prelude to the restoration of sinusoidal marrow [l-3, 81. Significantly, a rather similar pattern is seen with a heterotopic autologous transplant of normal bone marrow [7]. In this instance, frankly hematopoietic elements disappear, reticular tissue develops, and bone is formed DISCUSSION and subsequently filled with marrow. The When marrow is missing or its structure is investigation reported here involves both sysdisrupted, regeneration depends upon the tems, i.e., marrow removal as an experimental interplay of several developmental processes. model and marrow grafting as a method of This is in contrast to the restoration of active assay. Hence, inferences relevant to each may marrow within an existing stroma and vas- be gleaned from the results. ments were never seen in the absence of bone. There is a striking qualitative similarity in development of implants of a 2 to 4 day regenerating tissue and normal marrow. In each case, marrow encapsulated by bone can be seen 5 weeks after subcutaneous implantation (fig. 3). Hence, although the frequency of growth with bone and marrow formation increases with age of the transplant, particularly during the first week, it is obvious that even a 2 day regenerating tissue may have already acquired the potential of normal marrow as assayedin this way.

Exptl Cell Res 71

Marrow regeneration

It is clear from the present studies that a regenerating medullary tissue can acquire the potential of normal marrow before there are any obvious marrow elements or hematopoietic colony-forming cells [lo]. The potential is revealed as early as 2 days after femur depopulation. The probability of successful transplantation increases significantly as an organized connective tissue appears. However, the latter is not a firm requirement; about 35 % transplants without obvious connective tissue grow in a subcutaneous site in contrast to over 90% takes when such tissue is present. We infer from this that the pertinent component is an undifferentiated (mesenchymal) element associated with the emerging connective tissue. This conclusion is supported by our previous finding of intense proliferative activity of perivascular connective tissue cells in the haversian canals of the adjacent bone during the first few days after marrow extrusion [8]. It is known that regeneration of marrow in a mechanically depleted medullary cavity is a local phenomenon which does not require the seeding of circulating stem cells [4, 51. The picture with extramedullary implants is less clear. Here, it is thought that growth is initiated by surviving reticulum cells, some of which first differentiate to osteoblasts and others subsequently to sinusoidal cells [7]. But the cells that lead to hematopoietic elements within the lattice of trabecular bone can have a different origin. Although there is some histologic evidence of a similar origin [l 11,chromosome marker studies of semisyngeneic marrow transplants under the renal capsule of mice indicate that the bone is donor-type while the hematopoietic cells associated with bone are host-type [12]. In experiments with a closed system, i.e., in vivo implanted millipore filter chambers containing a piece of bone marrow or a suspension of bone marrow cells, bone for-

311

mation was not associated with hematopoiesis 30 days after transplantation [13]. This finding, however, does not necessarily support the conclusion that bone and marrow development in a heterotopic graft always involves different initiating cell types. The formation and integrity of marrow is strongly dependent upon the microenvironment and it is not known whether the requisite conditions, e.g., an appropriate bone structure, can be achieved in a closed system such as a millipore chamber. In this context, it is of interest that extensive bone formation and resorption with associated marrow has been seen in a single instance when a millipore chamber culture was implanted for 3 months [14]. In other millipore chamber studies only isolated marrow cells persisted for a time, bone was apparently not formed, and the implant eventually became fibroblastic [ 151. A certain number and density of the initial packing of marrow cells is necessary for bone formation [13] which could account for the failure to detect bone in this instance. Although marrow can be regenerated without the formation of cancellous bone when stromal and hematopoietic cells are present in their natural environment [ 161,bone formation is an invariant feature of the regenerative process when such cells are removed, injured, or placed in a foreign setting [l-3, 7, 8, 171.It has been suggested that the shortlived trabecular bone could contribute stimulating factors, provide a scaffolding for reconstruction of the sinusoidal marrow, or perhaps even provide a further source of progenitor cells. It should be emphasized that the production of bony tissue per se is not necessarily a forerunner of marrow development. However, it seemsthat the formation of a critical mass of cancellous bone and its resorption is almost always associatedwith the formation of bone marrow. This can be seen in extramedullary as well asin medullary sites. Exptl Cell Res 71

312 H. M. Patt & Mary A. Maloney

We may recall that there was a somewhat lower incidence of marrow than of bone in the growth derived from implants of early regenerating medullary tissue. Implants of older regenerating tissue were similar to those of normal marrow in this respect. The difference in yield of bone and marrow could perhaps be attributed to the fact that implants of early regenerating tissue were generally smaller, and possibily not as well vascularized. A certain massof bone must be formed before the process of bone resorption is activated. Significantly, the absence of marrow was restricted to those cases where only a small island of bone was apparent in the implant site. Marrow was always present when bone resorption was evident as judged by the beginning formation of an ossicle. It never appeared in the absenceof bone. It is still an open question whether bone marrow contains a discrete line of committed osteogenic cells (preosteoblasts) or a line of uncommitted cells which retains the potential to differentiate along various pathways, e.g., bone, stroma, and hematopoiesis. Reticulum cells have often been considered to fulfill the latter role as the mesenchymal cell population of marrow. Apparently, mesenchymal cell populations of many connective tissues can modulate in the presence of a suitable environment, such as that provided by a decalcified bone matrix, and produce new bone, which eventually contains new bone marrow [18]. Thus, it is perhaps not surprising that mesenchymal elements of bone can reconstitute bone marrow in a depleted medullary cavity without recourse to bloodborne stem cells [4, 51. Nor is it surprising that an autologous transplant of the early regenerating tissue which contains such modulated elements has the potential of normal bone marrow in an extramedullary locale. Mesenchymal cell populations in variExptl Cell Res 71

ous sites are known to differ in their osteogenetic competence [ 181. Moreover, since various sites differ in respect to their microenvironment and vascularity, one might anticipate a spectrum of effects and a variable contribution by the host to the growth and development of an implant [6, 19, 201. This work was performed under the auspices of the US Atomic Energy Commission. The authors gratefully acknowledge the technical assistance of Mm Margaret Miller and Miss Georgina Dunn.

REFERENCES 1. Riihlich, D, Z mikroskop anat Forsch 49 (1941) 425. 2. Steinberg, B & Hufford, V, Arch pathol (Chicago) 43 (1947) 117. 3. Br?inema&, P; Breine, U, Johansson, B, Roylance, P J, Rbckert, H & Yoffey, J M, Acta anat 59 (1964) 1. 4. Maloney, M A & Patt, H M, Science 165 (1969) 71. 5. Fong, P L, Maloney, M A & Patt, H M, Blood 37 (1971) 413. 6. Jacob, S W, Maloney, W C, Barsamian, E M, Owen, 0 E & Dunphy, J E, Surg gynec obstet 109 (1959) 697. 7. Tavassoli, M & Crosby, W H, Science 161 (1968) 54. 8. Patt, H M & Maloney, M A, Hemopoietic cellular proliferation (ed F Stohlman, jr) p. 56. Grune & Stratton, New York (1970). 9. Maloney, M A & Patt, H M, Cell tissue kinet 2 (1969) 29. 10. Maloney, M A, Proc sot exptl biol med 135 (1970) 412. 11. Bdnemark, P I & Breine, U, Blut X (1964) 236. 12. Friedenstein, A J, Petrakova, K V, Kurolesova, A I & Frolova, G P, Transplantation 6 (1968) 230. 13. Friedenstein, A J, Piatetzky-Shapiro, S & Petrakova, K V, J embryo1 exptl morph01 16 (1966) 581. 14. Rosin, A, Freiberg, H & Zajicek, G, Exptl cell res 29 (1963) 176. Berman, I & Kaplan, H, Blood 14 (1959) 1040. :2: Knospe, W H, Blom, J & Crosby, W H, Blood 31 (1968) 400. 17. Amsel, S, Maniatis, A, Tavassoli, M & Crosby, W H, Anat ret 164 (1969) 101. 18. Urist, M R, Hay, P H, Dubuc, F & Buring, K, Clin orthopaed 64 (1969) 194. 19., Tavassoli, M, Maniatis, A & Crosby, W H, Proc sot exptl biol med 133 (1970) 878. 20. Boyne, P J, Clin orthopaed 73 (1970) 199. Received September 29, 1971