Austin, J C

English

Wits Institutional Repository on DSPACE

■S'. Afr. J. met). Sci. (1973), 38, 95-98
THE INDUCTION OF BONE IN ORGANIZING HAEMATOMATA
IN RATS
by
J. C. AUSTIN
Denial Research Unit o f  the University o f  the Witwatersrand and the 
South African Medical Research Council, Johannesburg.
[received 20tii November, 1973]
The induction of ectopic bone formation has been described in a wide range of 
experimental systems. This lias emphasized the fact that undifferentiated osteogenic 
precursor cells persist in post foetal life after embryonic differentiation has ceased. 
A key observation in the studies on the transformation of these cells into osteoblasts 
was made by Urist [1965] when he discovered that the decalcified organic matrices 
of bone and dentine had osteoinductive properties. This phenomenon is attributed 
to the physicochemical effect of an inductor present in these matrices which induces 
competent mesenchymal cells to differentiate into osteoblasts [Urist et al., 1967]. 
The osteogenetic competence of mesenchymal cell populations was shown to be 
restricted in post-foetal life to specific areas of the body and in these areas mesenchy­
mal cells showed dilferent levels of readiness to differentiate into bone in response 
to implants of decalcified allogenic bone matrix (D.A.B.M.) [Urist et a!., 1967].
The use of an induction system to assist refractory skeletal regeneration is dependent 
upon it being able to initiate osteogenesis and maintain it until bone continuity is 
restored. Extraskeletal implants of D.A.B.M. however become encapsulated in 
fibrous connective tissue soon after implantation and this forms a barrier which 
confines the yield of induced bone to the limits of the enclosed matrix [Urist, 1965],
The rapid unrestricted ingrowth of woven bone into organizing fracture haemato- 
mata suggested that the cell population in granulation tissue may provide a favourable 
environment for bone induction.
This study was undertaken to determine whether implants of D.A.B.M. would 
induce osteogenesis in the granulation tissue of organizing haematomata held in 
perforated silicone rubber chambers implanted subcutaneously in rats.
MATERIAL AND METHODS 
Preparation of silicone rubber chambers
Fifteen cylindrical silicone rubber chambers 12 mm long by 4 mm in diameter 
were prepared in order to hold blood clots and D.A.B.M. implants under standardized 
conditions in a soft tissue environment in rats (Fig. 1). The chambers were prepared 
using 250 /xm thick silicone polymer sheeting (Silastic—Dow Corning, Michigan, 
U.S.A.). This material is biologically inert and has no inhibitory effects on cellular 
growth or differentiation [Basset, 1962], The transparent sheeting was laid over a 
sheet of graph paper ruled with 1 mm squares and points situated 2 mm apart marked 
on to it in a square pattern. Using a dental rubber dam punch, holes 0.75 mm in 
diameter were punched through the rubber sheeting. The sheeting was then cut into 
12 mm squares, rolled into tubes and the apposing edges held together by means of 
a thin strip of adhesive tape applied internally along the seam. A bead of self curing 
silicone polymer liquid (Medical Adhesive Type A—Dow Corning) was applied ex­
ternally over the apposed edges of each tube and left to cure for 24 hours at room 
temperature. The adhesive tape on the inner surfaces was then removed and the open 
ends of the tubes closed with discs of silicone rubber 5 mm in diameter, applied with
96
the adhesive over the tube ends. After the adhesive had cured the chambers were 
washed in distilled water and autoclaved.
Preparation of D.A.B.M. implants
Four Long Evans rats weighing 150 g were killed with an overdose of pentobar­
bitone sodium administered intraperitoncally and their metatarsal bones were 
removed under aseptic conditions. The bones were scraped to remove all adhering 
soft tissues and the epiphyses were cut off. The remaining diaphyseal portions were 
decalcified in 250 ml of 0.6 N HC1 for 24 hours at 4°C. After decalcification the 
bones were cut into 6 mm lengths, rinsed in several changes of physiological saline 
and then stored in 70% ethanol. Just prior to their use the D.A.B.M. implants were 
rinsed in physiological saline to remove the ethanol.
Surgical procedure
Fifteen 260 g male Long Evans rats were anaesthetized in turn with a 
neuroleptanalgesic preparation (Thalamonal-Janssens Pharmaceutica, Belgium) 
containing 0.05 mg/ml fentanyl and 2.5 mg/ml droperidoi at a dosage rate of 2.5 
ml/Kg administered intramuscularly. Each rat was positioned lying on its right side 
and the skin over the right inner thigh and left flank shaved and cleansed with an 
alcoholic solution of chlorhcxidinc (Hibitanc I.C.I.).
A skin incision was made in the hind leg overlying the right femoral vein which 
was exposed by blunt dissection. 0.25 ml of blood was withdrawn from the vein in a 
syringe. A sterile silicone rubber chamber was filled with the blood and a D.A.B.M. 
implant placed in the chamber through a flapped open end of the chamber.
The filled chamber was placed in a petri dish and whilst the blood dotted, the leg 
wound was sutured. The left external oblique abdominal muscles were exposed through 
a flank incision and the chamber was sutured at each end to the muscle surface. 
The wound was then flooded with the residual blood held in the syringe filling the 
space surrounding the implanted chamber. The subcutaneous fat and skin was 
reapposed and sutured.
Retrieval o f specimens
Groups of 3 rats were killed at intervals of 1, 2, 3, 4 and 6 weeks after operation 
and the implanted chambers were carefully dissected out enclosed in a small amount 
of surrounding soft tissue.
Tissue preparation
The specimens were fixed in buffered formo! saline, dehydrated through serial 
alcohols, cleared in methyl salicylate and embedded in Paraplast (Sherwood Medical 
Industries Inc.). Serial sections, 7 /on thick, were cut transversely through the 
embedded chambers and stained with haematoxylin and eosin. In specimens in 
which newly calcified tissue was encountered, surface decalcification of the exposed 
end of the wax embedded tissue was performed in 5% nitric acid to enable a few 
decalcified sections to be prepared.
The South African Journal o f Medical Sciences
RESULTS
The operative wounds all healed by primary intention. At one week granulation 
tissue consisting of thin walled capillaries and young fibroblasts infiltrated with 
wandering macrophages and leucocytes had surrounded the chamber (Fig. 2). 
Granulation tissue had developed through the openings in the chamber walls and was 
proliferating into the blood clot within, along the inner surface of the chamber 
(Fig. 3). No changes had occurred in the D.A.B.M. implant at this stage.
At two weeks the granulation tissue had completely filled the chambers and 
enveloped the implants and filled their medullary cavities. Erosion chambers produced
97Austin: The induction o f bone in organizing haematomata in rats
by the proteolytic activities of wandering macrophages had appeared in the matrices 
of the implants and these were occupied by capillaries and mesenchymal cells (Fig. 4). 
At this stage the granulation tissue surrounding the implants had become less vascular 
and more fibrous, enclosing the implant in loose fibrous connective tissue.
At 3 weeks deposits of calcified tissue appeared in the implant matrices (Fig. 5). 
In partially decalcified sections induced osteoblasts were seen lying in contact with 
the newly formed matrix on the walls of excavation chambers situated around 
centrally placed capillaries (Fig. 6).
Deposits of new bone occurred in increasing amounts in the four and six weeks 
specimens. This only formed within the inner areas of the implanted matrix (Figs. 7 
and 8).
DISCUSSION
The induction of bone within organizing extraskeletal haematomata has indicated 
that osteogenetically competent cells are present in the cell population of granulation 
tissue.
The presence of clotted blood in the implanted chambers invited an ingrowth of 
granulation tissue which initially is highly vascular but soon becomes more fibrous. 
The D.A.B.M. implants become surrounded in a thick fibrous connective tissue 
capsules before deposits of new bone appeared. Urist et al. [1967] showed that 
intramuscular implants of D.A.B.M. were enclosed in a thin fibrous membrane 
within three to four days of implantation. This restricted any subsequent bone forma­
tion to the enclosed area.
For bone induction to occur directly in granulation tissue, an inductive substrate 
would have to act on mesenchymal cells present before they differentiate into connec­
tive tissue cells. Encapsulation of the inductive substrate occurred long before 
osteoblast differentiation began, effectively isolating the substrate from any surround­
ing cells with osteogenetic potential.
The implantation of allogenic human dentin matrix in gingival papillae to assist 
the regeneration of osseous defects in periodontal disease [Register et al., 1972] 
resulted in the formation of isolated ossicles of bone surrounded by connective tissue 
which did not attach to the alveolar bone.
For D.A.B.M. grafts to be used effectively direct contact between the inductive 
substrate and host bone is advisable to avoid encapsulation of the graft.
The restoration of experimental periodontal defects in dogs with D.A.B.M. by 
Narang and Wells [1972] showed that contact resulted in bony union between the 
graft and the host bone and stimulated the formation of new bone attached to host 
bone at the site of implantation.
The presence of clotted blood around decalcified allogenic bone matrix substrates 
creates conditions which are favourable for the isolation of induced bone in fibrous 
tissue.
SUMMARY
1. The induction of bone in extraskeletal haematomata with implants of decalcified 
allogenic matrix has been studied in rats.
2. The formation of new bone within the implants has indicated a source of 
osteogenetically competent mesenchymal cells in granulation tissue.
3. The implants were enclosed within a capsule of fibrous connective tissue within 
14 days of implantation.
98
4. This surrounding connective tissue capsule confined new bone formation to the 
area of the implanted matrix.
The South African Journal o f Medical Sciences
I am indebted to the stafl' of the Dental Research Unit for their guidance and encouragement 
and for their technical assistance with the histological preparations and photography.
REFERENCES
Basset, C. A. L. (1962). Current concepts o f bone formation. J. Done & Joint Sing., 44A, 1217-1244 
N akang, R. and Wells, H. (1972). Bone induction in experimental periodontal bony defects in 
dogs with decalcified allogenic bone matrix grafts. Oral Sin);., Oral M ed, Oral Path., 33, 306-313 
R egister, A. A., Scoim', W., Kassouny, D. Y., Beau, F. R. and Pesskin, D. (1972). Human bone 
induction by allogenic dentin matrix. J. Periodontal., 43, 459-467 
U rist, M. R. (1965). Bone formation by autoinduction. Science, 150, 893-899 
U r 1ST, M. R., H ay, P. I-I., Duauc, F. and Buring, K. (1969). Osteogenic competence. CUn. Ortho/)., 
64, 194-220
U rist, M. R., Silderman, B. F., Dunuc, F. L. and Rosenberg, .1. M. (1967). The bone induction 
principle. Clin. Ortho/)., 53, 243-283
PLATE 1
Fif/. 1. A silicone rubber implant chamber. Note the perforated walls to allow for granulation 
tissue ingrowth. (X 3)
Fif/. 2. Photomicrograph showing a section through an implanted chamber at I week. Lying in 
the centre of a film meshwork (F) is the D.A.B.M. implant (I). Granulation tissue (G) is seen growing 
through sectioned holes in the chamber walls (arrowed). The clear areas between the granulation 
tissue ingrowth (S) arc occupied by the chamber walls (X 100)
Fig. 3. Photomicrograph showing the granulation tissue ingrowth at 1 week, composed of thin 
walled capillaries (arrowed) and young fibroblasts, macrophages and lymphocytes growing into the 
film meshwork (F). (X 100)
Fig. 4. Photomicrograph of a section through the D.A.B.M. implant (1) at 2 weeks. Excavation 
chambers (arrowed) within it arc occupied by capillaries and mesenchymal cells. (X 230)
PLATE 2
Fig. 5. Photomicrograph of a section through the implanted matrix at 3 weeks showing deposits 
of calcified bone (N) within the implant. (X 290)
Fig. 6. Photomicrograph of a section through a partially decalcified specimen at 3 weeks showing 
young osteoblasts (O) lying on newly deposited bone on the walls of excavation chambers in the 
implanted matrix. Chondroblasts (C) arc also present in excavation chambers. (X 275)
Fig. 1. Photomicrograph of a section through an undccalcificd specimen at 6 weeks showing a 
large deposit of new bone (N) lying with the transplanted matrix (I) which is enclosed in a loose 
fibrous connective tissue. (X 70)
Fig. 8. Photomicrograph of a section through a partially decalcified specimen at 6 weeks. A cement 
line marks the interface between the new bone (N) and implanted D.A.B.M. matrix (I). A line of 
osteoblasts (O) differentiated from mesenchymal cells can be seen on the surface of newly formed 
bone. (X 165)


The induction of bone in organizing haematomata in rats

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The induction of bone in organizing haematomata in rats

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