1. The effects of frozen irradiated homogenous bone grafts on the healing of circumscribed defects in the mandibles and femora of forty albino rats have been studied.






2. The grafts were well tolerated by the host animals and did not appear in any way either to stimulate or to inhibit osteogenesis. The rate of healing of the defects containing the homografts compared favourably with that of the control defects.KIM201

Cleaton-Jones, P. E.

Maier, G.

Retief, D. H.

English

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THE EFFECT OF FROZEN IRRADIATED BONE HOMOGRAFTS ON THEHEALING OF DEFECTS IN THE MANDIBLE AND FEMUR OF THE RATP. E. CLEATON-JONES, D. H. RETIEF and G. MAIER*, JOHANNESBURG, SOUTH AFRICAFronl the Dental Research Unit ofthe University ofthe Witwatersrand and South African Medical Research Col11lciand the * Tissue Bank aiwl Transplantation Research Unit of the South African Institute for Medical Research,Johannesburg, South AfricaNumerous studies have been carried out to determine the effect of various types of bonetransplants on the healing pattern of experimentally produced defects in animals (Maatz,Lentz and Graf 1954; Burwell 1963; Heipel, Chase and Herndon 1963; Melcher and Irving1963; Buring and Urist 1967; Raina 1968). These have repeatedly shown that autogenousbone grafts are better than homogenous and heterogenous bone grafts.In clinical practice it is not always possible to use autogenous grafts, and in such instanceshomogenous grafts are the next best variety. The advent of tissue banks has made readilyavailable stored homogenous bone which is often irradiated for sterilisation. Since March1964 some 1,000 cases have been treated using frozen homogenous bone prepared by theJohannesburg Tissue Bank.This study is an experimental investigation of the effect of frozen irradiated homogenousbone prepared by the Johannesburg Tissue Bank on the healing mechanism in circumscribeddefects in albino rats.MATERIALS AND METHODSPreparation of the experimental homografts-Wistar strain albino rats, selected at random,were killed by coal gas. Their femora were immediately excised, cleaned of all soft tissueand cartilage and cut into conveniently sized portions. These were repeatedly washedin normal saline at 37 degrees Centigrade until completely clean. After this, the fragmentswere individually packed and sealed in polyethylene bags, placed in dry ice at minus 79 degreesCentigrade and gamma irradiated* at this temperature using a 60Co source to a total dose ofbetween four and seven megarads. The irradiated frozen bone was then stored in a deepfreeze at minus 30 degrees Centigrade until required. The grafts were thawed at roomtemperature immediately before use.Operative procedures-The recipients were forty Wistar strain albino rats of a mean weightof 150 grammes. Under neurolept anaesthesia (a fluanisone-phentanyl mixture, Philips-Duphar, 02 millilitre per 100 grammes body weight) circumscribed defects were made in themandibles and femora (Fig. 1) as follows.Mandibles-A longitudinal midline incision two centimetres long was made in the skin ofthe ventral aspect of the neck, extending from the symphysis menti towards the sternum.The underlying digastrie and superficial masseter muscles were separated by blunt dissectionand the tendinous insertion of the deep masseteric muscle on the oblique masseteric ridgewas exposed. This was then cut and reflected to expose the lateral aspect of the mandible.With a slowly running water-cooled round dental burr, defects approximately two millimetresin diameter were made in the mandible on both sides just above the masseteric ridge and inline with the distal root of the first molar. The depth of the defect was approximately one* Gamma irradiation was performed by the Nuclear Physics Research Unit ofthe University ofthe Witwatersrandusing a 60Co source donated by the National Council for the Care of Cripples.532 THE JOURNAL OF BONE AND JOINT SURGERYEFFECT OF FROZEN IRRADIATED BONE HOMOGRAFTS ON HEALING OF DEFECTS 533VOL. 53 B, NO. 3, AUGUST 1971millimetre, which ensured that it extended through the cortex into the meduliary bone. Thistechnique required only a single skin incision.Fernora-A longitudinal incision was made over the lateral aspect of the right femur. Theaponeurotic insertion of the biceps femoris muscle was exposed and separated by bluntdissection. A circumscribed defect was then made in the midshaft in the femur in the samemanner as described for the mandible.FIG. 1Diagram showing the position of the defects in the mandibleand femur.Into all the femoral defects and into all but twelve of the mandibular defects frozenirradiated bone was transplanted. The overlying muscles were then approximated and theskin incisions sutured with silk. The recipient rats were placed in well aired cages andallowed routine laboratory rations and as much water as they desired.Retrieval of specimens-The rats were killed in groups by coal gas, at weekly intervals up totwo months and then at three-monthly intervals until nine months. The bones containing thegrafts were excised en bloc together with some overlying soft tissue.Tissue preparation-The specimens were fixed in 10 per cent buffered formaiin, decalcified ineither 5 per cent formic acid or de Castro’s solution and embedded in paraffin wax. Serialsections were cut at 7 micra using a rotary microtome and stained with haematoxylin and eosin.RESULTSMandibular defects-The healing pattern of the twelve control defects followed that describedby Retief and Cleaton-Jones (1970). The defects containing the homografts showed thefollowing healing pattern.At one s’eek, new bone was already in contact with the graft itself. This new bone arose fromthe cut margins and intact endosteal medullary bone trabeculae in the floor of the defect.No periosteal callus was seen, nor was any foreign-body reaction evident. The spaces betweenthe new bony trabeculae were filled with a fine loose fibrous tissue (Fig. 2).At two tt’eeks, the endosteal callus was increased in amount, the trabeculae being larger andthicker. Periosteal callus was now evident on either side of the defect, extending over theunderlying callus and graft but not completely bridging the defect. A greater area of thegraft was now in contact with new bone (Fig. 3).At three weeks, the periosteal callus had bridged the defect so that the graft was completelysurrounded by bone. Remodelling of the callus had now begun and blood vessels were evidentin the graft (Fig. 4). Active haemopoietic bone marrow could be seen between many of thenew bony trabeculae.FIG. 2Photomicrograph of homograft (I-f) in a mandibular defect at one week showing endosteal callus(EC). Arrows indicate the margins of the defect. ( - 40.)FIG. 3Photomicrograph of homograft (H) in a mandibular defect at two weeks showing endosteal callus(EC) and periosteal callus (PC). ( - 40.)534 p. E. CLEATON-JONES, D. H. RETIEF AND G. MAIERTHE JOURNAL OF BONE AND JOINT SURGERYAt six iieeks, remodelling of the callus was continuing. Perivascular bone deposition andresorption were seen within the graft itself.At tito mont/is, much remodelling had occurred, particularly of the periosteal callus, and amature trabecular pattern was present. The grafts had now decreased in size and active bonemarrow was present throughout the healing defect (Fig. 5).FIG. 5Photomicrograph of homograft (I-f) in mandibular defect at two months showing a thick periostealcallus (PC) and active bone marrow (BM). ( -: 40.)EFFECT OF FROZEN IRRADIATED BONE HOMOGRAFTS ON HEALING OF DEFECTS 535VOL. 53B, NO. 3, AUGUST 1971At nine months, the defect was well healed and covered with a thick cortical bone plate. Theoutline of the original defect could be discerned with difficulty in a number of sections. Smallgraft fragments were still evident in all specimens (Fig. 6).Femorai defects-The normal healing pattern of circumscribed defects in the femora of albinorats as described by Meleher and Irving (1962) was used as a control.At one week, the graft was surrounded by a marked proliferation of callus arising from theF,G. 6Photomicrograph of a healed mandibular defect at nine months showing fragments of ilomograft (H). ( > 40.)! FIG. 7536 P. E. CLEATON-JONES, D. H. RETIEF AND G. MAIERTHE JOURNAL OF BONE AND JOINT SURGERYendosteal surface and cut margins of the defect. This callus protruded outwards beyond themargin of the defect. The trabeculae were thin and orientated at right angles to the surface,,‘‘._ . , /4#{231}t   4.., ),! v: :;:i..: :t Photomicrograph of homograft (H) in a fenloral defect at one week showing endosteal callus (EC) and periostealcallus (PC). ( < 40.)of the defect. A fine fibrous tissue filled the spaces between the trabeculae. New bone waslaid down directly on to the surface of the graft. Subperiosteal callus was seen which wasPhotomicrograph of homograft (H) in a femoral defect at two weeks showing increased periostealcallus (PC) growing over thickened endosteal callus (EC). ( > 40.)7   -,“ 4)‘ 4v_‘4.4_p #{149}‘ S... ‘- :- High power view of the specimen shown in Figure 8. New blood vessels (BV) are seen invadingthe homograft (H). New bone is in contact with the graft. ( : 320.)VOL. 53 B, NO. 3, AUGUST 1971EFFECT OF FROZEN IRRADIATED BONE HOMOGRAFTS ON HEALING OF DEFECTS 537small in amount and well away from the edges of the defect. No foreign-body reaction wasseen (Fig. 7).At tito iteeks, the bony trabeculae filling the defect had increased in thickness. Periostealcallus had increased in amount and was growing over the underlying endosteal trabeculae.538 p. F. CLEATON-JONES, D. H. RETIEF AND G. MAIERActive bone marrow was present between some of the trabeculae in the deeper layers (Fig. 8).New blood vessels were seen invading the graft. New bone was laid down directly on to allsurfaces of the graft (Fig. 9).FIG. 10Photomicrograph of homograft (H) in a healed femoral defect at six weeks. ( - 40.)‘S - .      . I,.,- #{149}- #{149}  ,. .‘ - .- ‘S..‘4THE JOURNAL OF BONE AND JOINT SURGERY.4 - .  . ‘   .  i,  .. J. : -   : . ...... ....T ‘ .,  I’ #{149}C1I ilL ----4FIG. 11Photomicrograph of a healed femoral defect at seven nlonths showing remnants of a homograft (H). ( #{149}64.)At si.v ;teeks, the defect was well healed with the graft incorporated into cortical bone. Thegraft was now much reduced in size and remodelling ofthe cortical plate had occurred (Fig. 10).EFFECT OF FROZEN IRRADIATED BONE HOMOGRAFTS ON HEALING OF DEFECTS 539At seven months, the margins of the defect were evident in some sections and small fragmentsof graft remained in all (Fig. 1 1).DISCUSSIONAn extra-oral approach to the production ofthe mandibular defects was used, as Mesrobianand Shklar (1969) showed that a marked inflammatory response caused by oral bacterialflora or food impaction influences healing. In the mandible careful positioning of the defectis necessary. If it is prepared too far above the masseteric ridge the underlying inferioralveolar nerve may be damaged. This adversely affects the healing pattern (Retief andDreyer 1967). On the other hand, if the defect is made too far posteriorly above the obliqueridge then the underlying continuously erupting incisor may be damaged. Melcher (1967)showed that the defect when penetrating that tooth was not occluded by callus six weeks later.In this study any specimen showing such nerve or tooth damage was discarded.Thompson (1958) showed that a burr speed of 500 revolutions per minute produced littledegeneration of osteocytes in the bone adjoining defects prepared in the mandibies of dogs.He showed that too slow a burr speed produced fragmentation of the margins of the defect,whereas higher speeds could produce severe cellular damage. Initially this was evidenced bylighter staining of the cytoplasm and pyknosis and eventually complete disintegration of theosteocytes, leaving empty lacunae. The burr speed and water cooling used in this study producedminimal cellular damage manifested by a few empty lacunae in the bone adjoining some ofthe defects.In both the mandibular and the femoral defects containing homografts no foreign-bodyreaction was seen and the homografts appeared well tolerated by the host tissues. Thesefindings are similar to those of Brooks, Heiple, Herndon and Powell (1963).The homografts did not appear in any way to inhibit osteogenesis other than serving asa mechanical barrier to the newly formed bone. The rate of new bone formation in themandibular grafts followed closely that in the controls while the femoral grafts followed thatdescribed by Melcher and Irving (1962). It is felt therefore that the homografts in no waystimulated new bone formation. This is in keeping with the findings of Buring (1968) thatgamma radiation used to sterilise organic bone matrix totally destroys the inductive capacityof the graft. The amount of new bone formed was less in amount than that in the controlsby the amount of unresorbed graft.Essentially then, our findings are similar to those of Heiple et al. (1963), who in theirstudy of healing after the implantation of various grafts in dogs found that frozen irradiatedgrafts compared favourably with the controls.SUMMARY1 . The effects of frozen irradiated homogenous bone grafts on the healing of circumscribeddefects in the mandibles and femora of forty albino rats have been studied.2. The grafts were well tolerated by the host animals and did not appear in any way either tostimuiate or to inhibit osteogenesis. The rate ofhealing ofthe defects containing the homograftscompared favourably with that of the control defects.REFERENCESBROOKS, D. B., HEIPLE, K. G., HERNDON, C. H., and POWELL, A. E. (1963): Immunological Factors inHomogenous Bone Transplantation. IV. The Effect of Various Methods of Preparation and Irradiationon Antigenicity. Jourizal of Bone and Joint Surgery, 45-A, 1617.BURING, K. (1968): Effects of Ionizing Radiation on the Matrix of Bone Implants. Cak,f-ied Tissue Research, 2,Supplement, 8.BURING, K., and URIST, M. R. (1967): Effects of Ionizing Radiation on the Bone Induction Principle in theMatrix of Bone Implants. Clinical Orthopaedics and Related Research, 55, 225.VOL. 53B, NO. 3, AUGUST 1971540 i. E. CLEATON-JONES, D. H. RETIEF AND G. MAIERBURWELL, R. G. (1963): Studies in the Transplantation of Bone. V. The Capacity of Fresh and TreatedHomografts of Bone to Evoke Transplantation Immunity. Journal of Bone and Joint Surgerv, 45-B, 386.HEIPLE, K. G., CHASE, S. W., and HERNDON, C. H. (1963): A Comparative Study of the Healing ProcessFollowing Different Types of Bone Transplantation. Journal of Bone and Joi,zt Surgery, 45-A, 1593.MAATZ, R., LENTZ, W., and GRAF, R. (1954): Spongiosa Test of Bone Grafts for Transplantation. Journal ofBone and Joint Surgery, 36-A, 721.MELCHER, A. H. (1967): Wound Repair in the Periodontium of the Rat Incisor. Archives of Oral Biology,12, 1645.MELCHER, A. H., and IRVING, J. T. (1962): The Healing Mechanism in Artificially Created CircumscribedDefects in the Femora of Albino Rats. Journal of Bone and Joint Surgery, 44-B, 928.MELCHER, A. H., and IRVING, J. T. (1963): The Effect oflmplanting Various Substances into Artificially CreatedCircumscribed Defects in Rat Femurs. Jourizal of Bone and Joint Surgerv, 45-B, I 62.MESROBIAN, A. Z., and SHKLAR, G. (1969): The Effects of Dietary Zinc Sulfate Supplements on the Healing ofExperimental Extraction Wounds. Oral Surgery, Oral Medicine and Oral Pathology, 28, 259.RAINA, A. M. (1968): Experimental and Clinical Studies on X-ray Irradiated Mandibular Bone Grafts. Bulleti,zof Tokyo Medical a,zd Dental University, 15, 313.RETIEF, D. H., and CLEATON-JONES, P. E. (1970): The Healing of Circumscribed Defects in the Mandibles ofAlbino Rats. Journal of the Dental Association of South Africa, 25, 34.RETIEF, D. H., and DREYER, C. J. (1 967) : Effects of Neural Damage on the Repair of Bony Defects in tile Rat.Archives ofOral Biology, 12, 1035.THOMPSON, H. C. (1958): Effect of Drilling into Bone. Journal ofOral Surgeri’, 16, 22.THE JOURNAL OF BONE AND JOINT SURGERY

The effect of frozen irradiated bone homografts on the healing of defects in the mandible and femur of the rat

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The effect of frozen irradiated bone homografts on the healing of defects in the mandible and femur of the rat

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