Bosworth, David

Frost, Harold M.

Sezgin, Ziya

English

Henry Ford Health System Scholarly Commons

Henry Ford Hospital Medical Journal Volume 10 Number 1 Article 22 3-1962 Effect Of Marsilid On Diaphyseal Lamellar Rabbit Bone Ziya Sezgin David Bosworth Harold M. Frost Follow this and additional works at: https://scholarlycommons.henryford.com/hfhmedjournal  Part of the Life Sciences Commons, Medical Specialties Commons, and the Public Health Commons Recommended Citation Sezgin, Ziya; Bosworth, David; and Frost, Harold M. (1962) "Effect Of Marsilid On Diaphyseal Lamellar Rabbit Bone," Henry Ford Hospital Medical Bulletin : Vol. 10 : No. 1 , 209-216. Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol10/iss1/22 This Part II is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Henry Ford Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons. Henry Ford Hosp. Med. Bull. Vol. 10, March, pl. 2, 1962 EFFECT OF MARSILID ON DIAPHYSEAL LAMELLAR RABBIT BONE Z1YA SEZGIN, M.D., DAVID BoswoRrn, M.D., and H AROLD M. FROST, 1.D. INTRODUCTION THESE INVESTIGAT IO s follo,"ed the observation by one of us 1hat the quality of bone in patients who had received Marsilld for long period'i or time appeared 10 differ al 1he opera1ing iable from 1ha1 or pa1ien1s who had nol received 1he drug. This series of experiments resulted and were approached wi1h certain questions: I ) Is 1here an effecl or Marsilid on rabbi l bone? Ir so, is 1he effocl exened o n bone forma tion, on bone resorption or on the state of already present bone? 2) Is !here an effecl or 1e1racyclincs on rabbi! bone? 3) Is there any mutual synergism or opposition between Mar ilid and tetracyclines? 4) Does Marsilid arrcc1 1he amoun1 of lelracycline dcposi1cd in rabbi! bone in vivo? It is interesting to note that for the most part our experiments did not yield answers to these questions. although there were some relevant finding~. The work emb races three separa te experiments performed seq uc n1ia lly. the laller experiments to some degree being designed on the basis of experience gained from the earlier ones. In the first experiment Marsilid was given for prolonged periods. In the second, 1arsilid was given with orally administered tetracycline for an inter-mediate length of lime. In the third, Marsi11d and cortisone \\-ere given in toxic doses for brief periods. concomirnntly "ith tctrac)'cline. Attention in these experiments was limited to lamellar bone formation in the diaphyscs or selec1ed long bones in New Zea lond " hile rabbi is. 01hcr errec1s or 1he drugs on rate of growth , rate of gai n in weight, enchondral o:-isifica tion, and so forth were ignored, with mino r exceptions which will be noted. EXPERIMlNTS Experimem I Ten three-month old rabbits were divided. two serving as conlrols and eight as ex-perimental animal.,: . The lat1er group wat,: .given 10 mg l..ilo Marsilid .daily by gavage for 1hree months. Then all animals were sacrificed. their ages then being s1, months. All were healthy. There was no gross or histological evidence of liver damage in c11hcr group. 209 SEZGI , BOSWORTH AND FROST Figure I ndccalcif1cd cro'is sect ion tibia, 6 mon1hs old rabb11, treated with Marsil id 10 mg/ kilo/ day for 90 duY''· Pcno<,tcum at top, cndosteum below. \ ) 1'otc the ,one of m1crope1rosi!t. nppcaring as the cle,trer bone between the series of vcnicn l India ml bar'i. Thi~ cond11ion results from oc lus1on of canali ulne w11 h nuncral and 1s accom-panied or preceded by death of the os1eocy1cs depending o n the,;e cnnalicu lac for nutri tion. B) otc lad, of mternal recon'itruction of cortex and lack of ll nversian sy,; tems. This bone in rahb1t'S at th1~ age normally contains myraid 1--lnH:rsinn systems. The defect illus1ratcd is a '\Clcc.:ted, e,1reme c,ample. In a larger series (E,cperiment II ) the depression of remodclhng "a about 401':'; of the actml) obscncd in conirols. STATI I . Fresh. undecalcificd. fuchsin-stained cross sec tions were made from the midd le Ih m.I. and Junctions of lhe third-,, of the 1ibia a nd femur of one hind limb.'' From the same bone, of 1hc other hind limb longi1udinal scc1ions were prepared. Eighty sections \\ ere u11ablc for ob-,crva1ion. OBSLRVA'l 10 : A) There was considerable micropctrosis5 in the Marsi lid treated group and little in the t\\O control animals. This was interpreted as a possible o~teocytoto~ic effect rfqu1ring confirmation by repetition of, and enlargement of. the experiment (Figure 1) . B) 1 here w,1s a decrc1. se in m1crnal cortical remodelling, no1ed on reviewing the materia l ;.after 1he darn from E,:periment II were :wailable. By inlernal remodelling is meant 1he production of rc,orption 1ipaccs in the confines of the cortex . and subsequent fi ll ing of these spJtc, "1th H,l\1Cr1iian ,y terns (Figures I, 2b, a nd 3). () n increase of dead o~tCOC)tes, in 1hc ~farsilid trea ted group of animals. was prescnl in 1hc micropc1ro1ic bone. Since osteocycc death appears 10 be the prelude 10 development of micropctr , 1,. the two observa1ions ould be re lated. 1·:cprr1ment II f1f1 y-l\\O ,1\-wed.. old rabbits ,1,,erc divided into 1wo groups, a control and an experimenta l ~roup. All \\ere g1\icn tclraqcline ora ll y by gavage for an initial period of two \\eeks. fol-lo.,..cJ by t'ftO 'fted., without 1e1raC)clinc. follo .... cd by a no1her two \\eeks on tetra yclinc. Thi multiple-ba nd tctraqclinc labelling. and in vivo labelling of bone "ith the tetracycline iroup of an11h1011c, rc:wlrmg from the rcpons of Milch , Rall . and Tobie, is discussed in dc1a1l elsewhere.•' 10 11 210 I I l EFFECT OF MARS/LID Figure 2a A) Cross section M/ 3 libial crest from a rabbit on M:1rs11id for 6 Y.eeks. E,pcrime~ll II. Pcrios1eum is at the top. Blue-light m1cro~uorescence. Although 1c1racycline was g,,.cn twice for a total or 4 Y.Celr..s on ly 1races of it arc v1s1ble as bright spoh near the penostcum. Note the lack of resorption spuccs which are normally present to some c,1cnt in this part of the ~~~~d. There has been less ll aversian system replacement of the cort1cal in1erior than 1s normally Figure 2b 8) Similar bone. location and exptriment as in Figure 2A but from a con1rol animal which did not receive Marsilid. A duster of resorption pace , some acti,·el) being filled •1th ne\\ Ha,et"\ian bone lies in the cortical interior near the end teal (loY.er) surface. There i also more re· modellmg going on close lo the periosleum th3n ob!ler\ed in Figure :? The occa.sion3J bnghl lines seen on the pcriosteal and resorption <ip3lC !iurfoccs arc tctr3C)d1nc rluorcscmg by blue-light technique. The presence of lhc drug prO\.C~ 1hc presence of a'-'.tne mmcrahzat1on of bone mntrix. As in Figure 2A, 1hc extreme ca!ICS have been photogrnphed. 21 I SEZG IN, BOSWOR TH AND FROST Figure 3 Cro ~. undccalcificd sec tion 1hrough midd le thi rd of tib ia of control rabbit. Experimen t II. Compare "-Ith Figure I Some resorption spaces may be seen as en larged, black areas . In the actual section m1co1d scam (Pre.bone of Lacroix and his school) ma1 be seen, indicating new bone formation 1s in progress. There I cons1dcrablc Havcrsian recons1ruc11on present, almost all of the small, circular blad areas being ll nvcrsian canals seen in cross section. Thi -.cc11on rcprc~ms the average, rather than the extreme appearance of no rma l rabbit tibia of the age nottd in the text . Marsilid. 3 mg/ l..ilo/ day, was given to the experimental group for the duration of the experiment . ST ATI T IC There "NCrc 26 control and 26 experimental animals. Sect ions were made a de ribcd in Expcrimen1 I from the tibias. femurs. and in addition from humerus and rib. Over 800 M!CllOn5 were suitable for study. OB ERVATIONS: A) It was noted that internal remodell ing of the bones was retarded, meaning that there was le Haversian reconstruction of the cortex. in the an imals rece iving Mur\1l1d In an effon 10 quantitate this observation the number of resorption spaces within the ortice \I.a, counted 10 a series of sections. There were 738 resorption spaces in 23 cro, \CCtion') from the Marsilid group and 980 in 19 sections from the control group. This 1s an average or 32 per section in the experimenta l. and S2 per section in the cont rol. groups. This finding was not anticipa ted. We believe it is rea l rather than a statistical fl u~e and It i, \I.Orthy of confirmation by others (Figures 2a. 2b. and 3). 8) Admm1strat1on of tetracycline was accompanied by an unequivocal decrease in eit her the rare of formauon of new osteoid, or the rate of its minera liza tion, or both. Th is was true m the control~ and the Marsi lid treated group. Re all that the duration of the periods when tetracycline was. given and the duration ~~:,~~ ~e~?,~~n .. s;r~~,h~:~~ ·~;cs:~~ !! ~encwfJ~t~hf ~~~e :n~~~111~darba~d :~1~:~ ~~:!~ What was actually ~en was also not anticipated: In eight animals the thickness of the m111al 1etr.1qhne band a\'craged SO micron ; of the second tetracycline band 30 microns; 212 I EFFECT OF MARS/LID but of the unlabeled band bet'4'CCn them 300 micron . It 1s unl.nown whether this effect 1s a direct result of the tetracyclines, or an indirect effect or this or some other factor in the experimental design. C) It was tentatively observed that Marsilid decreased the rate of circumferential lamellar apposition. An average of 381 microns was added to the outside diameter of the femur in two control animals and 270 microns in si:c: Marsilid treated animals over a two week period. This sou nds better than it is because the scatter in individual cases was too large to permit confidence in the meaning of the averaged values. D) There was no significant difference in the omount of micropetrosis noted in lhc control and Marsi lid treated animals in this experiment. The duration of the experiment was admittedly less but one has more confidence in a negative resu lt involving study or 52 animals than from a positive result involving only ten. In addition, the animals in the present experiment were still immature at time of sacrifice, while those in experiment one were mature. Micropetrosis appears to be a common feature of adult rodent bone. E) The information sought for when this experiment was designed could not be obtained. Mutual effects of tetracycline and Marsilid could not be evaluated because oral labelling proved to be a completely unsat isfactory method of labelling rabbit bone. In only eigh1 of the 52 animals could enough tetracycline be seen in the sections to pe.-mit measurements. The equipment used was orthodox and efficient: a Zeiss (Oberl..ochen) 200 watt b_igh pressure mercury source used with Zeiss barrier filters. Russe in vienna ha experienced the same difficulty with labelling rabbits.12 No such difficulties have been noted in labelling man, rats, or dogs.' Experiment JI/ Six week o ld rabbits were again used , a triple band design was aga in c;elected. the period between tetracycline labels being a weel... the labels being intraperitoneally injected tetraqcline, 200 mg/ l..ilo, given on the first day of three successive wcel..s. Six or these animals were placed on a toxic dose of Marsilid, 25 mg/ kilo/ day for a week. Three animals were placed On 50 mg/ kilo/ day of cortisone acetate for a week and six animals served as controls. Since no tetracycline was administered during the actua l times the other drugs were given there wa.; no question of mutual effects. ~ -• -. . ., ' i , • ' . • . J ·" -~ .. • -~ . ;,. ... " Figure 4 Undccalcified cross section tibia of rabbit given 50 mg/ .. ilo of cortisone during its last wed. of life, as described in Experiment Ill . Bright field . Periosteum is at top. 01e irregular periostcal urface which appears to be the result of resorption. ote that most or the remainder of the cortex (which was formed before cortisone was tarted) e)(h1b1ts much Ha"en.ian reconstruction. 213 SEZGIN, BOSWORTH AND FROST STATI TIC ; T"'o of the 1arsilid group died during the experiment while receiving this drug. The rema1mng animals were sacrificed from on 10 nine days after ending the C).pcnmenl and sec1ions made from bones as described in Experiment 11. OB ER ATIO S: A) The two Marsllid treated animals that died prematurely exhibited h\ier damage gro ly and histologically. B) In the surviving Marsilid treated animals there was a n increase in death of ostcocytcs. but only in bone formed during the experiment. Thii. distribution of osteocyte death is different from that seen in micrope.trosis and as the result of increase in age. C) In th cort15,onc 1reated animals sacrificed immediately afler stoppage of the drug. there was an almosr total arrest of new bone formation with an accompanying lack of osteoid seam~ and decrease in lhe amounL of fuchsin permeable bone (Figure 4). By way of c"<planation it may be no1ed that recen1ly formed bone is fuchsin permeable because 11 has not hnd time 10 mineralize to the point that diffusion of the dye is cffec1ively prevented. Wi1h increasing lime after formation bone normall y becomes fuchsin impermeable. Whe n new bone formation is arrested. no new fuchsin permeable material is formed but that already prc~ent continues to mineralize. so that nf1er a sufficient period of time little or no fuchsin permeable bone will be obscrved.1 D) If cortisone treated a nimals are not sacrificed until a week or so after stoppage of the drug there is a distinct "bounce" in new bone formation during this week and very active pcrios1eal appositional activity may be observed. as though nature were trying to maleup for lo~t ume. E) Intraperitoneally injcct~d tetracycline produce~ intense labels easily observed by ultrav1olc1' or bluc•light m1crofluorescence.' 11 However. the formation of new periosteal bone i a very irregular, sporadic process in rabbits with the result that often a single dose of tetracycline doc°' not show up on sections: no bone was actively being formed when the drug was given (Figure 5). F) In tarsilid treated animals sacrificed at the conclusion of the experiment there was little acuvc perio~tcal bone formation observed. few osteoid seams. and little fuchsin permeable bone. G) In Marsilid treated animals sacri ficed a week or more after concl usion of the e'\perimcnl very a 11ve pcriostcal new bone formation was appare nt , indicaling that there was a "bounce" in bone formation just as was seen in the cort isone treated, but le~s numeroui. group of anima ls (Figure 6). CONCLUSIONS Subject 10 independent confirmation the fo llowing statements about this work appear reasonable: I) Marsilid seems to inhibit internal cortical reconstruction in rabbit diaphysis. The histology suggests that this is the result of interference with the initial phase of reconscruction: the formation of resorption spaces. 2) There is no definite evidence that Marsi lid in reasonable doses exerts any effect on osteOC}tes. There i::, definite evidence that in toxic doses Marsi lid has a n o~lcOC} totoA1c effect on the osteOC) tes in recently formed bone. 3) Toxic dosages of larsilid produce bone effects similar to those produced by cor11sone. It is therefore possible that the observed effects of Marsilid were the rct,ult of an endogenous increase in cortisone secre tion resulting from the slrcss of the experiment It al~ is possible that larsi lid in some manner potentiatcs the effect of endogenou,I) secreted corticoids. 4) The rebound acceleration an bone formation noted in Experiment Ill has been obscf'ed by other onve tigators. notably Collins, Garrett, and Johnston' using 214 I EfFECT OF MARS/ LID Figure 5 ndc :llciricd cross sec1ion 1ibia of rabbit gi\CO 25 mg L1lo of M arsilid in the last v.ccl of its life. i~1:;~N;ht1hen~~~~~n~~~j\=c~~;;~~l'::a:~o~1 o}h~c:p~a~~xe d~r:fn~1 11~~~ :r:el.;~u;~~~~"fo 1!!:~~l~~.ne and Note !he irregular pcriostc:d surface, cspccitll ly nt the right. simil::lr 10 but not as marked as that illustrated in Figure 4. Only t"o of the 3 tetra9chnc bands arc 1dent1f1ablc. The morphololty suggests 1ha1 lhe third band 1s lhe missing one. Sinc.:c ths band 1~ al~o m,~smg m 1110.ll of the comrol no conclus,ons may be drawn. Figu re 6 Undccalcifled eras ~ction 11bia from a rabbit ~,,en \1 :milid 25 m~ lilo for 7 days, but not sacri-ficed until 9 day after stoppage of the drug. \licrofluorcsccncc. Penosteum at top. The top 1rrctubr layer i'> periosteum. J ust under this layer lies n thin lamina of more d:ukly \t;1imng bone. This layer i.!t fuch,in permeahlc and 1s 1he resuh of mten1oe new bone forming :u:muy follov. m~ stoppage of the drug. Compare with Figure 5. None of the 3 te1racyc:lme label :1re present m 1h1s bone! 2 15 SEZGI , BOSWORTH AND FROST calcium. It has been variously interpreted by investigators who did not have access to direct histological observation. 5) Rabbits appear lo be a poor animal for sludy of ralc of bone formalion due to their unpredictable, irregular rate of bone formation . There appears to be an adverse effect of tetracycline on rabbit bone formation which has not been noted in other mammals after birth and has not been noted in man . REFEREN ES I . Collins, E. J., Garrett. .. and Johnston, R. : Effect of steroid regimen on ea•, distribution, citcrct1on and resorp11on from bone in young bcogle . Paper submitted to the Gordon onferencc on truc1urc and Physiology or Bones and Teeth, Mcridan, N. H., July 1961. 2. J-rost , H. 1.: Preparation o f thin, undecalcificd bone sec tio ns by rnpid manual method, Stnin Technol. 33:273, 1958. 3. Frost. 1-1 . M.: Feat hering : A theory of genesis, Henry Ford Hosp. Med. Bull. 9: 103, 1961. 4. Frost , H. M.: Staining or fresh, undecaJcirir.d, thin bone sections, Stain Technol. 34: 135, 1959. S. host, H. M.: ficropctrosis, J. Bone & faint urg. 42A : 144, 1960. 6. t-ro t, H. M .. Roth, H., Villanueva, A. R .• nnd Sianisavljevic, S. : E~perimental muh1band 1etra ychne measurement of Jamellar ostcoblnstic activity, Henry Ford Hosp. Med. Bull. 9 ;312, 1961. 1. Fros~JI. 11~~~r~\~i~~uecv~~ti~SeRi~ a~i~o~o~~~i nll~cc::~~~cj~~i~;51st~~nJ~~ of newly forming bone 8. Le Blond, . P. Lacroi1t, P , Ponlot, R, and Ohem, A : Les ctudcs m1t1au1t de l'osteogcncse: nou\'cllcs donnccs h1stochcm1ques ct .1u1oradiograph1qucs. Bull . l"A cad. Roy Med. Belg 24 :421, 1959. 9. lellors, R. C.: Annl)licnl Cytology, ed. 2, cw York, Blakiston, 1955. 10. 111ch, R. A., Rall, D. P., nnd Tobie , J. E.: Bone locnliza1ion of the tetracyclines, J. Nat. Cancer Ins!. 19 :87, 1957. I I. 111 h. R. A., Rall, 0 . P., and Tobie, J. E. : Fluorescence of 1e1racycline antibiotics in bone, J. Bone & Joint urg. 40A :897, 1958. 12 Ru sc, 0 : Persona l communication ( Primnrius, Unfollkrankenhnus, Wien XII) . 13 Vm~cn1, J. : Re her he sur la constitution du 1issu osscux compa t, Arch. de Biol. 65:531, 1954. 216 l I 

Effect Of Marsilid On Diaphyseal Lamellar Rabbit Bone

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Effect Of Marsilid On Diaphyseal Lamellar Rabbit Bone

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