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Thesis Submitted in Part Fulfilment for Under the Supervis'ion Of 4f 11-l't AN INVES TIGATION INTO THE EFFECTS OF AN ANTIMETABOLITE I ( METHOTREXATE ) ON BONE HEALING I t R.H.B. J0NES, B.D.S. B.Sc. (Dent. ) Thesis submitted in part fulfilment for the Degree of Master of Dental Surgery Under the supervis'ion of K.K. Chau M.D.S. F.R.A.C.D.S I I DEPARTMENT OF ORAL PATHOLOGY AND ll { ORAL SURGERY I j UNIVERSITY OF ADELAIDE I 'l I 1976 i t i TABLE OF CONTENTS Page PRECIS i DECLARATION ii ACKNOI^lLEDGEMENTS iii INTRODUCTI ON iv DEFINITION OF TERMS V CHAPTER I THE TREATMENT OF CANCER l.l A. METHODS OF CANCER THERAPY 1.2 (l ) Surgery 1.2 (2) Radiotherapy 1.3 (3) Chemotherapy 1.3 't B. THE CHEMOTHERAPEUTIC AGENTS .3 (l ) The Al ky'latì ng Agents 1.5 (2) The Anti -metabol i tes 1.6 ( i) Anti-purineagents 1.7 ( ii) Ant'i-pyrimidine agents 1.8 (iii) Folic Acid inhibjtors 1.8 (3 ) The Ant j b'ioti cs 1.ll (4) Hormones l.ll (5) Plant Extracts and Others t.1l C. THE ACTION OF CYTOTOXIC AGENTS ON THE CELL CYCLE 1.12 D. GENERAL PRINCiPLES OF CANCER CHEMOTHERAPY l.l5 E. THE ADMINISTRATION OF CYTOTOXIC AGENTS I .16 CHAPTER 2 IHE BIOLOGY OF BONE HEALING A. GENERAL ASPECTS OF I^IOUND HEALING 2.1 B. OSTEOGENESIS IN BONE REPAIR: AN HISTORICAL PERSPECTIVE 2.3 C. THE ROLE OF THE SKELETAL TISSUES IN OSTEOGENESIS 2.5 (l ) The Orig'in of the 0steoblast 2.5 (2) The Role of the Periosteum in Bone Healing 2.6 TABLE 0F CONTENTS (cont'd) Pa ge CHAPTER 2 THE BIOLOGY OF BONE HEALING (cont'd) .INDUCTION' D. THE ROLE OF IN OSTEOGENTSIS 2.7 (l ) The Development of the 'Induction Hypothes'is' 2.8 (2) The Stimulus for 0steogenic Induction 2.9 E. THE BI-PHASIC THEORY OF OSTEOGENESIS 2.12 F. FUNCT-IONAL ASPECTS OF THE OSTEOBLAST 2.13 r) The Pl euri -potenti al of the ' Progeni tor Cel I ' 2.14 2) Osteoblasts and Ground Substance 2.14 3) 0steoblasts and Bone Matrix Formation 2.15 4) 0steobl asts and Cal ci fìcati on 2.16 CHAPTER 3 THE HISTOLOGY OF BONE REPAIR A. HEALING OF FRACTURES IN LONG BONTS 3.1 ( I ) The Periosteal Reacti on 3.2 (2) The 0steocytes 3.3 (3) The Endosteum and Marrow Reaction 3.4 (4) Cartilage Formation within the Fracture 3.5 (5) Quantitat'ive aspects of Callus Formation in 3.5 Fracture Heal ing B. THE HEALING OF CORTICAL BONE DTFECTS 3.6 ( I ) The H'i stol ogy of Heal ì ng Cortì cal Defects 3.6 (2) The External vs the Internal Cal I us 3.8 j (3) Carti 1 age Formati on 'in the Heal ng of 3..l0 Corti cal Dèfects CHAPTER 4 THE EFFECTS OF CYTOTOXIC AGENTS AND RADIOTHERAPY 4.1 ON WOUND HEALING A THT EFFECTS OF IRRADIATION 4.1 (l ) The Effects of Irradiatìon on 'Fibroplasia' 4..l (2) The Effects of Irradiation on Vascular Tissues 4.2 i) Moderate doses 4.2 ii) Large doses 4.2 1'r1) Late effects 4.3 TABLE 0F CONTENTS (cont'd) Pa ge CHAPTER 4 THE EFFECTS OF CYTOTOXIC AGENTS AND RADIOTHERAPY ON hJO UND HEALING ( con t 'd (3) The tffects of Irradiation on Bone 4.4 i ) Di sturbance of bone growth 4.4 ì ) Degenerati ve changes 4.5 i ) D'i sturbance of bone repa i r 4.6 B. THE EFFECTS OF CYTOTOXIC AGENTS ON WOUND HEALING 4.7 (l ) The Effects of Cytotoxic Agents on'Fibrop'lasia' 4.7 (2) The Effects of Cytotoxic Agents on Blood Vessels 4.9 (3) The tffects of Cytotoxic Agents on Bone 4.9 (4) Studies on Indiv'idual Cytotoxìc Agen ts 4.9 ( i) The Alkyìatìng Agents 4.9 ( 'ii ) The Anti -metabol'ites 4.ll a. 5-Fl uorouraci I 4.I b. Methotrexate 4.12 a C A COMPARISON BTTWEEN THE EFFECTS OF IRRADIATION 4.12 and CYTOT0XIC AGENTS 0N W0UND HEALING CHAPTER 5 OBJECTS AND METHODOLOGY OF THE PRESENT INVESTIGATION A OBJ TCTS 5.1 UÞ METHODOLOGY 5.2 (l ) The Experimental Model 5.2 (2) Choice of the Experìmental Anirnal 5.3 (3) Choice of the Anti-cancer Chemotherapeutic Agent 5.3 (4) Method of Administrat'ion 5.3 (5) Dosage 5.4 (6) Schedul e of Drug Adm'in j strat'ion 5.5 (7) Establishment of a Control 5.7 C. THE HISTOLOGICAL INVESTIGATION 5.7 (l ) The "lag phase" Mucopolysaccharide Production 5.7 (2) Cel I ul ar Prol i feration 5.10 (3) Coì I agen Formation 5.il (4) New Bone Formation 5.12 TABLE OF CONTENTS ( cont' d ) Pa ge CHAPTER 6 MATERIALS AND METHODS A WOUND PRODUCTION 6.1 B EXPERIMENTAL DTTAILS 6.2 (l) Group I-Control 6.2 (2) Group II - Operation and then Methotrexate 6.2 Administration (0M) (3) Group III - Methotrexate Administration and 6.2 then Operat'ion (M0) CHAPTER 7 RESULTS A. INTRODUCTION 7.1 B. THE CONTROL SERIES 7.8 C. IXPERIMENTAL SERIES - OM 7 .35 (l ) Bl ood Cl ot Format'ion 7.35 (2) The Inflammatory Reaction 7 .35 (3) Granulation Tissue Formatjon 7.35 ( i) Fibroblasts 7.35 ( ii) Ground substance 7. 38 (ijj) Extracellular fibre formation 7. 38 (4) Bone Formation 7 .40 ( i) Endosteal callus 7 .40 'ii ( ) Subperìosteal cal I us 7 .43 (iji) Internal callus 7 .45 (5) Remode'l1ing 7. 50 D. EXPERIMENTAL SERIES - MO 7.52 CHAPTER 8 DISCUSSION A. HEALING OF THE CORTICAL DEFECT IN THT CONTROL SERIES 8.1 I ) Granuìatjon T'issue Formation B.l 2 ) The fate of the Bone Filìngs and the defect 8.3 margr ns ( 3) 0steogenesì s 8.4 ( i ) Endosteal cal I us formatj on 8.4 ( 'ij ) External cal I us format'ion 8.5 (ii'i ) Internal callus fornrat'ion 8.5 ( i v) Remodel 1 ing 8.5 TABLE 0F CONTENTS ( cont'd ) Pa ge CHAPTER 9 SUMI'4ARY AND CONCLUSIONS 9.1 APPENDI CIS I. THE GROUND SUBSTANCE IN WOUND HEALING app l II. THE SYNTHESIS OF COLLAGEN ii 'i III. EXPERI MENTAL METHODOLOGY ì'i IV. DI ET iv V. HISTOLOGICAL AND HISTOCHEMICAL TECHNIQUES V RE FE REN CES Ref I -36 TABLES I. CONTROL C - GROUP I 6.3 II. OPERATION - METHOTREXATE - OM SERIES - GROUP II 6.4 III. METHOTREXATE OPERATED - MO SERIES - GROUP III 6.5 COMPARISON OF CONTROL AND EXPERII\4ENTAL SERIES - TABLES T.l Number of fibroblasts per high power field 7 .59 T.2 P.A.S. Staining 7 .60 T.3 Sjlver Staining 7.62 T.4 Van Gieson Staining 7 .64 T.5 Endosteal Callus (d') 7.66 T.6 Perjosteal CalIus (d") 7 .68 f.7 Internal Cal I us Fornation 7.70 COMPARISON OF CONTROL AND EXPERIMENTAL SERIES - HIST OGRAMS H.I P.A.S. 7 .61 H.2 Si I ver 7.63 H. 3 Van Gi eson - Co1 1 agen 7.65 H.4 Endosteal Cal I us 7 .67 H.5 Periosteal Cal I us 7 .69 H.6 Bone Formation (Internal Callus) 7.71 i PRECIS The use of cytotoxic agents is now an accepted adiunct to the management of nlalignant disease. It has been suggested that these agents also cause retarded wound healìng. However, a Search of the literature has not revealed any experimental study on the effects of these drugs on healing in bone. The present investìgation 'is to determine the effects of the cytotoxic agent Methotrexate, on the healing of a bone wound. A drill hole was made'in the mid-shaft of the femur in the Sprague Dawley rat and the subsequent healing that took pìace was studjed by using routine histological methods, augmented by some hi stochemi cal observati ons. Three experimental serjes were established: l. A control group with no drug admìnjstration. 2. An experìmental group with Methotrexate administered after the operat'ion (0M series ). 3. An experìmental group with Methotrexate adm'inistered prior to operation (M0 serjes). The result of the control group jnd'icated that the defects healed in a manner s'imilar to that described by previous investìgators - by the formation of 'periosteal','endosteal' and' ìnternal' calluses. The effects of intraperitoneal adm'inistration of Methotrexate with the dosage used in the present investigat'ion did not appear to have any significant effect on bone healing. There were some minor al- teratjons in the pattern of healing in the 'inl'tìal stages in the 0M series, but recovery soon took pìace followjng the cessation of drug admi ni strati on . 'l 'l DECLARATI ON This thesis is submitted in part fulfilment of the requirements for" the Degree of l4aster of Dental Surgery in the University of Adelaide. Candidature for the degree was satisfied by obta'ining the Degree of Bachelor of Science in Dentistry in 1974. This thesis contains no material which, except where due mention is made, has been accepted for the award of any other degree or djploma ìn any Unìversity. To the best of my knowledge, this thesjs contains no material prevìous'ly published or written by another person, except where due reference has been made in th ext. R.H.B. JONES l'll ACKNOl^lLE DGEMENTS Firstly, I would l'ike to thank Dr. K.K. Chau, Senjor Lecturer 'in the Department of Oral Pathology and Ora1 Surgery, for his patìent guidance and help durìng the preparation of this thesjs.
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