206 Abstracts From the Bone & Tooth Society,

OSTEOlOlNDUCTlON IN HUMAN DEMINERALIZED MALLEUS AND INCUS ALLO-IMPLANTS USED TO RECONSTRUCT THE OSSICULAR CHAIN - A PRELIMINARY REPORT

N.J. Frootko and J.T Triffitt Department of Otolaryngology, Radcliffe Infirmary, Oxford, and MRC Bone Research Laboratory, Nuffield Department of Orthopaedic Surgery, Oxford

Cadaver acquired undemineralized ossicular bone allo- implants are frequently used to reconstruct ossicular chain defects in the middle ear (ossiculoplasty). These allo-implants are currently preserved preoperatively using a variety of tech- niques including deep-freezing, freeze drying, autoclaving and preservation in chemical agents such as formaldehyde, Cialit* and alcohol. New bone formation in these implants has been shown to be minimal or absent. This lack of bone morpho- genesis together with reduction in the size of the implant is a major cause of ossiculoplasty failure. Previously we have shown in the rat that specimens of demineralized ossicular bone matrix have a much higher capacity than undemineral- ized specimens to induce bone formation in the middle ear, (Frootko, N.J., and Triffitt, J.T.; J. Bone Jt. Surg., 658(l), 89 (1983). This process of bone induction has been studied in a prospective clinical ossiculoplasty trial. Malleuses and in- cuses, acquired post mortem from refrigerated unembalmed cadavers within twelve hours of death, were immediately demineralized in 0.6 M hydrochloric acid at 4OC. This was followed by sequential extraction in absolute alcohol and anhy- drous ether, rehydration in water, freeze drying and sterilization by ethylene oxide gas. These allo-implants were used in staged tympanoplasties in 20 patients, [I 0 malleus-stapes assemblies (MSA), and IO tympanic membrane-stapes assemblies (TSA)]. After 9 to 18 months postoperative follow-up, all TSA’s and 8 MSA’s have maintained closure of the Air-Bone gap (preoperative bone conduction and post operative air conduc- tion) to 15 dB or less at 0.5 KHz to 2 KHz. In two MSA’s implant displacement off the stapes head occurred. These implants were removed at revision surgery at six months and eight months postoperative. There was no obvious evidence of reduction in size or change in shape of these implants. On histological examination there was no evidence of an immune response, both implants were well vascularized and most of the implanted matrix was replaced by viable new woven bone, which was becoming lamellar in some areas.

Conclusion

Demineralized human incus and malleus allo-implants are osteoinductive in the middle ear and may prove to be a useful alternative to preserved undemineralized ossicular bone im- plants currently used in middle ear reconstructive surgery. Longer follow up will be required to determine whether resorp- tion of induced new bone leading to gross reduction in size has been avoided in these allo-implants.

THE MEASUREMENT OF TISSUE pH AT CALCIFYING FRONTS BY a’P NMR SPECTROSCOPY

Raymond J. Newman N&field Department of Orthopaedic Surgery, Nuffield Orthopaedic Centre, Oxford OX3 7LD

The measurement of pH at calcifying fronts is difficult and micro-electrode techniques have not been as helpful as initially

‘Cialtt (sodium P-ethylmercur~thiobenzoxazole-5carboxylate)

expected. The recent introduction of high resolution phos- phorus nuclear magnetic resonance spectroscopy (31P NMR) is an important advance since the spectral position of the inor- ganic phosphate peak(s) can be used to monitor the intra- and extracellular pH in vivo. In addition the relative levels of the major phosphate-containing metabolites can be measured non-invasively. Unilateral tibia1 shaft fractures were induced under general anaesthesia in the rat and 31P NMR spectra recorded from the calf region. During healing the inorganic phosphate peak was split and further studies showed that the two components of the signal originated in the fracture haematoma and muscle cytoplasm respectively. Intramyocel- lular pH remained constant throughout healing (7.06 + 0.03 SEM, n = 12) but the pH of the fracture haematoma changed progressively from 7.19 f 0.15 two days after fracture to 7.50 + 0.15 on day twenty. Deposition of radio-opaque callus occurred predominantly when the pH was relatively alkaline. In a second group of ten rats fracture union was delayed by about five days by the parenteral administration of prednisolone (0.25 mg kg- ‘). Again the pH of the fracture haematoma changed progressively from acid to alkaline but in the prednisolone- treated animals (plasma prednisolone level 9.22 f 0.83 ng ml-‘) the extent and rate of pH change was less than in saline- treated controls (plasma prednisolone level 4.74 + 0.26 ng ml-‘). The deposition of radio-opaque callus was delayed in the prednisolone-treated rats but still occurred predominantly when the pH of the fracture haematoma was relatively alkaline. These results indicate that 31 P NMR spectroscopy can be used to monitor noninvasively and sequentially the local pH changes that undoubtedly occur during fracture repair. Future forms of therapy for delayed or non-union may be usefully directed at modifying the rate at which these pH changes occur.

EFFECT OF BONE MATRIX CONSTITUENTS ON OSTEOGENESIS

Elaine Green, Cathy Hinton and James T Triffitt MRC Bone Research Laboratory, Nuffield Department of Orthopaedic Surgery, University of Oxford, Nuffield Orthopaedic Centre, Oxford

Decalcified bone matrix surgically implanted into extraskeletal sites results in the differentiation of certain cells present in the connective tissues such that they become chondrogenic and osteogenic. The factor or factors responsible for the induction of bone differentiation appear to be proteinaceous and are diffusible through filters of 0.22 pm pore diameter (1). Rabbit bone matrix proteins have been solubilized using 4M GuHCI, and the osteoinductive activity recovered in a protein fraction soluble in GuHCl but insoluble in water. Ion exchangechroma- tography on DEAE-sepharose in 6M urea, pH 7.2, resulted in the recovery of the biological activity in the breakthrough frac- tion with the bone collagen. Chromatography of this fraction on Sepharose CLGB allowed the isolation of the osteoinductive factor in a low molecular weight fraction, distinct from collagen. The osteoinductive factor has also been identified as a low molecular weight component (M, 14000-50000) after separa- tion by HPLC using gel permeation columns eluted with 4M GuHCI. The separation of fractions exhibiting the biological activity has been followed by implantation of the lyophilized proteins into abdominal muscle pouches in allogeneic rabbits. After 28 days the formation of ectopic bone at the implantation site is assessed by radiographic and histological methods. Bone matrix extracts with osteoinductive activity affect the proliferation and differentiation of cells cultured both in vivo and in vitro. Rabbit bone marrow cells cultured in vivo in diffusion

Abstracts From the Bone & Tooth Society 207

chambers in the peritoneal cavity of allogeneic hosts differenti- ated to produce increased yields of bone in the presence of bone matrix when compared with control chambers. Cells from bone pieces and omentum, previously cultured in vitro for several passages, when subsequently implanted intraper- itoneally in diffusion chambers together with bone matrix were induced to differentiate to form bone. In vitro, bone matrix extracts affected proliferation of these cultured cells. These observations may allow the development of a reliable and rapid in vitro assay system for detection of the osteoinductive factor during extensive purification procedures. In addition, the ob- served stimulatory effect of bone matrix on bone formation from cell populations than contain osteogenic precursor cells may indicate a physiological control of osteogenesis by components present in the matrix.

(1) Urist, M.R. (1965). Science 150, 893

EUTOPlCANOECTOPlCEXPRESSlONOFTHEHUMAN CALCITONIN GENE M.R. Edbrooke, J.H. McVey, D. Parker and R.K. Craig Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London Wl N 8AA

Analysis of the human calcitonin gene has revealed many fea- tures in common with the now well characterized rat calcitonin gene. Here we describe the identification of an alternative human gene product, the calcitonin gene related peptide (CGRP). We also describe the eutopic and ectopic expression of the two separate human gene products in the BEN cell-line (derived from a large cell carcinoma of the lung) and in medullary carcinoma of the thyroid.

RAClALOlFFERENCESINCALClTONlNSECRETlON J.C. Stevenson, Clare H. Myers and A.B. Ajdukiewicz Endocrine Unit, Royal Postgraduate Medical School, Hammersmith Hospital, London WI2 and MRC Laboratories, Fajara, Nr. Banjul, The Gambia, West Africa

Postmenopausal osteoporosis is a major problem in the Western world yet it is almost exclusively a disease of light- skinned races. The reasons for this are probably two-fold. Firstly, blacks have a denser skeleton than whites. Secondly, it is possible that postmenopausal bone loss is greater in whites than in blacks. However, the underlying mechanisms for these differences have not been established. We therefore took the opportunity to study levels of calcitonin, together with its flanking peptide katacalcin, in Gambian black people and compared the results to those of British white people. Twenty- three black patients with various disorders who had been admitted into hospital in The Gambia were studied. Eleven patients had disorders known to be associated with elevated calcitonin levels. The remaining 12 adult patients (6 males, age range 22-52; 6 females, age range 26-65 years) were assumed to be ‘normal” for calcitonin secretion. Age and sex matched control values were randomly selected from existing data on British whites for comparison. Calcitonin and katacalcin levels were measured on fasting plasma samples by radioimmunoas- say. There was a close correlation between circulating calcitonin and katacalcin levels (r = 0.80). Sex differences were observed with males having higher levels (X mg/l) than

females in both blacks (124 v 59) and whites (46 v 22). Overall, the levels of calcitonin, and of katacalcin, were higher in blacks than in whites (p < 0.001, and p < 0.02). However, the most striking finding was that calcitonin levels were by far lowest in white women, a finding reflected in the katacalcin levels. We suggest that the low calcitonin levels found in white women will render their skeletons more sensitive to the actions of bone- resorbing hormones. This would allow excessive bone loss at the menopause when calcitonin levels fall further. It might also contribute to a lower bone mass throughout life. Whether ge- netic or environmental factors are responsible for this racial difference in calcitonin levels remains to be determined. These findings may explain, at least in part, racial differences in the incidence of osteoporosis.

MECHANlSMOFACTlONOFSTANOZOLOLlNTHETREATMENT OFOSTEOPOROSIS A.J.P. Yates, R.E.S. Gray, R.C. Percival, K.I. Muncy, J.H. Galloway, M. Couch, R. Vaishnav, J.A. Gallagher, J.N. Beresford, R.G.G. Russell and J.A. Kanis Dept. of Human Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, 30 2RX

Stanozolol has been shown to increase total body calcium in osteoporotic patients. However, the mechanisms involved have not been elucidated. We have studied several bio- chemical indices of bone turnover in 22 postmenopausal osteoporotic women treated with stanozolol5 mg daily for up to 2 years. There was no significant change either in serum cal- cium or in 47Ca intestinal absorption. In contrast, there was a marked and consistent fall in the daily urinary excretion of calcium (3.22 mmollday to 1.76 mmollday; p < 0.001) and the fasting urinary calcium excretion expressed as a ratio to creati- nine (0.38 mmollmmol to 0.14 mmollmmol p < 0.005) after 3 months of treatment, an effect which was sustained for the duration of the study. The unchanged calcium absorption, reduced calcium excretion and unchanged serum calcium suggest that stanozolol induced net bone formation. However, both serum alkaline phosphatase and osteocalcin values decreased during the early phase of treatment, and urinary excretion of hydroxyproline did not change. The effect on fast- ing calcium excretion, together with the lackof effect on plasma calcium indicate that stanozolol increased renal tubular reab- sorption of calcium. However this was not associated with a change in mean serum immunoreactive PTH throughout the period of treatment (85 pmolll pre-treatment; 84 pmol/l at 3 months). This suggests that the changes in tubular reabsorp- tion may be mediated either by an increased renal sensitivity to PTH or by a non-PTH mechanism. Theformer mechanism may be more likely since there were small but significant decreases observed in tubular reabsorption of phosphate. Using a human bone cell culture system we have also demonstrated that stanozolol directly stimulated both cell division and bone matrix production in vitro. In contrast, in these experiments stanozolol antagonized the effects of vitamin D to stimulate synthesis of alkaline phosphatase and osteocalcin. These ef- fects are similar to the observations made in patients whereby both alkaline phosphatase and osteocalcin decreased rather than increased. Thus, a dual mechanism for the action of sta- nozolol with direct effects on both bone formation and renal tubular calcium reabsorption appears to best explain the observed data.