Análisis del modelo de ingreso local para los municipios de la categoría

3.6 a Bar graph showing the mean Ca/P ratios measured on cryo-sections of the roxyapatite (HA) standard, normal bone, and pooled 01 types. Each error bar represents standard error o f the mean.

1. 75 n

1 . 6 5 -

1. 55 -

1. 45

H A std. Norm Adult Norm Foetal OI all types

Fig. 3.6 b Bar graph showing the mean Ca/P ratios measured on resin sections of the hydroxyapatite (HA) standard, normal bone, and pooled 01 types. Each error bar represents the standard error of the mean.

1. 75 1

1. 65 J

1. 55 -

1.45

HA std. Norm Adult Norm Foetal 0 1 all types

Fig. 3.7 Bar graph showing the mean Ca/P ratios of normal and 01 bone measured on resin sections and compared by age groups. Each error bar represents the standard error of the mean.

1.8-

« u

3.4 Discussion

After careful standardization and calibration in the electron microscope, the Ca/P ratio of 01 bone mineral demonstrated lower values (1.58) than the normal controls (1.69). When the results were divided into clinical types o f 01 they appeared to reflect the severity of disease with 01 type II bone sections giving the lowest value (1.55). The results o f the cryo-sections were even more emphatic (for 01 type II, Ca/P ratio =1.49). Other 01 types demonstrated slightly lower Ca/P ratio values (01 type I = 1.63, type III = 1.65, type IV = 1.62) than normal bone (1.70) and the hydroxyapatite (HA) standard (1.66), but showed no significant statistical difference.

This study confirms the findings o f Cassella and Ali (1992) and Cassella et al (1995) that the 01 bone Ca/P ratio is lower than normal bone. Furthermore this answers all the criticisms o f artefactual demineralisation during tissue processing. This is the first report on Ca/P ratios determined on cryo-sections of 01 and normal bone, and the first one to express them separately according to clinical types. The 01 type II bone mineral shows the lowest Ca/P ratio, and clearly shows a significant difference in Ca/P ratios from the normal foetal and adult bone mineral and also from the other 01 types. The lower bone mineral composition in severe forms of 01 could be due to several factors.

As mentioned in the introduction, HA is the main inorganic crystalline constituent of bone and dentine. HA is a more or less ideal crystalline component with a Ca/P ratio of 1.66. Materials deviating from ideal HA composition are referred to as non- stoichiometric apatite (Brown 1966). There are several proposals to account for non- stoichiometric apatite but the most widely accepted ones are as follows:

a) calcium deficiency in the HA lattice. b) substitution in the HA lattice.

c) intracrystalline mixtures of HA and octacalcium phosphate (OCP) and tetracalcium phosphate (TCP).

d) increase in phosphate ions.

Any of the above mentioned factors can be responsible for making the HA crystals non- stoichiometric. According to Brown (1966) intracrystalline mixtures o f HA and OCP and TCP are the major cause of stoichiometric variability in apatite. There is evidence that OCP initially precipitates and then hydrolyses to HA. Nancollas et al (1989) demonstrated the overgrowth of OCP on HA by using high resolution TEM at 2.5 Â resolution. Posner and Perloff (1957) gave two explanations for lower Ca/P ratio in non- stoichiometric apatite. The first is the adsorption of excess phosphate ions onto the surface of the HA crystal; the second is the statistical absence of calcium ions from the interior lattice sites. However they discount the surface adsorption theory because surface areas o f some synthetic apatites are too low to account for their non-

Stoichiometry. As it was not possible to test any o f the theories that account for non­ stoichiometry in this study, the low Ca/P ratio in 01 type II and type II/III could be due to any o f the above mentioned factors. The growing body of evidence that 01 type II contains shorter crystals favours the lattice substitution theory as a possible explanation for the lower Ca/P ratio observed in the type II and II/III 01 bone mineral. This reduction in crystal size may separate the crystallites from one other, and make them more soluble. Hence the calcium ions can be easily substituted by sodium ions or potassium ions or replaced by H2O or HgO"*" ions thus making the HA crystals non- stoichiometric. Alternatively an increase in phosphate ions could lower the Ca/P ratio. The evidence for this change in composition is illustrated in Fig. 3.4c, where the XRMA spectrum generated by 01 type II bone was overlaid on a spectrum generated by normal foetal bone. The difference in the peak height can be observed only in the phosphorus peak with no observed difference in the calcium peak. There appears to be no change in calcium but an increase in phosphorus in 01 bone which could be due to the presence of pyrophosphates in 01 bone (Professor Ali - personal communication). This is supported by the work o f Solomons and Styner (1969) who found elevated levels of serum and urinary pyrophosphate in twenty eight 01 patients by chemical analyses. According to Ryan and McCarthy (1995) pyrophosphate can be generated by the breakdown of adenosine triphosphate to adenosine monophosphate in the presence of the enzyme nucleoside triphosphate pyrophosphohydrolase. Cassella et al (1994b) carried out a Fourier Transform Infrared Spectroscopy (FTIR) study on bone mineral from transgenic mice resembling 01 and found that it was apatite in nature despite the lower Ca/P ratio. Landis (1995) studied the oim/oim mouse model that resembles moderately severe OI by using high voltage electron microscopic tomography (3D) and reported various sizes and shapes of hydroxyapatite crystals. The orientation, location and alignment of these crystals with respect to the collagen were distinctly different from the normal calcified tissues. In normal calcified tissue there was a regularity in the staggering o f collagen fibres. In the oim/oim mouse, collagen assembly in parts of the tissue was disorganised and many collagen fibrils were twisted and kinked and the characteristic 67 nm D-period was out of register across some adjacent fibrils.

However, apart from these abnormalities there may be other factors affecting the normal mineralisation. The thinner collagen fibres observed in 01 type II (chapter 4), the abnormal amounts o f proteoglycan found in some of the type II/III bone (chapter 7) and other ultrastructural abnormalities (chapter 2) could be playing an important role in abnormal mineral formation. Undoubtedly the lower Ca/P ratio o f 01 type II bone mineral observed in this study may be a key factor with regards to the bone fragility in 01. Future work should clarify the causes of this abnormal mineralisation especially in 01 type II.

Chapter 4

H istom orphom etry of Type I collagen fibrils in the osteoid o f OI