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Abnormal collagen and mineral formation in osteogenesis imperfecta
Bone and Mineral, 17 (1992) 123-128
123
Elsevier This paper was presented at the Fifth International Conference on Cell-Medialed Calcification and Matrix Vesicles, held November 16-20.1991, Hilton Head, South Carolina.
J.P. Cassella and S. Yousuf Ali Department of Experimental Pathology, institute of Orthopaedics, (University of London) Royal National Orthopaedic Hospital, Stanmore, Middlesex, HA74LP,
UK
Osteogenesis Imperfecta (O.I.) is a genetically and biochemically heterogeneous group of disorders affecting both mineralised and non-mineralised connective tissue. The ease and frequency with which fractures occur, blue sclera and affected dentition (Dentinogenesis Imperfecta) are common clinical features. Studies have shown that O.I. can be divided into at least four groups with two groups inherited as autosomal dominant and two as autosomal recessive traits [ 11. A numerical classification has been proposed [2] which has been correlated with numerous biochemical and morphological studies; this classification will be used to describe the patients in this study. The expanding genetic diversity in 0.1. as determined at the level of molecular biology [3] has not been matched by any in-depth study to show possible differences in collagen, mineral or cellular architecture which can be visualised by electron microscopy or associated analytical techniques.
aterials an Electron microscopy
Specimens of bone from 12 patients with 0.1. and 7 normal controls were removed during orthopaedic surgery. The bone was fixed in 2.5% gluteraldehyde solution (18 hours) at 4°C. The specimens were cut into small pieces (l-3 mm3) during the first hour of fixation. The tissue was washed (2 x 30 minutes) in a 0.1 M sodium cacodylate buffer (pN 7.4), followed by secondary fixation in a 1% osmium tetroxide solution diluted in 0.1 sodium cacodylate. The tissue was dehydrated through a graded series of ethanols before vacuum infiltration and embedding in Spurr’s resin for 18 hours at 60°C. Ultrathin sections (60-100 nm) were collected on pioloform
124
coated 200 mesh copper grids and stained using uranyl acetate and lead citrate. Specimens were viewed in a Philips CM12 transmission electron microscope, (TEM). Areas of osteoid were randomly chosen and photographed at 53,000~ magnification. X-ray microanalysis Bone specimens were processed as above but osmium tetroxide was omitted.
Chemically pure hydroxyapatite (HA, reference 4), tricalcium phosphate (TCP, reference 4), calcium pyrophosphate CaPyro, Sigma Ltd) and calcium tetra-hydrogen orthophosphate (TOP, BDH Ltd) were processed as for the bone specimens. Ultrathin sections (100 nm) were cut and floated on 0.085 M sodium cacodylate buffer. Sections were collected on pioloform coated nickel grids. Energy dispersive XRMA was performed using an EDAX 9800 microanalysis system with a specimen tilt angle of 20” and a take-off angle of 40”, the count time was 200 live seconds. Imuge analysis
TEM negatives were placed on a light box and a video image captured on a Cambridge Instruments ‘Quantimet 520’ image analyser. Using a digitablet the image of the collagen fibrils were drawn around and the system calibrated to express equivalent diameters in nanometres. In order to confirm that the accuracy of measurement was maintained throughout the study, a reproducibility study was performed. This involved image analysing 10 negatives each from 3 patients chosen at random. The same negatives were later re-analysed and the equivalent diameters from both sets of measurements compared using a related t-test. A Mann-Whitney U-test was used to determine if there was a difference between 0.1. and normal control collagen fibrils. A Kruskall-Wallis test was performed to determine of there was a difference in collagen fibril diameter in the four different clinical types of 0.1. and if there was a relationship between the age and sex of the patient or the site of biopsy. X-my powder criffraction (XR PD) Specimens of fresh bone tissue were cooled to -70°C and then freeze dried at -40°C for 48 hours. The bone was powdered and packed into a 0.3 mm Lindemann glass tube. Specimens were then placed in a Gandolfi X-ray powder camera and placed on a Philips PWlOlO x-ray generator. The samples were subjected to filtered copper (Ka) radiation (wavelength 1.5411(A)using an exposure time of three hours, at 40 kV and 20 mA. The x-ray powder diffraction pattern was recorded using the Debye-Scherrer method. Fourier transform infra-red spectroscopy (FTIR)
Specimens of powdered bone and potassium bromide (KBr) powder (spectroscopic grade, BDH Ltd.), were placed in a 13 mm evacuable dye. A KBr disk was pressed at 10 tons pressure for 5 minutes. The disk was placed in a Nicolet SDXC FTIR spectrophotometer. Spectra were collected as transmission spectra at 4 wavenumbes resolution with 32 scans performed per specimen. A background reading was obtained using an empty cell compartment.
125
Image analysis
The statistical data obtained from the image analysis results indicated that: normal type I collagen fibrils had a mean diameter of 53.3 nm, whereas collagen fibrils from 0.1. osteoid were larger with a mean diameter of60.4 nm, (Fig. 1). There was a relationship between the type of 0.1. and the diameter of the collagen fibrils, with Type I 0.1. tending towards the larger fibril diameter and Type 4 0.1. tending towards the smaller diameter (Table 1). COLLAGEN FIBR FOR 0.1 AND b6 7 5
64
Kruskall Wallis Test -~------
62 60
Compared by: .-_---.
56
‘rype
56
d
54
Site of biopsy Age
Sex Oi
- --
p Value p = 0.05 0.1 cpco.375 0.05 < p < 0.02 0. I < p < 0.375
NO&AL
X-ray microanalysis
A ‘standard’ graph (Fig. 2) of calcium-phosphate ratios observed using the EDAX system (Cliff-Lorimer ratio model) was plotted against the empirical calcium-phosphate standards. There was a significant difference between the Ca/P ratios of normal and 0.1. bone with the 0.1. bone demonstrating a lower Ca/P ratio, (Fig. 3).
WF (observed) VS Cal’P (empirical) MOLAR RATIOS FOR Ca-P STANDARDS, NORMAL AND 0.1. BONE 201 I
p..cp
MEAN Cd VALUE5 FOR NORMAL, BON?3 AND Ca-P STANDARDS
0.1.
c..”
I.B
HA
CaPyro TOP / 0.0 02 04 06 06 1.0 I2 1.4 16 1.E 2.0 Crdl’ MOLAR RATIO (RMPIRICAL)
NORMAL. 0.1. HA T.CP Cd’yro T.0.P
X-ray powder diffraction
A plot of relative intensity against 2 8 was plotted from the powder diffraction pattern and the results for normal, 0.1. bone and the calcium-phosphate standards compared. All specimens analysed had an apatitic crystal structure.
126 Fourier transform infra-red spectroscopy
The vibrational spectra values for the calcium-phosphates were obtained from a ‘peak-picker’ programme and the characteristic peaks compared. XRPD data correlated with the FTIR results. Carbonate groups were detected in normal and 0.1. bone but not in the standards analysed. The standards demonstrated good crystallinity while the bone mineral from all specimens was more diffuse. FUR and XRPD did not yield any significant differences between non-pathological or synthetic samples of HA. UItrastructurulobservations
The ultrastructure of bone in 0.1. has shown to be as heterogeneous as its clinical and genetic aspects. Mineralising mitochondria have demonstrated needle-like crystals or spherules, (Fig. 4). XRMA*has shown these crystals to contain calcium and phosphorus, and uncontrasted spectra have shown electron dense needles in mitochondria.
Fibres of unknown genesis have been found in six patients with various types of 0.1. Two siblings (Fig. 5) demonstrated similar fibres but there was a degree of heterogeneity between the other patients (Fig. 6). Possibilities of the presence of altered elastin, reticulin, amyloid and amianthoid fibres have all been discounted on the basis of their characteristic morphology. Calcified cartilage was found throughout the growth plate from a number of Type I 0.1. neonatal specimens, the longitudinal septae and zone of neo-vascularisation were extremely disrupted. A stroma1 pattern of calcification intermixed with woven bone was found where endochondral bone formatiqm was expected (Figs. 7 and 8). Neo-chondrogenesis was an interesting feature in the iliac crest of a patient with type I 0.1. Matrix vesicles could clearly be seen as the primary initiator of calcification, (Fig. 9).
127
The statistical data indicated that collagen fibril diameters in 0.1. are larger (60.4 nm) than in normal, age, sex and site matched controls (53.3 nm). This is contradictory to previous studies 151.One explanation for the difference in results between this and previous studies may be the larger number of fibrils measured in this study, on average two to three times more fibrils were measured than in previous studies. The experimental design was aimed at measuring 2000 fibrils per patient in a random scan over one ultrathin specimen. In a few cases there were insufficient fibrils present to reach this target. The decreased synthesis and transport of altered type I pro-collagen has been suggested to allow an increase in the ratio of type 1:type III collagen thus resulting in thinner diameter fibrils, however in the light of new data this cannot be the only controlling factor in fibril diameter. One possibility for these fibrous bodies is that they are collagenous in nature, being either ‘breakdown’ products or de nova fibrillogenesis in an altered form. The mitochondrial calcification would suggest some cellular dysfunction associated with the ionic calcium and phosphate pumps. FTIR and XRPD did not yield any significant differences between non-pathologicai or synthetic samples of HA. XRPD results indicated that there was a significant difference between the Ca/P ratios of normal and O.I. bone with the 0.1. bone demanstrating a lower Ca/P ratio. Since HA is the main mineral component found in normal human bone tissue,
128
the presence of a mineral with a calcium-phosphate ratio lower than HA in some patients with certain types of 0.1. demonstrates that the mineral is abnormal. This may in part explain the fragility and associated fracture of bone.
References 1 Sillence DO, Rimoin DL, Danks DM. Clinical variability in osteogenesis imperfecta. Variable cxpressivity or genetic heterogeneity. Birth Defects 1979;Y13:lS (SB). 2 Sillence DO. Osteogenesis imperfecta: an expanding panorama of variants. Clinical Orthopaedics and Related Research 19gl;lS9:11-25. 3 Rowe DW. osteogenesis imperfecta. Bone and Mineral Research 1991;7:209-241. 4 Klein CPAT, Patka P, den Hollander W. A histological comparison between hydroxylapatite and Bwhitlockite macroporous ceramics implanted in dog femora. The Third World Biomaterials Congress 1988;p. 67. S Stag H, Freisingcr P. Proceedings of Vth international conference on osteogenesis imperfecta 1990; p. 36.
123
Elsevier This paper was presented at the Fifth International Conference on Cell-Medialed Calcification and Matrix Vesicles, held November 16-20.1991, Hilton Head, South Carolina.
J.P. Cassella and S. Yousuf Ali Department of Experimental Pathology, institute of Orthopaedics, (University of London) Royal National Orthopaedic Hospital, Stanmore, Middlesex, HA74LP,
UK
Osteogenesis Imperfecta (O.I.) is a genetically and biochemically heterogeneous group of disorders affecting both mineralised and non-mineralised connective tissue. The ease and frequency with which fractures occur, blue sclera and affected dentition (Dentinogenesis Imperfecta) are common clinical features. Studies have shown that O.I. can be divided into at least four groups with two groups inherited as autosomal dominant and two as autosomal recessive traits [ 11. A numerical classification has been proposed [2] which has been correlated with numerous biochemical and morphological studies; this classification will be used to describe the patients in this study. The expanding genetic diversity in 0.1. as determined at the level of molecular biology [3] has not been matched by any in-depth study to show possible differences in collagen, mineral or cellular architecture which can be visualised by electron microscopy or associated analytical techniques.
aterials an Electron microscopy
Specimens of bone from 12 patients with 0.1. and 7 normal controls were removed during orthopaedic surgery. The bone was fixed in 2.5% gluteraldehyde solution (18 hours) at 4°C. The specimens were cut into small pieces (l-3 mm3) during the first hour of fixation. The tissue was washed (2 x 30 minutes) in a 0.1 M sodium cacodylate buffer (pN 7.4), followed by secondary fixation in a 1% osmium tetroxide solution diluted in 0.1 sodium cacodylate. The tissue was dehydrated through a graded series of ethanols before vacuum infiltration and embedding in Spurr’s resin for 18 hours at 60°C. Ultrathin sections (60-100 nm) were collected on pioloform
124
coated 200 mesh copper grids and stained using uranyl acetate and lead citrate. Specimens were viewed in a Philips CM12 transmission electron microscope, (TEM). Areas of osteoid were randomly chosen and photographed at 53,000~ magnification. X-ray microanalysis Bone specimens were processed as above but osmium tetroxide was omitted.
Chemically pure hydroxyapatite (HA, reference 4), tricalcium phosphate (TCP, reference 4), calcium pyrophosphate CaPyro, Sigma Ltd) and calcium tetra-hydrogen orthophosphate (TOP, BDH Ltd) were processed as for the bone specimens. Ultrathin sections (100 nm) were cut and floated on 0.085 M sodium cacodylate buffer. Sections were collected on pioloform coated nickel grids. Energy dispersive XRMA was performed using an EDAX 9800 microanalysis system with a specimen tilt angle of 20” and a take-off angle of 40”, the count time was 200 live seconds. Imuge analysis
TEM negatives were placed on a light box and a video image captured on a Cambridge Instruments ‘Quantimet 520’ image analyser. Using a digitablet the image of the collagen fibrils were drawn around and the system calibrated to express equivalent diameters in nanometres. In order to confirm that the accuracy of measurement was maintained throughout the study, a reproducibility study was performed. This involved image analysing 10 negatives each from 3 patients chosen at random. The same negatives were later re-analysed and the equivalent diameters from both sets of measurements compared using a related t-test. A Mann-Whitney U-test was used to determine if there was a difference between 0.1. and normal control collagen fibrils. A Kruskall-Wallis test was performed to determine of there was a difference in collagen fibril diameter in the four different clinical types of 0.1. and if there was a relationship between the age and sex of the patient or the site of biopsy. X-my powder criffraction (XR PD) Specimens of fresh bone tissue were cooled to -70°C and then freeze dried at -40°C for 48 hours. The bone was powdered and packed into a 0.3 mm Lindemann glass tube. Specimens were then placed in a Gandolfi X-ray powder camera and placed on a Philips PWlOlO x-ray generator. The samples were subjected to filtered copper (Ka) radiation (wavelength 1.5411(A)using an exposure time of three hours, at 40 kV and 20 mA. The x-ray powder diffraction pattern was recorded using the Debye-Scherrer method. Fourier transform infra-red spectroscopy (FTIR)
Specimens of powdered bone and potassium bromide (KBr) powder (spectroscopic grade, BDH Ltd.), were placed in a 13 mm evacuable dye. A KBr disk was pressed at 10 tons pressure for 5 minutes. The disk was placed in a Nicolet SDXC FTIR spectrophotometer. Spectra were collected as transmission spectra at 4 wavenumbes resolution with 32 scans performed per specimen. A background reading was obtained using an empty cell compartment.
125
Image analysis
The statistical data obtained from the image analysis results indicated that: normal type I collagen fibrils had a mean diameter of 53.3 nm, whereas collagen fibrils from 0.1. osteoid were larger with a mean diameter of60.4 nm, (Fig. 1). There was a relationship between the type of 0.1. and the diameter of the collagen fibrils, with Type I 0.1. tending towards the larger fibril diameter and Type 4 0.1. tending towards the smaller diameter (Table 1). COLLAGEN FIBR FOR 0.1 AND b6 7 5
64
Kruskall Wallis Test -~------
62 60
Compared by: .-_---.
56
‘rype
56
d
54
Site of biopsy Age
Sex Oi
- --
p Value p = 0.05 0.1 cpco.375 0.05 < p < 0.02 0. I < p < 0.375
NO&AL
X-ray microanalysis
A ‘standard’ graph (Fig. 2) of calcium-phosphate ratios observed using the EDAX system (Cliff-Lorimer ratio model) was plotted against the empirical calcium-phosphate standards. There was a significant difference between the Ca/P ratios of normal and 0.1. bone with the 0.1. bone demonstrating a lower Ca/P ratio, (Fig. 3).
WF (observed) VS Cal’P (empirical) MOLAR RATIOS FOR Ca-P STANDARDS, NORMAL AND 0.1. BONE 201 I
p..cp
MEAN Cd VALUE5 FOR NORMAL, BON?3 AND Ca-P STANDARDS
0.1.
c..”
I.B
HA
CaPyro TOP / 0.0 02 04 06 06 1.0 I2 1.4 16 1.E 2.0 Crdl’ MOLAR RATIO (RMPIRICAL)
NORMAL. 0.1. HA T.CP Cd’yro T.0.P
X-ray powder diffraction
A plot of relative intensity against 2 8 was plotted from the powder diffraction pattern and the results for normal, 0.1. bone and the calcium-phosphate standards compared. All specimens analysed had an apatitic crystal structure.
126 Fourier transform infra-red spectroscopy
The vibrational spectra values for the calcium-phosphates were obtained from a ‘peak-picker’ programme and the characteristic peaks compared. XRPD data correlated with the FTIR results. Carbonate groups were detected in normal and 0.1. bone but not in the standards analysed. The standards demonstrated good crystallinity while the bone mineral from all specimens was more diffuse. FUR and XRPD did not yield any significant differences between non-pathological or synthetic samples of HA. UItrastructurulobservations
The ultrastructure of bone in 0.1. has shown to be as heterogeneous as its clinical and genetic aspects. Mineralising mitochondria have demonstrated needle-like crystals or spherules, (Fig. 4). XRMA*has shown these crystals to contain calcium and phosphorus, and uncontrasted spectra have shown electron dense needles in mitochondria.
Fibres of unknown genesis have been found in six patients with various types of 0.1. Two siblings (Fig. 5) demonstrated similar fibres but there was a degree of heterogeneity between the other patients (Fig. 6). Possibilities of the presence of altered elastin, reticulin, amyloid and amianthoid fibres have all been discounted on the basis of their characteristic morphology. Calcified cartilage was found throughout the growth plate from a number of Type I 0.1. neonatal specimens, the longitudinal septae and zone of neo-vascularisation were extremely disrupted. A stroma1 pattern of calcification intermixed with woven bone was found where endochondral bone formatiqm was expected (Figs. 7 and 8). Neo-chondrogenesis was an interesting feature in the iliac crest of a patient with type I 0.1. Matrix vesicles could clearly be seen as the primary initiator of calcification, (Fig. 9).
127
The statistical data indicated that collagen fibril diameters in 0.1. are larger (60.4 nm) than in normal, age, sex and site matched controls (53.3 nm). This is contradictory to previous studies 151.One explanation for the difference in results between this and previous studies may be the larger number of fibrils measured in this study, on average two to three times more fibrils were measured than in previous studies. The experimental design was aimed at measuring 2000 fibrils per patient in a random scan over one ultrathin specimen. In a few cases there were insufficient fibrils present to reach this target. The decreased synthesis and transport of altered type I pro-collagen has been suggested to allow an increase in the ratio of type 1:type III collagen thus resulting in thinner diameter fibrils, however in the light of new data this cannot be the only controlling factor in fibril diameter. One possibility for these fibrous bodies is that they are collagenous in nature, being either ‘breakdown’ products or de nova fibrillogenesis in an altered form. The mitochondrial calcification would suggest some cellular dysfunction associated with the ionic calcium and phosphate pumps. FTIR and XRPD did not yield any significant differences between non-pathologicai or synthetic samples of HA. XRPD results indicated that there was a significant difference between the Ca/P ratios of normal and O.I. bone with the 0.1. bone demanstrating a lower Ca/P ratio. Since HA is the main mineral component found in normal human bone tissue,
128
the presence of a mineral with a calcium-phosphate ratio lower than HA in some patients with certain types of 0.1. demonstrates that the mineral is abnormal. This may in part explain the fragility and associated fracture of bone.
References 1 Sillence DO, Rimoin DL, Danks DM. Clinical variability in osteogenesis imperfecta. Variable cxpressivity or genetic heterogeneity. Birth Defects 1979;Y13:lS (SB). 2 Sillence DO. Osteogenesis imperfecta: an expanding panorama of variants. Clinical Orthopaedics and Related Research 19gl;lS9:11-25. 3 Rowe DW. osteogenesis imperfecta. Bone and Mineral Research 1991;7:209-241. 4 Klein CPAT, Patka P, den Hollander W. A histological comparison between hydroxylapatite and Bwhitlockite macroporous ceramics implanted in dog femora. The Third World Biomaterials Congress 1988;p. 67. S Stag H, Freisingcr P. Proceedings of Vth international conference on osteogenesis imperfecta 1990; p. 36.