X-ray diffraction study of the mineralization of turkey leg tendon

X-ray diffraction studies were conducted on calcified turkey leg tendon to establish the effect of mineralization on some of the structural properties of collagen. The principal finding was that the first equatorial reflection of collagen in freshly-excised calcified tendon had a d-spacing intermediate between the values for dried collagen and fresh unmineralized collagen. Since this spacing is a sensitive monitor of moisture levels in collagen, the data suggest that mineralization reduces the amount of water than can associatein vivo with the collagen component in tissue. It was also found that the presence of mineral appears to increase the resistance of collagen to permanent thermal denaturation.

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Zusammenfassung Es wurden Röntendiffraktionsuntersuchungen an calcifizierten Truthahnbeinsehnen durch geführt, um die Wirkung der Mineralisation auf einige der strukturellen Eigenschaften von Collagen festzustellen. Die wichtigste Beobachtung war, daß die erste äquatoriale Reflexion von Collagen in frish herauspräparierten calcifizierten Sehnen einen d-Wert aufwies, der zwischen den Werten für trockenes Collagen und frisches, nicht mineralisiertes Collagen liegt. Da dieser d-Wert empfindlich auf den Feuchtigkeitsgrad des Collagens reagiert, lassen diese Resultate vermuten, daß die Mineralisation die Wassermenge reduziert, welche sich in vivo mit der Collagenkomponente im Gewebe verbinden kann. Es wurde auch beobachtet, daß die Anwesenheit von Mineral den Widerstand von Collagen gegen bleibende thermale Denaturierung heraufzusetzen scheint.

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Résumé Des études de diffraction aux rayons X sont réalisées au niveau du tendon calcifié de la patte de dinde pour déterminer l'effet de la minéralisation sur certaines propriétés structurales du collagène. Le résultat le plus important indique que la première raie équatoriale du collagène de tendon calcifié, fraichement prélevé, présente une équidistance intermédiaire entre les valeurs de collagène déssèché et du collagène frais non calcifié. Comme cette équidistance parait traduire fidèlement le degré d'humidité du collagène, il semble que la minéralisation réduit la quantité d'eau associéein vivo avec le composant collagènique du tissu. La présence du minéral semble augmenter la résistance du collagène à une dénaturation thermique permanente.

     

The loci of mineral in turkey leg tendon as seen by atomic force microscope and electron microscopy

Transmission electron micrographs of fully mineralized turkey leg tendon in cross-section show the ultrastructure to be more complex than has been previously described. The mineral is divided into two regions. Needlelike-appearing crystallites fill the extrafibrillar volume whereas only platelike crystallites are found within the fibrils. When the speciment is tilted through a large angle, some of the needlelike-appearing crystallites are replaced by platelets, suggesting that the needlelike crystallites are platelets viewed on edge. If so, these platelets have their broad face roughly parallel to the fibril surface and thereby the fibril axis, where the intrafibrillar platelets are steeply inclined to the fibril axis. The projection of the intrafibrillar platelets is perpendicular to the fibril axis. The extrafibrillar volume is at least 60% of the total, the fibrils occupying 40%. More of the mineral appears to be extrafibrillar than within the fibrils. Micrographs of the mineralized tendon in thickness show both needlelike-appearing and platelet crystallites. Stereoscopic views show that the needlelike-appearing crystallites do not have a preferred orientation. From the two-dimensional Fourier transform of a selected area of the cross-sectional image, the platelike crystallites have an average dimension of 58 nm. The needlelike-appearing crystallites have an average thickness of 7 nm. The maximum length is at least 90 nm. Atomic force microscopy (AFM) of unstained, unmineralized turkey leg tendon shows collagen fibrils very much like shadow replicas of collagen in electron micrographs. AFM images of the mineralized tendon show only an occasional fibril. Mineral crystallites are not visible. Because the collagen is within the fibrils, the extrafibrillar mineral must be embedded in noncollagenous organic matter. When the tissue is demineralized, the collagen fibrils are exposed. The structure as revealed by the two modalities is a composite material in which each component is itself a composite. Determination of the properties of the mineralized tendon from the properties of its elements is more difficult than considering the tendon to be just mineral-filled collagen.     

Nonmineralized and mineralized bone collagen in bone of immobilized monkeys

Summary Monkey bones from different time periods of immobilization and reambulation of the monkeys were assessed histologically. The bone was also assessed biochemically for nonmineralized collagen in bone. Results indicate that more non-mineralized bone is present in monkeys that have been immobilized, and upon reambulation, these values tend to be normalized to control bone.     

The distribution of water in calcified turkey leg tendon

X-ray diffraction and water sorption data are presented which show that the extracellular water in calcified turkey leg tendon is associated principally with the collagen component.     

On the solid state of bone

Photovoltaic effect (infrared and visible light) is observed in bone and its two major components, collagen and apatite, at room temperature. A dimunition in the magnitude of photovoltage is observed after exposure to ultraviolet light in all the cases. The drift mobility of the charge carriers is obtained by measuring I versus V relationships in sandwich samples and relating them to the permitivity of the medium. Lifetime of the injected carriers is measured in the usual way. The data are consistent with the hypothesis that the effects are due to protonic conduction phenomena.     

Vectorial sequence of mineralization in the turkey leg tendon determined by electron microscopic imaging

Summary Turkey leg tendons were used as a model tissue to study the spatial and temporal relationships of mineral deposition between matrix vesicles and collagen fibrils by various electron microscopic techniques—bright field, selected-area dark field (SADF), and electron spectroscopic imaging (ESI). These latter imaging techniques enabled the direct localization and spatial distributions of both apatite crystals and atomic elements (Ca, P) within matrix vesicles and collagen. In longitudinal planes of section, a consistent vectorial gradient of mineralization was observed which started with the first localization of apatite mineral in matrix vesicles; with further development, the mineral spread from the vesicle to the extravesicular interstices and then into the adjacent collagen fibrils. Once intrafibrillar, the mineral was observed to advance both laterally and axially. The association of vesicle/collagen mineral was examined by ESI analysis of Ca and P elemental maps and appeared as a continuum between the vesicles and the adjacent collagen fibrils. Similarly, an intimate spatial relationship was observed between the mineral of vesicles and collagen in transversely cut sections of tendon. The sequential development of this mineralized matrix is discussed in light of matrix vesicle/collagen interactions.     

Fixation and demineralization of bone tissue for immunohistochemical staining of neuropeptides

Summary The present study in the rat demonstrates the feasibility of applying immunohistochemical staining techniques on bone tissue for studies of substances such as neuropeptides contained in nerve fibers. Two fixation procedures, as well as the influence of demineralization on neuropeptide antigenicity, were studied in bone and for comparison in small intestine.In vivo perfusion with paraformaldehyde and picric acid, followed by demineralization in a solution of either EDTA-cacodylate or buffered EDTA-sucrose, proved to be the most appropriate with respect to preserved substances were tested. In the bone tissue, immunoreactivity was found to four neuropeptides: substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y, and also to the catecolamine-synthesizing enzyme tyrosine hydroxylase. The described method for identifying intraosseal neuropeptides offers a new means of studying skeletal innervation and bioactive substances in bone tissue.     

The distribution of trace metal ions in bone and tendon

The natural trace metal compositions of human bone mineral and demineralized bone were measured by emission spectroscopy and compared with those of the original whole bone and human tendon. Cu, Fe and Zn remained in the collagenous matrix of bone and were found in similar quantities in tendon. It is suggested, therefore, that these ions are chemically bound to the collagen matrix in these tissuesin vivo, and that the binding has a part in their activity. Most of the Pb, Si, Sr, and V in bone remained with the mineral portion, probably as substituted or interstitial ions. Zn is divided between the organic and mineral phases.This work was supported in part by Grant No. AMO 7626, National Institutes of Health, United States Public Health Service and by the Veterans Administration Research Service.     

重组胶原矿化骨负载骨形态发生蛋白2活性多肽复合材料的制备及其异位成骨研究

目的 构建重组胶原矿化骨/BMP2活性多肽新型仿生骨修复材料,并评价其异位成骨能力.方法 将48只成年SD大鼠随机分为4组,在A、B、C组的大鼠背部肌肉内分别植入含3、2、1 nag BMP2活性多肽的重组胶原矿化骨;在D组的大鼠背部肌肉内植入单纯重组胶原矿化骨,于4、8和12周时间点将大鼠处死,经CT三维重建、组织学观察及钙含龟测定检测植入材料的成骨情况.结果 A、B.C组,在各时间点的CT三维重建、组织学观察及钙含量检测结果均表明其异位成骨能力优于D组,差异有统计学意义(P<0.01),其中A、B两组的异位成骨能力优于C组,差异有统计学意义(P<0.01).结论 BMP2活性多肽町以显著增强重组胶原矿化骨的骨诱导活性,这种骨诱导性存在一定的剂量效应关系.     

Fourier transform infrared microscopy of calcified turkey leg tendon

Fourier transform infrared microscopy (FT-IRMS) was used to monitor spatial variations in the quality and quantity of the mineral phase in calcified turkey tendon. Spectral maps were generated by analysis of 50 μm ×~ 50 μm areas within different regions of the tendon. Spectra of the transitional region, where nonmineralized matrix ends and mineralized matrix begins, revealed marked changes in the spectrally determined mineral-to-matrix ratio, whereas regions deeper into the mineralization front showed a relatively constant ratio. Since spectra of EDTA-demineralized matrix were similar to those of nonmineralized matrix, the nonmineralized regions of the tendon were used for spectral subtraction. The broad, relatively featureless contour of the mineral ν₁,ν₃ phosphate region (900–1200 cm⁻¹) showed only subtle changes at different stages of mineralization. Second derivatives of these spectra were calculated and compared with those of synthetic, poorly crystalline hydroxyapatite (HA). The peak positions seen in second-derivative spectra of the mineral near the transitional region were within ±2 cm⁻¹ of the least mature synthetic HAs whereas spectra of the mineral deeper into the mineralization front were within ±2 cm⁻¹ of the most mature synthetic HAs. Spectra from cross- and longitudinal sections at equivalent positions in the tendon, and polarized FT-IRMS data were analyzed to determine the effect of mineral orientation on the parameters used to characterize the mineral. Spectra of cross- and longitudinal sections of the tendon showed no major differences in either the ν₁,ν₃ phosphate region or the amide I, II, or III components (1200–1800 cm⁻¹). However, polarized FT-IR spectra revealed dramatic differences in both of these regions. Despite these differences, second-derivative analysis of the ν₁,ν₃ regions revealed no significant changes in the positions of the underlying bands used to characterize the environments of the phosphate ion in poorly crystalline HA. The results of this study demonstrate the power of FT-IRMS to monitor spatial variations of the mineral phase in calcified tissue. Also, the incorporation of polarized radiation provides a method capable of assessing the molecular orientation of the mineral phase relative to the collagen matrix. Received: 17 January 1995 / Accepted: 26 May 1995     

The effect of different mineral frames on ectopic bone formation in mouse hind leg muscles induced by native reindeer bone morphogenetic protein

Introduction Bone morphogenetic proteins (BMPs) require carrier material for slow release and framing material for osteoconduction.Materials and methods The effect of a frame on early bone formation induced by partially purified native reindeer BMP in composite implants containing 3 mg of BMP, type IV collagen and tricalcium phosphate (TCP/Col/BMP) or hydroxyapatite (HA/Col/BMP) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA/Col/BMP) or biocoral (NC/Col/BMP) was evaluated using a mouse hind leg muscle pouch model. Collagen with native reindeer BMP (Col/BMP) and corresponding implants without native reindeer BMP served as controls. Evaluation was done by incorporation of ⁴⁵Ca, radiographically and histologically 3 weeks after the implantation.Results None of the implants without native reindeer BMP were able to induce new bone visible on radiographs. The area of new bone formation in the Col/BMP (p=0.026) and TCP/HA/Col/BMP (p=0.012) groups was significantly greater than in the TCP/Col/BMP group. The optical density of the new bone area was significantly greater in the TCP/HA/Col/BMP group than in the TCP/Col/BMP (p=0.036) or Col/BMP (p=0.02) groups. ⁴⁵Ca incorporation was many times greater in all the groups containing native reindeer BMP than in the corresponding groups without BMP. In the Col/BMP (p=0.046) and TCP/HA/Col/BMP (p=0.046) groups, ⁴⁵Ca incorporation was significantly greater than in the TCP/Col/BMP group. No significant differences were found in any parameters between HA/Col/BMP and NC/Col/BMP groups and the other BMP-containing groups.Conclusions Hydroxyapatite, biocoral and biphasic tricalciumphosphate-hydroxyapatite are equally good as framing material for native reindeer BMP, while tricalciumphosphate is somewhat worse. Osteoinduction of native reindeer BMP works well with collagen alone.     

Demineralization of bone matrix: Observations from electron microscope and electron-probe analysis

Displacement or removal of mineral during the processing of calcified tissues for electron microscopy is a recognized phenomenon. An electron microscope analysis has been made of artefactual mineral loss during ultramicrotomy of osteogenic tissue. It is concluded from morphological investigation and the use of electron diffraction that this loss of crystalline mineral during sectioning can considerably change the morphology of calcified tissues and may lead to inaccurate interpretation of cell and matrix morphology. Electron probe X-ray microanalysis has been used to demonstrate in a semi-quantitative manner, the artefactual loss of calcium and phosphorus. Problems of specimen preparation for such analytical work are discussed.     

The effect of the microscopic and nanoscale structure on bone fragility

Bone mineral density is the gold-standard for assessing bone quantity and diagnosing osteoporosis. Although bone mineral density measurements assess the quantity of bone, the quality of the tissue is an important predictor of fragility. Understanding the macro- and nanoscale properties of bone is critical to understanding bone fragility in osteoporosis. Osteoporosis is a disease that affects more than 75 million people worldwide. The gold standard for osteoporosis prognosis, bone mineral density, primarily measures the quantity of bone in the skeleton, overlooking more subtle aspects of bone's properties. Bone quality, a measure of bone's architecture, geometry and material properties, is evaluated via mechanical, structural and chemical testing. Although decreased BMD indicates tissue fragility at the clinical level, changes in the substructure of bone can help indicate how bone quality is altered in osteoporosis. Additionally, mechanical properties which can quantify fragility, or bone's inability to resist fracture, can be changed due to alterations in bone architecture and composition. Recent studies have focused on examination of bone on the nanoscale, suggesting the importance of understanding the interactions of the mineral crystals and collagen fibrils and how they can alter bone quality. It is therefore important to understand alterations in bone that occur at the macro-, micro- and nanoscopic levels to determine what parameters contribute to decreased bone quality in diseased tissue.     

The effects of stress- relaxation plate on disorganization and repair of regional bone structure: an experimental study

Objective:To study the influence of stress-relaxation plate on disorganization and repair of the cortex beneath the plate.Methods:A washer made of viscoelastic polyethylene was placed between the screw and the screw hole of conventional stainless rigid plate (RP) to produce a stressrelaxation plate (SRP).Both SRP and RP were applied to osteotomized tibia in 48 New Zealand rabbits.Healing process of the fracture with either SRP or RP fixation (control) was comparatively studied with polarized light microscopy,in situ hybridization of collagen mRNA and immunohistochemical technique from 2 to 36 weeks postoperatively.Results:The study of plated bone remodeling showed that the degree of cortex osteoporosis beneath the plate was similar between the SRP and RP group within 12 weeks postoperatively.In comparison,the disorganization of bone structure in SRP group happened later and milder than that of RP group,and the repair process began at 12 weeks after implantation.As a consequence,the absorption cavities became smaller and the structure of collagen fibers became well oriented along with these changes by polarized light microscopy.In addition to these,the in situ hybridization analysis of collagen genes and the immunohistochemical study of type I,Ⅲ collagen at 8 to 12 weeks after implantation.from this time on ,the changes above became more evident significantly before most of cavities were repaired by 36 weeks.In contrast to the changes in the SRP group,no expression and synthesis of any kind of collagen could be observed during 12 to 36 weeks after implantation in RP group.Conclusions:Without removal of the bone plate,the SRP fixation not only reduces the degree of plated bone osteoporosis,but also makes the disorganized bone structure restored to normal in terms of the expression and synthesis of type I collagen mRNA of osteoblasts lying on the surface of absorption cavities.     

Alterations in the collagen framework of osteoarthritic cartilage and subchondral bone

Summary Specimens obtained from human osteoarthritic knee joints and dog knees with experimentally induced osteoarthritis were used to study the collagenous framework of articular cartilage and subchondral bone in relation to osteoarthritic changes using scanning electron microscopy and light microscopy. Degenerative articular cartilage in osteoarthritic joints showed radial orientation of the collagen fibrils, which were usually discernible as fibrillar bundle formations. Cartilage with extensive lesions often showed cleavages or fissures down to the calcified layer. The cartilagenous collagen fibrils in osteoarthritic specimens merged into the subchondral bone plate making the tidemark and the osteochondral junction irregular or obscure. The trabecular orientation of subchondral bone changed with alteration in the articular cartilage and with reactive changes in the subchondral bone, showing the effect of cartilaginous degeneration on its ultrastructure.

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Résumé Des prélèvements obtenus à partir de genoux humains arthrosiques et de genoux de chien porteurs de lésions arthrosiques expérimentales ont permis l'étude de la charpente de collagène du cartilage articulaire et de l'os sous-chondral, par microscopie conventionnelle et par microscopie électronique.Dans les lésions dégénératives, les fibrilles de collagène du cartilage ont une orientation radiale, alors qu'elles sont habituellement groupées en faisceaux. En cas de lésions étendues, le cartilage présente souvent des clivages ou des fissures menant jusqu'aux couches calcifiées. Les fibrilles de collagène du cartilage arthrosique se fondent dans la plaque osseuse souschondrale, rendant la jonction ostéo-cartilagineuse irrégulière ou mal définie.L'orientation trabéculaire de l'os sous-chondral se modifie en fonction de l'altération du cartilage articulaire. Ces modifications réactionnelles montrent que, même au niveau ultrastructural, l'architecture de l'os sous-chondral reflète la dégénérescence cartilagineuse.

     

Size and Shape of Mineralites in Young Bovine Bone Measured by Atomic Force Microscopy

Atomic force microscopy (AFM) was used to obtain three-dimensional images of isolated mineralites extracted from young postnatal bovine bone. The mean mineralite size is 9 nm × 6 nm × 2.0 nm, significantly shorter and thicker than the mineralites of mature bovine bone measured by the same technique. Mineralites of the young postnatal bone can be accommodated within the hole zone regions of a quasi-hexagonally packed collagen fibril in the fashion described by Hodge [9] in which laterally adjacent hole zone regions form continuous channels across the diameter of a fibril for a distance of at least 10 nm. Deposition of mineralites of the size noted above in this void volume of the fibrils would result in little or no distortion of the collagen molecules or supramolecular structure of the collagen fibril. The new AFM data supporting this claim is consistent with findings obtained by electron microscopy and low-angle x-ray and neutron diffraction that mineralites formed within collagen fibrils during initial stages of calcification occur within the hole zone region. However, the deposition of additional mineralites in the intermolecular spaces between collagen molecules in the overlap region of the fibrils would significantly distort the fibrils since the space available between adjacent molecules is considerably less than even the smallest dimension of the mineralites.     

The further purification and characterization of mouse bone collagenase

Mouse bone collagenase, a specific tissue collagenase, was isolated from the tissue culture media of 5 day old mouse tibiae. By a combination of (NH₄)₂SO₄ precipitation, molecular sieve filtration and ion exchange chromatography a fraction was obtained with a yield of approximately 11% which had a specific enzyme activity 120-fold greater than the original crude extract. The molecular weight of the fraction containing the enzyme activity was approximately 41000 as determined by calibrated molecular sieve filtration studies. There were at least two distinct major fractions and one minor fraction possessing collagenase activity which had distinct isoelectric points. It is not clear whether these fractions represent isoenzymes from bone or from bone and cartilage, or are derived from a single enzyme which has been minimally degraded by the extraction and purification techniques used. Collagenase activity was inhibited by EDTA, cysteine and horse serum, but not by phenylmethylsulfonylfluoride, epsilon amino caproic acid or soybean trypsin inhibitor.

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Zusammenfassung Mäuseknochen-Kollagenase, eine spezifische Gewebe-Kollagenase, wurde aus Gewebekulturmedien von 5 Tage alten Mäusetibiae isoliert. Durch eine Kombination von (NH₄)₂SO₄-Ausfällung, Gelfiltration und Ionenaustausch-Chromatographie wurde mit ungefähr 11% Ausbeute eine Fraktion erhalten, die eine spezifische Enzymaktivität besaß, welche 120mal größer war als diejenige des ursprünglichen unbehandelten Extraktes. Das Molekulargewicht der die Enzymaktivität enthaltenden Fraktion war ungefähr 41000, was durch kalibrierte Gelfiltrations-Untersuchungen bestimmt wurde. Mindestens zwei verschiedene Hauptfraktionen und eine kleinere Fraktion mit Kollagenaseaktivität wurden erhalten, welche verschiedene isoelektrische Punkte hatten. Es ist unklar, ob diese Fraktionen Isoenzyme aus Knochen oder aus Knochen und Knorpel darstellen, oder ob sie von einem einzelnen Enzym stammen, welches durch die verwendeten Extraktions- und Reinigungstechniken minimal degradiert wurde. Die Kollagenaseaktivität wurde durch EDTA, Cystein und Pferdeserum gehemmt, nicht aber durch Phenylmethylsulfonylfluorid, Epsilon-amino-capronsäure oder Soyabohnen-Trypsin-Hemmer.

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Résumé De la collagénase osseuse de souris, une collagénase tissulaire spécifique, est isolée du milieu de culture tissulaire d'un tibia de souris de 5 jours. En combinant une précipitation de (NH₄)₂SO₄, une filtration moléculaire sur tamis et la chromatographie par échangeurs d'ions, on obtient une fraction d'un rendement d'environ 11%, qui possède une activité enzymatique spécifique 120 fois plus élevée que l'extrait original total. Le poids moléculaire de la fraction, contenant l'activité enzymatique, est d'environ 41000, en se basant sur des études de filtration par tamis moléculaire calibré. Il existe au moins deux fractions principales distinctes et une fraction plus faible, ayant des activités en collagénase et des points isoélectriques distincts. Il n'est pas démontré que ces fractions constituent des isoenzymes de l'os ou de l'os et du cartilage, ou sont dérivés d'une seule enzyme, peu dégradée par l'extraction et la purification. L'activité de la collagénase est inhibée par l'EDTA, la cystéine, le sérum de cheval; mais elle résiste au fluorure de phénylméthyle-sulfonyle, à l'acide epsilon aminocaproique et à l'inhibiteur de trypsine de graine de soja.

     

The discrepancy in the carbonate found in fish bone and blood

It has been reported in the literature that fish, to get sufficient oxygen, must overventilate with respect to CO₂ and therefore exhibit a low circulating level of bicarbonate.Accordingly, the bones of trout and carp were analyzed to learn if the composition of their bone mineral reflects the low serum level of bicarbonate. It was found that the CO₂ content of fish bone is not significantly different from that of normal mammalian bone.Synthetic apatite crystals, made under comparable conditions of T, , and (HCO ₃ ^– ) were found to contain only 1/7th to 1/8th the CO₂ of fish bone.These data strongly suggest that the composition of the fluids in bone does not reflect, in a simple way, the composition of the circulating serum as is generally assumed.This work was supported in part by the United States Public Health Service Training Grant No. 1 T1 DE 175-02 and in part by the United States Atomic Energy Commission Contract No. W-7401-Eng-49 and has been assigned Report No. UR-49-922.     

Affinity of bone sialoprotein and several other bone and dentin acidic proteins to collagen fibrils

Summary Bone and dentin contain several kinds of mineral-binding proteins and cell-attachment proteins. The authors examined the affinity of these proteins to type I collagen, a major matrix protein of the tissue. Bone sialoprotein (BSP), bone Gla protein (BGP), bone small proteoglycan II (PG II), osteonectin (ON), and dentin phosphophoryn (DPP) were labeled with fluorescein isothiocyanate and incubated with reconstituted type I collagen fibril. DPP, BGP, BSP, and PG II were absorbed significantly to the collagen fibril at physiological ionic strength with dissociation constants of 10^-6–10^-7 M. BSP and PG II enhanced the fibrillogenesis of collagen. These acidic proteins can affect the surface properties of collagen fibril, and BSP, having the cell-attachment sequence Arg-Gly-Asp, possibly mediates interaction between collagen fibril and cells.     

Image analysis of collagen-associated mineral distribution in cryogenically prepared turkey leg tendons

Summary This study is concerned with the cryogenic preservation of intrafibrillar apatite distribution in type I collagen of turkey leg tendons. Cryogenic specimen preparations by the rapid freezing of nonfixed and noncryoprotected leg tendons were performed by two different protocols: (1) low temperature substitution, fixation and staining followed by low temperature embedment; (2) frozen hydrated and air-dried cryosections were examined with the electron microscope at −165°C and normal operating temperatures, respectively. These protocols revealed the axial periodicity for mineralized collagen to have a 65–69 nm range with a mean value of 67 nm as determined by point-to-point measurements. Mineral distributions and specific apatite visualization were examined by electron microscopic imaging in bright field and selected-area dark field, respectively. Fourier filtered images and image subtraction were used to separate the axial repeating and nonrepeating intrafibrillar mineral domains of collagen. The removal of these axial repeats revealed an underlying and integrated mineral distribution, demonstrating that apatite is not confined to axial periodicities such as those of the gap zone.