Collagen Fibril Density Research Articles

Control of the Collagen Fibril Diameter in the Equine Superficial Digital Flexor Tendon in Horses by Decorin

The distribution pattern of collagen fibril diameter in the equine superficial digital flexor tendon (SDFT) is known to differ in central and peripheral areas of some regions. This study reports the essence of collagen fibril differences among different regions of the equine SDFT by transmission electron microscopic (TEM) and high-voltage electron microscopic observations and biochemical analysis. The distribution of large collagen fibrils increased but the density of collagen fibrils decreased from the proximal metacarpal region to the distal metacarpal region. Large collagen fibrils with an irregular cross-sectional profile were found more frequently in the middle metacarpal region than in other regions. Three-dimensional reconstruction of images of irregularly shaped collagen fibrils revealed that these fibrils are formed through fusion of small collagen fibrils with large ones. The amount of decorin, which reportedly inhibits the lateral fusion of collagen fibrils, decreased in the direction of the distal metacarpal region. On the other hand, the size of decorin gradually increased in the direction of the distal metacarpal region. These results suggest that regional differences in collagen fibril distribution and density of collagen fibrils in the SDFT are due, at least in part, to fusion of collagen fibrils and the concomitant regional differences in the amount and size of decorin.

Extracellular matrix (ECM) microstructural composition regulates local cell-ECM biomechanics and fundamental fibroblast behavior: a multidimensional perspective

The extracellular matrix (ECM) provides the principal means by which mechanical information is communicated between tissue and cellular levels of function. These mechanical signals play a central role in controlling cell fate and establishing tissue structure and function. However, little is known regarding the mechanisms by which specific structural and mechanical properties of the ECM influence its interaction with cells, especially within a tissuelike context. This lack of knowledge precludes formulation of biomimetic microenvironments for effective tissue repair and replacement. The present study determined the role of collagen fibril density in regulating local cell-ECM biomechanics and fundamental fibroblast behavior. The model system consisted of fibroblasts seeded within collagen ECMs with controlled microstructure. Confocal microscopy was used to collect multidimensional images of both ECM microstructure and specific cellular characteristics. From these images temporal changes in three-dimensional cell morphology, time- and space-dependent changes in the three-dimensional local strain state of a cell and its ECM, and spatial distribution of beta1-integrin were quantified. Results showed that fibroblasts grown within high-fibril-density ECMs had decreased length-to-height ratios, increased surface areas, and a greater number of projections. Furthermore, fibroblasts within low-fibril-density ECMs reorganized their ECM to a greater extent, and it appeared that beta1-integrin localization was related to local strain and ECM remodeling events. Finally, fibroblast proliferation was enhanced in low-fibril-density ECMs. Collectively, these results are significant because they provide new insight into how specific physical properties of a cell's ECM microenvironment contribute to tissue remodeling events in vivo and to the design and engineering of functional tissue replacements.

Relationship between collagen fibrils, glycosaminoglycans, and stress relaxation in mitral valve chordae tendineae.

The tensile properties of mitral valve chordae tendineae derive from their structural make-up. The objectives of this study were to compare the stress relaxation properties of different types of chordae and relate their variation to structural features. Fifty chordae from eight hearts were subjected to stress relaxation tests. The percent stress relaxation and the relaxation rates were found to increase in the order of marginal. basal, and strut chordae. The water content of the three types of chordae was the same (marginal 77.1+/-5.9%, basal 77.0+/-3.4%, strut 78.0+/-2.3% wet weight). The collagen, elastin, and glycosaminoglycan (GAG) content in chordae were quantified using hydroxyproline assay, fastin elastin assay, and fluorophore-assisted carbohydrate electrophoresis, respectively. Collagen content of marginal chordae was only slightly less than that of basal and strut chordae (marginal 56.6+/-8.2%, basal 61.4+/-5.6%, strut 63.8+/-3.9% dry weight). There was also no significant difference in elastin content between the chordae (marginal 5.3+/-3.2%, basal 5.4+/-2.7%, strut 4.6+/-1.7% dry weight). However, the concentrations of unsulfated chondroitin/dermatan sulfate, 6-sulfated chondroitin sulfate, and 4-sulfate chondroitin sulfate significantly decreased in the order of marginal, basal, and strut. The total GAG-content also decreased in the order of marginal, basal, and strut (p = 0.06). The greater amount of GAGs in marginal versus strut chordae is consistent with our previous observations that marginal chordae have a greater collagen fibril density and thus more GAG-mediated, fibril-to-fibril linkages. The greater number of proteoglycan linkages may prevent the slippage of fibrils with respect to each other, and thus reduce stress relaxation. The different viscoelastic properties of mitral valve chordae can thus be explained morphologically.

Collagen fibril diameter distribution does not reflect changes in the mechanical properties of in vitro stress-deprived tendons

The purpose of this study was to determine if an association exists between the tensile properties and the collagen fibril diameter distribution in in vitro stress-deprived rat tail tendons. Rat tail tendons were paired into two groups of 21 day stress-deprived and 0 time controls and compared using transmission electron microscopy ( n=6) to measure collagen fibril diameter distribution and density, and mechanical testing ( n=6) to determine ultimate stress and tensile modulus. There was a statistically significant decrease in both ultimate tensile strength (control: 17.95±3.99 MPa, stress-deprived: 6.79±3.91 MPa) and tensile modulus (control: 312.8±89.5 MPa, stress-deprived: 176.0±52.7 MPa) in the in vitro stress-deprived tendons compared to controls. However, there was no significant difference between control and stress-deprived tendons in the number of fibrils per tendon counted, mean fibril diameter, mean fibril density, or fibril size distribution. The results of this study demonstrate that the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons is not correlated with the collagen fibril diameter distribution and, therefore, the collagen fibril diameter distribution does not, by itself, dictate the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons.

Characterization of statically loaded tissue-engineered mitral valve chordae tendineae.

Chordae tendineae are essential to the proper function of the mitral valve. Native chordae contain a dense collagenous core and an outer elastin sheath. We have been using the principle of directed collagen gel shrinkage to fabricate tissue-engineered mitral valve chordae. Because the microstructure of biologic tissues determines their mechanical behavior, the morphology of collagen and elastin in tissue-engineered chordae should mimic that of native chordae. The objective of this study, therefore, was to examine the morphology of our tissue-engineered constructs in comparison to native chordae. A collagen-cell suspension was cast into silicon rubber wells with microporous anchors at the ends and cultured in an incubator. The anchors allowed shrinkage to occur only transverse to the long axis of the wells, thus creating highly aligned collagen fibril constructs. The collagen constructs were cultured for 8 weeks and characterized mechanically, histologically, and biochemically at different culture time points. Histologic sections showed that in all mature constructs collagen fibers were oriented parallel to the long axis of the constructs. At the edge of the tissue collagen fibers were in general straight, whereas in the middle of the tissue they were wavy. Transmission electron microscopy showed a progressive increase in the density and longitudinal orientation of collagen fibrils with culture time. Light and scanning electron microscopy showed the presence of an elastin sheath around the collagen core. Immunostaining demonstrated that smooth muscle cells differentiate during tissue development and TUNEL assay showed that cells in the interior of the constructs undergo apoptosis. This study has demonstrated that collagen-cell constructs, with material properties and microstructure similar to native mitral valve chordae, can be developed using static culture.

Collagen fibrils appear more closely packed in the prepupillary cornea: optical and biomechanical implications.

The size and organization of stromal collagen fibrils influence the biomechanical and optical properties of the cornea and hence its function. How fibrillar structure varies with position across the cornea has not been fully characterized. The present study was designed to quantify the collagen fibril spacing and diameter across the normal human cornea and to relate this to the properties of the tissue. Small-angle x-ray diffraction was used to map in detail the variation in fibril spacing and fibril diameter along orthogonal medial-lateral and inferior-superior meridians of five normal human corneoscleral discs. Mean fibril diameters remained constant across all corneas up to the limbus, whereupon a sharp increase was observed. However, mean fibril spacing across the central 4 x 3 mm (prepupillary) cornea measured 5% to 7% lower than in the peripheral cornea. Collagen fibrils in the prepupillary cornea appear to be more closely packed than in the peripheral cornea. Anisotropy in fibril packing across the cornea has potential implications for the transparency and refractive index of the tissue. Biomechanically, it is possible that the higher packing density of stress-bearing collagen fibrils in the prepupillary cornea is necessary for maintaining corneal strength, and hence curvature, in a region of reduced tissue thickness. By inference, these results could have important implications for the development of corneal models for refractive surgery.

The effect of cranial cruciate ligament insufficiency on caudal cruciate ligament morphology: An experimental study in dogs.

To investigate the effect of cranial cruciate ligament (CrCL) insufficiency on morphology of the canine caudal cruciate ligament (CdCL). In vivo experimental study. Five adult foxhounds. Two years after CrCL transection, the histologic appearance of CdCLs from CrCL-deficient and unoperated contralateral control (CrCL-intact) stifle joints were evaluated using light and transmission electron microscopy. CdCLs from CrCL-deficient joints had extracellular matrix changes, characterized by chondroid metaplasia and disruption of cell architecture. Percent of small-diameter fibrils in CdCLs from CrCL-deficient joints was significantly greater (P <.05) than that in CdCLs from CrCL-intact joints. Collagen fibril density in CdCLs from CrCL-deficient joints (41.09 +/- 5.39%) tended to be less than that in CdCLs from CrCL-intact joints (52.96 +/- 6.92%); however, this difference was not significant (P =.056). Mean eccentricity (ratio of minor to major diameters) of collagen fibrils was significantly (P <.0001) lower for CdCLs from CrCL-deficient joints (0.85 +/- 0.016) when compared with that for CdCLs from CrCL-intact joints (0.87 +/- 0.015). Significant alterations were found in the morphology of CdCLs from CrCL-deficient joints. These changes may be associated with repetitive microtrauma to the CdCL secondary to instability or enzymatic degradation in the hostile synovial environment of an unstable joint. Regardless of the cause, the switch to a predominantly small-diameter collagen fibril profile may reflect compromised material properties of the CdCL. This should be taken into account when considering surgical techniques that rely on the CdCL to stabilize CrCL-deficient stifles.

Collagen fibril diameters increase and fibril densities decrease in skin subjected to repetitive compressive and shear stresses

Understanding microstructural changes that occur in skin subjected to repetitive mechanical stress is crucial towards the development of therapies to enhance skin adaptation and load tolerance in patients at risk of skin breakdown (e.g. prosthesis users, wheelchair users). To determine if collagen fibril diameter, collagen fibril density, dermal thickness, epidermal thickness, basement membrane length, and dermal cell density changed in response to repetitive stress application, skin subjected to moderate cyclic compressive and shear stresses for 1 h/d, 5 d/week, for 4 week was compared with skin from an unstressed contralateral control. The lateral aspects of the hind limbs of 12 Landrace/Yorkshire pigs were used. Skin from under the stressed site and a contralateral control site was processed for electron microscopy and light microscopy analysis. Electron microscopy results demonstrated significant ( p<0.01) increases in collagen fibril diameter of 15.9%, 22.4%, and 22.9% for the upper, mid, and lower layers of the dermis, respectively, for the stressed skin compared with the control skin. Collagen fibril density (fibrils/unit cross-sectional area) decreased significantly for stressed vs. control by 19.8%, 29.2%, and 31.8% for the upper, mid, and lower layers, respectively. Light microscopy results demonstrated trends of a decrease in dermal thickness and an increase in cell density for stressed vs. control samples, but the differences were not significant. Differences in epidermal thickness and basement membrane length were not significant. These results demonstrate that quantifiable changes occur in collagen fibril architecture but not in the gross tissue morphology following in vivo cyclic loading of pig skin.

Collagen Fibrils in Nanophthalmic Sclerae

To examine collagen fibrils in 3 nanophthalmos sclerae and to compare them with normal control sclerae morphometrically. Three cases of nanophthalmos associated with uveal effusion were studied. When sclerectomy was performed, scleral specimens were collected and fixed with 3% glutaraldehyde/2.5% paraformaldehyde. After epon-embedding and ultrathin sectioning, they were examined by transmission electron microscopy. Collagen fibrils from both nanophthalmos and normal control sclerae were compared in diameters and numbers per micron 2 areas. All scleral tissues from the three cases were associated with irregularly woven and unclear collagen bundles. Several abnormal findings, such as twisting or fraying, were also detected in a few collagen fibrils. The diameter and density of normal-appearing collagen fibrils that occupied most areas of nanophthalmos sclerae were the same as those from normal control sclerae morphometrically. Although nanophthalmos sclerae even with uveal effusion showed thick irregular collagen bundles and a few abnormal collagen fibrils, most collagen fibrils appeared the same as normal controls.

Morphological and biochemical re-evaluation of the process of cavitation in the rat knee joint: cellular and cell strata alterations in the interzone.

To assess the contribution of apoptosis to the mechanism of synovial joint cavitation, and to clarify morphological cellular changes during cavitation, we investigated the development of the rat knee joint by light and electron microscopy, TUNEL methods, and electrophoresis of DNA fragments. Although cavitation occurred within the interzone, which consists of 2 outer and a middle layer termed the intermediate zone, no morphological or biochemical signs of cell death, in particular apoptosis, were seen in the interzone at any embryonic stage. Microscopic and ultrastructural alterations affecting cell differentiation were clearly observed in the interzone, i.e. mesenchymal cells gradually showed elongation, cytoplasmic vacuolation and pyknosis in the intermediate zone where the elongated cells were arranged in parallel in some strata. Some of these cells were further flattened into spindle cells and the number of strata decreased to 2. The rest of the cells were incorporated secondarily into the outer layers, becoming chondroblasts. Collagen fibrils were arranged in a network structure in the outer layers, which obviously differed from the directional pattern parallel to the long axis of elongated cells in the intermediate zone. In addition, the density of collagen fibrils was higher in the outer layers than in the intermediate zone. During cavitation, the initial separation was detected between the elongated cells in the intermediate zone in paraffin sections at E16.5 and the spindle cells in epoxy sections at E18.5. The spindle cells lining the cavity, namely, the surfaces of the epiphysis and meniscus, finally became chondrocytes. The diminution of proteoglycans and collagen fibrils and the synthesis of hyaluronan in the extracellular matrix are now generally believed to be parts of the mechanism for cavitation based on the concept of 'loss of cohesion'. The microscopic and ultrastructural alterations in the interzone seemed to reflect differences in the arrangement and density of collagen fibrils and the developmental condition of the extracellular matrix between layers. Also it did not seem likely that these alterations inhibit the synthesis of hyaluronan at the presumptive joint line because this synthesis takes place at the plasma membrane. Separation between spindle cells should therefore represent the mechanism for developmentally programmed cavitation. Reorganization of the extracellular matrix is probably necessary for the cellular metamorphoses in the interzone involved in the process of cavitation.

Content and Distribution of Noncollagenous Matrix Proteins in Bone and Cementum: Relationship to Speed of Formation and Collagen Packing Density

The organic matrix of collagen-based calcified tissues consists of a supporting collagen meshwork and various noncollagenous matrix proteins (NCPs). Together, they contribute to determining the structure and biomechanical properties of the tissue. Their respective organization and interrelation can advantageously be examined by immunocytochemistry, an approach which allows correlation of composition with structure. The aim of this article is to review postembedding immuno- and lectin–gold-labeling data on the characterization of the noncollagenous compartment in rat and human bone and cementum, and on its relationship to collagen. The two major NCPs, bone sialoprotein and osteopontin, generally codistribute and accumulate in cement lines and in the spaces among the mineralized collagen fibrils. However, there are variations in their distribution and density of labeling throughout the tissue. Indeed, bone and cementum can form in environments that are either poor or enriched in NCPs. The amount of NCPs generally correlates with bone and cementum types and with speed of formation of the tissue and packing density of collagen fibrils. Taken together, the data suggest that production of both collagenous and noncollagenous constituents can be “modulated” during formation of collagen-based calcified tissues. It is concluded that, in addition to structural and compositional parameters, tissue dynamics must be taken into consideration in order to understand the significance of the apparent accumulation of NCPs at some sites and to determine the mechanisms of normal and pathological calcified tissue formation.

Electron microscopic evaluation of the effects of stress-shielding on maturation of the mid-substance and ligament-bone junction of the reconstructed anterior cruciate ligament in rabbits.

To analyze the effects of stress-shielding on graft maturation after the anterior cruciate ligament (ACL) reconstruction, autogenous ACL reconstruction using Achilles tendon was performed in rabbits. Two-end fixation with a ligament augmentation device (LAD), as a stress-shielding model (SS group), and pull-out fixation with Leeds-Keio artificial ligaments (L-K ligament), as a non-stress-shielding model (non-SS group), were investigated. Intact ACL was used as the control. Specimens were harvested 6 mon postoperatively, and the analysis was focused on collagen fibril maturation in the mid-substance and bone-anchoring pattern in the bone tunnel, under light and transmission electron microscopy. The density of collagen fibrils in the control was lower than that in both experimental groups (p<0.01). The per cent collagen area in the control was higher than that in the SS (p<0.01) and the non-SS group (p< 0.05). The per cent collagen area in the non-SS group was higher than that in the SS group (p<0.05). At the ligament-bone interface, intracellular calcification of the degenerated chondrocytes occurred in the non-SS group, while in the SS group hydroxyapatite deposits were observed only in the extracellular matrix. These results show unfavorable influence of stress-shielding on graft maturation not only in the mid-substance but also at the ligament-bone junction.

Analysis of Collagen and Elastic Fibers in Shoulder Capsule in Patients with Shoulder Instability

This study examined collagen cross-links, collagen fibril diameter and density, amino acid composition, and elastic fibers in shoulder capsule and skin in four patient groups: 1) unidirectional anterior instability (N = 8); 2) multidirectional instability/primary surgery (N = 6); 3) multidirectional instability/revision surgery (N = 6); and 4) no history of instability (N = 5). Compared with normal capsule, capsule from groups 1 and 2 had more stable and reducible collagen cross-links, significantly greater mean collagen fibril diameter, more cysteine, and a higher density of elastin staining. Compared with shoulder capsule in groups 1 and 2, shoulder capsule from group 3 contained significantly more reducible cross-links, smaller-diameter collagen fibrils, decreased collagen fibril density, and an increased density of elastin staining. There were no significant differences in any parameters between groups 1 and 2. We hypothesized that repeated capsular deformation in patients with shoulder instability results in changes in the capsule that increase its strength and resistance to stretching. Skin analyses demonstrated a significantly smaller mean collagen fibril diameter in skin from group 2 compared with group 1, suggesting the possibility of an underlying connective tissue abnormality.

Differential expression of type XIV collagen/undulin by human mammary gland intralobular and interlobular fibroblasts.

Immunolocalisation of type XIV collagen/undulin in the human mammary gland revealed greater deposition in the interlobular stroma than in the intralobular stroma. The interlobular stroma is located between the breast lobules and their associated intralobular stroma. Fibroblasts isolated from the interlobular stroma synthesised 3- to 5-fold more type XIV collagen/undulin than intralobular fibroblasts, but synthesised type I and type IV collagens in similar amounts. The differential expression of type XIV collagen/undulin was maintained with passage in culture. The results suggest a role for type XIV collagen/undulin in stabilising dense collagen fibrils. The maintenance of two types of structurally distinct stromas may be important during developmental processes in the mammary gland.

Electron microscopic investigation of the effects of diabetes mellitus on the Achilles tendon

Fine structural changes in the Achilles tendons of patients with long-term diabetes mellitus were investigated. All patients had clinical and electrophysiological evidence of diabetic neuropathy and had ulceration and/or Charcot neuroarthropathy. Several differences between tendons of diabetic (n = 12) and nondiabetic (n = 5) individuals were observed by electron microscopy. In diabetics, these differences included increased packing density of collagen fibrils, decreases in fibrillar diameter, and abnormal fibril morphology. In one diabetic patient, individual collagen fibrils were tightly apposed so that many areas of tendon appeared as a single mass of closely adhering fibrillae. In addition, foci in which collagen fibrils appeared twisted, curved, overlapping and otherwise highly disorganized were common in specimens from most patients (11 of 12). These morphologic abnormalities in the Achilles tendons of diabetics appear to reflect a poorly known process of structural reorganization that may be the result of nonenzymatic glycation expressed over many years. Such structural changes could contribute to the tightening of the Achilles tendor a phenomenon consistent with clinical observations of extreme shortening of the Achilles tendon-gastrocnemius-soleus complex common in advanced diabetic neuropaths. In patients with diabetic neuropathy, tendon shortening causes severe equinus that may precipitate serious ulceration, stress fractures, and Charcot collapse of the foot. However, in nondiabetics, the fine structure of the Achilles tendon appears normal, consistent with the finding that the ultrastructural changes result from diabetes rather than neuropathy.

Decreased birefringence of the superficial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training, detected by quantitative polarised light microscopy.

To investigate the effects of a one year programme of running training (up to 40 km/day for 15 weeks) on the spatial orientation pattern of collagen and glycosaminoglycans in articular cartilage in different parts of the knee (stifle) and shoulder joints of young beagle dogs. Area specific measurements of the optical path difference (= retardation, gamma) and the cartilage zone thickness were performed using conventional procedures and a new computer based quantitative polarised light microscopy method. Transmission electron microscopy was used to determine the zonal volume density of collagen fibrils. The concentrations of collagen and hydroxypyridinium crosslinks were investigated biochemically. Running training decreased gamma by 24-34% (p < 0.05) in the superficial zone of the lateral femoral condyle articular cartilage and at the centre of the tibial condyles. Gamma of glycosaminoglycans decreased by 26% (p < 0.05) in the superficial zone of the lateral condyle of the femur, but at the same site the volume density of collagen fibrils was unchanged. Neither the collagen concentration nor the concentration of hydroxypyridinium crosslinks was altered as a result of running. In both control and runner dogs, the thickness and gamma values of the superficial zone were greater in the humerus and the femur than in the tibia. Endurance type running exercise in beagles caused a reduction in the superficial zone birefringence of the articular cartilage, which indicates either a disorganisation or a reorientation of the superficial zone collagen network. Articular cartilage showed marked variability of collagen network organisation over the different knee (stifle) joint articular surfaces.

The development of preatherosclerotic coronary artery lesions in perinatal piglets.

The fine structure and ultrastructure of the anterior descending coronary artery were studied in a series of perinatal piglets at 1 week prior to birth, and at 8, 24, 72 and 168 h after birth. In the anterior descending coronary artery, at or just distal to the branch point of the left circumflex artery, early plaque-like intimal lesions were present in the majority of animals. These consisted of subendothelial edema, fragmentation and dissolution of the internal elastic lamella, and the appearance of intimal myoid cells known as modified smooth muscle cells (MSMCs). These changes were present in all piglets at and before 8 h of age. They persisted and progressed during the first week of life in about half of the piglets. Beginning at 72 h and continuing through 168 h, there was an increase in MSMCs and the appearance of fibroblasts. Both fibroblasts and MSMCs were associated with the elaboration of dense collagen fibrils. Foamy macrophages appeared within the subendothelial intima having the appearance of lipophages. While the prevalence of these changes at birth indicates that they may be part of normal development, their persistence in half the piglets and structural features suggest reaction to intimal injury beginning prenatally. The lesions may be precursive of coronary atherosclerosis later in life and may parallel the early stages of atherogenesis in humans.

Modulation by hypergravity of extracellular matrix macromolecules in in vitro human dermal fibroblasts

In vitro human dermal fibroblasts were submitted to normal gravity (1 g) or to chronic hypergravity (20 g) over a period of 8 days. Changes in organization of extracellular matrix molecules were seen by indirect immunofluorescence. In the fibronectin layer, bundles of fibrils were gathered together leading to a disorganisation of the normal parallel pattern of fibers seen in control cultures. Type I collagen fibrils appeared with wooly outlines in controls whereas thick fibers were closely packed in 20-g cultures. A moderate increase of type III collagen fibril density was observed. No elastic fibers were seen in control or in 20-g cultures. In the culture medium, the release of soluble elastin (ELISA) and type I and III collagens (RIA) was undisturbed. Assays of enzymes involved in the remodeling of extracellular matrix showed an increase of cellular elastase activity (10%) and a decrease of the spontaneously active collagenase. Nevertheless, the total collagenase activity, (activated by trypsin), was increased by up to 30%. These data show a significant rise of the latent collagenase activity and suggest that release of the tissue inhibitor of metalloproteinase (TIMP1) was enhanced by hypergravity.

Structure of corneal scar tissue: an X-ray diffraction study

Full-thickness corneal wounds (2 mm diameter) were produced in rabbits at the Schepens Eye Research Institute, Boston. These wounds were allowed to heal for periods ranging from 3 weeks to 21 months. The scar tissue was examined using low- and wide-angle x-ray diffraction from which average values were calculated for 1) the center-to-center collagen fibril spacing, 2) the fibril diameter, 3) the collagen axial periodicity D, and 4) the intermolecular spacing within the collagen fibrils. Selected samples were processed for transmission electron microscopy. The results showed that the average spacing between collagen fibrils within the healing tissue remained slightly elevated after 21 months and there was a small increase in the fibril diameter. The collagen D-periodicity was unchanged. There was a significant drop in the intermolecular spacing in the scar tissues up to 6 weeks, but thereafter the spacing returned to normal. The first-order equatorial reflection in the low-angle pattern was visible after 3 weeks and became sharper and more intense with time, suggesting that, as healing progressed, the number of nearest neighbor fibrils increased and the distribution of nearest neighbor spacings reduced. This corresponded to the fibrils becoming more ordered although, even after 21 months, normal packing was not achieved. Ultrastructural changes in collagen fibril density measured from electron micrographs were consistent with the increased order of fibril packing measured by x-ray diffraction. The results suggest that collagen molecules have a normal axial and lateral arrangement within the fibrils of scar tissue. The gradual reduction in the spread of interfibrillar spacings may be related to the progressive decrease in the light scattered from the tissue as the wound heals.

虚弱下顎骨に対する高カルシウム食, カルシトニンの併用療法に関する実験的研究 : 歯槽骨の変化

There is no fixed treatment method for debilitated bone at the growth stage. In general, measures taken are symptomatic. Male Wistar rats aged three weeks old which corresponds to human childhood were used. Calcium-deficient food was given and bone debility was induced in them. A treatment method combining high calcium food (mixed food of standard food and cuttlebone in the ratio of 2 to 1) and calcitonin was used. The results were as follows : 1. Analytical findings on the X-ray microanalyzer Relative Ca ratio and relative P ratio were determined from the results of quantification analysis by point analysis. In relative Ca ratio, values were shown in the descending order of the control group, calcium-deficient food + high calcium food group, combined treatment group, and calcium-deficient food group. In relative P ratio, the valued were shown in the descending order of the control group, combined treatment group, calcium-deficient food + high calcium food group, and calcium-deficient food group. 2. Histopathological findings In the calcium-deficient food + high calcium food group as compared with the calcium-deficient food group, osseous quantity in the whole osseous layer increased, and bone remodeling mechanism of transition from bone resorption to bone fromation was actively accelerated. Furthermore, in the combined treatment group, fasciculated bone showed increase in thickness and spongy bone showed increase in Haversian system markedly. 3. Findings on scanning electron microscope In the combined treatment group, dense collagen fibrils running in a given direction increased and findings of bone matrix united with collagen fibril network increased. 4. Hematological findings In the combined treatmet group as compared with the control group, no significant differences were found in blood electrolyte or in blood biochemistry. Examination of endocrinic functions showed low values for CT in the combined treatment group as compared with the control group. 1, 25(OH)_2VD showed high values in the combined treatment group as compared with the control group. On the basis of the foregoings findings, in the combined treatment group, calcification and bone construction were accelerated, but the osseous layer as a whole was not completely recovered as compared with the control group.