Validation of quantitative backscattered electron imaging for the measurement of mineral density distribution in human bone biopsies.

The measurement of bone mineral density (BMD) using X-rays is usually employed to monitor the mineral content in a given portion of bone. However, this method cannot differentiate between changes in bone volume or in degree of mineralization of the bone matrix. In contrast to BMD, bone mineral density distribution (BMDD), as measured on bone sections by quantitative backscattered electron imaging (qBEI), is able to distinguish differences in the degree of mineralization. For routine clinical research, we have validated the method of calibration and standardization of the backscattered electron (BE) signal. Carbon and aluminum were used as reference materials for BE gray levels and osteoid and apatite for calcium concentration. Experiments were performed to get knowledge about precision (intraassay variance-instrumental stability and interassay variance-reproducibility) and accuracy (standardization) of this method as well as the biological variance (intraindividual and interindividual) in human bone. On transiliac biopsies or necropsies from 20 individuals having had accidental death (13 females, 7 males, age 30-85 years) BMDD measurements were conducted. The patients' medical history as well as the histomorphology of these bones showed no evidence of metabolic bone disease. For instance, the standard deviations of the weighted mean calcium concentrations were <0.3%, <0.4%, <0.9%, and <2.6% of the mean for the intraassay, interassay, intraindividual, and interindividual variations, respectively. In addition, a mean BMDD histogram for transiliac bone specimens was calculated from the 20 aforementioned individuals. The method used allows detection of the degree of mineralization independently from the actual bone volume, a result that seems to be of special interest in the assessment of the effect of treatments for osteoporosis. The power of this technique is demonstrated by using bone from a patient with a metabolic bone disease. In this case of osteomalacia due to celiac disease, the mean calcium concentration in the bone matrix was reduced by 19.3% as compared with normal.

[Radiologic and histologic observations in central talus fractures]. / Radiologische und histologische Beobachtungen an zentralen Talusfrakturen.

In a prospective study of central fractures of the talus, radiological data (plain radiography, CT, MRI) were scrutinized in terms of correlation with histological findings (biopsy taken at time of screw removal). The study looked at nine fractures in eight patients aged 14-48 years. There was one fracture of Hawkins type I, one fracture of type II, four fractures of type III, and three corpus fractures. All were operated on within 24 h after injury; seven had screw fixation, two had adaptation with K wires. The patients were kept non-weight-bearing for 14-53 weeks. Duration of follow-up was 2 years or more in all but one patient. In the final radiograph the talus was intact in four patients, while four showed a partial avascular collapse; in one patient, a partial collapse was doubtful. Histology taken 16-52 weeks after injury showed osteocyte-free original bone in all cases, with deposition of new bone varying in extent. Hawkins sign was partially positive in six patients, positive in one, and negative in one. Decalcification as well as fracture healing can be better followed by CT than by plain radiography. MRI appeared normal in five patients, partially pathological in two, and abnormal in one. A positive Hawkins sign and normal signal behavior can be seen as prognostically favorable signs. However, in the case of a central talus fracture, even with a favorable course osteonecrosis of at least a part of the talus with gradual replacement by new bone occurred. Vascularization of the central talus seems to be disturbed more frequently than is reflected radiologically.

Vascular anatomy of the ovine anterior cruciate ligament. A macroscopic, histological and radiographic study.

Sheep are being extensively utilized in animal models for orthopaedic research, but the vascular anatomy of their anterior cruciate ligament (ACL) has not yet been thoroughly described. This study demonstrates the blood supply to the ACL. Vascular injection with plastogen G, lead oxide and India ink was performed in 12 back limbs of Styrian mountain sheep, and gross observations, microradiography and routine histology were done. The large vessel and the microvascular anatomy are similar to those described for humans. The middle genicular artery and the descending genicular artery contribute vessels that supply the ACL. Epiligamentous vascular plexuses give off capillaries which penetrate the ligament substance and supply numerous, longitudinally oriented intraligamentous vessels. These findings make the Styrian mountain sheep a potential animal model for biologic investigations of ACL pathology.

Histomorphology and bone morphometry of the bone marrow edema syndrome of the hip.

From a prospective study of patients with MR imaging proven bone marrow edema syndrome of the hip, bone biopsies that were retrieved at core decompression treatment of 32 femoral heads (from 28 men and 3 women; age range, 25-63 years) were evaluated microscopically. The undecalcified microtome sections showed diffuse or spotty areas of interstitial and intrasinusoidal fluid in the marrow cavities, together with fat cell destruction or fibrovascular regeneration or both in exactly the regions exhibiting the magnetic resonance signals for bone marrow edema. The vital bone trabeculae in these edematous regions showed more or less continuous, partly osteoblast covered osteoid seams, and often, formation of irregular woven bone (microcallus), pointing to increased bone formation activity. Preceding or active osteoclastic resorption was rarely seen. Computer assisted bone morphometry revealed age related normal to elevated bone volume densities (above 20% bone volume of tissue volume); thus, no evidence for osteoporosis was present. In addition to increased osteoid volumes, a decreased maximal hydroxyapatite content and a shift to undermineralized bone was found by mineral densitometry of corresponding microradiographs, when compared with age matched femoral heads without bone pathology. These bone mineral changes, but not transient bone loss, could be the explanation for the more or less subtle and transient radiolucency in hips affected by bone marrow edema syndrome. Live trabeculae and active bone formation, however, point to increased repair capacity, which seems the key for the spontaneously reversible course of this syndrome. There is still controversy whether the bone marrow edema syndrome represents a distinct transient disease or an early reversible phase of avascular necrosis, but because of the similarities in histopathology reported for early classic avascular necrosis and bone marrow edema in the literature and in the authors' own material, a common pathophysiology is discussed for these seemingly different diseases.

A new scanning electron microscopy approach to the quantification of bone mineral distribution: backscattered electron image grey-levels correlated to calcium K alpha-line intensities.

The introduction of backscattered electron (BSE) imaging in scanning electron microscopy (SEM) has led to new possibilities for the evaluation of mineral distributions in bone on a microscopic level. The different grey-levels seen in the BSE-images can be used as a measure for the local mineral content of bone. In order to calibrate these BSE-grey-levels (BSE-GL) and correlate them to mineral contents, various attempts, using reference samples with known weighted mean atomic number and/or using simulated bone tissues with known hydroxyapatite concentrations, have been made. In contrast, a new approach is presented here based on measurements of the X-ray intensities of the calcium K alpha-line on selected areas of real bone samples; the measured intensities are then related to the corresponding BSE-GL. A linear positive correlation between weight percent (wt%) calcium and BSE-GL was found. When the BSE-mode is standardized using carbon and aluminum as references, the different mineral contents in bone samples can be recorded as BSE-GL, calibrated to wt% of calcium or hydroxyapatite (HA), respectively. The resulting mineral concentration histograms have a dynamic range from 0 to 89 wt% HA and have a binwidth resolution of 0.45 wt% HA. The presented modifications of the BSE method strongly enhance its feasibility in the field of bone research and its application as a special diagnostic tool for bone diseases.

Abnormal bone mineralization after fluoride treatment in osteoporosis: a small-angle x-ray-scattering study.

Sodium fluoride treatment of osteoporosis is known to stimulate bone formation and to increase bone mass, but recent clinical trials failed to prove its antifracture effectiveness. The formation of bone with abnormal structure and, therefore, increased fragility is discussed as a possible explanation. Until now, however, exact information on the mineral structure of osteoporotic bone after fluoride treatment has been lacking. Bone biopsies were taken from three patients with postmenopausal osteoporosis before and after fluoride treatment (60 mg NaF/day for 1-2 years), from one patient with iatrogenic fluorosis, as well as from three normal controls. The mineral in these samples was investigated by a combination of backscattered electron imaging and small-angle x-ray scattering. Depending on the total dose of fluoride, an increasing amount of new bone is laid down on the surface of preexisting trabeculae. Its mineral structure is identical to that of heavy fluorosis and is characterized by the presence of additional large crystals, presumably located outside the collagen fibrils. These large crystals, which are not present in the controls or in osteoporotic bone before fluoride treatment, contribute to increase the mineral density without significantly improving the biomechanical properties of the bone. The possible success of fluoride treatment depends not only on the amount of newly formed bone but also on the rate of bone turnover. Indeed, as soon as significant amounts of fluoride are present, bone turnover leads to the replacement of old (normal) bone by new (pathologically mineralized) bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative assessment of fracture healing. The value of computerised sonometry in a correlative animal experiment in sheep.

Usually follow-up of the healing process of bone fractures is by subjective assessment of standard X-rays, based on the clinician's personal experience. It is therefore impossible to completely avoid misjudgements in the evaluation of the mechanical stability. Other authors have reported that the mechanical response of bony structures to a knock impulse allows a definitive statement about the stiffness of a fracture. The practicability of computerised sonometry in computer-aided evaluation of the mechanical vibration reaction and the acoustic transmission of sound through osseous structures for assessing stability has been proved in experimental and clinical studies. We designed an animal study that would allow a systematic correlation between the diagnostic investigations in general use. As a main goal, this study tries to correlate sonography with other quantitative techniques, even if they are not used under everyday clinical conditions. Our results show that methods based on direct assessment of the mechanical stability between fracture fragments allow a more exact evaluation of fracture healing. This study helps to classify the results of computerised sonometry together with other diagnostic procedures used for the evaluation of fracture healing, and furthermore provides a basis for clinical interpretation of the findings of a new, non-invasive technique for precise quantitative assessment of fracture healing.

Early osteoinduction in rats is not altered by fibrin sealant.

The angiogenic and fibrogenic activities of fibrin sealant (Tisseel) suggest that it could enhance osteoinductivity during the first days after implantation. To test this hypothesis, the osteoinductivity of sealed and unsealed bone matrix gelatin was tested by implantation into rat abdominal wall muscle pouches for two to ten days. A sponge of collagen with or without fibrin sealant was used for control. Alkaline phosphatase (ALPase) was employed to quantify osteoinduction, and myeloperoxidase (MPO) was used to determine the inflammatory reaction. Plain histologic analysis of bone matrix gelatin implants at Days 2, 4, and 10 did not demonstrate any significant differences between sealed and nonsealed implants. Histologic analysis at Day 21 confirmed the osteoinductivity of the implant. The combination of bone matrix gelatin and fibrin sealant induced an early significant increase in ALPase activity, which could be interpreted as early induction of histologically undetectable osteogenesis. A simultaneous increase in the level of MPO suggested that the bone matrix gelatin/fibrin sealant-induced ALPase reaction might be caused by an inflammatory process, but neutrophil counting did not correlate to the MPO data. Morphometry, however, seems to be insufficient to assay a heterogenous histologic distribution of singular cells. It is proposed that the ALP/MPO ratio may be used as an inflammation-independent marker of osteogenesis. This study does not support the notion of an osteoinductive potency of fibrin sealant per se; however, it is clear that fibrin sealant does not impair bone matrix gelatin-dependent osteoinduction.

Mineral crystals in calcified tissues: a comparative study by SAXS.

The shape, the typical orientation, and the average size of mineral crystals in different types of mineralized tissues were investigated by means of small-angle x-ray scattering (SAXS). To rule out eventual artifacts due to sample preparation, four different standard preparation techniques were used and a comparison showed that the SAXS results were identical for all four methods. In mineralized turkey leg tendon, a frequently used model system for bone, the crystals were found to be typically plate-like with a thickness of the order of 2 nm. This stands in contrast to the case of bone (calvaria, femur, and iliac crest) from mouse, rat, and dog, where mainly needle-like crystals were found. The thickness of these crystals ranged from 3 to 4 nm but was remarkably constant for different bones of a given animal. The preferred orientation of the needle-like crystals was along the main axis of the femur and within the surface of the calvaria (for mouse, rat, and dog). The mineral plates in turkey leg tendon were located inside the hole zone and oriented along the fibril axis. Finally, no periodic arrangement of the crystals inside the hole zone of the collagen could be found.

Fresh autogeneic, frozen allogeneic, and decalcified allogeneic bone grafts in dogs.

In fully-grown mongrel dogs, diaphyseal ulnar defects 25 mm long were stabilised by screws and plates, and were temporarily filled with silicone rubber blocks. After eight weeks the block was replaced either by fresh autogeneic cancellous bone, allogeneic deep-frozen cancellous bone, allogeneic decalcified bone matrix, or bone matrix gelatin. After 24 weeks the implants were evaluated by radiography, histology, and measurements of new bone volume, using computer-assisted density registration on microradiographs. Only the autogeneic bone grafts led to healing in all instances. Bone regeneration in the other groups was not significantly better than in the sham group in which no graft was employed. Decalcified bone matrix proved ineffective.

Decalcified and undecalcified cancellous bone block implants do not heal diaphyseal defects in dogs.

UNLABELLED: In a previous study it has been shown that granulae of decalcified bone matrix do not induce bony healing of 8-week-old mid-diaphyseal defects in dogs. The aim of this study was to test whether osteoinduction combined with the osteoconductive mechanisms provided by the natural structure of cancellous bone blocks would yield better results. A 30-mm-long diaphyseal defect of the left ulna was created in eight adult mongrel dogs and the bone was stabilized with a plate. A Silastic spacer was inserted in the defect for 8 weeks, followed by implantation of frozen undecalcified or decalcified allogeneic cancellous bone blocks for 16 weeks. Healing was analyzed using morphologic methods. At 16 weeks after implantation all grafts had been resorbed. In the decalcified group one defect healed, while none in the other group did so. The implant material was bioassayed in nude rats for osteoinductivity, which was found to be low in decalcified matrix and not detectable in undecalcified bone. CONCLUSION: Allogeneic cancellous bone blocks, demineralized or not, have no osteoinductive capacity and no osteoconductive function that promotes healing of mid-diaphyseal bone defects in dogs.

[Partial resection of the ulna as a diaphyseal bone defect model in the dog]. / Die Ellenteilresektion als diaphysäres Knochendefektmodell beim Hund.

A 25-mm periosteal cylinder was removed from the middle of the ulna in fully grown mongrel dogs. A silastic block was interposed and the defect was fixed by a plate. Eight weeks later, the block was removed. During the following 24 weeks, spontaneous bone regeneration was evaluated by radiologic and histomorphometric evidence. In a second series, three screws were placed transversely to the longitudinal axis from the ulna into the radius to stabilize the partial resected ulna. The observation period after silastic removal was 16 weeks. Evaluation was based on radiology and on Indian ink stain of the vessels. Plate fixation provides an essentially stable defect model, with partly considerable bone apposition to the proximal area of resection. This is explained by the entry of the only nutrient artery into the proximal diaphyseal third. No instance of spontaneous defect healing following plate fixation was observed. The transverse fixation of the ulna revealed early radiologic signs of instability.

[Preradiologic stage of idiopathic femur head necrosis demonstrated by intraosseous pressure measurement]. / Das präradiologische Stadium der idiopathischen Hüftkopfnekrose intraossären Druckmessung.

Early identification is the focal point of efforts aimed toward efficient diagnosis and treatment of osteonecrosis. With the intraosseous pressure measurement method of Ficat and Arlet it is possible to determine the onset of this disease at the preradiological stage. At the Vienna-Speising Orthopedic Clinic 11 out of a total of 66 cases of idiopathic necroses of the femoral head were diagnosed at the preradiological stage. The only treatment given in these cases was decompression by removal of a bone cylinder. The follow-up examination after an average of 22 months revealed very good results in all cases, both subjectively and roentgenologically (stage deterioration did not occur in a single case).

Evidence for negative correlation between quantitative histological studies and microradiography of iliac crest bone and forearm osteodensitometry in elderly women with osteoporosis.

Quantitative histomorphometric and microradiographic analysis of iliac crest bone biopsy specimens of 10 unselected and untreated postmenopausal women with osteoporosis was performed and the results were correlated with the values measured by osteodensitometry on the right forearm. No positive correlation between histomorphometry or microradiography and osteodensitometry was observed. Moreover, the data of various histomorphometric parameters and those of the bone mineral content measured by microradiography revealed a significant negative correlation when compared to the values obtained by osteodensitometry. Our results suggest, that evaluation of one skeletal site is not necessarily representative of the entire skeleton in patients with postmenopausal osteoporosis.

Experimental investigations into the clinical significance of bone growth at viscerocranial sutures.

The development of a viscerocranial suture under physiological conditions was investigated in a total of 37 pigs and the effects of various traumas on expected sutural bone growth were observed. Different operations were carried out on one side only in the vicinity of the zygomaticotemporal suture in 18-25 day old piglets. The animals were sacrificed six months after the operation. The skulls of some of the animals were macerated and subjected to macroscopic and radiological investigation. Both zygomatic arches were removed from each of the remaining animals and used for the production of undecalcified microtome and ground sections. Intravital fluorochrome labelling was used to determine the asymmetric growth at this suture; this is interpreted as being the formation of new bone following passive expansion of the sutural region. After injury or transplantation of the periosteum and after the healing of a narrow bone defect or fracture we observed either a continuous suture from the outset or the formation of a new fissure after osseous regeneration. Similar findings were also made after the transplantation of compact rib fragments into a sutural defect without subsequent bridging of the defect. Only after the transplantation of autologous pieces of cancellous rib into an osseous defect in the sutural region was sutural ossification, accompanied by distinct deformation of the skull observed. As a clinical consequence of this it may be presumed that trauma or operation in the region of a suture has a considerable effect on the growth of the visceral cranium only if it results in premature synostosis.

[Fundamentals of echographic diagnosis of acute and chronic sinusitis (author's transl)]. / Grundlagen der echopgraphischen diagnose akuter und chronischer Sinusitiden.

Changes of sound permeability in facial bones are caused by inflammatory or tumorous reaction in the mucosa of the sinuses; echo-orbitography (8-10 MHz) shows in this case extraorbital echo signals if bony walls are thin enough (e.g. Lamina papyracea, anterior and upper wall of the antrum). Further examinations of facial bones show early inflammatory changes of bony tissue in sinusitis without influencing the echographic pattern; the amount of hydroxylapatite remains unchanged.