Is retrieval of bone material from multiorgan donors effective enough to cover demand for biostatic bone tissue grafts in Poland?

In Poland there is growing demand for biostatic allogeneic bone transplantation mostly for traumatologic operations and orthopedic reconstructions. The bone material is primarily harvested during postmortem examinations in forensic and pathology laboratories. Nevertheless, the collected amounts are not sufficient, so that material needs to be acquired from alternative sources, such as multiorgan donors. Between 1998 and 2003, 2331 potential donors were registered by the Transplantation Coordinating Center in Warsaw, which was adjusted to 1794 donors who would have been accepted as donors of the bone tissue. Unfortunately, due to denials from family members and public prosecutors, the sample was only 1416 donors, which would cover about 40% of the clinical orthopedic demand in Poland.

Factors determining the contamination of bone tissue procured from cadaveric and multiorgan donors.

There is an increasing demand for bone allografts that are widely used in orthopedic reconstructive procedures. The bone tissue may be harvested from two sources: cadavers and multiorgan donors. Providing safe and valuable bone allografts is of paramount importance. Contamination of allografts during bone retrieval seems to be one of the most important problems since pathogenic microorganisms might be responsible for postoperative infections and complications in the healing process. The purpose of our study was to identify all factors contributing to bacteriological contamination of harvested bones. Therefore, we have considered factors such as harvesting environment, explantation techniques, storage and preparation of allografts, number of preceding procurements from the same donor, procurement duration, and time interval between death and tissue procurement. The microbiological evaluation of allografts has been performed by taking cultures from all collected bones. Our study revealed significantly greater contamination rates of bone allografts harvested from morgue than from multiorgan donors. According to this observation, we suggest that orthopedic surgeons should pay particular attention to obtain more bones of the highest quality, personally participating in multiorgan procurements.

Amnion allografts prepared in the Central Tissue Bank in Warsaw.

Applications of allogenic amnion grafts range from wound dressing of severe burns, dermabrasions and lower extremity ulcer treatments to plastic surgery, laryngology and ophthalmology. The aim of the present study was to elaborate the method of processing, preservation and sterilization of human amnion allografts prepared as wound dressing used mainly for burned patients. During the amniotic sac processing (after separation of chorion) special attention was paid to ensure that the epithelial side of amnion is placed directly on polyester net used as a support. After application on the wound, the epithelial side with the basement membrane is facing outwards; this will promote migration, attachment and spreading of the host cells encouraging epithelialization. Human amnion allografts were preserved by lyophilization or deep-freezing and subsequently radiation-sterilized with a dose of 35 kGy. It has been observed, however, that lyophilized irradiated allografts are resorbed within a few days, while frozen irradiated ones better adhere to wound and persist even 3 weeks after grafting, therefore, it has been decided to preserve amnion by deep-freezing. Since the beginning of 1998 over 400 preserved radiation-sterilized amnion allografts (with a total surface area over 40,000 cm2) have been prepared at the Central Tissue Bank in Warsaw and distributed to clinics and hospitals throughout the country.

Alteration of mineral crystallinity and collagen cross-linking of bones in osteopetrotic toothless (tl/tl) rats and their improvement after treatment with colony stimulating factor-1.

A common feature of various types of mammalian osteopetroses is a marked increase in bone mass accompanied by spontaneous bone fractures. The toothless (tl/tl) rat osteopetrotic mutation is characterized by drastically reduced bone resorption due to a profound deficiency of osteoclasts and their precursors. An altered bone morphology has also been observed. The mutants cannot be cured by bone marrow transplantation, but skeletal defects are greatly reduced after treatment with colony stimulating factor 1 (CSF-1). The objectives of this study were to characterize mineral and collagen matrices in cancellous and compact bone isolated from long bones of 6-week-old normal littermates, tl/tl osteopetrotic mutants and mutants (tl/tl) treated with CSF-1. There were no differences in bone mineral content, but a significant decrease in the crystallinity of mineral evaluated by the method based on electron paramagnetic resonance spectrometry was observed in all bones of tl/tl mutants as compared to that of controls. Within the collagen matrix, slight decreases in the labile cross-links, but significant increases in the content of the stable cross-links, pyridinoline, and deoxypyridinoline, were observed in both cancellous and compact bone of osteopetrotic mutants. In tl/tl mutants treated with human recombinant CSF-1, the normalization of the crystallinity of bone mineral as well as collagen cross-links was found. Our results indicate that remodeling of bone matrix in tl/tl mutants is highly suppressed, but that after treatment with CSF-1, this activity recovers significantly. Taken together, these data provide further support for the hypothesis that CSF-1 is an essential factor for normal osteoclast differentiation and bone remodelling.

Optical diffractometry in bone research.

NASA: A new method of quantitative evaluation and comparison for bone tissue histological studies is described. Optical diffractometry allows numerical evaluation of dimensions of structures being studied and also permits study by discriminant analysis.^ieng

Nuragic bone collagen retains its molecular structure.

The molecular structure of bone collagen was found to be preserved in Nuragic bone samples several centuries after burial. Enhanced birefringence of bone and dentin collagen fibres observed in polarized light after Sirus red staining was used to demonstrate this fact.

Bone powder exposed to the action of 12C and 25Mg ion beams as investigated by electron paramagnetic resonance spectroscopy.

Bovine compact bone powder was irradiated with alpha-particles of 241Am, 12C6+ and 35Mg12+ ion beams, then examined by the e.p.r. spectroscopy. The stable e.p.r. signal found is of the same characteristic as that observed in gamma-irradiated bone (g perpendicular = 2.0017, g parallel = 1.9973, delta Hpp = 0.85 mT). Thus, the structure of radiation induced paramagnetic center in bone does not depend on the LET of radiation used. The radiolytic yields of the formation of stable paramagnetic centres in bone powder irradiated with gamma-rays are G gamma = 0.08 +/- 0.01 while with 12C6+ ion beams roughly about Gc = 0.02, respectively.

Applications of EPR spectroscopy to radiation treated materials in medicine, dosimetry, and agriculture.

Survey is given of the EPR studies on irradiated bone mineral referred to transplantology, physiology, pathology, dosimetry and to the detection of irradiated food. Stability, dose-dependency and nature of radiation-induced EPR signal in bone mineral (hydroxyapatite) are discussed. Paramagnetic centers produced by radiation as "labels" as applied to biomedical experiments and dosimetry are evaluated. Results on the detection of irradiated fruits, vegetables, and other dry food products are presented.

Decreased heterotopic osteogenesis in vitamin-D-deficient, but normocalcemic guinea pigs.

The effect of vitamin D deficiency unhampered by hypocalcemia on de novo bone formation was studied in guinea pigs. Heterotopic induction of osteogenesis was evaluated 4 weeks after intramuscular transplantation of allogenic urinary bladder transitional epithelium from vitamin-D-repleted (+D) donors into +D and -D recipients. In -D recipients the frequency of osteogenesis and the amount of induced bone were significantly diminished; induced bone was less mature, scantly cellular woven bone poorly repopulated with bone marrow. No effect of vitamin D deficiency on orthotopic bone growth and on mineralization of orthotopic and heterotopically induced bone was observed. It is proposed that in addition to inducing factors (BMPs, growth factors) which may be responsible for transformation of mesenchymal cells to osteoprogenitor cells, normal concentrations of 1,25-(OH)2D3 may be required for proliferation and further differentiation of these cells into osteoblasts and for expression of genes engaged in extracellular matrix formation and maturation.

Radiation-induced paramagnetic centers in research on bone physiopathology.

It has been found that ionizing radiation induces stable paramagnetic centers in the crystalline lattice of bone hydroxyapatite (HA). These defects, which can be detected and measured by electron spin resonance (ESR) spectrometry, can be treated as a new kind of biologic marker and used for the following: (1) evaluation of crystallinity of bone mineral, because the yield of these centers depends on the size of HA crystals; (2) quantification of the rebuilding process of radiation-sterilized bone grafts, because of the stability of the centers; and (3) estimation of the dose of ionizing radiation absorbed by living organisms, because of the linear relation between the dose of ionizing radiation and the yield of radiation-induced paramagnetic centers in bone mineral.

Effect of radiation sterilization on the osteoinductive properties and the rate of remodeling of bone implants preserved by lyophilization and deep-freezing.

The effect of various doses of ionizing radiation on the osteoinductive properties of decalcified bone matrices implanted heterotopically and on the rate of remodeling of nondecalcified bone grafts implanted orthotopically in allogeneic systems was studied. Decalcified bone matrices and nondecalcified bone grafts were preserved by lyophilization or by deep-freezing and were subsequently irradiated with appropriate doses at room temperature or at -72 degrees. Lyophilized matrices irradiated at room temperature with 35 and 50 kGy, respectively, were completely resorbed five weeks after heterotopic implantation into the muscles and did not induce osteogenesis, whereas the resorption of deep-frozen ones irradiated with the same doses at -72 degrees was slower and new bone formation was induced. The preservation of the osteoinductive capacity of irradiated, deep-frozen matrices may depend on two factors: reduction of radiation damage on the inducing agents and collagen irradiated in the presence of water, which may diminish the rate of matrix resorption. The rate of remodeling of undecalcified deep-frozen bone implants irradiated at -72 degrees and grafted orthotopically was higher than that of lyophilized ones irradiated at room temperature. It is possible that the temperature during irradiation plays a critical role in protection against radiation damage.

Early and transient osteopetrosis in microphthalmic MIB-rats.

Microphtalmic blanc mutation (mib/mib) displays a very mild form of osteopetrosis in rats. The autosomal recessive mib mutation shows pleiotropic expressions in homozygotes. Microphtalmia, absence of eye and skin pigmentation, retardation in the tooth eruption were observed in the mutants. Most bone abnormalities occurred in newborns. An increased radiological opacity of long bones, persistence of primitive bone in medullary cavities, reduced number of poorly differentiated osteoclasts in mandibulae, reduced number of mononuclear peritoneal cells as well as reduced number of mononuclear osteoclast precursors in peritoneal cell population were found. In 3 weeks old and in adult mutants, both bone structure and the number of mandible osteoclasts appear normal, but the number of blood monocytes, peritoneal cells and mononuclear osteoclast precursors in peritoneal cell population remain significantly lower than in the healthy littermates. These observations indicate that the early failure of osteoclast differentiation and maturation is transient in the mib/mib form of osteopetrosis.

Spreading of cells on various substrates evaluated by Fourier analysis of shape.

In order to evaluate in mathematical terms the morphological changes occurring in the course of cell spreading, Fourier analysis of shape was applied. Human urothelial Hu 961 b cells plated on type IV collagen, fibronectin, laminin, glass and bovine serum albumin (BSA) were studied. Fourier parameters describing cell shape as well as surface areas covered by the cells on the substrate were subjected to statistical analysis. Using analysis of variance and discriminant analysis it was found that parameters describing cell shape (both gross shape of cells and their fine scale contour foldings) possessed a higher power of discrimination between the cells spread on various substrates than the differences in cell surface areas. In the course of observation (75 and 150 min) the highest number of attached cells and highest degree of spreading were found when cells were plated on type IV collagen. Moderate alterations in cell shape and moderate increase of surface area were seen in the group of cells seeded on fibronectin, whereas the cells plated on laminin, glass and BSA revealed a moderate increase of surface area, but no changes in their shape were observed. The differences in attachment of cells and in the degree of their spreading might be due to the variation in expression of plasma membrane receptors for various substrates. The Fourier analysis of cell shape coupled with measurement of surface area is a good tool for quantitative evaluation of cell spreading and can be used for discrimination between cells spread on different substrates.

Differences in the kinetics of the mineralization process in endochondral and intramembranous osteogenesis in human fetal development.

About 300 samples of bone tissue from femoral diaphyses and from parietal bones of calvaria were collected from human fetuses aged from 6-8 to 40-41 weeks in order to compare the kinetics of the mineralization process in endochondral and intramembranous bone formation. The crystallinity of bone mineral was evaluated by the method based on electron spin resonance (ESR) spectrometry, and mineral content was measured after ashing of the samples. Highly significant differences in the kinetics of deposition of mineral (ash content) and maturation of deposited mineral (crystallinity coefficient) were found between parietal bones and femoral diaphyses in the course of fetal development. In the early stages of development (6-8 to 10-12 weeks), the content of mineral and its crystallinity in parietal bones were significantly lower than in femoral diaphyses. In the parietal bones a gradual increase in both values was observed until they reached a plateau at the age of 30 weeks. On the other hand, in femoral diaphyses of 6-8-week-old fetuses mineral content was high and deposited mineral showed a high degree of crystallinity. Both these values did not change significantly until birth. The differences in kinetics of the mineralization process between the weight-bearing bones and bones that are not exposed to the influence of strong external forces are most probably genetically conditioned. The collected data supply embryological information and might be used in the future for evaluation of pathologic changes in skeletal development.

Evaluation of sperm motility by optical diffractometry of spermatograms.

Evaluation of sperm motility based on analysis of spermatograms by optical diffractometry was performed. Spermatograms are defined as images of sperm tracks obtained on microphotographs by dark field illumination with long exposure time. Single track analysis proved that the details concerning single tracks are "seen" and recognized by the technique of optical diffractometry. Normal "linear", as well as abnormal "irregular", including "circular", tracks were numerically described. Multitrack analysis: The general pattern of all tracks contained in particular spermatograms derived from two groups of semen classified by hospital laboratory as "high" (control) and "low" ("mediocre") motility of spermatozoa, respectively, were analysed by optical diffractometry. The hospital laboratory was asked to provide for diffractometric analysis samples of the "mediocre" semens of quality similar enough to the "control" ones what concerns the percentage of motile spermatozoa (40 percent) and spermatozoan concentration. Diffraction patterns of 102 spermatograms of control sperm and of 103 spermatograms of sperm qualified as "mediocre" by subjective microscopic evaluation of sperm motility, were analysed and compared. Using discriminant analysis based on parameters describing radial distribution of light intensity in diffraction patterns of control and "mediocre" spermatograms, it was possible to classify correctly 71 cases as high motility sperms out of 102 spermatograms evaluated beforehand by hospital laboratory criteria as control ones. In the group of 103 spermatograms classified as "mediocre" sperms by hospital laboratory criteria 69 cases were correctly recognized as "mediocre" by diffractometric analysis. The overlapping of the analysed two groups of sperm by 30 percent might be explained by the fact that in the control spermatograms many trajectories are formed by inefficient, slow moving spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone tissue.

The effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone in guinea pig was studied. Heterotopic bone formation was induced by transplantation of allogenic urinary bladder epithelium. Starting from the day of transplantation the animals were treated subcutaneously with HEBP and Cl2MBP with a dose of 12.5 mg P/kg/day during 35 days. The control group was injected with 0.9% NaCl solution. The advantage of heterotopic bone induction as an experimental model is the fact that the applied drugs act on de novo bone formation. Collagen fibers were treated as markers of bone because their size and spatial arrangement reflect the structure and maturity of organic matrix of this tissue. Decalcified histological sections of induced bone, taken 35 days after implantation of inductor, were stained by the picrosirius method. This staining enhances the natural birefringency of collagen fibers and allows for better and specific visualization of collagen fibers bundles under polarizing microscope. In this way the amount of information in the analysed image is increased. Thirty five microphotographs were analysed from each of the investigated groups with the use of optical diffractometry. The radial distribution of light intensity in diffraction patterns was analysed what allowed to evaluate spatial frequencies connected with the width of collagen bundles in induced bone tissue. Since the spatial arrangement of collagen fibers in newly formed bone is random, analysis of angular distribution of light intensity in diffractograms was not performed. Using discriminant analysis the significant differences between all three studied groups of animals were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective evaluation of fibrosis in human testicular biopsies by analysis based on optical diffractometry.

Bilateral testicular biopsies were obtained from 27 patients and submitted to two different treatments for each of them: firstly, a classical fixation and colouring, permitting the histopathological diagnosis and subjective appreciation of the degree of fibrosis; secondly, a new objective technique based on optical diffractometry: this method is based on the analysis of light intensity distribution in the diffraction pattern of an original image. Using two different methods of discriminant analysis, we observed some errors due to the subjective examination; in particular, several fibrosed samples had been judged as 'normal' by the histopathologist. Moreover, we showed the existence of some heterogeneity between different slides from the same original biopsy.

Fourier analysis of the cell shape of paired human urothelial cell lines of the same origin but of different grades of transformation.

The rationale of the present investigation is the observations made by many authors of changes in the molecular structure of the cell surface during the multistep process of malignant transformation. These changes may influence cell-matrix and cell-cell interactions and thereby cause changes in cell adhesiveness and cell shape. The aim of the present work was to investigate whether the development of various grades of transformation in vivo and in vitro of human urothelial cells is accompanied by significant changes in cell shape as measured by Fourier analysis. The following transformation grades (TGr) have been defined (Christensen et al. 1984; Kieler 1984): TGr I = nonmalignant, mortal cell lines that grow independently of fibroblasts and have a prolonged life span. TGr II = nonmalignant cell lines with an infinite life span. TGr III = malignant and immortal cell lines that grow invasively in co-cultures with embryonic chick heart fragments and possess tumorigenic properties after s.c. injection into nude mice. Comparisons of 4 pairs of cell lines were performed; each pair was of the same origin. Two pairs--each including a TGr I cell line (Hu 961b and Hu 1703S) compared to a TGr III cell line (Hu 961a or Hu 1703He)--were derived from two transitional cell carcinomas (TCC) containing a heterogeneous cell population. Two additional cell lines classified as TGr II (HCV-29 and Hu 609) were compared to two TGr III sublines (HCV-29T and Hu 609T, respectively) which arose by "spontaneous" transformation during propagation in vitro of the respective maternal TGr II-cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)