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PubMed2005

[The influence of mesenchymal stem cells on bone tissue regeneration upon implantation of demineralized bone matrix].

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC are able to differentiate into chondroblasts, adipocytes, neurons, glia, cardiomyocytes, or osteoblasts. The problem of MSC usage in cell therapy of bone defects is widely discussed at present. The experiments were carried out using rats of inbred line Wistar-Kyoto. MSC were isolated from bone marrow and cultivated in vitro. Demineralized bone matrices (DBM) were obtained from parietal bones of rats and hens. Part of DBM was loaded with MSC. Bone defects were made in cranium parietal regions. DBM with or without MSC or metal plates were transplanted in these regions. It was shown that the application of MSC increased angiogenesis and osteogenesis in the damaged bone. The implantation of rat's DBM with MSC led to the formation of a full value bone. MSC suppressed inflammation, when transplantation of hen's DBM was carried out. The application of MSC always improved bone tissue regeneration.

Animals↗
PubMed1998

The role of alpha2 beta1 and alpha3 beta1 integrin receptors in the initial anchoring of MDA-MB-231 human breast cancer cells to cortical bone matrix.

Molecular mechanisms involved in the rapid attachment of the human breast cancer cell line MDA-MD-231 to cortical bone matrix were studied. The attachment of MDA-MD-231 cells to cortical bone disks could be blocked by 75% when cells were pretreated with a monoclonal antibody to the beta1-subunit of the integrin family. Monoclonal antibodies against the alpha2, alpha3, and alpha5 integrin subunits inhibited the attachment by 76, 26, and 8 % respectively. Collagenase type I and collagen type I antibody blocked the cell attachment by 45 and 50 % whereas pretreatment of the cells with soluble collagen type I blocked the attachment by 85 %. Our study with a panel of cancer cell lines further showed a close correlation between alpha2 beta1 and alpha3 beta1 integrin receptor expression and the capability to attach to cortical bone. These receptors appear to be the key receptors utilized by cancer cells for the initial attachment to cortical bone, and this could facilitate the localization of alpha2 beta1 and alpha3 beta1 expressing cancer cells to the skeleton.

Animals↗
PubMed1990

Attainment of hermetic seal with decalcified allogenic bone matrix grafts--surgical vs. conservative approach.

Twenty four non vital upper anterior teeth from twelve healthy patients, between the ages of 18-30 years, having definite radiolucent areas at the periapex, were taken for this study. A comparison was made between the surgical and conservative approach, using decalcified allogenic bone matrix (DABM) grafts to achieve a physiologic seal at the root apex. The period of observation were 60 days, 120 days and 180 days. The clinical and radiological finding of this study strongly support the observation that DABM accelerates osteogenesis and cementogenesis which seal the root apex through a physiological process. The process of healing was quicker when the surgical approach was used. However a longer period of observation and histological study is required before any conclusion can be drawn, as regard the choice of a surgical or conservative approach.

Adult↗
PubMed1999

Mineralization processes in demineralized bone matrix grafts in human maxillary sinus floor elevations.

For reconstruction of the severely resorbed lateral maxilla for dental implant placement, one of the successful procedures is to elevate the maxillary sinus floor by implanting demineralized bone matrix (DBM). We studied bone formation in DBM grafts in the lateral maxilla in humans by means of histology and histomorphometry. Six months after grafting, at the time of dental implantation biopsies were taken from the grafted areas of seven patients. All biopsies contained mineralized matrix (MM) in the grafted area. At close inspection, three types of mineralization were found. First, lamellar biomineralization was seen in and near the maxillary host bone. Second, remineralization was observed in some particles that probably had not been completely demineralized. In the area connecting the graft and host bone, where woven bone was formed against DBM particles, a third mechanism was detected. In this case many dotlike foci of remineralization appeared close to the bone-DBM interface. The remineralized DBM and woven bone were both subsequently remodeled. Bone formation was most active in the area adjoining the maxillary host bone. We conclude that in human sinus floor elevation, allogenic DBM increases mineralized tissue volume by osteoconduction that is supported by the remineralization processes. Osteoinduction by this material seems questionable.

Adult↗
PubMed1995

Increased expression of osteonectin and osteopontin, two bone matrix proteins, in human breast cancer.

Microcalcifications are a common phenomenon associated with breast cancer and are often the only mammographic sign of a malignant breast disease. Although microcalcifications are not restricted to breast cancer and can be also associated with benign lesions, it is noteworthy that they are composed exclusively of hydroxyapatite in breast carcinoma. Hydroxyapatite is the bone-associated phosphocalcic crystal the deposition of which in bone tissue requires the coordinated expression of several molecules such as osteonectin (OSN) and osteopontin (OPN), synthesized by cells of the osteoblastic lineage. In this study, we evaluated the expression of these two bone matrix proteins, using an immunoperoxidase technique and specific antibodies, in 79 breast lesions including 28 benign and 51 cancerous specimens. We found that normal mammary tissue associated with the lesions examined expressed generally undetectable or lightly detectable (0 or 1+) amounts of OSN and OPN (92 and 81%, respectively). Benign breast lesions, including fibroadenoma and fibrocystic dysplasia, were generally weakly stained (0 or 1+) with both anti-OSN and anti-OPN antibodies (96.4 and 60.7%, respectively). Interestingly, the majority of both in situ and invasive breast carcinoma lesions showed a strong expression (2+ or 3+) for OSN or OPN (74.5 and 84.3%, respectively). High expression of these two bone matrix proteins was associated with frequent microcalcification deposition in the lesion. This study is the first extensive study of OSN and OPN expression in mammary cancers. Our data suggest that OSN and OPN could play a role in the formation of ectopic microcalcifications often associated with breast cancer. It is also tempting to speculate that the expression of these two glycoproteins by breast cancer cells play a role in the preferred bone homing of breast metastases.

Amino Acid Sequence↗
PubMed2002

Natural variation in the extent of phosphorylation of bone phosphoproteins as a function of in vivo new bone formation induced by demineralized bone matrix in soft tissue and bony environments.

Implants of allogenic demineralized bone matrix were placed in distinct in vivo environments, i.e. calvarial (bony) and subcutaneous (soft tissue) sites. Detailed analyses of the biochemical components were performed. Quantitative levels of osteopontin (OPN), bone sialoprotein (BSP) and calcium phosphate (Ca-P) deposition within each implant environment varied as a function of new bone formation, and were substantially different in samples from calvarial and subcutaneous sites. Quantification of the extent of phosphorylation of affinity-purified OPN and BSP from such implants indicated that: (i) the number of mols of phosphoserine (P-Ser)/mol of affinity-purified OPN or BSP varied as a function of implant time and bone formation within both implant sites, and (ii) the 'effective P-Ser concentration' provided by the total OPN and BSP within each implant site varied and increased as a function of time, being approx. 5-fold higher for BSP in calvarial compared with subcutaneous implants. Peak levels of mols of P-Ser/mol of BSP coincided with maximum rates of Ca-P deposition in calvarial implants. Levels of OPN phosphorylation from both calvarial and subcutaneous implants also indicated fluctuations as a function of bone formation. Hence the present study, for the first time, provides direct evidence of natural variation in the extent of phosphorylation of both OPN and BSP as a function of time of mineralized tissue formation. Further evaluation of the data provides the first evidence of a direct and linear relationship between the rate of Ca-P deposition and the ratio of P-Ser-BSP/P-Ser-OPN for calvarial implants. Data for subcutaneous implants failed to provide such correlation. Overall, the present work demonstrates that the natural biological progression of the process of biomineralization follows strict criteria consistent with the anatomical location. Biomineralization fails to proceed in the same way in a soft tissue environment.

Animals↗
PubMed1983

Histological investigation of bone induction by demineralized allogeneic bone matrix: a natural biomaterial for osseous reconstruction.

The objective of the study was to delineate the histological sequence of events associated with bone formation induced in extraosseous sites by implants of demineralized allogeneic bone matrix (DABM) in order to provide a more complete basis for the clinical use of the natural biomaterial for osseous reconstruction. The rat was the experimental animal model. Implants were prepared by demineralization of diaphyseal cortical rat bone in 0.6N HCl for 18-24 h. A total of 104 specimens, 4 mm square, were implanted both subcutaneously and intramuscularly in 26 rats for periods of 3 days to 22 weeks. Histological evaluation revealed a sequence of events that at 3 days displayed an inflammatory response to surgical trauma and after 6 weeks exhibited the formation of a mature lamellar ossicle with central marrow which was maintained through 22 weeks. Resorption of the DABM, first evident at 1 week, was accompanied by the appearance of chondroblast in vascular canals and in crevices in the implant. Osteoblastic activity, initially seen at 2 weeks, appeared to be associated with capillary and blood vessel formation around the implant and within enlarged resorptive cavities within the specimens. Calcification occurred in the cartilaginous matrix and osteoid and within the implanted matrix. The osteoinductive process comprises (1) the inflammation/granulation/scar formation processes elicited by implantation of a material, (2) the resorptive response elicited by DABM, and (3) the process of induction of nonosseous mesenchymal cells to differentiate to chondroblasts and osteoblasts. The results of the present study support previous investigations demonstrating the bone-inducing property of DABM and provide further evidence for the potential value of this natural biomaterial for use in osseous reconstruction.

Animals↗
PubMed1986

Preliminary studies of the secondary structure in solution of two phosphoproteins of chicken bone matrix by circular dichroism and fourier transform-infrared spectroscopy.

The secondary structures of two phosphoproteins from chicken bone matrix of Mr approximately 15kDa and approximately 28kDa, rich in Asx, Glx, and Ser, and containing Ser(P) and Thr(P) residues, have been investigated in solution by Circular Dichroism (CD) and Fourier Transform-Infrared Spectroscopy (FT-IR). CD spectroscopy, which yields useful information on the backbone conformation of polypeptides and proteins, suggests a predominantly beta-sheet structure for the two phosphoproteins. The FT-IR spectra of the approximately 15kDa protein, which is sensitive to secondary structure and hence provides complimentary information to CD spectroscopy, are consistent with the results obtained by CD studies.

Amino Acids↗
PubMedDate not supplied

[Changes in the osteoinductive activity of the bone matrix in ontogeny].

Correlation between the animal's age, degree of mineralization of its bone tissue and the osteoinductive activity of the bone organic matrix was established in experiments with rats of the same litter at an age of 2, 4, 8, 16 weeks. The osteoinductive activity was estimated by the capacity of matrix to induce ectopic osteogenesis using biochemical methods. Bone mineralization increased and the capacity for osteoinduction decreased roughly 1,5-fold with the age of donor animals. It is suggested that the reduction of osteoinductive potencies is based on the decrease in lability of the bond between the protein osteoinducer and the collagen matrix, as shown by unequal sensitivity of bone matrix of rats of different age to the doubling of the shortest possible time of bone demineralization.

Aging↗
PubMed1998

Effect of allogenic freeze-dried demineralized bone matrix on guided tissue regeneration in dogs.

This randomized, split-mouth study was designed to evaluate the adjunctive effect of allogenic, freeze-dried, demineralized bone matrix (DBM) to guided tissue regeneration (GTR). Contralateral fenestration defects (6 x 4 mm) were created 6 mm apical to the buccal alveolar crest on maxillary canine teeth in 6 beagle dogs. DBM was implanted into one randomly selected fenestration defect. Expanded polytetrafluoroethylene (ePTFE) membranes were used to provide bilateral GTR. Tissue blocks including defects with overlying membranes and soft tissues were harvested following a four-week healing interval and prepared for histometric analysis. Differences between GTR+DBM and GTR defects were evaluated using a paired t-test (N = 6). DBM was discernible in all GTR+DBM defects with limited, if any, evidence of bone metabolic activity. Rather, the DBM particles appeared solidified within a dense connective tissue matrix, often in close contact to the instrumented root. There were no statistically significant differences between the GTR+DBM versus the GTR condition for any histometric parameter examined. Fenestration defect height averaged 3.7+/-0.3 and 3.9+/-0.3 mm, total bone regeneration 0.8+/-0.6 and 1.5+/-0.8 mm, and total cementum regeneration 2.0+/-1.3 and 1.6+/-1.7 mm for GTR+DBM and GTR defects, respectively. The histologic and histometric observations, in concert, suggest that allogenic freeze-dried DBM has no adjunctive effect to GTR in periodontal fenestration defects over a four-week healing interval. The critical findings were 1) the DBM particles remained, embedded in dense connective tissue without evidence of bone metabolic activity; and 2) limited and similar amounts of bone and cementum regeneration were observed for both the GTR+DBM and GTR defects.

Alveolar Bone Loss↗
PubMed1994

Effect of glass bioactivity on new bone development induced by demineralized bone matrix in a rat extraskeletal site.

The effects of two kinds of bioactive glass and two kinds of phosphate-free glass on new bone development induced by demineralized bone matrix (DBM) were studied in the rat abdominal muscle pouch model. After 8 weeks' implantation histomorphometric analysis revealed that the amount of new bone in DBM combined with bioactive glass was comparable to DBM without bioactive glass. DBM grafts combined with phosphate-free glass showed significantly less new bone formation. Scanning electron microscopic examination confirmed that new bone bonded to the surface of bioactive glass. The release of ions from the glass seemed to slow down after new bone had bonded to it. Exclusion of phosphate from a bioactive glass resulted in loss of ability to develop the Ca,P-rich surface layer needed for bone bonding.

Animals↗
PubMed1993

Role of bone matrix in osteoclast recruitment in cultured fetal rat calvariae.

In cultured 19 day fetal rat calvaria, osteoclasts first appear after 48 h, more rapidly than with other cultured embryonic long bone rudiments. This may be because the calvarial osteoclast precursors are more differentiated or intramembraneous bone is a more powerful stimulus for osteoclast maturation than endochondral bone. To investigate this further, 19 day calvariae were stripped of their endocranial membranes, devoiding them of osteoclast precursors, and cocultured with the membranes or with other sources of these cells, such as bone marrow, fetal liver, spleen, and blood. There was similar recruitment of mature osteoclasts onto the surface of the "stripped" calvariae from the endocranial membranes and from the hematopoietic tissues after 48 h culture. Intact 19 day fetal calvariae were cultured with human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) or with 1,25-dihydroxyvitamin D3, [1,25-(OH)2D3], each thought to influence different stages of osteoclast maturation. They stimulated osteoclast recruitment, although 1,25-(OH)2D3 was effective only in the first 24 h of culture. They also increased osteoclast recruitment from fetal liver onto stripped calvariae. When intact bones were cultured with hrGM-CSF and 1,25-(OH)2D3 together, osteoclast number decreased but their area increased. Calvariae therefore appear to contain osteoclast precursors at earlier (GM-CSF-sensitive) and later [1,25-(OH)2D3-sensitive] stages. As recruitment onto stripped calvariae was similar whichever source of precursors was used, it is likely that calvarial bone matrix is an important influence on rapid osteoclast maturation in these bones in vitro.

Animals↗
PubMed1989

An electron microscopical study on the presence of proteoglycans in the calcified bone matrix by use of cuprolinic blue.

With Cuprolinic Blue (CBl) as contrasting agent, PGs could be demonstrated in mouse fetal bone matrix. Large CBl-positive rod-like structures proved to be present in and outside the calcification nodules in regions of beginning mineralization. In further developed bone also smaller rods were present in the mineralized matrix. The CBl-positive rods were sensitive to chondroitinase ABC and hyaluronidase. Under the circumstances we chose, this indicates that these structures are PGs containing chondroitin and/or dermatan sulphate. The fine filamentous and granular material in the nodules was still present after digestion with these enzymes, but disappeared after treatment with pronase. This is an indication that this material mainly contains proteins.

Animals↗
PubMed2003

Histologic evaluation of human biopsies after dental augmentation with a demineralized bone matrix putty.

To solve some of the problems inherent in bone regeneration, various types of graft materials, matrix, putty, and gel delivery systems have been developed. These deliver demineralized bone matrix (DBM) to a graft site and maintain it in an appropriate position to achieve favorable results. This prospective study reviewed 10 cases of extraction immediate grafting with a putty-DBM delivery system. Five patients were male and 5 female. At intervals ranging from 4 to 21 months postextraction grafting, bone cores were harvested at the time of surgical insertion of Replace endosteal two-stage implants. All patients were restored with single-tooth self-standing prostheses. Bone quality and quantity in each of the bone cores were evaluated. All 10 cases were completed with favorable outcomes.

Adult↗
PubMed2006

Osteoinductivity of demineralized bone matrix in immunocompromised mice and rats is decreased by ovariectomy and restored by estrogen replacement.

The osteoinduction potential of human demineralized bone matrix (DBM) in females with low estrogen (E2) is unknown. Moreover, the osteoinductivity of commercial human DBM is tested in male athymic rats and mice, but DBM performance in these animals may not reflect performance in female animals or provide information on E2's role in the process. To gain insight, human DBM was implanted bilaterally in the gastrocnemius of twenty-four athymic female mice (10 mg/implant) and twenty-four athymic female rats (15 mg/implant). Eight animals in each group were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized with E2-replacement (OVX+E2) via subcutaneous slow release capsules of 17beta-estradiol. OVX and OVX+E2 animals were pair-fed to SHAM animals. Four animals from each group were euthanized at 35 days and four at 56 days. Animal weight, uterine weight, and blood estrogen levels confirmed that pair feeding, ovariectomy, and E2 replacement were successful. Histological sections of implanted tissues were evaluated qualitatively for absence or presence of DBM, ossicle formation, and new bone or cartilage using a previously developed qualitative scoring system (QS) and by histomorphometry to obtain a quantitative assessment of osteoinduction. OVX mice had a small but significant QS decrease at 35 days compared to SHAM mice, confirmed by quantitative measurement of ossicle, marrow space, and new bone areas. The QS in rats was not affected by OVX but histomorphometry showed decreased new bone in OVX rats, which was restored by E2. The QS indicated that the number of new bone sites was not reduced by OVX in rats or mice at 56 days, but the relative amount of new bone v. marrow space was affected and differed with animal species. Residual DBM was less in OVX animals, indicating that DBM resorption was affected. Cartilage was present in rats but not in mice, suggesting that endochondral ossification was slower and indicating that bone graft studies in these species are not necessarily comparable. These results show the importance of E2 in human DBM-induced bone formation and suggest that E2 may be needed for clinical effectiveness in post-menopausal women.

Animals↗
PubMed2000

The expression of bone matrix protein mRNAs around beta-TCP particles implanted into bone.

Tissue response around beta-tricalcium phosphate (beta-TCP) particles (150-300 microm in diameter) implanted into rat tibiae was analyzed by in situ hybridization with digoxigenin-labeled procollagen alpha1(I) (COL), osteonectin, osteocalcin, and osteopontin (OPN) RNA probes. Specimens were collected at 3, 5, 7, and 10 days after the operation. Holes without implantation were used as control. In both the beta-TCP implanted and control groups, new bone was formed centripetally and all four kinds of mRNA were expressed in activated osteoblasts. A COL signal was expressed most strongly and widely, and was detected at the peripheral region of the hole at day 3. The other three mRNAs were also expressed in bone forming osteoblasts by day 7. However, in the earlier cell reaction stage, OPN expression in the beta-TCP implanted group was different than that in the control group: OPN mRNA was seen exclusively in the cells on the particles, and an OPN signal was detected not only in COL-positive cells, but also in COL-negative cells. The former cells may be osteoblasts and reflect the early process of bone formation on biomaterials. The latter cells may be macrophages and reflect foreign body reactions. Expression of these OPN mRNAs induced by implantation of beta-TCP may play a role in bone formation on the materials and in determining their biocompatibility.

Animals↗
PubMed2000

Augmenting local bone with Grafton demineralized bone matrix for posterolateral lumbar spine fusion: avoiding second site autologous bone harvest.

Mineralization and integrity of the bone graft mass were evaluated among patients having posterolateral fusion. Grafting consisted of a composite of Grafton and "local" autologous bone (n=56) or iliac crest autograft alone (n=52). Mineralization was rated radiographically at baseline and at 3, 6, 12, and 24 months. Integrity was judged as fused or not fused. Mineralization ratings did not differ significantly between groups at any postoperative interval (P values of .25-1.00). The percentage of patients fused was similar in both groups (60% and 56% for Grafton and controls, respectively; P=.83). Fifteen control patients reported donor site pain. These findings warrant further evaluation of this composite.

Adult↗