Expression of bone morphogenetic protein-2 and -7 in urinary bladder cancer predicts time to tumor recurrence.

INTRODUCTION: Urinary bladder cancer patients who have undergone transurethral resection of bladder tumor (TURBT) are at risk of recurrence. This study aims to correlate the level of bone morphogenetic protein (BMP) expression with urothelial carcinoma invasiveness, TNM stage and time to recurrence after TURBT. MATERIAL AND METHODS: In 33 specimens of healthy transitional epithelium and 42 of urothelial carcinoma, BMP2, BMP4 and BMP7 expression was determined by real-time polymerase chain reaction. Patients who underwent TURBT were followed up for 1 year. RESULTS: BMP2 and BMP7 were downregulated in infiltrating urothelial carcinoma, the relative expression being 0.76 (p = 0.04) and 0.28 (p = 0.025) respectively, while BMP4 was downregulated in non-invasive tumors. High expression of BMP2 and BMP7 correlated with prolonged time to recurrence (log-rank: p = 0.01 and p = 0.03 respectively). CONCLUSIONS: Low expression of BMP2 and BMP7 is associated with shorter time to recurrence. The BMP expression levels are not indicative of tumor stage.

Regulation of Bone Homeostasis by Osteocytes.

New data concerning the function of osteocytes as the central regulators of bone homeostasis are briefly outlined. It is established that osteocytes are the main target cells for parathormone. They are a rich source of sclerostin, the main inhibitor of osteoblast activity, and of the RANKL cytokine, the most important regulator of osteoclastogenesis. Under shear stress causing microinjury, osteocytes enter programmed cell death (apoptosis) and osteocyte apoptosis is a signal for nearby healthy osteocytes to activate osteoclasts to resorb bone.

[The importance of lactoferrin in bone regeneration]. / Znaczenie laktoferryny w regeneracji kosci.

Lactoferrin is an iron-binding protein secreted by mammary gland, thus present in milk and in colostrum, which are a cheap and easy to obtain sources of this protein. Lactoferrin is also present in specific granules of neutrophils. Lactoferrin is a multifunctional agent involved, among others in the immune response and in the regulation of bone metabolism. Lactoferrin actives of osteoblast proliferation and bone matrix secretion, and inhibits apoptosis of osteoblast and osteoclastogenesis. Lactoferrin administered to rodents accelerates bone healing and prevents bone loss induced by ovariectomy. Therefore the use of lactoferrin or milk whey in osteoporosis treatment and prevention is postulated.

Are chondroclasts and osteoclasts identical?

Brief characteristics of cells termed "osteoclasts" and "chondroclasts" are outlined and reasons to consider them as the same cell type, able to resorb calcified matrix, are discussed.

Sclerostin, an osteocytes-derived bone-forming inhibitor.

Sclerostin is a recently identified glycoprotein expressed and synthesized by osteocytes. It is a powerful inhibitor of osteoblasts proliferation and differentiation. Sclerostin inhibits the Wnt signaling, the main trigger of osteoblasts activity. Osteocytes on response to a mechanical loading decrease the synthesis of sclerostin enabling in osteoblasts the Wnt signaling and promote their bone-forming activity. This explains why mechanical loading induces bone formation. Monoclonal antibodies directed against sclerostin reverses sclerostin induced bone catabolic effect and are promising tool in prevention and treatment of osteoporosis in human.

Osteogenic efficiency of demineralized and lyophylized xenogeneic bone and syngeneic dentine implants in mice.

Intramuscular implantation of demineralized and lyophilized rat bone matrix and murine lower incisors into thigh muscles of BALB/c mice results in deposits of bone adjacent to the implants, a phenomenon termed as ectopic osteogenesis. The yield of induced bone does not critically depend on the mass of implanted matrices, and thus on the quantity of bone morphogenetic proteins (BMPs) present in the implants. A positive correlation between bone matrix implant weight and the yield of induced bone was observed only 28 days post grafting, i.e. when endochondreal osteogenesis is completed and bone resorption has not advanced. A more consistent yield of bone induction wasobserved in the case of demineralized tooth implants. It is postulated that chondro/osteoinduction by demineralized, lyophilized matrix implants is not determined by the range of BMPs presumably released in proportion to implant size, but is rather limited by the population of responsive host mesenchymal cells.

Long-term preservation of Bone Morphogenetic Activity in stored demineralized murine incisors.

Demineralized bone or dentine implanted intramuscularly induce endochondral bone formation. This phenomenon, termed "bone induction" is triggered by non-collagenous signal molecules, named "Bone Morphogenetic Proteins" (BMPs), released from bone or dentine. Demineralization of bone/dentine prior their implantation facilitates the release of BMPs from the extracellular matrix allowing to reach a BMP threshold level needed to initiate the process of differentiation of mesenchymal cells towards an osteogenic/chondrogenic lineage. Unprocessed, mineralized tissues usually fail to induce cartilage/bone. Isolated BMPs are commercially available, and in clinical practice are an alternative for demineralized tissues, however, in many cases demineralized bone has advantages over soluble BMPs, as it combines both bone inducing principles and mechanical properties, a feature important for bridging bone fracture and filling bone defects. Demineralized bones are an inexpensive source of bone forming agents for bone-fracture healing or filling bone defects. In this report we demonstrated that storage of lyophilized demineralized murine incisors for 30 months does not deteriorate its osteoinductive potency and colonizing induced bone by bone marrow. Lyophylized incisors, stored for 0-30 months at refrigator were implanted intramuscularly and recovered, together with surrounding tissues at various time intervals ranging 10-450 days. Bone closely associated with implant was observed in about 87% of cases, regardless the storage duration. It is concluded that storage of demineralized and lyophilized incisor matrices for at least 30 months does not change their osteoinductive potency.

The adipocyte component of bone marrow in heterotopic bone induced by demineralized incisor grafts.

The relative proportion of adipocytes to hematopoietic elements in the marrow of heterotopically induced bone evaluated 4-42 weeks post implantation of demineralized murine incisors was estimated by histological analysis of hematoxylin-eosin stained tissue sections. Using computerized image analysis of microphotographs,the proportion of nuclear cells vs. adipocytes was ascertained. The percentage of adipocytes in marrow increases over time. Such an effect, the replacement of myelopoietic marrow by adipogenic (yellow) marrow and the resorption of induced bone, is observed in human osteoporosis. A decline in the non-adipogenic cell compartments of bone marrow accompanying induced bone begins in the fourth week of induction, gradually progresses until the 26th week, and does not change after that. The luminosity, a parameter used in image analysis and proportional to the number of nuclear cells, was 124 ± 3 in hematopoietic femoral bone marrow, and that of bone marrow of the induced bone was of a similar value (117 ± 8) in the fourth week. An evident decline in luminosity of bone marrow filling the foci of heterotopic bone was observed in samples taken at nine weeks (82 ± 20). This process progressed until the 26th week, reaching a luminosity of 70 ± 21. At the 42nd week, the luminosity remained at the same level (71 ± 27). This indicates that the replacement of hematopoietic bone marrow of heterotopically induced bone by unilocular adipocytes begins relatively early (the fourth week) and is persistent.

Adipose mesenchymal stem cells. Their characteristics and potential application in tissue repair.

Mesenchymal stem cells, derived from adipose tissue do not differ substantially from mesenchymal stem cells isolated from bone marrow stroma. They are able to differentiate in differentiating culture medium into various cell type of mesodermal lineage, but also into cells of ectodermal type. Their potency to differentiate toward osteogenic and adipogenic lineage is promising to be a ready source of cells for tissue regeneration.

Haematopoietic and osteogenic bone marrow stem cells.

This article discusses the concept of a common stem cell for bone marrow stromal cells and haematopoietic cells. Until recently it was generally accepted that bone marrow contains two types of stem cells. One is the haemopoietic stem cell; the second one, the mesenchymal stem cell or stromal stem cell, gives rise to the stromal compartment of the marrow. The mesenchymal stem cells can differentiate into osteoblasts, chondroblasts, adipocytes, fibroblasts, and smooth muscle cells. Although the interplay between haemopoietic and stromal cells is well established, no transition of cells from the haemopoietic compartment into cells of the stromal compartment has been demonstrated. Recent data, based on grafting of genetically-marked haemopoietic cells points to the possibility of the generation of adipocytes from haemopoietic stem cells. These findings support the hypothesis postulating a common precursor cell for both bone and bone marrow.There is evidence that osteoblasts can differentiate into adipocytes, and that mesenchymal cells derived from subcutaneous adipose tissue can differentiate into osteogenic cells. The possibility of transdifferentiation of adipocytes into osteoblasts has also been demonstrated.

Peripheral blood and lymphatic organs in BALB/c mice during the progressive and regressive phases of Moloney sarcoma development.

Blood cell counts, differential blood cell counts and weights of the spleen and peripheral lymph nodes draining the area of lesions induced by Moloney sarcoma virus inoculation into the quadriceps shank muscles of inbred BALB/c mice were examined at various stages of tumor development and regression. The blood cell count remained constant through the observation period up to 27 days post tumor development and regression. Differential counts revealed some changes in the cellular composition of the peripheral blood. The most pronounced was an increase of neutrophiles at the stage of tumor development, and their decline with tumour regression. The enlargement of the spleen and of the popliteal lymph nodes draining the tumour site, at the peak of tumor development on day 13 post MSV inoculation, involved at least a doubling of splenic weight, and a much greater weight increase for lymph nodes. This was a long-lasting, although declining event, extending beyond tumor regression.

Heterotopic osteogenesis by murine demineralized incisors at lesions sites induced by concanavalin a in mice.

The aim of this study was to examine the effects of Concanavalin A (Con A) administration on the early (preosteogenic) and late stages of osteogenesis induced by implantation of demineralized murine incisors into syngeneic mice. Local administration of Con A resulted in an increased yield of demineralized incisor-induced bone when injected during the preosteogenic stage of induction. This effect was not observed when Con A was injected after heterotopic osteogenesis had been established. This suggests that Con A recruits osteoprogenitor cells, but does not stimulate differentiated chondroblasts and osteoblasts.

Bioactive composites for bone regeneration. Review.

Beside auto- and allogeneic bone chips used for filling bone lesions or to enhance of bone regeneration, various types of ceramics, natural and synthetized bone mineral hydroxyapatite and tricalcium phosphate, as well as numerous polymers and co-polymers of alfa-hydroxy acids are widely used. These materials in a pure form demonstrate good osteoconductivity, but very rarely are osteoinductive. However, deposition of osteogenic cells on their surface, most frequently derived from bone marrow stroma enables such "constructs" or "composites" to maintain osteogenic differentiation, both in vitro, where expression of several types of osteogenic markers, such as alkaline phosphatase expression, collagen type I synthesis, osteocalcin expression, mineralization is detected, and in vivo, where they form sites of histogenesis of bone and later of bone marrow.

Senescence of osteogenic cells. Review.

In older individuals the regeneration of bone tissue is delayed due to the diminished activity of osteoblasts, while osteogenic potency of human bone marrow stromal cells, also capable of forming bone, does not change with age. The number of osteoprogenitor cells is not reduced in ageing subjects, but their proliferating rate and the activity of their derivatives--osteoblasts, is markedly diminished, since the self-renewing potential of these cells is hindered. The senescence of osteoblasts in ageing people is also accelerated. Changes within the bone marrow microenvironment reduce osteogenic potential of osteoprogenitor cells leading to impaired bone formation seen in senility, while the potency for ectopic osteogenesis does not change with age. This review summarizes documented mechanisms of changes in osteogenic activity of cells in elderly.

Fluvastatin does not elevate periosteal osteogenesis induce by Moloney sarcoma virus (MSV) in mice.

Several studies have demonstrated the pleiotropic effects of statins. Since Wang and associates reported that in rabbits lovastatin reduced steroid-induced bone loss, numerous authors have confirmed these data, however, others have reported conflicting results. In this study, the effects of fluvastatin on bone formation were investigated in early and late phase of osteogenesis. In the first set of experiments (early phase of osteogenesis) CFW/Ll mice were randomly divided into three groups. Two groups were injected with Moloney-murine sarcoma virus (Mo-MSV) into right thighs to induce orthotopic bone formation. Mice in the experimental group received fluvastatin for 11 consecutive days. Thirty days after Mo-MSV inoculation, total serum cholesterol, triglycerides, high- and low-density lipoprotein cholesterol, alkaline phosphatase (AP) were measured and bone mineral increase was calculated. In the second set of experiments (late phase of osteogenesis), fluvastatin was administered from day 11 after Mo-MSV inoculation for 20 consecutive days. Fluvastatin administration in the early phase of osteogenesis made no significant difference in average bone increase compared with mice receiving placebo. Lipid profile and AP were not significantly affected. During late phase of osteogenesis, the average increase in femural dry mass was significantly lower in the group of mice receiving fluvastatin than in the control group. Also, Mo-MSV-initiated tumors disappeared earlier in mice treated with fluvastatin. This may be attributed to the antioncogenic potential of fluvastatin. These results also point out that orthotopic bone formation at the sites of Mo-MSV inoculation in mice seems to be a useful model to examine the pleiotropic effects of statins.

Metaplasia of chondrocytes into osteoblasts.

Hypertrophic chondrocytes, commonly considered as terminal cells responsible for apoptotic elimination in endochondral osteogenesis, have the potential to switch their metabolic role and enter osteoblastic differentiation, based on histochemical, immunohistochemical, biochemical and cytological analysis. During endochondral osteogenesis, some osteocytes are derived from hypertrophic chondrocytes. Also non-hypertrophic chondrocytes are able to transform into osteogenic cells, and the bone thus formed is termed "transchondroid bone". In this review a summary and discussion of reports on chondrocyte transdifferentiation is given.

Osteogenic potential of bone marrow stromal cells.

Bone marrow is the point of origin of several cell types, including stromal cells. Adherent bone marrow stromal cells can differentiate into chondrocytes, adipocytes and osteoblasts. Several substances which modulate the dynamics and differentiation of bone marrow stromal cells have been identified. Recently it has been discovered that those bone marrow cells which are non-adherent in tissue culture for 3 days have osteogenic potential comparable to that of whole bone marrow cells. In the future, implantation of non-adherent bone marrow stromal cells may be of use as an aid in bone fracture healing, similar to whole marrow or adherent stromal cell grafting at present.

Implantation of natural hydroxyapatite from porcine bone into soft tissues in mice.

The natural origin of hydroxyapatite (HA) derived from pig bones (Polish patent No.P-359 960 pending from 5th May 2003) was histologically examined for its biocompatibility following implantation into mouse muscles. The implanted ceramic was encapsulated by well vascularized connective tissue and very slowly resorbed by multinucleated cells. This material did not elicit an immune reaction and adjacent bones were unaffected. This ceramic could be safely used as a filling material alone, or as a composite graft.

The limitation of DEXA analysis for bone mass determination in mice.

An increase in femoral and tibio/fibular bone mass following periosteal membrane stimulation by Moloney sarcoma virus inoculation into thigh muscles of mice was measured in situ on formalin fixed excised hind limbs using a Hologic 4500A Fan Beam X-ray bone densitometer adapted for small bone samples. These results were verified by measurements of constant dry bone mass of the same bones liberated from soft limb tissues by NaOH hydrolysis. There was no consistent data correlation found between the DEXA scan and dry bone mass evaluations. It is concluded that the sensitivity of the DEXA measurement is unsuitable when assessing very small bone samples, weighing merely 20-30 mg.

Influence of osteoprotegerin (OPG) on experimentally induced ectopic bone.

In this paper the effect of osteoprotegerin (OPG) on slowing down the resorption process of heterotopically induced bone tissue is described. The induced ossicle is resorbed ex inactivitate. This system is analogous to osteoporosis in immobilised skeletal bones. Bone induction was achieved in BALB/c mice after injection of a suspension of 3 x l0(6) HeLa cells into thigh muscle of animals immuno-suppressed by a single dose ofhydrocortisone. To slow down the process of induced bone resorption, OPG was administered and the effect was measured quantitatively by weighing the mass of the induced ossicle after hydrolysis of soft tissues surrounding the induced ossicles. As an effect of the application of OPG, dry bone mass of the induced ossicles exceeded 340-540% of the values of the control specimens following 9 applications of0.05 mg OPG per mouse every second day or 14 doses every day.