Three-dimensional printed polycaprolactone-based scaffolds provide an advantageous environment for osteogenic differentiation of human adipose-derived stem cells.

The capacity of bone grafts to repair critical size defects can be greatly enhanced by the delivery of mesenchymal stem cells (MSCs). Adipose tissue is considered the most effective source of MSCs (ADSCs); however, the efficiency of bone regeneration using undifferentiated ADSCs is low. Therefore, this study proposes scaffolds based on polycaprolactone (PCL), which is widely considered a suitable MSC delivery system, were used as a three-dimensional (3D) culture environment promoting osteogenic differentiation of ADSCs. PCL scaffolds enriched with 5% tricalcium phosphate (TCP) were used. Human ADSCs were cultured in osteogenic medium both on the scaffolds and in 2D culture. Cell viability and osteogenic differentiation were tested at various time points for 42 days. The expression of RUNX2, collagen I, alkaline phosphatase, osteonectin and osteocalcin, measured by real-time polymerase chain reaction was significantly upregulated in 3D culture. Production of osteocalcin, a specific marker of terminally differentiated osteoblasts, was significantly higher in 3D cultures than in 2D cultures, as confirmed by western blot and immunostaining, and accompanied by earlier and enhanced mineralization. Subcutaneous implantation into immunodeficient mice was used for in vivo observations. Immunohistological and micro-computed tomography analysis revealed ADSC survival and activity toward extracellular production after 4 and 12 weeks, although heterotopic osteogenesis was not confirmed - probably resulting from insufficient availability of Ca/P ions. Additionally, TCP did not contribute to the upregulation of differentiation on the scaffolds in culture, and we postulate that the 3D architecture is a critical factor and provides a useful environment for prior-to-implantation osteogenic differentiation of ADSCs. Copyright © 2016 John Wiley & Sons, Ltd.

Osteocyte Lacunae Density in Dentine-Induced Ectopic Bone.

The frequency of osteocytic lacunae, expressed as mean lacunae number per 1000 µm2 of measured bone, evaluated 65 days post intramuscular implantation of demineralized incisors is higher (1.10 ± 0.19) than in femoral (orthotopic) bone (0.91 ± 0.16). The surface of evaluated bones was measured by means of the "weight of bone picture". These results provide new data on the biology of ectopic bone.

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.

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.

[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.

Fluvastatin influences hair color in C57BL/6 mice.

Our recent in vitro experiments suggest that fluvastatin may influence tyrosinase (key enzyme of melanogenesis) synthesis. The aim of the present study was to verify those findings in experiments, in vitro, in melanoma cell line, and in vivo, in mice. The expression of tyrosinase in B16F10 melanoma cell line, after induction of melanogenesis by UVB irradiation, was examined by Western blot analysis. Afterwards, the effect of fluvastatin on melanin synthesis in hair follicles of C57Bl/6 mice was investigated. The expression of tyrosinase was reduced in the presence of fluvastatin. In mice after anagen induction over the dorsal skin, gel containing fluvastatin in various concentrations was injected subcutaneously, while in part of control groups of mice, gel with placebo was injected. In addition, gel with fluvastatin was injected to four week-old mice (mice in first postnatal anagen) without anagen induction. In extension, injections of gel with fluvastatin or placebo were performed in mice without anagen induction (but after first postnatal anagen). In part of study group of mice (mice after anagen induction and injection of fluvastatin) regrowth of depigmented hair was observed, while in all control groups (mice after injection of placebo), such hair depigmentation over the skin area was not found. In conclusion, this study, for the first time, shows that fluvastatin might affect melanin synthesis in vivo.

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.

Skeletal muscle-derived fibroblast-like cells fail to enable HeLa cells to induce bone in the murine kidney.

HeLa cells fail to induce bone in murine kidneys, despite being highly chondro/osteogenic when implanted into thigh muscles. Bone induction in the kidneys failed also when HeLa cells were grafted together with skeletal-muscle-derived cell cultures. It is postulated that kidney parenchyma releases unidentified factcor(s) which prevent activation by inducer of cells termed Friedenstein's (1976) "inducible osteoprogenitor cells", while this hypothetical factor does not prevent further differentiation of"determined osteoprogenitor cells", thus allowing bone to form in the renal parenchyma.

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.

[Regulation of the differentiation of bone marrow mesenchymal stem cells into osteoblastic and adipogenic lineage]. / Regulacja adipocytogenezy i osteoblastogenezy z mezenchymatycznych komórek macierzystych szpiku.

The role of transcription factors and signalling pathways important for differentiation of bone marrow mesenchymal stem cell differentiation toward osteogenic and adipogenic lineage are briefly outlined.

Effects of time of initial exposure to MSV sarcoma on bone induction by dentine matrix implants and on orthotopic femora.

HCl-demineralized murine lower incisors were implanted intramuscularly into syngeneic BALB/c mice to induce heterotopic osteogenesis. Implants were exposed at the early, preosteogenic stage (4), or at the later, osteogenic stage (12) to the Moloney sarcoma virus (MSV), which within 3-4 days results in a sarcoma. The yield of bone induction was determined by weight of dry bone mass following NaOH hydrolysis of soft tissues. To verify the effect of this sarcoma on orthotopic local femoral bone, the dry mass of the tumor-exposed femora was measured and compared with the weight of MSV-unexposed contralateral controls. MSV-sarcoma or cells involved with their spontaneous rejection have a stimulatory effect on the periosteal membrane of the tumor-adjacent femoral bones, increasing their dry mass on average by 18%. No stimulatory effect on heterotopic bone induction was observed when the MSV sarcoma grew during the early, preosteogenic stage (4 onward), but when the tooth matrix had been exposed to such tumor at the already bone-forming stage, (12 onward), the yield of bone induction was enhanced. Thus, it is postulated that lesions induced by MSV during the early, preosteogenic stage inhibit recruitment of osteoprogenitor cells or degrade Bone Morphogenetic Proteins (BMPs) released by matrix resorbing inflammatory cells, whereas when acting on already existing bone they have a stimulatory effect.

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.

Fluvastatin increases tyrosinase synthesis induced by alpha-melanocyte-stimulating hormone in B16F10 melanoma cells.

The aim of this study was to evaluate the effect of fluvastatin on the alpha-melanocyte-stimulating hormone-mediated increase in tyrosinase activity in the melanoma B16F10 cell line and to establish whether Akt and extracellular signal-regulated kinase (Erk) inhibition is involved in tyrosinase synthesis after fluvastatin administration. Fluvastatin modulates alpha-melanocyte-stimulating hormone induced melanogenesis by increasing tyrosinase mRNA production, as shown by real time PCR, or tyrosinase protein synthesis, as presented by western blot technique. The stimulatory effect of fluvastatin on melanogenesis was, in part, induced by modulation of cell proliferation (decreased melanoma cell proliferation in G2/M phase) and possibly decrease of Akt. These findings indicate that fluvastatin increases tyrosinase synthesis induced by alpha-melanocyte-stimulating hormone in B16F10 cells and reveal an unknown effect of statin use: their influence on melanin production.

Aggressive osteoblastoma of the sphenoid bone.

For osteoblastoma, with its predilection for the spinal column and appendicular skeleton, the skull is an unusual site, and paranasal sinus involvement is very rare. Herein, we report on a case in which the disease was located within the sphenoid bone. To the best of our knowledge, this is the 4th reported case of osteoblastoma with a sphenoid origin (1). We report an osteoblastoma of the sphenoid sinus in a 12-year-old girl who presented with exophthalmos. Computed tomography (CT) demonstrated an expansile lesion of the sphenoid which caused the orbital contents to be compressed and deviated to the right. In the magnetic resonance imaging scan, the lesion was found to invade the cranial base in the frontal and temporal region, approximating to the cavernous sinus and internal carotid artery on the right. Bilateral fronto-orbital craniotomy was performed. Histologically, the lesion was composed of proliferating osteoblasts along with vascular stroma. The tumor was described as an aggressive osteoblastoma. In the follow-up CT four months later, a pathological mass was observed in the area of the nasal septum, and a signal void was present on all sequences in the densely sclerotic areas. A second resection was performed. The patient has been disease-free for 61 months. Herein, we present the diagnosis and management of this unusual lesion. The histopathology and the imaging characteristics are shown.

Leptin as a modulator of osteogenesis.

Leptin, a hormone secreted by adipose tissue, but also by several other tissues, has a dual effect on bone, acting by two independent mechanisms. As a signal molecule with growth factor characteristics, leptin is able to stimulate osteoblastic cells and to inhibit osteoclast formation and activity, thus promoting osteogenesis. However, as a molecule which stimulates sympathetic neurons in the hypothalamus, leptin indirectly inhibits bone formation. This inhibitory effect of leptin mediated by activation of sympathetic nervous system can be abrogated by application of blood pressure-reducing beta-blockers, which also inhibit receptors of hypothalamic adrenergic neurons.