(223)Ra and other bone-targeting radiopharmaceuticals-the translation of radiation biology into clinical practice

Osseous metastases are a source of significant morbidity for patients with a variety of cancers. Radiotherapy is well established as an effective means of palliating symptoms associated with such metastases. The role of external beam radiotherapy is limited where sites of metastases are numerous and widespread. Low linear energy transfer (LET) radionuclides have been utilized to allow targeted delivery of radiotherapy to disparate sites of disease, with evidence of palliative benefit. More recently, the bone targeting, high LET radionuclide (223)Ra has been shown to not only have a palliative effect but also a survival prolonging effect in metastatic, castration-resistant prostate cancer with bone metastases. This article reviews the different radionuclide-based approaches for targeting bone metastases, with an emphasis on (223)Ra, and key elements of the underlying radiobiology of these that will impact their clinical effectiveness. Consideration is given to the remaining unknowns of both the basic radiobiological and applied clinical effects of (223)Ra as targets for future research.

Studies on hemopoietic chimerism following allogeneic bone marrow transplantation in the molecular biology era

Donor hematopoiesis or donor chimerism in the host following allogeneic bone marrow transplantation (BMT) has appeared crucial to the engraftment process. However, as molecular techniques exploiting neutral variation in human genetic material have been used in the study of chimerism, the detection of residual host cells or mixed hemopoietic chimerism has indicated that donor chimerism is not obligatory following BMT. This review focuses on the detection and significance of mixed chimerism (MC) in patients transplanted for both malignant and non-malignant hemopoietic disease and attempts to tease out the contribution of MC to engraftment, leukemia relapse, graft rejection and long-term disease-free survival.

Biology of Bone Metastases in Prostate Cancer

Advanced-stage prostate cancer (PCa) patients are often diagnosed with bone metastases. Bone metastases remain incurable and therapies are palliative. PCa cells prevalently cause osteoblastic lesions, characterized by an excess of bone formation. The prevailing concept indicates that PCa cancer cell secrete an excess of paracrine factors stimulating osteoblasts directly or indirectly, thereby leading to an excess of bone formation. The exact mechanisms by which bone formation stimulates PCa cell growth are mostly elusive. In this review, the mechanisms of PCa cancer cell osteotropism, the cancer cell-induced response within the bone marrow/bone stroma, and therapeutic stromal targets will be summarized.

Implication of 3D culture system for study of prostate cancer (CaP) mediated bone metastasis

Introduction : For the past decade, three dimensional (3D) culture has served as a foundation for regenerative medicine study. With an increasing awareness of the importance of cell-cell and cell-extracellular matrix interactions which are lacking in 2D culture system, 3D culture system has been employed for many other applications namely cancer research. Through development of various biomaterials and utilization of tissue engineering technology, many in vivo physiological responses are now better understood. The cellular and molecular communication of cancer cells and their microenvironment, for instance can be studied in vitro in 3D culture system without relying on animal models alone. Predilection of prostate cancer (CaP) to bone remains obscure due to the complexity of the mechanisms and lack of proper model for the studies. In this study, we aim to investigate the interaction between CaP cells and osteoblasts simulating the natural bone metastasis. We also further investigate the invasiveness of CaP cells and response of androgen sensitve CaP cells, LNCaP to synthetic androgen.----- Method : Human osteoblast (hOB) scaffolds were prepared by seeding hOB on medical grade polycaprolactone-tricalcium phosphate (mPLC-TCP) scaffolds and induced to produce bone matrix. CaP cell lines namely wild type PC3 (PC3-N), overexpressed prostate specific antigen PC3 (PC3k3s5) and LNCaP were seeded on hOB scaffolds as co-cultures. Morphology of cells was examined by Phalloidin-DAPI and SEM imaging. Gelatin zymography was performed on the 48 hours conditioned media (CM) from co-cultures to determine matrix metalloproteinase (MMP) activity. Gene expression of hOB/LNCaP co-cultures which were treated for 48 hours with 1nM synthetic androgen R1881 were analysed by quantitative real time PCR (qRT-PCR).----- Results : Co-culture of PCC/hOB revealed that the morphology of PCCs on the tissue engineered bone matrix varied from homogenous to heterogenous clusters. Enzymatically inactive pro-MMP2 was detected in CM from hOBs and PCCs cultured on scaffolds. Elevation in MMP9 activity was found only in hOB/PC3N co-culture. hOB/LNCaP co-culture showed increase in expression of key enzymes associated with steroid production which also corresponded to an increase in prostate specific antigen (PSA) and MMP9.----- Conclusions : Upregulation of MMP9 indicates involvement of ECM degradation during cancer invasion and bone metastases. Expression of enzymes involved in CaP progression, PSA, which is not expressed in osteoblasts, demonstrates that crosstalk between PCCs and osteoblasts may play a part in the aggressiveness of CaP. The presence of steroidogenic enzymes, particularly, RDH5, in osteoblasts and stimulated expression in co-culture, may indicate osteoblast production of potent androgens, fuelling cancer cell proliferation. Based on these results, this practical 3D culture system may provide greater understanding into CaP mediated bone metastasis. This allows the role of the CaP/hOB interaction with regards to invasive property and steroidogenesis to be further explored.

Cryoreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell-–biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate–fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell–biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreser-vation of “ready-to-use” cell–biomaterial constructs.

Runx2 overexpression in bone marrow stromal cells accelerates bone formation in critically-sized femoral defects

The repair of large non-unions in long bones remains a significant clinical problem due to high failure rates and limited tissue availability for auto- and allografts. Many cell-based strategies for healing bone defects deliver bone marrow stromal cells to the defect site to take advantage of the inherent osteogenic capacity of this cell type. However, many factors, including donor age and ex vivo expansion of the cells, cause bone marrow stromal cells to lose their differentiation ability. To overcome these limitations, we have genetically engineered bone marrow stromal cells to constitutively overexpress the osteoblast specific transcription factor Runx2. In the present study, we examined Runx2-modified bone marrow stromal cells, delivered via poly(caprolactone) scaffolds loaded with type I collagen meshes, in critically-sized segmental defects in rats compared to unmodified cells, cell-free scaffolds and empty defects. Runx2 expression in bone marrow stromal cells accelerated healing of critically-sized defects compared to unmodified bone marrow stromal cells and defects receiving cell-free treatments. These findings provide an accelerated method for healing large bone defects which may reduce recovery time and the need for external fixation of critically-sized defects.

Osteoarthritic cartilage chondrocytes alter subchondral bone osteoblast differentiation via MAPK signalling pathway involving ERK1/2

Osteoarthritic subchondral bone is characterized by abnormal bone density and enhanced production of bone turnover markers, an indication of osteoblast dysfunction. Several studies have proposed that pathological changes in articular cartilage influence the subchondral bone changes, which are typical of the progression of osteoarthritis; however, direct evidence of this has yet to be reported. The aim of the present study was to investigate what effects articular cartilage cells, isolated from normal and osteoarthritic joints, may have on the subchondral bone osteoblast phenotype, and also the potential involvement of the mitogen activated protein kinase (MAPK) signalling pathway during this process. Our results suggest that chondrocytes isolated from a normal joint inhibited osteoblast differentiation, whereas chondrocytes isolated from an osteoarthritic joint enhanced osteoblast differentiation, both via a direct and indirect cell interaction mechanisms. Furthermore, the interaction of subchondral bone osteoblasts with osteoarthritic chondrocyte conditioned media appeared to significantly activate ERK1/2 phosphorylation. On the other hand, conditioned media from normal articular chondrocytes did not affect ERK1/2 phosphorylation. Inhibition of the MAPK–ERK1/2 pathways reversed the phenotype changes of subchondral bone osteoblast, which would otherwise be induced by the conditioned media from osteoarthritic chondrocytes. In conclusion, our findings provide evidence that osteoarthritic chondrocytes affect subchondral bone osteoblast metabolism via an ERK1/2 dependent pathway.

Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

Repair of calvarial defects with customized tissue-engineered bone grafts - I. Evaluation of osteogenesis in a three-dimensional culture system

Bone generation by autogenous cell transplantation in combination with a biodegradable scaffold is one of the most promising techniques being developed in craniofacial surgery. The objective of this combined in vitro and in vivo study was to evaluate the morphology and osteogenic differentiation of bone marrow derived mesenchymal progenitor cells and calvarial osteoblasts in a two-dimensional (2-D) and three-dimensional (3-D) culture environment (Part I of this study) and their potential in combination with a biodegradable scaffold to reconstruct critical-size calvarial defects in an autologous animal model [Part II of this study; see Schantz, J.T., et al. Tissue Eng. 2003;9(Suppl. 1):S-127-S-139; this issue]. New Zealand White rabbits were used to isolate osteoblasts from calvarial bone chips and bone marrow stromal cells from iliac crest bone marrow aspirates. Multilineage differentiation potential was evaluated in a 2-D culture setting. After amplification, the cells were seeded within a fibrin matrix into a 3-D polycaprolactone (PCL) scaffold system. The constructs were cultured for up to 3 weeks in vitro and assayed for cell attachment and proliferation using phase-contrast light, confocal laser, and scanning electron microscopy and the MTS cell metabolic assay. Osteogenic differentiation was analyzed by determining the expression of alkaline phosphatase (ALP) and osteocalcin. The bone marrow-derived progenitor cells demonstrated the potential to be induced to the osteogenic, adipogenic, and chondrogenic pathways. In a 3-D environment, cell-seeded PCL scaffolds evaluated by confocal laser microscopy revealed continuous cell proliferation and homogeneous cell distribution within the PCL scaffolds. On osteogenic induction mesenchymal progenitor cells (12 U/L) produce significantly higher (p < 0.05) ALP activity than do osteoblasts (2 U/L); however, no significant differences were found in osteocalcin expression. In conclusion, this study showed that the combination of a mechanically stable synthetic framework (PCL scaffolds) and a biomimetic hydrogel (fibrin glue) provides a potential matrix for bone tissue-engineering applications. Comparison of osteogenic differentiation between the two mesenchymal cell sources revealed a similar pattern.

Clonal characterization of bone marrow derived stem cells and their application for bone regeneration

Tissue engineering allows the design of functionally active cells within supportive bio-scaffolds to promote the development of new tissues such as cartilage and bone for the restoration of pathologically altered tissues. However, all bone tissue engineering applications are limited by a shortage of stem cells. The adult bone marrow stroma contains a subset of nonhematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). BMSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate single- cell-derived colonies. These cells have the capacity to undergo extensive replication in an undifferentiated state ex vivo. In addition, BMSCs have the potential to develop either in vitro or in vivo into distinct mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Thus, BMSCs are an attractive cell source for tissue engineering approaches. However, BMSCs are not homo- geneous and the quantity of stem cells decreases in the bone marrow in aged population. A sequential loss of lineage differentiation potential has been found in the mixed culture of bone marrow stromal cells due to a heterogenous popu- lation. Therefore, a number of studies have proposed that homogenous bone marrow stem cells can be generated from clonal culture of bone marrow cells and that BMSC clones have the greatest potential for the application of bone regeneration in vivo