Effects of Oxygen and Glucose on Bone Marrow Mesenchymal Stem Cell Culture.

This study determines whether the viability of mesenchymal stem cell (MSC) in vitro is most sensitive to oxygen supply, energetic substrate supply, or accumulation of lactate. Mouse unmodified (wild type (WT)) and erythropoietin (EPO) gene-modified MSC is cultured for 7 days in normoxic (21%) and anoxic conditions. WT-MSC is cultured in anoxia for 45 days in high and regular glucose media and both have similar viability when compared to their normoxic controls at 7 days. Protein production of EPO-MSC is unaffected by the absence of oxygen. MSC doubling time and post-anoxic exposure is increased (WT: 32.3-73.3 h; EPO: 27.2-115 h). High glucose leads to a 37% increase in cell viability at 13 days and 17% at 30 days, indicating that MSC anoxic survival is affected by supply of metabolic substrate. However, after 30 days, little difference in viability is found, and at 45 days, complete cell death occurs in both the conditions. This death cannot be attributed to lack of glucose or lactate levels. MSC stemness is retained for both osteogenic and adipogenic differentiations. The absence of oxygen increases the doubling time of MSC but does not affect their viability, protein production, or differentiation capacity.

In vitro ion adsorption and cytocompatibility of dicalcium phosphate ceramics.

BACKGROUND: In vitro cell testing of degradable bioceramics such as brushite or monetite is often challenging due to the ion release into or adsorption from the culture medium. These ionic changes are then mostly responsible for cell proliferation and activity, which prohibits the investigation of effects originating from surface topography or further material modifications. METHODS: Here, we aimed to solve this problem by developing a pre-conditioning regime following the repeated immersion of brushite and monetite samples in various Ca2+, Mg2+ and PO43- containing electrolytes, followed by studying ion adsorption / release as well as changes in phase composition and in vitro cytocompatibility with MG63 cells. RESULTS: The results demonstrated that by using DMEM cell culture medium in a ratio of 10 ml/sample was sufficient to minimize changes of ionic composition after 7 d with a daily change of the medium. This leads to changes of the surface composition with dissolution of the brushite phase. In turn, this also positively influences the in vitro cytocompatibility with a 2-3 fold higher cell number and cell activity on the DMEM pretreated surfaces. CONCLUSIONS: Controlled sample washing prior to cell testing using DMEM medium seems to be a valuable procedure not only to stabilize the pH during cell culture but also to maintain ion concentrations within a cell friendly range.

Intra-tumor delivery of zoledronate mitigates metastasis-induced osteolysis superior to systemic administration.

Bisphosphonates (BPs) have recently been shown to have direct anti-tumor properties. Systemic treatment with BPs can have multiple adverse effects such as osteonecrosis of the jaw and BP induced bone fracturing and spine instability. While benefits of systemic BP treatments may outweigh risks, local treatment with BPs has been explored as an alternate strategy to reduce unwarranted risk. In the present study, we examined whether local delivery of BPs inhibits tumor-induced osteolysis and tumor growth more effectively than systemic treatment in an animal model of tumor-induced bone disease. Following establishment of an intra-tibial model of bone metastases in athymic mice, the experimental group was treated by local administration of zoledronate into the tibial lesion. A comparison of the effect of local versus systemic delivery of zoledronate on the formation of tumor-induced osteolysis was also carried out. A significant increase in mean bone volume/tissue volume % (BV/TV) of the locally treated group (12.30±2.80%) compared to the control group (7.13±1.22%) (P<0.001). Additionally, there was a significant increase in the BV/TV (10.90±1.25%) in the locally treated group compared to the systemically treated group (7.53±0.75%) (P=0.005). These preliminary results suggest that local delivery of BPs outperforms both systemic and control treatments to inhibit tumor-induced osteolysis.

Mimicking oxygen delivery and waste removal functions of blood.

In addition to immunological and wound healing cell and platelet delivery, ion stasis and nutrient supply, blood delivers oxygen to cells and tissues and removes metabolic wastes. For decades researchers have been trying to develop approaches that mimic these two immediately vital functions of blood. Oxygen is crucial for the long-term survival of tissues and cells in vertebrates. Hypoxia (oxygen deficiency) and even at times anoxia (absence of oxygen) can occur during organ preservation, organ and cell transplantation, wound healing, in tumors and engineering of tissues. Different approaches have been developed to deliver oxygen to tissues and cells, including hyperbaric oxygen therapy (HBOT), normobaric hyperoxia therapy (NBOT), using biochemical reactions and electrolysis, employing liquids with high oxygen solubility, administering hemoglobin, myoglobin and red blood cells (RBCs), introducing oxygen-generating agents, using oxygen-carrying microparticles, persufflation, and peritoneal oxygenation. Metabolic waste accumulation is another issue in biological systems when blood flow is insufficient. Metabolic wastes change the microenvironment of cells and tissues, influence the metabolic activities of cells, and ultimately cause cell death. This review examines advances in blood mimicking systems in the field of biomedical engineering in terms of oxygen delivery and metabolic waste removal.

Local delivery of iron chelators reduces in vivo remodeling of a calcium phosphate bone graft substitute.

UNLABELLED: Iron chelators are known activators of the Hypoxia Includible Factor-1α (HIF-1α) pathway, a critical cellular pathway involved in angiogenic responses to hypoxia. Local delivery of these chelators has shown promise in bone tissue engineering strategies by inducing angiogenesis and osteogenesis. Hypoxic microenvironments are also a stimulus for osteoclast differentiation and resorptive activity, a process likely mediated by HIF-1α. In vitro, low doses of the iron chelator Deferoxamine (DFO) has shown to induce HIF-1α mediated osteoclast formation and function. However other studies have proposed an opposite in vitro effect likely through HIF independent mechanisms. To investigate use of these medications in bioceramic based bone tissue engineering strategies this study aimed to determine the in vivo effect of local delivery of iron chelators on bioceramic remodeling. A non-weight bearing cranial onlay model was used to assess monetite resorption and new bone formation in the presence or absence of a repeated delivery of two iron chelators, DFO and 1,10 Phenanthroline (PHT) at doses known to induce HIF. We found a marked reduction graft resorption and remodeling associated with iron chelation. This was correlated to a 3-fold reduction in osteoclast number at the bone graft interface. Iron is needed for mitochondrial biogenesis during osteoclastic differentiation and reducing extracellular iron levels may inhibit this process and possibly overpower any HIF induced osteoclast formation. Our findings suggest that these inexpensive and widely available molecules may be used to locally reduce bioceramic scaffold resorption and encourages future investigations of iron chelators as bone anti-resorptive agents in other clinical contexts. STATEMENT OF SIGNIFICANCE: Low doses of iron chelators can induce angiogenesis and osteogenesis in repairing bone by stimulating the oxygen sensitive gene; hypoxia inducible factor. These medications have potential to augment bioceramic based bone tissue engineering strategies without the downsides of protein-based growth factors. HIF activation is also known to stimulate osteoclast-mediated resorption and could potentially accelerate remodeling of biocermaics, however we have shown that the local delivery of iron chelation at doses known to induce HIF resulted in a reduction of monetite resorption and a significant decrease in osteoclast number at the bone graft interface. This maybe due to HIF independent mechanism. This is the first study to show a local effect of iron chelators in vivo on osteoclast-mediated resorption. This opens the potential of further study of these bifunctional medications to modulate resorption of biocermaics in environments where a prolonged presence of material is desired for graft site stability. Moreover these safe widely used medications can be explored to locally reduce osteoclasts in pathological bone resorption.

Newly identified interfibrillar collagen crosslinking suppresses cell proliferation and remodelling.

Copper is becoming recognised as a key cation in a variety of biological processes. Copper chelation has been studied as a potential anti-angiogenic strategy for arresting tumour growth. Conversely the delivery of copper ions and complexes in vivo can elicit a pro-angiogenic effect. Previously we unexpectedly found that copper-stimulated intraperitoneal angiogenesis was accompanied by collagen deposition. Here, in hard tissue, not only was healing accelerated by copper, but again enhanced deposition of collagen was detected at 2 weeks. Experiments with reconstituted collagen showed that addition of copper ions post-fibrillogenesis rendered plastically-compressed gels resistant to collagenases, enhanced their mechanical properties and increased the denaturation temperature of the protein. Unexpectedly, this apparently interfibrillar crosslinking was not affected by addition of glucose or ascorbic acid, which are required for crosslinking by advanced glycation end products (AGEs). Fibroblasts cultured on copper-crosslinked gels did not proliferate, whereas those cultured with an equivalent quantity of copper on either tissue culture plastic or collagen showed no effect compared with controls. Although non-proliferative, fibroblasts grown on copper-cross-linked collagen could migrate, remained metabolically active for at least 14 days and displayed a 6-fold increase in Mmps 1 and 3 mRNA expression compared with copper-free controls. The ability of copper ions to crosslink collagen fibrils during densification and independently of AGEs or Fenton type reactions is previously unreported. The effect on MMP susceptibility of collagen and the dramatic change in cell behaviour on this crosslinked ECM may contribute to shedding some light on unexplained phenomena as the apparent benefit of copper complexation in fibrotic disorders or the enhanced collagen deposition in response to localised copper delivery.

Elucidating the individual effects of calcium and phosphate ions on hMSCs by using composite materials.

The biological performance of bone graft substitutes based on calcium phosphate bioceramics is dependent on a number of properties including chemical composition, porosity and surface micro- and nanoscale structure. However, in contemporary bioceramics these properties are interlinked, therefore making it difficult to investigate the individual effects of each property on cell behavior. In this study we have attempted to investigate the effects of calcium and inorganic phosphate ions independent from one another by preparing composite materials with polylactic acid (PLA) as a polymeric matrix and calcium carbonate or sodium phosphate salts as fillers. Clinically relevant bone marrow derived human mesenchymal stromal cells (hMSCs) were cultured on these composites and proliferation, osteogenic differentiation and ECM mineralization were investigated with time and were compared to plain PLA control particles. In parallel, cells were also cultured on conventional cell culture plates in media supplemented with calcium or inorganic phosphate to study the effect of these ions independent of the 3D environment created by the particles. Calcium was shown to increase proliferation of cells, whereas both calcium and phosphate positively affected alkaline phosphatase enzyme production. QPCR analysis revealed positive effects of calcium and of inorganic phosphate on the expression of osteogenic markers, in particular bone morphogenetic protein-2 and osteopontin. Higher levels of mineralization were also observed upon exposure to either ion. Effects were similar for cells cultured on composite materials and those cultured in supplemented media, although ion concentrations in the composite cultures were lower. The approach presented here may be a valuable tool for studying the individual effects of a variety of soluble compounds, including bioinorganics, without interference from other material properties.

A new class of bioactive glasses: calcium-magnesium sulfophosphates.

Low-melting ionic sulfophosphate glasses from the system P2O5-SO4-MO-Na2O (M = Zn(2+), Ca(2+) or Mg(2+)) have been previously shown by us to allow tuneable aqueous dissolution and also enable processing temperatures well below 400°C. Sulfate ions are extremely safe for use in the body as decades of use of calcium sulfate bone grafts testifies and there is no known limit on their adult oral toxicity. This glass system therefore offers great potential for use as biomaterials, especially in organic-inorganic hybrid systems such as glass-polymer composites for tissue engineering or drug encapsulation and delivery applications. A compositional region was identified where stable sulfophosphates of the type P2O5-SO4-(Ca, Mg, Zn)O-Na2O can be fabricated. For these glasses, the viscosity-temperature-dependence, glass transformation temperatures (Tg ) and the onset of crystallization were evaluated as the primary processing parameters. As a first step in exploring their potential as a biomaterial, in this study we examine the bioactivity of several compositions of these glasses using fibroblast, monocyte, and osteoclast cell culture models to determine cellular responses in terms of attachment, proliferation, differentiation, and toxicity.

The effect of amorphous pyrophosphate on calcium phosphate cement resorption and bone generation.

Pyrophosphate ions are both inhibitors of HA formation and substrates for phosphatase enzymes. Unlike polyphosphates their hydrolysis results simultaneously in the complete loss of mineral formation inhibition and a localised elevation in orthophosphate ion concentration. Despite recent advances in our knowledge of the role of the pyrophosphate ion, very little is known about the effects of pyrophosphate on bone formation and even less is known about its local delivery. In this work we first developed a self setting pyrophosphate based calcium cement system with appropriate handling properties and then compared its in vivo degradation properties with those of a non-pyrophosphate containing control. Contrary to expectation, the presence of the pyrophosphate phase in the cement matrix did not inhibit mineralisation of the healing bone around the implant, but actually appeared to stimulate it. In vitro evidence suggested that enzymatic action accelerated dissolution of the inorganic pyrophosphate ions, causing a simultaneous loss of their mineralisation inhibition and a localised rise in supersaturation with respect to HA. This is thought to be a rare example of a biologically responsive inorganic material and these materials seem to be worthy of further investigation. Bioceramics to date have mainly been limited to orthophosphate, silicate and carbonate salts of calcium, here we report the successful application of a pyrophosphate material as a degradable osteoconductive bone repair cement.

Silk fibroin derived polypeptide-induced biomineralization of collagen.

Silk fibroin (SF) is extensively investigated in osteoregenerative therapy as it combines extraordinary mechanical properties and directs calcium-phosphate formation. However, the role of the peptidic fractions in inducing the protein mineralization has not been previously decoded. In this study, we investigated the mineralization of fibroin-derived polypeptides (FDPs), which were obtained through the chymotryptic separation of the hydrophobic crystalline (Cp) fractions and of the hydrophilic electronegative amorphous (Cs) fractions. When immersed in simulated body fluid (SBF), only Cs fragments demonstrated the formation of carbonated apatite, providing experimental evidence that the mineralization of SF is dictated exclusively by its electronegative amino-acidic sequences. The potential of Cs to conceptually mimic the role of anionic non-collagenous proteins in biomineralization processes was investigated via their incorporation (up to 10% by weight) in bulk osteoid-like dense collagen (DC) gels. Within 6 h in SBF, apatite was formed in DC-Cs hybrid gels, and by day 7, carbonated hydroxylapatite crystals were extensively formed. This accelerated 3-D mineralization resulted in a nine-fold increase in the compressive modulus of the hydrogel. The tailoring of the mineralization and mechanical properties of hydrogels through hybridization with FDPs could potentially have a significant impact on cell delivery and bone regenerative medicine.

Silver-doped calcium phosphate cements with antimicrobial activity.

There is a current need for the localised delivery of antibiotics in order to treat implant-based bacterial infections. Existing treatments use non-resorbable materials such as poly(methyl methacrylate) beads loaded with antibiotics; unfortunately, as they are not resorbable, these beads require secondary surgery for removal. Calcium phosphate cements have considerable potential for the localised delivery of drugs since they can be resorbed to some extent within the body, eliminating the need for a secondary surgical procedure. Therefore, in this study, the efficacy of both hydroxyapatite and brushite cements in the delivery of silver ions has been investigated. The activity of the Ag(+) released from the cements was assessed against the growth of both Staphylococcus aureus and Staphylococcus epidermidis; the brushite cement exhibited excellent antibacterial properties and also showed an increase in compressive strength of over 30%. In this study we have found that with a few changes in Ag(+) concentration it should be possible to produce a fully resorbable bone replacement material that is combined with an antibacterial scaffold with controlled release over a period of time, which is likely to inhibit bacterial infections associated with implantation procedures.

Mesenchymal stem cell-seeded multilayered dense collagen-silk fibroin hybrid for tissue engineering applications.

Tissue engineering of multilayered constructs that model complex tissues poses a significant challenge for regenerative medicine. In this study, a three-layered scaffold consisting of an electrospun silk fibroin (SF) mat sandwiched between two dense collagen (DC) layers was designed and characterized. It was hypothesized that the SF layer would endow the DC-SF-DC construct with enhanced mechanical properties (e.g., apparent modulus, tensile strength, and toughness), while the surrounding DC layers provide an extracellular matrix-like environment for mesenchymal stem cell (MSC) growth. MSC-seeded DC-SF-DC hybrids were produced using the plastic compression technique and characterized morphologically, chemically, and mechanically. Moreover, MSC viability was assessed for up to 1 wk in culture. Scaffold analyses confirmed compaction and integration of the meso-scaled multilayered DC-SF-DC hybrid, which was reflected in a significantly higher toughness value when compared to DC and SF alone. MSCs directly incorporated into the DC layers remained viable for up to day 7. The ease of multilayered construct fabrication, enhanced biomechanical properties, along with uniformity of cell distribution confirmed the possibility for the incorporation and segregation of different cell types within distinct layers for the regeneration of complex tissues, such as skin, or central nervous system dura mater.

Sustained steroid release in pulmonary inflammation model.

There is a need for particles which exhibit controlled release of therapeutic agents delivered via the inhalational route, for tissue specific applications such as anti-cancer, bronchodilators and antiviral agents as well as drugs for systemic action. The aim of this study was to assess the acute toxicity, distribution and capacity of the microspheres to exhibit controlled release properties in an in vivo model of airway inflammation. Calcium pyrophosphate nanofibrous microspheres were loaded with dexamethasone phosphate (Dex-P); the profile of drug release was studied in vitro and validated in vivo. Unloaded microspheres were administered intra-tracheally (i.t.) to rats to assess the tissue reaction. The anti-inflammatory properties of the Dex-P loaded microspheres against an inflammatory agent (compound 48/80), were evaluated in vivo. Unloaded microspheres did not cause an inflammatory response when given at doses below 3mg, and appeared to be eliminated through mucus clearance mechanisms. Microspheres loaded with Dex-P but not Dex-P alone, were capable of inhibiting eosinophil and total inflammatory cell increases in bronchoalveolar lavage fluid for 42 h following a single application. These observations demonstrated that calcium pyrophosphate nanofibrous microspheres displayed in vivo controlled release properties, were well tolerated and did not accumulate in the lung.

Ascorbic acid accelerates osteoclast formation and death.

Ascorbic acid (AA) plays a key role in bone formation. However controversy remains about the effect of AA on cells responsible for bone destruction, osteoclasts. We investigated the effect of AA on osteoclastogenesis using primary mouse bone marrow cultures and monocytic RAW 264.7 cells treated with osteoclastogenic factors RANKL and MCSF. Treatment with AA resulted in significant increase in osteoclast number, size and nucleation. To assess osteoclast oxidative stress level, a ratio of reduced (GSH) to oxidized (GSSG) glutathione and the total glutathione content (GSH(t)) were evaluated. Osteoclast differentiation was associated with a decrease in GSH/GSSG and GSH(t). AA induced further decrease in both parameters, and resulted in significant production of H(2)O(2), indicating its pro-oxidant action. At low concentration, H(2)O(2) induced similar effects to AA, although less potently, and catalase partially inhibited AA-induced osteoclastogenesis. To assess the modification in osteoclast metabolism, the mitochondrial activity was evaluated using JC-1 and the ATP levels were assessed. Osteoclast formation was associated with the increase in mitochondrial activity and ATP concentration, which were further increased in the presence of AA. Importantly, the stimulatory effect of AA was only evident at early phase of osteoclastogenesis, whereas at the late stage AA significantly accelerated osteoclast death. Thus, during osteoclastogenesis AA acts as an oxidant, first stimulating osteoclast formation, but later limiting osteoclast lifespan. This duality of AA action allows reconciling the stimulatory action of AA on osteoclastogenesis observed in vitro with an overall attenuation of bone resorption in the presence of AA observed in vivo.

Craniofacial vertical bone augmentation: a comparison between 3D printed monolithic monetite blocks and autologous onlay grafts in the rabbit.

Onlay autografting is amongst the most predictable techniques for craniofacial vertical bone augmentation, however, complications related to donor site surgery are common and synthetic alternatives to onlay autografts are desirable. Recent studies have shown that the acidic calcium phosphates, brushite and monetite, are osteoconductive, osteoinductive and resorb faster in vivo than hydroxyapatite. Moreover, they can be 3D printed allowing precise host bone-implant conformation. The objectives of this study were to confirm that craniofacial screw fixation of 3D printed monetite blocks was possible and to compare the resulting vertical bone augmentation with autograft. 3D printed monolithic monetite onlay implants were fixed with osteosynthesis screws on the calvarial bone surface of New Zealand rabbits. After 8 weeks, integration between the implant and the calvarial bone surface was observed in all cases. Histomorphometry revealed that 42% of the monetite was resorbed and that the new bone formed within the implant occupied 43% of its volume, sufficient for immediate dental implant placement. Bone tissue within the autologous onlay occupied 60% of the volume. We observed that patterns of regeneration within the implants differed throughout the material and propose that this was due to the anatomy and blood supply pattern in the region. Rapid prototyped monetite being resorbable osteoconductive and osteoinductive would appear to be a promising biomaterial for many bone regeneration strategies.

Minimally invasive maxillofacial vertical bone augmentation using brushite based cements.

An ideal material for maxillofacial vertical bone augmentation procedures should not only be osteoconductive, biocompatible and mechanically strong, but should also be applied using minimally invasive procedures and remain stable with respect to the original bone surfaces. This way, implant exposure and infection might be reduced and good mechanical stability may be achieved. Calcium phosphate cements are proven biocompatible and osteoconductive materials that can be injected using minimally invasive procedures. Among these cements, brushite based cements have the added advantage of being biodegradable in vivo. Therefore, this material has the potential for use in the aforementioned procedures. An in vivo study was performed in rabbits to evaluate the potential use of brushite cements in minimally invasive maxillofacial vertical bone augmentation procedures. In this study, we injected self-setting brushite cements on the subperiosteal bone surface using a minimally invasive tunnelling technique. The cement pastes were stable on the bone surface and hardened soon after they were injected thereby negating the need for additional supports such as membranes or meshes. The animals were sacrificed 8 weeks after the intervention and histological observations revealed signs of successful vertical bone augmentation. Therefore, we have demonstrated a minimally invasive vertical bone augmentation procedure that is an attractive alternative to current surgical procedures in terms of increased simplicity, reduced trauma, and lower cost of surgery.

Bioactivity of bone resorptive factor loaded on osteoconductive matrices: stability post-dehydration.

Since calcium phosphate cements were proposed two decades ago, extensive research has been realized to develop and improve their properties. They have proved their efficiency as bone graft substitutes and their ability to incorporate and release drugs. However, to date, all 'resorbable' osteoconductive synthetic biomaterials are in fact simply soluble. In order to investigate a synthetic material capable of inducing osteoclast remodelling post-implantation, a formulation of calcium phosphate cement loaded with a pro-resorptive cytokine (RANKL) was studied. Many prior release studies on calcium phosphates did not confirm that the matrix had no detrimental effect on the molecule to be released during storage prior to use or that bioactivity was maintained during storage. In this report, the stability of our protein was tested after loading onto the cement, and various regimens to improve stability were compared. The presence of trehalose was shown to stabilize the bioactivity of RANKL adsorbed to brushite cement. The reduction of both moisture and oxygen in the storage vessel improved osteoclastogenic potential of the matrix compared with that stored in ambient atmosphere and temperature. No loss in activity was observed over the study period for the loaded matrix stored in dry nitrogen.

Osteopontin functions as an opsonin and facilitates phagocytosis by macrophages of hydroxyapatite-coated microspheres: implications for bone wound healing.

Osteopontin (OPN) is a secreted protein abundant in mineralized tissue extracellular matrices and bodily fluids. Previously we have shown that mineralized debris at surgical wound sites in bone and teeth are coated by macrophage-derived OPN and phagocytosed. Here, we have performed opsonophagocytosis assays to determine whether OPN acts as an opsonin and facilitates phagocytosis by macrophages of protein- and hydroxyapatite mineral-coated microspheres. Moreover, we have examined the opsonization effects of monomer OPN versus OPN polymerized (crosslinked) by tissue transglutaminase 2. Murine macrophages J774A.1 were exposed to polystyrene-latex microspheres having different surface chemistries (non-ionic, aldehyde amidine, carboxyl and aliphatic amine) which were coated with either serum albumin, immunoglobulin, monomer OPN or polymer OPN. Similar experiments with the same protein coatings were performed using hydroxyapatite-covered microspheres. Internalization of microspheres by phagocytosis into macrophages was confirmed by co-localization with the (phago)lysosomal markers lysosome-associated membrane protein-1 (Lamp-1) and LysoTracker, and by light microscopy and transmission electron microscopy after serial sectioning of plastic/resin-embedded cells containing microspheres. OPN significantly increased phagocytosis of both microspheres and hydroxyapatite-covered microspheres compared to negative controls (albumin-coated and uncoated microspheres), with phagocytic indices similar to, or greater than, those of the positive control (IgG-coated). The effect of OPN and hydroxyapatite on microsphere phagocytosis was synergistic. Polymer OPN further enhanced the phagocytosis of aliphatic amine and aldehyde amidine microspheres. Taken together, these results indicate that OPN is an effective opsonin able to facilitate particle uptake (including mineralized particles) by macrophages.

The use of RANKL-coated brushite cement to stimulate bone remodelling.

Calcium phosphate cements were first proposed as synthetic bone substitutes over two decades ago, however, they are characterised by slow chemical or cellular resorption and a slow osteointegration. In contrast, bone autograft has been shown to stimulate osteoclastogenesis and angiogenesis resulting in active bone remodelling and rapid graft incorporation. Therefore, we aimed to develop a biomaterial able to release a key stimulator of the bone remodelling process, cytokine RANKL. Cylinders of brushite cement, hydroxyapatite cement and sodium alginate were loaded with RANKL either by incorporation into the cement or by coating the material with soluble RANKL. To test the biological activity of these formulations, we assessed their effectiveness in inducing osteoclast formation from RAW 264.7 monocytic cell line. Only brushite and hydroxyapatite cements coated with RANKL allowed for retaining sufficient biological activity to induce osteoclast formation. Most efficient was coating 40 mg cylinder of brushite cement with 800 ng RANKL. We have found that RANKL-coated brushite cement exhibits osteoclastogenic activity for at least 1 month at 37 degrees C. Thus, we developed a formulation of brushite cement with RANKL - a synthetic bone graft that is similar to autografts in its ability to actively induce osteoclastogenesis.

Influence of calcium phosphate crystal assemblies on the proliferation and osteogenic gene expression of rat bone marrow stromal cells.

Calcium phosphates (CaPs) have been investigated as substrates to promote bone formation both in vitro and in vivo. The aim of this study was to examine the proliferation and differentiation of rat bone marrow stromal cells (BMSCs) cultured on three-dimensional (3D) octacalcium phosphate (OCP) crystal assemblies. The cytotoxicity of OCP crystal assemblies was evaluated by measuring the lactate dehydrogenase (LDH) release from BMSCs during 10h of incubation with OCP crystal assemblies. The proliferation of BMSCs on OCP crystal assemblies in medium with or without osteogenic supplements was also investigated using the MTT assay with tissue culture treated plastic (TP) as the control. The tissues formed by BMSCs cultured on OCP crystal assemblies for 24 days were examined following staining with haematoxylin and eosin (H&E), alkaline phosphatase (ALP) and Van Gieson's techniques. The influence of OCP crystal assemblies on mRNA expression of alpha chain of collagen type I (Coll-Ia), ALP and osteocalcin (OC), osteonectin (ON), osteopontin (OP), lumican, Cbfa1, EST317 and EST350 by the BMSCs were also investigated using semi-quantitative RT-PCR. Although OCP crystals were relatively cytotoxic compared with TP, proliferation of BMSCs occurred when seeded onto OCP crystal assemblies. BMSCs cultured on OCP demonstrated similar proliferation rates as found on the control and no significant difference (P<0.05) in the number of cells cultured in medium supplemented with or without osteogenic additives on TP and OCP. The deposition of collagen and ALP were detected in tissue synthesised by BMSCs cultured on OCP crystals assemblies. OCP crystal assemblies down-regulated basal bone ECM proteins, including Coll-Ia, ON and lumican, in the first week of culture, whilst up-regulation of the same genes was observed after 24 days of culture. The observed down-regulation of Cbfa1 on OCP substrates was consistent with the negative effect of OCP crystal assemblies on the genes encoding bone ECM proteins. The up-regulation of OC mRNA expression by OCP crystal assemblies could be related to the requirement for synthesis of more OC proteins to control the concentration of calcium ions in culture medium.