Beneficial effects of desferrioxamine on encapsulated human islets--in vitro and in vivo study.

As many as 2000 IEQs (islet equivalent) of encapsulated human islets are required to normalize glucose levels in diabetic mice. To reduce this number, encapsulated islets were exposed to 100 µM desferrioxamine (DFO) prior to transplantation. Cell viability, glucose-induced insulin secretion, VEGF (Vascular endothelial growth factor), HIF-1α (Hypoxia inducible factor-1 alpha), caspase-3 and caspase-8 levels were assessed after exposure to DFO for 12, 24 or 72 h. Subsequently, 1000, 750 or 500 encapsulated IEQs were infused into peritoneal cavity of diabetic mice after 24 h exposure to DFO. Neither viability nor function in vitro was affected by DFO, and levels of caspase-3 and caspase-8 were unchanged. DFO significantly enhanced VEGF secretion by 1.6- and 2.5-fold at 24 and 72 h, respectively, with a concomitant increase in HIF-1α levels. Euglycemia was achieved in 100% mice receiving 1000 preconditioned IEQs, as compared to only 36% receiving unconditioned IEQs (p < 0.001). Similarly, with 750 IEQ, euglycemia was achieved in 50% mice receiving preconditioned islets as compared to 10% receiving unconditioned islets (p = 0.049). Mice receiving preconditioned islets had lower glucose levels than those receiving unconditioned islets. In summary, DFO treatment enhances HIF-1α and VEGF expression in encapsulated human islets and improves their ability to function when transplanted.

Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways.

The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES+/CD133+) and their differentiated (diff) progeny cells (NES-/CD133-). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFß1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53ß in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.

Serotonin decreases HIV-1 replication in primary cultures of human macrophages through 5-HT(1A) receptors.

BACKGROUND AND PURPOSE: 5-HT (serotonin) is known to be involved in neuroinflammation and immunoregulation. The human immunodeficiency virus (HIV) targets cells such as monocytes/macrophages, which colocalize with 5-HT-releasing cell types, mostly platelets. In this study, we investigated the effects of 5-HT on HIV-1-infected macrophages in vitro. EXPERIMENTAL APPROACH: Human macrophages cultured in serum-free medium were treated over 7 days with 5-HT at three concentrations (0.01, 1 and 100 microM) with or without agonists and antagonists of 5-HT(1A) and 5-HT(2) receptors. After 7 days of treatment, macrophages were infected with HIV-1/Ba-L and virus replication was monitored over 16 days and expression of proviral HIV DNA was investigated by PCR after 24 h of infection. Cell surface expression of HIV-1/Ba-L receptor (CD4) and coreceptor (CCR5) was investigated by flow cytometry. The CCR5 ligand, macrophage inflammatory protein-1alpha (MIP-1alpha), was quantified by ELISA in cell culture supernatants and MIP-1alpha mRNA expression was assessed by reverse transcriptase-PCR. KEY RESULTS: In vitro, 5-HT downregulated the membranous expression of CCR5 and led to a decrease of HIV-1 infection, probably through its action on 5-HT(1A) receptors. 5-HT (100 microM) was also able to induce overexpression of MIP-1alpha mRNA leading to an increase of MIP-1alpha secretion by human macrophages. CONCLUSIONS AND IMPLICATIONS: The effects of 5-HT on HIV infection could be a consequence of the increase in MIP-1alpha concentrations and/or CCR5 receptor downregulation. These results suggest that 5-HT can inhibit the replication of HIV-1 in primary culture of human macrophages through its action on 5-HT(1A) receptors.

Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds.

Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z' value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.

Mechanisms of L-Serine Neuroprotection in vitro Include ER Proteostasis Regulation.

ß-N-methylamino-L-alanine (L-BMAA) is a neurotoxic non-protein amino acid produced by cyanobacteria. Recently, chronic dietary exposure to L-BMAA was shown to trigger neuropathology in nonhuman primates consistent with Guamanian ALS/PDC, a paralytic disease that afflicts Chamorro villagers who consume traditional food items contaminated with L-BMAA. However, the addition of the naturally occurring amino acid L-serine to the diet of the nonhuman primates resulted in a significant reduction in ALS/PDC neuropathology. L-serine is a dietary amino acid that plays a crucial role in central nervous system development, neuronal signaling, and synaptic plasticity and has been shown to impart neuroprotection from L-BMAA-induced neurotoxicity both in vitro and in vivo. We have previously shown that L-serine prevents the formation of autofluorescent aggregates and death by apoptosis in human cell lines and primary cells. These effects are likely imparted by L-serine blocking incorporation of L-BMAA into proteins hence preventing proteotoxic stress. However, there are likely other mechanisms for L-serine-mediated neuroprotection. Here, we explore the molecular mechanisms of L-serine neuroprotection using a human unfolded protein response real-time PCR array with genes from the ER stress and UPR pathways, and western blotting. We report that L-serine caused the differential expression of many of the same genes as L-BMAA, even though concentrations of L-serine in the culture medium were ten times lower than that of L-BMAA. We propose that L-serine may be functioning as a small proteostasis regulator, in effect altering the cells to quickly respond to a possible oxidative insult, thus favoring a return to homeostasis.

L-Serine-Mediated Neuroprotection Includes the Upregulation of the ER Stress Chaperone Protein Disulfide Isomerase (PDI).

The unfolded protein response (UPR) is a highly evolutionarily conserved response to endoplasmic reticulum (ER) stress, which functions to return cells to homeostasis or send them into apoptosis, depending on the degree of cellular damage. ß-N-methylamino-L-alanine (L-BMAA) has been shown to induce ER stress in a variety of models and has been linked to several types of neurodegenerative disease including Guamanian amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). L-Serine, an amino acid critical for cellular metabolism and neurological signaling, has been shown to be protective against L-BMAA-induced neurotoxicity in both animal and cell culture models. While the mechanisms of L-BMAA neurotoxicity have been well characterized, less is known about L-serine neuroprotection. We recently reported that L-serine and L-BMAA generate similar differential expression profiles in a human ER stress/UPR array, despite L-serine being neuroprotective and L-BMAA being linked to neurodegenerative disease. Here, we further investigate the mechanism(s) of L-serine-induced UPR dysregulation by examining key genes and proteins in the ER stress/UPR pathways. We report that L-serine selectively increased protein disulfide isomerase (PDI) protein translation, an ER chaperone involved in refolding misfolded proteins, suggesting it may be modulating the UPR to favor recovery from ER stress. This constitutes a new mechanism for L-serine-mediated neuroprotection and has implications for its use as a therapy for neurodegenerative illnesses.

Tissue-engineered bone regeneration.

Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs when the defects were filled with the tissue-engineered bone.

Progesterone Alters Kynurenine Pathway Activation in IFN-γ-Activated Macrophages - Relevance for Neuroinflammatory Diseases.

We have previously demonstrated that the kynurenine pathway (KP), the major biochemical pathway for tryptophan metabolism, is dysregulated in many inflammatory disorders that are often associated with sexual dimorphisms. We aimed to identify a potential functional interaction between the KP and gonadal hormones. We have treated primary human macrophages with progesterone in the presence and absence of inflammatory cytokine interferon-gamma (interferon-γ) that is known to be a potent inducer of regulating the KP enzyme. We found that progesterone attenuates interferon-γ-induced KP activity, decreases the levels of the excitotoxin quinolinic acid, and increases the neuroprotective kynurenic acid levels. We also showed that progesterone was able to reduce the inflammatory marker neopterin. These results may shed light on the gender disparity in response to inflammation.

Kynurenines, Gender and Neuroinflammation; Showcase Schizophrenia.

Schizophrenia has a clear sexual dimorphism in age of onset and progression. The underlying mechanisms of this dimorphism are not known, but may be found in the interactions of sex hormones with the tryptophan catabolising kynurenine pathway. Schizophrenia is associated with general inflammation and disruption of glutamatergic and dopaminergic signalling. Metabolites of the kynurenine pathway have been shown to be immunomodulatory and have effects on glutamatergic and dopaminergic signalling. This review discusses the currently available literature on sex hormones and their effect on the kynurenine pathway in the context of the glutamatergic, dopaminergic and immunological features of schizophrenia.

An enzyme in the kynurenine pathway that governs vulnerability to suicidal behavior by regulating excitotoxicity and neuroinflammation.

Emerging evidence suggests that inflammation has a key role in depression and suicidal behavior. The kynurenine pathway is involved in neuroinflammation and regulates glutamate neurotransmission. In the cerebrospinal fluid (CSF) of suicidal patients, levels of inflammatory cytokines and the kynurenine metabolite quinolinic acid (QUIN), an N-methyl-d-aspartate receptor agonist, are increased. The enzyme amino-ß-carboxymuconate-semialdehyde-decarboxylase (ACMSD) limits QUIN formation by competitive production of the neuroprotective metabolite picolinic acid (PIC). Therefore, decreased ACMSD activity can lead to excess QUIN. We tested the hypothesis that deficient ACMSD activity underlies suicidal behavior. We measured PIC and QUIN in CSF and plasma samples from 137 patients exhibiting suicidal behavior and 71 healthy controls. We used DSM-IV and the Montgomery-Åsberg Depression Rating Scale and Suicide Assessment Scale to assess behavioral changes. Finally, we genotyped ACMSD tag single-nucleotide polymorphisms (SNPs) in 77 of the patients and 150 population-based controls. Suicide attempters had reduced PIC and a decreased PIC/QUIN ratio in both CSF (P<0.001) and blood (P=0.001 and P<0.01, respectively). The reductions of PIC in CSF were sustained over 2 years after the suicide attempt based on repeated measures. The minor C allele of the ACMSD SNP rs2121337 was more prevalent in suicide attempters and associated with increased CSF QUIN. Taken together, our data suggest that increased QUIN levels may result from reduced activity of ACMSD in suicidal subjects. We conclude that measures of kynurenine metabolites can be explored as biomarkers of suicide risk, and that ACMSD is a potential therapeutic target in suicidal behavior.

De novo reconstruction of functional bone by tissue engineering in the metatarsal sheep model.

Large bone defects are still a challenge to orthopedic surgeons. In this study, a massive bone defect with a clinically relevant volume was efficiently reconstructed by transplanting an engineered bone in which mesenchymal stem cells (MSCs) expanded in autologous serum (AS) were combined with a porous scaffold. In the first step, we established that the way in which the MSCs are distributed over the scaffold affects the ultimate bone-forming ability of the transplant: constructs consisting of a natural coral scaffold and a pseudo-periosteal layer of MSCs surrounding the implant (coral-MSC3D) formed significantly more bone than constructs in which the MSCs were distributed throughout the implant (p = 0.01). However, bone healing occurred in only one sheep, owing to the high resorption rate of natural coral scaffold. To overcome this problem, constructs in which MSCs were combined with a porous coralline-based hydroxyapatite (CHA) scaffold having the same architecture as natural coral but a lower resorption rate were prepared. After their implantation, these constructs were found to have the same osteogenic potential as autologous bone grafts in terms of the amount of newly formed bone present at 4 months (p = 0.89) and to have been completely replaced by newly formed, structurally competent bone within 14 months. Nevertheless, although the rate of bone healing was strikingly improved when CHA-MSC3D constructs were used (five of seven animals healed) as compared with the coral-MSC3D construct (one of seven healed), it was still less satisfactory than that obtained with autografts (five of five healed).

Cytotoxic activity of the MK2 inhibitor CMPD1 in glioblastoma cells is independent of MK2.

MAPK-activated protein kinase 2 (MK2) is a checkpoint kinase involved in the DNA damage response. MK2 inhibition enhances the efficacy of chemotherapeutic agents; however, whether MK2 inhibition alone, without concurrent chemotherapy, would attenuate survival of cancer cells has not been investigated. CMPD1 is a widely used non-ATP competitive inhibitor that prevents MK2 phosphorylation. We employed CMPD1 together with MK2 knock-down and ATP-competitive MK2 inhibitor III (MK2i) in a panel of glioblastoma cells to assess whether MK2 inhibition could induce cancer cell death. While CMPD1 was effective at selective killing of cancer cells, MK2i and MK2 knock-down had no effect on viability of glioblastoma cells. CMPD1 treatment induced a significant G2/M arrest but MK2i-treated cells were only minimally arrested at G1 phase. Intriguingly, at doses that were cytotoxic to glioblastoma cells, CMPD1 did not inhibit phosphorylation of MK2 and of its downstream substrate Hsp27. These results suggest that CMPD1 exhibits cytotoxic activity independently of MK2 inhibition. Indeed, we identified tubulin as a primary target of the CMPD1 cytotoxic activity. This study demonstrates how functional and mechanistic studies with appropriate selection of test compounds, combining genetic knock-down and pharmacological inhibition, coordinating timing and dose levels enabled us to uncover the primary target of an MK2 inhibitor commonly used in the research community. Tubulin is emerging as one of the most common non-kinase targets for kinase inhibitors and we propose that potential tubulin-targeting activity should be assessed in preclinical pharmacology studies of all novel kinase inhibitors.

The p38-MK2-HuR pathway potentiates EGFRvIII-IL-1ß-driven IL-6 secretion in glioblastoma cells.

The microenvironment of glioblastoma (GBM) contains high levels of inflammatory cytokine interleukin 6 (IL-6), which contributes to promote tumour progression and invasion. The common epidermal growth factor receptor variant III (EGFRvIII) mutation in GBM is associated with significantly higher levels of IL-6. Furthermore, elevated IL-1ß levels in GBM tumours are also believed to activate GBM cells and enhance IL-6 production. However, the crosstalk between these intrinsic and extrinsic factors within the oncogene-microenvironment of GBM causing overproduction of IL-6 is poorly understood. Here, we show that EGFRvIII potentiates IL-1ß-induced IL-6 secretion from GBM cells. Importantly, exacerbation of IL-6 production is most effectively attenuated in EGFRvIII-expressing GBM cells with inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) and MAPK-activated protein kinase 2 (MK2). Enhanced IL-6 production and increased sensitivity toward pharmacological p38 MAPK and MK2 inhibitors in EGFRvIII-expressing GBM cells is associated with increased MK2-dependent nuclear-cytoplasmic shuttling and accumulation of human antigen R (HuR), an IL-6 mRNA-stabilising protein, in the cytosol. IL-1ß-stimulated activation of the p38 MAPK-MK2-HuR pathway significantly enhances IL-6 mRNA stability in GBM cells carrying EGFRvIII. Further supporting a role for the p38 MAPK-MK2-HuR pathway in the development of inflammatory environment in GBM, activated MK2 is found in more than 50% of investigated GBM tissues and correlates with lower grade and secondary GBMs. Taken together, p38 MAPK-MK2-HuR signalling may enhance the potential of intrinsic (EGFRvIII) and extrinsic (IL-1ß) factors to develop an inflammatory GBM environment. Hence, further improvement of brain-permeable and anti-inflammatory inhibitors targeting p38 MAPK, MK2 and HuR may combat progression of lower grade gliomas into aggressive GBMs.

Chronic exposure of human neurons to quinolinic acid results in neuronal changes consistent with AIDS dementia complex.

OBJECTIVE: Concentrations of quinolinic acid, an N-methyl-D-aspartate agonist, are often elevated for long periods of time in the cerebrospinal fluid (CSF) and brain tissue of patients with AIDS dementia complex (ADC). This study was designed to test the hypothesis that chronic exposure of human neurons to quinolinic acid levels equivalent to those in the CSF of ADC patients is neurotoxic. DESIGN AND METHODS: Human fetal brain 14-16 weeks post-menses was cultured in medium with no detectable levels of quinolinic acid. After 4 weeks, 350 or 1200 nmol/l quinolinic acid was added to the feeding medium for a further 5 weeks. Neurotoxicity was evaluated using immunohistochemistry, transmission and scanning electron microscopy, and image analysis. RESULTS: A total of 1200 nmol/l quinolinic acid caused altered cell associations, a decrease in cell density and decreased microtubule-associated protein (MAP)-2 immunoreactivity compared with cultures exposed to 350 nmol/l quinolinic acid or controls. Image analysis of neurons in randomly selected fields revealed significantly swollen cells (P < 0.0001) compared with those treated with 350 nmol/l quinolinic acid or controls. Dendritic varicosities and discontinuous microtubular arrays were present in neurons exposed to both quinolinic acid concentrations, but not in control cultures. CONCLUSIONS: This study is the first to assess quinolinic acid levels in the experimental medium, and demonstrates that chronic exposure of human neurons to concentrations of quinolinic acid equivalent to those in the CSF of patients with ADC leads to alterations in dendritic ultrastructure and MAP-2 immunoreactivity, which is consistent with ADC pathology.

IFN-beta1b induces kynurenine pathway metabolism in human macrophages: potential implications for multiple sclerosis treatment.

Interferon-beta(1b) (IFN-beta(1b)) has limited efficacy in the treatment of relapsing-remitting multiple sclerosis (RRMS). The kynurenine pathway (KP) is chiefly activated by IFN-gamma and IFN-alpha, leading to the production of a variety of neurotoxins. We sought to determine whether IFN-beta(1b) induces the KP in human monocyte-derived macrophages, as one explanation for its limited efficacy. Serial dilutions of IFN-beta(1b) (at concentrations comparable to those found in the sera of IFN-beta(1b)-treated patients) were added to human macrophage cultures. Supernatants were collected at various time points and assayed for the KP end product, quinolinic acid (QUIN). The effect of IFN-beta(1b) on the KP enzymes indoleamine 2,3-dioxygenase (IDO), 3-hydroxyanthranilate dioxygenase (3HAO), and quinolinate phosphoribosyltransferase (QPRTase) mRNA expression was assessed by semiquantitative RT-PCR. IFN-beta(1b) (> or =10 IU/ml) led to increased mRNA expression of both IDO and QUIN production (7901 +/- 715 nM) after 72 h at 50 IU/ml IFN-beta(1b) (p < 0.0001). This study demonstrates that IFN-beta(1b), in pharmacologically relevant concentrations, induces KP metabolism in human macrophages and may be a limiting factor in its efficacy in the treatment of MS. Inhibitors of the KP may be able to augment the efficacy of IFN-beta in MS.

Effects of chitosan on rat knee cartilages.

The aim of this work is to study the effects of chitosan on rat knee cartilages. 0.2 ml of 0.1% chitosan solution pH = 6.9 were injected inside rat knees articular cavity. One, three and six weeks after injection, histological and histomorphometric studies were performed on undecalcified samples embedded in polymethylmetacrylate. Results show that after 1 and 6 weeks: (i) chitosan slows significantly (P < 0.005) the decrease in epiphyseal cartilage thicknesses and (ii) increases significantly articular cartilage chondrocyte densities (P < 0.002). However chitosan solution induces a proliferation of fibrous tissue with abundant fibroblasts, fibrocytes and monocytes inside the joint and this proliferation is still present after 6 weeks. This study suggests that chitosan could act on the growth of epiphyseal cartilage and wound healing of articular cartilage.

Evaluation of the osteogenic potential of biomaterials implanted in the palatal connective tissue of miniature pigs using undecalcified sections.

Calcium phosphate or calcium carbonate biomaterials are widely used as bone substitutes in periodontal surgery. This study evaluates the osteogenic potential of five different alloplastic biomaterials implanted in the connective tissue of the palatal papilla in miniature pigs. A porous hydroxyapatite (PHA), a dense hydroxyapatite (DHA), a semi-porous hydroxyapatite (SPHA), a tricalcium phosphate (TCP) and a calcium carbonate natural coral (NC) were implanted in a tunnel in the palatal papillae of seven miniature pigs. Undecalcified sections were examined histologically at 1, 2, 3, 4, 8, 12 and 24 wk intervals. Resorbable materials (TCP and NC) were totally resorbed by 24 wk. DHA, PHA and HA showed very limited resorption, although there were multinucleated giant cells in contact with PHA and SPHA. There was no histologically detectable bone formation in contact with or near any of the biomaterials tested. However, several particles of NC, and sometimes of PHA, were surrounded by a dense, mineralized matrix. It is concluded that none of these biomaterials, in their presently available forms, has any bone inducing capacity.

Rapid diagnosis of gram negative urinary infections: identification and antimicrobial susceptibility testing in 24 hours.

A rapid method for diagnosing urinary tract infections, using identification and antimicrobial susceptibility testing that can be carried out in 24 hours, was devised. The method relies on direct inoculation of diluted urine (1/500) in the API 20 E and API ATB systems. Urine was simultaneously cultured on Columbia blood agar and on Drigalski agar to control the purity and for purposes of comparison. The results of this method and those obtained with a conventional method were compared by analysing 1352 urines. The results showed that all of the organisms were correctly identified using the conventional method, and susceptibility testing (rapid method) gave results that agreed with those of the classical method in 94% of cases, with major discrepancies in only 0.08% of cases. The rapid method applies only to monomicrobial infections.

Madreporic coral: a new bone graft substitute for cranial surgery.

Since 1985, the authors have been using madreporic coral fragments (genera Porites) as a bone graft substitute. Of the 167 coral grafts implanted, 150 were coral "corks" used to obliterate burr holes (diameter 10 mm), five were large implants (length 20 to 40 mm) to repair skull defects, and 12 were coral blocks to reconstruct the floor of the anterior cranial fossa. Previous experimental studies suggested that coral grafts would be well tolerated and become partially reossified as the calcific skeleton was resorbed. The authors describe their experience and detail the main biological properties of these materials, which appear to be very promising for use in cranial reconstructive surgery.