Pediatric Mandibular Defect Rehabilitation by Human Transforming Growth Factor-ß3 with an Implant-Supported Prosthesis.

Large mandibular defects in children are an uncommon but challenging problem for surgeons to solve. The time-honored options of autologous bone grafts are seldom a viable option, as suitable donor sites are unavailable. Osteoinductive morphogens may yet provide a solution in these cases. A large mandibular tumor in a child 10 years of age necessitated the resection of the entire dentate portion of the mandible. The defect was reconstructed at a second stage with a composite graft of human transforming growth factor-ß3 (hTGF-ß3), human demineralized bone matrix, and 12 g of autologous bone harvested from the posterior iliac crest. A mature ossicle suitable for the placement of osseointegrated implants developed in the erstwhile defect, and an implant-supported dental prosthesis was placed. The patient has been followed up into adulthood. Facial growth has proceeded unhindered, and the patient has maintained full oral and dental function. This case reports the long-term result of an uncommon condition treated with a novel method. The longterm follow-up of this patient provides evidence to dispel some of the concerns for the use of osteoinductive proteins in children. A composite graft of osteogenic morphogens, osteocompetent autologous cells, and mineralized and demineralized matrices-as opposed to osteogenic morphogens used solo-may improve clinical bone regeneration.

Osteogenic Competence and Potency of the Bone Induction Principle: Inductive Substrates That Initiate "Bone: Formation by Autoinduction".

ABSTRACT: The de novo induction of bone has always been a fascinating phenomenon, keeping skeletal reconstructionists and cellular developmental biologists continuously engaged to finally provide a molecular and cellular approach to the induction of bone formation. A significant advancement was made by the purification and cloning of the human recombinant bone morphogenetic proteins, members of the transforming growth factor-ß supergene family. Human bone morphogenetic proteins are powerful inducers of bone in animal models including nonhuman primates. Translation in clinical contexts has however, proven to be surprisingly difficult. This review also describes the significant induction of bone formation by the human transforming growth factor-ß3 when implanted in heterotopic intramuscular sites of the Chacma baboon Papio ursinus. Large mandibular defects implanted with 250 mg human transforming growth factor-ß3 in human patients showed significant osteoinduction; however, the induction of bone was comparatively less than the induction of bone in P ursinus once again highlighting the conundrum of human osteoinduction: is the bone induction principle failing clinical translation?

Mortality after cardioverter-defibrillator replacement: Results of the DECODE survival score index.

BACKGROUND: Device replacement is the ideal time to reassess health care goals regarding continuing implantable cardioverter-defibrillator (ICD) therapy. Only few data are available on the decision making at this time. OBJECTIVES: The goals of this study were to identify factors associated with poor prognosis at the time of ICD replacement and to develop a prognostic index able to stratify those patients at risk of dying early. METHODS: DEtect long-term COmplications after implantable cardioverter-DEfibrillator replacement (DECODE) was a prospective, single-arm, multicenter cohort study aimed at estimating long-term complications in a large population of patients who underwent ICD/cardiac resynchronization therapy - defibrillator replacement. Potential predictors of death were investigated, and all these factors were gathered into a survival score index (SUSCI). RESULTS: We included 983 consecutive patients (median age 71 years (63-78)); 750 (76%) were men, 537 (55%) had ischemic cardiomyopathy; 460 (47%) were implanted with cardiac resynchronization therapy - defibrillator. During a median follow-up period of 761 days (interquartile range 628-904 days), 114 patients (12%) died. In multivariate Cox regression analysis, New York Heart Association class III/IV, ischemic cardiomyopathy, body mass index < 26 kg/m2, insulin administration, age ≥ 75 years, history of atrial fibrillation, and hospitalization within 30 days before ICD replacement remained associated with death. The survival score index showed a good discriminatory power with a hazard ratio of 2.6 (95% confidence interval 2.2-3.1; P < .0001). The risk of death increased according to the severity of the risk profile ranging from 0% (low risk) to 47% (high risk). CONCLUSION: A simple score that includes a limited set of variables appears to be predictive of total mortality in an unselected real-world population undergoing ICD replacement. Evaluation of the patient's profile may assist in predicting vulnerability and should prompt individualized options, especially for high-risk patients.

Long Term Follow-Up of Pediatric Mandibular Reconstruction With Human Transforming Growth Factor-ß3.

Translating bone regeneration induced by recombinant human bone morphogenetic proteins from animal models to human patients has proven inexplicably inconsistent. This prompted us to test in 5 pediatric patients, an alternative osteoinductive morphogen, recombinant human transforming growth factor ß3 (hTGF-ß3), to reconstruct mandibular defects of such a size to preclude reconstruction with autologous bone. An osteoinductive implant of human demineralized bone matrix (DBM) loaded with 125 µg hTGF-ß3 per gram of DBM was implanted into one defect, and 250 µg hTGF-ß3 per gram of DBM in another. Thereafter in 3 patients limited amounts of particulate cortico-cancellous bone graft harvested from the posterior iliac crest were combined with 250 µg hTGF-ß3 per gram of DBM. Patients were followed up for 3 to 6 years. Three patients achieved clinically significant osteoinduction, 1 patient with hTGF-ß3 only, and 2 by combining hTGF-ß3 with a small supplement of autologous bone. One patient with hTGF-ß3 only and followed up for 5 years retains a viable reconstruction but has had sub-optimal bone regeneration. One patient had osteoinductive failure due to sepsis although the plate reconstruction remains viable. Recombinant human TGF-ß3 initiates osteoinduction in humans and potentiates autologous bone graft activity allowing the reconstruction of large mandibular defects in pediatric patients.

Donor CYP3A5 genotype influences tacrolimus disposition on the first day after paediatric liver transplantation.

AIM: The aim of the present study was to investigate the influence of the cytochrome P450 (CYP) 3A4/5 genotype in paediatric liver transplant recipients and donors, and the contribution of age and gender to tacrolimus disposition on the first day after transplantation. METHODS: The contribution of the CYP3A4/5 genotype in paediatric liver transplant recipients and donors to the tacrolimus blood trough concentrations (C0 ) and the tacrolimus concentration/weight-adjusted dose ratio on day 1 was evaluated in 67 liver-transplanted children: 33 boys and 34 girls, mean age 4.5 years. RESULTS: Donor CYP3A5 genotype appears to be significantly associated with tacrolimus disposition on the first day after liver transplantation (P < 0.0002). Other physiological factors, such as recipient age and donor gender may also play a role and lead to significant differences in tacrolimus C0 and tacrolimus concentration/weight-adjusted dose ratio on day 1. However, according to the general linear model, only recipient age appears to be independently associated with tacrolimus disposition on the first day after liver transplantation (P < 0.03). Indeed, there was a faster tacrolimus metabolism in children under 6 years of age (P < 0.02). CONCLUSIONS: Donor CYP3A5 genotype, recipient age and, to a lesser extent, donor gender appear to be associated with tacrolimus disposition on day 1 after transplant. This suggests that increasing the starting tacrolimus doses in paediatric patients under 6 years of age who receive a graft from a male extensive metabolizer may enhance the possibility of their tacrolimus levels reaching the therapeutic range sooner.

GSH-targeted nanosponges increase doxorubicin-induced toxicity "in vitro" and "in vivo" in cancer cells with high antioxidant defenses.

Several reports indicate that chemo-resistant cancer cells become highly adapted to intrinsic oxidative stress by up-regulating their antioxidant systems, which causes an increase of intracellular GSH content. Doxorubicin is one of the most widely used drugs for tumor treatment, able to kill cancer cells through several mechanisms. However, doxorubicin use is limited by its toxicity and cancer resistance. Therefore, new therapeutic strategies able to reduce doses and to overcome chemo-resistance are needed. A new class of glutathione-responsive cyclodextrin nanosponges (GSH-NS), is able to release anticancer drugs preferentially in cells having high GSH content. Doxorubicin-loaded GSH-NS, in the cancer cells with high GSH content, inhibited clonogenic growth, cell viability, topoisomerase II activity and induced DNA damage with higher effectiveness than free drug. Moreover, GSH-NS reduced the development of human tumor in xenograft models more than free drug. These characteristics indicate that GSH-NS can be a suitable drug delivery carrier for future applications in cancer therapy.

Mitochondrial Dysfunction in Cancer and Neurodegenerative Diseases: Spotlight on Fatty Acid Oxidation and Lipoperoxidation Products.

In several human diseases, such as cancer and neurodegenerative diseases, the levels of reactive oxygen species (ROS), produced mainly by mitochondrial oxidative phosphorylation, is increased. In cancer cells, the increase of ROS production has been associated with mtDNA mutations that, in turn, seem to be functional in the alterations of the bioenergetics and the biosynthetic state of cancer cells. Moreover, ROS overproduction can enhance the peroxidation of fatty acids in mitochondrial membranes. In particular, the peroxidation of mitochondrial phospholipid cardiolipin leads to the formation of reactive aldehydes, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), which are able to react with proteins and DNA. Covalent modifications of mitochondrial proteins by the products of lipid peroxidation (LPO) in the course of oxidative cell stress are involved in the mitochondrial dysfunctions observed in cancer and neurodegenerative diseases. Such modifications appear to affect negatively mitochondrial integrity and function, in particular energy metabolism, adenosine triphosphate (ATP) production, antioxidant defenses and stress responses. In neurodegenerative diseases, indirect confirmation for the pathogenetic relevance of LPO-dependent modifications of mitochondrial proteins comes from the disease phenotypes associated with their genetic alterations.

Reconstruction of 56 mandibular defects with autologous compressed particulate corticocancellous bone grafts.

We retrospectively evaluated the results of particulate corticocancellous bone grafting of mandibular defects. Patients with deficits of mandibular continuity as a result of injuries or resection of disease had the affected segment debrided or resected, followed by placement of a patient-specific reconstruction plate. Eight weeks after resection, it was reconstructed with an autotransplant from the posterior iliac crest. Grafts were deemed successful if the regenerated ossicle (after 6 months' maturation) was adequate to take an osseointegrated fixture at least 10mm long. Fifty-six patients were treated, of whom 5 were lost to follow-up. The remaining 51 patients were followed up for a mean (SD) of 29 (18) months. The mean (SD) length of the defect was 12.4 (8.4) cm. Of the 51 reconstructions, 43 healed uneventfully and the grafts were deemed successful. Two healed grafts developed recurrent tumour, which required resection of the entire reconstructed area in one, and partial resection in the other. Three patients lost the complete graft from sepsis, and five developed sepsis that required debridement with partial loss of the graft. Two patients in the latter group required a second graft. One patient required an augmentation graft, as the ossicle was not sufficient to take an implant. The technique of staged grafting with particulate corticocancellous bone after moulding of the recipient site with a spacer produces unmatched restitution of mandibular anatomy with low morbidity.

Improved Anti-Tumoral Therapeutic Efficacy of 4-Hydroxynonenal Incorporated in Novel Lipid Nanocapsules in 2D and 3D Models.

4-hydroxynonenal (HNE), a lipid peroxidation product, is a promising anti-neoplastic drug due to its remarkable anti-cancer activities. However, this possibility has not been explored, because the delivery of HNE is very challenging as a result of its low solubility and its poor stability. This study intentionally designed a new type of lipid nanocapsules specifically for HNE delivery. They consist of a medium chain triglyceride liquid oil core surrounded by a polymer shell. A ß-cyclodextrin-poly(4-acryloylmorpholine) conjugate was selected as the shell component. HNE-loaded nanocapsules were about 350 nm in size with a negative surface charge. They were stable for two years when stored in suspensions at 4 degrees C. In vitro experiments showed that HNE was released from the nanocapsules at a considerable rate. Nanocapsule uptake into cells was evaluated using a fluorescent formulation that revealed rapid internalisation. Cytotoxicity studies demonstrated the safety of the formulation. Enhanced anti-tumoral activity against various cell lines, depending on increased HNE stability, was obtained by using HNE-loaded nanocapsules. In particular, we have demonstrated an increase in anti-proliferative, pro-apoptotic and differentiative activity in several tumour cell lines from different tissues. Moreover, we evaluated the effects of these new nanocapsules on a three-dimensional human reconstructed model of skin melanoma. Interestingly, the encouraging results obtained with topical administration on the epidermal surface could open new perspectives in melanoma treatments.

Role of 4-hydroxynonenal-protein adducts in human diseases.

SIGNIFICANCE: Oxidative stress provokes the peroxidation of polyunsaturated fatty acids in cellular membranes, leading to the formation of aldheydes that, due to their high chemical reactivity, are considered to act as second messengers of oxidative stress. Among the aldehydes formed during lipid peroxidation (LPO), 4-hydroxy-2-nonenal (HNE) is produced at a high level and easily reacts with both low-molecular-weight compounds and macromolecules, such as proteins and DNA. In particular, HNE-protein adducts have been extensively investigated in diseases characterized by the pathogenic contribution of oxidative stress, such as cancer, neurodegenerative, chronic inflammatory, and autoimmune diseases. RECENT ADVANCES: In this review, we describe and discuss recent insights regarding the role played by covalent adducts of HNE with proteins in the development and evolution of those among the earlier mentioned disease conditions in which the functional consequences of their formation have been characterized. CRITICAL ISSUES: Results obtained in recent years have shown that the generation of HNE-protein adducts can play important pathogenic roles in several diseases. However, in some cases, the generation of HNE-protein adducts can represent a contrast to the progression of disease or can promote adaptive cell responses, demonstrating that HNE is not only a toxic product of LPO but also a regulatory molecule that is involved in several biochemical pathways. FUTURE DIRECTIONS: In the next few years, the refinement of proteomical techniques, allowing the individuation of novel cellular targets of HNE, will lead to a better understanding the role of HNE in human diseases.

Re-evaluating the induction of bone formation in primates.

The molecular cloning of the osteogenic proteins of the transforming growth factor-ß (TGF-ß) supergene family and the results of numerous pre-clinical studies in several mammalian species including non-human primates, have prematurely convinced molecular biologists, tissue engineers and skeletal reconstructionists alike to believe that single recombinant human bone morphogenetic/osteogenic proteins (hBMPs/OPs) would result in tissue induction when translated in clinical contexts. This theoretical potential has not been translated to acceptable clinical results. Clinical trials in craniofacial and orthopedic applications such as mandibular reconstruction and sinus-lift operations have indicated that supra physiological doses of a single recombinant human protein are needed to induce unacceptable tissue regeneration whilst incurring significant costs without achieving equivalence to autogenous bone grafts. The acid test for clinically relevant bone tissue engineering should now become the concept of clinically significant osteoinduction, whereby the regenerated bone is readily identifiable on radiographic examination by virtue of its opacity and trabecular architecture. The need for alternatives to the hBMPs/OPs is now felt more acutely following reported complications and performance failure associated with the clinical use of hBMP-2 and hOP-1 (BMP-7). Because of the often substandard regeneration of clinical defects implanted with hBMPs/OPs, we now need to finally deal with the provocative question: are the hBMPs/OPs the only initiators of the induction of bone formation in pre-clinical and clinical contexts? The rapid induction of bone formation by the hTGF-ß3 isoform in heteropic intramuscular sites of the Chacma baboon Papio ursinus together with TGF-ß1, TGF-ß3, BMP-2, BMP-3, OP-1, RUNX-2 and Osteocalcin up-regulation and expression, hyper cellular osteoblastic activity, osteoid synthesis, angiogenesis and capillary sprouting are the molecular and morphological foundation for the induction of bone formation in clinical contexts. The induction of bone as initiated by hTGF-ß3 when implanted in the rectus abdominis muscle of P. ursinus is via the BMPs/OPs pathway with hTGF-ß3 controlling the induction of bone formation by regulating the expression of BMPs/OPs via Noggin expression, eliciting the induction of bone formation by up-regulating endogenous BMPs/OPs and it is blocked by hNoggin, providing insights into performance failure of hBMPs/OPs in clinical contexts. Physiological expression of BMPs/OPs genes upon implantation of hTGF-ß3 may escape the antagonist expression of Noggin and other inhibitors, whereas direct application of hBMPs/OPs, representing a later by-product step of the bone induction cascade as set by the TGF-ß3 master gene in primates, sets into motion Noggin' antagonist action, as shown by the limited effectiveness of hBMPs/OPs in clinical contexts. The unprecedented induction of bone formation by 250 µg hTGF-ß3 when combined with coral-derived macroporous constructs is the novel molecular and morphological frontier for the induction of bone formation in man. The induction of bone by hTGF-ß3 has been thus translated in clinical contexts to treat a large mandibular defect in a pediatric patient; 30 months after implantation of 250 µg hTGF-ß3 per gram of human demineralized bone matrix, radiographic analyses show the reconstruction of the avulsed large mandibular segment including the induction of the avulsed coronoid process.

Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral-derived macroporous constructs.

Coral-derived calcium carbonate/hydroxyapatite macroporous constructs of the genus Goniopora with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) initiate the induction of bone formation. Which are the molecular signals that initiate pattern formation and the induction of bone formation? To evaluate the role of released calcium ions and osteoclastogenesis, 7% HA/CC was pre-loaded with either 500 µg of the calcium channel blocker, verapamil hydrochloride, or 240 µg of the osteoclast inhibitor, biphosphonate zoledronate, and implanted in the rectus abdominis muscle of six adult Chacma baboons Papio ursinus. Generated tissues on days 15, 60 and 90 were analysed by histomorphometry and qRT-PCR. On day 15, up-regulation of type IV collagen characterized all the implanted constructs correlating with vascular invasion. Zoledronate-treated specimens showed an important delay in tissue patterning and morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal, if any, bone formation by induction. 7% HA/CC pre-loaded with the Ca(++) channel blocker verapamil hydrochloride strongly inhibited the induction of bone formation. Down-regulation of bone morphogenetic protein-2 (BMP-2) together with up-regulation of Noggin genes correlated with limited bone formation in 7% HA/CC pre-loaded with either verapamil or zoledronate, indicating that the induction of bone formation by coral-derived macroporous constructs is via the BMPs pathway. The spontaneous induction of bone formation is initiated by a local peak of Ca(++) activating stem cell differentiation and the induction of bone formation.

Interim reconstruction and space maintenance of mandibular continuity defects preceding definitive osseous reconstruction.

The use of space maintenance in mandibular defects as an interim measure before definitive osseous reconstruction may prevent problems associated with delayed reconstruction including increased technical difficulty, contracture of soft tissues that limits the volume of the final reconstruction, and the potential for iatrogenic injury to adjacent anatomical structures. The use of a condyle/ramus spacer made of medical grade, ultrahigh-molecular-weight polyethylene, and a flexible body spacer made of high quality, inert, non-toxic medical and food grade silicone rubber, was tested in 38 patients with mandibular defects after the resection of benign but locally aggressive disease, advanced osteomyelitis, and injuries. The spacer was retained for a maximum of 8 weeks, and was then removed through an extraoral approach before definitive reconstruction with a particulate corticocancellous bone graft. One of the 38 patients failed to attend for follow up and returned 7 months later with severe, generalised sepsis that required removal of the spacer and exclusion from the study. Of the remaining 37 patients, 32 healed uneventfully, 1 required removal of the spacer 2 weeks after implantation for intraoral wound dehiscence, and 4 had mild to moderate disturbances of wound healing that required either minor revision or local wound care until removal at the time of reconstruction. The use of a spacer promotes wound healing and simplifies and expedites secondary reconstruction of mandibular defects.

Rosiglitazone and AS601245 decrease cell adhesion and migration through modulation of specific gene expression in human colon cancer cells.

PPARs are nuclear receptors activated by ligands. Activation of PPARγ leads to a reduction of adhesion and motility in some cancer models. PPARγ transcriptional activity can be negatively regulated by JNK-mediated phosphorylation. We postulated that the use of agents able to inhibit JNK activity could increase the effectiveness of PPARγ ligands. We analysed the effects of rosiglitazone (PPARγ ligand) and AS601245 (a selective JNK inhibitor) alone or in association on adhesion and migration of CaCo-2, HT29, and SW480 human colon cancer cells and investigated, through microarray analysis, the genes involved in these processes. Cell adhesion and migration was strongly inhibited by rosiglitazone and AS601245. Combined treatment with the two compounds resulted in a greater reduction of the adhesion and migration capacity. Affymetrix analysis in CaCo-2 cells revealed that some genes which were highly modulated by the combined treatment could be involved in these biological responses. Rosiglitazone, AS601245 and combined treatment down-regulated the expression of fibrinogen chains in all three cell lines. Moreover, rosiglitazone, alone or in association with AS601245, caused a decrease in the fibrinogen release. ARHGEF7/ß-PIX gene was highly down-regulated by combined treatment, and western blot analysis revealed that ß-PIX protein is down-modulated in CaCo-2, HT29 and SW480 cells, also. Transfection of cells with ß-PIX gene completely abrogated the inhibitory effect on cell migration, determined by rosiglitazone, AS601245 and combined treatment. Results demonstrated that ß-PIX protein is involved in the inhibition of cell migration and sustaining the positive interaction between PPARγ ligands and anti-inflammatory agents in humans.

A Macroporous Bioreactor Super Activated by the Recombinant Human Transforming Growth Factor-ß(3).

Macroporous single phase hydroxyapatite (HA) and biphasic HA/ß-tricalcium phosphate with 33% post-sinter hydroxyapatite (HA/ß-TCP) were combined with 25 or 125 µg recombinant human transforming growth factor-ß3 (hTGF-ß(3)) to engineer a super activated bioreactor implanted in orthotopic calvarial and heterotopic rectus abdominis muscle sites and harvested on day 30 and 90. Coral-derived calcium carbonate fully converted (100%) and partially converted to 5 and 13% hydroxyapatite/calcium carbonate (5 and 13% HA/CC) pre-loaded with 125 and 250 µg hTGF-ß(3), and 1:5 and 5:1 binary applications of hTGF-ß(3): hOP-1 by weight, were implanted in the rectus abdominis and harvested on day 20 and 30, respectively, to monitor spatial/temporal morphogenesis by high doses of hTGF-ß(3). Bone formation was assessed on decalcified paraffin-embedded sections by measuring the fractional volume of newly formed bone. On day 30 and 90, single phase HA implants showed greater amounts of bone when compared to biphasic specimens; 5 and 13% HA/CC pre-loaded with 125 and 250 µg hTGF-ß(3) showed substantial induction of bone formation; 250 µg hTGF-ß(3) induced as yet unreported massive induction of bone formation as early as 20 days prominently outside the profile of the macroporous constructs. The induction of bone formation is controlled by the implanted ratio of the recombinant morphogens, i.e., the 1:5 hTGF-ß(3):hOP-1 ratio by weight was greater than the inverse ratio. The unprecedented tissue induction by single doses of 250 µg hTGF-ß(3) resulting in rapid bone morphogenesis of vast mineralized ossicles with multiple trabeculations surfaced by contiguous secreting osteoblasts is the novel molecular and morphological frontier for the induction of bone formation in clinical contexts.

AS601245, an Anti-Inflammatory JNK Inhibitor, and Clofibrate Have a Synergistic Effect in Inducing Cell Responses and in Affecting the Gene Expression Profile in CaCo-2 Colon Cancer Cells.

PPARαs are nuclear receptors highly expressed in colon cells. They can be activated by the fibrates (clofibrate, ciprofibrate etc.) used to treat hyperlipidemia. Since PPARα transcriptional activity can be negatively regulated by JNK, the inhibition of JNK activity could increase the effectiveness of PPARα ligands. We analysed the effects of AS601245 (a JNK inhibitor) and clofibrate alone or in association, on proliferation, apoptosis, differentiation and the gene expression profile of CaCo-2 human colon cancer cells. Proliferation was inhibited in a dose-dependent way by clofibrate and AS601245. Combined treatment synergistically reduced cell proliferation, cyclin D1 and PCNA expression and induced apoptosis and differentiation. Reduction of cell proliferation, accompanied by the modulation of p21 expression was observed in HepG2 cells, also. Gene expression analysis revealed that some genes were highly modulated by the combined treatment and 28 genes containing PPRE were up-regulated, while clofibrate alone was ineffective. Moreover, STAT3 signalling was strongly reduced by combined treatment. After combined treatment, the binding of PPARα to PPRE increased and paralleled with the expression of the PPAR coactivator MED1. Results demonstrate that combined treatment increases the effectiveness of both compounds and suggest a positive interaction between PPARα ligands and anti-inflammatory agents in humans.