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?

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.

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.

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.

Biomimetic matrices self-initiating the induction of bone formation.

The new strategy of tissue engineering, and regenerative medicine at large, is to construct biomimetic matrices to mimic nature's hierarchical structural assemblages and mechanisms of simplicity and elegance that are conserved throughout genera and species. There is a direct spatial and temporal relationship of morphologic and molecular events that emphasize the biomimetism of the remodeling cycles of the osteonic corticocancellous bone versus the "geometric induction of bone formation," that is, the induction of bone by "smart" concavities assembled in biomimetic matrices of macroporous calcium phosphate-based constructs. The basic multicellular unit of the corticocancellous bone excavates a trench across the bone surface, leaving in its wake a hemiosteon rather than an osteon, that is, a trench with cross-sectional geometric cues of concavities after cyclic episodes of osteoclastogenesis, eventually leading to osteogenesis. The concavities per se are geometric regulators of growth-inducing angiogenesis and osteogenesis as in the remodeling processes of the corticocancellous bone. The concavities act as a powerful geometric attractant for myoblastic/myoendothelial and/or endothelial/pericytic stem cells, which differentiate into bone-forming cells. The lacunae, pits, and concavities cut by osteoclastogenesis within the biomimetic matrices are the driving morphogenetic cues that induce bone formation in a continuum of sequential phases of resorption/dissolution and formation. To induce the cascade of bone differentiation, the soluble osteogenic molecular signals of the transforming growth factor ß supergene family must be reconstituted with an insoluble signal or substratum that triggers the bone differentiation cascade. By carving a series of repetitive concavities into solid and/or macroporous biomimetic matrices of highly crystalline hydroxyapatite or biphasic hydroxyapatite/ß-tricalcium phosphate, we were able to embed smart biologic functions within intelligent scaffolds for tissue engineering of bone. The concavities assembled in the bioceramic constructs biomimetize the remodeling cycle of the corticocancellous bone and are endowed with multifunctional pleiotropic self-assembly capacities, initiating angiogenesis and bone formation by induction without the exogenous applications of the osteogenic-soluble molecular signals of the transforming growth factor ß supergene family. The incorporation of specific biologic activities into biomimetic matrices by manipulating the geometry of the substratum, defined as geometric induction of bone formation, is now helping to engineer therapeutic osteogenesis in clinical contexts.

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.

PPARgamma ligands inhibit telomerase activity and hTERT expression through modulation of the Myc/Mad/Max network in colon cancer cells.

In human cells the length of telomeres depends on telomerase activity. This activity and the expression of the catalytic subunit of human telomerase reverse transcriptase (hTERT) is strongly up-regulated in most human cancers. hTERT expression is regulated by different transcription factors, such as c-Myc, Mad1 and Sp1. In this study, we demonstrated that 15d-PG J2 and rosiglitazone (an endogenous and synthetic peroxisome proliferators activated receptor gamma (PPARgamma) ligand, respectively) inhibited hTERT expression and telomerase activity in CaCo-2 colon cancer cells. Moreover, both ligands inhibited c-Myc protein expression and its E-box DNA binding activity. Additionally, Mad1 protein expression and its E-box DNA binding activity were strongly increased by 15d-PG J2 and, to a lesser extent, by rosiglitazone. Sp1 transcription factor expression and its GC-box DNA binding activity were not affected by both PPARgamma ligands. Results obtained by transient transfection of CaCo-2 cells with pmaxFP-Green-PRL plasmid constructs containing the functional hTERT core promoter (including one E-box and five GC-boxes) and its E-box deleted sequences, cloned upstream of the green fluorescent protein reporter gene, demonstrated that 15d-PG J2, and with minor effectiveness, rosiglitazone, strongly reduced hTERT core promoter activity. E-boxes for Myc/Mad/Max binding showed a higher activity than GC-boxes for Sp1. By using GW9662, an antagonist of PPARgamma, we demonstrated that the effects of 15d-PG J2 are completely PPARgamma independent, whereas the effects of rosiglitazone on hTERT expression seem to be partially PPARgamma independent. The regulation of hTERT expression by 15d-PG J2 and rosiglitazone, through the modulation of the Myc/Max/Mad1 network, may represent a new mechanism of action of these substances in inhibiting cell proliferation.

Transforming growth factor-beta isoforms and the induction of bone formation: implications for reconstructive craniofacial surgery.

Craniofacial skeletal reconstruction remains a challenging problem despite major molecular and surgical developments in the understanding of bone formation by induction. The induction of bone formation has been a critical topic of research across the planet. The bone induction principle identified important cues for tissue engineering of bone, namely, osteogenic soluble molecular signals, the bone morphogenetic and osteogenic proteins, and insoluble signals or substrata including biomimetic bioactive matrices and responding stem cells. In primates, and in primates only, the osteogenic soluble molecular signals that initiate the induction of bone formation additionally include the 3 mammalian transforming growth factor-beta (TGF-beta) isoforms, members of the TGF-beta supergene family. The mammalian TGF-beta isoforms, when implanted in the rectus abdominis muscle of the nonhuman primate Papio ursinus, induce rapid and substantial endochondral bone formation resulting in large corticalized ossicles by day 30 after heterotopic implantation; in calvarial defects of the same nonhuman primates, identical or higher doses of the TGF-beta protein do not induce bone formation because of the overexpression of Smad-6 and Smad-7, gene product inhibitors of the TGF-beta signaling pathway. The addition of minced fragments of autogenous rectus abdominis muscle partially restores the osteoinductive activity of the human TGF-beta3 isoform resulting in the induction of bone formation in the treated calvarial defects. Recombinant human TGF-beta3 delivered by Matrigel matrix and implanted in class II and III furcation defects of mandibular molars of P. ursinus induce periodontal tissue regeneration. The addition of minced fragments of autogenous rectus abdominis muscle significantly enhances cementogenesis. This review highlights the induction of bone formation by the osteogenic proteins of the TGF-beta superfamily in the nonhuman primate P. ursinus and reviews combinatorial applications of myoblastic/myogenic stem cell-based therapeutics for bone induction and morphogenesis. The recruitment of myoendothelial cells is also discussed in the light of the intrinsic and spontaneous induction of bone formation by smart biomaterial matrices that induce bone differentiation in heterotopic extraskeletal sites of P. ursinus without the exogenous application of the osteogenic soluble molecular signals of the TGF-beta superfamily.

A prospective audit over a six month period of condylar fractures at Chris Hani Baragwanath Hospital.

OBJECTIVE: To determine the frequency of occurrence, patterns, aetiology and outcome of treatment of mandibular condylar fractures at Chris Hani Baragwanath Hospital. MATERIALS AND METHODS: All patients with condylar fractures seen at Chris Hani Baragwanath Hospital over a six-month period from January to June 2003 were included in the study. Data was recorded on sex, age, date of injury, cause of trauma, status of the occlusion and presence of associated facial injuries. The type of condylar fracture was recorded and classified following the radiographic examination according to Spiessl and Schroll. The type of treatment rendered and its outcome were also documented. Patients were followed up from 1 week to 6 months post-treatment. RESULTS: The sample comprised 84 patients with 95 condylar fractures; 69 (82%) males and 15(18%) females. 73.8% of the fractures were caused by interpersonal violence followed by road traffic accidents (16,67%) and falls (7.14%). 73 (87%) patients had unilateral fractures and 11 (13%) had bilateral condylar fractures. Of the 95 condylar fractures, there were 16 medially dislocated condylar fractures in 13 patients. Type I, Type II and Type III fractures accounted for 31%, 26% and 25% of the total fractures respectively. CONCLUSION: A relationship exists between the type of condylar fracture and the cause of the fracture: road traffic accidents and falls produce more bilateral and dislocated fractures than interpersonal violence. Prolonged intermaxillary fixation, failure to use training elastics, absence of active physiotherapy and poor patient compliance results in increased risk of hypomobility.

4-Hydroxynonenal and PPARgamma ligands affect proliferation, differentiation, and apoptosis in colon cancer cells.

PPARgamma ligands inhibit growth and induce apoptosis of various cancer cells. 4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation and induces differentiation or apoptosis in neoplastic cells. The aim of this work was to investigate the effects of PPARgamma ligands (rosiglitazone and 15-deoxy-prostaglandin J2 (15d-PGJ2)) and HNE, alone or in association, on proliferation, apoptosis, differentiation, and growth-related and apoptosis-related gene expression in colon cancer cells (CaCo-2 cells). PPARgamma ligands inhibited cell proliferation (IC50 was 37.47+/-6.6 microM, for 15d-PGJ2, and 170.34+/-20 microM for rosiglitazone). HNE (1 microM) inhibited cell growth by 70%. Apoptosis was induced by 15d-PGJ2 and HNE and, to a minor extent, rosiglitazone. Differentiation was induced by rosiglitazone and by 15d-PGJ2, but not by HNE. PPARgamma ligands inhibited c-myc expression. HNE induced a transitory increase in c-myc expression and a subsequent down-regulation. HNE induced p21 expression, whereas PPARgamma ligands did not. Expression of the bax gene was increased by HNE and 15d-PGJ2, but not by rosiglitazone. No synergism or antagonism was found between HNE and PPARgamma ligands. Both apoptosis and differentiation induction may be responsible for the inhibition of proliferation by PPARgamma ligands; apoptosis and c-myc and p21 expression seem to be involved in the inhibition of proliferation by HNE.

Anterior open bite correction by Le Fort I or bilateral sagittal split osteotomy.

Some of the most challenging dentofacial deformities facing surgeons and orthodontists are anterior open bite malocclusions. Determining the cause for the development of an anterior open bite and formulating a diagnosis are complicated by the role of neuromuscular and genetic influences. Long-term skeletal and dental stability has been a concern because of the influence that the neuromusculature has on the repositioned jaws and stability of teeth after vertical orthodontic mechanics required for closing open bites.

Bone morphogenetic proteins and the induction of bone formation: from laboratory to patients.

This article revisits the induction of bone by the osteogenic proteins of the transforming growth factor-beta superfamily in nonhuman and human primates and proposes that the translation in clinical contexts of the phenomenon of bone; formation by autoinduction, is predictably achievable by the binary application of relatively low doses of transforming growth factor-beta proteins with a recombinant human osteogenic protein. The synergistic induction of bone formation is a cost-effective clinical strategy because published data in nonhuman primates have shown that doses of recombinant human recombinant osteogenic protein-1 can be reduced at least fivefold and still increase bone formation compared with higher doses of single applications of human recombinant osteogenic protein-1.

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.

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.

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.

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.

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.