Microfragmented Fat and Biphasic Calcium Phosphates for Alveolar Cleft Repair: Protocol for a Prospective, Nonblinded, First-in-Human Clinical Study.

BACKGROUND: Biphasic calcium phosphates (BCP) may serve as off-the-shelf alternatives for iliac crest-derived autologous bone in alveolar cleft reconstructions. To add osteoinductivity to the osteoconductive BCPs to achieve similar regenerative capacity as autologous bone, a locally harvested buccal fat pad will be mechanically fractionated to generate microfragmented fat (MFAT), which has been shown to have high regenerative capacity due to high pericyte and mesenchymal stem cell content and a preserved perivascular niche. OBJECTIVE: Our primary objectives will be to assess the feasibility and safety of the BCP-MFAT combination. The secondary objective will be efficacy, which will be evaluated using radiographic imaging and histological and histomorphometric evaluation of biopsies taken 6 months postoperatively, concomitant with dental implant placement. METHODS: Eight patients with alveolar cleft (≥15 years) will be included in this prospective, nonblinded, first-in-human clinical study. MFAT will be prepared intraoperatively from the patient's own buccal fat pad. Regular blood tests and physical examinations will be conducted, and any adverse events (AEs) or serious EAs (SAEs) will be meticulously recorded. Radiographic imaging will be performed prior to surgery and at regular intervals after reconstruction of the alveolar cleft with the BCP-MFAT combination. Biopsies obtained after 6 months with a trephine drill used to prepare the implantation site will be assessed with histological and histomorphometric analyses after methylmethacrylate embedding and sectioning. RESULTS: The primary outcome parameter will be safety after 6 months' follow-up, as monitored closely using possible occurrences of SAEs based on radiographic imaging, blood tests, and physical examinations. For efficacy, radiographic imaging will be used for clinical grading of the bone construct using the Bergland scale. In addition, bone parameters such as bone volume, osteoid volume, graft volume, and number of osteoclasts will be histomorphometrically quantified. Recruitment started in November 2019, and the trial is currently in the follow-up stage. This protocol's current version is 1.0, dated September 15, 2019. CONCLUSIONS: In this first-in-human study, not only safety but also the histologically and radiographically assessed regenerative potential of the BCP-MFAT combination will be evaluated in an alveolar cleft model. When an SAE occurs, it will be concluded that the BCP-MFAT combination is not yet safe in the current setting. Regarding AEs, if they do not occur at a higher frequency than that in patients treated with standard care (autologous bone) or can be resolved by noninvasive conventional methods (eg, with analgesics or antibiotics), the BCP-MFAT combination will be considered safe. In all other cases, the BCP-MFAT combination will not yet be considered safe. TRIAL REGISTRATION: Indonesia Clinical Trial Registry INA-EW74C1N; https://tinyurl.com/28tnrr64. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/42371.

Contextualizing sociocultural beliefs about cleft, knowledge of the treatments, and factors associated with delay of treatment in Eastern Indonesia.

INTRODUCTION: Indonesia is a multicultural Asian country with a high incidence of cleft. This study contextualizes how patients' sociocultural backgrounds hinder cleft management in a diverse nation. MATERIAL AND METHODS: This study involved 202 families of cleft patients attending six tertiary care hospitals in South Sulawesi between 2021 and 2022. A mixed-methods, descriptive cross-sectional study employed semi-structured interviews and focus group discussions. Thematic content analysis was done using Murdock's causal attribution of illness. Knowledge of the treatments and surgery expectations used open coding. We held medical team focus group discussions to validate education on treatments. Cleft management education was thematically analyzed based on Indonesia's Minister of Health Decree. RESULTS: Two hundred-two families and ten medical teams participated. Thematic content analysis revealed common beliefs and factors that hinder medical treatments. The participants were 109 Buginese, 57 Makassarese, 16 Durinese, 8 Luwunese, 8 Torajanese, and 4 Mandarese. 22.3 % were unaware of causation, while 29.2 % attributed it to natural causes. About half of the interviewees believed in supernatural attribution. Even though 40 % of participants knew little about the surgery, they agreed that surgery improves appearance and speech. Medical treatments are delayed due to a lack of treatment knowledge, parents' concerns about surgical safety, and beliefs about causes. DISCUSSION: Indigenous societies in South Sulawesi believe in supernatural causes of cleft. Most had incomplete surgical treatment information. An intensive educational health program about causes, treatments, medical specialists, and treatment goals is warranted to enhance patient compliance with medical treatment, ultimately leading to improved health outcomes.

A narrative review of the role of Eph receptors in head and neck squamous cell carcinoma.

Tyrosine kinase receptors (TKR) coordinate a variety of pathological processes in head and neck squamous cell carcinoma (HNSCC), and eventually play a role in patient outcomes. In this review, the role of Eph receptors in HNSCC progression and the possibility of targeting these receptors are illustrated. All relevant studies were identified through a comprehensive search of four electronic databases, including PubMed, Scopus, web of science, and Embase till August 2022. EphA2 and EphB4, along with ephrin-B2, were the most extensively studied proteins in this family. However, overexpression of EphB4 and its ligand ephrin-B2 were the only proteins that consistently showed association with a poor outcome, indicating that these proteins might serve as valuable prognostic markers in HNSCC. High expression of EphA3 and EphB4 was found to play a crucial role in radioresistance of HNSCC. EphB4 loss, in particular, was observed to induce an immunosuppression phenotypic HNSCC. Currently, ongoing clinical trials are investigating the benefits of EphB4-ephrin-B2 blockade in combination with standard of care treatment in HNSCC. Further efforts are needed to explore the biological role and behavioral complexity of this family of TKR in HNSCC with great attention to avoid heterogeneity of HNSCC subsites.

A network meta-analysis assessing the effectiveness of various radical and conservative surgical approaches regarding recurrence in treating solid/multicystic ameloblastomas.

Multiple treatment approaches have been undertaken to reduce the incidence of recurrence in solid/multicystic ameloblastoma (SMA), both conservative and radical. A network meta-analysis (NMA) was conducted to assess and compare the effectiveness of these various treatment approaches concurrently. This study was reported based on the Preferred Reporting Items for Systematic Reviews for Network Meta-Analysis (PRISMA-NMA) statement. PubMed (MEDLINE), ScienceDirect, Scopus, and Web of Science were searched until August 10, 2021. The NMA was conducted using the STATA program. Of 1153 records identified in the search, seven observational studies with 180 patients were included. Six different treatment approaches were identified. Segmental resection ranked highest for reducing the recurrence rate with the highest SUCRA score (77.7), followed by curettage with cryotherapy (66.9) and marginal resection (49.3). Network inconsistencies and publication bias appeared to be absent. According to the Confidence in Network Meta-Analysis (CINeMa) method, the evidence's certainty was low for all comparisons due to imprecision and within-study bias. In conclusion, this study is the first NMA in the field of ameloblastoma. Segmental resection seemed to be the most effective treatment approach for minimizing recurrence in SMA patients. Nevertheless, weak certainty of evidence makes that the results must be regarded with caution.

Postoperative Daycare as a Safe and Cost-Effective Option for Secondary Alveolar Bone Graft (SABG) Surgery: A Retrospective Comparative Cohort Study.

OBJECTIVE: To evaluate the outcomes of Secondary Alveolar Bone Grafting (SABG) in patients treated either in daycare or with multiple day hospitalization (MDH) in relation to costs and complication rates. DESIGN: Retrospective comparative cohort study. SETTING: The data was collected from two settings: Postoperative daycare or MDH after oral cleft surgery in an Academic Medical Center in The Netherlands. PATIENTS: Data of 137 patients with unilateral Cleft lip, alveolus, and palate (CLAP) treated between 2006-2018 were evaluated. Registered clinical variables: age, gender, cleft subtype, bone donor site, type of hospitalization, length of stay, additional surgery, complications, surgeons, and costs. INTERVENTIONS: Closure of the alveolar cleft with/without closure of the anterior palate. MAIN OUTCOME MEASURES: Univariate analyses. RESULTS: Of the 137 patients, 46.7% were treated in MDH, and 53.3% in daycare. Total costs for daycare were significantly lower (P < .001). All patients treated in daycare received mandibular symphysis bone, whereas in MDH, 46.9% received iliac crest bone instead. Bone donor site was associated with postoperative care type. Complication rates were slightly but not significantly higher in daycare (26%) vs. MDH (14.1%) (P = .09). Most were Grade I (minor) according to Clavien Dindo classification. CONCLUSIONS: Daycare after alveolar cleft surgery is about as safe as MDH, but significantly cheaper.

Radiation modulates expression and related activities of c-Met protein in oral tongue squamous cell carcinoma cell lines.

OBJECTIVES: c-Met, a receptor tyrosine kinase, is involved in the growth, invasion and metastasis of a variety of cancers. In a set of cell lines from several solid tumors, a five-fold increase in c-Met expression after irradiation has been reported. This study aimed to assess if c-Met is likewise abundantly expressed in oral tongue squamous cell carcinoma (OTSCC) upon exposure to irradiation, followed by a Met-induced biological response. MATERIALS AND METHODS: Six OTSCC cell lines were exposed to gamma radiation doses of 2, 4, and 6 Gray. The changes in c-Met protein levels were assessed by western blot and flow cytometry. c-Met gene expression, cell migration, proliferation and cell cycle assays were performed as phenotypic readouts. RESULTS: Irradiation resulted in upregulation of c.Met in all cell lines with different time kinetics. On average the cells displayed minimal c-Met expression on their surface ranging from 5 to 30% of total protein. Abrupt downregulation of c-Met surface expression occurred one hour after radiation but recovered 48 h post-radiation. Intracellularly, the highest level of expression was found on day 5 after radiation exposure. Irradiation induced aggressive invasive potential of the cells as determined in cell migration assays, particularly in cell lines with the highest c-Met expression. CONCLUSIONS: These results provide novel insights into both intracellular and extracellular dynamics of c-Met expression profiles upon irradiation of OTSCC cells in vitro. It might also suggest that radiation enhances cell migration, indicative of invasiveness, through c-Met up-regulation, at least for certain types of OTSCC cells.

WEE1 kinase inhibitor MK-1775 sensitizes oral tongue squamous cell carcinoma cells to radiation irrespective of TP53 status.

OBJECTIVE: Oral tongue squamous cell carcinoma (OTSCC) frequently harbors non-functional p53 and depends on G2/M checkpoint mediated by WEE1. WEE1 suppression has been identified as a promising anti-tumor strategy. This study investigated the capacity of WEE1 kinase inhibitor (MK-1775) and its underlying mechanisms in enhancing radiation responses of OTSCC cells in vitro. MATERIALS AND METHODS: WEE1 kinase expression and its downstream target (CDK1) were investigated in OTSCC versus normal oral tissue. A synergistic combination of MK-1775 with radiation on OTSCC cell lines with different p53 statuses was assessed by viability assay. The radio-sensitizing effects of MK-1775 on apoptosis, cell cycle, DNA damage, and mitotic entry were also determined. RESULTS: Irradiation enhanced CDK1 expression in all tested cell lines, though the effect was far more pronounced in p53 mutated cell lines. MK-1775 exhibited inhibitory effects against the survival of all cell lines and enhanced their response to the radiation. These effects were strongly elicited by induction of apoptosis and lethal mitosis, but less likely by abrogation of radiation-induced G2 arrest. CONCLUSION: These results demonstrate the efficacy of MK-1775 in enhancing the radiation effect on OTSCC in vitro associated with a significant apoptotic death rate, identifying WEE1 inhibitor as a potent radiosensitizer in OTSCC irrespective of p53 mutational status.

Virtual and traditional surgical planning in orthognathic surgery - systematic review and meta-analysis.

Traditional surgical planning (TSP) and virtual surgical planning (VSP) have been used in bimaxillary osteotomy planning. The time is taken in the planning and operating stages, and the working/doctor/total time of either approach are useful determinants of the efficiency of the operating method and quality of care. This systematic review and meta-analysis examined if VSP has a comparative advantage over TSP in the bimaxillary osteotomy. Cochrane Library, PubMed, EMBASE, and Google Scholar were used as databases to collect studies that met the outlined inclusion criteria based on PRISMA. Eight of 759 studies were considered to meet the eligibility criteria, and six fit for meta-analysis. The findings demonstrated significant VSP advantage over TSP in planning time (Z = 3.97 (p < 0.00001), WMD = -5.29 (CI -7.90 to -2.68)). While more time-efficient than TSP, the difference with VSP was not significant during surgery (Z = 0.44 (p = 0.66), WMD = -0.10 (CI -0.51 to 0.34)). The study used random effects due to the high I2 of the planning mean differences. The continued evolution of VSP and improved application knowledge will be important in reducing the time of planning and surgery, thus improving the outcomes of the complex bimaxillary osteotomy. The current evidence shows that VSP significantly performs better than TSP in reducing the bimaxillary osteotomy planning time, but the timing difference is not significant during surgery. Future analysis will benefit from using studies with standard research and reporting metrics and procedures, thus improving evidence-based clinical practice.

Age-group-specific trend analyses of oropharyngeal squamous cell carcinoma incidence from 1989 to 2018 and risk factors profile by age-group in 2015-2018: a population-based study in The Netherlands.

Incidence of oropharyngeal squamous cell carcinoma (OPSCC) is increasing globally and the human papillomavirus (HPV) has been linked to this increase. This study aimed to present a comprehensive overview of OPSCC trends in incidence rates by age group and investigate differences in risk factors profile. Netherlands Cancer Registry data from 1989-2018 were analyzed to calculate the annual percentage change (APC) over European standardized incidence rates by gender and age group using joinpoint regression software. Smoking, alcohol drinking and HPV-status were available for 2015-2018. During 1989-2018, 13 048 cases of OPSCC were reported with a male-to-female ratio of 2.1:1. The overall incidence rate increased by 5.4% (APC) annually from 1989 to 1996 but slowed thereafter by 1.2%. Significant declines were found in patients of 35-44 years (APCs -3.7%). Adults aged 45-59 years displayed significant increases from 1989 to 2001, followed by a significant decline. In patients ≥60 years, the incidence rates increased overall, with APC for women being consistently higher than men. The data on HPV status was available for 69% of the patients, of whom 47% were HPV+. Smoking and alcohol consumption were more prevalent, that is 75 and 76 % respectively. The declining trends of OPSCC for Dutch people aged 35-44 years from 1989 to 2018 and for those aged 45-59 years from 2002 onwards are inconsistent to trends reported elsewhere in the developed countries. The prevalence of smoking and drinking alcohol was quite high in all age groups, whereas the proportion of HPV-positivity was relatively low.

Safety and feasibility study of using polyphosphate (PolyP) in alveolar cleft repair: a pilot study.

BACKGROUND: Bone grafting is an important surgical procedure to reconstruct alveolar bone defects in patients with cleft lip and palate. Polyphosphate (PolyP) is a physiological polymer present in the blood, primarily in platelets. PolyP plays a role as a phosphate source in bone calcium phosphate deposition. Moreover, the cleavage of high-energy bonds to release phosphates provides local energy necessary for regenerative processes. In this study, polyP is complexed with calcium to form Calcium polyP microparticles (Ca-polyP MPs), which were shown to have osteoinductive properties in preclinical studies. The aim of this study was to evaluate the feasibility, safety, and osteoinductivity of Ca-polyP MPs, alone or in combination with BCP, in a first-in-human clinical trial. METHODS: This single-blinded, parallel, prospective clinical pilot study enrolled eight adolescent patients (mean age 18.1: range 13-34 years) with residual alveolar bone cleft. Randomization in two groups (four receiving Ca-polyP MPs only, four a combination of Ca-polyP MPs and biphasic calcium phosphate (BCP)) was performed. Patient follow-up was 6 months. Outcome parameters included safety parameters and close monitoring of possible adverse effects using radiographic imaging, regular blood tests, and physical examinations. Osteoinductivity evaluation using histomorphometric analysis of biopsies was not possible due to COVID restrictions. RESULTS: Due to surgical and feasibility reasons, eventually, only 2 patients received Ca-polyP MPs, and the others the combination graft. All patients were assessed up to day 90. Four out of eight were able to continue with the final assessment day (day 180). Three out of eight were unable to reach the hospital due to COVID-19 restrictions. One patient decided not to continue with the study. None of the patients showed any allergic reactions or any remarkable local or systematic side effects. Radiographically, patients receiving Ca-polyP MPs only were scored grade IV Bergland scale, while patients who got the BCP/Ca-polyP MPs combination had scores ranging from I to III. CONCLUSIONS: Our results indicate that Ca-polyP MPs and the BCP/Ca-polyP MPs combination appear to be safe graft materials; however, in the current setting, Ca-polyP MPs alone may not be a sufficiently stable defect-filling scaffold to be used in alveolar cleft repair. TRIAL REGISTRATION: Indonesian Trial Registry under number INA-EW74C1N by the ethical committee of Faculty of Medicine, Hasanuddin University, Makassar, Indonesia with code number 1063/UN4.6.4.5.31/PP36/2019 .

Inlet flow rate of perfusion bioreactors affects fluid flow dynamics, but not oxygen concentration in 3D-printed scaffolds for bone tissue engineering: Computational analysis and experimental validation.

Fluid flow dynamics and oxygen-concentration in 3D-printed scaffolds within perfusion bioreactors are sensitive to controllable bioreactor parameters such as inlet flow rate. Here we aimed to determine fluid flow dynamics, oxygen-concentration, and cell proliferation and distribution in 3D-printed scaffolds as a result of different inlet flow rates of perfusion bioreactors using experiments and finite element modeling. Pre-osteoblasts were treated with 1 h pulsating fluid flow with low (0.8 Pa; PFFlow) or high peak shear stress (6.5 Pa; PFFhigh), and nitric oxide (NO) production was measured to validate shear stress sensitivity. Computational analysis was performed to determine fluid flow between 3D-scaffold-strands at three inlet flow rates (0.02, 0.1, 0.5 ml/min) during 5 days. MC3T3-E1 pre-osteoblast proliferation, matrix production, and oxygen-consumption in response to fluid flow in 3D-printed scaffolds inside a perfusion bioreactor were experimentally assessed. PFFhigh more strongly stimulated NO production by pre-osteoblasts than PFFlow. 3D-simulation demonstrated that dependent on inlet flow rate, fluid velocity reached a maximum (50-1200 µm/s) between scaffold-strands, and fluid shear stress (0.5-4 mPa) and wall shear stress (0.5-20 mPa) on scaffold-strands surfaces. At all inlet flow rates, gauge fluid pressure and oxygen-concentration were similar. The simulated cell proliferation and distribution, and oxygen-concentration data were in good agreement with the experimental results. In conclusion, varying a perfusion bioreactor's inlet flow rate locally affects fluid velocity, fluid shear stress, and wall shear stress inside 3D-printed scaffolds, but not gauge fluid pressure, and oxygen-concentration, which seems crucial for optimized bone tissue engineering strategies using bioreactors, scaffolds, and cells.

Global incidence and profile of ameloblastoma: A systematic review and meta-analysis.

OBJECTIVES: To evaluate the global incidence of ameloblastoma and to provide a profile of ameloblastoma patients. MATERIAL AND METHODS: A systematic review and meta-analysis was conducted. Searches were performed in PubMed, EMBASE, SCOPUS, and Web of Science for articles published from 1969 to 2018 for the global incidence and from 1995 to 2018 for the profile of ameloblastoma patients. RESULTS: Seven studies on the incidence rate of ameloblastoma were included in the meta-analysis. These studies only covered Europe, Africa, and Australia. The pooled incidence rate was 0.92 per million person-years (95% CI: 0.57-1.49), with significant heterogeneity between studies. Forty-two articles provided profile data of 6,446 ameloblastoma patients. Mean age was 34 years and the peak age incidence in the third decade of life. In Europe and North America, ameloblastoma mostly occurred at an older age when compared to Africa and South America. A slight male preference (53%) was found, and the mandible appeared to be the preferred site. The most common type of ameloblastoma was multicystic. The histopathologic patterns were mostly follicular and plexiform. CONCLUSIONS: This is the first study assessing the global incidence of ameloblastoma. The pooled incidence rate was determined to be 0.92 per million person-years.

Radical vs conservative treatment of intraosseous ameloblastoma: Systematic review and meta-analysis.

OBJECTIVES: The aim of the present study was to assess the outcomes of radical and conservative treatment approaches of solid/multicystic and unicystic ameloblastoma in terms of recurrence rates. MATERIAL AND METHODS: A systematic review and meta-analysis was conducted based on the PRISMA statement. Search was performed using PubMed, Embase, SCOPUS, and Web of Science for articles published from January 1969 until March 2018. Quality assessment of the selected articles was conducted using the Quality Appraisal of Case Series Studies Checklist. The meta-analysis was performed using the MedCalc program. RESULTS: The search strategy yielded 6,984 articles; 20 studies met the eligibility criteria and were included in the meta-analysis. The pooled recurrence rate of solid/multicystic ameloblastomas following radical treatment was 8%, while conservative treatment caused recurrences in 41%. For unicystic ameloblastomas, these values were 3% and 21%, respectively. The risk of recurrences in both types of ameloblastomas following radical treatment was lower than following conservative treatment. CONCLUSIONS: The present study showed statistically significant differences in recurrence favoring radical treatment for both unicystic and solid/multicystic ameloblastoma. The solid/multicystic type showed more recurrences than the unicystic type. Unfortunately, since only retrospective studies were available, the evidence is less strong as wished for.

StemBell therapy stabilizes atherosclerotic plaques after myocardial infarction.

BACKGROUND AIMS: After a myocardial infarction (MI) atherosclerosis is accelerated leading to destabilization of the atherosclerotic plaque. mesenchymal stromal cells are a promising therapeutic option for atherosclerosis. Previously, we demonstrated a novel stem cell delivery technique, with adipose stem cells coupled to microbubbles (i.e., StemBells) as therapy after MI. In this study, we aim to investigate the effect of StemBell therapy on atherosclerotic plaques in an atherosclerotic mouse model after MI. METHODS: MI was induced in atherosclerotic Apolipoprotein E-deficient mice that were fed a high-fat Western diet. Six days post-MI, the mice received either 5 × 105/100 µL StemBells or vehicle intravenously. The effects of StemBell treatment on the size and stability of aortic root atherosclerotic plaques and the infarcted heart were determined 28 days post-MI via (immuno)histological analyses. Moreover, monocyte subtypes and lipids in the blood were studied. RESULTS: StemBell treatment resulted in significantly increased cap thickness, decreased intra-plaque macrophage density and increased percentage of intra-plaque anti-inflammatory macrophages and chemokines, without affecting plaque size and serum cholesterol/triglycerides. Furthermore, StemBell treatment significantly increased the percentage of anti-inflammatory macrophages within the infarcted myocardium but did not affect cardiac function nor infarct size. Finally, also the average percentage of anti-inflammatory monocytes in the circulation was increased after StemBell therapy. DISCUSSION: StemBell therapy increased cap thickness and decreased intra-plaque inflammation after MI, indicative of stabilized atherosclerotic plaque. It also induced a shift of circulating monocytes and intra-plaque and intra-cardiac macrophages towards anti-inflammatory phenotypes. Hence, StemBell therapy may be a therapeutic option to prevent atherosclerosis acceleration after MI.

A review of the most promising biomarkers for early diagnosis and prognosis prediction of tongue squamous cell carcinoma.

BACKGROUND: There is a great interest in developing biomarkers to enhance early detection and clinical management of tongue squamous cell carcinoma (TSCC). However, the developmental path towards a clinically valid biomarker remains extremely challenging. Ideally, the initial key step in moving a newly discovered biomarker towards clinical implementation is independent replication. Therefore, the focus of this review is on biomarkers that consistently showed clinical relevance in two or more publications. METHODS: We searched PubMed database for relevant papers across different TSCC sample sources, i.e., body fluids (saliva, serum/plasma) and tissues. No restriction regarding the date of publication was applied except for immunohistochemistry (IHC); only studies published between 2010 and June 2017 were included. RESULTS: The search strategy identified 1429 abstracts, of which 96 papers, examining 150 biomarkers, were eventually included. Of these papers, 66% were exploratory studies evaluating single or a panel of biomarkers in one publication. Ultimately, based on studies that had undergone validation for their clinical relevance in at least two independent studies, we identified 10 promising candidates, consisting of different types of molecules (IL-6, IL-8, and Prolactin in liquid samples; HIF-1α, SOX2, E-cadherin, vimentin, MALAT1, TP53, and NOTCH1 in tissue biopsies) CONCLUSIONS: Although more exploratory research is needed with newer methods to identify biomarkers for TSCC, rigorous validation of biomarkers that have already shown unbiased assessment in at least two publications should be considered a high priority. Further research on these promising biomarkers or their combination in multi-institutional studies, could provide new possibilities to develop a specific panel for early diagnosis, prognosis, and individualized treatments.

Codelivery of doxorubicin and JIP1 siRNA with novel EphA2-targeted PEGylated cationic nanoliposomes to overcome osteosarcoma multidrug resistance.

PURPOSE: Osteosarcoma (OS) mostly affects children and young adults, and has only a 20%-30% 5-year survival rate when metastasized. We aimed to create dual-targeted (extracellular against EphA2 and intracellular against JNK-interacting protein 1 [JIP1]), doxorubicin (DOX)-loaded liposomes to treat OS metastatic disease. MATERIALS AND METHODS: Cationic liposomes contained N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and distearoyl-phosphatidylethanolamine-methyl-poly(ethylene glycol) (DSPE-mPEG) conjugate. EphA2 targeting was accomplished by conjugating YSA peptide to DSPE-mPEG. Vesicles were subsequently loaded with DOX and JIP1 siRNA. RESULTS: Characteristics assessment showed that 1) size of the bilayered particles was 109 nm; 2) DOX loading efficiency was 87%; 3) siRNA could be successfully loaded at a liposome:siRNA ratio of >24:1; and 4) the zeta potential was 18.47 mV. Tumor-mimicking pH conditions exhibited 80% siRNA and 50.7% DOX sustained release from the particles. Stability studies ensured the protection of siRNA against degradation in serum. OS cell lines showed increased and more pericellular/nuclear localizations when using targeted vesicles. Nontargeted and targeted codelivery caused 70.5% and 78.6% cytotoxicity in OS cells, respectively (free DOX: 50%). Targeted codelivery resulted in 42% reduction in the siRNA target, JIP1 mRNA, and 46% decrease in JIP1 levels. CONCLUSION: Our dual-targeted, DOX-loaded liposomes enhance toxicity toward OS cells and may be effective for the treatment of metastatic OS.

Preparation of PEGylated cationic nanoliposome-siRNA complexes for cancer therapy.

Cationic liposomes have been investigated as non-viral vectors for gene delivery for more than a decade to overcome challenges associated with viral gene delivery. However, due to instability of liposomes, siRNA delivery is still a serious problem. In this study, we developed stealth PEGylated liposome formulations and focused on the effects of PEGylated liposomes on parameters related to size, zeta potential, polydispersity index, siRNA-loading efficiency and long-term stability of the siRNA-liposome complex. We were able to generate siRNA lipoplexes that could be very efficiently loaded, did not aggregate, could be stored at 4 °C for at least 6 months with only marginal release (1-5%) of siRNA and enhanced intracellular delivery of siRNA. Moreover, we could demonstrate that PEGylation positively contributed to all these parameters compared to liposomes, which were not PEGylated. The prepared lipoplex was successfully silenced J1P1 expression in MG-63 osteosarcoma cell line. In conclusion, our novel PEGylated liposomes have high potential for systemic delivery of siRNA and can improve in vivo stability of free siRNA and also siRNA lipoplexes.

Osteocyte morphology and orientation in relation to strain in the jaw bone.

Bone mass is important for dental implant success and is regulated by mechanoresponsive osteocytes. We aimed to investigate the relationship between the levels and orientation of tensile strain and morphology and orientation of osteocytes at different dental implant positions in the maxillary bone. Bone biopsies were retrieved from eight patients who underwent maxillary sinus-floor elevation with ß-tricalcium phosphate prior to implant placement. Gap versus free-ending locations were compared using 1) a three-dimensional finite-element model of the maxilla to predict the tensile strain magnitude and direction and 2) histology and histomorphometric analyses. The finite-element model predicted larger, differently directed tensile strains in the gap versus free-ending locations. The mean percentage of mineralised residual native-tissue volume, osteocyte number (mean ± standard deviations: 97 ± 40/region-of-interest), and osteocyte shape (~90% elongated, ~10% round) were similar for both locations. However, the osteocyte surface area was 1.5-times larger in the gap than in the free-ending locations, and the elongated osteocytes in these locations were more cranially caudally oriented. In conclusion, significant differences in the osteocyte surface area and orientation seem to exist locally in the maxillary bone, which may be related to the tensile strain magnitude and orientation. This might reflect local differences in the osteocyte mechanosensitivity and bone quality, suggesting differences in dental implant success based on the location in the maxilla.

A comprehensive mathematical model of drug release kinetics from nano-liposomes, derived from optimization studies of cationic PEGylated liposomal doxorubicin formulations for drug-gene delivery.

This study focuses on the development of a universal mathematical model for drug release kinetics from liposomes to allow in silico prediction of optimal conditions for fine-tuned controlled drug release. As a prelude for combined siRNA-drug delivery, nanoliposome formulations were optimized using various mole percentages of a cationic lipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) in the presence or absence of 3 mol% distearoyl phosphoethanolamine, polyethylene glycol (PEG-2000mDSPE). Outcome parameters were particle size, zeta potential, entrapment efficiency, in vitro drug release, and tumor cell kill efficiency. The optimized formula (containing 20% DOTAP with 3% DSPE-mPEG(2000) was found to be stable for six months, with round-shaped particles without aggregate formation, an average diameter of 71 nm, a suitable positive charge, and 89% drug encapsulation efficiency (EE). The 41% drug release during 6 h confirmed controlled release. Furthermore, the release profiles as functions of pH and temperature were investigated and the kinetics of the drug release could adequately be fitted to Korsmeyer-Peppas' model by multiple regression analysis. The statistical parameters confirmed good conformity of final models. Functionality of the novel cationic liposome formulations (± DOX) was tested on osteosarcoma (OS) cell lines. Increased OS cell toxicity (1.3-fold) was observed by the DOX-loaded vs. the free DOX. A feasibility pilot showed that siRNA could be loaded efficiently as well. In conclusion, we have established a predictive mathematical model for the fine-tuning of controlled drug release from liposomal formulations, while creating functional drug-delivery liposomes with potential for siRNA co-delivery to increase specificity and efficacy.

EphA2 Targeted Doxorubicin-Nanoliposomes for Osteosarcoma Treatment.

PURPOSE: To employ Doxorubicin-loaded liposomes, modified with YSA-peptide to target EphA2, to reduce adverse effects against primary bone cells and maximize toxicity against Saos-2 osteosarcoma cells. METHODS: PEGylated liposomes were prepared by thin film method using Dipalmitoylphosphatidylcholine (DPPC), cholesterol and distearylphosphatidylethanolamine-polyethyleneglycol conjugate (DSPE-mPEG) in 67.9:29.1:3 M ratios, and loaded with DOX (L-DOX) by pH-gradient method. Targeted liposomes (YSA-L-DOX), were prepared by conjugating YSA-peptide to DSPE-mPEG. Liposomes were physicochemically characterized and tested in cellular toxicity assays. RESULTS: YSA conjugation efficiency was >98%. Size and polydispersity index of both L-DOX and YSA-L-DOX were around 88 nm and 0.188, respectively. Both had similar zeta potential, and 85% DOX loading efficiencies. DOX release kinetics followed the Korsmeyer-Peppa model, and showed comparable release for both formulations from 1-8 h, and a plateau of 29% after 48 h. Both formulations could be stably stored for ≥6 months at 4°C in the dark. Toxicity assays showed a significant 1.91-fold higher cytotoxicity compared to free DOX in the Saos-2 cells, and 2-fold lesser toxicity in primary bone cells compared to the Saos-2 cells. Cellular uptake studies showed higher and more nuclear uptake in YSA-L-DOX compared to L-DOX treated cells. CONCLUSIONS: YSA-L-DOX vesicles might be effective for targeted treatment of osteosarcoma.