In Vitro microRNA Expression Profile Alterations under CDK4/6 Therapy in Breast Cancer.

BACKGROUND: Breast cancer is the most common type of cancer worldwide. Cyclin-dependent kinase inhibition is one of the backbones of metastatic breast cancer therapy. However, there are a significant number of therapy failures. This study evaluates the biomarker potential of microRNAs for the prediction of a therapy response under cyclin-dependent kinase inhibition. METHODS: This study comprises the analysis of intracellular and extracellular microRNA-expression-level alterations of 56 microRNAs under palbociclib mono as well as combination therapy with letrozole. Breast cancer cell lines BT-474, MCF-7 and HS-578T were analyzed using qPCR. RESULTS: A palbociclib-induced microRNA signature could be detected intracellularly as well as extracellularly. Intracellular miR-10a, miR-15b, miR-21, miR-23a and miR-23c were constantly regulated in all three cell lines, whereas let-7b, let-7d, miR-15a, miR-17, miR-18a, miR-20a, miR-191 and miR301a_3p were regulated only in hormone-receptor-positive cells. Extracellular miR-100, miR-10b and miR-182 were constantly regulated across all cell lines, whereas miR-17 was regulated only in hormone-receptor-positive cells. CONCLUSIONS: Because they are secreted and significantly upregulated in the microenvironment of tumor cells, miRs-100, -10b and -182 are promising circulating biomarkers that can be used to predict or detect therapy responses under CDK inhibition. MiR-10a, miR-15b, miR-21, miR-23a and miR-23c are potential tissue-based biomarkers.

Tobamovirus infection aggravates gray mold disease caused by Botrytis cinerea by manipulating the salicylic acid pathway in tomato.

Botrytis cinerea is the causative agent of gray mold disease, and infects more than 1400 plant species, including important crop plants. In tomato, B. cinerea causes severe damage in greenhouses and post-harvest storage and transport. Plant viruses of the Tobamovirus genus cause significant damage to various crop species. In recent years, the tobamovirus tomato brown rugose fruit virus (ToBRFV) has significantly affected the global tomato industry. Most studies of plant-microbe interactions focus on the interaction between the plant host and a single pathogen, however, in agricultural or natural environments, plants are routinely exposed to multiple pathogens. Here, we examined how preceding tobamovirus infection affects the response of tomato to subsequent infection by B. cinerea. We found that infection with the tobamoviruses tomato mosaic virus (ToMV) or ToBRFV resulted in increased susceptibility to B. cinerea. Analysis of the immune response of tobamovirus-infected plants revealed hyper-accumulation of endogenous salicylic acid (SA), upregulation of SA-responsive transcripts, and activation of SA-mediated immunity. Deficiency in SA biosynthesis decreased tobamovirus-mediated susceptibility to B. cinerea, while exogenous application of SA enhanced B. cinerea symptoms. These results suggest that tobamovirus-mediated accumulation of SA increases the plants' susceptibility to B. cinerea, and provide evidence for a new risk caused by tobamovirus infection in agriculture.

Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.

Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.

Dermatology and medical law: the effect of legal issues on the treatment of patients in dermatological practices in southern Germany

Background: Usage of modern therapies in the treatment of chronic dermatological diseases has proven to be effective but associated with high costs. High therapy costs might raise legal issues and even lead to recourse claims. Objectives: To evaluate dermatologists' interests and knowledge in medical law and the occurrence of recourse claims, and to assess the impact of medical law on clinical practice. Materials & Methods: Dermatologists of the "Psoriasis-Praxisnetz Süd-West e.V." participated in a web-based questionnaire study investigating the relationship between medical law and usage of modern therapies. The questionnaire was separated in two sub-polls carried out from 11/2016 to 12/2016 and 02/2017 to 03/2017, respectively. The first addressed general topics of medical law and the second specific legal topics, particularly recourse claims. Results: Overall, 76 dermatologists participated in the first and 66 in the second sub-poll. In the first sub-poll, 27.6% of participants attended a seminar on medical law within the last 12 months. Furthermore, 28.8% of the participants of the second sub-poll already experienced a previous recourse claim, and 26.3% of those stated feeling confident or rather confident on legal topics. This proportion was lower among those who had not experienced a recourse claim (17.0%). Overall, 73.7% of those who had a previous recourse claim changed their prescription behaviour as a direct consequence thereof. Conclusion: The study demonstrates a close relationship between medical law issues and the prescription behaviour of dermatologists working in private practices in southern Germany. Regular legal education would thus be beneficial for patient-centred care.

The impact of the first COVID-19 wave on office-based dermatological care in Germany: a focus on diagnosis, therapy and prescription of biologics

Background: The COVID-19 pandemic has led to widespread changes in medical care. However, it is still unclear to what extent the care of patients suffering from moderate-to-severe psoriasis, chronic spontaneous urticaria or atopic dermatitis has been affected. Objectives: This study was conducted to determine the impact of the SARS-CoV-2 pandemic on medical care in dermatological practices, focusing on physicians' concerns related to susceptibility to infections in combination with different treatment modalities. Materials & Methods: Dermatologists working in medical offices in the German federal states of Bavaria and Lower Saxony participated in a cross-sectional, non-interventional, questionnaire-based study investigating the influence of COVID-19 on dermatological care. The study was performed after the first wave of the coronavirus pandemic in July/August, 2020. Results: A total of 195 dermatologists participated in the study. Almost one in five practices were closed for at least one week during the pandemic. The care of patients with chronic inflammatory skin diseases was impaired, affecting diagnostic investigation. Physicians stated that the pandemic substantially influenced systemic therapy. Nearly half of physicians surveyed were concerned about increased susceptibility to infections under biological therapy. No significant differences were identified between the German federal states of Bavaria and Lower Saxony in the south and north of Germany, respectively. Conclusion: This study reveals a significant impact of the COVID-19 pandemic on the care of dermatological patients in medical offices in Germany. New management modalities and continuous education are needed to improve care in pandemic situations.

Evaluation of circulating microRNAs as non-invasive biomarkers in the diagnosis of ovarian cancer: a case-control study.

PURPOSE: Ovarian cancer is the seventh most frequent form of malignant diseases in women worldwide and over 150,000 women die from it every year. More than 70 percent of all ovarian cancer patients are diagnosed at a late-stage disease with poor prognosis necessitating the development of sufficient screening biomarkers. MicroRNAs displayed promising potential as early diagnostics in various malignant diseases including ovarian cancer. The presented study aimed at identifying single microRNAs and microRNA combinations detecting ovarian cancer in vitro and in vivo. METHODS: Intracellular, extracellular and urinary microRNA expression levels of twelve microRNAs (let-7a, let-7d, miR-10a, miR-15a, miR-15b, miR-19b, miR-20a, miR-21, miR-100, miR-125b, miR-155, miR-222) were quantified performing quantitative real-time-PCR. Therefore, the three ovarian cancer cell lines SK-OV-3, OAW-42, EFO-27 as well as urine samples of ovarian cancer patients and healthy controls were analyzed. RESULTS: MiR-15a, miR-20a and miR-222 showed expression level alterations extracellularly, whereas miR-125b did intracellularly across the analyzed cell lines. MicroRNA expression alterations in single cell lines suggest subtype specificity in both compartments. Hypoxia and acidosis showed scarce effects on single miRNA expression levels only. Furthermore, we were able to demonstrate the feasibility to clearly detect the 12 miRNAs in urine samples. In urine, miR-15a was upregulated whereas let-7a was down-regulated in ovarian cancer patients. CONCLUSION: Intracellular, extracellular and urinary microRNA expression alterations emphasize their great potential as biomarkers in liquid biopsies. Especially, miR-15a and let-7a qualify for possible circulating biomarkers in liquid biopsies of ovarian cancer patients.

Anticarcinogenic Effects of Odorant Substances Citral, Citrathal R and Cyclovertal on Breast Cancer in vitro.

PURPOSE: In 2020, breast cancer still represents the most common type of cancer in women worldwide. Depending on the specific molecular subtype, clinical breast cancer management comprises surgery, radiotherapy, chemotherapy and targeted therapy. Furthermore, there are some therapeutic approaches from the field of complementary and alternative medicine. Current research focuses on the elucidation of new therapeutic targets for treatment development. Odorant substances affect apoptosis, proliferation and cell cycle in healthy and cancerous cells. Exact signalling pathways involved are not entirely clear. The present study aims to analyse their therapeutic potential in breast cancer. METHODS: This study focuses on the effect of commonly used odorant substances (citral, citrathal R, cyclovertal, para-cymol, hexylacetat, herbavert, dihydromyrcerol and limonen) on the breast cancer cell lines MDA-MB-231, T47-D and BT474. Methodologically, this study applied cell culturing, MTT assay for detection of IC50 of the odorant substance, RNA purification followed by qRT-PCR, protein isolation and Western Blot, as well as immunocytochemistry. Further, this study investigates the role of transient receptor potential channel V1 (TRPV1), involved in the mechanisms of action for some odorant substances. Therefore, capsazepine, a TRPV1 antagonist, was used. RESULTS: The odorant substances citral, citrathal R and cyclovertal have significant pro-apoptotic (p < 0.001), anti-proliferative (p < 0.001) and cell cycle-arresting effects measurable in RNA expression as well as in protein levels and immunocytochemical staining. The combination of citral and capsazepine no longer showed significant pro-apoptotic, antiproliferative, and cell cycle inhibitory effects compared to the compounds alone. This indicates that TRPV1 is necessary for the signal transduction of citral. CONCLUSION: This present study reveals three odorant substances with effects on cell viability, indicating their potential use in breast cancer therapy.

Prescription Behaviour and Barriers to Prescription of Biologicals for Treatment of Chronic Inflammatory Skin Diseases in Dermatological Practice in Two German Federal States.

The globally increasing prevalence of chronic inflammatory skin diseases has substantial costs. Biologicals have become available as therapeutic options, but are encumbered with barriers to prescription. The aim of this study was to evaluate the barriers to prescription of biologicals in the treatment of chronic dermatological diseases. Dermatologists working in private practices in the German federal states of Bavaria and Lower Saxony participated in a cross-sectional study. Economic and legal aspects, including "high therapy costs", "low reimbursements", and "fear of regress claims", were identified as the most prevalent barriers. Significant differences between dermatologists from Bavaria and Lower Saxony were found only regarding the treatment of atopic dermatitis. This study demonstrates the prevalence of barriers to the prescription of biologicals in the treatment of chronic dermatological diseases. Overcoming these barriers could improve the usage of modern therapies and thereby expand patient-centred care for chronic skin diseases.

Detection of microRNA in urine to identify patients with endometrial cancer: a feasibility study.

OBJECTIVE: To find dysregulated urinary microRNAs associated with endometrial cancer as a first step in finding a non-invasive new diagnostic biomarker. The second objective is to determine the correlation of urinary microRNAs with clinicopathological characteristics. METHODS: A prospective cohort study of patients presenting with abnormal bleeding between March and November 2019 was performed at the Royal Cornwall Hospital Trust Truro. Urine samples were obtained from women diagnosed with endometrial cancer and benign endometrial sampling. MicroRNA was isolated and quantitative real time PCR was used to detect expression levels of microRNAs. RESULTS: A total of 61 women were included in this study: 24 endometrial cancer patients, and 37 controls. Median age was 64 years (range 45-94) and median body mass index was 29 kg/m2 (range 17-54). MiR-223 was significantly up-regulated in urine of endometrial cancers patients (p=0.003). Furthermore, let7-i, miR-34a, and miR-200c were significantly down-regulated and miR-424 was up-regulated in obese women. In addition, miR-148a and miR-222 were significantly down-regulated in elderly women, and miR-16, miR-26b, and miR-200c were significantly deregulated in women with multiple comorbidities. CONCLUSION: MicroRNA expression levels in urine can potentially be used as a non-invasive diagnostic test for endometrial cancer. Furthermore, aberrant microRNA expression in urine is associated with patient characteristics. Further research in larger trials is needed to validate the potential utility of urinary microRNAs.

Usefulness of microRNA detection in the diagnostics of endometrial cancer.

INTRODUCTION: MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression and contribute to the development of cancer. They have been shown to be stable in tissue samples and may be promising diagnostic biomarkers for endometrial cancer. MATERIAL AND METHODS: A retrospective cohort study of women diagnosed with endometrial cancer between January 2017 and December 2017 was performed at the Royal Cornwall Hospital. Archived formalin-fixed paraffin-embedded samples were obtained from patients with endometrial cancer and healthy women. MicroRNA was isolated and quantitative real-time polymerase chain reaction was used to detect expression levels of miRNAs. RESULTS: A total of 76 women were included: 36 endometrial cancer patients, 40 healthy controls. A distinct panel of miR-200a, miR-200b, miR-200c, miR-205, and miR-182 showed an area under the curve of 0.958, sensitivity 92%, specificity 89%, positive predictive value of 89% (95% CI 82%-94%) and negative predictive value of 91% (95% CI 85%-96%) in diagnosing endometrial cancer. High miR-182 expression levels were significantly related to high-grade endometrioid tumors compared with low-grade tumors. CONCLUSIONS: We demonstrated high diagnostic accuracy of miRNA for detecting endometrial cancer. In addition, miRNA contributed to an improvement in distinguishing between high-grade and low-grade endometrioid tumors.

Mutually distinguishing microRNA signatures of breast, ovarian and endometrial cancers in vitro.

Early diagnosis and therapy in the first stages of a malignant disease is the most crucial factor for successful cancer treatment and recovery. Currently, there is a high demand for novel diagnostic tools that indicate neoplasms in the first or pre­malignant stages. MicroRNAs (miRNA or miR) are small non­coding RNAs that may act as oncogenes and downregulate tumor­suppressor genes. The detection and mutual discrimination of the three common female malignant neoplasia types breast (BC), ovarian (OC) and endometrial cancer (EC) could be enabled by identification of tumor entity­specific miRNA expression differences. In the present study, the relative expression levels of 25 BC, EC and OC­related miRNAs were assessed by reverse transcription­quantitative PCR and determined using the 2­ΔΔCq method for normalization against the mean of four housekeeping genes. Expression levels of all miRNAs were analyzed by regression against cell line as a factor. An expression level­based discrimination between BC and OC cell types was obtained for a subgroup of ten different miRNA types. miR­30 family genes, as well as three other miRNAs, were found to be uniformly upregulated in OC cells compared with BC cells. BC and EC cells could be distinguished by the expression profiles of six specific miRNAs. In addition, four miRNAs were differentially expressed between EC and OC cells. In conclusion, miRNAs were identified as a potential novel tool to detect and mutually discriminate between BC, OC and EC. Based on a subset of 25 clinically relevant human miRNA types, the present study could significantly discriminate between these three female cancer types by means of their expression levels. For further verification and validation of miRNA­based biomarker expression signatures that enable valuable tumor detection and characterization in routine screening or potential therapy monitoring, additional and extended in vitro analyses, followed by translational studies utilizing patients' tissue and liquid biopsy materials, are required.

Urinary Exosomal MicroRNAs as Potential Non-invasive Biomarkers in Breast Cancer Detection.

INTRODUCTION: Breast cancer (BC) is the most frequent malignant disease in women worldwide and is therefore challenging for the healthcare system. Early BC detection remains a leading factor that improves overall outcome and disease management. Aside from established screening procedures, there is a constant demand for additional BC detection methods. Routine BC screening via non-invasive liquid biopsy biomarkers is one auspicious approach to either complete or even replace the current state-of-the-art diagnostics. The study explores the diagnostic potential of urinary exosomal microRNAs with specific BC biomarker characteristics to initiate the potential prospective application of non-invasive BC screening as routine practice. METHODS: Based on a case-control study (69 BC vs. 40 healthy controls), expression level quantification and subsequent biostatistical computation of 13 urine-derived microRNAs were performed to evaluate their diagnostic relevance in BC. RESULTS: Multilateral statistical assessment determined and repeatedly confirmed a specific panel of four urinary microRNA types (miR-424, miR-423, miR-660, and let7-i) as a highly specific combinatory biomarker tool discriminating BC patients from healthy controls, with 98.6% sensitivity and 100% specificity. DISCUSSION: Urine-based BC diagnosis may be achieved through the analysis of distinct microRNA panels with proven biomarker abilities. Subject to further validation, the implementation of urinary BC detection in routine screening offers a promising non-invasive alternative in women's healthcare.

Discovery of potential serum and urine-based microRNA as minimally-invasive biomarkers for breast and gynecological cancer.

BACKGROUND: Deregulated microRNAs (miRNAs) in breast and gynecological cancer might contribute to improve early detection of female malignancies. OBJECTIVE: Specification of miRNA types in serum and urine as minimally-invasive biomarkers for breast (BC), endometrial (EC) and ovarian cancer (OC). METHODS: In a discovery phase, serum and urine samples from 17 BC, five EC and five OC patients vs. ten healthy controls (CTRL) were analyzed with Agilent human miRNA microarray chip. Selected miRNA types were further investigated by RT-qPCR in serum (31 BC, 13 EC, 15 OC patients, 32 CTRL) and urine (25 BC, 10 EC, 10 OC patients, 30 CTRL) applying two-sample t-tests. RESULTS: Several miRNA biomarker candidates exhibited diagnostic features due to distinctive expression levels (serum: 26; urine: 22). Among these, miR-518b, -4719 and -6757-3p were found specifically deregulated in BC serum. Four, non-entity-specific, novel biomarker candidates with unknown functional roles were identified in urine (miR-3973; -4426; -5089-5p and -6841). RT-qPCR identified miR-484/-23a (all p⩽ 0.001) in serum as potential diagnostic markers for EC and OC while miR-23a may also serve as an endogenous control in BC diagnosis. CONCLUSIONS: Promising miRNAs as liquid biopsy-based tools in the detection of BC, EC and OC qualified for external validation in larger cohorts.

Circulating non­coding RNA­biomarker potential in neoadjuvant chemotherapy of triple negative breast cancer?

Due to the positive association between neoadjuvant chemotherapy (NACT) and the promising early response rates of patients with triple negative breast cancer (TNBC), including probabilities of pathological complete response, NACT is increasingly used in TNBC management. Liquid biopsy­based biomarkers with the power to diagnose the early response to NACT may support established monitoring tools, which are to a certain extent imprecise and costly. Simple serum­ or urine­based analyses of non­coding RNA (ncRNA) expression may allow for fast, minimally­invasive testing and timely adjustment of the therapy regimen. The present study investigated breast cancer­related ncRNAs [microRNA (miR)­7, ­9, ­15a, ­17, ­18a, ­19b, ­21, ­30b, ­222 and ­320c, PIWI­interacting RNA­36743 and GlyCCC2] in triple positive BT­474 cells and three TNBC cell lines (BT­20, HS­578T and MDA­MB­231) treated with various chemotherapeutic agents using reverse transcription­quantitative PCR. Intracellular and secreted microvesicular ncRNA expression levels were analysed using a multivariable statistical regression analysis. Chemotherapy­driven effects were investigated by analysing cell cycle determinants at the mRNA and protein levels. Serum and urine specimens from 8 patients with TNBC were compared with 10 healthy females using two­sample t­tests. Samples from the patients with TNBC were compared at two time points. Chemotherapeutic treatments induced distinct changes in ncRNA expression in TNBC cell lines and the BT­474 cell line in intra­ and extracellular compartments. Serum and urine­based ncRNA expression analysis was able to discriminate between patients with TNBC and controls. Time point comparisons in the urine samples of patients with TNBC revealed a general rise in the level of ncRNA. Serum data suggested a potential association between piR­36743, miR­17, ­19b and ­30b expression levels and an NACT­driven complete clinical response. The present study highlighted the potential of ncRNAs as liquid biopsy­based biomarkers in TNBC chemotherapy treatment. The ncRNAs tested in the present study have been previously investigated for their involvement in BC or TNBC chemotherapy responses; however, these previous studies were restricted to patient tissue or in vitro models. The data from the present study offer novel insight into ncRNA expression in liquid samples from patients with TNBC, and the study serves as an initial step in the evaluation of ncRNAs as diagnostic biomarkers in the monitoring of TNBC therapy.

Hypoxia- and acidosis-driven aberrations of secreted microRNAs in endometrial cancer in vitro.

Due to their post-transcriptional regulatory impact on gene expression, microRNAs (miRNA, miRs) influence decisively cellular processes of differentiation, proliferation and apoptosis. In oncogenic pathways various miRNAs exert either oncogenic or tumor suppressor activities in a stage-specific manner. Dysregulation of miRNA expression pattern has been associated with several human cancers including endometrial cancer (EC). In the present study, expression profile alterations of EC associated secreted miRNAs were determined under the microenvironmental stress situations hypoxia and acidosis occurring in tumor progression and metastasis. The potential influence of hypoxia and acidosis vs. control conditions on the expression levels of 24 EC-relevant miRNA types was quantitatively accessed via real-time PCR in three established EC in vitro models. Expression data were analyzed statistically. In vitro application of hypoxia resulted in downregulation of miR-15a, miR-20a, miR-20b and miR-128-1 in Ishikawa cells (type I EC) and upregulation of miR-21 in EFE-184 cells (type I EC). Acidosis triggered upregulation of tumor promoting miR-125b in AN3-CA cell (type II EC), whereas in Ishikawa cells (type I EC) miRNAs with tumor suppressive function were found altered in divergent directions, both up- (let-7a) and down- (miR-22) regulated. Our current findings emphasize the functional importance of secreted miRNAs in the immediate response of EC cells to exogenic stress situations such as the typical tumor epiphenomena hypoxia and acidosis. Focusing on the specific potential of secreted, thus circulating miRNA molecules, alterations in expression levels not only influence intracellular gene expression and signaling cascades, but also transfer the induction of (tumor)biological cellular changes to adjacent cells.

Amifostine protects vascularity and improves union in a model of irradiated mandibular fracture healing.

BACKGROUND: Pathologic fractures of the mandible can be devastating to cancer patients and are due in large part to the pernicious effects of irradiation on bone vascularity. The authors' aim was to ascertain whether amifostine, a radioprotective drug, will preserve vascularity and improve bone healing in a murine model of irradiated mandibular fracture repair. METHODS: Rats were randomized into three groups: nonirradiated fracture (n = 9), irradiation/fracture (n = 5), and amifostine/irradiation/fracture (n = 7). Animals in the irradiation groups underwent a human equivalent dose of radiation directed at the left hemimandible. Animals treated in the amifostine group received amifostine concomitantly with radiation. All animals underwent unilateral left mandibular osteotomy with external fixation set to a 2.1-mm fracture gap. Fracture healing was allowed for 40 days before perfusion with Microfil. Vascular radiomorphometrics were quantified with micro-computed tomography. RESULTS: When compared with the irradiated/fractured group, amifostine treatment more than doubled the rate of fracture unions to 57 percent. Amifostine treatment also resulted in an increase in vessel number (123 percent; p < 0.05) and a corresponding decrease in vessel separation (55.5 percent; p < 0.05) there was no statistical difference in the vascularity metrics between the amifostine/irradiation/fracture group and the nonirradiated/fracture group. CONCLUSIONS: Amifostine prophylaxis during radiation maintains mandibular vascularity at levels observed in nonirradiated fracture specimens, corresponding to improved unions. These results set the stage for clinical exploration of this targeted therapy alone and in combination with other treatments, to mitigate the effects of irradiation on bone healing and fracture repair.

The effect of Amifostine prophylaxis on bone densitometry, biomechanical strength and union in mandibular pathologic fracture repair.

BACKGROUND: Pathologic fractures (Fx) of the mandibles are severely debilitating consequences of radiation (XRT) in the treatment of craniofacial malignancy. We have previously demonstrated Amifostine's effect (AMF) in the remediation of radiation-induced cellular damage. We posit that AMF prophylaxis will preserve bone strength and drastically reverse radiotherapy-induced non-union in a murine mandibular model of pathologic fracture repair. MATERIALS AND METHODS: Twenty-nine rats were randomized into 3 groups: Fx, XRT/Fx, and AMF/XRT/Fx. A fractionated human equivalent dose of radiation was delivered to the left hemimandibles of XRT/Fx and AMF/XRT/Fx. AMF/XRT/Fx was pre-treated with AMF. All groups underwent left mandibular osteotomy with external fixation and setting of a 2.1mm fracture gap post-operatively. Utilizing micro-computed tomography and biomechanical testing, the healed fracture was evaluated for strength. RESULTS: All radiomorphometrics and biomechanical properties were significantly diminished in XRT/Fx compared to both Fx and AMF/XRT/Fx. No difference was demonstrated between Fx and AMF/XRT/Fx in both outcomes. CONCLUSION: Our investigation establishes the significant and substantial capability of AMF prophylaxis to preserve and enhance bone union, quality and strength in the setting of human equivalent radiotherapy. Such novel discoveries establish the true potential to utilize pharmacotherapy to prevent and improve the treatment outcomes of radiation-induced late pathologic fractures.

An isogenic model of murine mandibular distraction osteogenesis.

The advent of stem cell-based therapies makes current models of mandibular distraction osteogenesis unwieldy. We thereby designed an isogenic model of distraction osteogenesis whose purpose was to allow for the free transfer of cells and components between rats. As immune response plays a significant role in healing and prevention of infection, an immune-competent mode is desirable rather than an athymic rat/xenograft model. The purposes of this study were as follows: (1) to replicate established models of distraction osteogenesis in a rodent model using an isogenic rat strain, and (2) to characterize the differences between inbred, isogenic rats and outbred rats in mandibular distraction osteogenesis via radiomorphometry and biomechanical response analysis. We demonstrated successful distraction osteogenesis to 5.1 mm in all Lewis (isogenic) rat mandibles as well as all Sprague-Dawley (outbred) rat mandibles, with no significant difference in volume-normalized radiomorphometrics, trending difference in non-volume-normalized radiomorphometrics and significant differences in biomechanical response parameters. We attribute the differences demonstrated to the decreased size of the Lewis rat mandible in comparison to Sprague-Dawley mandibles. We also provide information with caring with the additional needs of the Lewis rat. Given these differences, we find that Lewis rats function as an excellent model for isogenic mandibular distraction osteogenesis, but data procured may not be comparable between isogenic and nonisogenic models.

Localized deferoxamine injection augments vascularity and improves bony union in pathologic fracture healing after radiotherapy.

BACKGROUND: Medically based efforts and alternative treatment strategies to prevent or remediate the corrosive effects of radiotherapy on pathologic fracture healing have failed to produce clear and convincing evidence of success. Establishing an effective pharmacologic option to prevent or treat the development of non-unions in this setting could have immense therapeutic potential. Experimental studies have shown that deferoxamine (DFO), an iron-chelating agent, bolsters vascularity and subsequently enhances normal fracture healing when injected locally into a fracture callus in long bone animal models. Since radiotherapy is known to impede angiogenesis, we hypothesized that the pharmacologic addition of DFO would serve to mitigate the effects of radiotherapy on new vessel formation in vitro and in vivo. MATERIALS AND METHODS: In vitro investigation of angiogenesis was conducted utilizing HUVEC cells in Matrigel. Endothelial tubule formation assays were divided into four groups: Control, Radiated, Radiated+Low-Dose DFO and Radiated+High-Dose DFO. Tubule formation was quantified microscopically and video recorded for the four groups simultaneously during the experiment. In vivo, three groups of Sprague-Dawley rats underwent external fixator placement and fracture osteotomy of the left mandible. Two groups received pre-operative fractionated radiotherapy, and one of these groups was treated with DFO after fracture repair. After 40 days, the animals were perfused and imaged with micro-CT to calculate vascular radiomorphometrics. RESULTS: In vitro, endothelial tubule formation assays demonstrated that DFO mitigated the deleterious effects of radiation on angiogenesis. Further, high-dose DFO cultures appeared to organize within 2h of incubation and achieved a robust network that was visibly superior to all other experimental groups in an accelerated fashion. In vivo, animals subjected to a human equivalent dose of radiotherapy (HEDR) and left mandibular fracture demonstrated quantifiably diminished µCT metrics of vascular density, as well as a 75% incidence of associated non-unions. The addition of DFO in this setting markedly improved vascularity as demonstrated with 3D angiographic modeling. In addition, we observed an increased incidence of bony unions in the DFO treated group when compared to radiated fractures without treatment (67% vs. 25% respectively). CONCLUSION: Our data suggest that selectively targeting angiogenesis with localized DFO injections is sufficient to remediate the associated severe vascular diminution resulting from a HEDR. Perhaps the most consequential and clinically relevant finding was the ability to reduce the incidence of non-unions in a model where fracture healing was not routinely observed.