Three-Dimensional Bioprinting of GelMA Hydrogels with Culture Medium: Balancing Printability, Rheology and Cell Viability for Tissue Regeneration.

Three-dimensional extrusion bioprinting technology aims to become a fundamental tool for tissue regeneration using cell-loaded hydrogels. These biomaterials must have highly specific mechanical and biological properties that allow them to generate biosimilar structures by successive layering of material while maintaining cell viability. The rheological properties of hydrogels used as bioinks are critical to their printability. Correct printability of hydrogels allows the replication of biomimetic structures, which are of great use in medicine, tissue engineering and other fields of study that require the three-dimensional replication of different tissues. When bioprinting cell-loaded hydrogels, a small amount of culture medium can be added to ensure adequate survival, which can modify the rheological properties of the hydrogels. GelMA is a hydrogel used in bioprinting, with very interesting properties and rheological parameters that have been studied and defined for its basic formulation. However, the changes that occur in its rheological parameters and therefore in its printability, when it is mixed with the culture medium necessary to house the cells inside, are unknown. Therefore, in this work, a comparative study of GelMA 100% and GelMA in the proportions 3:1 (GelMA 75%) and 1:1 (GelMA 50%) with culture medium was carried out to determine the printability of the gel (using a device of our own invention), its main rheological parameters and its toxicity after the addition of the medium and to observe whether significant differences in cell viability occur. This raises the possibility of its use in regenerative medicine using a 3D extrusion bioprinter.

Fabrication and characterisation of bioglass and hydroxyapatite-filled scaffolds.

Tissue engineering is a continuously evolving field. One of the main lines of research in this field focuses on the replacement of bone defects with materials designed to interact with the cells of a living organism in order to provide the body with a structure on which new tissues can easily grow. Among the most commonly used materials are bioglasses, which are frequently used due to their versatility and good properties. This article discusses the results of the production of an injectable paste of Bioglass® 45S5 and hydroxyapatite on a 3D printed porous structure by additive manufacturing, using a thermoplastic (PLA). The results were evaluated in a specific application of the paste, so the mechanical and bioactive properties were studied to show the multiple possibilities of using this combination for its application in regenerative medicine and more specifically in bone implants.

Comparison of the potential for bioprinting of different 3D printing technologies.

26Additive manufacturing technologies offer a multitude of medical applications due to the advances in the development of the materials used to reproduce customized model products. The main problem with these technologies is obtaining the correct cell viability values, and it is where three-dimensional (3D) bioprinting emerges as a very interesting tool that should be studied extensively, as it has significant disadvantages with respect to printability. In this work, the comparison of 3D bioprinting technology in hydrogels and thermoplastics for the development of biomimetic parts is proposed. To this end, the study of the printability of different materials widely used in the literature is proposed, to subsequently test and analyze the parameters that indicate whether these materials could be used to obtain a biomimetic structure with structural guarantees. In order to analyze the materials studied, different tools have been designed to facilitate the quantitative characterization of their printability using 3D printing. For this purpose, different structures have been developed and a characterization methodology has been followed to quantify the printability value of each material in each test to subsequently discard the materials that do not obtain a minimum value in the result. After the study, it was found that only gelatin methacryloyl (GelMA) 5% could generate biomimetic structures faithful to the designed 3D model. Furthermore, by comparing the printing results of the different materials used in 3D bioprinting and consequently establishing the approach of different strategies, it is shown that hydrogels need to be further developed to match the results achieved by thermoplastic materials used for bioprinting.

Evolution of bioprinting and current applications.

Bioprinting is a very useful tool that has a huge application potential in different fields of science and biotechnology. In medicine, advances in bioprinting are focused on the printing of cells and tissues for skin regeneration and the manufacture of viable human organs, such as hearts, kidneys, and bones. This review provides a chronological overview of some of the most relevant developments of bioprinting technique and its current status. A search was carried out in SCOPUS, Web of Science, and PubMed databases, and a total of 31,603 papers were found, of which 122 were finally chosen for analysis. These articles cover the most important advances in this technique at the medical level, its applications, and current possibilities. Finally, the paper ends with conclusions about the use of bioprinting and our expectations of this technique. This paper presents a review on the tremendous progress of bioprinting from 1998 to the present day, with many promising results indicating that our society is getting closer to achieving the total reconstruction of damaged tissues and organs and thus solving many healthcare-related problems, including the shortage of organ and tissue donors.

Methodology for characterizing the printability of hydrogels.

280Currently, the characterization techniques for hydrogels used in bioprinting are extensive, and they could provide data on the physical, chemical, and mechanical properties of hydrogels. While characterizing the hydrogels, the analysis of their printing properties is of great importance in the determination of their potential for bioprinting. The study of printing properties provides data on their capacity to reproduce biomimetic structures and maintain their integrity after the process, as it also relates them to the possible cell viability after the generation of the structures. Current hydrogel characterization techniques require expensive measuring instrument that is not readily available in many research groups. Therefore, it would be interesting to propose a methodology to characterize and compare the printability of different hydrogels in a fast, simple, reliable, and inexpensive way. The aim of this work is to propose a methodology for extrusion-based bioprinters that allows determining the printability of hydrogels that are going to be loaded with cells, by analyzing cell viability with the sessile drop method, molecular cohesion with the filament collapse test, adequate gelation with the quantitative evaluation of the gelation state, and printing precision with the printing grid test. The data obtained after performing this work allow the comparison of different hydrogels or different concentrations of the same hydrogel to determine which one has the most favorable properties to carry out bioprinting studies.

Relationship between shear-thinning rheological properties of bioinks and bioprinting parameters.

Three-dimensional bioprinting is a technology in constant development, mainly due to its extraordinary potential to revolutionize regenerative medicine. It allows fabrication through the additive deposition of biochemical products, biological materials, and living cells for the generation of structures in bioengineering. There are various techniques and biomaterials or bioinks that are suitable for bioprinting. Their rheological properties are directly related to the quality of these processes. In this study, alginate-based hydrogels were prepared using CaCl2 as ionic crosslinking agent. Their rheological behavior was studied, and simulations of the bioprinting processes under predetermined conditions were carried out, looking for possible relationships between the rheological parameters and the variables used in the bioprinting processes. A clear linear relationship was found between the extrusion pressure and the flow consistency index rheological parameter, k, and between the extrusion time and the flow behavior index rheological parameter, n. This would allow simplification of the repetitive processes currently applied to optimize the extrusion pressure and dispensing head displacement speed, thereby helping to reduce the time and material used as well as to optimize the required bioprinting results.

Design of Industrial Standards for the Calibration of Optical Microscopes.

One of the most important fields of study in material science is surface characterization. This topic is currently a field of growing interest as many functional properties depend on the surface texture. In this paper the authors, after a short a review of different methods for surface topography characterization and the determination of the traceability problems that arise in this type of measurements, propose four different designs of material standards that can be used to calibrate the most common optical measuring instruments used for these tasks, such as measuring microscopes, metallurgical microscopes, confocal microscopes, focus variation microscopes, etc. The authors consider that the use of this type of standards (or others similar to them) could provide a step forward in assuring metrological traceability for different metrological characteristics that enables a more precise measurement of surface features with optical measuring instruments. In addition, authors expect that this work could lay the groundwork for the development of custom standards with specialized features tuned to gain a better metrological control when measuring specific geometrical surface properties.

VITAL phase 2 study: Upfront 5-fluorouracil, mitomycin-C, panitumumab and radiotherapy treatment in nonmetastatic squamous cell carcinomas of the anal canal (GEMCAD 09-02).

AIM: VITAL, a phase II single-arm study, aimed to evaluate efficacy and safety of panitumumab addition to 5-fluorouracil (5-FU), mitomycin-C (MMC) and radiotherapy (RT) in patients with localized squamous cell carcinoma of the anal canal (SCCAC). METHODS: Adult, treatment-naïve SCCAC patients (Stage T2-T4, any N, M0) and ECOG-PS ≤2, received panitumumab (6 mg/kg, day 1 and Q2W; 8 weeks), 5-FU (1000 mg/m2 /d, days 1-4 and 29-32), MMC (10 mg/m2 , days 1 and 29) and RT 45 Gy (1.8 Gy/fraction) to the primary tumor and mesorectal, iliac and inguinal lymph nodes, plus 10-15 Gy boost dose to the primary tumor and affected lymph nodes. The primary objective was disease free survival rate (DFS) at 3-years (expected 3-year DFS rate: 73.7 ± 12%). RESULTS: Fifty-eight patients (31 women; median age: 59 years; ECOG-PS 0-1:98%; TNM II [29%] (T2 or T3/N0/M0)/IIIA (T1-T3/N1/M0 or T4/N0/M0) [21%]/IIIB (T4/N1/M0 or any T/N2 or N3/M0) [47%]/nonevaluable [4%]) were included. The median follow-up was 45 months. The 3-year DFS rate was 61.1% (95% CI: 47.1, 72.4). The 3-year overall survival rate was 78.4% (95% CI: 65.1, 87.1). Eighteen patients (31.0%) required a colostomy within 2 years posttreatment. Grade 3-4 toxicities were experienced by 53 (91%) patients. Most common grade 3-4 treatment-related events were radiation skin injury (40%) and neutropenia (24%). No toxic deaths occurred. Improved efficacy in colostomy-free survival and complete response rate was observed in human papilloma virus positive patients. CONCLUSIONS: Panitumumab addition to MMC-5FU regimen in SCCAC patients increases toxicity and does not improve patients' outcomes. RT plus MMC-5FU remains the standard of care for localized SCCAC patients.

Products based on olive oil, betaine, and xylitol in the post-radiotherapy xerostomia.

AIM: The objective of this study was determining if the use of products based in olive oil, betaine and xylitol are efficacious to decrease the impact of the dry mouth in the quality of life of the patients with xerostomia due to radiotherapy treatment. BACKGROUND: Following therapeutic irradiation of the head and neck, patients with profound xerostomia have complaints associated with oral dryness, speech, and taste. There is no strong evidence that any topical therapy is effective for relieving the symptom of dry mouth. MATERIAL AND METHODS: 40 patients who had been treated with radiotherapy for head and neck carcinoma and reported symptoms of dry mouth were included in the study. A xerostomia-related quality of life questionnaire, visual analogue scale questionnaire for subjective assessment of salivary dysfunction and salivary flow were reported before and 15 days after the use of topical products based on olive oil, betaina and xylitol. RESULTS: The four primary quality of life areas demonstrated significantly greater improvement after the use of topical products and all eight VAS items had favourable changes. The reduction of symptoms was statistically significant in 7 of the 8 items. After the use of the products, there were improvements in salivary flow in 45%. CONCLUSIONS: The use of products based on olive oil, betaine and xylitol, shaped like collutory, toothpaste, gel and spray significantly improved most symptoms and the quality of life limitations produced by dry mouth in patients treated with radiotherapy.

Evaluation of tumor recurrences after radical prostatectomy using 18F-Choline PET/CT and 3T multiparametric MRI without endorectal coil: a single center experience.

BACKGROUND: To evaluate and compare the utility of 18F-fluorocholine (18F-CH) PET/CT versus 3-Tesla multiparametric MRI (mpMRI) without endorectal coil to detect tumor recurrences in patients with biochemical relapse following radical prostatectomy (RP). Secondarily, to identify possible prognostic variables associated with mpMRI and 18F-CH PET/CT findings. METHODS: Retrospective study of 38 patients who developed biochemical recurrence after RP between the years 2011 and 2015 at our institution. PET/CT and mpMRI were both performed within 30 days of each other in all patients. The PET/CT was reviewed by a nuclear medicine specialist while the mpMRI was assessed by a radiologist, both of whom were blinded to outcomes. RESULTS: The median prostate-specific antigen (PSA) value pre-MRI/PET-CT was 0.9 ng/mL (interquartile range 0.4-2.2 ng/mL). There were no differences in the detection rate between 18F-CH PET/CT and mpMRI for local recurrence (LR), lymph node recurrence (LNR) and bone metastases (BM). Separately, mpMRI and 18F-CH PET/CT were positive for recurrence in 55.2% and 52.6% of cases, respectively, and in 65.7% of cases when findings from both modalities were considered together. The detection of LR was better with combined mpMRI and choline PET/CT versus choline PET/CT alone (34.2% vs 18.4%, p = 0.04). Salvage treatment was modified in 22 patients (57.8%) based on the imaging findings. PSA values on the day of biochemical failure were significantly associated with mpMRI positivity (adjusted odds ratio (OR): 30.9; 95% confidence interval (CI): 1.5-635.8). Gleason score > 7 was significantly associated with PET/CT positivity (OR: 13.9; 95% CI: 1.5-125.6). A significant association was found between PSA doubling time (PSADT) (OR: 1.3; 95% CI: 1.0-1.7), T stage (OR: 21.1; 95% CI: 1.6-272.1), and LR. CONCLUSIONS: Multiparametric MRI and 18F-CH PET/CT yield similar detection rates for LR, LNR and pelvic BM. The combination of both imaging techniques provides a better LR detection versus choline PET/CT alone. The initially planned salvage treatment was modified in 57.8% of patients due to imaging findings. In addition to PSA values, Gleason score, T stage, and PSADT may provide valuable data to identify those patients that are most likely to benefit from undergoing both imaging procedures.

Tumor staging using 3.0 T multiparametric MRI in prostate cancer: impact on treatment decisions for radical radiotherapy.

To assess and validate the incorporation of the multiparametric magnetic resonance imaging (mpMRI) tumour category (mT-category) to the conventional clinical tumour category (cT-category), in order to guide the radiotherapy (RT) treatment decisions in prostate cancer. In addition, to identify the clinical factors associated to the technique reliability. mpMRI was performed in 274 prostate cancer patients in order to refine the treatment decisions according to PSA, Gleason Score (GS) and cT-category. Comparisons between the cT and mT-category were performed, as well as the impact on the RT treatment [target volume, doses and hormonal therapy (HT)] independently if it was finally performed. Changes in HT indication for intermediate risk were also analyzed. mpMRI validation was performed with pathological staging (n = 90 patients finally decided to join surgery). The mpMRI upstaging range was 86-94 % for any PSA value or GS. Following mpMRI, 32.8 % of the patients (90/274) were assigned to a different risk group. Compared to cT-category, mpMRI identified more intermediate-risk (46.4 vs. 59.5 %) and high-risk (19.0 vs. 28.8 %) prostate cancer patients. This resulted in a higher indication (p < 0.05) of seminal vesicle irradiation (63.5 vs. 70.0 %), inclusion of any extracapsular disease (T3-T4) within the target volume (1.8 vs. 18.2 %), higher doses (65.3 vs. 88.3 %) and HT associated to RT (45.6 vs. 62.4 %). Global accuracy for mpMRI was higher compared to DRE/TRUS (8.9 vs. 71.1 %, p < 0.05). mpMRI reliability was independent of PSA or GS. mpMRI tumor staging significantly modified the RT treatment decisions in all prostate cancer risk groups.

Role of 3T multiparametric magnetic resonance imaging without endorectal coil in the detection of local recurrent prostate cancer after radical prostatectomy: the radiation oncology point of view.

OBJECTIVE: The aims of this study were to evaluate the role of 3 tesla multiparametric magnetic resonance imaging (3TmMRI) without endorectal coil in the detection of radiographic local recurrences (rLRs) in a contemporary cohort of patients with prostate cancer who presented with biochemical recurrence after radical prostatectomy (RP) with low prostate-specific antigen (PSA) levels, and to identify clinical parameters associated with the 3TmMRI findings. MATERIALS AND METHODS: Between 2009 and 2013, 57 patients with biochemical recurrence of prostate cancer after RP who were considered for salvage radiation therapy (SRT) were included. 3TmMRI with T2-weighted imaging, diffusion weighted imaging (DWI) and dynamic contrast-enhanced imaging without endorectal coil was carried out in all patients before treatment. RESULTS: In 14 out of 57 patients (24.56%) local recurrence was detected through 3TmMRI. Median pre-SRT PSA was 0.40 ng/ml (interquartile range 0.30-2.05 ng/ml). The recurrence was perianastomotic in eight out of 14 patients (57.14%) and retrovesical in six out of 14 patients (42.86%). The median size of the local recurrence was 15.2 mm (range 8.0-46.0 mm). The probability of rLR was significantly higher in patients with PSA levels above 0.5 ng/ml [adjusted odds ratio (OR) 6.25, 95% confidence interval (CI) 1.27-30.79, p = 0.02] or PSA doubling time (PSADT) over 14 months (adjusted OR 7.12, 95% CI 1.40-36.25, p = 0.01). CONCLUSIONS: This is the first study to find a significant relationship between the PSADT and the rLR through MRI. Patients with PSADT longer than 14 months or pre-SRT PSA above 0.5 ng/ml benefited most from 3TmMRI. Its routine use could have significant clinical implications for SRT.

Bystander effects and radiotherapy.

Radiation-induced bystander effects are defined as biological effects expressed after irradiation by cells whose nuclei have not been directly irradiated. These effects include DNA damage, chromosomal instability, mutation, and apoptosis. There is considerable evidence that ionizing radiation affects cells located near the site of irradiation, which respond individually and collectively as part of a large interconnected web. These bystander signals can alter the dynamic equilibrium between proliferation, apoptosis, quiescence or differentiation. The aim of this review is to examine the most important biological effects of this phenomenon with regard to areas of major interest in radiotherapy. Such aspects include radiation-induced bystander effects during the cell cycle under hypoxic conditions when administering fractionated modalities or combined radio-chemotherapy. Other relevant aspects include individual variation and genetics in toxicity of bystander factors and normal tissue collateral damage. In advanced radiotherapy techniques, such as intensity-modulated radiation therapy (IMRT), the high degree of dose conformity to the target volume reduces the dose and, therefore, the risk of complications, to normal tissues. However, significant doses can accumulate out-of-field due to photon scattering and this may impact cellular response in these regions. Protons may offer a solution to reduce out-of-field doses. The bystander effect has numerous associated phenomena, including adaptive response, genomic instability, and abscopal effects. Also, the bystander effect can influence radiation protection and oxidative stress. It is essential that we understand the mechanisms underlying the bystander effect in order to more accurately assess radiation risk and to evaluate protocols for cancer radiotherapy.

Incidence of human papillomavirus-related oropharyngeal cancer and outcomes after chemoradiation in a population of heavy smokers.

BACKGROUND: Incidence of human papillomavirus (HPV)-related oropharyngeal carcinomas is increasing worldwide. The purpose of this study was to report the incidence in our region, and to determine the influence of HPV status on survival among a heavy smoking population. METHODS: p16 expression was analyzed in 102 patients with stage II to IV treated with chemoradiation. Overall survival (OS), locoregional control, and disease-free survival (DFS) were compared for HPV+ and HPV- status. RESULTS: The majority of patients were smokers (86%). p16 positivity was found in 26.7%. Patients who were HPV+ were younger (56 vs 59 years old; p = .052). No differences were observed regarding tumor stage, sex, or smoking between HPV+ and HPV-. Three-year OS was 67.4% for patients who were HPV+ versus 49.7% for HPV- (hazard ratio [HR], 0.55; p = .095). CONCLUSION: Incidence of HPV-related oropharyngeal carcinoma in Spain is similar to that reported in other European countries. In this sample of heavy smokers, we observed a nonsignificant trend for better outcomes in patients who were HPV+.

Current treatment of rectal cancer adapted to the individual patient.

Preoperative radiochemotherapy and total mesorectal excision surgery is a recommended standard therapy for patients with locally advanced rectal cancer. However, some subgroups of patients benefit more than others from this approach. In order to avoid long-term complications of radiation and chemotherapy, efforts are being made to subdivide T3N0 stage using advanced imaging techniques, and to analyze prognostic factors that help to define subgroup risk patients. Long-course radiochemotherapy has the potential of downsizing the tumor before surgery and may increase the chance of sphincter preservation in some patients. Short-course radiotherapy (SCRT), on the other hand, is a practical schedule that better suits patients with intermediated risk tumors, located far from the anal margin. SCRT is also increasingly being used among patients with disseminated disease, before resection of the rectal tumor. Improvements in radiation technique, such as keeping the irradiation target below S2/S3 junction, and the use of IMRT, can reduce the toxicity associated with radiation, specially long-term small bowel toxicity.

Epidemiology and prevalence of oropharyngeal candidiasis in Spanish patients with head and neck tumors undergoing radiotherapy treatment alone or in combination with chemotherapy.

OBJECTIVE: To describe the oropharyngeal candidiasis (OPC) prevalence in Spanish patients with head and neck cancer undergoing radiotherapy, alone or combined with chemotherapy. Secondary objectives were to determine the prevalence of Candida species colonization, and to explore whether different Candida species colonizing the oral cavity and the treatment were associated with a higher prevalence of OPC. METHODS: This is an observational, cross-sectional, multicentre study, conducted in Spanish radiation oncology units. Patients were diagnosed with head and neck cancer and started a radiotherapy treatment alone or combined with chemotherapy at the moment of their inclusion (N = 92). RESULTS: The OPC prevalence was 26 %. The identification of colonizing pathogens was performed in 49 patients, and Candida albicans was the dominant yeast (69 %), while non-albicans Candida was only found in 15 patients (31 %). Patients with C. albicans colonization had a significant higher prevalence of OPC compared to patients colonized by non-albicans Candida (p = 0.0273), but no difference was found regarding the OPC prevalence in patients receiving only radiotherapy compared to patients with both radiotherapy and chemotherapy treatments. CONCLUSIONS: Our data represent a step further in the knowledge of Candida species present in Spanish patients with head and neck tumors under radiation therapy. This is an essential step to manage the prophylaxis and treatment of OPC, since it might lead to severe clinical complications causing treatment interruption and, thus, representing a reduction in anti-tumor efficacy.

[The Alliance for the Prevention of Colorectal Cancer in Spain. A civil commitment to society]. / Alianza para la Prevención del Cáncer de Colon en España: un compromiso cívico con la sociedad.

Colorectal cancer (CRC) is the most common malignant tumor in Spain, when men and women are considered together, and the second leading cause of cancer death. Every week in Spain over 500 cases of CRC are diagnosed, and nearly 260 people die from the disease. Epidemiologic estimations for the coming years show a significant increase in the number of annual cases. CRC is a perfectly preventable tumor and can be cured in 90% of cases if detected in the early stages. Population-based screening programs have been shown to reduce the incidence of CRC and mortality from the disease. Unless early detection programs are established in Spain, it is estimated that in the coming years, 1 out of 20 men and 1 out of 30 women will develop CRC before the age of 75. The Alliance for the Prevention of Colorectal Cancer in Spain is an independent and non-profit organization created in 2008 that integrates patients' associations, altruistic non-governmental organizations and scientific societies. Its main objective is to raise awareness and disseminate information on the social and healthcare importance of CRC in Spain and to promote screening measures, early detection and prevention programs. Health professionals, scientific societies, healthcare institutions and civil society should be sensitized to this highly important health problem that requires the participation of all sectors of society. The early detection of CRC is an issue that affects the whole of society and therefore it is imperative for all sectors to work together.