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Background: To study neoadjuvant chemoradiotherapy (nCRT) and potential predictive factors for response in locally advanced oral cavity cancer (LA-OCC).
Methods: The INVERT trial is an ongoing single-center, prospective phase 2, proof-of-principle trial. Operable patients with stage III-IVA squamous cell carcinomas of the oral cavity were eligible and received nCRT consisting of 60 Gy with concomitant cisplatin and 5-fluorouracil. Surgery was scheduled 6-8 weeks after completion of nCRT. Explorative, multiplex immunohistochemistry (IHC) was performed on pretreatment tumor specimen, and diffusion-weighted magnetic resonance imaging (DW-MRI) was conducted prior to, during nCRT (day 15), and before surgery to identify potential predictive biomarkers and imaging features. Primary endpoint was the pathological complete response (pCR) rate.
Results: Seventeen patients with stage IVA OCC were included in this interim analysis. All patients completed nCRT. One patient died from pneumonia 10 weeks after nCRT before surgery. Complete tumor resection (R0) was achieved in 16/17 patients, of whom 7 (41%, 95% CI: 18-67%) showed pCR. According to the Clavien-Dindo classification, grade 3a and 3b complications were found in 4 (25%) and 5 (31%) patients, respectively; grade 4-5 complications did not occur. Increased changes in the apparent diffusion coefficient signal intensities between MRI at day 15 of nCRT and before surgery were associated with better response (p=0.022). Higher abundances of programmed cell death protein 1 (PD1) positive cytotoxic T-cells (p=0.012), PD1+ macrophages (p=0.046), and cancer-associated fibroblasts (CAFs, p=0.036) were associated with incomplete response to nCRT.
Conclusion: nCRT for LA-OCC followed by radical surgery is feasible and shows high response rates. Larger patient cohorts from randomized trials are needed to further investigate nCRT and predictive biomarkers such as changes in DW-MRI signal intensities, tumor infiltrating immune cells, and CAFs.
Objectives: The aim of the present study was to characterize the cellular reaction to a xenogeneic resorbable collagen membrane of porcine origin using a subcutaneous implantation model in Wistar rats over 30 days.
Materials and methods: Ex vivo, liquid platelet-rich fibrin (PRF), a leukocyte and platelet-rich cell suspension, was used to evaluate the blood cell membrane interaction. The material was implanted subcutaneously in rats. Sham-operated rats without biomaterial displayed physiological wound healing (control group). Histological, immunohistological, and histomorphometric analyses were focused on the inflammatory pattern, vascularization rate, and degradation pattern.
Results: The membrane induced a large number of mononuclear cells over the observation period, including lymphocytes, macrophages, and fibroblasts. After 15 days, multinucleated giant cells (MNGCs) were observed on the biomaterial surface. Their number increased significantly, and they proceeded to the center of the biomaterial on day 30. These cells highly expressed CD-68, calcitonin receptor, and MMP-9, but not TRAP or integrin-ß3. Thus, the membrane lost its integrity and underwent disintegration as a consequence of the induction of MNGCs. The significant increase in MNGC number correlated with a high rate of vascularization, which was significantly higher than the control group. Physiological wound healing in the control group did not induce any MNGCs at any time point. Ex vivo blood cells from liquid-PRF did not penetrate the membrane.
Conclusion: The present study suggests a potential role for MNGCs in biomaterial degradation and questions whether it is beneficial to accept them in clinically approved biomaterials or focus on biomaterials that induce only mononuclear cells. Thus, further studies are necessary to identify the function of biomaterial-induced MNGCs.
Clinical relevance: Understanding the cellular reaction to biomaterials is essential to assess their suitability for specific clinical indications and outline the potential benefit of specific group of biomaterials in the respective clinical indications.
Different tissue engineering techniques are used to support rapid vascularisation. A novel technique is the use of platelet-rich fibrin (PRF), an autologous source of growth factors. This study was the first to investigate the influence of PRF matrices, isolated following different centrifugation protocols, on human dermal vascular endothelial cells (ECs) in mono-culture and co-culture with human primary fibroblasts (HFs) as an in vitro model for tissue regeneration. Focus was placed on vascular structure formation and growth factor release. HFs and ECs were cultivated with PRF prepared using a high (710 ×g) or low (44 ×g) relative centrifugation force (RCF) over 14 d. Immunofluorescence staining and immunohistochemistry were used to evaluate the microvascular formation. Cell culture supernatants were collected for evaluation of growth factor release. The results showed a PRF-mediated effect on the induction of angiogenesis in ECs. Microvessel-like structure formation was promoted when ECs were combined with low-RCF PRF as compared to high-RCF PRF or control group. The percentage of vascular lumen area was significantly higher in low-RCF PRF, especially at day 7, which coincided with statistically significantly higher growth factor [vascular endothelial factor (VEGF), transforming growth factor β1 (TGF-β1) and platelet derived growth factor (PDGF)] concentration measured in low-RCF PRF as compared to high-RCF PRF or control group. In conclusion, reducing the RCF according to the low-speed centrifugation concept (LSCC) resulted in increased growth factor release and angiogenic structure formation with EC mono-culture, suggesting that PRF may be a highly beneficial therapeutic tool for tissue engineering applications.
Purpose: The aim of this study was to analyze systematically the influence of the relative centrifugation force (RCF) on leukocytes, platelets and growth factor release within fluid platelet-rich fibrin matrices (PRF).
Materials and methods: Systematically using peripheral blood from six healthy volunteers, the RCF was reduced four times for each of the three experimental protocols (I–III) within the spectrum (710–44 g), while maintaining a constant centrifugation time. Flow cytometry was applied to determine the platelets and leukocyte number. The growth factor concentration was quantified 1 and 24 h after clotting using ELISA.
Results: Reducing RCF in accordance with protocol-II (177 g) led to a significantly higher platelets and leukocytes numbers compared to protocol-I (710 g). Protocol-III (44 g) showed a highly significant increase of leukocytes and platelets number in comparison to -I and -II. The growth factors’ concentration of VEGF and TGF-β1 was significantly higher in protocol-II compared to -I, whereas protocol-III exhibited significantly higher growth factor concentration compared to protocols-I and -II. These findings were observed among 1 and 24 h after clotting, as well as the accumulated growth factor concentration over 24 h.
Discussion: Based on the results, it has been demonstrated that it is possible to enrich PRF-based fluid matrices with leukocytes, platelets and growth factors by means of a single alteration of the centrifugation settings within the clinical routine.
Conclusions: We postulate that the so-called low speed centrifugation concept (LSCC) selectively enriches leukocytes, platelets and growth factors within fluid PRF-based matrices. Further studies are needed to evaluate the effect of cell and growth factor enrichment on wound healing and tissue regeneration while comparing blood concentrates gained by high and low RCF.
The present study evaluated the tissue response toward a resorbable collagen membrane derived from bovine achilles tendon (test group) in comparison to physiological wound healing (control group). After subcutaneous implantation in Wistar rats over 30 days, histochemical and immunohistochemical methods elucidated the cellular inflammatory response, vascularization pattern, membrane protein and cell absorbance capacity. After 30 days, the test-group induced two different inflammatory patterns. On the membrane surface, multinucleated giant cells (MNGCs) were formed after the accumulation of CD-68-positive cells (macrophages), whereas only mononuclear cells (MNCs) were found within the membrane central region. Peri-implant vascularization was significantly enhanced after the formation of MNGCs. No vessels were found within the central region of the membrane. Physiological wound healing revealed no MNGCs at any time point. These dynamic changes in the cellular reaction and vascularization within the test-group are related typical indications of a foreign body reaction. Due to the membrane-specific porosity, mononuclear cells migrated into the central region, and the membrane maintained its integrity over 30 days by showing no breakdown or disintegration. The ex vivo investigation analyzed the interaction between the membrane and a blood concentrate system, liquid platelet-rich fibrin (liquid PRF), derived from human peripheral blood and consisting of platelets, leukocytes and fibrin. PRF penetrated the membrane after just 15 min. The data question the role of biomaterial-induced MNGCs as a pathological reaction and whether this is acceptable to trigger vascularization or should be considered as an adverse reaction. Therefore, further pre-clinical and clinical studies are needed to identify the types of MNGCs that are induced by clinically approved biomaterials.
Background: With the aging population and a rising incidence of squamous cell carcinoma of the head and neck (SCCHN), there is an emerging need for developing strategies to treat elderly patients.
Patients and Methods: We retrospectively analyzed 158 patients treated with definitive, concurrent chemoradiotherapy (CRT) for SCCHN. Clinicopathological characteristics, acute toxicities, and oncological outcomes were compared between patients younger and older than (or of age equal to) 65, 70, and 75 years.
Results: RT dose, chemotherapy regimen, and total chemotherapy dose were balanced between the groups. After a median follow-up of 29 months, overall survival (OS), progression-free survival (PFS), local control rate, and distant metastasis-free survival stratified by age of ≥65, ≥70, or ≥75 years revealed no differences. The rate of acute toxicities was also not higher for older patients. Worse ECOG performance score (ECOG 2-3) was associated with impaired OS () and PFS ().
Conclusion: Definitive treatment with CRT for SCCHN is feasible and effective; even in advanced age treatment decisions should be made according to general condition and comorbidity, rather than calendar age alone.
Cellular attachment plays a vital role in the differentiation of pheochromocytoma (PC12) cells. PC12 cells are noradrenergic clonal cells isolated from the adrenal medulla of Rattus norvegicus and studied extensively as they have the ability to differentiate into sympathetic neuron-like cells. The effect of several experimental parameters including (i) the concentration of nerve growth factor (NGF); (ii) substratum coatings, such as poly-L-lysine (PLL), fibronectin (Fn), and laminin (Lam); and (iii) double coatings composed of PLL/Lam and PLL/Fn on the differentiation process of PC12 cells were studied. Cell morphology was visualised using brightfield phase contrast microscopy, cellular metabolism and proliferation were quantified using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, and the neurite outgrowth and axonal generation of the PC12 cells were evaluated using wide field fluorescence microscopy. It was found that double coatings of PLL/Lam and PLL/Fn supported robust adhesion and a two-fold enhanced neurite outgrowth of PC12 cells when treated with 100 ng/mL of NGF while exhibiting stable metabolic activity, leading to the accelerated generation of axons.
Tumor-associated macrophages, angiogenesis, and tumor cell migration in oral squamous cell carcinoma
(2017)
Objective: To investigate the relationship between tumor-associated macrophages (TAMs), neovascularization, and tumor cell migration in oral squamous cell carcinoma (OSCC) of an African subpopulation.
Materials and Methods: Twenty OSCC paraffin blocks underwent immunohistochemistry to TAM1 (CCR7), TAM2 (CD206), Twist, E-cadherin, N-cadherin, and CD34. The relative percentage of CCR7 + and CD206 + cells to overall immune cell population was calculated for three high power fields and an average was taken. TAM-related microvessel density (MVD) was determined as the mean of the three recorded values. Cases that had no CD34 + vessels adjacent to the TAMs region were regarded as having an MVD score of 0.
Results: Ten cases (50%) expressed greater CCR7 activity than CD206, seven cases (35%) expressed approximately equal activity of CCR7 and CD206, while three cases (15%) expressed greater activity of CD206 than CCR7. Twist expression was strong in some cases with strong N-cadherin and weak E-cadherin, but the expression of Twist was not consistently high in all cases that expressed strong N-cadherin and weak E-cadherin.
Conclusions: TAMs distribution suggested antitumor activity and the potential for tumor metastasis was only partly due to Twist-mediated epithelial–mesenchymal transition.
Platelet-rich fibrin (PRF) is a blood concentrate derived from venous blood that is processed without anticoagulants by a one-step centrifugation process. This three-dimensional scaffold contains inflammatory cells and plasma proteins entrapped in a fibrin matrix. Liquid-PRF was developed based on the previously described low-speed centrifuge concept (LSCC), which allowed the introduction of a liquid-PRF formulation of fibrinogen and thrombin prior to its conversion to fibrin. Liquid-PRF was introduced to meet the clinical demand for combination with biomaterials in a clinically applicable and easy-to-use way. The aim of the present study was to evaluate, ex vivo, the interaction of the liquid-PRF constituents with five different collagen biomaterials by histological analyses. The results first demonstrated that large variability existed between the biomaterials investigated. Liquid-PRF was able to completely invade Mucograft® (MG; Geistlich Biomaterials, Wolhusen, Switzerland) and to partly invade Bio-Gide® (BG; Geistlich Biomaterials, Wolhusen, Switzerland) and Mucoderm® (MD; Botiss Biomaterials, Berlin, Germany), and Collprotect® (CP; Botiss Biomaterials, Berlin, Germany) showed only a superficial interaction. The BEGO® collagen membrane (BCM; BEGO Implant Systems) appeared to be completely free of liquid-PRF. These results were confirmed by the different cellular penetration and liquid-PRF absorption coefficient (PAC) values of the evaluated membranes. The present study demonstrates a system for loading biomaterials with a complex autologous cell system (liquid-PRF) in a relatively short period of time and in a clinically relevant manner. The combination of biomaterials with liquid-PRF may be clinically utilized to enhance the bioactivity of collagen-based biomaterials and may act as a biomaterial-based growth factor delivery system.
The permeability and inflammatory tissue reaction to Mucomaix® matrix (MM), a non- cross-linked collagen-based matrix was evaluated in both ex vivo and in vivo settings. Liquid platelet rich fibrin (PRF), a blood concentrate system, was used to assess its capacity to absorb human proteins and interact with blood cells ex vivo. In the in vivo aspect, 12 Wister rats had MM implanted subcutaneously, whereas another 12 rats (control) were sham-operated without biomaterial implantation. On days 3, 15 and 30, explantation was completed (four rats per time-point) to evaluate the tissue reactions to the matrix. Data collected were statistically analyzed using analysis of variance (ANOVA) and Tukey multiple comparisons tests (GraphPad Prism 8). The matrix absorbed the liquid PRF in the ex vivo study. Day 3 post-implantation revealed mild tissue inflammatory reaction with presence of mononuclear cells in the implantation site and on the biomaterial surface (mostly CD68-positive macrophages). The control group at this stage had more mononuclear cells than the test group. From day 15, multinucleated giant cells (MNGCs) were seen in the implantation site and the outer third of the matrix with marked increase on day 30 and spread to the matrix core. The presence of these CD68-positive MNGCs was associated with significant matrix vascularization. The matrix degraded significantly over the study period, but its core was still visible as of day 30 post-implantation. The high permeability and fast degradation properties of MM were highlighted.