Generation of 3D melanoma models using an assembloid-based approach.

While 3D tumor models have greatly evolved over the past years, there is still a strong requirement for more biosimilar models which are capable of recapitulating cellular crosstalk within the tumor microenvironment while equally displaying representative levels of tumor aggressiveness and invasion. Herein, we disclose an assembloid melanoma model based on the fusion of individual stromal multicellular spheroids (MCSs). In contrast to more traditional tumor models, we show that it is possible to develop self-organizing, heterotypic melanoma models where tumor cells present stem-cell like features like up-regulated pluripotency master regulators SOX2, POU5F1 and NANOG. Additionally, these assembloids display high levels of invasiveness while embedded in 3D matrices as evidenced by stromal cell promotion of melanoma cell invasion via metalloproteinase production. Furthermore, sensitivity to anticancer drug doxorubicin was demonstrated for the melanoma assembloid model. These findings suggest that melanoma assembloids may play a significant role in the field of 3D cancer models as they more closely mimic the tumor microenvironment when compared to more traditional MCSs, opening the doors to a better understanding of the role of tumor microenvironment in supporting tumor progression. STATEMENT OF SIGNIFICANCE: The development of complex 3D tumor models that better recapitulate the tumor microenvironment is crucial for both an improved comprehension of intercellular crosstalk and for more efficient drug screening. We have herein developed a self-organizing heterotypic assembloid-based melanoma model capable of closely mimicking the tumor microenvironment. Key features recapitulated were the preservation of cancer cell stemness, sensitivity to anti-cancer agents and tumor cell invasion promoted by stromal cells. The approach of pre-establishing distinct stromal domains for subsequent combination into more complex tumor constructs provides a route for developing superior tumor models with a higher degree of similarity to native cancer tissues.

Modelling the complex nature of the tumor microenvironment: 3D tumor spheroids as an evolving tool.

Cancer remains a serious burden in society and while the pace in the development of novel and more effective therapeutics is increasing, testing platforms that faithfully mimic the tumor microenvironment are lacking. With a clear shift from animal models to more complex in vitro 3D systems, spheroids emerge as strong options in this regard. Years of development have allowed spheroid-based models to better reproduce the biomechanical cues that are observed in the tumor-associated extracellular matrix (ECM) and cellular interactions that occur in both a cell-cell and cell-ECM manner. Here, we summarize some of the key cellular interactions that drive tumor development, progression and invasion, and how successfully are these interactions recapitulated in 3D spheroid models currently in use in the field. We finish by speculating on future advancements in the field and on how these can shape the relevance of spherical 3D models for tumor modelling.

Spongy-like hydrogels prevascularization with the adipose tissue vascular fraction delays cutaneous wound healing by sustaining inflammatory cell influx.

In vitro prevascularization is one of the most explored approaches to foster engineered tissue vascularization. We previously demonstrated a benefit in tissue neovascularization by using integrin-specific biomaterials prevascularized by stromal vascular fraction (SVF) cells, which triggered vasculogenesis in the absence of extrinsic growth factors. SVF cells are also associated to biological processes important in cutaneous wound healing. Thus, we aimed to investigate whether in vitro construct prevascularization with SVF accelerates the healing cascade by fostering early vascularization vis-à-vis SVF seeding prior to implantation. Prevascularized constructs delayed re-epithelization of full-thickness mice wounds compared to both non-prevascularized and control (no SVF) groups. Our results suggest this delay is due to a persistent inflammation as indicated by a significantly lower M2(CD163+)/M1(CD86+) macrophage subtype ratio. Moreover, a slower transition from the inflammatory to the proliferative phase of the healing was confirmed by reduced extracellular matrix deposition and increased presence of thick collagen fibers from early time-points, suggesting the prevalence of fiber crosslinking in relation to neodeposition. Overall, while prevascularization potentiates inflammatory cell influx, which negatively impacts the cutaneous wound healing cascade, an effective wound healing was guaranteed in non-prevascularized SVF cell-containing spongy-like hydrogels confirming that the SVF can have enhanced efficacy.

Highly tailorable gellan gum nanoparticles as a platform for the development of T cell activator systems.

BACKGROUND: T cell priming has been shown to be a powerful immunotherapeutic approach for cancer treatment in terms of efficacy and relatively weak side effects. Systems that optimize the stimulation of T cells to improve therapeutic efficacy are therefore in constant demand. A way to achieve this is through artificial antigen presenting cells that are complexes between vehicles and key molecules that target relevant T cell subpopulations, eliciting antigen-specific T cell priming. In such T cell activator systems, the vehicles chosen to deliver and present the key molecules to the targeted cell populations are of extreme importance. In this work, a new platform for the creation of T cell activator systems based on highly tailorable nanoparticles made from the natural polymer gellan gum (GG) was developed and validated. METHODS: GG nanoparticles were produced by a water in oil emulsion procedure, and characterized by dynamic light scattering, high resolution scanning electronic microscopy and water uptake. Their biocompatibility with cultured cells was assessed by a metabolic activity assay. Surface functionalization was performed with anti-CD3/CD28 antibodies via EDC/NHS or NeutrAvidin/Biotin linkage. Functionalized particles were tested for their capacity to stimulate CD4+ T cells and trigger T cell cytotoxic responses. RESULTS: Nanoparticles were approximately 150 nm in size, with a stable structure and no detectable cytotoxicity. Water uptake originated a weight gain of up to 3200%. The functional antibodies did efficiently bind to the nanoparticles, as confirmed by SDS-PAGE, which then targeted the desired CD4+ populations, as confirmed by confocal microscopy. The developed system presented a more sustained T cell activation over time when compared to commercial alternatives. Concurrently, the expression of higher levels of key cytotoxic pathway molecules granzyme B/perforin was induced, suggesting a greater cytotoxic potential for future application in adoptive cancer therapy. CONCLUSIONS: Our results show that GG nanoparticles were successfully used as a highly tailorable T cell activator system platform capable of T cell expansion and re-education.

Integrin-specific hydrogels for growth factor-free vasculogenesis.

Integrin-binding biomaterials have been extensively evaluated for their capacity to enable de novo formation of capillary-like structures/vessels, ultimately supporting neovascularization in vivo. Yet, the role of integrins as vascular initiators in engineered materials is still not well understood. Here, we show that αvß3 integrin-specific 3D matrices were able to retain PECAM1+ cells from the stromal vascular fraction (SVF) of adipose tissue, triggering vasculogenesis in vitro in the absence of extrinsic growth factors. Our results suggest that αvß3-RGD-driven signaling in the formation of capillary-like structures prevents the activation of the caspase 8 pathway and activates the FAK/paxillin pathway, both responsible for endothelial cells (ECs) survival and migration. We also show that prevascularized αvß3 integrin-specific constructs inosculate with the host vascular system fostering in vivo neovascularization. Overall, this work demonstrates the ability of the biomaterial to trigger vasculogenesis in an integrin-specific manner, by activating essential pathways for EC survival and migration within a self-regulatory growth factor microenvironment. This strategy represents an improvement to current vascularization routes for Tissue Engineering constructs, potentially enhancing their clinical applicability.

Maxillary Interdental Osteotomies Have Low Morbidity for Alveolar Crestal Bone and Adjacent Teeth: A Cone Beam Computed Tomography-Based Study.

PURPOSE: Maxillary segmentation involving interdental osteotomies can have an adverse effect on the interdental crestal bone and adjacent teeth. The purpose of the present study was to evaluate the effect of interdental osteotomies on surrounding osseous and dental structures, including adjacent teeth, using cone beam computed tomography (CBCT), in patients who underwent segmental maxillary osteotomies. PATIENTS AND METHODS: The present retrospective cohort study evaluated interdental osteotomy (IDO) sites between the lateral incisors and canines in patients treated with 3-piece Le Fort I osteotomies. CBCT scans were assessed using Kodac Dental Imaging software at specific intervals: T0 (before surgery), T1 (immediately after surgery), and T2 (a minimum of 11 months after surgery). The statistical analysis using a linear regression model was adjusted to compare the variables at the different intervals. Injury to the dental structures was assessed by radiological evidence of dental damage, the requirement for endodontic treatment, or tooth loss. RESULTS: We evaluated 94 IDO sites in 47 patients in the present study. The mean inter-radicular distance at T0 was 2.5 mm. A statistically significant increase was seen in the inter-radicular distance (between T1 and T0) of 0.72 mm, with a reduction of the alveolar bone crest height (between T2 and T0) of 0.19 mm (P < .001) for the group that underwent IDO. A weak correlation was found for this increase in the inter-radicular distance, with changes in the alveolar crest bone height. The potential complications associated with interdental osteotomies such as iatrogenic damage to the tooth structure, the need for endodontic treatment, and tooth loss were not encountered in any patients. CONCLUSIONS: We found very low morbidity for the interdental alveolar crest and the integrity of teeth adjacent to interdental osteotomies for patients who underwent maxillary segmentation between the lateral incisors and canines.

Dendrimers: Breaking the paradigm of current musculoskeletal autoimmune therapies.

The advances achieved by cell-based therapies to treat autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS), despite promising, are still insufficient for the current demands. RA and MS therapeutic approaches follow world guidelines to use disease modifying drugs and biological agents that, regardless of some good results in clinical outcomes, are well known for several systemic secondary side effects. Dendrimers are custom-made nanoparticles with proved clinical potential, displaying proper size, chemistries, immunomodulatory, and anti-inflammatory properties. This has directed their potential use as drug delivery systems for cancer therapy, for instance. This review manuscript discloses the hidden potential behind dendrimers as alternative viable solutions to treat RA and MS, by focusing in the most recent reports describing the use of dendrimers for suppressing inflammation and possibly preventing disease progression. The advantages of their use as compared with current applied therapies is also discussed herein.

Effect of extracellular calcium on regucalcin expression and cell viability in neoplastic and non-neoplastic human prostate cells.

Extracellular calcium (Ca2+o) and its receptor, the Ca2+-sensing receptor (CaSR), play an important role in prostate physiology, and it has been shown that the deregulation of Ca2+ homeostasis and the overexpression of CaSR are involved in prostate cancer (PCa). Regucalcin (RGN), a Ca2+-binding protein that plays a relevant role in intracellular Ca2+ homeostasis, was identified as an under-expressed protein in human PCa. Moreover, RGN was associated with suppression of cell proliferation, suggesting that the loss of RGN may favor development and progression of PCa. This work aims to unveil the role of Ca2+o on RGN expression and viability of non-neoplastic (PNT1A) and neoplastic (LNCaP) prostate cell lines. It was demonstrated that Ca2+o up-regulates RGN expression in both cell lines, but important differences were found between cells for dose- and time-responses to Ca2+o treatment. It was also shown that high [Ca2+]o triggers different effects on cell proliferation of neoplastic and non-neoplastic PCa cells, which seems to be related with RGN expression levels. This suggests the involvement of RGN in the regulation of cell proliferation in response to Ca2+o treatment. Also, the effect of Ca2+o on CaSR expression seems to be dependent of RGN expression, which is strengthened by the fact that RGN-knockdown in PNT1A cells increases the CaSR expression, whereas transgenic rats overexpressing RGN exhibit low levels of CaSR. Overall, our results highlighted the importance of RGN as a regulatory protein in Ca2+-dependent signaling pathways and its deregulation of RGN expression by Ca2+o may contribute for onset and progression of PCa.

Condylar hyperplasia of the temporomandibular joint: types, treatment, and surgical implications.

Condylar hyperplasia (CH) is a progressive and pathologic overgrowth of either or both mandibular condyles, which can affect the neck, ramus, or body of the mandible. It may lead to facial asymmetry, malocclusion, speech, and masticatory problems. Identifying the specific type of condylar hyperplasia is crucial. Serial radiographs, dental models, clinical evaluations, and bone scan techniques are usually the best diagnostic methods to determine the type of CH and if the growth process is still active. The protocol of surgical procedures recommended in this article for CH has been proven to treat the condylar pathology and correct the jaw deformity.

Concomitant treatment of mandibular ameloblastoma and bilateral temporomandibular joint osteoarthritis with bone graft and total joint prostheses.

Temporomandibular joint (TMJ) osteoarthritis is a degenerative disease that can create clinical problems in the masticatory musculature, jaws, occlusion, and other associated structures and is commonly accompanied by inflammatory changes and pain. Many cases of TMJ dysfunction can be managed with nonsurgical therapies, but patients with irreversible TMJ damage may require surgical intervention for repair or reconstruction. Despite various methods of TMJ reconstruction, the patient-fitted total joint prostheses may be the best option to achieve good outcomes. Multicystic ameloblastoma is a benign odontogenic neoplasm of the jaws that is found most often in the mandible, in the region of the molars, and the ramus. Ameloblastomas usually progress slowly, but are locally invasive and may cause significant morbidity and sometimes death. This report describes a case of concomitant treatment of recurrent mandibular ameloblastoma and severe bilateral TMJ osteoarthritis treated by resection of the tumor, reconstruction with bone grafting, and bilateral TMJ reconstruction in a 63-year-old woman.

Management of ankylosed maxillary canine with single-tooth osteotomy in conjunction with orthognathic surgery.

Ankylosis of teeth is the abnormal adherence of alveolar bone to dentin or cementum. Ankylosis of a submerged maxillary canine can be challenging when striving for an optimal occlusal and esthetic treatment outcome with orthognathic surgery. If an ankylosed tooth does not respond to orthodontic forces, surgical procedures may be indicated to facilitate movement of the tooth to the correct position including a single-tooth segmental osteotomy to reposition the alveolar bone including the ankylosed tooth. The objective of this case report is to describe the treatment of a patient with an ankylosed submerged maxillary right canine, with a single-tooth osteotomy performed to reposition the tooth into its correct position concomitant with double-jaw orthognathic surgery and TMJ surgery.

Orthognathic and TMJ surgery: postsurgical patient management.

Postsurgical patient management is a critical factor for high-quality patient treatment and predictable outcomes in orthognathic and temporomandibular joint (TMJ) surgery. Lack of understanding of proper patient management on the part of the surgeon and orthodontist can result in compromised or even disastrous results. Surgeons and orthodontists must have the knowledge and ability to implement postsurgical management protocols and strategies to provide the best care and outcomes possible for their orthognathic and TMJ surgery patients. Information is presented here in reference to postsurgical orthodontics, healing process, dietary considerations, activities, and potential complications to aid the clinician in understanding the postsurgical processes that patients will encounter.

Autogenous grafts/allografts/conduits for bridging peripheral trigeminal nerve gaps.

Nerve repairs and grafting techniques have been around for many years. Autogenous nerve grafts have worked reasonably well in the right circumstances but are associated with difficulties in achieving a proper donor-host match and with postsurgical sequelae at the donor site. Vein grafts seem to work almost as well as autogenous nerve grafts in digital nerve repairs that require a graft less than 3 cm in length. Currently, the most promising nerve graft materials are the polyglycolic acid tubes and processed decellularized allografts, which have shown good results without the morbidity of autogenous nerve grafts. However, more research studies using these materials for TN repairs are essential to validate the superiority of these procedures.

Management of the infected temporomandibular joint total joint prosthesis.

PURPOSE: Patients with specific temporomandibular joint (TMJ) conditions and pathology may benefit from TMJ reconstruction by use of total joint prostheses. A potential risk to patients receiving TMJ total joint prostheses is infection. The purpose of this study was to present our experience in treating infected TMJ total joint prostheses over a 12-year period, as well as the protocol we have developed to manage acute infections and the protocol we follow for chronic infections of TMJ total joint prostheses. PATIENTS AND METHODS: This retrospective study evaluated the records of 316 consecutive patients (290 female and 26 male patients) who had TMJ reconstruction from 1997 to 2009 by 1 surgeon, using patient-fitted TMJ Concepts total joint prostheses (TMJ Concepts, Ventura, CA), with a total of 579 prostheses placed, to determine the occurrence and management methods of postoperative infections. Postoperative infections involving the TMJ prostheses developed in 8 of 316 patients (2.5%) and 9 of 579 prostheses (1.6%): 7 patients unilaterally and 1 patient bilaterally. Patients were divided into 2 groups: group 1 (n = 5) had acute infections in 6 joints and group 2 (n = 3) had chronic infections in 3 joints. Patient 5 began in group 1 but was transferred to group 2. One patient with Munchausen syndrome and self-induced infections was eliminated from the study. Patients were treated by our protocol for management of acute or chronic infections. RESULTS: In group 1 (n = 5) the onset of infection symptoms averaged 12 days after surgery (range, 5-24 days). The time from onset of symptoms to surgical intervention was 3.4 days (range, 2-5 days). We found that 4 of 5 patients (80%) and 5 of 6 joints (83%) were successfully treated with retention of the prostheses. Patient 5 varied from the protocol and maintained a chronic infection of her right TMJ prosthesis that transferred her to group 2. In group 2 (n = 3) all 3 patients (including patient 5) had chronic infections with draining fistulas that were successfully treated by the chronic infection protocol including prosthesis removal and replacement. CONCLUSIONS: TMJ total joint prostheses can become infected. The earlier the diagnosis is made and the acute infection protocol initiated (within 2-5 days), the greater the chance of salvage of the prosthesis. For chronic infections, the treatment protocol has likewise been very successful, but it does require 2 surgical stages for removal and replacement of the prosthesis. Management of infected total joint prosthesis can be challenging, but with aggressive treatment following the appropriate protocol, infected prostheses can be successfully managed.