A Cadaver-based Comparison of Sleeve-guided Implant-drill and Dynamic Navigation Osteotomy and Root-end Resections.

INTRODUCTION: This study compared the accuracy and efficiency of fully guided static and dynamic computer-assisted surgical navigation techniques for osteotomy and root-end resection (RER). METHODS: Fifty roots from cadaver heads were divided into two groups: fully guided static computer-assisted endodontic microsurgery (FG sCAEMS) and dynamic computer-assisted endodontic microsurgery (dCAEMS) (all, n = 25). Cone-beam computed tomography scans were taken pre and postoperatively. The osteotomy and RER were planned virtually in the preoperative cone-beam computed tomography scan and guided using 3D-printed surgical guides in the FG sCAEMS and 3D-dynamic navigation system in the dCAEMS. The 2D and 3D deviations and angular deflection were calculated. The osteotomy volume, resected root length, and resection angle were measured. The osteotomy and RER time and the number of procedural mishaps were recorded. RESULTS: FG sCAEMS was as accurate as dCAEMS, with no difference in the 2D and 3D deviation values or angular deflection (P > .05). The osteotomy and RER time were shortened using FG sCAEMS (P < .05). The FG sCAEMS showed a greater number of incomplete RERs than dCAEMS. Osteotomy volume, RER angle, and root length resected were similar in both groups (P > .05). FG sCAEMS and dCAEMS were feasible for osteotomy and RER. CONCLUSIONS: Within the limitations of this cadaver-based study, FG sCAEMS was as accurate as dCAEMS. Both FG sCAEMS and dCAEMS were time-efficient for osteotomy and RER.

Comparison of a Novel Static Computer-aided Surgical and Freehand Techniques for Osteotomy and Root-end Resection.

INTRODUCTION: This study compared the accuracy and efficiency of a novel static computer-aided surgical technique using a 3-dimensional (3D)-printed surgical guide (3D-SG) with a fully guided drill protocol (3D-SG FG) to the freehand (FH) osteotomy and root-end resection (RER). METHODS: Forty-six roots from 2 cadaver heads were divided into 2 groups: 3D-SG FG (n = 23) and FH (n = 23). Cone-beam computed tomographic scans were taken preoperatively and postoperatively. The endodontic microsurgery was planned in Blue Sky Bio software, and the 3D-SG was designed and 3D printed. The osteotomy and RER were conducted using a guided twist drill diameter of 2 mm and an ascending tapered drill with diameters of 2.8/3.2, 3.2/3.6, 3.8/4.2, and 4.2 mm with respective guided drill guides. Two-dimensional and three-dimensional virtual deviations and angular deflection were calculated. Linear osteotomy measures and root resection angle were obtained. The osteotomy and RER time and the number of mishaps were recorded. RESULTS: Two-dimensional and three-dimensional accuracy deviations and angular deflection were lower in the 3D-SG FG protocol than in the FH technique (P < .05). The height, length, and depth of the osteotomy and root resection angle were less in the 3D-SG FG protocol than in the FH technique (P < .05). The osteotomy and RER time with the 3D-SG FG protocol were less than the FH method (P < .05). CONCLUSIONS: Within the limitations of this cadaver-based study using denuded maxillary and mandibular jaws, 3D-SG FG protocol showed higher accuracy than FH osteotomy and RER. Moreover, the 3D-SG FG drill protocol significantly reduced the surgical time.

Architecture, Chromatin and Gene Organization of Toxoplasma gondii Subtelomeres.

Subtelomeres (ST) are chromosome regions that separate telomeres from euchromatin and play relevant roles in various biological processes of the cell. While their functions are conserved, ST structure and genetic compositions are unique to each species. This study aims to identify and characterize the subtelomeric regions of the 13 Toxoplasma gondii chromosomes of the Me49 strain. Here, STs were defined at chromosome ends based on poor gene density. The length of STs ranges from 8.1 to 232.4 kbp, with a gene density of 0.049 genes/kbp, lower than the Me49 genome (0.15 kbp). Chromatin organization showed that H3K9me3, H2A.X, and H3.3 are highly enriched near telomeres and the 5' end of silenced genes, decaying in intensity towards euchromatin. H3K4me3 and H2A.Z/H2B.Z are shown to be enriched in the 5' end of the ST genes. Satellite DNA was detected in almost all STs, mainly the sat350 family and a novel satellite named sat240. Beyond the STs, only short dispersed fragments of sat240 and sat350 were found. Within STs, there were 12 functional annotated genes, 59 with unknown functions (Hypothetical proteins), 15 from multigene FamB, and 13 from multigene family FamC. Some genes presented low interstrain synteny associated with the presence of satellite DNA. Orthologues of FamB and FamC were also detected in Neospora caninum and Hammondia hammondi. A re-analysis of previous transcriptomic data indicated that ST gene expression is strongly linked to the adaptation to different situations such as extracellular passage (evolve and resequencing study) and changes in metabolism (lack of acetyl-CoA cofactor). In conclusion, the ST region of the T. gondii chromosomes was defined, the STs genes were determined, and it was possible to associate them with high interstrain plasticity and a role in the adaptability of T. gondii to environmental changes.

Selected renal cells harbor nephrogenic potential.

Selected renal cells (SRCs), a renal epithelial cell-enriched platform, are being advanced as an autologous cell-based therapy for the treatment of chronic kidney disease. However, the mechanism underlying its renal reparative and restorative effects remains to be fully elucidated. In this study, we coupled knowledgebase data with empirical findings to demonstrate that genes differentially expressed by SRCs form interactomes within tubules and glomeruli and mediate a suite of renal developmental activities including epithelial cell differentiation, renal vasculature development, and glomerular and nephron development. In culture, SRCs form organoids which self-assemble into tubules in the presence of a scaffold. Implanted into the kidneys of subtotally nephrectomized rats, SRCs are associated with comma- and S-shaped body cell formation and glomerular development, and improvement in renal filtration indices and renal microarchitecture. These data suggest that SRCs harbor nephrogenic potential, which may explain, at least in part, their therapeutic activity.

Evaluation of Toxoplasma gondii recombinant antigens for early diagnosis of congenital toxoplasmosis.

The performance of Toxoplasma rGra8, rMic1, and the chimeric rGra4-Gra7 antigens for early congenital toxoplasmosis (CT) diagnosis was evaluated. Sera from CT patients showed high IgG reactivity to rMic1, rGra8, and rGra4-Gra7. The seroreactivity of samples from uninfected infants was lost within 2 months of age.

Toxoplasma gondii seropositivity associated to peri-urban living places in pregnant women in a rural area of Buenos Aires province, Argentina.

Infection with Toxoplasma gondii is very common in humans throughout the world, the intake of raw or undercooked meat with tissue cysts and fruits, vegetables and water contaminated with parasite oocysts being the main routes of infection. Here, we analyzed the seroprevalence of anti-T. gondii antibodies in pregnant females (age 13-44 years; n = 920) between April 2014 and December 2017 from Chascomús (Argentina), a city immersed in a rural area. Altogether 320 tested positive for immunoglobulin G antibodies, yielding an overall seroprevalence of 34.8% (CI 95%: 31.7-37.9). No association was observed between seropositivity and age. In addition, by using the QGIS 3.2.1 software we analyzed the geographical distribution of 769 (83.6%) pregnant females in two main areas of the city: Urban (n = 157) and Peri-urban (n = 612) with a seroprevalence of 26.8% (CI 95%: 19.8-33.7) and 36.4% (CI 95%: 32.6-40.3) respectively, and this difference was statistically significant (p = 0.023). Furthermore, we assessed through a questionnaire survey, between April 2016 to December 2017, possible risk factors such as activity (urban and rural), home water supply, animal husbandry, presence of cats as pets, gardening and consumption of meat and its derivatives (pork, sheep meat and sausages) and their frequencies (consumption per week), not finding significant association with seropositivity. Significant differences was found when the seroprevalence was analyzed between the urban and peri-urban neighborhoods of the city of Chascomús. The higher seroprevalence in peri-urban neighborhoods could be due to an unfavorable socioeconomic situation and/or to undeveloped peri-urban environments, which is a risk factor that should be taken into account when planning the health care of pregnant females.

Selected renal cells modulate disease progression in rodent models of chronic kidney disease via NF-κB and TGF-ß1 pathways.

AIM: Identification of mechanistic pathways for selected renal cell (SRC) therapeutic bioactivity in rodent models of chronic kidney disease. MATERIALS & METHODS: In vivo and in vitro functional bioassays applied to investigate regenerative outcomes associated with delivery of SRC to diseased rodent kidney. RESULTS: In vivo, SRC reduces chronic infiltration by monocytes/macrophages. SRC attenuates NF-κB and PAI-1 responses while simultaneously promoting host tubular cell expansion through trophic cues. In vitro, SRC-derived conditioned media attenuates TNF-α-induced NF-κB response, TGF-ß-mediated PAI-1 response and increases expression of transcripts associated with cell cycle regulation. Observed bioactive responses were from vesicle and nonvesicle-associated factors, including specific miRNAs. CONCLUSION: We identify a paracrine mechanism for SRC immunomodulatory and trophic cues on host renal tissues, catalyzing long-term functional benefits in vivo.

Cross-linked gelatin microspheres with continuously tunable degradation profiles for renal tissue regeneration.

Collagen and gelatin-based biomaterials are widely used in tissue engineering applications. Various methods have been reported for the cross-linking of these macromolecules for the purpose of delaying their biodegradation to prolong their in vivo residence (in tissue engineering applications) or tailoring their drug releasing capacity (when used as drug carriers). In this study, a carbodiimide-based cross-linking method, also used in the production of United States Food and Drug Administration-approved products, was employed to obtain differentially cross-linked gelatin beads. The colorimetric determination of the in vitro enzymatic susceptibility of the beads indicated that the resistance to degradation linearly correlated with the concentration of carbodiimide used for the cross-linking reaction. This result was also confirmed in vivo by the histological evaluation of the residence time of orthotopically injected cell-seeded beads. These data would indicate that the production of gelatin-based microbeads with tunable degradation profiles might be applicable toward the development of products that catalyze regeneration of kidney and other solid organs.

Potency evaluation of tissue engineered and regenerative medicine products.

Methodologies for the rigorous and quantitative evaluation of biological activity or potency are an essential aspect of the developmental pathway for all biologic product candidates. Such assays typically leverage key mechanistic pathways demonstrated to mediate observed therapeutic outcomes. Tissue engineered/regenerative medicine (TE/RM) therapeutics include cell based therapies as well as engineered tissues and neo-organs for which clarity regarding the mechanism or mechanisms of action may not be forthcoming. Here, we discuss how strategies for the development of potency assays for TE/RM product candidates may harness potential mechanisms of action or other therapeutically relevant bioactivity along with cell number and viability. As the pipeline for TE/RM product candidates expands through 2014 and beyond, the establishment of a defined framework for potency assays will facilitate successful translational outcomes.

Calidad de la ejecución del subprograma de atención comunitaria al adulto mayor del Policlínico Universitario "José Martí" / Quality of implementation of the community care subprogram for older adult at "José Martí" University Polyclinic

Se realizó una investigación en sistemas y servicios de salud para evaluar la calidad de la ejecución del Subprograma de Atención Comunitaria al Adulto Mayor en el área de salud correspondiente al Policlínico Universitario "José Martí Pérez" de Santiago de Cuba, desde noviembre del 2010 hasta igual mes del 2011. El universo estuvo constituido por 72 profesionales y 63 ancianos frágiles del área antes citada. Se evaluaron los elementos estructura (recursos humanos, materiales), proceso (competencia profesional) y resultados (satisfacción de los proveedores y usuarios e indicadores del programa). La calidad de la ejecución del subprograma fue inadecuada, debido a la insuficiente competencia e insatisfacción de los profesionales de la salud evaluados, por lo cual se evidenciaron dificultades tanto en el proceso como en los resultados. Por tales razones se recomendó efectuar un estudio de intervención relacionado con las deficiencias encontradas en la evaluación de la calidad de la ejecución del subprograma.

A research in health systems and services was made to assess the quality of implementation of the Community Care Subprogram for Older Adult in the health area corresponding to "José Martí Pérez" University Polyclinic of Santiago de Cuba, from November 2010 to the same month of 2011. The sample was formed by 72 professionals and 63 fragile elderly of that area. Elements such as structure (human resources, material), process (professional competence) and outcomes (satisfaction of users and providers and program indicators) were assessed. Implementation quality of the subprogram was inadequate due to insufficient competence and dissatisfaction of health professionals evaluated, thus difficulties were evident in both the process and the outcomes. For these reasons it was recommended to make an intervention study related to the deficiencies in the quality assessment of the subprogram implementation.

Fabrication of a multi-layer three-dimensional scaffold with controlled porous micro-architecture for application in small intestine tissue engineering.

Various methods can be employed to fabricate scaffolds with characteristics that promote cell-to-material interaction. This report examines the use of a novel technique combining compression molding with particulate leaching to create a unique multi-layered scaffold with differential porosities and pore sizes that provides a high level of control to influence cell behavior. These cell behavioral responses were primarily characterized by bridging and penetration of two cell types (epithelial and smooth muscle cells) on the scaffold in vitro. Larger pore sizes corresponded to an increase in pore penetration, and a decrease in pore bridging. In addition, smaller cells (epithelial) penetrated further into the scaffold than larger cells (smooth muscle cells). In vivo evaluation of a multi-layered scaffold was well tolerated for 75 d in a rodent model. This data shows the ability of the components of multi-layered scaffolds to influence cell behavior, and demonstrates the potential for these scaffolds to promote desired tissue outcomes in vivo.

Tissue engineering of esophagus and small intestine in rodent injury models.

Regenerative constructs composed of synthetically sourced, biodegradable biomaterials seeded with smooth muscle-like cells have been leveraged to mediate regeneration of bladder and bladder-like neo-organs. Here, we describe how such constructs may be applied to catalyze regeneration of esophagus and small intestine in preclinical rodent models.

Histological evaluation of tissue regeneration using biodegradable scaffold seeded by autologous cells for tubular/hollow organ applications.

The accurate interpretation of histological outcomes is a critical endpoint in preclinical studies. Thus, the toxicologic pathologist plays a vital role in conducting a comprehensive microscopic evaluation that would ultimately help defining the safety and functionality in Tissue Engineering/Regenerative Medicinal (TERM) products. In spite of many advances in regenerative medicine, there are no specific guidelines for the histological assessment of TERM products (Jayo et al. Toxicol Pathol 36:92-96, 2008). In this chapter, we describe the methodology designed to facilitate the detection of structural and functional changes when conducting a histological assessment including tissue collection (test article extraction), sampling, processing and fixation, special stains, statistical analysis, and morphometry.

A population of selected renal cells augments renal function and extends survival in the ZSF1 model of progressive diabetic nephropathy.

New treatment paradigms that slow or reverse progression of chronic kidney disease (CKD) are needed to relieve significant patient and healthcare burdens. We have shown that a population of selected renal cells (SRCs) stabilized disease progression in a mass reduction model of CKD. Here, we further define the cellular composition of SRCs and apply this novel therapeutic approach to the ZSF1 rat, a model of severe progressive nephropathy secondary to diabetes, obesity, dyslipidemia, and hypertension. Injection of syngeneic SRCs into the ZSF1 renal cortex elicited a regenerative response that significantly improved survival and stabilized disease progression to renal structure and function beyond 1 year posttreatment. Functional improvements included normalization of multiple nephron structures and functions including glomerular filtration, tubular protein handling, electrolyte balance, and the ability to concentrate urine. Improvements to blood pressure, including reduced levels of circulating renin, were also observed. These functional improvements following SRC treatment were accompanied by significant reductions in glomerular sclerosis, tubular degeneration, and interstitial inflammation and fibrosis. Collectively, these data support the utility of a novel renal cell-based approach for slowing renal disease progression associated with diabetic nephropathy in the setting of metabolic syndrome, one of the most common causes of end-stage renal disease.

Extension of bladder-based organ regeneration platform for tissue engineering of esophagus.

Recent successes in regenerative medicine and tissue engineering of bladder and bladder-like neo-organs have leveraged regenerative constructs composed of a biodegradable scaffold seeded with a population of smooth muscle cells. We have shown that such smooth muscle cells are isolatable from adipose and other sources alternate to the primary organ. We hypothesize that this regenerative platform is not limited to regeneration of bladder and bladder-like neo-organs, but rather represents a foundational technology platform broadly applicable for regeneration of laminarly organized hollow organs. Using esophagus as an illustrative example in support of this hypothesis, we demonstrate that patch constructs composed of adipose-derived smooth muscle cells seeded on a biodegradable matrix catalyze complete regeneration of the esophageal wall in a rodent model of esophageal injury. By implication, such regenerative constructs may potentially be used to mediate the regeneration of any laminarly organized tubular organ.

Regeneration of native-like neo-urinary tissue from nonbladder cell sources.

Urinary pathology requiring urinary diversion, partial or full bladder replacement, is a significant clinical problem affecting ~14,000 individuals annually in the United States alone. The use of gastrointestinal tissue for urinary diversion or bladder reconstruction/replacement surgeries is frequently associated with complications. To try and alleviate or reduce the frequency of these complications, tissue engineering and regenerative medicine strategies have been developed using bio-absorbable materials seeded with cells derived from the bladder. However, bladder-sourced cells may not always be suitable for such applications, especially in patients with bladder cancer. In this study, we describe the isolation and characterization of smooth muscle cells (SMCs) from porcine adipose and peripheral blood that are phenotypically and functionally indistinguishable from bladder-derived SMCs. In a preclinical Good Laboratory Practice study, we demonstrate that autologous adipose- and peripheral blood-derived SMCs may be used to seed synthetic, biodegradable tubular scaffold structures and that implantation of these seeded scaffolds into a porcine cystectomy model leads to successful de novo regeneration of a tubular neo-organ composed of urinary-like neo-tissue that is histologically identical to native bladder. The ability to create urologic structures de novo from scaffolds seeded by autologous adipose- or peripheral blood-derived SMCs will greatly facilitate the translation of urologic tissue engineering technologies into clinical practice.

Regeneration of rodent small intestine tissue following implantation of scaffolds seeded with a novel source of smooth muscle cells.

AIMS: To apply an organ regeneration platform technology of autologous smooth muscle cell/biomaterial combination products, previously demonstrated to be successful for urinary tissue regeneration, to the regeneration of the small intestine. MATERIALS & METHODS: Patch and tubular constructs were implanted in rodent small intestines and histologically evaluated over a time course for evidence of regeneration of the laminarly organized neo-mucosa and muscle layers. RESULTS: Laminarly organized neo-mucosa and muscle layer bundles are demonstrated as early as 8 weeks postimplantation. CONCLUSION: An organ regeneration technology platform of autologous smooth muscle cell/biomaterial combination products can be extended to the regeneration of the small intestine.

Functional evaluation of primary renal cell/biomaterial neo-kidney augment prototypes for renal tissue engineering.

Development of a tissue-engineered neo-kidney augment (NKA) requires evaluation of defined, therapeutically relevant cell and cell/biomaterial composites (NKA constructs) for regenerative potential in mammalian kidney. Previous work identified primary renal cell populations that extended survival and improved renal function in a rodent model of chronic kidney disease (CKD). This study extends that work toward the goal of developing NKA by (i) screening in vivo inflammatory and fibrotic responses to acellular biomaterials delivered to healthy rodent renal parenchyma, (ii) evaluating the functionality of renal cell/biomaterial combinations in vitro, (iii) generating NKA constructs by combining therapeutically relevant cell populations with biocompatible biomaterial, and (iv) evaluating in vivo neokidney tissue development in response to NKA constructs delivered to healthy rodent renal parenchyma. Gelatin and hyaluronic acid (HA)-based hydrogels elicited the least inflammatory and fibrotic responses in renal parenchyma relative to polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA) beads or particles and were associated with neovascularization and cellular infiltration by 4 weeks postimplantation. Renal cell populations seeded onto gelatin or HA-based hydrogels were viable and maintained a tubular epithelial functional phenotype during an in vitro maturation of 3 days as measured by transcriptomic, proteomic, secretomic, and confocal immunofluorescence assays. In vivo delivery of cell-seeded NKA constructs (bioactive renal cells + gelatin hydrogels) to healthy rodent renal parenchyma elicited neokidney tissue formation at 1 week postimplantation. To investigate a potential mechanism by which NKA constructs could impact a disease state, the effect of conditioned media on TGF-ß signaling pathways related to tubulo-interstitial fibrosis associated with CKD progression was evaluated. Conditioned medium was observed to attenuate TGF-ß-induced epithelial-mesenchymal transition (EMT) in vitro in a human proximal tubular cell line (HK2).

Isolation, characterization, and expansion methods for defined primary renal cell populations from rodent, canine, and human normal and diseased kidneys.

Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlights the need for new treatments to restore renal function. Regenerative medicine is a promising approach from which treatments for organ-level disorders (e.g., neurogenic bladder) have emerged and translated to clinics. Regenerative templates, composed of biodegradable material and autologous cells, isolated and expanded ex vivo, stimulate native-like organ tissue regeneration after implantation. A critical step for extending this strategy from bladder to kidney is the ability to isolate, characterize, and expand functional renal cells with therapeutic potential from diseased tissue. In this study, we developed methods that yield distinct subpopulations of primary kidney cells that are compatible with process development and scale-up. These methods were translated to rodent, large mammal, and human kidneys, and then to rodent and human tissues with advanced CKD. Comparative in vitro studies demonstrated that phenotype and key functional attributes were retained consistently in ex vivo cultures regardless of species or disease state, suggesting that autologous sourcing of cells that contribute to in situ kidney regeneration after injury is feasible, even with biopsies from patients with advanced CKD.

Tubular cell-enriched subpopulation of primary renal cells improves survival and augments kidney function in rodent model of chronic kidney disease.

Established chronic kidney disease (CKD) may be identified by severely impaired renal filtration that ultimately leads to the need for dialysis or kidney transplant. Dialysis addresses only some of the sequelae of CKD, and a significant gap persists between patients needing transplant and available organs, providing impetus for development of new CKD treatment modalities. Some postulate that CKD develops from a progressive imbalance between tissue damage and the kidney's intrinsic repair and regeneration processes. In this study we evaluated the effect of kidney cells, delivered orthotopically by intraparenchymal injection to rodents 4-7 wk after CKD was established by two-step 5/6 renal mass reduction (NX), on the regeneration of kidney function and architecture as assessed by physiological, tissue, and molecular markers. A proof of concept for the model, cell delivery, and systemic effect was demonstrated with a heterogeneous population of renal cells (UNFX) that contained cells from all major compartments of the kidney. Tubular cells are known contributors to kidney regeneration in situ following acute injury. Initially tested as a control, a tubular cell-enriched subpopulation of UNFX (B2) surprisingly outperformed UNFX. Two independent studies (3 and 6 mo in duration) with B2 confirmed that B2 significantly extended survival and improved renal filtration (serum creatinine and blood urea nitrogen). The specificity of B2 effects was verified by direct comparison to cell-free vehicle controls and an equivalent dose of non-B2 cells. Quantitative histological evaluation of kidneys at 6 mo after treatment confirmed that B2 treatment reduced severity of kidney tissue pathology. Treatment-associated reduction of transforming growth factor (TGF)-ß1, plasminogen activator inhibitor (PAI)-1, and fibronectin (FN) provided evidence that B2 cells attenuated canonical pathways of profibrotic extracellular matrix production.