Gene-Activated Materials in Regenerative Dentistry: Narrative Review of Technology and Study Results

Treatment of a wide variety of defects in the oral and maxillofacial regions requires the use of innovative approaches to achieve best outcomes. One of the promising directions is the use of gene-activated materials (GAMs) that represent a combination of tissue engineering and gene therapy. This approach implies that biocompatible materials will be enriched with gene-carrying vectors and implanted into the defect site resulting in transfection of the recipient’s cells and secretion of encoded therapeutic protein in situ. GAMs may be presented in various designs depending on the type of material, encoded protein, vector, and way of connecting the vector and the material. Thus, it is possible to choose the most suitable GAM design for the treatment of a particular pathology. The use of plasmids for delivery of therapeutic genes is of particular interest. In the present review, we aimed to delineate the principle of work and various designs of plasmid-based GAMs and to highlight results of experimental and clinical studies devoted to the treatment of periodontitis, jaw bone defects, teeth avulsion, and other pathologies in the oral and maxillofacial regions

Refinement of Animal Experiments: Replacing Traumatic Methods of Laboratory Animal Marking with Non-Invasive Alternatives

Reliable methods for identifying rodents play an important role in ensuring the success of preclinical studies. However, animal identification remains a trivial laboratory routine that is not often discussed, despite the fact that more than 6 million rodents are used in animal studies each year. Currently, there are extensive regulations in place to ensure adequate anesthesia and to reduce animal suffering during experiments. At the same time, not enough attention is paid to the comfort of rodents during routine identification procedures, which can be painful and cause some complications. In order to achieve the highest ethical standards in laboratory research, we must minimize animal discomfort during the identification phase. In this article, we discuss traumatic methods of identification and describe several painless methods for marking in long-term experimental studies. The use of non-traumatic and non-invasive methods requires the renewal of marks as they fade and additional handling of the rodents. Laboratory personnel must be trained in stress-minimizing handling techniques to make mark renewal less stressful

Post-Implantation Inflammatory Responses to Xenogeneic Tissue-Engineered Cartilage Implanted in Rabbit Trachea: The Role of Cultured Chondrocytes in the Modification of Inflammation

Immune responses to tissue-engineered grafts made of xenogeneic materials remain poorly studied. The scope of current investigations is limited by the lack of information on orthotopically implanted grafts. A deeper understanding of these processes is of great importance since innovative surgical approaches include the implantation of xenogeneic decellularized scaffolds seeded by cells. The purpose of our work is to study the immunological features of tracheal repair during the implantation of tissue-engineered constructs based on human xenogeneic scaffolds modified via laser radiation in rabbits. The samples were stained with hematoxylin and Safranin O, and they were immunostained with antibodies against tryptase, collagen II, vimentin, and CD34. Immunological and inflammatory responses were studied by counting immune cells and evaluating blood vessels and collagen. Leukocyte-based inflammation prevailed during the implantation of decellularized unseeded scaffolds; meanwhile, plasma cells were significantly more abundant in tissue-engineered constructs. Mast cells were insignificantly more abundant in tissue-engineered construct samples. Conclusions: The seeding of decellularized xenogeneic cartilage with chondrocytes resulted in a change in immunological reactions upon implantation, and it was associated with plasma cell infiltration. Tissue-engineered grafts widely differed in design, including the type of used cells. The question of immunological response depending on the tissue-engineered graft composition requires further investigation

Assessment of Immunological Responses - A Novel Challenge in Tissue Engineering and Regenerative Medicine

The number of articles on tissue engineering and regenerative medicine has increased dramatically in the last decade; however, the number of clinically implemented techniques remains small. Possible reasons include insufficient investigation of immune reactions on implanted tissue-engineered grafts and cells or a lack of consensus regarding which immunological tests must be performed to evaluate immunological responses. To provide an example of insufficiency in the assessment of immunological reactions, we analyzed three papers published between 2020 and 2021 and discussed the possibility of creating a standardized assay palette for the assessment of immunological responses in different types of implants

Implantation orthotopique expérimentale d'une greffe trachéale créée par génie tissulaire à partir d'un échafaudage dévitalisé ensemencé de cellules mésenchymateuses et épithéliales

International audienceAim. To study the viability of the tissue-engineered graft (TEG) based on the devitalized tracheal scaffold (DTS), seeded by mesenchymal stromal and epithelial cells in the experiment on rabbits with the assessment of the cytocompatibility and biocompatibility in vivo. Materials and methods. Syngeneic bone marrow-derived mesenchymal stromal cells (BM-MSCs) and lung epithelial cells of rabbit were obtained. Morphology and phenotype of the BMSCs culture were confirmed by immunofluorescent staining on CD90 and CD271 markers. Pulmonary epithelium cells obtained by enzymatic treatment of shredded lung tissue of rabbit were stained with CKPan, CK8/18 and CK14 markers of epithelial cells. The donor trachea was devitalized in three freeze-thawing cycles. Double-layer cell seeding of DTS was performed under static and dynamic culturing. Orthotopic implantation of TEG was performed at the site of the anterolateral wall defect in the rabbit trachea formed as a result of tracheal resection over four rings. The results were evaluated by computed tomography, histological and immunohisto- chemical analyses. Results. TEG implant based on DTS with two-layer cell cultures of rabbit BM-MSCs and epithelial cells was obtained. Three months after the implantation, TEG engraftment was observed, no tracheal wall stenosis was observed, but a slight narrowing of the lumen in the implantation area was observed. Viability of the tissue-engineered graft was confirmed by histological method 6 months after implantation. Epithelialization and vascularization of the tracheal wall, absence of signs of purulent inflammation and aseptic necrosis were shown. Small narrowing of the tracheal lumen was caused by chronic inflammation produced by irritation of the mucous suture material. Conclusion. The new model for evaluating the viability of a tissue engineering implant during critical airway defect closure was obtained. Two-layer vitalization of DTS with lung epithelial cells and BM-MSCs allows to create a tissue engineered structure for replacement of non-long tracheal defects in the experiment in vivo. The use of the tracheal tissue-engineered graft for orthotopic implantation showed a biocompatibility with minimal tissue reaction.Objectif : étudier la viabilité d'une greffe tissulaire (TEG) basée sur un échafaudage trachéal dévitalisé (DTS) ensemencé de cellules stromales et épithéliales mésenchymateuses dans une expérience sur des lapins avec évaluation de la cytocompatibilité et de la biocompatibilité in vivo. Matériels et méthodes. Des cellules de moelle osseuse mésenchymateuses syngéniques (MSBMC) et des cellules épithéliales syngéniques de poumon de lapin ont été obtenues. La morphologie et le phénotype de la culture de MSBMC ont été confirmés par une coloration d'immunofluorescence pour les marqueurs CD90 et CD271. Les cellules épithéliales pulmonaires obtenues par traitement enzymatique de tissu pulmonaire de lapin haché ont été colorées avec les marqueurs CKPan, CK8/18 et CK14 caractéristiques des cellules épithéliales. La trachée du donneur a été dévitalisée en trois cycles successifs de congélation-décongélation. L'ensemencement des cellules en double couche de DTS a été effectué en culture statique et dynamique. L'implantation orthotopique de DTS a été réalisée au niveau de la malformation de la paroi antérolatérale chez le lapin, formée à la suite d'une résection trachéale sur quatre anneaux. Les résultats ont été évalués par tomographie assistée par ordinateur et par des analyses histologiques et immunohistochimiques. Résultats. Un implant TEG, basé sur le DTS, avec colonisation bicouche par des cultures de cellules MSBMC et de cellules épithéliales de lapin a été obtenu. Trois mois après l'implantation, une greffe de TEG a été constatée, aucune sténose de la paroi trachéale n'a été observée. Cependant, un léger rétrécissement de la lumière au niveau du site d'implantation a été noté. Six mois après l'implantation, la viabilité du TEG a été confirmée par une méthode histologique. L'épithélialisation et la vascularisation de la paroi trachéale, l'absence de signes d'inflammation purulente et de nécrose aseptique ont été mises en évidence. Le petit rétrécissement de la lumière de la trachée s'est avéré avoir été causé par une inflammation chronique due à l'irritation de la muqueuse avec le matériel de suture. Conclusion. Un nouveau modèle a été créé pour évaluer la viabilité d'un implant d'ingénierie tissulaire lors de la fermeture d'un défaut critique des voies respiratoires. Le TEG développé, basé sur le DTS ensemencé (bicouche) par les cellules épithéliales du poumon et le BMSC, a été utilisé avec succès pour remplacer des défauts trachéaux non étendus dans une expérience in vivo. L'utilisation d'une greffe trachéale issue du génie tissulaire pour une implantation orthotopique a montré une biocompatibilité avec une réponse tissulaire minimale.Цель. Изучить жизнеспособность тканеинженерной конструкции (ТИК) на основе девитализированного трахеального матрикса (ДТМ), заселенного мезенхимальными стромальными и эпителиальными клетками, на модели оценки жизнеспособности тканеинженерного имплантата при закрытии критического дефекта дыхательных путей у кроликов. Оценить потенциал ТИК к поддержанию стабильного просвета трахеи в области имплантации. Материалы и методы. Получены сингенные мезенхимальные стромальные клетки костного мозга (МСК КМ) и сингенные эпителиоциты легкого кролика. Морфологию и фенотип культуры МСК КМ подтверждали иммунофлюоресцентным окрашиванием на маркеры CD90 и CD271. Клетки легочного эпителия, полученные методом энзиматической обработки измельченной ткани легкого кролика, были окрашены на характерные для эпителиальных клеток маркеры CKPan, CK8/18 и CK14. Девитализация донорской трахеи проведена тремя последовательными циклами замораживания–оттаивания. Двухслойное заселение ДТМ клетками выполнено в условиях статичного и динамического культивирования. Проведена ортотопическая имплантация ТИК на место дефекта переднебоковой стенки трахеи кролика, сформированного в результате резекции трахеи на протяжении четырех колец. Оценка результатов выполнена методами компьютерной томографии, гистологического и иммуногистохимического анализов. Результаты. Получен имплантат ТИК на основе ДТМ с двухслойным заселением клеточными культурами МСК КМ и эпителиоцитов кролика. Через 3 мес. после имплантации отмечалось приживление ТИК, стенозирования стенки трахеи не наблюдалось, однако отмечалось незначительное сужение просвета в области имплантации. На 6-й мес. после имплантации жизнеспособность тканеинженерной конструкции подтверждалась гистологическим методом. Показана эпителизация и васкуляризация стенки трахеи, отсутствие признаков гнойного воспаления и асептического некроза. Определена причина небольшого сужения просвета трахеи хроническое воспаление, вызванное раздражением слизистой шовным материалом. Заключение. Получена модель для оценки жизнеспособности тканеинженерного имплантата при закрытии критического дефекта дыхательных путей. Разработанная ТИК на основе ДТМ, двухслойно заселенного эпителиоцитами легкого и МСК КМ, была успешно применена для замещения непротяженных дефектов трахеи в эксперименте in vivo. Минимальная тканевая реакция на ТИК трахеи была обусловлена биосовместимостью имплантата

Refinement of Animal Experiments: Replacing Traumatic Methods of Laboratory Animal Marking with Non-Invasive Alternatives

Reliable methods for identifying rodents play an important role in ensuring the success of preclinical studies. However, animal identification remains a trivial laboratory routine that is not often discussed, despite the fact that more than 6 million rodents are used in animal studies each year. Currently, there are extensive regulations in place to ensure adequate anesthesia and to reduce animal suffering during experiments. At the same time, not enough attention is paid to the comfort of rodents during routine identification procedures, which can be painful and cause some complications. In order to achieve the highest ethical standards in laboratory research, we must minimize animal discomfort during the identification phase. In this article, we discuss traumatic methods of identification and describe several painless methods for marking in long-term experimental studies. The use of non-traumatic and non-invasive methods requires the renewal of marks as they fade and additional handling of the rodents. Laboratory personnel must be trained in stress-minimizing handling techniques to make mark renewal less stressful

Adverse events, side effects and complications in mesenchymal stromal cell-based therapies

Numerous clinical studies have shown a wide clinical potential of mesenchymal stromal cells (MSCs) application. However, recent experience has accumulated numerous reports of adverse events and side effects associated with MSCs therapy. Furthermore, the strategies and methods of MSCs therapy did not change significantly in recent decades despite the clinical impact and awareness of potential complications. An extended understanding of limitations could lead to a wider clinical implementation of safe cell therapies and avoid harmful approaches. Therefore, our objective was to summarize the possible negative effects observed during MSCs-based therapies. We were also aimed to discuss the risks caused by weaknesses in cell processing, including isolation, culturing, and storage. Cell processing and cell culture could dramatically influence cell population profile, change protein expression and cell differentiation paving the way for future negative effects. Long-term cell culture led to accumulation of chromosomal abnormalities. Overdosed antibiotics in culture media enhanced the risk of mycoplasma contamination. Clinical trials reported thromboembolism and fibrosis as the most common adverse events of MSCs therapy. Their delayed manifestation generally depends on the patient’s individual phenotype and requires specific awareness during the clinical trials with obligatory inclusion in the patient’ informed consents. Finally we prepared the safety checklist, recommended for clinical specialists before administration or planning of MSCs therapy

Intraoperative Creation of Tissue-Engineered Grafts with Minimally Manipulated Cells: New Concept of Bone Tissue Engineering In Situ

Transfer of regenerative approaches into clinical practice is limited by strict legal regulation of in vitro expanded cells and risks associated with substantial manipulations. Isolation of cells for the enrichment of bone grafts directly in the Operating Room appears to be a promising solution for the translation of biomedical technologies into clinical practice. These intraoperative approaches could be generally characterized as a joint concept of tissue engineering in situ. Our review covers techniques of intraoperative cell isolation and seeding for the creation of tissue-engineered grafts in situ, that is, directly in the Operating Room. Up-to-date, the clinical use of tissue-engineered grafts created in vitro remains a highly inaccessible option. Fortunately, intraoperative tissue engineering in situ is already available for patients who need advanced treatment modalities