Proliferative Layer Research Articles

A multiphysics hybrid continuum - agent-based model of in vitro vascularized organoids.

Organoids are 3D in vitro models that fulfill a hierarchical function, representing a small version of living tissues and, therefore, a good approximation of cellular mechanisms. However, one of the main disadvantages of these models is the appearance of a necrotic core due to poor vascularization. The aim of this work is the development of a numerical framework that incorporates the mechanical stimulation as a key factor in organoid vascularization. Parameters, such as fluid velocity and nutrient consumption, are analyzed along the organoid evolution. The mathematical model created for this purpose combines continuum and discrete approaches. In the continuum part, the fluid flow and the diffusion of oxygen and nutrients are modeled using a finite element method approach. Meanwhile, the growth of the organoid, blood vessel evolution, as well as their interaction with the surrounding environment, are modeled using agent-based methods. Continuum model outcomes include the distribution of shear stress, pressure and fluid velocity around the organoid surface, in addition to the concentration of oxygen and nutrients in its interior. The agent models account for cell proliferation, differentiation, organoid growth and blood vessel morphology, for the different case studies considered. Two main conclusions are achieved in this work: (i) the results of the study quantitatively predict in vitro data, with an enhanced blood vessel invasion under high fluid flow and (ii) the diffusion and consumption model parameters of the organoid cells determine the thickness of the proliferative, quiescent, hypoxic and necrotic layers.

The combination of odontogenic stem cells and mandibular advancement promotes the length of the mandible in adult rats by facilitating the development of condylar cartilage

BackgroundMandibular retraction is a prevalent dental and maxillofacial deformity that negatively affects patients' functional health and facial aesthetics. It has been challenging to achieve optimal outcomes for patients who have passed the peak of growth and development using only functional orthopedic treatment. There is a pressing need to explore innovative methods to promote the adaptive remodeling of adult condylar cartilage and the mandible in response to external stimuli. This study aimed to investigate the impact of varying injection frequencies of stem cells from the apical papilla (SCAPs) on the growth and development of condylar cartilage and the mandible, as well as their potential for adaptive remodeling.MethodsThe study was conducted on 8-week-old adult male Sprague–Dawley rats. The effects of SCAPs injection and different durations of mandibular advancement (MA) on the adaptive remodeling of condylar cartilage and the mandible were assessed. After the initial experimental findings, various injection frequencies of SCAPs were applied to determine the most effective conditions for promoting the growth and adaptive remodeling of condylar cartilage and the mandible during an 8-week period of mandibular advancement.ResultsThe study found that rats with extended mandibular lead times (8 weeks) or an appropriately increased frequency of mandibular leading time (once every 2 weeks or once every 1 week) exhibited increased lengths of the mandibular body and ascending branch, and a thickened full layer of condylar cartilage. The highest proportions of the proliferative layer, mature layer, and hypertrophic layer were observed in these rats. Additionally, there was a significant increase in the expression levels of SOX9 and COL2A1.ConclusionThe data from this study suggest that adult rats, even after missing their peak growth period, retain the potential for continued growth and development of their condylar cartilage. By prolonging the duration of mandibular advancement and administering injections of stem cells from the apical papilla (SCAPs), it is possible to stimulate the growth and development of the mandibular condyle.

How malocclusion interferes with tissue inhibitor of metalloproteinase-1 expression and morphology of the articular cartilage of the mandible in female rats

ObjectiveThe purpose of this study was to investigate morphological alterations and tissue inhibitor of metalloproteinase-1 expression in the articular cartilage of the mandible under conditions of experimentally induced malocclusion. DesignTwenty-four 8-week-old female Wistar rats were used and divided into control and experimental groups with two different treatment periods (2 and 4 weeks). Sagittal malocclusions were orthodontically created, causing mesial movement of the first molars and distalization of the third molars unilaterally and on opposite sides of the arches. Sagittal sections of the articular cartilage of the mandible were subjected to hematoxylin and eosin and immunohistochemistry for tissue inhibitor of metalloproteinase-1. Chi-square and MannWhitney U tests were applied. ResultsAnimals treated for 2 and 4 weeks showed morphological alterations in articular cartilage of the mandible. The main findings were thickening of the posterior third, layer derangement, osteoclast activity and osteophyte formation. Among the cellular aspects, the presence of chondrocytes with condensed nuclei and cytoplasm reduction were observed. The enzyme in control animals was observed only in the mature layer. Treated animals showed immunopositive cells in the proliferative and mature layers, and in the 2-week treated group, the posterior third of the cartilage had more immunolabeled cells than control (P=0.0291). ConclusionsThe occlusal disorder caused morphological changes in articular cartilage of the mandible, probably due to the attempt to adapt to the new condition. Tissue inhibitor of metalloproteinase-1 expression may play a role as an initial modulator in the biological events observed in articular cartilage of the mandible.

Effects of unilateral nasal obstruction on mandibular condyle in mice of different ages: An exploration based on H-type angiogenesis coupling osteogenesis.

Nasal obstruction leads to a hypoxia condition throughout the entire body. In this study, the unilateral nasal obstruction (UNO) mouse model was established by blocking the left nostril of mice. The aim of this study was to investigate the effects of UNO-induced hypoxia on mandibular condyle in juvenile (3-week-old), adolescent (6-week-old) and adult (12-week-old) male C57BL/6J mice from the perspective of H-type angiogenesis coupling osteogenesis. Firstly, UNO exerted a significant inhibitory effect on weight gain in mice of all ages. However, only in adolescent mice did UNO have an obvious detrimental effect on femoral bone mass accrual. Subsequently, micro-computed tomography (CT) analysis of mandibular condylar bone mass revealed that UNO significantly retarded condylar head volume gain but increased condylar head trabecular number (Tb.N) in juvenile and adolescent mice. Furthermore, UNO promoted the ratio of proliferative layer to cartilage layer in condylar cartilage and facilitated the chondrocyte-to-osteoblast transformation in juvenile and adolescent mice. Moreover, although UNO enhanced the positive expression of hypoxia-inducible factor (HIF)-1α in the condylar subchondral bone of mice in all ages, an increase in H-type vessels and Osterix+ cells was only detected in juvenile and adolescent mice. In summary, on the one hand, in terms of condylar morphology, UNO has a negative effect on condylar growth, hindering the increase in condylar head volume in juvenile and adolescent mice. However, on the other hand, in terms of condylar microstructure, UNO has a positive effect on condylar osteogenesis, promoting the increase of condylar Tb.N, chondrocyte-to-osteoblast transformation, HIF-1α expression, H-type angiogenesis and Osterix+ cells in juvenile and adolescent mice. Although the changes in condylar morphology and microstructure caused by UNO have not yet been fully elucidated, these findings improve our current understanding of the effects of UNO on condylar bone homeostasis.

A Facile, Transfection-Free Approach to siRNA Delivery in In Vitro 3D Spheroid Models.

Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two-dimensional (2D) cell culture fails to faithfully model the complexity found in vivo, and novel drug candidates that show promising results in 2D models often do not translate to the clinic. More recently, three-dimensional (3D) cell culture models have gained popularity owing to their greater physiological relevance to in vivo biology. In particular, 3D spheroid models are becoming widely used due to their ability to mimic solid tumors, both in architecture and gradation of nutrients distributed from the outer, proliferative layers into the inner, quiescent layers of cells. Similar to in vivo tumors, cell lines grown in 3D spheroid models tend to be more resistant to antitumor drug treatments than their 2D cultured counterparts, though distinct signaling pathways and gene targets conferring this resistance have yet to be fully explored. RNA interference (RNAi) is an effective tool to elucidate gene function and discover novel druggable targets in 2D models; however, only a few studies have successfully performed RNAi in complex 3D models to date. Here, we demonstrate efficient RNAi-mediated knockdown using "transfection-free" Dharmacon Accell siRNAs in three spheroid culture models, in the presence or absence of the extracellular matrix. This methodology has the potential to be scaled up for complex arrayed screening experiments, which may aid in the identification of novel druggable targets with greater clinical relevance than those identified in 2D experiments. © 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D spheroids in matrix-free ULA plates Alternate Protocol 1: Generation of Matrigel matrix-embedded 3D spheroids Alternate Protocol 2: Generation of GrowDex hydrogel-embedded 3D spheroids Basic Protocol 2: Delivery of siRNA and collection of matrix-free 3D spheroids Alternate Protocol 3: Delivery of siRNA and collection of matrix-embedded spheroids Basic Protocol 3: RNA and protein extraction from spheroids for characterization of gene knockdown.

The transcription factor BMI1 increases hypoxic signaling in oral cavity epithelia

The tongue epithelium is maintained by a proliferative basal layer. This layer contains long-lived stem cells (SCs), which produce progeny cells that move up to the surface as they differentiate. B-lymphoma Mo-MLV insertion region 1 (BMI1), a protein in mammalian Polycomb Repressive Complex 1 (PRC1) and a biomarker of oral squamous cell carcinoma, is expressed in almost all basal epithelial SCs of the tongue, and single, Bmi1-labelled SCs give rise to cells in all epithelial layers. We previously developed a transgenic mouse model (KrTB) containing a doxycycline- (dox) controlled, Tet-responsive element system to selectively overexpress Bmi1 in the tongue basal epithelial SCs. Here, we used this model to assess BMI1 actions in tongue epithelia. Genome-wide transcriptomics revealed increased levels of transcripts involved in the cellular response to hypoxia in Bmi1-overexpressing (KrTB+DOX) oral epithelia even though these mice were not subjected to hypoxia conditions. Ectopic Bmi1 expression in tongue epithelia increased the levels of hypoxia inducible factor-1 alpha (HIF1α) and HIF1α targets linked to metabolic reprogramming during hypoxia. We used chromatin immunoprecipitation (ChIP) to demonstrate that Bmi1 associates with the promoters of HIF1A and HIF1A-activator RELA (p65) in tongue epithelia. We also detected increased SC proliferation and oxidative stress in Bmi1-overexpressing tongue epithelia. Finally, using a human oral keratinocyte line (OKF6-TERT1R), we showed that ectopic BMI1 overexpression decreases the oxygen consumption rate while increasing the extracellular acidification rate, indicative of elevated glycolysis. Thus, our data demonstrate that high BMI1 expression drives hypoxic signaling, including metabolic reprogramming, in normal oral cavity epithelia.

Progress of the study of pericytes and their potential research value in adenomyosis.

Pericytes (PCs) are versatile cells integral to the microcirculation wall, exhibiting specific stem cell traits. They are essential in modulating blood flow, ensuring vascular permeability, maintaining homeostasis, and aiding tissue repair process. Given their involvement in numerous disease-related pathological and physiological processes, the regulation of PCs has emerged as a focal point of research. Adenomyosis is characterized by the presence of active endometrial glands and stroma encased by an enlarged and proliferative myometrial layer, further accompanied by fibrosis and new blood vessel formation. This distinct pathological condition might be intricately linked with PCs. This article comprehensively reviews the markers associated with PCs, their contributions to angiogenesis, blood flow modulation, and fibrotic processes. Moreover, it provides a comprehensive overview of the current research on adenomyosis pathophysiology, emphasizing the potential correlation and future implications regarding PCs and the development of adenomyosis.

Nanog, Stat-3, and Sox-5 involvement in human fetal temporomandibular joint late development

Background and aimThe temporomandibular joint (TMJ) is a synovial joint that allows the complex movements essential for life. It connects the jawbone to the skull, working as a sliding hinge. Moreover, pluripotent stem cells are a source of precursors and tissue-specific cells in developing organisms, however, their biodistribution in developing fetal tissues is weakly studied. The aim of our study was analyse immunohistochemical expression of Nanog, Oct-4, Sox-2 and Stat-3 and Sox-5, in TMJ tissue samples from human fetuses aged between the 12th and 20th weeks of intrauterine life. Materials and methodsWe fixed and processed TMJ tissue samples from human fetuses, histological sections and immunohistochemical procedures were carried out. ResultsTMJ histological studies examination did not reveal any difference in the tissue organization between the samples in the studied periods. Immunohistochemical analysis demonstrated that Oct-4 and Sox-2 lack their expression in TMJ. In contrast, Nanog was expressed in nucleous of proliferative layer of mandibular condyle, Stat-3 was expressed in nuclear cells of articular disc, Stat-3 and Sox-5 showed positive nuclear and cytoplasmic immunostaining in codrocyte layers and in ossification areas. ConclusionsNanog acts in maintanence of pluripotency, Stat-3 in articular disc acts as a transcriptional factor. Stat-3 and Sox-2 act in chondrocyte and osteoblast diferentiation. Distribution of the cells, which express Nanog, Stat-3, and Sox-5 in TMJ tissue during fetal development, can help further understand its physiology, pathology, and repairing capacities.

Mouth breathing induces condylar remodelling and chondrocyte apoptosis via both the extrinsic and mitochondrial pathways in male adolescent rats

The prevalence of mouth breathing is high in children and adolescents. It causes various changes to the respiratory tract and, consequently, craniofacial growth deformities. However, the underlying mechanisms contributing to these effects are obscure. Herein, we aimed to study the effects of mouth breathing on chondrocyte proliferation and death in the condylar cartilage and morphological changes in the mandible and condyle. Additionally, we aimed to elucidate the mechanisms underlying chondrocyte apoptosis and investigate any variations in the related pathways. Subchondral bone resorption and decreased condylar cartilage thickness were observed in mouth-breathing rats; further, mRNA expression levels of Collagen II, Aggrecan, and Sox 9 were lower in the mouth breathing group, while those of matrix metalloproteinase 9 increased. TdT-mediated dUTP nick end labelling staining and immunohistochemistry analyses showed that apoptosis occurred in the proliferative and hypertrophic layers of cartilage in the mouth breathing group. TNF, BAX, cytochrome c, and cleaved-caspase-3 were highly expressed in the condylar cartilage of the mouth-breathing rats. These results suggest that mouth breathing leads to subchondral bone resorption, cartilage layer thinning, and cartilage matrix destruction, inducing chondrocyte apoptosis via both the extrinsic and mitochondrial apoptosis pathways.

Comparison of part-time and full-time mandibular advancement: enlightenment based on type H vessel coupling osteogenesis.

This study was aimed at resolving the controversy over the wearing time of mandibular advancement (MA) appliances by comparing the differences between part-time and full-time MA (PTMA and FTMA) from the perspective of promoting H-type vessel coupling osteogenesis in the condylar heads. Thirty 30-week-old male C57BL/6J mice were randomly divided into 3 groups: the control (Ctrl), PTMA, and FTMA groups. The mandibular condyles were analyzed by morphology, micro-computed tomography, histological staining, and immunofluorescence staining to investigate the changes of the condylar heads in the PTMA and FTMA groups after 31 days. Both PTMA and FTMA models promoted condylar growth and achieved stable mandibular advancement at day 31. However, compared with PTMA, FTMA has the following characteristics. First, new bone formation in the condylar head was detected in the retrocentral region in addition to the posterior region. Second, the condylar proliferative layer was thicker, and the number of pyknotic cells in the hypertrophic and erosive layers was higher. Moreover, endochondral osteogenesis of the condylar head was more active. Finally, the retrocentral and posterior regions of the condylar head had more vascular loops or arcuate H-type vessel coupling Osterix+ osteoprogenitors. While both PTMA and FTMA induced new bone formation in the condylar heads of middle-aged mice, FTMA promoted more osteogenesis by volume and region. Furthermore, FTMA presented more H-type vessel coupling Osterix+ osteoprogenitors in both the retrocentral and posterior regions of the condylar head. FTMA is better at promoting condylar osteogenesis, especially in non-growing patients. We suggest that enhancing H-type angiogenesis could be an effective strategy to achieve favorable MA outcomes, especially for patients unable to meet the FT-wearing requirement or being non-growing.

Therapeutic effects of kartogenin on temporomandibular joint injury by activating the TGF-β/SMAD pathway in rats.

Patients with temporomandibular dysfunction (TMD) usually suffer from pathology or malpositioning of the temporomandibular joint (TMJ) disk, leading to the degenerative lesion of condyles. Kartogenin can promote the repair of damaged cartilage. This study aimed to explore whether intra-articular injection of kartogenin could alleviate the TMJ injury induced by type II collagenase. We measured the head withdrawal threshold and found that kartogenin alleviated the pain around TMD induced by type II collagenase. We observed the morphology of the condylar surface and found that kartogenin protected the integration of the condylar surface. We analyzed the density of the subchondral bone and found that kartogenin minimized the damage of TMJ injury to the subchondral bone. We next explored the histological changes and found that kartogenin increased the thickness of the proliferative layer and more collagen formation in the superficial layer. Then, to further ensure whether kartogenin promotes cell proliferation in the condyle, we performed immunohistochemistry of proliferating cell nuclear antigen (PCNA). The ratio of PCNA-positive cells was significantly increased in the kartogenin group. Next, immunofluorescence of TGF-β1 and SMAD3 was performed to reveal that kartogenin activated the TGF-β/SMAD pathway in the proliferative layer. In conclusion, kartogenin may have a therapeutic effect on TMJ injury by promoting cell proliferation in cartilage and subchondral bone. Kartogenin may be promising as an intra-articular injection agent to treat TMD.

High nuclear expression of DNMT1 in correlation with inactivation of TET1 portray worst prognosis among the cervical carcinoma patients: clinical implications.

In this study, we aimed to understand the interplay of the epigenetic modifier genes DNMT1 and TET1 along with HPV infection in the cervical epithelium and how it changes during tumorigenesis. For this purpose, initially the bioinformatical analysis (methylation and expression profile) of DNMT1 and TET1 was analyzed in the TCGA dataset. Next genetic (deletion) and epigenetic profiling (promoter methylation) of DNMT1 and TET1 were done in our sample pool and also validated in CACX cell lines as well. The results were further correlated with different clinicopathological parameters. Our data revealed that HPV infection in basal/parabasal layers of cervical epithelium actually disrupts the epigenetic homeostasis of DNMT1 and TET1 proteins which ultimately leads to the high expression of DNMT1 along with further reduction in TET1 protein during the development of carcinoma. Further, in-depth look into the results revealed that comparatively low methylation frequency of DNMT1 coupled with high promoter methylation and deletion frequency [22-46%] of TET1 were the plausible reasons of their antagonistic expression profile during the progression of the disease. Interestingly, the prevalence of DNMT1 [9.1%] and TET1 promoter methylation [22.7%] found in both the plasma DNA of the respective CACX patients implicated its diagnostic importance in this study. Lastly, molecular alteration of TET1 alone or in combination with DNMT1 showed the worst overall survival among the patients. Hence, it may be concluded that an inverse molecular profile of DNMT1 and TET1 genes seen in the proliferative basal-parabasal layers of the cervical epithelium was aggravated during the development of CACX along with genetic and epigenetic changes due to HPV infection.

Visualization of temporomandibular joint articular cartilage using synchrotron-radiation X-ray phase-contrast imaging

X-ray phase-contrast imaging using synchrotron radiation was applied to visualize non-invasively the fine structures of articular cartilage of temporomandibular joint (TMJ). Among various X-ray phase-contrast imaging methods, we chose diffraction enhanced imaging (DEI) and carried out experiments using synchrotron radiation, aiming to visualize TMJ articular cartilage of rats and to identify the optimum X-ray energy. For comparison, the same sample was observed by a conventional laboratory X-ray source and X-ray micro-CT apparatus. DEI was able to visualize the articular cartilage of the mandibular condyle, which was not possible with the conventional laboratory X-ray source and X-ray micro-CT apparatus. DEI could also visualize the fibrous and proliferative layers in the articular cartilage of the mandibular condyle. We found that the optimum imaging energy was 20 keV for our sample. It was shown that DEI is a promising method for non-invasive visualization of TMJ articular cartilage. Our findings will open up new possibilities for applications of DEI with synchrotron radiation for basic medical and preclinical research.

The basement membrane in epidermal polarity, stemness, and regeneration.

The epidermis is a specialized epithelium that constitutes the outermost layer of the skin, and it provides a protective barrier against environmental assaults. Primarily consisting of multilayered keratinocytes, the epidermis is continuously renewed by proliferation of stem cells and the differentiation of their progeny, which undergo terminal differentiation as they leave the basal layer and move upward toward the surface, where they die and slough off. Basal keratinocytes rest on a basement membrane at the dermal-epidermal junction that is composed of specific extracellular matrix proteins organized into interactive and mechanically supportive networks. Firm attachment of basal keratinocytes, and their dynamic regulation via focal adhesions and hemidesmosomes, is essential for maintaining major skin processes, such as self-renewal, barrier function, and resistance to physical and chemical stresses. The adhesive integrin receptors expressed by epidermal cells serve structural, signaling, and mechanosensory roles that are critical for epidermal cell anchorage and tissue homeostasis. More specifically, the basement membrane components play key roles in preserving the stem cell pool, and establishing cell polarity cues enabling asymmetric cell divisions, which result in the transition from a proliferative basal cell layer to suprabasal cells committed to terminal differentiation. Finally, through a well-regulated sequence of synthesis and remodeling, the components of the dermal-epidermal junction play an essential role in regeneration of the epidermis during skin healing. Here too, they provide biological and mechanical signals that are essential to the restoration of barrier function.

Longitudinal effects of estrogen on mandibular growth and changes in cartilage during the growth period in rats

Estrogen is a steroid hormone that induces skeletal growth and affects endochondral ossification of the long tubular bone growth plate during the growth period. However, the effects of estrogen on endochondral ossification of the mandibular condylar cartilage are unclear. In this study, ovariectomized Wistar/ST rats were used to investigate the longitudinal effects of estrogen on mandibular growth. The rats were administered different doses of estrogen. Longitudinal micro-computed tomographic scanning, histological staining and ELISA on plasma growth hormone were performed to examine the effects of estrogen on mandibular growth. The results showed that mandibular growth was suppressed throughout the growth period by estrogen in a dose-dependent manner. In addition, long-term administration of a high dose of estrogen to the rats resulted in significant increase in growth hormone throughout the growth period, significant circularization of cell nuclei in the proliferative layer, intensely staining cartilage matrix in the subchondral bone, and significant suppression of estrogen receptor (ER) alpha and beta expression in the mandibular cartilage. However, regardless of estrogen concentration, in the posterior part of the mandibular cartilage, ER expression extended to both the hypertrophic and proliferative layers. These results indicate that estrogen suppresses mandibular growth throughout the growth period. Additionally, it influences endochondral ossification via its effect on ERs.

MTORC1 Activity in Psoriatic Lesions Is Mediated by Aberrant Regulation through the Tuberous Sclerosis Complex.

In the basal, proliferative layer of healthy skin, the mTOR complex 1 (mTORC1) is activated, thus regulating proliferation while preventing differentiation. When cells leave the proliferative, basal compartment, mTORC1 signaling is turned off, which allows differentiation. Under inflammatory conditions, this switch is hijacked by cytokines and prevents proper differentiation. It is currently unknown how mTORC1 is regulated to mediate these effects on keratinocyte differentiation. In other tissues, mTORC1 activity is controlled through various pathways via the tuberous sclerosis complex (TSC). Thus, we investigated whether the TS complex is regulated by proinflammatory cytokines and contributes to the pathogenesis of psoriasis. TNF-α as well as IL-1β induced the phosphorylation of TSC2, especially on S939 via the PI3-K/AKT and MAPK pathway. Surprisingly, increased TSC2 phosphorylation could not be detected in psoriasis patients. Instead, TSC2 was strongly downregulated in lesional psoriatic skin compared to non-lesional skin of the same patients or healthy skin. In vitro inflammatory cytokines induced dissociation of TSC2 from the lysosome, followed by destabilization of the TS complex and degradation. Thus, we assume that in psoriasis, inflammatory cytokines induce strong TSC2 phosphorylation, which in turn leads to its degradation. Consequently, chronic mTORC1 activity impairs ordered keratinocyte differentiation and contributes to the phenotypical changes seen in the psoriatic epidermis.

ALPK1 Aggravates TMJOA Cartilage Degradation via NF-κB and ERK1/2 Signaling

Temporomandibular joint osteoarthritis (TMJOA) is a common degenerative joint disease without effective intervention strategies. Previous research implied that alpha-kinase 1 (ALPK1) is involved in the inflammatory responses of gout, a chronic arthritis. Herein, we found the main distribution of ALPK1 in a proliferative layer of condylar cartilage and marrow cavity of subchondral bone, as well as a lining layer of synovial tissues in human temporomandibular joint. Moreover, the expression of ALPK1 was augmented in degraded condylar cartilage of monosodium iodoacetate (MIA)–induced TMJOA mice. After MIA induction, ALPK1 knockout mice exhibited attenuated damage of cartilage and subchondral bone, as well as synovitis, as compared with wide type mice. In contrast, intra-articular administration of recombinant human ALPK1 aggravated the pathology of MIA-induced TMJOA. Furthermore, ex vivo study demonstrated that ALPK1 exacerbated chondrocyte catabolism by upregulating matrix metalloproteinase 13 and cyclooxygenase 2 by activating NF-κB (nuclear factor–kappaB) signaling and suppressed anabolism by downregulating aggrecan by inhibiting ERK1/2 (extracellular signal–regulated kinase 1/2) in articular chondrocytes. Taken together, ALPK1 exacerbates the degradation of condylar cartilage during TMJOA through the NF-κB and ERK1/2 signaling pathway. This study provides a new insight regarding the role of ALPK1 during TMJOA pathology.

Dia1 coordinates differentiation and cell sorting in a stratified epithelium.

Although implicated in adhesion, only a few studies address how the actin assembly factors guide cell positioning in multicellular tissues. The formin, Dia1, localizes to the proliferative basal layer of the epidermis. In organotypic cultures, Dia1 depletion reduced basal cell density and resulted in stratified tissues with disorganized differentiation and proliferative markers. Since crowding induces differentiation in epidermal tissues, we hypothesized that Dia1 is essential to reach densities amenable to differentiation before or during stratification. Consistent with this, forced crowding of Dia1-deficient cells rescued transcriptional abnormalities. We find Dia1 promotes rapid growth of lateral cell-cell adhesions, necessary for the construction of a highly crowded monolayer. In aggregation assays, cells sorted into distinct layers based on Dia1 expression status. These results suggest that as basal cells proliferate, reintegration and packing of Dia1-positive daughter cells is favored, whereas Dia1-negative cells tend to delaminate to a suprabasal compartment. This work elucidates the role of formin expression patterns in constructing distinct cellular domains within stratified epithelia.

A Case Report of Ultrasonographic “Endometrial Three Layer” Pattern: A Unique Finding in Ectopic Pregnancy Recent Perspective

Although diagnostic ultrasonography is playing an increasing role in the investigation of the patient with suspected ectopic pregnancy (EP), it has significant limitations in the very early stages of pregnancy. By sonographically exploring the intrauterine echoes in 45 cases of documented EP, we demonstrated a unique pattern in 28 cases (62.2%). A well-defined spheric structure forming an endometrial three-layer (ETL) pattern was seen, probably formed by a midline echo between the two adjacent edematous proliferative layers of the endometrium, the latter resembling the late proliferative endometrium. In 17 patients with a proved EP (37.8%), the ETL pattern was not demonstrated. However, the ETL was not demonstrated in all 40 cases of early intrauterine pregnancy and all 50 cases of miscarriage. These findings suggest 100% specificity and a sensitivity of 62.2% for the ETL pattern in the diagnosis of EP.

Therapeutic application of 3B-PEG injectable hydrogel/Nell-1 composite system to temporomandibular joint osteoarthritis

This study aims to construct a composite system of the tri-block polyethylene glycol injectable hydrogel (3B-PEG IH) and neural epithelial growth factor-like protein 1 (Nell-1), and to analyze its therapeutic effect on temporomandibular joint osteoarthritis (TMJOA). Sol-gel transition temperature was measured via inverting test. The viscoelastic modulus curves was measured by rheometer. Degradation and controlled release profiles of 3B-PEG IH were drawn in vitro. In vivo gel retention and biocompatibility were completed subcutaneously on the back of rats. After primary chondrocytes were extracted and identified, the cell viability in 3B-PEG IH was measured. Evaluation of gene expression in hydrogel was performed by real-time polymerase chain reaction. TMJOA rabbits were established by intra-articular injection of type II collagenase. Six weeks after composite systems being injected, gross morphological score, micro-CT, histological staining and grading were evaluated. The rusults showed that different types of 3B-PEG IH all reached a stable gel state at 37 °C and could support the three-dimensional growth of chondrocytes, but poly(lactide-co-caprolactone)-block-poly(ethyleneglycol)-block-poly(lactide-co-caprolactone) (PLCL-PEG-PLCL) hydrogel had a wider gelation temperature range and better hydrolytic stability for about 4 weeks. Its controlled release curve is closest to the zero-order release kinetics. In vitro, PLCL-PEG-PLCL/Nell-1 could promote the chondrogenic expression and reduce the inflammatory expression. In vivo, TMJOA rabbits were mainly characterized by the disorder of cartilage structure and the destruction of subchondral bone. However, PLCL-PEG-PLCL/Nell-1 could reverse the destruction of the subchondral trabecula, restore the fibrous and proliferative layers of the surface, and reduce the irregular hyperplasia of fibrocartilage layer. In conclusion, by comparing the properties of different 3B-PEG IH, 20 wt% PLCL-PEG-PLCL hydrogel was selected as the most appropriate material. PLCL-PEG-PLCL/Nell-1 composite could reverse osteochondral damage caused by TMJOA, Nfatc1-Runx3 signaling pathway may play a role in it. This study may provide a novel, minimally-invasive therapeutic strategy for the clinical treatment of TMJOA.