Collagen Density Research Articles

The Possible Protective Effect of Resveratrol on Naphthalene Induced Nephrotoxicity in Adult Male Albino Rat

Abstract Background Resveratrol, a natural polyphenolic compound that is extensively stemmed from grapes, berries, red wine, and peanut skins, is reported with its pharmacological activities against numerous cardiovascular and renal diseases, inflammation, anti-oxidation and cancers. Naphthalene (NA) is a common environmental hazardous contaminant and is abundant in tobacco smoking, in addition to various exposures. Aim of Work the aim of the present work was to study the possible protective effect of Resveratrol on Naphthalene induced nephrotoxicity in adult male albino rat. Materials and Methods 36 Adult male albino rats were assigned randomly into three groups; control group, it was further subdivided into four subgroups (IA received no treatment, IB received Resveratrol 10 mg/kg/day, IC received corn oil 5 mg/kg/day and ID received Propylene glycol), NA group (rats received 200 mg/kg of NA dissolved in 5 ml/kg corn oil daily for four weeks, orally), and Resveratrol protected group (rats received Resveratrol dissolved in propylene glycol at a dose 10 mg/kg followed after 60 min with Naphthalene at a dose 200 mg/kg dissolved in 5 ml/kg corn oil daily for four weeks). At the end of experiment (30 days), rats were euthanized, and kidney specimens were processed into paraffin blocks for light microscopic examination. Morphometric study and statistical analysis were done. Results The present work demonstrated that NA induced several histopathological changes of the kidneys in terms of cortex and medulla. Distorted renal capsule, thickened glomerular basement membrane, scattered small-sized and hypercellular glomeruli with apoptotic cells and narrow Bowman’s spaces. Proximal tubules and the distal tubules showed markedly apoptotic epithelial lining with pyknotic nuclei, partial loss of brush borders, vacuolations and intra-tubular hyaline casts. Congested interstitial blood vessels, peri-vascular edema, massive inflammatory infiltrate and dense collagen distribution. Renal medulla showed collecting tubules with apoptotic epithelial lining with pyknotic nuclei and intratubular hyaline casts. Findings were demonstrated by histomorphometry. Resveratrol protected group showed histopathological findings significantly reduced compared with NA group. Reduced number of scattered small-sized glomeruli with less thickened glomerular basement membrane. The proximal tubules and distal tubules appeared with less apoptotic epithelial lining, fewer pyknotic nuclei and preserved brush borders and interstitium with fair collagen deposition. The medullary regions showed collecting tubules with mostly normal epithelial lining and reduced inflammatory infiltrate. Conclusion The present study demonstrated the deleterious effects of NA on the structure of the kidney. It also suggested the protective role of Resveratrol against the nephrotoxicity.

Wound healing of gaster perforation with Human Dried Amniotic Membrane (H-DAM) as biomaterial patch viewing from the fibroblasts counts and collagen density

Link of Video Abstract: https://youtu.be/UldBMnzIelE Background: Gastric perforation in neonates is an emergency in neonatology with a mortality rate of up to 75%. Many studies have been carried out to improve tissue healing, including using various surgical techniques and materials, one of which is using H-DAM technology. Therefore, this study aims to evaluate the differences in the number of fibroblasts and collagen density in gastric perforation repair with H-DAM as a biomaterial patch compared to using an omental patch in New Zealand white rabbits. Methods: A true-experimental study was conducted among 30 samples of rabbits and none of them dropped out. The inclusion criteria for this study are New Zealand White Rabbit male sex, age 6-9 months with a weight of 2-3 kg, healthy and active. The Exclusion criteria for this study are rabbits not fasted for 12 hours. During the 12-hour fasting period, it behaved aggressively, attacked other rabbits, and found surgical site infection. Data were analyzed using SPSS version 25.0 for Windows. Results: From the examination results, there was a significant difference in gastric healing in terms of examining the number of fibroblasts with the Kruskal-Wallis test (p=0.000. The One-way ANOVA analysis found that the three data groups had no significant difference (p=0.234). Conclusion: There is an increase in the number of fibroblasts and collagen density in gastric perforation repair with H-DAM as a biological dressing compared to primary repair of gastric perforation using an omental patch in rabbit models so that H-DAM can be the repair technique of choice in gastric perforation in the rabbit.

Guanosine and Deoxyinosine Structural Analogs Extracted from Chick Early Amniotic Fluid Promote Cutaneous Wound Healing.

Wound healing is a complex, dynamic process supported by a myriad of cellular events that must be tightly coordinated to efficiently repair damaged tissue. These wounds are a significant socioeconomic burden due to their high prevalence and recurrence, which is why the phenomenon of wounds has also been labeled as a "Silent Epidemic". Most of these wounds become "chronic", with around 15% of them remaining unresolved 1-year post incidence, which results in a prolonged yet avoidable burden to patients, families, and the health system. In this experimental study, we tried to purify the potent components in chick early amniotic fluid (ceAF) and applied these components to the wound healing mechanism. We first subjected ceAF to a series of purifications, including an HPLC purification system along with ion-exchange chromatography technology to purify other potential components. Upon narrowing down, we found two structural analogs: guanosine and deoxyinosine. We performed these components' cell scratch and trans-well migration assays to validate the accurate dosage. We also assessed these components via topical administration on the skin of murine model wounds. For this, we randomly divided C57BL/6 (all black, male, 5 weeks old) mice into groups. The wound model was established through excising the skin of mice and treated the wounds with different fractions of guanosine and deoxyinosine continuously for 8-10 day intervals. Once the healing was complete, the skin was excised to determine the inflammatory response and other biochemical parameters of the healed skin, including epidermal thickness, collagen density, macrophages, and neutrophil infiltration at the wounded site. Quantitative real-time PCR and immunoblot assays were performed to determine active gene expression and protein expression of proinflammatory molecules, growth factors, and cytokines. All these findings support our data indicating the promising healing properties of guanosine and deoxyinosine isolated from ceAF.

Organization of collagen fibers and tissue hardening: Markers of fibrotic scarring after spinal cord injury in mice revealed by multiphoton-atomic force microscopy imaging

Spinal cord injury is a dramatic disease leading to severe motor, sensitive and autonomic impairments. After injury the axonal regeneration is partly inhibited by the glial scar, acting as a physical and chemical barrier. The scarring process involves microglia, astrocytes and extracellular matrix components, such as collagen, constructing the fibrotic component of the scar. To investigate the role of collagen, we used a multimodal label-free imaging approach combining multiphoton and atomic force microscopy. The second harmonic generation signal exhibited by fibrillar collagen enabled to specifically monitor it as a biomarker of the lesion. An increase in collagen density and the formation of more tortuous fibers over time after injury are observed. Nano-mechanical investigations revealed a noticeable hardening of the injured area, correlated with collagen fibers' formation. These observations indicate the concomitance of important structural and mechanical modifications during the fibrotic scar evolution.

The size, number, and distribution of nerve endings around and within the human epiglottis, focusing on tracheal intubation maneuvers.

The aim of this study was to examine the distribution of nerve endings in the mucosa, submucosa, and cartilage of the epiglottis and the vallecula area and to quantify them. The findings could inform the choice of laryngoscope blades for intubation procedures. Fourteen neck slices from seven unembalmed, cryopreserved human cadavers were analyzed. The slices were stained, and cross and longitudinal sections were obtained from each. The nerve endings and cartilage were identified. The primary metrics recorded were the number, area, and circumference of nerve endings located in the mucosa and submucosa of the pharyngeal and laryngeal sides of the epiglottis, epiglottis cartilage, and epiglottic vallecula zone. The length and thickness of the epiglottis and cartilage were also measured. The elastic cartilage of the epiglottis was primarily continuous; however, it contained several fragments. It was covered with dense collagen fibers and surrounded by adipose cells from the pharyngeal and laryngeal submucosa. Nerve endings were found within the submucosa of pharyngeal and laryngeal epiglottis and epiglottic vallecula. There were significantly more nerve endings on the posterior surface of the epiglottis than on the anterior surface. The epiglottic cartilage was twice the length of the epiglottis. The study demonstrated that the distribution of nerve endings in the epiglottis differed significantly between the posterior and anterior sides; there were considerably more in the former. The findings have implications for tracheal intubation and laryngoscope blade selection and design.

Idiopathic Sclerosing Encapsulating Peritonitis: A Case Reports

Sclerosing encapsulating peritonitis is a disease characterized by a chronicle inflammatory process in which part or all of the digestive tract is enveloped by a dense fibro collagen membrane. The clinical picture is not very telling and nonspecific, however imaging, in particular injected computed tomography, easily allows the diagnosis, but the idiopathic form remains very rare. In this article, we report the case of a 44-years-old man with no particular history, who presents to the emergency room for stopping materials and gases, an abdomino-pelvic calculated tomography injected confirms the diagnosis of an intestinal obstruction consecutive to idiopathic sclerosing encapsulating peritonitis.

The effect of bevacizumab, 5-fluorouracil, and triamcinolone on the healing modulation of surgical wounds in rats.

In this study, we aimed to analyze the effect of 5-fluorouracil, triamcinolone, and bevacizumab on scar modulation in an experimental rat model of surgical lesions. Rats (Rattus norvegicus albinus) were divided into four groups: bevacizumab, 5-fluorouracil + triamcinolone, bevacizumab + 5-fluorouracil + triamcinolone, and control (received no medication) groups. A linear, dorsal incision was created and sutured for the first intention wound healing, mimicking the surgical incision of upper blepharoplasty. Treatments were initiated on day 7, and the rats were euthanized on day 14. Only in the 5-fluorouracil + triamcinolone group was there a difference in the number of infiltrated monocytes. There was 56%, 86%, and 85% decrease in the number of neovessels in the bevacizumab, 5-fluorouracil + triamcinolone, and bevacizumab + 5-fluorouracil + triamcinolone groups, respectively, compared with the control. Picrosirius red staining showed higher collagen density and more organized collagen in the treatment groups than in the control group. Scar modulation was observed in all groups, but the 5-fluorouracil + triamcinolone group presented the best results. To our knowledge, this is the first study to evaluate the influence of three medications in combination on healing. When used together, these medications can prevent the development of unsightly scars, and are therefore promising alternatives to corticosteroids.

The Effectiveness of Sungkai Leaf (Peronema canescens Jack.) Extract Gel on the Collagen Density of Incision Wounds in Vivo

An incision is an open wound resulting in skin injury and underlying tissues. The wound-healing process involves a proliferative phase that produces collagen. Sungkai leaves (Peronema canescens Jack.) can be used as an alternative therapy for wound healing because it contains flavonoids, tannins, and saponins that stimulate fibroblasts to synthesize collagen. This study aimed to determine the effectiveness of sungkai leaf extract gel in healing incision wounds in vivo based on collagen density scoring. Twenty-five Wistar rats (Rattus norvegicus) were randomly divided into five groups, the negative control group (K1) was given a gelling agent CMC 1%, the positive control group (K2) was given bioplacenton gel 1%, the treatment group (K3, K4, K5) was given a gel sungkai leaf extract concentration of 5, 10, and 15. The treatment is given topically at the incision site of the incision site of 0.1 g once a day for 14 days. The rats were euthanized on the fifteenth day, and the incision area was excised for histopathological preparation. The data obtained were analyzed using Kruskal-Wallis with SPSS 26. The test results showed p=0.010, meaning there is a significant difference (p<0.05) between the entire treatment. Based on these results, the K4 group (Sungkai leaf extract gel concentration of 10%) showed the highest collagen density scoring results compared to other treatments. This study concludes that sungkai leaves effectively heal incision wounds in Wistar rats based on collagen density scoring parameter.

Multifaced roles of desmoplastic reaction and fibrosis in pancreatic cancer progression: Current understanding and future directions.

Desmoplastic reaction is a fibrosis reaction that is characterized by a large amount of dense extracellular matrix (ECM) and dense fibrous stroma. Fibrotic stroma around the tumor has several different components, including myofibroblasts, collagen, and other ECM molecules. This stromal reaction is a natural response to the tissue injury process, and fibrosis formation is a key factor in pancreatic cancer development. The fibrotic stroma of pancreatic cancer is associated with tumor progression, metastasis, and poor prognosis. Reportedly, multiple processes are involved in fibrosis, which is largely associated with the upregulation of various cytokines, chemokines, matrix metalloproteinases, and other growth factors that promote tumor growth and metastasis. Fibrosis is also associated with immunosuppressive cell recruitment, such as regulatory T cells (Tregs) with suppressing function to antitumor immunity. Further, dense fibrosis restricts the flow of nutrients and oxygen to the tumor cells, which can contribute to drug resistance. Furthermore, the dense collagen matrix can act as a physical barrier to block the entry of drugs into the tumor, thereby further contributing to drug resistance. Thus, understanding the mechanism of desmoplastic reaction and fibrosis in pancreatic cancer will open an avenue to innovative medicine and improve the prognosis of patients suffering from this disease.

Repurposing Tamoxifen for Tumor Microenvironment Priming and Enhanced Tumor-Targeted Drug Delivery.

The dense stromal matrix in fibrotic tumors hinders tumor-targeted drug delivery. Tamoxifen (TMX), an estrogen receptor modulator that is clinically used for the treatment of breast cancer, has been shown to reprogram the tumor microenvironment (TME) and to alleviate desmoplasia. We here investigated if TMX, administered in free and nano-formulated form, can be repurposed as a TME remodeling agent to improve tumor accumulation of nano-formulations in pancreatic ductal adenocarcinoma and triple-negative breast cancer mouse models, evaluated using clinical-stage Cy7-labeled core-crosslinked polymeric micelles (CCPM). Under control conditions, we found higher levels of Cy7-CCPM in PANC-1 tumors (16.7 % ID g-1 at 48 h post i.v. injection) than in 4T1 tumors (11.0 % ID g-1). In both models, free and nano-formulated TMX failed to improve CCPM delivery. These findings were congruent with the results from histopathological immunofluorescence analysis of tumor tissue, which indicated that TMX treatment did not significantly change vascularization, perfusion, macrophage infiltration, collagen density, and collagen fiber thickness. Altogether, our results demonstrate that in PANC-1 and 4T1 mouse models, TMX treatment does not contribute to beneficial TME priming and enhanced tumor-targeted drug delivery.

Polychlorinated Biphenyls (PCBs) Impact Prostatic Collagen Density and Bladder Volume in Young Adult Mice Exposed during in Utero and Lactational Development.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants linked to deleterious health outcomes, including voiding dysfunction in developmentally exposed mice. Changes in prostate volume and/or extracellular matrix composition are associated with voiding dysfunction in men and animal models. Whether PCB-induced changes in voiding function in male mice occur in part via alterations to the prostate or an alternate mechanism is unclear. Therefore, we tested whether developmental exposure to the MARBLES PCB mixture altered prostate morphology in young adult offspring. C57Bl/6J female mice were dosed daily with the MARBLES PCB mixture at 0, 0.1, 1 or 6 mg/kg/d for two weeks prior to mating and through gestation and lactation, offspring were collected at 6 weeks of age. Ventral prostate mass was decreased in the 1 mg/kg/d PCB group compared to other PCB groups. There were no PCB-induced changes in prostate smooth muscle thickness, apoptosis, proliferation, or testes mass. PCBs impacted the prostate extracellular matrix; anterior prostate collagen density was decreased in the 1 mg/kg/d PCB group compared to all other groups. Normalized bladder volume was increased in male and female offspring in the 6 mg/kg/d PCB group compared to control. No change in water consumption, bladder mass or bladder smooth muscle thickness accompanied changes in bladder volume. Urine and serum creatinine concentrations were elevated but only in male mice. Together, these results suggest that developmental exposure to PCBs can influence prostate wet weight and prostate/bladder morphology, but PCBs do not promote prostate enlargement. Whether these changes persist throughout adult life and how they contribute to voiding function in animal models and humans is of future interest.

Effect of matrix properties on transmission and reflectance mode division-of-focal-plane Stokes polarimetry.

Division-of-focal-plane Stokes polarimetry is emerging as a powerful tool for the microstructural characterization of soft tissues. How individual extracellular matrix (ECM) properties influence polarimetric signals in reflectance or transmission modes of quantitative polarized light imaging (QPLI) is not well understood. We aimed to investigate how ECM properties affect outcomes obtained from division-of-focal-plane polarimetric imaging in reflectance or transmission modes. Tunable collagen gel phantoms were used to modulate ECM properties of anisotropy, collagen density, crosslinking, and absorber density; the effects of degree of linear polarization (DoLP) and angle of polarization (AoP) on polarimetry outcomes were assessed. A model biological tissue (i.e., bovine tendon) was similarly imaged and evaluated using both reflectance and transmission modes. Reflectance QPLI resulted in decreased DoLP compared with transmission mode. A 90deg shift in AoP was observed between modes but yielded similar spatial patterns. Collagen density had the largest effect on outcomes besides anisotropy in both imaging modes. Both imaging modes were sufficiently sensitive to detect structural anisotropy differences in gels of varying fiber alignment. Conclusions drawn from phantom experiments should carry over when interpreting data from more complex tissues and can help provide context for interpretation of other Stokes polarimetry data.

Administration of Arabica Coffee Bean (Coffea Arabica) Cream Extract from Wamena Papua Can Reduce MMP-1 And Increase Collagen in Wistar Male Rats (Rattus Norvegicus) Exposed to Ultraviolet-B

Introduction: The aim of the study was to determine the effectiveness of Arabica coffee bean extract cream (Coffea arabica) from Wamena in reducing MMP-1 levels and increasing collagen density in Wistar rats (Rattus norvegicus) exposed to UV-B light. Methods: This is a post-test only control group design experimental study using male rats as subjects. Each group was exposed to UV light and smeared with coffee bean cream extract from Wamena every day. Then the back skin tissue was taken and the amount of collagen was examined by Picro Sirius Red staining and MMP1 levels were examined by ELISA staining. Results: The mean MMP-1 levels in groups P1, P2, and P3 respectively 7.57 ± 0.70, 5.99 ± 0.60, and 4.57 ± 0.36. The average percentage of collagen in the P1, P2, and P3 groups was 72.23 ± 4.42, 77.80 ± 2.73, and 81.05 ± 2.77, respectively. The average MMP-1 level decreased with the increasing dose of Wamena arabica coffee bean extract cream. The mean percentage of collagen increased with increasing doses of Wamena arabica coffee bean extract cream in experimental animals. Conclusion: Administration of 3% and 6% Wamena Arabica coffee bean extract cream can inhibit the increase in MMP-1 levels and can increase the amount of collagen in male wistar rats (Rattus norvegicus) exposed to UV-B light. Keywords: Arabica coffee bean, MMP-1, Collagen

Collagen-nanoclay hydrogels for stiffness enhancement of the extracellular matrix: the effect on spheroid growth

The extracellular matrix (ECM) is crucial for regulating cellular functions and maintaining tissue integrity. Alterations in ECM stiffness are associated with various pathological conditions, including cancer and cardiovascular disease. Therefore, being able to tailor and customize ECM stiffness could revolutionize personalized research and therapies in tissue engineering. Clay nanomaterials are an emerging class of biomaterials with unique properties that make them promising candidates for biomedical applications.
 In this study, we investigated the potential of laponite nanoclays (LapNC) to enhance the mechanical properties of collagen hydrogels without increasing the collagen amount. Collagen hydrogels of different concentrations (2.5, 4, and 6 mg/ml) were supplemented with LapNC, and the resulting hydrogels were thoroughly characterized using rheology, scanning electron microscopy (SEM) and other assays to quantify additional physical parameters, such as the permeability and swelling potential. In this study, we investigated the potential application of the customized hydrogels as 3D cancer culture systems for tumor spheroid growth. To this end, we quantified the spheroid area over time by culturing three cancer cell lines in the supplemented and non-supplemented matrices. Moreover, we assessed the migration capacity of the spheroids with respect to the initial cluster formation.
 Our results confirmed that LapNC solution could be successfully incorporated into the culture media without clay aggregation. The supplemented hydrogels exhibited enhanced mechanical properties without compromising cellular viability or causing collagen collapse. Regardless of the collagen concentration, LapNC-supplemented hydrogels showed higher storage moduli and lower permeability than control hydrogels, which was qualitatively corroborated by SEM visualization of the porosity. Furthermore, the addition of LapNC resulted in the formation of larger spheroids at every collagen concentration. These findings have significant implications for tissue engineering applications, particularly in cancer spheroid analysis.
 The successful incorporation of LapNC into collagen hydrogels as nanofillers to modify the rheological and structural properties highlights their potential for tissue engineering applications. Our results demonstrate that the mechanical properties of collagen hydrogels can be enhanced by the addition of LapNC without increasing collagen density. This may provide a powerful method for personalized research and therapies in multiple fields of tissue engineering. In particular, our study suggests that LapNC-supplemented hydrogels can serve as 3D cancer culture systems for stiffness-dependent tumor spheroid growth, opening up new avenues for cancer research and therapy.

Up-Cycling of Olea europaea L. Ancient Cultivars Side Products: Study of a Combined Cosmetic-Food Supplement Treatment Based on Leaves and Olive Mill Wastewater Extracts.

In recent years, a reversal of the global economic framework has been taking place: from the linear model, there has been a gradual transition to a circular model where by-products from the agri-food industry are taken and transformed into value products (upcycling) rather than being disposed of. Olive tree pruning represents an important biomass currently used for combustion; however, the leaf part of the olive tree is rich in phenolic substances, including hydroxytyrosol. Mill wastewater is also discarded, but it still contains high amounts of hydroxytyrosol. In this study, cosmetic and food supplement formulations were prepared using biophenols extracted from leaves and wastewater and were tested in a placebo-controlled study on healthy volunteers using a combined cosmetic and food supplement treatment. A significant improvement in skin health indicators (collagen density, elasticity, etc.) and a 17% improvement against Photo-induced Irritative Stimulus was observed.

PFKFB3 Inhibitor 3PO Reduces Cardiac Remodeling after Myocardial Infarction by Regulating the TGF-β1/SMAD2/3 Pathway.

Adverse cardiac remodeling, including cardiac fibrosis, after myocardial infarction (MI) is a major cause of long-term heart failure. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), an enzyme that regulates glucose metabolism, also plays an important role in various fibrotic and cardiovascular diseases. However, its effects on MI remain unknown. Here, PFKFB3 inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) and a permanent left anterior descending ligation mouse model were used to explore the functional role of PFKFB3 in MI. We showed that PFKFB3 expression increased significantly in the area of cardiac infarction during the early phase after MI, peaking on day 3. 3PO treatment markedly improved cardiac function, accompanied by decreased infarction size and collagen density in the infarct area. Meanwhile, 3PO attenuated cardiac fibrosis after MI by reducing the expression of collagen and fibronectin in murine hearts. Notably, 3PO reduced PFKFB3 expression and inhibited the transforming growth factor-beta 1/mothers against the decapentaplegic homolog 2/3 (TGF-β1/SMAD2/3) signaling pathway to inhibit cardiac fibrosis after MI. Moreover, PFKFB3 expression in neonatal rat cardiac fibroblasts (NRCFs) increased significantly after MI and under hypoxia, whereas 3PO alleviated the migratory capacity and activation of NRCFs induced by TGF-β1. In conclusion, 3PO effectively reduced fibrosis and improved adverse cardiac remodeling after MI, suggesting PFKFB3 inhibition as a novel therapeutic strategy to reduce the incidence of chronic heart failure following MI.

Identification of resibufogenin, a component of toad venom, as a novel senolytic compound in vitro and for potential skin rejuvenation in male mice.

Senescent cells that accumulate with age have been shown to contribute to age-related diseases and organ dysfunction and have attracted attention as a target for anti-aging therapy. In particular, the use of senescent cell-depleting agents, or senolytics, has been shown to improve the aging phenotype in animal models. Since senescence has been implicated in the skin, particularly in fibroblasts, this study used aged human skin fibroblasts to investigate the effects of resibufogenin. A component of the traditional Chinese medicine toad venom, resibufogenin was investigated for senolytic and/or senomorphic activity. We found that the compound selectively caused senescent cell death without affecting proliferating cells, with a marked effect on the suppression of the senescence-associated secretory phenotype. We also found that resibufogenin causes senescent cell death by inducing a caspase-3-mediated apoptotic program. Administration of resibufogenin to aging mice resulted in an increase in dermal collagen density and subcutaneous fat, improving the phenotype of aging skin. In other words, resibufogenin ameliorates skin aging through selective induction of senescent cell apoptosis without affecting non-aged cells. This traditional compound may have potential therapeutic benefits in skin aging characterized by senescent cell accumulation.

Bone beads enveloped with vascular endothelial cells for bone regenerative medicine.

The transplantation of pre-vascularized bone grafts is a promising strategy to improve the efficacy of engraftment and bone regeneration. We propose a hydrogel microbead-based approach for preparing vascularized and high-density tissue grafts. Mesenchymal stem cell-encapsulated collagen microgels (2µL), termed bone beads, were prepared through spontaneous constriction, which improved the density of the mesenchymal stem cells and collagen molecules by more than 15-fold from the initial day of culture. Constriction was attributed to cell-attractive forces and involved better osteogenic differentiation of mesenchymal stem cells than that of spheroids. This approach was scalable, and ∼2000 bone beads were prepared semi-automatically using a liquid dispenser and spinner flask. The mechanical stimuli in the spinner flask further improved the osteogenic differentiation of the mesenchymal stem cells in the bone beads compared with that in static culture. Vascular endothelial cells readily attach to and cover the surface of bone beads. The in vitro assembly of the endothelial cell-enveloped bone beads resulted in microchannel formation in the interspaces between the bone beads. Significant effects of endothelialization on in vivo bone regeneration were shown in rats with cranial bone defects. The use of endothelialized bone beads may be a scalable and robust approach for treating large bone defects. STATEMENT OF SIGNIFICANCE: A unique aspect of this study is that the hMSC-encapsulated collagen microgels were prepared through spontaneous constriction, leading to the enrichment of collagen and cell density. This constriction resulted in favorable microenvironments for the osteogenic differentiation of hMSCs, which is superior to conventional spheroid culture. The microgel beads were then enveloped with vascular endothelial cells and assembled to fabricate a tissue graft with vasculature in the interspaces among the beads. The significant effects of endothelialization on in vivo bone regeneration were clearly demonstrated in rats with cranial bone defects. We believe that microgel beads covered with vascular endothelial cells provide a promising approach for engineering better tissue grafts for bone-regenerative medicine.

Extracellular matrix remodelling and stiffening contributes to tumorigenesis of salivary carcinoma ex pleomorphic adenoma——A study based on patient-derived organoids

BackgroundSalivary carcinoma ex pleomorphic adenoma (CXPA) is defined as a carcinoma that develops from benign pleomorphic adenoma (PA). Abnormally activated Androgen signaling pathway and amplification of HER-2/neu(ERBB-2) gene are known to be involved in CXPA tumorigenesis. Recent progress in tumour microenvironment research has led to identification that extracellular matrix (ECM) remodelling and increased stiffness act as critical contributing role in tumour carcinogenesis. This study examined ECM modifications to elucidate the mechanism underlying CXPA tumorigenesis.ResultsPA and CXPA organoids were successfully established. Histological observation, immunohistochemistry (IHC), and whole-exome sequencing demonstrated that organoids recapitulated phenotypic and molecular characteristics of their parental tumours. RNA-sequencing and bioinformatic analysis of organoids showed that differentially expressed genes are highly enriched in ECM-associated terms, implying that ECM alternations may be involved in carcinogenesis. Microscopical examination for surgical samples revealed that excessive hyalinized tissues were deposited in tumour during CXPA tumorigenesis. Transmission electron microscopy confirmed that these hyalinized tissues were tumour ECM in nature. Subsequently, examination by picrosirius red staining, liquid chromatography with tandem mass spectrometry, and cross-linking analysis indicated that tumour ECM was predominantly composed of type I collagen fibers, with dense collagen alignment and an increased level of collagen cross-linking. IHC revealed the overexpression of COL1A1 protein and collagen-synthesis-related genes, DCN and IGFBP5 (p < 0.05). Higher stiffness of CXPA than PA was demonstrated by atomic force microscopy and elastic imaging analysis. We utilized hydrogels to mimic ECM with varying stiffness degrees in vitro. Compared with softer matrices (5Kpa), CXPA cell line and PA primary cells exhibited more proliferative and invasive phenotypes in stiffer matrices (50Kpa, p < 0.01). Protein-protein interaction (PPI) analysis of RNA-sequencing data revealed that AR and ERBB-2 expression was associated with TWIST1. Moreover, surgical specimens demonstrated a higher TWIST1 expression in CXPA over PA. After knocking down TWIST1 in CXPA cells, cell proliferation, migration, and invasiveness were significantly inhibited (p < 0.01).ConclusionDeveloping CXPA organoids provides a useful model for cancer biology research and drug screening. ECM remodelling, attributed to overproduction of collagen, alternation of collagen alignment, and increased cross-linking, leads to increased ECM stiffness. ECM modification is an important contributor in CXPA tumorigenesis.

The Comparative Dermal Stimulation Potential of Constant-Volume and Constant-Amount Diluted Calcium Hydroxylapatite Injections Versus the Concentrated Form.

Biostimulation properties of diluted and hyperdiluted calcium hydroxylapatite (CaHA) injections have become increasingly popular. However, the existing data are insufficient to certify a particular dose-response pattern. To assess and compare the dermal stimulation potentials of different concentrations of CaHA injections. Two independent experiments (Experiment-1: constant injection volume vs Experiment-2: constant CaHA amount) included 4 study groups each, and these experimental groups were placed consecutively on the abdominal skin of a juvenile Yorkshire pig. Histopathological and immunohistochemical stainings were performed on punch biopsy materials collected 4 months after the injection day. The fibroblast count significantly decreased upon dilution from 1:3 to 1:19 in experiment 1 ( p = .000) but still higher than the control group. In experiment 1, the collagen density of the concentrated form was more elevated than the 1:19 dilution and the negative control groups ( p = .034 and .000, respectively) but similar to the 1:3 dilution ( p = .123). No significant difference was observed between the groups regarding collagen density with a standard amount of CaHA (0.2 mL, 30%) ( p > .05). Despite the efficacy being more pronounced till 1:3 dilution, hyperdiluted CaHA at any dilution ratio up to 1:19 can provide a higher fibroblast count than the negative control group.