Rat Subcutaneous Tissue Research Articles

Gum Arabic and Eugenol in a Silica-Induced Inflammatory Angiogenesis Model in Wistar Rat

This work aims to evaluate the effects of gum arabic and eugenol on inflammatory angiogenesis in a subcutaneous silica inoculum model. Ninety-six Wistar rats were distributed over 16 groups. The substances administered by gavage included distilled water, gum arabic (500 mg/100g weight), and eugenol (10mg/100g weight). Control groups received 0.2 mL of sterile distilled water subcutaneously. The experimental groups were subjected to an inoculum of 0.2 mL of silica solution (50 mg/mL) subcutaneously. For seven or 14 days, the groups received distilled water or gum arabic or eugenol or gum arabic plus eugenol. There was no difference between the histological analysis of slides stained with hematoxylin-eosin. There was a reduction in vascular density in animals that received only gum arabic for 7 (0.09 µm² ± 0.02) or 14 (0.08 µm² ± 0.03) days and only eugenol for seven days (0.08 µm² ± 0.03), but this was not statistically significant. The microvascular density significantly increased in the group treated with eugenol for 14 days (0.14 µm² ± 0.02). Eugenol reduced inflammatory angiogenesis when administered for seven days and stimulated it when administered for 14 days. Gum arabic had a potential inhibitory effect on this model. The study of inflammatory angiogenesis, induced by a silica inoculum in the subcutaneous tissue of rats, is a new and reproducible model for evaluating angiogenesis and inflammation.

A validated LC-MS/MS method for the quantitation of cefazolin in human adipose tissue: Application of EMR-Lipid sorbent as an efficient sample clean-up before mass spectrometric analyses

A novel, simple, rapid, and sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed to determine cefazolin concentrations in human adipose tissue. Sample preparation was performed by protein precipitation followed by using Captiva EMR-Lipid plates. The mobile phase consisted of 5 mM ammonium formate and 0.1% formic acid in water and 0.1% formic acid in ACN, and was pumped through a Synergi Fusion-RP column with a gradient elution program at a flow rate of 0.3 mL/min. The mass spectrometer was operated in a positive ion mode. Cloxacillin was used as an internal standard due to the observed cross-signal contribution between cefazolin and 13C2,15N-cefazolin. The method was validated according to the FDA and EMA guidelines and passed all the acceptance criteria. The calibration range was 0.05–50 µg/mL in adipose tissue homogenate (0.15–150 µg/g in adipose tissue), precision CV < 4.5%, accuracy within 93.1–100.4%. The carry-over was negligible, recovery of the method was high, and no significant matrix effect was present. Rat subcutaneous adipose tissue was demonstrated to be a suitable surrogate matrix for human adipose tissue. The validated method was successfully applied in a pilot pharmacokinetic study and will further be used in a large cohort of non-obese and obese patients dosed prophylactically with cefazolin before surgeries.

Effect of ethanol washing on porcine pulmonary artery wall decellularization using sodium dodecyl sulfate.

To determine the effectiveness of ethanol (EtOH) washing on porcine pulmonary artery (PA) wall decellularization using sodium dodecyl sulfate (SDS), we compared three different washing methods (phosphate-buffered saline [PBS], pH 9 alkali, and EtOH washing). Fresh porcine PA walls were decellularized using 0.5% SDS and 0.5% sodium deoxycholate (SDC). The decellularized tissues were rinsed using three different washing techniques. Histological, biochemical, and mechanical analyses were conducted. Implantation into the subcutaneous tissue of rats and patch implantation into the carotid artery of dogs were performed as preliminary in vivo studies. The decellularization protocol based on SDS and SDC effectively removed the cells. The major extracellular matrix (ECM) structures (collagen, elastic fiber, and glycosaminoglycan) were properly preserved with the 75% EtOH-washing method. Significantly reduced residual SDS content was identified in EtOH-washed tissues compared to that in the other methods. No significant difference in the mechanical strength test was observed between the washing methods, and the EtOH-washing method showed better results in the metabolic activity test compared to the PBS-washing method. In the rat study model, no acute rejection or massive calcification was observed. The in vivo preliminary canine study showed better cell repopulation in the EtOH-washed group. EtOH washing of SDS-based decellularized porcine PA wall can reduce the residual SDS content and preserve ECM structures, especially the elastin content, and could also enhance cell repopulation after re-implantation.

Hepatic enzymes and immunoinflammatory response to Bio-C Temp bioceramic intracanal medication implanted into the subcutaneous tissue of rats

Our purpose was to evaluate the biocompatibility and hepatotoxicity of a new bioceramic intracanal medicament, Bio-C Temp (BIO). The biological properties of BIO were compared with calcium hydroxide-based intracanal medicament (Calen; CAL), used as gold pattern. Polyethylene tubes filled with BIO or CAL, and empty tubes (control group, CG) were implanted into subcutaneous tissue of rats. After 7, 15, 30 and 60 days, the samples were embedded in paraffin for morphological, quantitative and immunohistochemistry analyses. At 7 and 60 days, blood samples were collected for analysis of serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels. The data were submitted to two-way ANOVA and Tukey’s test (p ≤ 0.05). No significant difference was detected in serum GOT and GPT levels among BIO, CAL and CG specimens. In all periods, BIO specimens exhibited lower number of inflammatory cells and immunoexpression of IL-6, a pro-inflammatory cytokine, than CAL specimens. The reduction of these parameters was accompanied by significant increase in the collagen content and in the immunoexpression of IL-10, a cytokine involved in the tissue repair, over time. Our findings indicate that Bio-C Temp is biocompatible and had no hepatotoxicity effect.

Living reactions of tissue-engineered bone derived from apatite-fiber scaffold in rat subcutaneous tissues

Living reactions of tissue-engineered bone derived from apatite-fiber scaffold in rat subcutaneous tissues

Expanding tubular microvessels on stiff substrates with endothelial cells and pericytes from the same adult tissue

Endothelial cells (ECs) usually form a monolayer on two-dimensional (2D) stiff substrates and a tubular structure with soft hydrogels. The coculture models using ECs and pericytes derived from different adult tissues or pluripotent stem cells cannot mimic tissue-specific microvessels due to vascular heterogeneity. Our study established a method for expanding tubular microvessels on 2D stiff substrates with ECs and pericytes from the same adult tissue. We isolated microvessels from adult rat subcutaneous soft connective tissue and cultured them in the custom-made tubular microvascular growth medium on 2D stiff substrates (TGM2D). TGM2D promoted adult microvessel growth for at least 4 weeks and maintained a tubular morphology, contrary to the EC monolayer in the commercial medium EGM2MV. Transcriptomic analysis showed that TGM2D upregulated angiogenesis and vascular morphogenesis while suppressing oxidation and lipid metabolic pathways. Our method can be applied to other organs for expanding organ-specific microvessels for tissue engineering.

Periodontal tissue engineering using an apatite/collagen scaffold obtained by a plasma- and precursor-assisted biomimetic process.

In the treatment of severe periodontal destruction, there is a strong demand for advanced scaffolds that can regenerate periodontal tissues with adequate quality and quantity. Recently, we developed a plasma- and precursor-assisted biomimetic process by which a porous collagen scaffold (CS) could be coated with low-crystalline apatite. The apatite-coated collagen scaffold (Ap-CS) promotes cellular ingrowth within the scaffold compared to CS in rat subcutaneous tissue. In the present study, the osteogenic activity of Ap-CS was characterized by cell culture and rat skull augmentation tests. In addition, the periodontal tissue reconstruction with Ap-CS in a beagle dog was compared to that with CS. The plasma- and precursor-assisted biomimetic process was applied to CS to obtain Ap-CS with a low-crystalline apatite coating. The effects of apatite coating on the scaffold characteristics (i.e., surface morphology, water absorption, Ca release, protein adsorption, and enzymatic degradation resistance) were assessed. Cyto-compatibility and the osteogenic properties of Ap-CS and CS were assessed in vitro using preosteoblastic MC3T3-E1 cells. In addition, we performed in vivo studies to evaluate bone augmentation and periodontal tissue reconstruction with Ap-CS and CS in a rat skull and canine furcation lesion, respectively. As previously reported, the plasma- and precursor-assisted biomimetic process generated a low-crystalline apatite layer with a nanoporous structure that uniformly covered the Ap-CS surface. Ap-CS showed significantly higher water absorption, Ca release, lysozyme adsorption, and collagenase resistance than CS. Cell culture experiments revealed that Ap-CS was superior to CS in promoting the osteoblastic differentiation of MC3T3-E1 cells while suppressing their proliferation. Additionally, Ap-CS significantly promoted (compared to CS) the augmentation of the rat skull bone and showed the potential to regenerate alveolar bone in a dog furcation defect. Ap-CS fabricated by the plasma- and precursor-assisted biomimetic process provided superior promotion of osteogenic differentiation and bone neoformation compared to CS.

A Novel Age-Related Circular RNA Circ-ATXN2 Inhibits Proliferation, Promotes Cell Death and Adipogenesis in Rat Adipose Tissue-Derived Stromal Cells.

Adipose tissue-derived stromal cells are promising candidates investigating the stem cell-related treatment. However, their proportion and utility in the human body decline with time, rendering stem cells incompetent to complete repair processes in vivo. The involvement of circRNAs in the aging process is poorly understood. Rat subcutaneous adipose tissue from 10-week-old and 27-month-old rats were used for hematoxylin and eosin (H and E) staining, TUNEL staining, and circRNA sequencing. Rat adipose tissue-derived stromal cells were cultured and overexpressed with circ-ATXN2. Proliferation was examined using xCELLigence real-time cell analysis, EdU staining, and cell cycle assay. Apoptosis was induced by CoCl2 and examined using flow cytometry. RT-PCR assay and Oil Red O staining were used to measure adipogenesis at 48 h and 14 days, respectively. H and E staining showed that the diameter of adipocytes increased; however, the number of cells decreased in old rats. TUNEL staining showed that the proportion of apoptotic cells was increased in old rats. A total of 4,860 and 4,952 circRNAs was detected in young and old rats, respectively. Among them, 67 circRNAs exhibited divergent expression between the two groups (fold change ≥2, p ≤ 0.05), of which 33 were upregulated (49.3%) and 34 were downregulated (50.7%). The proliferation of circ-ATXN2-overexpressing cells decreased significantly in vitro, which was further validated by xCELLigence real-time cell analysis, EdU staining, and cell cycle assay. Overexpression of circ-ATXN2 significantly increased the total apoptotic rate from 5.78 ± 0.46% to 11.97 ± 1.61%, early apoptotic rate from 1.76 ± 0.22% to 5.50 ± 0.66%, and late apoptosis rate from 4.02 ± 0.25% to 6.47 ± 1.06% in adipose tissue-derived stromal cells. Furthermore, in circ-ATXN2-overexpressing cells, RT-PCR assay revealed that the expression levels of adipose differentiation-related genes PPARγ and CEBP/α were increased and the Oil Red O staining assay showed more lipid droplets. Our study revealed the expression profile of circRNAs in the adipose tissue of old rats. We found a novel age-related circular RNA—circ-ATXN2—that inhibits proliferation and promotes cell death and adipogenesis in rat adipose tissue-derived stromal cells.

Analysis of N15-rat growth hormone after incubation with rat subcutaneous tissue and immune cells using ultra-pressure chromatography-mass spectrometry

Therapeutic proteins (TPs) are exposed to various immune cells like macrophages and neutrophils, especially after subcutaneous (SC) administration. It is well known that the immune cells can generate reactive oxygen species (ROS) and this may lead to oxidation of TPs. The oxidation can occur in the SC tissue after SC administration, during distribution to the immune organs like lymph nodes and spleen, and even in the blood circulation. The oxidation can lead to alteration of their pharmacokinetics and efficacy. Therefore, it is important to study the oxidation of TPs in the biological matrices using ultra-pressure chromatography-mass spectrometry. Rat growth hormone (rGH) was selected as a test protein due to its similarity with human growth hormone (hGH), which is widely used for treatment of growth hormone deficiency. In this manuscript, we have summarized sample processing strategy and ultra-pressure chromatography-mass spectrometry methodology to identify rGH and its degradation products after ex-vivo incubation with rat SC tissue, and in vitro incubation with rat splenocytes and canine peripheral blood mononuclear cells (cPBMCs) as a model foreign host species. We did not observe oxidation of rGH in these biological matrices. This could be due to very minor yields of oxidation products, lack of sensitivity of the mass spectrometry method, loss of protein during sample processing, rapid turnover of oxidized protein or a combination of all factors.

Synthesis and Characterization of a Ti–Zr‐Based Alloy with Ultralow Young's Modulus and Excellent Biocompatibility

Recently, metastable β‐type Ti alloys have attracted attention as promising materials for medical implants due to low Young's modulus and superior biocompatibility. The former is dependent on texture developed during the thermomechanical process. Herein, new Ti–Zr–Nb–Sn–Mo alloys with ultralow Young's modulus and excellent biocompatibility are developed as promising materials for medical implants. The effect of Mo on the microstructure and mechanical properties of the Ti–18Zr–5Nb–3Sn system is investigated. X‐ray diffraction (XRD) and scanning electron microscope (SEM) results reveal that Mo acts as a β‐phase stabilizer. Moreover, mechanical behavior and recrystallization texture show strong composition dependence; the 2.5Mo alloy shows the lowest Young's modulus of 40 GPa. This is due to the formation of a recrystallization texture with strong Goss component of &lt;001&gt;. Corrosion behavior of the 2.5Mo alloy in the cell culture medium is similar to that of the commercially pure titanium (cp‐Ti), suggesting excellent corrosion resistance in the biological environment. Both murine fibroblasts and osteoblastic cells show good growth on the 2.5Mo alloy as the same level of that on the cp‐Ti. Implantation into rat subcutaneous tissue confirms no significant difference in the tissue reaction between the 2.5Mo alloy and the cp‐Ti.

Intraprostatic injection of exosomes isolated from adipose-derived mesenchymal stem cells for the treatment of chronic non-bacterial prostatitis.

Mesenchymal stem cells (MSCs) own the capacity to secrete trophic factors as exosomes which play significant roles in regulating the functions of other cells and preventing inflammation. Due to the inflammatory process in chronic non-bacterial prostatitis (CNP) and the ambiguity in the treatment of this disease, the present study was aimed to investigate the therapeutic use of adipose-derived MSC exosomes in an animal model of CNP. MSCs were first isolated from rat subcutaneous adipose tissue, and exosomes were extracted from them. Specific features of exosomes were characterized by a scanning electron microscope, western blot technique, and Dynamic Light Scattering methods. To establish CNP in rats, intraprostatic injection of Freund's complete adjuvant was done. After confirmation of prostatitis, intraprostatic injections of exosomes were performed for treatment. Histological evaluation revealed that treatment with exosomes resulted in a relative improvement of lesions caused by CNP. The expression of p-NF-κB and p-IκBα proteins along with inflammatory markers was significantly increased in the CNP group, which treatment with exosomes significantly reduced their expression as well as IL-1β and TNF-α proteins. The antioxidant effects of exosomes were also determined by significantly regulating glutathione peroxidase and superoxide dismutase activity and malondialdehyde levels in these animals. Our results cautiously suggest the therapeutic effects of MSC-derived exosomes against CNP-induced prostatitis through their antioxidant and anti-inflammatory activities, which should be further considered in the future.

Mesenchymal adipose stem cells maintain the capacity for differentiation and survival in culture beyond the long term

ABSTRACT Mesenchymal cells (MSCs) are considered to be cellular populations of common embryological origin. For clinical research applications, MSCs are expanded and increased with cells obtained from a primary culture. By extracting cells from tissue and encouraging them to reproduce, the stem cell population ends up dominating the culture due to a high proliferation rate and self-renewal. The first subcultures between the third and sixth are chosen in order to obtain the maximum number of cells with optimal differentiation capacity. However, few studies have reported long-term cultivation of MSCs. The objective of this study was to advance the knowledge on the characteristics of MSCs by assessing their capacity for self-renewal and phenotypic maintenance beyond 50 cell subcultures, which is defined as the normal limit for cellular survival. Rat subcutaneous adipose tissue was the source of mesenchymal adipose stem cells (MASCs) cultured over 175 subcultures. Early 1 to 5 and late 25 to 30 subcultures were used to induce cellular differentiation to become adipogenic, chondrogenic and osteogenic connective tissue cells. MASCs characteristics were studied using flow cytometry, transmission electron microscopy (TEM), and immunohistochemical and reverse transcription polymerase chain reaction (RT-qPCR) assays. The MASCs maintained cell differentiation capacity for more than 30 subcultures but lost potentiality starting at 60 up to 175 subcultures. MASCs showed the embryonic phenotypes OCT3/4 and Nanog indefinitely, and developed compensatory mechanisms, such as autophagy, to achieve cell survival over a long time period. Therefore, long-term subcultures showed that MASCs could maintain their potential for clinical research use.

Anticoagulant Hydrogel Tubes with Poly(ɛ-Caprolactone) Sheaths for Small-Diameter Vascular Grafts.

Small-diameter vascular grafts (inner diameter<6mm) are useful in treating cardiovascular diseases. The off-the-shelf small-diameter vascular grafts for clinical applications remain a great limitation owing to their thrombogenicity or intimal hyperplasia. Herein, bilayer anticoagulant hydrogel tubes with poly(ε-caprolactone) (PCL) sheaths are prepared by freeze-thawing and electrospinning, which contain nanofibrillated cellulose (NFC)/poly(vinyl alcohol) (PVA)-heparin/poly-L-lysine nanoparticles tube as an inner layer and PCL sheath as an outer layer. The structure, anticoagulant property, and biocompatibility of the inner layer are studied. The effects of thickness of the outer layer on perfusion performance and mechanical property of hydrogel tubes with PCL sheaths (PCL-NFC/PVA-NPs tubes) are investigated. The effect of compliance of PCL-NFC/PVA-NPs tubes on their blood flow is studied by numerical simulation. The tissue compatibility and the patency of PCL-NFC/PVA-NPs tubes are evaluated by implantation in subcutaneous tissue of rats and carotid artery of rabbits. PCL-NFC/PVA-NPs tubes have prominent anticoagulation, sufficient burst pressure and good compliance similar to native arteries. PCL-NFC/PVA-NPs tubes facilitate infiltration of host cells and achieve active proliferation of recruited cells, which will be a promising candidate for small-diameter vascular grafts.

Enhancement of Migration and Tenogenic Differentiation of Macaca Mulatta Tendon-Derived Stem Cells by Decellularized Tendon Hydrogel.

Decellularized tendon hydrogel from human or porcine tendon has been manufactured and found to be capable of augmenting tendon repair in vivo. However, no studies have clarified the effect of decellularized tendon hydrogel upon stem cell behavior. In the present study, we developed a new decellularized tendon hydrogel (T-gel) from Macaca mulatta, and investigated the effect of T-gel on the proliferation, migration and tenogenic differentiation of Macaca mulatta tendon-derived stem cells (mTDSCs). The mTDSCs were first identified to have universal stem cell characteristics, including clonogenicity, expression of mesenchymal stem cell and embryonic stem cell markers, and multilineage differentiation potential. Decellularization of Macaca mulatta Achilles tendons was confirmed to be effective by histological staining and DNA quantification. The resultant T-gel exhibited highly porous structure or similar nanofibrous structure and approximately swelling ratio compared to the collagen gel (C-gel). Interestingly, stromal cell-derived factor-1 (SDF-1) and fibromodulin (Fmod) inherent in the native tendon extracellular matrix (ECM) microenvironment were retained and the values of SDF-1 and Fmod in the T-gel were significantly higher than those found in the C-gel. Compared with the C-gel, the T-gel was found to be cytocompatible with NIH-3T3 fibroblasts and displayed good histocompatibility when implanted into rat subcutaneous tissue. More importantly, it was demonstrated that the T-gel supported the proliferation of mTDSCs and significantly promoted the migration and tenogenic differentiation of mTDSCs compared to the C-gel. These findings indicated that the T-gel, with its retained nanofibrous structure and some bioactive factors of native tendon ECM microenvironment, represents a promising hydrogel for tendon regeneration.

Continuous measurement of surface electrical potentials from transplanted cardiomyocyte tissue derived from human-induced pluripotent stem cells under physiological conditions in vivo.

Recording the electrical potentials of bioengineered cardiac tissue after transplantation would help to monitor the maturation of the tissue and detect adverse events such as arrhythmia. However, a few studies have reported the measurement of myocardial tissue potentials in vivo under physiological conditions. In this study, human-induced pluripotent stem cell-derived cardiomyocyte (hiPSCM) sheets were stacked and ectopically transplanted into the subcutaneous tissue of rats for culture in vivo. Three months after transplantation, a flexible nanomesh sensor was implanted onto the hiPSCM tissue to record its surface electrical potentials under physiological conditions, i.e., without the need for anesthetic agents that might adversely affect cardiomyocyte function. The nanomesh sensor was able to record electrical potentials in non-sedated, ambulating animals for up to 48h. When compared with recordings made with conventional needle electrodes in anesthetized animals, the waveforms obtained with the nanomesh sensor showed less dispersion of waveform interval and waveform duration. However, waveform amplitude tended to show greater dispersion for the nanomesh sensor than for the needle electrodes, possibly due to motion artifacts produced by movements of the animal or local tissue changes in response to surgical implantation of the sensor. The implantable nanomesh sensor utilized in this study potentially could be used for long-term monitoring of bioengineered myocardial tissue in vivo under physiological conditions.

Osteoinduction within adipose tissue fragments by heterodimeric bone morphogenetic Proteins-2/6 and -2/7 versus homodimeric bone morphogenetic protein-2: Therapeutic implications for bone regeneration.

Fragments of subcutaneous adipose tissue that have been genetically modified to express bone morphogenetic protein-2 (BMP-2) regenerate large segmental osseous lesions in rodents. Gene-activated adipose tissue can be implanted into osseous defects without prior cell extraction and cell culture. The present study aimed to explore whether the heterodimers BMP-2/6 or BMP-2/7 exceed the osteoinductive effect of BMP-2 on adipose tissue. In an in vitro tissue culture system, freshly harvested rat subcutaneous adipose tissue was cultivated in the presence of either BMP-2 or BMP-2/6 or BMP-2/7 at a high (200 ng/ml) and low (50 ng/ml) concentration. Gene expression analysis as well as histological and immunohistochemical methods were applied to test for osteoinduction. A concentration of 200 ng/ml of homodimeric BMP-2 induced osteogenic differentiation most potently, showing more calcification and a higher expression level of bone markers than both concentrations of BMP-2/6 or -2/7. A concentration of 50 ng/ml of BMP-2 was a significantly stronger osteogenic inducer than both concentrations of BMP-2/6 and the low concentration of BMP-2/7. The most potent heterodimeric driver of osteoinduction was BMP-2/7 at a high concentration, demonstrating effects similar to those of BMP-2 at a low concentration. Homodimeric BMP-2 evoked osteoinduction within adipose tissue more potently and at a lower concentration than heterodimeric BMP-2/6 or BMP-2/7. This result agrees well with the fact that it might be easier to translate adipose grafts activated by homodimeric BMP-2 clinically. Preclinical in vivo gene transfer studies are necessary to confirm the results of the present study.

Oral Use of Phenytoin to Reduce Calcification in Bovine Pericardium and Porcine Aortic Leaflets Implants in Rats.

Introduction:This study aims to test the effect of phenytoin as an inhibitor of the process of dystrophic calcification in bovine pericardium and porcine leaflets implanted in the subcutaneous tissue of rats.Methods:Isolated segments of biomaterials were implanted subcutaneously in young rats. The study groups received 500 mg phenytoin per kilogram of diet per day. After 90 days, samples were collected and quantitative calcification assessment by optical microscopy, radiological studies with mammography, and atomic emission spectrometry were performed.Results:Inflammatory reaction was a frequent finding in all groups when analyzed by optical microscopy. The calcium level assessed by atomic absorption spectrophotometry was significantly lower in the study groups using phenytoin compared to the control groups (control bovine pericardium group X=0.254±0.280 µg/mg; study bovine pericardium group X=0.063±0.025 µg/mg; control porcine aortic leaflets group X=0.640±0.226 µg/mg; study porcine aortic leaflets group X=0.056±0.021 µg/mg; P<0.05). Radiologic studies revealed a statistically significant difference between the groups treated with and without phenytoin (not only regarding the bovine pericardium but also the porcine leaflets).Conclusion:The results obtained suggest that phenytoin reduces the calcification process of bovine pericardium segments and porcine aortic leaflets in subdermal implants in rats; also, the incidence of calcification in bovine pericardium grafts was similar to that of porcine aortic leaflets.

Biomineralization of three calcium silicate-based cements after implantation in rat subcutaneous tissue.

ObjectivesThe aim of this study was to evaluate the dystrophic mineralization deposits from 3 calcium silicate-based cements (Micro-Mega mineral trioxide aggregate [MM-MTA], Biodentine [BD], and EndoSequence Root Repair Material [ESRRM] putty) over time after subcutaneous implantation into rats.Materials and MethodsForty-five silicon tubes containing the tested materials and 15 empty tubes (serving as a control group) were subcutaneously implanted into the backs of 15 Wistar rats. At 1, 4, and 8 weeks after implantation, the animals were euthanized (n = 5 animals/group), and the silicon tubes were removed with the surrounding tissues. Histopathological tissue sections were stained with von Kossa stain to assess mineralization. Scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM/EDX) were also used to assess the chemical components of the surface precipitates deposited on the implant and the pattern of calcium and phosphorus distribution at the material-tissue interface. The calcium-to-phosphorus ratios were compared using the non-parametric Kruskal-Wallis test at a significance level of 5%.ResultsThe von Kossa staining showed that both BD and ESRRM putty induced mineralization starting at week 1; this mineralization increased further until the end of the study. In contrast, MM-MTA induced dystrophic calcification later, from 4 weeks onward. SEM/EDX showed no statistically significant differences in the calcium- and phosphorus-rich areas among the 3 materials at any time point (p > 0.05).ConclusionsAfter subcutaneous implantation, biomineralization of the 3-calcium silicate-based cements started early and increased over time, and all 3 tested cements generated calcium- and phosphorus-containing surface precipitates.

Biological response to lyophilized demineralized dentin matrix implanted in the subcutaneous tissues of rats

AimLyophilized demineralized dentin matrix (LDDM) consists of a type 1 collagen complex matrix containing growth factors and no mineral crystals. Although the efficacy of LDDM for bone grafting is well known, there is limited evidence on the biological response to human lyophilized DDM. Therefore, the aim of this study was to evaluate the biological response of subcutaneous tissues in rats to powdered LDDM, mineral trioxide aggregate (MTA), and Biodentine implanted using polyethylene tubes. MethodsForty Wistar rats were divided into four groups (n = 10 each) depending on the experimental time intervals and were placed in polyethylene tubes containing LDDM, MTA, biodentine, and one empty control. After 3, 7, 15, and 30 observation days, the animals were sacrificed and quantitative and qualitative analysis of the subcutaneous tissue samples was carried out. The intensity of the inflammatory response was scored from 0 (no response) to 3 (severe response), and the data were statistically analyzed using ANOVA and Bonferroni tests (p < 0.05). ResultsAll groups exhibited moderate inflammation after 3 and 7 days of observation, with presence of inflammatory infiltrate predominantly consisting of macrophages and angioblastic proliferation being observed. After 15 observation days, the control group exhibited mild inflammation and a predominance of fibroblasts, and this differed significantly from the remaining cement groups that exhibited moderate inflammation. After 30 days of observation, all groups exhibited a mild inflammatory response, predominance of fibroblasts, and a greater amount of collagen fibers. ConclusionWithin the limitations of this study, it can be concluded that LDDM exhibited an acceptable biological response similar to MTA and Biodentine in the subcutaneous tissues of rats.

Improvement of the mechanical and biological properties of bioactive glasses by the addition of zirconium oxide (ZrO2 ) as a synthetic bone graft substitute.

In this study, mechanical properties of bioactive glass (BAG) synthetic bone graft substitute was improved by containing ZrO2 (ZrO2 -BAG), while maintaining advantageous biological properties of BAG such as osteoinductive and osteoconductive properties. The ZrO2 -BAG was produced by adding ZrO2 in the following proportions to replace Na2 O in 45S5 BAG: 1% (Zr1-BAG), 3% (Zr3-BAG), 6% (Zr6-BAG), and 12% (Zr12-BAG). Properties including XRD, XPS, SEM, DSC, fracture toughness, and Vickers microhardness were evaluated. To assess the biological properties, Ca/P apatite formation, ion release, degradation rate, cell proliferation, ALP activity (ALP), and alizarin red S staining assay (ARS) were evaluated. Also, expression of osteogenic differentiation markers, Osteopontin (OPN), confirmed by immunofluorescence staining. Finally, an in vivo test was carried out to by implanting ZrO2 -BAG into the subcutaneous tissue of rats. The results of each test were statistically analyzed with one-way ANOVA followed by Tukey's post hoc statistical test. Amorphous ZrO2 -BAG was successfully produced with increased mechanical properties as the ZrO2 content was increased. Additionally, ZrO2 -BAG exhibited a slower ion release and degradation rate compare to BAG without ZrO2 . Bioactivity of ZrO2 -BAG was confirmed with apatite layer formed on the surface, significantly higher proliferation rate and significantly enhanced ALP and the degree of ARS of the cells compare to respective controls. The tissue reactions observed in the in vivo study showed neo-formed vessels after implantation of ZrO2 -BAG.