Preservation Of Microarchitecture Research Articles

Decellularization of kidney tissue: comparison of sodium lauryl ether sulfate and sodium dodecyl sulfate for allotransplantation in rat.

An automatic decellularization device was developed to perfuse and decellularize male rats' kidneys using both sodium lauryl ether sulfate (SLES) and sodium dodecyl sulfate (SDS) and to compare their efficacy in kidney decellularization and post-transplantation angiogenesis. Kidneys were perfused with either 1% SDS solution for 4h or 1% SLES solution for 6h. The decellularized scaffolds were stained with hematoxylin and eosin, periodic acid Schiff, Masson's trichrome, and Alcian blue to determine cell removal and glycogen, collagen, and glycosaminoglycan contents, respectively. Moreover, scanning electron microscopy was performed to evaluate the cell removal and preservation of microarchitecture of both SDS and SLES scaffolds. Additionally, DNA quantification assay was applied for all groups in order to measure residual DNA in the scaffolds and normal kidney. In order to demonstrate biocompatibility of the decellularized scaffolds, human umbilical cord mesenchymal stromal/stem cells (hUC-MSCs) were seeded on the scaffolds. In addition, the allotransplantation was performed in back muscle and angiogenesis was evaluated. Complete cell removal in both SLES and SDS groups was observed in scanning electron microscopy and DNA quantification assays. Moreover, the extracellular matrix (ECM) architecture of rat kidney in the SLES group was significantly preserved better than the SDS group. The hUC-MSCs were successfully migrated from the cell culture plate surface into the SDS and SLES decellularized scaffolds. The formation of blood vessels was observed in the kidney in both SLES and SDS decellularized kidneys. The better preservation of ECM than SDS introduces SLES as the solvent of choice for kidney decellularization.

Identification of effective elastic modulus using modal analysis; application to canine cancellous bone

Mechanical properties of cancellous bone is of increasing interest due to its involvement in aging pathologies and oncology. Characterization of fragile bone tissue is challenging and available methodologies include quasi-static compressive tests of small size specimens, ultrasound and indentation techniques. We hypothesized that modal analysis of flexure beams could be a complementary methodology to obtain Young modulus.The sampling methodology was adapted such that the uniqueness of the linear dynamic response was available to determine the elastic modulus from natural frequencies and mode shapes. In a first step, the methodology was validated using a synthetic bone model as control. Then, water-jet cutting allowed collecting fourteen small beam-like specimens in canine distal femurs. X-ray microtomography confirmed the microarchitecture preservation, the homogeneity and the isotropy at the specimen scale to derive effective properties. The first natural frequency in clamped-free boundary conditions was used to obtain mean values of Young modulus, which ranged from 210 MPa to 280 MPa depending on the specimen collection site. Experimental tests were rapid and reproducible and our preliminary results were in good agreement with literature data. In conclusion, beam modal analysis could be considered for exploring mechanical properties of fragile and scarce biological tissues.

Utilization of Natural Detergent Potassium Laurate for Decellularization in Lung Bioengineering.

Recent advances in tissue engineering using decellularized organ scaffolds have expanded the possibilities for organ replacement therapy. However, detergent-based decellularization itself damages the extracellular matrix (ECM), which results in failure associated with the transplanted bioengineered organ. This study determined that potassium laurate (PL), a natural detergent, significantly reduces lung ECM damage during the decellularization process compared with protocols using sodium dodecyl sulfate. PL-decellularized lungs showed better microarchitecture preservation and low biological reactions after subcutaneous implantation. PL-decellularized scaffolds supported rat lung endothelial cell attachment/proliferation and the bioengineered lungs significantly reduced lung congestion after transplantation.

The Benefit of Menopausal Hormone Therapy on Bone Density and Microarchitecture Persists After its Withdrawal.

Menopausal hormone therapy (MHT) favorably affects bone mineral density (BMD). Whether MHT also affects bone microarchitecture, as assessed by trabecular bone score (TBS), has never been evaluated. Our objective was to assess the effect of MHT on TBS and BMD before and after its withdrawal. This was a cross-sectional study. This study included the general community. Data were collected from the OsteoLaus cohort (1500 women aged 50-80 years). After exclusion of women with bone-modulating treatments, 1279 women were categorized according to MHT status into current (CU), past (PU), and never (NU) users. Spine TBS and BMD at lumbar spine, femoral neck, and total hip were assessed by dual X-ray absorptiometry. Age- and body mass index-adjusted analysis showed higher TBS values in CU vs PU or NU (1.31 ± 0.01, 1.29 ± 0.01, and 1.27 ± 0.01, respectively; P < .001). All BMD values were significantly higher in CU vs PU or NU. Compared to NU, PU exhibited higher lumbar spine (0.94 ± 0.01 vs 0.91 ± 0.01 g/cm2; P = .017) and total hip (0.86 ± 0.01 vs 0.84 ± 0.01 g/cm2; P = .026) BMD and a trend for higher TBS (P = .066). The 10-year loss of TBS and BMD at lumbar spine and total hip was significantly lower for both CU and PU vs NU. MHT duration had no effect on bone parameters. In PU, the residual effect on TBS and BMD was significantly more prominent in early discontinuers (<2 years). MHT is associated with bone microarchitecture preservation, as assessed by TBS. The effect of MHT on TBS and BMD persists at least 2 years after withdrawal.

Optimization of Liver Decellularization Maintains Extracellular Matrix Micro-Architecture and Composition Predisposing to Effective Cell Seeding.

Hepatic tissue engineering using decellularized scaffolds is a potential therapeutic alternative to conventional transplantation. However, scaffolds are usually obtained using decellularization protocols that destroy the extracellular matrix (ECM) and hamper clinical translation. We aim to develop a decellularization technique that reliably maintains hepatic microarchitecture and ECM components. Isolated rat livers were decellularized by detergent-enzymatic technique with (EDTA-DET) or without EDTA (DET). Histology, DNA quantification and proteomics confirmed decellularization with further DNA reduction with the addition of EDTA. Quantification, histology, immunostaining, and proteomics demonstrated preservation of extracellular matrix components in both scaffolds with a higher amount of collagen and glycosaminoglycans in the EDTA-DET scaffold. Scanning electron microscopy and X-ray phase contrast imaging showed microarchitecture preservation, with EDTA-DET scaffolds more tightly packed. DET scaffold seeding with a hepatocellular cell line demonstrated complete repopulation in 14 days, with cells proliferating at that time. Decellularization using DET preserves microarchitecture and extracellular matrix components whilst allowing for cell growth for up to 14 days. Addition of EDTA creates a denser, more compact matrix. Transplantation of the scaffolds and scaling up of the methodology are the next steps for successful hepatic tissue engineering.

Micromorphometric analysis of bone blocks harvested with eight different ultrasonic and sonic devices for osseous surgery.

The aim of this study was to analyse invitro the main features of osteotomies performed by means of different ultrasonic and sonic systems for bone surgery. Six ultrasonic and two sonic devices for osseous surgery were evaluated during block harvesting on bovine bone. After measuring cutting speed, images of the blocks were acquired by light stereo-microscope and E-SEM, in order to measure the osteotomy thickness and to evaluate the presence of intra-trabecular bone debris and signs of thermal injuries on the bone. Roughness evaluation was performed using a profilometer. All the ultrasonic instruments required a shorter time than sonic systems to perform the block harvesting (p<0.05). Piezomed was found to be the most efficient in terms of cutting speed (20.5mm(2)/min), even if not significantly different from most of the devices here tested (p>0.05). K-Bisonic and Variosurg 3 showed the smallest percentage variance between tip thickness and osteotomy width. Intra-trabecular debris was found to occur in inverse proportion with the width of the osteotomy: the tighter the track, the higher the amount of debris. Sonicflex Bone, Piezotome 2 and Sonosurgery showed almost no signs of thermal injuries on the osteotomised surfaces. No single ultrasonic or sonic device combined all the best features of speed, precision and bone micro-architecture preservation.

Panax notoginseng saponins mitigate ovariectomy-induced bone loss and inhibit marrow adiposity in rats.

Previous data have suggested that Panax notoginseng saponins (PNS) can prevent estrogen deficiency-induced bone loss by dual action: stimulation of new bone formation and inhibition of bone resorption. Marrow adipogenesis has been identified as a negative indicator of skeletal strength and integrity. This study assessed the effects of early PNS supplementation on bone microarchitecture preservation and marrow fat content in an ovariectomized rat model. Forty adult female Sprague-Dawley rats were randomly assigned to four equal groups for 12 weeks of treatment: (1) sham operation (SHAM) + vehicle; (2) ovariectomy (OVX) + vehicle; (3) OVX + 17β-estradiol (25 μg/kg); (4) OVX + PNS (300 mg/kg/d, PO). Marrow fat content of the femur was determined, using fat/water magnetic resonance imaging (MRI), at baseline and 6 and 12 weeks after operation. At the end of the experiment, bone turnover, trabecular microarchitecture, and marrow adipocytes were assessed by serum biomarkers, micro-computed tomography (micro-CT), and histopathology, respectively. The effects of PNS on adipocytic differentiation were reflected by expression levels of the adipogenic genes PPARγ2 and C/EBPα, as determined by reverse transcription-polymerase chain reaction. Ovariectomized rats experienced remarkable increases in marrow fat content across time points, which were accompanied by elevated rate of bone turnover, global volumetric bone density, and trabecular microarchitecture deterioration. These OVX-induced pathological changes are reversible in that most of them could be mostly corrected upon 17β-estradiol treatment. PNS treatment significantly reduced marrow adipogenesis (adipocyte density, -27.2%; size, -22.7%; adipocyte volume-to-tissue volume ratio, -53.3%; all P < 0.01) and adipocyte marker gene expression, and prevented bone mass loss and microarchitecture deterioration. Moreover, PNS enhanced osteoblast activity but suppressed osteoclast turnover, as evidenced by decreased levels of serum C-terminal telopeptides of type I collagen and elevated levels of alkaline phosphatase. PNS mitigates estrogen deficiency-induced deterioration of trabecular microarchitecture and suppresses marrow adipogenesis.

Lung tissue engineering and preservation of alveolar microstructure using a novel casting method

We used a rat model to decellularize and seed alveolar cells on a three-dimensional lung scaffold to preserve alveolar microarchitecture. We verified the preservation of terminal respiratory structure by casting and by scanning electron microscopy (SEM) of the casts after decellularization. Whole lungs were obtained from 12 healthy Sprague-Dawley rats, cannulated through the trachea under sterile conditions, and decellularized using a detergent-based method. Casting of both natural and decellularized lungs was performed to verify preservation of the inner microstructure of scaffolds for further cell seeding. Alveolar cell seeding was performed using green fluorescent protein (GFP) lung cells and non-GFP lung cells, and a peristaltic pump. We assessed cell seeding using histological and immunohistochemical staining, and enzymatic evaluation. All cellular components were removed completely from the scaffolds, and histological staining and SEM of casts were used to verify the preservation of tissue structure. Tensile tests verified conservation of biomechanical properties. The hydroxyproline content of decellularized lungs was similar to native lung. Histological and immunohistochemical evaluations showed effective cell seeding on decellularized matrices. Enzymatic measurement of trypsin and alpha 1 antitrypsin suggested the potential functional properties of the regenerated lungs. Casts produced by our method have satisfactory geometrical properties for further cell seeding of lung scaffolds. Preservation of micro-architecture and terminal alveoli that was confirmed by SEM of lung casts increases the probability of an effective cell seeding process.

Preservation of micro-architecture and angiogenic potential in a pulmonary acellular matrix obtained using intermittent intra-tracheal flow of detergent enzymatic treatment

Tissue engineering of autologous lung tissue aims to become a therapeutic alternative to transplantation. Efforts published so far in creating scaffolds have used harsh decellularization techniques that damage the extracellular matrix (ECM), deplete its components and take up to 5 weeks to perform. The aim of this study was to create a lung natural acellular scaffold using a method that will reduce the time of production and better preserve scaffold architecture and ECM components. Decellularization of rat lungs via the intratracheal route removed most of the nuclear material when compared to the other entry points. An intermittent inflation approach that mimics lung respiration yielded an acellular scaffold in a shorter time with an improved preservation of pulmonary micro-architecture. Electron microscopy demonstrated the maintenance of an intact alveolar network, with no evidence of collapse or tearing. Pulsatile dye injection via the vasculature indicated an intact capillary network in the scaffold. Morphometry analysis demonstrated a significant increase in alveolar fractional volume, with alveolar size analysis confirming that alveolar dimensions were maintained. Biomechanical testing of the scaffolds indicated an increase in resistance and elastance when compared to fresh lungs. Staining and quantification for ECM components showed a presence of collagen, elastin, GAG and laminin. The intratracheal intermittent decellularization methodology could be translated to sheep lungs, demonstrating a preservation of ECM components, alveolar and vascular architecture. Decellularization treatment and methodology preserves lung architecture and ECM whilst reducing the production time to 3 h. Cell seeding and in vivo experiments are necessary to proceed towards clinical translation.