Biomedical engineering – Page 2

Adhesive anti-fibrotic interfaces on diverse organs

[ad_1]

Preparation of adhesive implants

The adhesive layer of the adhesive implant was prepared using a previously reported method^23,24. To prepare an adhesive stock solution, 35% w/w acrylic acid, 7% w/w poly(vinyl alcohol) (PVA; M_w = 146,000–186,000, 99+% hydrolysed), 0.2% w/w α-ketoglutaric acid and 0.05% w/w N,N′-methylenebisacrylamide were added into nitrogen-purged deionized water. Next, 30 mg of acrylic acid N-hydroxysuccinimide ester was dissolved in each 1 ml of the above stock solution to prepare the adhesive precursor solution. The chitosan-based adhesive layer was prepared by replacing PVA with 2% w/w chitosan (Mw = 250–300 kDa, degree of deacetylation > 90%; ChitoLytic). The precursor solution was poured onto a glass mould with a spacer (100-µm thickness) and placed in a UV chamber (354 nm, 12 W power) for 30 min to prepare the adhesive hydrogel. The adhesive hydrogel was dried thoroughly under airflow and a vacuum desiccator to prepare the dry adhesive layer. A mock device of the adhesive implant was introduced by spin-coating a polyurethane resin (HydroThane, AdvanSource Biomaterials) onto the dry adhesive layer.

Preparation of non-adhesive implants

To prepare the non-adhesive implant, the adhesive implant was immersed in a sterile 1× phosphate-buffered saline (PBS; pH 7.4, 144 mg l⁻¹ potassium phosphate monobasic, 9,000 mg l⁻¹ sodium chloride and 795 mg l⁻¹ sodium phosphate dibasic) bath at room temperature overnight. During this process, the adhesive layer of the implant reached the equilibrium swollen state and became non-adhesive by losing the capability to form physical (hydrogen bonds) and covalent (amide bonds) crosslinking with tissues²⁶.

Preparation of implantable electrodes

To prepare the implantable electrodes, gold electrodes (thickness, 50 µm) were integrated between the polyurethane layer (thickness, 100 µm) and the adhesive or non-adhesive layer (thickness, 100 µm; Supplementary Fig. 6a). The surface of the gold electrode was treated with oxygen plasma for 3 min (30 W power, Harrick Plasma) to activate the surface functionalization, followed by immersion in cysteamine hydrochloride solution (50 mM in deionized water) for 1 h at room temperature. After the functionalization, the gold electrode was thoroughly washed with deionized water and dried with nitrogen flow. The functionalized gold electrode was cut into 2-mm-diameter circles and placed on the adhesive hydrogel (two electrodes per implant). An electrode lead wire (AS633, Cooner Wire) was connected to the gold electrodes and the polyurethane insulation layer (HydroThane, AdvanSource Biomaterials) was introduced to the gold electrodes. The assembled implant was thoroughly dried under airflow and in a vacuum desiccator to prepare the adhesive implantable electrodes. To prepare the non-adhesive implantable electrodes, the adhesive implantable electrodes were immersed in a sterile PBS bath overnight. All samples were prepared in an aseptic manner and were further disinfected under UV for 1 h before use.

Mechanical characterization

Either the chitosan-based adhesive implant or the PVA-based adhesive implant was applied to ex vivo porcine skin with a gentle pressure for 5 s. Interfacial toughness was measured on the basis of the T-peel test (ASTM F2256). Shear strength was measured on the basis of the lap-shear test (ASTM F2255). Tensile strength was measured on the basis of the tensile test (ASTM F2258). All tests were conducted using a mechanical testing machine (2.5-kN load cell, Zwick/Roell Z2.5). Aluminium fixtures were applied using cyanoacrylate glue to provide grips for tensile tests. All mechanical characterizations were carried out three times using independently prepared samples.

In vitro protein adsorption assay

A gelatin hydrogel (10% w/v, 300 g Bloom, Sigma-Aldrich) was used as the substrate for in vitro protein adsorption assay. The adhesive and non-adhesive implants were cut into 5-mm-diameter circles by using a biopsy punch and placed on the gelatin hydrogel. The samples were then incubated in a solution with 5 mg ml⁻¹ fluorescently tagged albumin (A13101, Thermo Fisher) or fibrinogen (F13191, Thermo Fisher) for 30 min. After the incubation, the samples were washed three times with fresh PBS to remove unadhered proteins. The samples were imaged using a confocal microscope (SP8, Leica), with the confocal plane set at the gelatin hydrogel–implant interface under a pitch model with excitation and emission at 495 nm and 515 nm (for albumin) and 495 nm and 635 nm (for fibrinogen). The relative fluorescence intensity of absorbed proteins was calculated by using ImageJ (version 2.1.0).

In vivo intraperitoneal implantation in rat model

All animal studies on rats were approved by the MIT Committee on Animal Care, and all surgical procedures and postoperative care were supervised by the MIT Division of Comparative Medicine (DCM) veterinary staff.

Sprague Dawley rats (female and male, 225 to 250 g, 12 weeks, Charles River Laboratories) were used for all in vivo rat studies. Before implantation, all samples were prepared using aseptic techniques and were further disinfected for 1 h under UV light. For in vivo intraperitoneal implantation, the animals were anaesthetized using isoflurane (2 to 3% isoflurane in oxygen) in an anaesthetizing chamber before the surgery, and anaesthaesia was maintained using a nose cone throughout the surgery. Abdominal hair was removed, and the animals were placed on a heating pad during the surgery. The abdominal wall, colon or stomach was exposed by means of a laparotomy. The adhesive implant (10 mm in width and 10 mm in length) was applied to the abdominal wall (n = 4 per time point), colon (n = 4) or stomach (n = 4) surface by gently pressing with a surgical spatula or fingertip. The non-adhesive implant (10 mm in width and 10 mm in length) was implanted on the abdominal wall (n = 4 per time point), colon (n = 4) or stomach (n = 4) surface using sutures at the corners of the samples (8-0 Prolene, Ethicon). For commercially available tissue adhesives, 0.5 ml of Coseal (n = 6) or Tisseel (n = 6) was used to adhere the non-adhesive implant (10 mm in width and 10 mm in length) to the abdominal wall surface. For the adhesive implant with sutures, the adhesive implant (10 mm in width and 10 mm in length) was applied to the abdominal wall surface (n = 6), and sutures (8-0 Prolene, Ethicon) were used at the corners of the samples²⁴. The abdominal wall muscle and skin incisions were closed with sutures (4-0 Vicryl, Ethicon). On days 3, 7, 14, 28 and 84 post-implantation, the animals were euthanized using CO₂ inhalation. Abdominal wall, colon or stomach tissues of interest were excised and fixed in 10% formalin for 24 h for histological and immunofluorescence analysis. All animals in the study survived and were kept in normal health conditions on the basis of daily monitoring by the MIT DCM veterinarian staff.

In vivo intrathoracic implantation in rat model

For in vivo intrathoracic implantation, the animals were anaesthetized using isoflurane (2 to 3% isoflurane in oxygen) in an anaesthetizing chamber before the surgery, and anaesthesia was maintained using a nose cone throughout the surgery. Chest hair was removed, and endotracheal intubation was carried out, connecting the animals to a mechanical ventilator (RoVent, Kent Scientific). The animals were placed on a heating pad for the duration of the surgery. The lung or heart was exposed by means of a thoracotomy. The pericardium was removed using fine forceps for the heart implantation. The adhesive implant (10 mm in width and 10 mm in length) was applied to the lung (n = 4) or heart (n = 4) surface by gently pressing with a surgical spatula or fingertip. The non-adhesive implant (10 mm in width and 10 mm in length) was implanted to the lung (n = 4) or heart (n = 4) surface by sutures at the corners of the samples (8-0 Prolene, Ethicon)²⁴. The muscle and skin incisions were closed with sutures (4-0 Vicryl, Ethicon). The animal was ventilated with 100% oxygen until normal breathing resumed. On days 28 and 84 post-implantation, the animals were euthanized by CO₂ inhalation. Lung or heart tissues of interest were excised and fixed in 10% formalin for 24 h for histological and immunofluorescence analysis. All animals in the study survived and were kept in normal health conditions on the basis of daily monitoring by the MIT DCM veterinarian staff.

In vivo intraperitoneal implantation in mouse model

All animal studies on mice were approved by the MIT Committee on Animal Care, and all surgical procedures and postoperative care were supervised by the MIT DCM veterinary staff. The mice housing room temperature was set at 21 °C with the room monitoring alarms set at ±2 °C, and relative humidity was maintained at 30–70% with a 12 h light/12 h dark cycle.

Immunocompetent C57BL/6 mice (female and male, 18–25 g, 6–8 weeks, Jackson Laboratory) or humanized HuCD34-NCG mice (female, 18–25 g, 16–18 weeks, Charles River Laboratories) were anaesthetized with 2–3% isoflurane, and then the abdomen was shaved and cleaned using betadine and 70% ethanol. A 1-cm incision was made along the abdomen midline and the abdominal wall was exposed by means of a laparotomy. The adhesive implant (5 mm in width and 5 mm in length) or non-adhesive implant (5 mm in width and 5 mm in length) was applied to the abdominal wall (n = 6 per group for C57BL/6 mice; n = 5 per group for HuCD34-NCG mice) by gently pressing. Both PVA-based and chitosan-based samples were used for C57BL/6 mice. Only PVA-based samples were used for HuCD34-NCG mice. The abdominal wall muscle and skin incisions were closed with sutures (5-0 Vicryl, Ethicon). On days 14 and 28 post-implantation, the abdominal wall of interest was excised and fixed in 10% formalin overnight for histological analysis.

In vivo intraperitoneal implantation in porcine model

All animal studies on pigs were approved by the Mayo Clinic institutional animal care and use committee at Rochester.

The female domestic pigs (female, 50 kg, 20 weeks, Manthei Hog Farm) were placed in dorsal recumbency, and the abdominal region was clipped and prepared aseptically. A blade was used to incise the ventral midline and extended using electrocautery when necessary. The linea alba was incised, and the peritoneum was bluntly entered, with the incision extended to match the skin incision. The small intestine was exteriorized and moist lap sponges were used for isolation. Then, the adhesive implant or non-adhesive implant was applied and adhered to the surface of the abdominal wall and small intestine (n = 4 for each group). The small intestine was thoroughly lavaged and returned to the abdomen. Then, the entire abdominal cavity was lavaged and suctioned, and the celiotomy incision was closed. On day 7 post-implantation, the animals were humanely euthanized, and the abdominal wall and small intestine of interest were excised and fixed in 10% formalin for 24 h for histological analyses. All animals in the study survived and were kept in normal health conditions on the basis of daily monitoring by the Mayo Clinic Rochester veterinarian staff.

In vivo electrophysiological study

Before implantation, the adhesive and non-adhesive implantable electrodes were prepared using aseptic techniques and were further disinfected for 1 h under UV. For in vivo epicardial electrode implantation, the animals were anaesthetized using isoflurane (2 to 3% isoflurane in oxygen) in an anaesthetizing chamber before the surgery, and anaesthesia was maintained using a nose cone throughout the surgery. Chest and back hair were removed, and endotracheal intubation was carried out, connecting the animals to a mechanical ventilator (RoVent, Kent Scientific). The animals were placed on a heating pad for the duration of the surgery. The heart was exposed by means of a thoracotomy and the pericardium was removed using fine forceps for the epicardial implantation. The adhesive implantable electrodes were applied to the left ventricular surface (n = 6) by gently pressing with a surgical spatula or fingertip. The non-adhesive implantable electrodes were implanted to the left ventricular surface (n = 6) by sutures at the corners of the samples (8-0 Prolene, Ethicon). The lead wire was then tunnelled subcutaneously from a ventral exit site close to the left fourth intercostal space to the dorsal side. The dorsal end of the lead wire was inserted through a subcutaneous port. The subcutaneous port was placed by interrupted sutures (4-0 Vicryl, Ethicon) between the shoulder blades of the animal and covered by a protective aluminium cap (VABRC, Instech Laboratories). The muscle and skin incisions were closed with sutures (4-0 Vicryl, Ethicon). The animal was ventilated with 100% oxygen until autonomous breathing was regained.

On days 0, 3, 7, 14, 28, 56 and 84 post-implantation, each animal was anaesthetized and connected to the data acquisition hardware (PowerLab, AD Instrument) and software (LabChart Pro 7, AD Instrument) for electrophysiological recording and stimulation by the implanted electrodes. For electrophysiological recording, the data acquisition hardware was connected to the implanted electrodes through the dorsal subcutaneous port. Epicardial signals were recorded to evaluate the R-wave amplitude. For electrophysiological stimulation, an external stimulator (FE180, AD Instrument) was connected to the implanted electrodes through the dorsal subcutaneous port. Unipolar rectangular current pulses (0.5 ms, 0–3 mA, 5–7 Hz) were used for continuous ventricular pacing and the surface electrocardiogram was monitored to evaluate the capture threshold at the same time. On days 28 and 84 post-implantation, the animals were euthanized by CO₂ inhalation. Heart tissues of interest were excised and fixed in 10% formalin for 24 h for histological analysis. All animals in the study survived and were kept in normal health conditions on the basis of daily monitoring by the MIT DCM veterinarian staff.

Immunofluorescence analysis

The expression of targeted markers (αSMA, CD68, CD3, CD206, iNOS, vimentin, neutrophil elastase) was analysed after the immunofluorescence staining of the collected tissues. Before the immunofluorescence analysis, the paraffin-embedded fixed tissues were sliced and prepared into slides. The slides were deparaffinized and rehydrated with deionized water. Antigen retrieval was carried out using the steam method during which the slides were steamed in IHC-Tek Epitope Retrieval Solution (IW-1100) for 35 min and then cooled for 20 min. Then the slides were washed in three changes of PBS for 5 min per cycle. After washing, the slides were incubated in primary antibodies (1:200 mouse anti-αSMA (ab7817, Abcam); 1:200 mouse anti-CD68 (ab201340, Abcam); 1:100 rabbit anti-CD3 (ab5690, Abcam); 1:1,000 rabbit anti-CD206 (ab64693, Abcam); 1:500 mouse anti-vimentin (ab8978, Abcam); 1:2,000 rabbit anti-iNOS (ab283655, Abcam); 1:200 mouse anti-iNOS (GTX60599, GeneTex); 1:50 rabbit anti-neutrophil elastase (bs-6982R, Bioss)) diluted with IHC-Tek antibody diluent for 1 h at room temperature. The slides were then washed three times in PBS and incubated with Alexa Fluor 488-labelled anti-rabbit or anti-mouse secondary antibody (1:200, Jackson Immunoresearch) or Alexa Fluor 594-labelled donkey anti-mouse secondary antibody (1:200, Jackson Immunoresearch) for 30 min. The slides were washed in PBS and then counterstained with propidium iodide solution for 20 min. A laser confocal microscope (SP8, Leica) was used for image acquisition. ImageJ (version 2.1.0) was used to quantify the number of cells in the collagenous layer at the implant–tissue interface from the immunofluorescence images³⁴ (500 µm width of the field of view). All analyses were blinded with respect to the experimental conditions.

Luminex quantification analysis

On days 3 and 7 post-implantation, the abdominal muscle wall of interest was collected. The collected samples were snap-frozen in liquid nitrogen and homogenized on a TissueLyser LT (Qiagen) following the manufacturer’s instructions. A Luminex multiplex assay was used to measure the concentrations of immune-response-related cytokines and chemokines (RECYTMAG-65K, Milliplex). Values per sample were normalized to the total protein content and expressed as picograms per total milligram of protein (Supplementary Table 1).

qPCR analysis

RNA was isolated from the samples snap-frozen in liquid nitrogen immediately after excision using the TRIzol protocol (Invitrogen). All samples were homogenized and normalized by loading 1 µg of total RNA in all cases for reverse transcription using a SuperScript First Strand cDNA Synthesis Kit (Invitrogen). Complementary DNA (1:20 dilution) was amplified by qPCR with the following primers: Mrc1 (5′-AACTTCATCTGCCAGCGACA-3′; reverse: 5′-CGTGCCTCTTTCCAGGTCTT-3′), Tgfb1 (5′-AGTGGCTGAACCAAGGAGAC-3′; reverse: 5′-CCTCGACGTTTGGGACTGAT-3′), Nos2 (5′-TGGTGAGGGGACTGGACTTT-3′; reverse: 5′-CCAACTCTGCTGTTCTCCGT-3′), Cd86 (5′-AGACATGTGTAACCTGCACCAT-3′; reverse: 5′-TACGAGCTCACTCGGGCTTA-3′), S100a8 (5′-CGAAGAGTTCCTTGTGTTGGTG-3′; reverse: 5′-AGCTCTGTTACTCCTTGTGGC-3′), Ly6c (5′-ACCTGGTCACAGAGAGGAAGT-3′; reverse: 5′-AGCAGTTAGCATTAAGTGGGACT-3′), Il10 (5′-TTGAACCACCCGGCATCTAC-3′; reverse: 5′-CCAAGGAGTTGCTCCCGTTA-3′), Cd11b (5′-GACTCCGCATTTGCCCTACT-3′; reverse: 5′-GCTGCCCACAATGAGTGGTA-3′) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (5′-CACCATCTTCCAGGAGCGAG-3′; reverse: 5′-CCACGACATACTCAGCACCA-3′). Samples were incubated for 10 min at 95 °C for 15 s and at 60 °C for 1 min in the real-time cycler Agilent MX3000P. Gapdh was used as the reference gene for normalization and analysis. The comparative CT (ΔΔCT) method was used for relative quantification of gene expression.

RNA-sequencing analysis

RNA extraction, library preparation and sequencing reactions were conducted at GENEWIZ. Total RNA was extracted using the Qiagen RNeasy Plus Universal mini kit following the manufacturer’s instructions (Qiagen). Extracted RNA samples were quantified using the Qubit 2.0 Fluorometer (Life Technologies) and RNA integrity was checked on Agilent TapeStation 4200 (Agilent Technologies). RNA-sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacturer’s instructions (NEB). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 min at 94 °C. First-strand and second-strand cDNAs were subsequently synthesized. cDNA fragments were end-repaired and adenylated at the 3′ ends, and universal adaptors were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies), and quantified using the Qubit 2.0 Fluorometer (Invitrogen) as well as by qPCR (KAPA Biosystems). The sequencing libraries were clustered on one lane of a flow cell. After clustering, the flow cell was loaded on the Illumina HiSeq 4000 instrument and the samples were sequenced using a 2 × 150-base-pair paired end configuration. Image analysis and base calling were conducted by the HiSeq Control Software. Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq files and de-multiplexed using Illumina’s bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.

Read quality was evaluated using FastQC, and data were pre-processed with Cutadapt³⁵ for adaptor removal following best practices³⁶. Gene expression against the mRatBN7.2 transcriptome (Ensembl release 104)³⁷ was quantified with STAR³⁸ and featureCounts³⁹. Differential gene expression analysis was carried out using DESeq2 (ref. ⁴⁰), and ClusterProfiler⁴¹ was used for functional enrichment investigations. Genes with log₂[fold change] ≥ 1 and false discovery rate ≤ 0.05 were considered statistically significant.

Statistical analysis

GraphPad Prism (version 9.2.0) was used to assess the statistical significance of all comparison studies in this work. Data distribution was assumed to be normal for all parametric tests, but not formally tested. In the statistical analysis for comparison between multiple groups, one-way analysis of variance followed by Bonferroni’s multiple comparison test was conducted with the significance thresholds at *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P < 0.0001. In the statistical analysis of two groups, the two-sided unpaired t-test was used with the significance thresholds at *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P < 0.0001.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

[ad_2]

Source link

Tag: Biomedical engineering

Transcranial volumetric imaging using a conformal ultrasound patch