The effect of tenocyte/hyaluronic acid therapy on the early recovery of healing Achilles tendon in rats

The aim of this study was to explore the potential for a better recovery outcome for the Achilles tendon at an early healing stage when a mixed biomaterial-tenocyte injection is used. The experimental animals underwent single limb Achilles tendon transection followed by suturing repair. A solution of either hyaluronic acid with or without tenocytes or normal saline was randomly chosen to be injected around the injury site after surgery. To obtain the comprehensive recovery condition of the rats on different management protocols, the animals were evaluated histologically, mechanically, and functionally. A significant difference in the recovery condition was found in the injured tendon injected with the hyaluronic acid solution with tenocytes compared with the other groups. Tendon stiffness and the locomotion abilities of the rats with healing Achilles tendons were improved in the hyaluronic acid with tenocyte transplantation group. The acceleration of the inflammatory phase in rats with the hyaluronic acid with tenocyte injections might be the major reason for the better functional outcomes.     

In vitro and in vivo research on using Antheraea pernyi silk fibroin as tissue engineering tendon scaffolds

In this paper, the feasibility of using Antheraea pernyi silk fibroin as tissue engineering tendon scaffold was investigated in vitro and in vivo, respectively, utilizing tenocytes and animal model. The animal model used here was an adult New Zealand White rabbit with a 15-mm gap defect in both sides of the Achilles tendon. The Achilles tendon defects in one side of hind legs were repaired using the braided A. pernyi silk fibroin scaffold in experimental group (n = 24), while the other side left untreated as negative group (n = 24). The recovery of the defect tendons were evaluated postoperatively at the 2nd, 6th, 12th, and 16th week using macroscopic, histological, immunohistochemical, scanning electron micrograph and biomechanical test techniques. In vitro results examined by scanning electron micrograph showed that A. pernyi silk fibroin promote the adhesion and propagation of the tenocytes. In vivo, at 16 weeks after implantation, morphological results showed that neo-tendons were formed, and bundles of collagen fibers in the neo-tendons were uniform and well oriented. Immunohistochemical results showed that collagen type in the regenerated tendons was predominantly type I. The maximum load of regenerated tendon at 16 weeks reached 55.46% of the normal tendon values. Preliminary, we concluded that A. pernyi silk fibroin promoted the recovery of Achilles tendon defect of rabbit and the application of A. pernyi silk fibroin as tissue engineering tendon scaffold is feasible.     

Changes in collagen fibril pattern and adhesion force with collagenase-induced injury in rat Achilles tendon observed via AFM

The Achilles tendon consists mainly of type I collagen fibers that contain collagen fibrils. When the Achilles tendon is injured, it is inflamed. The collagenase-induced model has been widely used to study tendinitis. The major advantages of atomic force microscopy (AFM) over conventional optical and electron microscopy for bio-imaging include its non-requirement of a special coating and vacuum, and its capability to perform imaging in all environments. AFM force-distance measurements have become a fundamental tool in the fields of surface chemistry, biochemistry and materials science. Therefore, the changes in the ultrastructure and adhesion force of the collagen fibrils on the Achilles tendons of rats with Achilles tendinitis were observed using AFM. The changes in the structure of the Achilles tendons were evaluated based on the diameter and D-banding of the collagen fibrils. Collagenase-induced Achilles tendinitis was induced with the injection of 30 microl crude collagenase into 7-week-old male Sprague-Dawley rats. The animals were each sacrificed on the first, second, third, fifth and seventh day after the collagenase injection. The normal and injured Achilles tendons were fixed in 4% buffered formalin and dehydrated with increasing concentrations of ethanol. AFM was performed using the non-contact mode at the resolution of 512 x 512 pixels, with a scan speed of 0.8 line/sec. The adhesion force was measured via the force-distance curve that resulted from the interactions between the AFM tip and the collagen fibril sample using the contact mode. The diameter of the collagen fibrils in the Achilles tendons significantly decreased (p < 0.05) after the collagenase injection, and the pattern of the D-banding of the collagen fibrils was similar to that of the diameter changes. The adhesion force decreased until the fifth day after the collagenase injection, but increased on the seventh day after the collagenase injection (p < 0.0001).     

Electron microscopic evaluation of the effects of stress-shielding on maturation of the mid-substance and ligament-bone junction of the reconstructed anterior cruciate ligament in rabbits

To analyze the effects of stress-shielding on graft maturation after the anterior cruciate ligament (ACL) reconstruction, autogenous ACL reconstruction using Achilles tendon was performed in rabbits. Two-end fixation with a ligament augmentation device (LAD), as a stress-shielding model (SS group), and pull-out fixation with Leeds-Keio artificial ligaments (L-K ligament), as a non-stress-shielding model (non-SS group), were investigated. Intact ACL was used as the control. Specimens were harvested 6 mon postoperatively, and the analysis was focused on collagen fibril maturation in the mid-substance and bone-anchoring pattern in the bone tunnel, under light and transmission electron microscopy. The density of collagen fibrils in the control was lower than that in both experimental groups (p<0.01). The per cent collagen area in the control was higher than that in the SS (p<0.01) and the non-SS group (p<0.01). The per cent collagen area in the non-SS group was higher than that in the SS group (p<0.05). At the ligament-bone interface, intracellular calcification of the degenerated chondrocytes occurred in the non-SS group, while in the SS group hydroxyapatite deposits were observed only in the extracellular matrix. These results show unfavorable influence of stress-shielding on graft maturation not only in the mid-substance but also at the ligament-bone junction. © 1999 Kluwer Academic Publishers     

Effects of elastin-derived peptide on Achilles' tendon healing: An experimental study

Different matrix macromolecules modulate the tendon healing process. Elastin contains sequences which exhibit chemotactic activity both in vitro and in vivo. We analyzed the effects of synthetic elastin-derived peptide Val-Gly-Val-Ala-Pro-Gly suspended in a gel solution on the healing process of Achilles' tendon in a rat model. A total tenotomy at the middle 3rd was performed in 32 rats. During the suture repair the gel with (Group A) or without (Group B) the elastin-derived peptide was applied to the tendon stumps. Four animals for each period and group were killed at 10, 30, 60 and 90 days after surgery. The scar tissue was processed for histochemical, immuno-histochemical and morphometric analysis. An improved healing process with increase in cellularity and vascularity, especially at the early stage of the Achilles' tendon healing process was observed in Group A compared to Group B. The fiber alignment was also positively influenced by the factor. Immunolabeling with HAM 56 and lisozyme revealed a stronger reaction for the presence of monocyte/macrophage in Group A vs Group B especially in early stages. Chondral metaplasia and endochondral ossification occurred in the healed tissue of both group at 60 and 90 days.     

Ultrasonic assessment of extracellular matrix content in healing Achilles tendon

Although several imaging modalities have been utilized to observe tendons, assessing injured tendons by tracking the healing response over time with ultrasound is a desirable method which is yet to be realized. This study examines the use of ultrasound for non-invasive monitoring of the healing process of Achilles tendons after surgical transection. The overall extracellular matrix content of the transection site is monitored and quantified as a function of time. B-mode images (built from successive A-scan signatures) of the injury site were obtained and compared to biomechanical properties. A quantitative measure of tendon healing using the extracellular matrix (ECM) content of the injury site was analyzed using linear regression with all biomechanical measures. Contralateral tendons were used as controls. The trend in the degree of ECM regrowth in the 4 weeks following complete transection of excised tendons was found to be most closely paralleled with that of linear stiffness (R(2) = 0.987, p < .05) obtained with post-ultrasound biomechanical tests. Results suggest that ultrasound can be an effective imaging technique in assessing the degree of tendon healing, and can be used to correlate structural properties of Achilles tendons.     

Effects of sterilization on an extracellular matrix scaffold: Part II. Bioactivity and matrix interaction

Small intestinal submucosa (SIS) has been successfully used to treat a variety of damaged or diseased tissues in human patients. As a biologic scaffold, SIS stimulates repair of damaged or diseased tissues and organs with tissue that is similar in structure and function to the material it was meant to replace. To meet clinical safety requirements, biologic materials from animal tissues must undergo processing treatments to minimize host immune response and to eliminate the possibility of disease transmission. The effect of peracetic acid disinfection, lyophilization, and ethylene oxide sterilization on the in vitro bioactivity of the processed SIS was therefore examined in murine fibroblasts and pheochromocytoma (PC12) cells. Specifically, the ability of processed SIS to support fibroblast attachment, to stimulate PC12 cell differentiation, and to upregulate fibroblast VEGF secretion was examined. Fibroblasts attach to the sterilized SIS, remain viable, and more than double their secretion of VEGF as a result of interacting with the SIS matrix components. Additionally, PC12 cells exhibit increased neurite outgrowth following stimulation by SIS matrix proteins versus controls. We conclude that a biologic scaffold can be prepared for human use and still retain significant bioactivity.     

In vitro characterization of a novel tissue engineered based hybridized nano and micro structured collagen implant and its in vivo role on tenoinduction,tenoconduction, tenogenesis and tenointegration

Surgical reconstruction of large tendon defects is technically demanding. Tissue engineering is a new option. We produced a novel tissue engineered, collagen based, bioimplant and in vitro characterizations of the implant were investigated. In addition, we investigated role of the collagen implant on the healing of a large tendon defect model in rabbits. A two cm length of the left rabbit’s Achilles tendon was transected and discarded. The injured tendons of all the rabbits were repaired by Kessler pattern to create and maintain a 2 cm tendon gap. The collagen implant was inserted in the tendon defect of the treatment group (n = 30). The defect area was left intact in the control group (n = 30). The animals were euthanized at 60 days post injury (DPI) and the macro- micro- and nano- morphologies and the biomechanical characteristics of the tendon samples were studied. Differences of P < 0.05 were considered significant. The host graft interaction was followed at various stages of tendon healing, using pilot animals. At 60 DPI, a significant increase in number, diameter and density of the collagen fibrils, number and maturity of tenoblasts and tenocytes, alignment of the collagen fibrils and maturity of the elastic fibers were seen in the treated tendons when compared to the control ones (P < 0.05). Compared to the control lesions, number of inflammatory cells, amount of peritendinous adhesions and muscle fibrosis and atrophy, were significantly lower in the treated lesions (P < 0.05). Treatment also significantly increased load to failure, tensile strength and elastic modulus of the samples as compared with the control ones. The collagen implant properly incorporated with the healing tissue and was replaced by the new tendinous structure which was superior both ultra-structurally and physically than the loose areolar connective tissue regenerated in the control lesions. The results of this study may be valuable in the clinical practice.     

Repair of goat tibial defects with bone marrow stromal cells and β-tricalcium phosphate

Tissue engineering techniques have been proven effective in bone regeneration and repairing load-bearing bone defects. Previous studies, however, have heretofore been limited to the use of slowdegradable or natural biomaterials as scaffolds. There are, however, no reports on using biodegradable, synthetic beta-tricalcium phosphate (β-TCP) as scaffolds to repair weight-bearing bone defects in large animals. In the present study, highly porous β-TCP scaffolds prepared by the polymeric sponge method were used to repair goat tibial defects. Fifteen goats were randomly assigned to one of three groups, and a 26 mm-long defect at the middle part of the right tibia in each goat was created. In Group A (six goats), a porous β-TCP ceramic cylinder that had been loaded with osteogenically induced autologous bone marrow stromal cells (BMSCs) was implanted in the defect of each animal. In Group B (six goats), the same β-TCP ceramic cylinder without any cells loaded was placed in the defect. In Group C (three goats), the defect was left untreated. In Group A, bony union can be observed by gross view, X-ray and micro-computed tomography (Micro-CT) detection, and histological observation at 32 weeks post-implantation. The implanted β-TCP scaffolds were almost completely replaced by tissue-engineered bone. Bone mineral density in the repaired area of Group A was significantly higher (p < 0.05) than that of Group B, in which scant new bone was formed in each defect and the β-TCP hadn’t been completely resorbed at 32 weeks. Moreover, the tissue-engineered bone of Group A had similar biomechanical properties as that of the normal left tibia in terms of bending strength and Young’s modulus (p > 0.05). In Group C, little or no new bone was formed, and non-union occurred, showing that the 26 mm segmental defect of the goat tibia was critical sized at 32 weeks. Thus, it can be concluded that the mechanical properties of the BMSCs/β-TCP composites could be much improved via tissue engineering approach and β-TCP might be used to repair the weight-bearing segmental defects of goat tibias. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protection by Chinese Herbs Against Doxorubicin-Induced Focal and Segmental Glomerulosclerosis in Rats

The objective of this study was to evaluate the efficacy of Chinese herbs on Doxorubicin-induced focal and segmental glomerulosclerosis (FSGS) in rats. Twenty age-matched male Wistar rats were divided into two groups: group A (n = 10) given only water ad libitum served as the control group and group B (n = 10) was given Chinese herbs (40 ml/kg with drug concentration 1.75 g/ml) beginning at day zero. All rats were administered doxorubicin (7 mg/kg) intravenously. All the rats were placed in metabolic cages at day 0, 7, 14, 21, and 28, and daily proteinuria was measured. At day 28, the animals were killed by cervical dislocation, followed by immediate organ collection for histologic analysis of kidneys; blood was collected by tail vein and cardiac puncture (at day 28) for the measurement of serum albumin. Body weight (BW) and food intake were recorded. The rats in groups A and B demonstrated severe susceptibility to doxorubicin injection with the onset of proteinuria (80–100 mg/24h) at day 7. The rats in group B were partly resistant to doxorubicin nephropathy with decreasing proteinuria and increasing serum albumin compared with group A (p < 0.05). All 10 rats in group A developed at least 5% glomerulosclerosis with tubular casts at day 28. In contrast, the rats in group B developed less severe histologic renal disease. The difference in histologic scores between the two groups were significant at day 28 (12 in group B vs. 20 in group A, p = 0.002). Food intake of Group B animals progressively increased to reach 67–73% of those observed before the doxorubicin administration with 28–43% in Group A. After the 4-wk experimental period, BW in Group A decreased more significantly than that in Group B (?20 ± 3 and ?16 ± 1%, respectively, p = 0.035, paired T test). Chinese herbs seem to reduce proteinuria and attenuate renal histologic severity in rats with doxorubicin-induced FSGS and may offer an alternative to the treatment of FSGS.     

Age-related maintenance versus reliability centred maintenance: a case study on aero-engines

Reliability Centred Maintenance (RCM) is a procedure carried out as part of the logistic support analysis (LSA) process and is described in the US Department of Defence Military Standards (Mil Std 2173). RCM allows logisticians the opportunity to determine the best maintenance policy for each component within a system. However, the only data that are available to carryout RCM using Mil Std 2173 are of MTBF. This implies that all the necessary mathematical models need to be based on the exponential distribution. This is a serious drawback to the whole concept of RCM as the exponential distribution cannot be used to model items that fail due to wear, or any other mode that is related to their age. In this paper, a new approach to RCM is proposed using the concepts of soft life and hard life to optimise the total maintenance cost. For simplicity, only one mode of failure is considered for each component. However, the model can be readily applied to multiple failure modes. The proposed model is applied to find the optimal maintenance policies in the case of military aero-engines using Monte Carlo simulation. The case study shows a potential benefit from setting soft lives on relatively cheap components that can cause expensive, unplanned engine rejections.     

Effects of photo-induced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine on physical properties of cross-linked polyethylene in artificial hip joints

Osteolysis caused by wear particles from polyethylene in the artificial hip joints is a serious issue. We have used photo-induced radical graft polymerization to graft 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer onto the surface of cross-linked polyethylene (CLPE-g-MPC) in order to reduce friction and wear at the bearing surface of the joint. The physical and mechanical properties of CLPE and CLPE-g-MPC were not significantly different, expect that the friction coefficient of untreated CLPE cups was 0.0075, compared with 0.0009 for CLPE-g-MPC cup, an 88% reduction. After 3.0 × 10⁶ cycles in the hip joint simulator test, we could not observe any wear of CLPE-g-MPC cups. We concluded that the advantage of photo-induced radical graft polymerization technique was that the grafted MPC polymer gave a high lubricity only on the surface and has no effect on the bulk properties of the CLPE substrate.     

Bioabsorbable poly-l/d-lactide (PLDLA) 96/4 triple-stranded bound suture in the modified Kessler repair: an ex vivo static and cyclic tensile testing study in a porcine extensor tendon model

Previously the biomechanical properties of the bioabsorbable poly-l/d-lactide (PLDLA) 96/4 suture were found suitable for flexor tendon repair. In this study, three PLDLA suture strands were bound together parallel to each other side-by-side to form a triple-stranded bound suture and the modified Kessler tendon repair was performed. The biomechanical properties of the PLDLA repair in porcine extensor tendons ex vivo were investigated with static and cyclic tensile testing. In both biomechanical tests, the strength of the PLDLA repair achieved the estimated forces needed to withstand active mobilization.     

Flexor tendon healing within the tendon sheath using bioabsorbable poly-l/d-lactide 96/4 suture. A histological in vivo study with rabbits

The bioabsorbable poly-l/d-lactide (PLDLA) 96/4 suture has good biomechanical and knot properties, and sufficient tensile strength half-life for flexor tendon repair. In the present study, the biocompatibility of PLDLA suture was compared with that of coated braided polyester suture in the rabbit flexor digitorum profundus tendon repaired within the tendon sheath. Postoperative unrestricted active mobilization was allowed. The tendons were studied histologically after 1-, 3-, 6-, 12-, 26-, and 52-week follow-ups. No differences were found in the biocompatibility between the suture materials, with only scattered multinuclear giant cells near the sutures in both groups from 6 weeks onwards. At 52 weeks, most of the PLDLA material was absorbed and the histological structure of the tendon appeared normal, whereas in the polyester repairs the suture knots filled the repair site, causing bulking of the tendon surface, and the collagen alignment appeared disoriented. The results suggest that the PLDLA 96/4 is a suitable suture material for flexor tendon repair.     

Water-Soluble C60 and Macrophages: Morphologic Features of FC4S-Treated Peritoneal Macrophages in vitro and in vivo - A Preliminary Report

Abstract

Morphologic investigation of water-soluble hexasulfonated C₆₀ (FC₄S) effects on rat peritoneal macrophages in vitro and in vivo was conducted by transmission electron microscopy. In the case of in vitro studies, two groups of spontaneous peritoneal macrophages were separately cocultured with FC₄S in a concentration of either 0, or 100 ng/ml for 24 h. The other two groups were cocultured with either FC₄S (100 ng/ml) for 12 h, or Pseudomonas aeruginosa exotoxin A (ETA) 100 ng/ml for 24 h. The former 12 h FC₄S-treated group was subsequently cultured with ETA for an another 12 h. All these 4 groups 24 h of cocultured monolayer cells were fixed in glutaraldehyde 2.5%, post fixed in 1% OsO₄ and processed for morphologic evaluation. The results revealed that the viability of FC₄S-treated cells in a healthy state, containing numerous cytoplasmic electron-dense (ED) bodies. The origin and nature of these ED bodies were not known. Comparable observation was obtained with the FC₄S-pretreated and ETA-treated the cells as viable and healthy. This suggested the existence of plausible antioxidative bio-reactions involving phagocytosed hexa(sulfobutyl)[60] fullerenes in cocultured macrophages. In the case of in vivo studies, the cells removed from rats that were ip injected with 60 mg/kg of FC₄S were found to be typical active macrophages that bore numerous lysosomes and phagolysosomes in a variable degree of phagocytosing state. The presence of phagolysosomes were attributable to the FC₄S exposure. The affected cells were diagnosed as “phagolysosomal macrophagopathy”.     

Grooved substrata facilitate in vitro healing of completely divided flexor tendons

Multiple grooved substrata with groove depth 5 m were found to facilitate the healing of completely divided rat flexor tendons in vitro. Sections of tendons cultured on plain substrata showed only partial healing with incompletely sealed epitenon layers and immature thin collagen fibres. Tendons cultured on patterned substrata healed with complete restoration of the epitenon layer and reconstitution of the internal structure of collagen fibres. Epitenon fibroblasts isolated from the surface of rat flexor tendons were shown to be more sensitive to topographical features than fibroblasts of the same size BHK fibroblasts. They remained more elongated and better aligned to the groove direction than BHK cells. Multiple grooved substrata facilitated epitenon cell movement. Cells were found to move with higher speed on patterned substrata than on plain substrata. In summary, we conclude that the use of multiple grooved substrata promotes tendon healing in vitro and may find application in clinical practice in tendon repair.     

A thermoplastic elastomer patch matrix for traditional Chinese medicine: design and evaluation

Objective: To design and evaluate a novel pressure sensitive adhesive (PSA) patch containing traditional Chinese medicine (TCM) using styrene–isoprene–styrene (SIS) copolymer.

Method: A mixture D-optimal design with ternary response surface diagram was employed in the optimization process. The proportions of SIS copolymer, tackifying resin and plasticizer were selected as the independent variables while tack force, peel strength of the patch and skin penetrability of methyl salicylate were selected as the dependent variables. The optimized patch was then evaluated including in vivo absorption, pharmacological activities and skin irritation, by comparing with a commercial patch based on natural rubber.

Results: The optimized patch, which comprised 30.0% SIS copolymer, 26.6% tackifying resin and 43.4% plasticizer, was superior to commercial patch in skin permeation, pharmacological activities and skin biocompatibility.

Conclusion: SIS copolymer was a suitable substitute to natural rubber in producing patches containing TCM formula.     

Metal Oxide Heterostructure Array via Spatially Controlled–Growth within Block Copolymer Templates

Nanofabrication is continuously searching for new methodologies to fabricate 3D nanostructures with 3D control over their chemical composition. A new approach for heterostructure nanorod array fabrication through spatially controlled–growth of multiple metal oxides within block copolymer (BCP) templates is presented. Selective growth of metal oxides within the cylindrical polymer domains of polystyrene‐block‐poly methyl methacrylate is performed using sequential infiltration synthesis (SIS). Tuning the diffusion of trimethyl aluminum and diethyl zinc organometallic precursors in the BCP film directs the growth of AlO_x and ZnO to different locations within the cylindrical BCP domains, in a single SIS process. BCP removal yields an AlO_x‐ZnO heterostructure nanorods array, as corroborated by 3D characterization with scanning transmission electron microscopy (STEM) tomography and a combination of STEM and energy‐dispersive X‐ray spectroscopy tomography. The strategy presented here will open up new routes for complex 3D nanostructure fabrication.     

A study of the cellular response to orientated fibronectin material in healing extensor rat tendon

3D orientated fibronectin (Fn) mats have been used as biocompatible and biodegradeable scaffolds to provide orientated cues using contact guidance for cell migration/adhesion and deposition of extracellular matrix. We have implanted Fn scaffolds in an established rat tendon(partial tenotomy) injury model to test its efficacy and monitor the early cellular and inflammatory response. Tendons were harvested at 0, 6 h, 1, 3, 5, 7 and 14 days for H&E, immunohistochemistry and TEM. Total cell counts within the window increased progressively with time with no significant differences between the Fn scaffolds and controls. CD45 (pan leukocyte) positive cell numbers peaked at 6 h and when expressed as a percentage of total cell counts as determined by H&E staining constituted 20% of the total cell number at 6 h but decreased to 5% of total number by 72 h. There were no significant differences in the inflammatory response between the control and implanted groups. Few CD44 (mesenchymal stem cell) positive cells identified had a surface location. A novel cell with long exaggerated cytoplasmic processes was identified by TEM. Our results show that the Fn scaffold did not degrade or elicit any untoward inflammatory response at the time points tested and has potential use in guiding the repair process.     

Thixotropy flow behaviour of solder and conductive adhesive pastes

This paper presents results on the thixotropic behavior of two suspensions; solder paste and isotropic conductive adhesives (ICAs). These materials are widely used as bonding medium in the electronics industry. Solder paste are metal alloys suspended in a flux medium while the ICAs consist of silver flakes dispersed in an epoxy resin. The thixotropy behavior was investigated through two rheological test; (i) hysteresis-loop test and (ii) steady shear rate test. In the hysteresis-loop test, the shear rate was increased from 0.01 to 10 s^–1 and then decreased from 10 to 0.01 s^–1. Meanwhile, in the steady shear rate test, the materials were subjected to a constant shear rate of 0.1, 1 and 10 s^–1 for a period of 1800 s. The solder paste exhibited a higher degree of structural breakdown compared to the isotropic conductive adhesives (ICAs). Both the suspensions showed a high degree of shear thinning behavior with time. Existing thixotropy model such as Weltman and Hahn were applied to understand the rate of structural change for solder paste and ICAs. The Weltman model (r0.95) showed a strong correlation with the experimental data compared to Hahn model (r0.95). The rate of structural breakdown increases with the value of B ₁, from Weltman model. The change in the microstructure of solder paste increases with the B ₁ but these trends were only observed for isotropic conductive adhesives at lower shear rates.