Successful surgical attenuation of portosystemic shunt in a dog with imaging-diagnosed portal vein aplasia.

An 8-month-old female Maltese dog was referred for examination with a history of circling, dullness, and drooling. Serum biochemical analysis revealed hyperammonemia, with microhepatica observed on radiography. Computed tomography angiography revealed a portosystemic shunt originating from the right gastric vein and inserting into the prehepatic caudal vena cava. Portal blood flow to the liver was not observed. Based on computed tomography angiography, the dog was tentatively diagnosed with portosystemic shunt with portal vein aplasia. An exploratory laparotomy was done to obtain a definitive diagnosis. The dog had no subjective clinical signs of portal hypertension during a temporary occlusion test of the portosystemic shunt. A thin-film band was placed around the portosystemic shunt to achieve partial attenuation. There was no evidence of hepatic encephalopathy in the long term after surgery, and the dog's liver volume increased over time. Computed tomography angiography at 6 mo after surgery identified well-visualized intrahepatic portal branches. Key clinical message: We inferred that a direct occlusion test is a reliable diagnostic technique that overcomes the limitations of diagnostic imaging methods, including computed tomography angiography, and is a good technique for determining whether surgical attenuation is possible in dogs with suspected portal vein aplasia.

Atténuation chirurgicale réussie d'un shunt porto-systémique chez un chien avec une aplasie de la veine porte diagnostiquée par imagerie. Une femelle bichon maltais âgée de 8 mois a été référée pour examen avec une histoire de tournis, apathie et salivation excessive. L'analyse biochimique du sérum a révélé une hyperammionémie, avec un petit foie observé lors des radiographies. Une angiographie par tomodensitométrie a révélé un shunt porto-systémique prenant son origine de la veine gastrique droite et s'insérant dans la veine cave caudale pré-hépatique. Le flot sanguin porte au foie n'était pas observé. Sur la base de l'angiographie par tomodensitométrie, un diagnostic présumé de shunt porto-systémique avec aplasie de la veine porte a été émis. Une laparotomie exploratoire a été effectuée afin d'obtenir un diagnostic définitif. Le chien ne présentait pas de signe clinique subjectif d'hypertension portale durant un test d'occlusion temporaire du shunt porto-systémique. Une bande de film mince a été placée autour du shunt porto-systémique pour causer une réduction partielle. Il n'y avait aucune évidence d'encéphalopathie hépatique à long terme après la chirurgie, et le volume du foie du chien a augmenté dans le temps. Une angiographie par tomodensitométrie effectuée 6 mo après la chirurgie a permis de bien visualiser des branches portes intra-hépatiques.Message clinique clé :Nous avons déduit qu'un test d'occlusion est une technique diagnostique fiable qui surpasse les limites des méthodes d'imagerie diagnostique, incluant l'angiographie par tomodensitométrie, et est une bonne technique pour déterminer si une réduction chirurgicale est possible chez des chiens chez qui on soupçonne une aplasie de la veine porte.(Traduit par Dr Serge Messier).

Beneficial Effect of Sirolimus-Pretreated Mesenchymal Stem Cell Implantation on Diabetic Retinopathy in Rats.

BACKGROUND: Diabetic retinopathy (DR) is a vision-threatening complication that affects virtually all diabetic patients. Various treatments have been attempted, but they have many side effects and limitations. Alternatively, stem cell therapy is being actively researched, but it faces challenges due to a low cell survival rate. In this study, stem cells were pretreated with sirolimus, which is known to promote cell differentiation and enhance the survival rate. Additionally, the subconjunctival route was employed to reduce complications following intravitreal injections. METHODS: Diabetes mellitus was induced by intraperitoneal injection of 55 mg/kg of streptozotocin (STZ), and DR was confirmed at 10 weeks after DM induction through electroretinogram (ERG). The rats were divided into four groups: intact control group (INT), diabetic retinopathy group (DR), DR group with subconjunctival MSC injection (DR-MSC), and DR group with subconjunctival sirolimus-pretreated MSC injection (DR-MSC-S). The effects of transplantation were evaluated using ERG and histological examinations. RESULTS: The ERG results showed that the DR-MSC-S group did not significantly differ from the INT in b-wave amplitude and exhibited significantly higher values than the DR-MSC and DR groups (p < 0.01). The flicker amplitude results showed that the DR-MSC and DR-MSC-S groups had significantly higher values than the DR group (p < 0.01). Histological examination revealed that the retinal layers were thinner in the DR-induced groups compared to the INT group, with the DR-MSC-S group showing the thickest retinal layers among them. CONCLUSIONS: Subconjunctival injection of sirolimus-pretreated MSCs can enhance retinal function and mitigate histological changes in the STZ-induced DR rat model.

Prevalence of feline chronic gingivostomatitis in feral cats and its risk factors.

OBJECTIVES: This study investigated the prevalence of feline chronic gingivostomatitis in urban feral cats in South Korea and analysed its risk factors. METHODS: Three hundred and forty-five feral cats that visited the hospital for neutering using a trap-neuter-return approach were screened for feline chronic gingivostomatitis based on clinical criteria. In addition, we determined if body weight, sex and the presence of tongue lesions are risk factors for feline chronic gingivostomatitis. The difference in severity due to the presence or absence of risk factors, and the relationship between gross findings and histopathological lesions, were analysed by grading lesion severity. RESULTS: Feline chronic gingivostomatitis was diagnosed in 92 cats. Disease prevalence did not significantly differ with body weight and sex but was significantly related to tongue lesions. CONCLUSIONS AND RELEVANCE: The prevalence of feline chronic gingivostomatitis in urban feral cats in South Korea was 26.6%. It was significantly more prevalent in cats that had tongue lesions. Severity was also significantly associated with tongue lesions. Feline chronic gingivostomatitis may be associated with an infectious agent that causes tongue lesions.

Development and Characterization of Cellulose/Iron Acetate Nanofibers for Bone Tissue Engineering Applications.

In tissue engineering, design of biomaterial with a micro/nano structure is an essential step to mimic extracellular matrix (ECM) and to enhance biomineralization as well as cell biocompatibility. Composite polymeric nanofiber with iron particles/ions has an important role in biomineralization and collagen synthesis for bone tissue engineering. Herein, we report development of polymeric cellulose acetate (CA) nanofibers (17 wt.%) and traces of iron acetates salt (0.5 wt.%) within a polymeric solution to form electrospinning nanofibers mats with iron nanoparticles for bone tissue engineering applications. The resulting mats were characterized using field emission scanning electron microscopy (FESEM), transmission electron microscope (TEM), Fourier transform infrared (FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The resulted morphology indicated that the average diameter of CA decreased after addition of iron from (395 ± 30) to (266 ± 19) nm and had dense fiber distributions that match those of native ECM. Moreover, addition of iron acetate to CA solution resulted in mats that are thermally stable. The initial decomposition temperature was 300 °C of CA/Fe mat > 270 °C of pure CA. Furthermore, a superior apatite formation resulted in a biomineralization test after 3 days of immersion in stimulated environmental condition. In vitro cell culture experiments demonstrated that the CA/Fe mat was biocompatible to human fetal-osteoblast cells (hFOB) with the ability to support the cell attachment and proliferation. These findings suggest that doping traces of iron acetate has a promising role in composite mats designed for bone tissue engineering as simple and economically nanoscale materials. Furthermore, these biomaterials can be used in a potential future application such as drug delivery, cancer treatment, and antibacterial materials.

Custom-made artificial eyes using 3D printing for dogs: A preliminary study.

Various incurable eye diseases in companion animals often result in phthisis bulbi and eye removal surgery. Currently, the evisceration method using silicone balls is useful in animals; however, it is not available to those with impaired cornea or severe ocular atrophy. Moreover, ocular implant and prostheses are not widely used because of the diversity in animal size and eye shape, and high manufacturing cost. Here, we produced low-cost and customized artificial eyes, including implant and prosthesis, using computer-aided design and three-dimensional (3D) printing technique. For 3D modeling, the size of the artificial eyes was optimized using B-mode ultrasonography. The design was exported to STL files, and then printed using polycaprolactone (PCL) for prosthesis and mixture of PCL and hydroxyapatite (HA) for ocular implant. The 3D printed artificial eyes could be produced in less than one and half hour. The prosthesis was painted using oil colors and biocompatible resin. Two types of eye removal surgery, including evisceration and enucleation, were performed using two beagle dogs, as a preliminary study. After the surgery, the dogs were clinically evaluated for 6 months and then histopathological evaluation of the implant was done. Ocular implant was biocompatible and host tissue ingrowth was induced after in vivo application. The custom-made prosthesis was cosmetically excellent. Although long-term clinical follow-up might be required, the use of 3D printed-customized artificial eyes may be beneficial for animals that need personalized artificial eye surgery.

Tissue engineered ultra-thin descemet stripping corneal endothelial layers using porcine cornea and stem cells.

Due to their very poor proliferative capacity, the dysfunction of corneal endothelial cells can sometimes lead to incurable eye diseases that require corneal transplantation. Although many studies have been performed to reconstruct corneal endothelial cells, corneal transplantation is still considered to be the established approach. In this study, we developed bio-engineered Descemet stripping endothelial (DSE) layers, using porcine cornea and induced pluripotent stem cell (iPSC)-derived corneal endothelial cells (iCECs). First, we optimized a protocol to prepare an ultra-thin and decellularized Descemet stripping (DS) scaffold from porcine cornea. Our DS layers show over 90% transparency compared to the control. Porcine-derived cells and xenogenic antigens disappeared, whereas the collagen matrix remained in the graft. Next, corneal endothelial cell lines or iCECs were seeded on the decellularized DS graft and cultured for 7 days. The drying method reduced graft rolling and edema, and increased transparency during culture. The reseeded cells were evenly distributed over the graft, and most of the cells survived. Although future clinical studies are warranted, engineered DSE tissues using xenogenic tissues and stem cells will be useful tools for the treatment of incurable corneal diseases.

Decellularized extracellular matrix-rich hydrogel-silver nanoparticle mixture as a potential treatment for acute liver failure model.

Acute liver failure (ALF) occurs due to severe liver damage that triggers rapid loss of normal liver function. Here, we investigate the usefulness of an injectable liver extracellular matrix (LECM)-rich hydrogel generated from an optimized decellularization protocol incorporated with silver nanoparticles (AgNPs) as a promising therapy for ALF. First, we optimized a non-destructive protocol for rat liver decellularization to obtain ECM-rich well-preserved scaffold. Then, LECM hydrogel generated from two commonly used decellularization protocols were compared by LECM hydrogel obtained from our optimized protocol. The ALF model was induced by an intraperitoneal (IP) thioacetamide (TAA) injection followed by the IP injection of LECM hydrogel, collagen-AgNP mixture, or LECM hydrogel-AgNP mixture. LECM-rich scaffold and hydrogel were successfully obtained using our optimized decellularization protocol. Use of the LECM hydrogel-AgNP mixture to treat TAA-induced ALF greatly improved liver injury and histological liver regeneration. Interleukin-6 and transforming growth factor-beta expressions were significantly reduced, while albumin, hepatocyte growth factor, and Ki67-positive cells were highly expressed. Moreover, aspartate transaminase and alanine transaminase plasma levels and liver homogenate nitric oxide level were significantly lowered. In conclusion, the LECM hydrogel-AgNP mixture has potential efficient therapeutic and regenerative effects on TAA-induced liver injury.

Conjugating homogenized liver-extracellular matrix into decellularized hepatic scaffold for liver tissue engineering.

The generation of a transplantable liver scaffold is crucial for the treatment of end-stage liver failure. Unfortunately, decellularized liver scaffolds suffer from lack of bioactive molecules and functionality. In this study, we conjugated homogenized liver-extracellular matrix (ECM) into a decellularized liver in a rat model to improve its structural and functional properties. The homogenized ECM was prepared, characterized, and subsequently perfused into ethyl carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) activated liver scaffolds. Various techniques were performed to confirm the improvements that were accomplished through the conjugation process; these included micro/ultra-structural analyses, biochemical analysis of ECM components, DNA quantification, swelling ratio, structural stability, calcification properties, platelet activation study, static and dynamic seeding with EAhy926 endothelial cells and HepG2 hepatocarcinoma cells, subcutaneous implantation and intrahepatic transplantation. The results showed that the conjugated scaffolds have superior micro- and ultrastructural and biochemical characteristics. In addition, DNA contents, swelling ratios, calcification properties, platelet reactions, and host inflammatory reactions were not altered with the conjugation process. The conjugated scaffolds revealed better cellular spreading and popularity compared to the non-conjugated scaffolds. Intrahepatic transplantation showed that the conjugated scaffold had higher popularity of hepatic regenerative cells with better angiogenesis. The conjugation of the decellularized liver scaffold with homogenized liver-ECM is a promising tool to improve the quality of the generated scaffold for further transplantation.

Surgical Ectrodactyly Repair Using Limb-lengthening and Bone Tissue Engineering Techniques in a Toy Dog Breed.

BACKGROUND/AIM: Bone tissue engineering is an emerging field of regenerative medicine that holds promise for the restoration of bones affected by trauma, neoplastic diseases, and congenital deformity. During the past decade, bone tissue engineering has evolved from the use of biomaterials that can only replace small areas of damaged bone, to the use of scaffolds in which grafts can be seeded before implantation. This case report proposes an alternative option for a veterinary patient suffering from ectrodactyly, which is one of several congenital deformities in dogs. A 2-month-old male toy poodle dog with ectrodactyly was treated using several stages of surgery involving pancarpal arthrodesis, limb lengthening, and bone tissue engineering techniques. RESULTS AND CONCLUSION: Over a period of 2 years, the operated limb gained almost the same function as the contralateral limb. Bone tissue engineering techniques can be used for the treatment of congenital deformities in dogs.

Evaluation of Auricular Cartilage Reconstruction Using a 3-Dimensional Printed Biodegradable Scaffold and Autogenous Minced Auricular Cartilage.

Auricular cartilage reconstruction represents one of the greatest challenges for otolaryngology-head and neck surgery. The native structure and composition of the auricular cartilage can be achieved by combining a suitable chondrogenic cell source with an appropriate scaffold. In reconstructive surgery for cartilage tissue, autogenous cartilage is considered to be the best chondrogenic cell source. Polycaprolactone is mainly used as a tissue-engineered scaffold owing to its mechanical properties, miscibility with a large range of other polymers, and biodegradability. In this study, scaffolds with or without autogenous minced auricular cartilage were implanted bilaterally in rabbits for auricular regeneration. Six weeks (n = 4) and 16 weeks (n = 4) after implantation, real-time quantitative reverse transcription polymerase chain reaction and histology were used to assess the regeneration of the auricular cartilage. Quantitative reverse transcription polymerase chain reaction analysis revealed that the messenger RNA expression of aggrecan, collagen I, and collagen II was higher in scaffolds with 50% minced cartilage than the scaffold-only groups or scaffolds with 30% minced cartilage (P < 0.05). Furthermore, histological analysis demonstrated significantly superior cartilage regeneration in scaffolds with the minced cartilage group compared with the scaffold-only and control groups (P < 0.05). Autogenous cartilage can be easily obtained and loaded onto a scaffold to promote the presence of chondrogenic cells, allowing for an improvement of the reconstruction of auricular cartilage. Here, the regeneration of auricular cartilage was also successful in the 50% minced cartilage group. The results presented in this study could have clinical implications, as they demonstrate the potential of a 1-stage process for auricular reconstruction.

cAMP/EPAC Signaling Enables ETV2 to Induce Endothelial Cells with High Angiogenesis Potential.

Although the generation of ETV2-induced endothelial cells (iECs) from human fibroblasts serves as a novel therapeutic strategy in regenerative medicine, the process is inefficient, resulting in incomplete iEC angiogenesis. Therefore, we employed chromatin immunoprecipitation (ChIP) sequencing and identified molecular mechanisms underlying ETV2-mediated endothelial transdifferentiation to efficiently produce iECs retaining appropriate functionality in long-term culture. We revealed that the majority of ETV2 targets in human fibroblasts are related to vasculature development and signaling transduction pathways, including Rap1 signaling. From a screening of signaling pathway modulators, we confirmed that forskolin facilitated efficient and rapid iEC reprogramming via activation of the cyclic AMP (cAMP)/exchange proteins directly activated by cAMP (EPAC)/RAP1 axis. The iECs obtained via cAMP signaling activation showed superior angiogenesis in vivo as well as in vitro. Moreover, these cells could form aligned endothelium along the vascular lumen ex vivo when seeded into decellularized liver scaffold. Overall, our study provided evidence that the cAMP/EPAC/RAP1 axis is required for the efficient generation of iECs with angiogenesis potential.

A placebo-controlled study comparing the efficacy of intra-articular injections of hyaluronic acid and a novel hyaluronic acid-platelet-rich plasma conjugate in a canine model of osteoarthritis.

BACKGROUND: The objective of this study was to assess the efficacy of intra-articular injections of hyaluronic acid (HA) and a novel, on-site conjugate of HA with autologous fibrinogen in platelet-rich plasma (HA-PRP) in a canine model of osteoarthritis (OA) METHODS: Twelve beagle dogs underwent a unilateral resection of the cranial cruciate ligament (CrCL) of the stifle joint. Clinical and radiographic signs of OA were confirmed in all dogs 8 weeks following CrCL resection and prior to treatment. The dogs were randomized into three groups: saline (n = 4), HA (n = 4), and HA-PRP (n = 4). Each dog received intra-articular injections of the respective substance into the affected joint at pre-determined time points. The dogs were assessed for adverse effects for 3 days after each injection and for lameness, pain, range of motion, kinetics, and radiographic OA severity prior to treatment and 3 months after injection. OA severity as determined by radiographic examination was not significantly different among the groups at any time point. The dogs were then humanely euthanatized and the stifle joint assessed by gross and histological examinations. RESULTS: Dogs treated with four weekly injections of HA or two biweekly injections of HA-PRP were significantly (p < 0.05) better than dogs treated with four weekly injections of saline at 2-, 4-, and 12-week time points based on a comfortable range of motion (CROM) and clinical lameness score. Gait analysis measuring symmetry and weight distribution on pressure sensor walkway showed significantly (p < 0.05) improved limb function for dogs treated with HA and HA-PRP compared with dogs treated with saline yet with better clinical outcome for the HA-PRP-treated group at 12 and 20 weeks follow-up. Gross and histological analysis of synovium and articular cartilage demonstrated significant (p < 0.05) improvement by both treatments groups compared to controls. There was however significantly (p < 0.05) less damage to the cartilage in the HA-PRP group compared to the HA-treated group. CONCLUSIONS: These data suggest that while injection of HA and HA-PRP may be sufficient for short-term amelioration of the symptoms associated with OA, treatment with HA-PRP conjugates may be superior, providing significantly better long-term cartilage preservation.

Characterization of silver nanoparticle-modified decellularized rat esophagus for esophageal tissue engineering: Structural properties and biocompatibility.

Decellularized esophageal matrices are ideal scaffolds for esophageal tissue engineering. Unfortunately, in order to improve transplantation possibilities, they require modification to reduce their degradation rate and immunogenicity. To date, no modifying agent has been approved to overcome these limitations. The objective of this study was to evaluate the ability of silver nanoparticles (AgNPs) to improve the structural stability and biocompatibility of decellularized rat esophagi. AgNPs have the advantage over currently used agents in that they bind with collagen fibers in a highly ordered manner, via non-covalent binding mechanisms forming multiple binding sites, while other agents provide only two-point connections between collagen molecules. Rat esophagi were decellularized, loaded with 5 µg/mL of AgNPs (100 nm), and then treated with an immobilization-complex buffer composed of ethyl carbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS). Then, they were evaluated in terms of ultra-structural morphology, water uptake, in vitro resistance to enzymatic and thermal degradation, indentation strength, in vitro anti-calcification, cytocompatibility with rat bone marrow derived stromal cells (rat-BMSCs), angiogenic properties, and in vivo biocompatibility, and compared to scaffolds modified using glutaraldehyde and EDC/NHS complex buffer alone. AgNP-modified scaffolds showed an improved ultrastructure, good water uptake, and considerable resistance against in vitro degradation and indentation, and a high resistance against in vitro calcification. Moreover, they were cytocompatible for allogeneic rat-BMSCs. Additionally, AgNPs did not alter the angiogenic properties of the modified scaffolds and decreased host immune responses after their subcutaneous implantation. The structural properties and biocompatibility of decellularized esophageal matrices could be improved by conjugation with AgNPs.

The degree of major histocompatibility complex matching between purebred Maltese and mongrel dogs using microsatellite markers.

Long-term maintenance of transplanted organs is one of the major factors that increases survival time of recipients. Although obtaining a major histocompatibility complex (MHC)-matched donor with the recipient is essential for successful organ transplantation, there have been limited reports on MHC matching between dogs. In this study, we analyzed the canine MHC matching rates using Maltese, one of the most popular purebred dogs, and mongrel dogs in Korea. Genomic DNA was extracted from blood leukocytes and DNA was amplified by polymerase chain reaction with primers specific to MHC microsatellite markers. The MHC matching degree was confirmed by the microsatellite markers using polyacrylamide gel electrophoresis. The MHC matching rates of each donor-recipient groups including Maltese-Maltese, mongrel-mongrel and Maltese-mongrel were 4.76%, 5.13% and 6.67%, respectively. There were no significant differences in the MHC matching degree between each group. These results demonstrate that MHC-matched donors could be selected from other breeds as much as from the same breed for transplantation. Knowledge of the MHC matching degree of purebred and mongrel dogs would offer valuable information not only for improving the success rate of organ transplantation surgery in canine patients but also for transplantation research using experimental canine models.

Application of alginate microbeads as a carrier of bone morphogenetic protein-2 for bone regeneration.

Bone morphogenetic protein-2 (BMP-2) is commonly used to enhance bone regeneration. The potential of BMP-2 for bone regeneration varies according to the concentration and release kinetics on the implanted site. Therefore, it is important to determine appropriate carriers of BMP-2. However, no optimal delivery vehicles have been identified. In the present study, we used alginate microbeads as a delivery vehicle for BMP-2. Alginate microbeads can be implanted onto the disease site through surgery or injection. The objective of this study was to evaluate that the osteoinductive properties of BMP-2 are effective in alginate microbeads as a carrier. In this study, the release kinetics of BMP-2 in alginate microbeads was evaluated using an enzyme-linked immunosorbent assay. BMP-2 released from alginate microbeads induced high alkaline phosphatase activity in canine adipose tissue-derived mesenchymal stem cells. Injection of alginate microbeads with BMP-2 into mouse subcutaneous tissue, as well as surgical implantation into the 5-mm circular calvarial defects in rats, was conducted and the results showed extensive new bone formation. In conclusion, alginate microbeads can be utilized as an effective BMP-2 delivery vehicle for use in orthopedic surgery and as an injectable vehicle for a minimally invasive therapy. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 286-294, 2019.

Incorporation of nanoparticles into transplantable decellularized matrices: Applications and challenges.

Decellularization of tissues can significantly improve regenerative medicine and tissue engineering by producing natural, less immunogenic, three-dimensional, acellular matrices with high biological activity for transplantation. Decellularized matrices retain specific critical components of native tissues such as stem cell niche, various growth factors, and the ability to regenerate in vivo. However, recellularization and functionalization of these matrices remain limited, highlighting the need to improve the characteristics of decellularized matrices. Incorporating nanoparticles into decellularized tissues can overcome these limitations because nanoparticles possess unique properties such as multifunctionality and can modify the surface of decellularized matrices with additional growth factors, which can be loaded onto the nanoparticles. Therefore, in this minireview, we highlight the various approaches used to improve decellularized matrices with incorporation of nanoparticles and the challenges present in these applications.

Silver nanoparticles improve structural stability and biocompatibility of decellularized porcine liver.

No ideal cross-linking agent has been identified for decellularized livers (DLs) yet. In this study, we evaluated structural improvements and biocompatibility of porcine DLs after cross-linking with silver nanoparticles (AgNPs). Porcine liver slices were decellularized and then loaded with AgNPs (100 nm) after optimization of the highest non-toxic concentration (5 µg/mL) using Human hepatocellular carcinoma (HepG2) and EAhy926 human endothelial cell lines. The cross-linking effect of AgNPs was evaluated and compared to that of glutaraldehyde and ethyl carbodiimide hydrochloride and N-hydroxysuccinimide. The results indicated that AgNPs improved the ultra-structure of DLs' collagen fibres with good porosity and increased DLs' resistance against in vitro degradation with good cytocompatibility. AgNPs decreased the host inflammatory reaction against implanted porcine DL slices in vivo and increased the polarization of M2 macrophages. Thus, structural and functional improvements of Porcine DLs could be achieved using AgNPs.

Impact of collagen-alginate composition from microbead morphological properties to microencapsulated canine adipose tissue-derived mesenchymal stem cell activities.

The purpose of this study was to identify the effect of collagen-alginate composition on the size and shape of microbeads and the proliferation and osteogenic properties of microencapsulated canine adipose-derived mesenchymal stem cells (ASCs) in vitro. Canine ASCs were microencapsulated in mixtures of various collagen-alginate compositions using a vibrational technologic encapsulator. The size and shape of the resultant microbeads were measured using a light field microscope and the viability of the microencapsulated canine ASCs was evaluated using a live/dead viability/cytotoxicity kit. Proliferation and osteogenic potentials of microencapsulated canine ASCs were evaluated using an alamarBlue proliferation assay and an alkaline phosphatase assay, respectively. As the collagen ratio increased, the size and size variation of microbeads increased and the shape of microbeads became more irregular. Nonetheless, homogeneous microbeads were created with no significant difference in size and shape, in the range of 0.75% alginate mixed with 0.099% collagen solution in 1.2% alginate solution. There were no significant differences in viability of the ASCs in the various collagen-alginate compositions. Both proliferation and osteogenic properties, in vitro, increased with increasing collagen ratio. Microencapsulation of canine ASCs with appropriate collagen-alginate composition increases cell proliferation and osteogenic properties, in vitro, without significant effects on the shape and size of microbeads and cell viability. Microencapsulation with adequate collagen-alginate composition may produce injectable microbeads that could enhance the therapeutic efficacy of stem cells.

Optimisation of a double-centrifugation method for preparation of canine platelet-rich plasma.

BACKGROUND: Platelet-rich plasma (PRP) has been expected for regenerative medicine because of its growth factors. However, there is considerable variability in the recovery and yield of platelets and the concentration of growth factors in PRP preparations. The aim of this study was to identify optimal relative centrifugal force and spin time for the preparation of PRP from canine blood using a double-centrifugation tube method. METHODS: Whole blood samples were collected in citrate blood collection tubes from 12 healthy beagles. For the first centrifugation step, 10 different run conditions were compared to determine which condition produced optimal recovery of platelets. Once the optimal condition was identified, platelet-containing plasma prepared using that condition was subjected to a second centrifugation to pellet platelets. For the second centrifugation, 12 different run conditions were compared to identify the centrifugal force and spin time to produce maximal pellet recovery and concentration increase. Growth factor levels were estimated by using ELISA to measure platelet-derived growth factor-BB (PDGF-BB) concentrations in optimised CaCl2-activated platelet fractions. RESULTS: The highest platelet recovery rate and yield were obtained by first centrifuging whole blood at 1000 g for 5 min and then centrifuging the recovered platelet-enriched plasma at 1500 g for 15 min. This protocol recovered 80% of platelets from whole blood and increased platelet concentration six-fold and produced the highest concentration of PDGF-BB in activated fractions. CONCLUSIONS: We have described an optimised double-centrifugation tube method for the preparation of PRP from canine blood. This optimised method does not require particularly expensive equipment or high technical ability and can readily be carried out in a veterinary clinical setting.

Injectable alginate-microencapsulated canine adipose tissue-derived mesenchymal stem cells for enhanced viable cell retention.

The purpose of this study was to establish an optimized protocol for the production of alginate-encapsulated canine adipose-derived mesenchymal stem cells (cASCs) and evaluate their suitability for clinical use, including viability, proliferation and in vivo cell retention. Alginate microbeads were formed by vibrational technology and the production of injectable microbeads was performed using various parameters with standard methodology. Microbead toxicity was tested in an animal model. Encapsulated cASCs were evaluated for viability and proliferation in vitro. HEK-293 cells, with or without microencapsulation, were injected into the subcutaneous tissue of mice and were tracked using in vivo bioluminescent imaging to evaluate the retention of transplanted cells. The optimized injectable microbeads were of uniform size and approximately 250 µm in diameter. There was no strong evidence of in vivo toxicity for the alginate beads. The cells remained viable after encapsulation, and there was evidence of in vitro proliferation within the microcapsules. In vivo bioluminescent imaging showed that alginate encapsulation improved the retention of transplanted cells and the encapsulated cells remained viable in vivo for 7 days. Encapsulation enhances the retention of viable cells in vivo and might represent a potential strategy to increase the therapeutic potency and efficacy of stem cells.