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Mesenchymal stem cells (MSCs) are applied in the treatment of spinal cord injury (SCI) because of their neural tissue restoring ability. In the clinical setting, intravenous injection of cryopreserved cells is essential for the immediate treatment of SCI, exhibiting the disadvantage of reduced cell properties.In this study, we potentiated the characteristics of cryopreserved MSCs by heat-shock (HS) treatment to induce the expression of HS protein (HSP) HSP70/HSP27 and further improved antioxidant capacity by overexpressing HSP32 (heme oxygenase-1 [HO-1]). We randomly assigned 12 beagle dogs with acute SCI into three groups and transplanted cells intravenously: (i) F-MSCs (MSCs in frozen/thawed conditions); (ii) F-HSP-MSCs (HS-treated MSCs in frozen/thawed conditions); and (iii) F-HSP-HO-MSCs (HO-1-overexpressing and HS-treated MSCs in frozen/thawed conditions).The potentiated MSCs exhibited increased growth factor-, anti-inflammatory-, antioxidant-, homing- and stemness-related gene expression. In the animal experiments, the HSP-induced groups showed significant improvement in hind-limb locomotion, highly expressed neural markers, less intervened fibrotic changes, and improved myelination. In particular, the HO-1-overexpression group was more prominent, controlling the initial inflammatory response with high antioxidant capabilities, suggesting that antioxidation was important to prevent secondary injury. Accordingly, HSPs not only successfully increased the ability of frozen MSCs but also demonstrated excellent neural protection and regeneration capacity in the case of acute SCI.The application of HSP-induced cryopreserved MSCs in first-aid treatment for acute SCI is considered to help early neural sparing and further hind-limb motor function restoration.

Background: The treatments of osteoarthritis (OA) are commonly conservative and multimodal to relieve pain and improve movement. Intra-articular injection of hyaluronic acid (IAHA) has been studied as a treatment option for OA in dogs. IAHA helps restore the viscoelasticity of the synovial fluid and relieves the clinical symptoms of OA. However, the efficacy of IAHA in dogs is still a controversial subject. This study aims to confirm the IAHA effect in dogs with spontaneous OA and to compare the effectiveness depending on the number of injections. Materials, Methods & Results: Thirty dogs with spontaneous OA were assigned to a single injection group (n=17) and a 3-weekly injections group (n=13). Dogs weighing less than 10 kg were injected 1 mL of HA to the OA joint, and more than 10 kg dogs were injected 2 mL of HA. In the case of the 3-weekly injections group, the same amount was administered 3 times at 1-week intervals. After the injection, physical and orthopedic examinations were performed to check for complications. Radiographic OA score was evaluated before and 3 months after the injection to confirm and to evaluate the progression of OA. Clinical symptom evaluations were performed on pre-injection, 1-, 2-, and 3-months post-injection. They consisted of the clinical lameness score by veterinarians and Canine Brief Pain Inventory (CBPI) by owners. Results were compared with unpaired t-test, repeated-measures ANOVA with Tukey’s or Sidak’s multiple comparison test, or Wilcoxon test, with P < 0.05. Patients had a median age of 9 years (range 3 to 16 years) and a bodyweight of 4.8 kg (range 2 to 48 kg). No systemic side effects or major complications were detected during the trial period. IAHA produced temporary pain and discomfort in 6 cases. There was no change in the radiographic OA score before and 3 months after injections in both groups, and the difference between groups was not confirmed. In both groups, the clinical lameness score significantly decreased at 1, 2, 3 months after injection compared with pre-injection. The score was lower at 3 months after the injection than at 1 month. The clinical lameness score had no significant difference between the groups. Similarly, CBPI was all decreased in the single injection group and 3-weekly injections group compared to pre-injection, and the score at 3 months post-injection was lower than at 1 month. No significant differences between the groups were found in CBPI. Discussion: Most studies on the efficacy of IAHA in canine OA have been conducted using an experimental model, so studies on spontaneous canine OA are insufficient. This study confirmed that IAHA improves clinical symptoms such as pain relief and movement improvement in spontaneous OA dogs using CBPI and clinical lameness score. In order to confirm the optimal IAHA protocol, a single IAHA and 3-weekly IAHA were compared. The result shows that clinical symptoms improved in both single and 3-weekly injections groups, but no significant difference was confirmed during the 3-month study period. These findings may suggest that a single IAHA may have a similar effect to multiple IAHA, and repeated injections are unnecessary. In humans and canine OA models, it is reported that the effect of IAHA was maintained for 6 months. This study showed that the effect of IAHA was maintained for 3 months study period and that clinical symptoms improved at 3 months than at 1 month. In conclusion, these findings suggested that IAHA improves clinical symptoms in dogs with spontaneous OA, and a single IAHA showed a similar effect to 3 weekly IAHA. Keywords: canine, treatment, hyaluronic acid, intra-articular injection, osteoarthritis.

Exogenous dual delivery of progenitor cell population and therapeutic growth factors (GFs) is one of alternative tissue engineering strategies for osteochondral tissue regeneration. In the present study, an implantable dual delivery platform was developed using coacervates (Coa) (i.e., a tertiary complex of poly(ethylene argininylaspartate diglyceride) (PEAD) polycation, heparin, and cargo insulin-like growth factor-1 (IGF-1), in thiolated gelatin (gelatin-SH)/ poly(ethylene glycol) diacrylate (PEGDA) interpenetrating network (IPN) hydrogels. Since Coa is able to protect cargo GF and maintain its long-term bioactivity, it is speculated that Coa-mediated delivery of chondrogenic factor IGF-1 with the aid of adipose-derived stem cells (ADSCs) would synergistically facilitate osteochondral tissue repair during physiological regeneration process. Our results indicate that gelatin-SH/PEGDA IPN hydrogels demonstrated biocompatibility and mechanical properties for a possible long-term transplantation, and PEAD-base Coa exhibited a sustained release of bioactive IGF-1 over 3 weeks. Subsequently, released IGF-1 from Coa could effectively induce chondrogenic differentiation of embedded ADSCs in the hydrogel, by showing enhanced glycosaminoglycan deposition and expression of chondrogenesis-associated genes. More importantly, at 12 weeks post-implantation in a rabbit full thickness osteochondral defect model, the quality of regenerative tissues in both chondral and subchondral layers was significantly improved in dual delivery of ADSC and IGF-1 in Coa encapsulated in gelatin-SH/PEGDA IPN hydrogels, as compared with a single delivery of ADSC only and a dual delivery without Coa. Therefore, we conclude that our Coa-embedded composite hydrogel platform could effectively augment osteochondral tissue regeneration holds promise for a feasible osteoarthritis therapeutic application.

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.

BACKGROUND Hyperbaric gaseous cryotherapy (HGC) is a type of cryotherapy used in human medicine for rehabilitation after orthopedic surgeries. Because HGC is known to reduce acute or chronic pain, research is needed to prove its effectiveness in veterinary medicine. OBJECTIVES To compare the effects of HGC between the HGC treatment group and the nontreatment (NT) group on postoperative swelling, range of motion, lameness score, postoperative pain, and kinetic measurements after stifle joint surgery in dogs. METHODS Dogs were randomized in an HGC group or NT groups. In the HGC group, HGC was applied once a day for a total of 2 days after surgery. All parameters were measured postoperatively and at 1, 2, 10, and 28 days after surgery. RESULTS Twenty dogs were enrolled: 10 in the HGC group and 10 in the NT group. Soft tissue swelling was not significantly different between groups at any time point. In the HGC group, pain scores decreased significantly 24 h after surgery and 48 h after surgery. Dogs in the HGC group showed a significantly decreased lameness and improvement for all kinetic measurements beginning 48 h after surgery. In addition, the HGC group indicated a significant increase in range of motion as compared with the NT group at 28 days after surgery. CONCLUSIONS HGC plays a powerful role in decreasing initial postoperative pain. Furthermore, the improvement in pain affects the use of the operated limb, and the continued use of the limb eventually assists in the quick recovery of normal function.

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.

In this study, we developed aptamer-conjugated hydroxyapatite (Apt-HA) that promotes bone regeneration and angiogenesis. The 3R02 bivalent aptamer specific to vascular endothelial growth factor (VEGF) was grafted to the hydroxyapatite (HA) surface. Apt-HA was tested for its VEGF protein capture ability to determine the optimal aptamer concentration immobilized on the HA. Apt-HA showed higher VEGF protein capture ability, and faster growth of human umbilical vein endothelial cell (HUVEC) compared to a neat HA with no cytotoxic effects on human osteoblasts. To examine in vivo angiogenesis and bone regeneration, Apt-HA and HA were bilaterally implanted into rabbit tibial metaphyseal defects and analyzed after eight weeks using micro-CT, histology, and histomorphometry. Apt-HA showed significantly increased the volume of new bones, the percentage of bone, and the density of bone mineral in cortical bone. Apt-HA also exhibited the enhanced bone formation at the cortical region in a histomorphometric analysis. Finally, Apt-HA showed significantly increased blood vessel number compared to a neat HA. In summary, the engineered Apt-HA has potential as a bone graft material that may simultaneously promote bone regeneration and angiogenesis. STATEMENT OF SIGNIFICANCE: This work presents a functional hydroxyapatite bone graft using a DNA-based aptamer which overcomes the limitations of existing bone graft materials, which use bound signaling peptides. DNA aptamer immobilized hydroxyapatite enhances the in vitro proliferation of human umbilical vascular endothelial cells as well as in vivo angiogenesis and bone regeneration. DNA aptamer immobilized hydroxyapatite shows no cytotoxic effect on human osteoblasts.

Decellularization of a whole organ is an attractive process that has been used to create 3D scaffolds structurally and micro-architecturally similar to the native one. Currently used decellularization protocols exhibit disrupted extracellular matrix (ECM) structure and denatured ECM proteins. Therefore, maintaining a balance between ECM preservation and cellular removal is a major challenge. The aim of this study was to optimize a multistep Triton X-100 based protocol (either using Triton X-100/ammonium hydroxide mixture alone or after its modification with DNase, sodium dodecyl sulfate or trypsin) that could achieve maximum decellularization with minimal liver ECM destruction suitable for subsequent organ implantation without immune rejection. Based on our findings, Triton X-100 multistep protocol was insufficient for whole liver decellularization and needed to be modified with other detergents. Among all Triton X-100 modified protocols, a Triton X-100/DNase-based one was considered the most suitable. It maintains a gradual but sufficient removal of cells to generate decellularized biocompatible liver scaffolds without any significant alteration to ECM micro- and ultra-structure.

Collecting blood from an indwelling arterial catheter may reduce stress from repeated venipuncture in patients requiring serial monitoring, but the use of arterial blood for hematological and biochemical testing remains understudied. Here, we compared hematological and biochemical results of arterial and venous blood and evaluated their clinical interchangeability. Blood samples from dogs who had recovered from anesthesia, collected by both arterial catheterization and venipuncture, were analyzed. To assess clinical acceptance between paired samples, the limit of agreement between the values derived from the arterial and venous blood samples was compared with the allowable total error (TEa) recommended for each parameter. We found no significant differences between the arterial and venous sample results for red/white blood cell and platelet counts and hematocrit, blood urea nitrogen, phosphate, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase, total bilirubin, sodium, potassium, and chloride levels, whereas hemoglobin, glucose, creatinine, and calcium levels differed significantly (p &lt, 0.05). Moreover, only gamma-glutamyl transpeptidase and potassium exceeded the recommended TEa. Hematological and biochemical results derived from venous and arterial blood samples are clinically interchangeable in post-anesthetic dogs, with the exception of gamma-glutamyl transpeptidase and potassium, thus, these values should be used with caution.

Background Mesenchymal stem cells (MSCs) are used for the treatment of osteoarthritis (OA), and MSC genetic engineering is expected to enhance cartilage repair. Here, we aimed to investigate the effect of MSCs overexpressing platelet-derived growth factor (PDGF) or heme oxygenase-1 (HO-1) in chondrocytes and synovial cells with an OA phenotype and assess the in vivo efficacy of intra-articular injections of these MSCs in canine OA models. Methods Canine adipose-derived MSCs were transfected with canine PDGF (PDGF-MSCs) or HO-1 (HO-1-MSCs) using lentiviral vectors. Canine chondrocytes or synovial cells were stimulated with lipopolysaccharide (LPS) to mimic the inflammatory OA model and then co-cultured with MSCs, PDGF-MSCs, or HO-1-MSCs for 24 h and 72 h. The mRNA levels of pro-inflammatory, extracellular matrix-degradative/synthetic, or pain-related factors were measured after co-culture by real-time PCR. Furthermore, a surgery-induced canine OA model was established and the dogs were randomized into four groups: normal saline (n = 4), MSCs (n = 4), PDGF-MSCs (n = 4), and HO-1-MSCs (n = 4). The OA symptoms, radiographic OA severity, and serum matrix metallopeptidase (MMP)-13 levels were assessed before and 10 weeks after treatment, to evaluate the safety and efficacy of the modified MSCs. Results PDGF or HO-1 overexpression significantly reduced the expression of pro-inflammatory factors, MMP-13, and nerve growth factor elicited by LPS and increased that of aggrecan and collagen type 2 in chondrocytes (P P P P Conclusions Genetically modifying PDGF and HO-1 in MSCs is an effective strategy for treating OA, suggesting that PDGF-MSCs can be novel therapeutic agents for improving OA symptoms.

A 5-month old Shih Tzu was diagnosed with congenital elbow luxation and uniapical complex angular deformity of the radius. Single radial oblique and dynamic ulnar osteotomies were performed, using patient-specific 3D-printed osteotomy guide. External skeletal fixation was maintained for three weeks to prevent re-luxation of elbow joint. Three months after the surgery, objective gait analysis indicated markedly improved limb function. In addition, radiograph showed improved congruity of elbow joint and appropriate bone healing. In dogs with congenital radial head luxation and concurrent complex angular deformity, a single oblique osteotomy might be a viable option to preserve bone length and correct the luxation of elbow joint.

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.

Direct administration of bone morphogenetic protein-2 (BMP-2) for bone regeneration could cause various clinical side effects such as osteoclast activation, inflammation, adipogenesis, and bone cyst formation. In this study, thiolated gelatin/poly(ethylene glycol) diacrylate (PEGDA) interpenetrating (IPN) composite hydrogels were developed for guided skull bone regeneration. To promote bone regeneration, either polycation-based coacervates (Coa) or gelatin microparticles (GMPs) were incorporated within IPN gels as BMP-2 carriers. Both BMP-2 loaded Coa and BMP-2 loaded GMPs showed significantly enhanced in vitro alkaline phosphate (ALP) activity of human mesenchymal stem cells (hMSCs) than non-BMP-2 treated control. Moreover, BMP-2 loaded GMPs group exhibited statistically increased ALP activity compared to both bolus BMP-2 administration and BMP-2 loaded Coa group, indicating that our carriers could protect and maintain biological activity of cargo BMP-2. Sustained release kinetics of BMP-2 from IPN composite hydrogels could be controlled by different formulations. For in vivo bone regeneration, various IPN gel formulations (i.e., (1) control, (2) only hydrogel, (3) hydrogel with bolus BMP-2, (4) hydrogel with BMP-2-loaded Coa, and (5) hydrogel with BMP-2-loaded GMPs) were bilaterally implanted into 5 mm-sized rat calvarial defects. After 4 weeks, micro-CT and histological analysis were performed to evaluate new bone formation. Significantly higher scores for bony bridging and union were observed in BMP-2-loaded Coa and BMP-2-loaded GMP groups as compared to other formulations. In addition, rats treated with BMP-2-loaded GMPs showed a significantly higher ratio of bone volume/total volume and lower trabecular separation scores than others. Finally, rats treated with either Coa or GMP groups exhibited a significant increase in bone formation area, as assessed via histomorphometric analysis. Taken together, it could be concluded that Coa and GMPs were effective carriers to maintain the bioactivity of cargo BMP-2 during its sustained release. Consequently, our IPN composite hydrogel system that combines such BMP-2 carriers could effectively promote skull bone regeneration.

Whole kidney decellularization is a promising approach in regenerative medicine for engineering a functional organ. The reaction of the potential host depends on the biocompatibility of these decellularized constructs. Despite the proven ability of decellularized kidney scaffolds to guide cell attachment and growth, little is known about biocompatibility and hemocompatibility of these scaffolds. Our aim is to prepare decellularized kidneys of a clinically relevant size and evaluate its biocompatibility and hemocompatibility. Porcine kidneys were cannulated via the renal artery, and then perfused with 0.1% sodium dodecyl sulfate solution. Hematoxylin and eosin as well as DAPI staining confirmed cellular clearance from native kidneys in addition to preservation of the microstructure. SEM confirmed the absence of any cellular content within the scaffold, which is maintained in a well-organized 3D architecture. Decellularized kidneys retained the intact renal vasculature upon examination with contrast radiography. The essential structural extracellular matrix molecules were well-preserved. Scaffolds were susceptible to enzymatic degradation upon collagenase treatment. Scaffolds showed a good hemocompatibility when exposed to porcine blood. Decellularization was efficient to remove 97.7% of DNA from native kidneys in addition to the immunogenic and pathogenic antigens. Scaffolds did not induce the human immune response in vitro. Decellularized kidneys were non-cytotoxic to pig kidney cells (PKs). PKs were able to grow and proliferate within the decellularized renal scaffolds with maintaining a higher function than cells grown as monolayers. Thus, we have developed a rapid decellularization technique for generating biocompatible kidney scaffolds that represents a step toward development of a transplantable organ. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2034-2047, 2018.

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.

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.

A 12-year-old neutered male domestic shorthair cat was presented with a 3-year history of multiple nonpruritic, ulcerated, plaque-like skin lesions but no other clinical signs. A systemic examination revealed mild lymphadenopathy. Histopathologic analysis of the skin lesions revealed multicentric basosquamous carcinoma (BSC). Immunohistochemical analysis, PCR, and sequencing detected Felis catus papillomavirus type 2 (FcaPV-2) within the tumors. As BSC is rare in cats, clinical behavior has not been established. To our knowledge, this is the first case report to demonstrate detection of FcaPV-2 within a BSC in a domestic cat.

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.

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) exhibit potency for the regeneration of infarcted hearts. Vascular endothelial growth factor (VEGF) is capable of inducing angiogenesis and can boost stem cell-based therapeutic effects. However, high levels of VEGF can cause abnormal blood vessel growth and hemangiomas. Thus, a controllable system to induce therapeutic levels of VEGF is required for cell therapy. We generated an inducible VEGF-secreting stem cell (VEGF/hUCB-MSC) that controls the expression of VEGF and tested the therapeutic efficacy in rat myocardial infarction (MI) model to apply functional stem cells to MI. To introduce the inducible VEGF gene cassette into a safe harbor site of the hUCB-MSC chromosome, the transcription activator-like effector nucleases system was used. After confirming the integration of the cassette into the locus, VEGF secretion in physiological concentration from VEGF/hUCB-MSCs after doxycycline (Dox) induction was proved in conditioned media. VEGF secretion was detected in mice implanted with VEGF/hUCB-MSCs grown via a cell sheet system. Vessel formation was induced in mice transplanted with Matrigel containing VEGF/hUCB-MSCs treated with Dox. Moreover, seeding of the VEGF/hUCB-MSCs onto the cardiac patch significantly improved the left ventricle ejection fraction and fractional shortening in a rat MI model upon VEGF induction. Induced VEGF/hUCB-MSC patches significantly decreased the MI size and fibrosis and increased muscle thickness, suggesting improved survival of cardiomyocytes and protection from MI damage. These results suggest that our inducible VEGF-secreting stem cell system is an effective therapeutic approach for the treatment of MI.

An eighteen-month-old female Humboldt penguin (Spheniscus humboldti) that was 50 cm in length and 4.5 kg in weight was presented with anorexia and vomiting. The hematological and blood biochemical profiles revealed no remarkable findings, and no Salmonella, Shigella or Vibrio spp. were isolated from the fecal culture. However, radiographic imaging revealed a long linear foreign body presenting from the lower esophagus to the stomach. To retrieve this foreign body, flexible endoscopic extraction was performed using flexible rat tooth grasping forceps. A long bamboo stick (29 × 1 cm) was removed from the stomach, and the penguin fully recovered.

In this study, gelatin-based cryogels were fabricated by mixing methacrylated gelatin (GelMA) with methacrylated hyaluronic acid (MeHA) or methacrylated chondroitin sulfate (MeCS) for cartilage tissue engineering. In vitro revealed that MeCS incorporated gelatin-based cryogel (G-MeCS) showed significant cartilaginous tissue stimulation. Furthermore, the cell-laden gelatin-based ECM cryogels were implanted into mouse subcutaneous tissue for 6 weeks and displayed uniform distribution of cells with normal phenotype maintenance. Finally, when these cryogels were implanted into osteochondral defect of New Zealand white rabbit, full integration with host tissue and increased cellularity were observed with G-MeCS cryogel.

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.

BackgroundThe 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)MethodsTwelve 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.ResultsDogs 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.ConclusionsThese 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.

Limb-sparing surgery is one of the surgical options for dogs with distal radial osteosarcoma (OSA). This case report highlights the novel application of a three-dimensional (3D)-printed patient-specific polycaprolactone/β-tricalcium phosphate (PCL/β-TCP) scaffold in limb-sparing surgery in a dog with distal radial OSA. The outcomes evaluated included postoperative gait analysis, complications, local recurrence of tumor, metastasis, and survival time. Post-operative gait evaluation showed significant improvement in limb function, including increased weight distribution and decreased asymmetry. The implant remained well in place and increased bone opacity was observed between the host bone and the scaffold. There was no complication due to scaffold or surgery. Significant improvement in limb function and quality of life was noted postoperatively. Local recurrence and pulmonary metastasis were identified at 8 weeks postoperatively. The survival time from diagnosis of OSA to death was 190 days. The PCL/β-TCP scaffold may be an effective alternative to cortical allograft in limb-sparing surgery for bone tumors.