Atlantoaxial joint stabilization using patient-specific 3-D-printed drill guides and 3-D-printed titanium plates or polymethyl methacrylate is effective in toy-breed dogs.

OBJECTIVE: To report the clinical outcomes in toy-breed dogs with atlantoaxial instability (AAI) stabilized with patient-specific 3-D-printed titanium plates or polymethyl methacrylate (PMMA), both with the assistance of 3-D-printed drill guides. ANIMALS: 15 client-owned dogs undergoing surgical treatment for AAI between January 1, 2020, and October 31, 2022. METHODS: The clinical characteristics, diagnostic images, and neurological outcomes of 15 dogs treated for AAI using 3-D-printing technology were reviewed. Postoperative CT images were examined to evaluate the screw placement accuracy in the atlas and axis. Clinical outcomes, including postoperative neurological improvement and screw loosening, were evaluated in dogs treated with a patient-specific titanium plate and those treated with PMMA. RESULTS: Patient-specific titanium plates (7 dogs) and PMMA (8 dogs) were used for AAI stabilization. The mean follow-up period was 15.2 months (range 7 to 22 months). A reduction of the axis without vertebral canal violation was confirmed on postoperative CT in 14 dogs. The mean deviation from the preoperative planning ranged from 0.30 to 1.27 mm at the insertion and exit points of 84 screws using this method. The neurological grade had improved in each dog postoperatively and at the final follow-up. Screw loosening was noted in 4 dogs in the titanium plates groups without neurological deterioration. CLINICAL RELEVANCE: Patient-specific 3-D-printed drill guides and titanium plates or PMMA are effective for AAI stabilization in toy-breed dogs, providing accurate guidance.

Surgical Reconstruction of Canine Nonunion Fractures Using Bone Morphogenetic Protein-2-loaded Alginate Microbeads and Bone Allografts.

BACKGROUND/AIM: Effective treatment of nonunion fractures is challenging as it requires a biological and mechanical environment to promote sufficient osteogenesis. Herein, we present a case series in which we evaluated the clinical efficacy of bone morphogenetic protein-2 (BMP-2)-loaded alginate microbeads and allografts in two dogs with nonunion fractures. CASE REPORT: A 3-year-old, 2.3-kg, spayed female Pomeranian (Case 1) presented with intermittent lameness of the left forelimb after radial and ulnar fracture repair 8 weeks prior. A 4-year-old, 4.8-kg, spayed female Pomeranian (Case 2) was referred for non-weight-bearing lameness of the left hindlimb due to implant failure following left tibial fracture repair. Both dogs had atrophic bone ends and no bridging calluses at the fracture site on radiographs, and were diagnosed with nonviable nonunion fractures of the radius/ulna and tibia, respectively. The surgical approach involved implant removal, debridement, and fracture gap reconstruction. BMP-2 was loaded into alginate microbeads for a prolonged release with bone allograft chips in both cases. In Case 1, bead grafts were applied directly at the fracture site, while in Case 2, they were implanted inside a frozen cortical bone allograft as a scaffold to fill the large gap. Postoperative radiography revealed excessive callus formation, early radiographic bone union, and cortical bone remodeling, in line with improved lameness scores. At the final follow-up, gait was improved and the desired bone length and shape were achieved in both cases. CONCLUSION: Simultaneous use of osteoinductive BMP-2 alginate microbeads and osteoconductive bone allografts yielded functionally and structurally favorable outcomes in canine nonunion fractures, without major complications.

Cervical spine reconstruction after total vertebrectomy using customized three-dimensional-printed implants in dogs.

BACKGROUND: Sufficient surgical resection is necessary for effective tumor control, but is usually limited for vertebral tumors, especially in the cervical spine in small animal neurosurgery. OBJECTIVE: To evaluate the primary stability and safety of customized three-dimensional (3D)-printed implants for cervical spine reconstruction after total vertebrectomy. METHODS: Customized guides and implants were designed based on computed tomography (CT) imaging of five beagle cadavers and were 3D-printed. They were used to reconstruct C5 after total vertebrectomy. Postoperative CT images were obtained to evaluate the safety and accuracy of screw positioning. After harvesting 10 vertebral specimens (C3-C7) from intact (group A) and implanted spines (group B), implant stability was analyzed using a 4-point bending test comparing with groups A and C (reconstituted with plate and pins/polymethylmethacrylate after testing in Group A). RESULTS: All customized implants were applied without gross neurovascular damage. In addition, 90% of the screws were in a safe area, with 7.5% in grade 1 (< 1.3 mm) and 2.5% in grade 2 (> 1.3 mm). The mean entry point and angular deviations were 0.81 ± 0.43 mm and 6.50 ± 5.11°, respectively. Groups B and C significantly decreased the range of motion (ROM) in C3-C7 compared with intact spines (p = 0.033, and 0.018). Both groups reduced overall ROM and neutral zone in C4-C6, but only group B showed significance (p = 0.005, and 0.027). CONCLUSION: Customized 3D-printed implants could safely and accurately replace a cervical vertebra in dog cadavers while providing primary stability.

Transplantation of Heat-Shock Preconditioned Neural Stem/Progenitor Cells Combined with RGD-Functionalised Hydrogel Promotes Spinal Cord Functional Recovery in a Rat Hemi-Transection Model.

BACKGROUND: Neural stem/progenitor cell (NSPC) transplantation in spinal cord injury (SCI) is a potential treatment that supports regeneration by promoting neuroprotection, remyelination, and neurite outgrowth. However, glial scarring hinders neuroregeneration and reduces the efficiency of cell transplantation. The present study aimed to enhance this neuroregeneration by surgically removing the glial scar and transplanting heat-shock (HS) preconditioned NSPCs in combination with Arg-Gly-Asp (RGD)-functionalised hydrogel in a rat spinal cord hemi-transection model. METHODS: Twelve Sprague-Dawley rats underwent spinal cord hemi-transection and were randomly divided into three treatment groups: hydrogel implantation (control group), NSPC-encapsulated hydrogel implantation, and HS-NSPC-encapsulated hydrogel implantation. HS preconditioning was applied to the NSPCs to reinforce cell retention and an RGD-functionalised hydrogel was used as a biomatrix. RESULTS: In vitro culture showed that preconditioned NSPCs highly differentiated into neurons and oligodendrocytes and exhibited higher proliferation and neurite outgrowth in hydrogels. Rats in the HS-NSPC-encapsulated hydrogel implantation group showed significantly improved functional recovery, neuronal and oligodendrocyte differentiation of transplanted cells, remyelination, and low fibrotic scar formation. CONCLUSIONS: The surgical removal of the glial scar in combination with HS-preconditioning and RGD-functionalised hydrogels should be considered as a new paradigm in NSPC transplantation for spinal cord regeneration treatment.

Three-dimensional-printed patient-specific guides for tibial deformity correction in small-breed dogs.

OBJECTIVE: To describe the use of patient-specific 3-D-printed osteotomy, reduction, and compression guides for tibial closing wedge osteotomy in small-breed dogs. ANIMALS: 6 dogs with unilateral tibial deformities. METHODS: Six small-breed dogs with 1 or a combination of tibial deformities, including excessive tibial plateau angle, valgus, and torsion, were scheduled to undergo tibial closing wedge osteotomy using patient-specific 3-D-printed osteotomy, reduction, and compression guides. The location and orientation of the wedge osteotomy were determined based on CT data using computer-aided design software. After the tibial deformities were corrected, postoperative CT or radiographs were obtained to compare the achieved tibial limb angles with the planned angles. Clinical evaluation and radiographic follow-up were performed on all dogs. RESULTS: Guides were successfully positioned at each specific location, and osteotomies were performed without radiation exposure or observer assistance in all dogs. Tibial deformities were corrected with angular errors of 1.8 ± 1.4°, 2.3 ± 2.1°, and 2.6 ± 1.3° in the sagittal, frontal, and transverse planes, respectively. Mild complications resolved within 1 month in 3 dogs, and revision surgery was not required. Five dogs improved to the normal gait (mean, 14.8 ± 6.6 weeks), and 1 dog recovered a satisfactory gait 24 weeks after surgery. All limbs healed 14 ± 4.7 weeks after surgery. CLINICAL RELEVANCE: Patient-specific 3-D-printed osteotomy, reduction, and compression guides can provide effective assistance allowing accurate correction of tibial deformities. Their use yields good clinical outcomes in small-breed dogs.

Clinical infection of Brucella canis in a companion dog with discospondylitis in the Republic of Korea.

A 2-year-old, spayed female, Bichon Frise dog was presented with reluctance to exercise, back pain, and frequent sitting down. Multiple osteolysis, periosteal proliferation, and sclerosis of the vertebral endplates of T11-13 were observed in the radiography, computed tomography, and magnetic resonance imaging. The bacterial culture of the urine specimen, the polymerase chain reaction (PCR) of the blood, and the antibody tests were positive for Brucella canis. Accordingly, discospondylitis caused by B. canis was diagnosed and doxycycline was administered. The clinical signs resolved and the culture and PCR results were negative afterwards. Doxycycline was discontinued after 6 months. The clinical signs recurred 2 weeks later, and the combination treatment of doxycycline and enrofloxacin was initiated. Though no clinical signs were observed after 9 months and the bacterial cultures and PCR were negative, the antibody titre remained at 1 : 200 or more. The dog will continue taking antibiotics until the antibody titre drops. To the best of our knowledge, this is the first case report of a clinical infection of B. canis associated with canine discospondylitis in the Republic of Korea. Although the clinical signs of brucellosis might improve with antibiotic treatment, the disease cannot be cured due to Brucella's various strategies to evade host immune systems. Specifically, it can proliferate and replicate within the host cells, resulting in an environment that makes treatment less effective. Furthermore, owing to its zoonotic potential, owners and veterinarians should consider lifelong management or euthanasia.

Neural stem/progenitor cells from adult canine cervical spinal cord have the potential to differentiate into neural lineage cells.

BACKGROUND: • Neural stem/progenitor cells (NSPCs) are multipotent self-renewing cells that can be isolated from the brain or spinal cord. As they need to be isolated from neural tissues, it is difficult to study human NSPCs. To facilitate NSPC research, we attempted to isolate NSPCs from dogs, as dogs share the environment and having many similar diseases with humans. We collected and established primary cultures of ependymal and subependymal cells from the central canal of the cervical spinal cord of adult dogs. To isolate pure NSPCs, we employed the monolayer culture and selective medium culture methods. We further tested the ability of the NSPCs to form neurospheres (using the suspension culture method) and evaluated their differentiation potential. RESULTS: • The cells had the ability to grow as cultures for up to 10 passages; the growth curves of the cells at the 3rd, 6th, and 9th passages showed similar patterns. The NSPCs were able to grow as neurospheres as well as monolayers, and immunostaining at the 3rd, 6th, and 9th passages showed that these cells expressed NSPC markers such as nestin and SOX2 (immunofluorescent staining). Monolayer cultures of NSPCs at the 3rd, 6th, and 9th passages were cultured for approximately 14 days using a differentiation medium and were observed to successfully differentiate into neural lineage and glial cells (astrocytes, neurons, and oligodendrocytes) at all the three passages tested. CONCLUSION: • It is feasible to isolate and propagate (up to at least 10 passages) canine cervical spinal cord-derived NSPCs with the capacity to differentiate into neuronal and glial cells. To the best of our knowledge this is the first study to successfully isolate, propagate, and differentiate canine NSPCs derived from cervical spinal cord in the adult canine, and we believe that these cells will contribute to the field of spinal cord regeneration in veterinary and comparative medicine.

A shape memory alloy implant can be an effective surgical treatment in the atlantoaxial joint stabilization using rabbits as substitutes for toy-breed dogs.

OBJECTIVE: To investigate the feasibility of using shape memory alloy (SMA) implants for atlantoaxial joint stabilization using a rabbit model as a substitute for canines. ANIMALS: 20 rabbit cadavers. METHODS: We prepared rabbit cadavers from the middle of the skull to the third cervical vertebra. The vertebral body and canal sizes of the atlas and axis were compared using CT data from rabbits, normal dogs, and dogs with atlantoaxial instability (AAI) to assess the feasibility of using rabbits as substitutes for toy-breed dogs. The shape memory alloy (SMA) implants were designed to stabilize the atlantoaxial joint without compromising the spinal canal passage for safety and were classified into SMA-1 and SMA-2 based on their design. To evaluate the strength, the ventrodorsal force was measured with atlantoaxial ligaments intact, after removing the ligaments, and after applying conventional wire or SMA implants to stabilize the atlantoaxial joint. The time taken for implant application was measured. RESULTS: No significant difference in vertebral body size of the atlas and axis was observed. A significant difference in vertebral canal size was observed between the animals. In biomechanical testing, the SMA-2 implant provided more stabilization, while the SMA-1 implant had lower strength than the conventional method using wires. The application time of wire was the longest, while that of SMA-1 was the shortest. CLINICAL RELEVANCE: SMA implants provide comparable strength and demonstrate superior efficacy compared to conventional dorsal wire fixation of atlantoaxial stabilization. Therefore, SMA implants can be an effective surgical option for AAI.

Cell-directed assembly of luminal nanofibril fillers in nerve conduits for peripheral nerve repair.

Graphene and its derivatives, graphene oxide (GO) and reduced graphene oxide (rGO), have attracted significant attention in the field of tissue engineering, particularly in nerve and muscle regeneration, owing to their excellent electrical conductivity. This paper reports the fabrication of cell-mixable rGO-decorated polycaprolactone (PCL) nanofibrils (NFs) to promote peripheral nerve repair with the assistant of electron transmission by rGO and cytokine paracrine by stem cells. Oxidized GO (GO-COOH) and branched polyethylenimine are layer-by-layer coated on hydrolyzed PCL NFs via electrostatic interaction, and the number of layering is manipulated to adjust the GO-COOH coating amount. The decorated GO-COOH is reduced in situ to rGO for electrical conductivity retrieval. PC12 cells cultivated with rGO-coated NF demonstrate spontaneous cell sheet assembly, and neurogenic differentiation is observed upon electrical stimulation. When transplant nerve guidance conduit containing the assembly of rGO-coated NF and adipose-derived stem cell to the site of neurotmesis injury of a sciatic nerve, animal movement is enhanced and autotomy is ameliorated for 8 weeks compared to transplanting the hollow conduit only. Histological analysis results reveal higher levels of muscle mass and lower levels of collagen deposition in the triceps surae muscle of the rGO-coated NF-treated legs. Therefore, the rGO-layered NF can be tailored to repair peripheral nerve injuries in combination with stem cell therapy.

Insertional biceps tendinopathy palliated successfully with intra-articular triamcinolone acetonide injection in a dog: A case report.

This report describes a dog diagnosed with insertional biceps tendinopathy that was palliated with intra-articular triamcinolone acetonide injections. The patient was a 6-year-old spayed female Chihuahua dog that had left thoracic limb lameness for 3 months before presentation. On physical examination, moderate pain was elicited by performing the biceps test and isolated full elbow extension on the left thoracic limb. Gait analysis showed asymmetrical peak vertical force and vertical impulse between thoracic limbs. Computed tomography (CT) revealed enthesophyte formation on the ulnar tuberosity of the left elbow joint. Ultrasonography showed a heterogeneous fibre pattern at the biceps tendon insertion site on the left elbow joint. These findings confirmed insertional biceps tendinopathy based on physical examination, CT and ultrasonography results. The dog received an intra-articular triamcinolone acetonide injection with hyaluronic acid in the left elbow joint. Clinical signs improved after the first injection, including a range of motion, pain and gait. A second injection was given in the same manner because of recurring mild lameness 3 months later. No clinical signs were observed during the follow-up period.

Hydroxyapatite microbeads containing BMP-2 and quercetin fabricated via electrostatic spraying to encourage bone regeneration.

BACKGROUND: Hydroxyapatite (HAp) possesses osteoconductive properties, and its granular form can serve as an effective drug delivery vehicle for bone regeneration. Quercetin (Qct), a plant-derived bioflavonoid, is known to promote bone regeneration; however, its comparative and synergistic effects with the commonly used bone morphogenetic protein-2 (BMP-2) have not been investigated. METHODS: We examined the characteristics of newly formed HAp microbeads using an electrostatic spraying method and analyzed the in vitro release pattern and osteogenic potential of ceramic granules containing Qct, BMP-2, and both. In addition, HAp microbeads were transplanted into a rat critical-sized calvarial defect and the osteogenic capacity was assessed in vivo. RESULTS: The manufactured beads had a microscale size of less than 200 µm, a narrow size distribution, and a rough surface. The alkaline phosphatase (ALP) activity of osteoblast-like cells cultured with the BMP-2-and-Qct-loaded HAp was significantly higher than that of either Qct- or BMP-2-loaded HAp groups. The mRNA levels of osteogenic marker genes such as ALP and runt-related transcription factor 2 were found to be upregulated in the HAp/BMP-2/Qct group compared to the other groups. In micro-computed tomographic analysis, the amount of newly formed bone and bone surface area within the defect was significantly higher in the HAp/BMP-2/Qct group, followed by the HAp/BMP-2 and HAp/Qct groups, which is consistent with the histomorphometrical results. CONCLUSIONS: These results imply that electrostatic spraying can be an efficient strategy to produce homogenous ceramic granules and that the BMP-2-and-Qct-loaded HAp microbeads can serve as effective implants for bone defect healing.

Zygomatic arch reconstruction with a patient-specific polycaprolactone/beta-tricalcium phosphate scaffold after parosteal osteosarcoma resection in a dog.

OBJECTIVE: To describe the surgical application of a 3D-printing-based, patient-specific, biocompatible polycaprolactone/beta-tricalcium phosphate (PCL/ß-TCP) scaffold to reconstruct the zygomatic arch after tumor resection in a dog. ANIMAL: A 13 year old female spayed Maltese. STUDY DESIGN: Case report METHODS: The dog's presenting complaint was swelling ventral to her right eye. A round mass arising from the caudal aspect of the right zygomatic arch was identified by computed tomography (CT). The histopathologic diagnosis was a low-grade spindle-cell tumor. Surgical resection was planned to achieve 5 mm margins. A patient-specific osteotomy guide and polycaprolactone/beta-tricalcium phosphate (PCL/ß-TCP) scaffold were produced. Osteotomy, including 30% of total zygomatic arch length, was performed using an oscillating saw aligned with the guide. The scaffold was placed in the defect. Parosteal osteosarcoma was diagnosed based on histopathological examination. Excision was complete, with the closest margin measuring 0.3 mm. RESULTS: Mild epiphora, due to surgical site swelling, subsided after 20 days. Tissue formation within and around the porous scaffold was noted on CT 10 months postoperatively, with no evidence of metastasis or local recurrence. Facial conformation appeared symmetrical, and no complications were noted 16 months postoperatively. CONCLUSION: The use of a 3D-printing-based, patient-specific, biocompatible PCL/ß-TCP scaffold successfully restored the structure and function of the zygomatic arch without complications, even following wide zygomectomy for complete tumor removal.

Heat-Shock Proteins Can Potentiate the Therapeutic Ability of Cryopreserved Mesenchymal Stem Cells for the Treatment of Acute Spinal Cord Injury in Dogs.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Directed Regeneration of Osteochondral Tissue by Hierarchical Assembly of Spatially Organized Composite Spheroids.

The use of engineered scaffolds or stem cells is investigated widely in the repair of injured musculoskeletal tissue. However, the combined regeneration of hierarchical osteochondral tissue remains a challenge due to delamination between cartilage and subchondral bone or difficulty in spatial control over differentiation of transplanted stem cells. Here, two types of composite spheroids are prepared using adipose-derived stem cells (hADSCs) and nanofibers coated with either transforming growth factor-ß3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis, respectively. Each type of spheroid is then cultured within a 3D-printed microchamber in a spatially arranged manner to recapitulate the bilayer structure of osteochondral tissue. The presence of inductive factors regionally modulates in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct without dedifferentiation. Furthermore, hADSCs from each spheroid proliferate and sprout and successfully connect the two layers mimicking the osteochondral interface without apertures. In vivo transplantation of the biphasic construct onto a femoral trochlear groove defect in rabbit knee joint results in 21.2 ± 2.8% subchondral bone volume/total volume and a cartilage score of 25.0 ± 3.7. The present approach can be an effective therapeutic platform to engineer complex tissue.

Hyperbaric gaseous cryotherapy for postoperative rehabilitation enhances functional recovery of canine stifle joint: a report on a short-term study.

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.

Sustained Release of Bone Morphogenetic Protein-2 through Alginate Microbeads Enhances Bone Regeneration in Rabbit Tibial Metaphyseal Defect Model.

Bone morphogenetic protein-2 (BMP-2) is widely used to enhance bone regeneration. However, because of its short half-life and rapid disappearance, large amounts of BMP-2 are needed, leading to unintended side effects. In this study, BMP-2-encapsulated alginate microbeads (AM) were used to enhance bone regeneration. Enzyme-linked immunosorbent assay confirmed the sustained release of BMP-2 from AM. Vascular endothelial growth factor (VEGF)-adsorbing aptamer-conjugated hydroxyapatite (Apt-HA) was used for osteoconduction and dual delivery of VEGF and BMP-2. For in vivo bone regeneration evaluation, the grafts (1) Apt-HA + phosphate-buffered saline (PBS), (2) Apt-HA + AM without BMP-2, (3) Apt-HA + BMP-2, and (4) Apt-HA + AM encapsulated with BMP-2 were implanted into rabbit tibial metaphyseal defects. After four weeks, micro-computed tomography (CT), histological, and histomorphometric analyses were performed to evaluate bone regeneration. The Apt-HA + AM with BMP-2 group revealed a significantly higher new bone volume and bone volume/total volume (BV/TV) in both cortical and trabecular bone than the others. Furthermore, as evaluated by histomorphometric analysis, BMP-2 AM exhibited a significantly higher bone formation area than the others, indicating that AM could be used to efficiently deliver BMP-2 through sustained release. Moreover, the combined application of BMP-2-encapsulated Apt-HA + AM may effectively promote bone regeneration.

Mesenchymal stem cells genetically engineered to express platelet-derived growth factor and heme oxygenase-1 ameliorate osteoarthritis in a canine model.

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 < 0.05). In addition, the expression of aggrecanases was significantly downregulated in synovial cells, whereas that of tissue inhibitor of metalloproteinases was upregulated (P < 0.05). Furthermore, the co-cultured MSCs highly expressed genes that contributed to the maintenance of joint homeostasis (P < 0.05). In vivo studies showed that OA symptoms improved after administration of all MSCs. Also, PDGF-MSCs significantly improved limb function and reduced pain (P < 0.05). The results of the radiographic assessment and serum MMP-13 levels did not vary significantly compared to those of the control. 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.

Preparation of Stretchable Nanofibrous Sheets with Sacrificial Coaxial Electrospinning for Treatment of Traumatic Muscle Injury.

Traumatic muscle injury with massive loss of muscle volume requires intramuscular implantation of proper scaffolds for fast and successful recovery. Although many artificial scaffolds effectively accelerate formation and maturation of myotubes, limited studies are showing the therapeutic effect of artificial scaffolds in animal models with massive muscle injury. In this study, improved myotube differentiation is approved on stepwise stretched gelatin nanofibers and applied to damaged muscle recovery in an animal model. The gelatin nanofibers are fabricated by a two-step process composed of co-axial electrospinning of poly(ɛ-caprolactone) and gelatin and subsequent removal of the outer shells. When stepwise stretching is applied to the myoblasts on gelatin nanofibers for five days, enhanced myotube formation and polarized elongation are observed. Animal models with volumetric loss at quadriceps femoris muscles (>50%) are transplanted with the myotubes cultivated on thin and flexible gelatin nanofiber. Treated animals more efficiently recover exercising functions of the leg when myotubes and the gelatin nanofiber are co-implanted at the injury sites. This result suggests that mechanically stimulated myotubes on gelatin nanofiber is therapeutically feasible for the robust recovery of volumetric muscle loss.

Comparison of Hematological and Biochemical Results Derived from Arterial and Venous Blood Samples in Post-Anesthetic Dogs.

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 < 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.

Intravenous Administration of Heat Shock-Treated MSCs Can Improve Neuroprotection and Neuroregeneration in Canine Spinal Cord Injury Model.

Transplantation of mesenchymal stem cells (MSCs) is a promising treatment for spinal cord injury (SCI). However, many transplanted cells die within a few days, eventually limiting the efficacy of cellular therapy. To overcome this problem, we focused on the potential of heat shock (HS) proteins in facilitating recovery from cell damage and protecting against cytotoxicity. PCR results showed that the expression of neurotrophic factor, anti-inflammatory, stemness, and homing genes increased in HS-treated MSCs. We investigated whether HS-treated MSCs could promote recovery of hindlimb function in an acute canine SCI model. We compared the effects of intravenous transplantation with (i) lactated Ringer's solution as a control, (ii) green fluorescent protein-expressing MSCs (MSCs-GFP), and (iii) GFP-expressing and HS-treated MSCs (MSCs-GFP-HS). Spinal cords were harvested at four weeks and used for Western blot and histopathological analyses. The MSCs-GFP-HS group showed significant improvements in hindlimb function from weeks 3 and 4 compared with the other groups. This group also showed higher expression of neural markers, fewer intervening fibrotic changes, and pronounced myelination. These results suggest that induction of an HS response in MSCs could promote neural sparing. In conclusion, transplantation of HS-treated MSCs could improve neuroprotection and neuroregeneration in acute SCI.