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.

Pilot study on the feasibility of shape memory alloy implantation for Vancouver type B1 periprosthetic femoral fractures in a canine model: a step toward advancing treatment modalities.

BACKGROUND: Cerclage wiring is commonly used for treating fractures; however, it has several limitations, including mechanical weakness, decreased blood circulation, and technical complexity. In this study, we developed an implant using a shape memory alloy (SMA) and tested its efficacy in treating Vancouver type B1 (VB1) periprosthetic femoral fractures (PFFs) in a canine model. METHODS: The mid-diaphyseal fracture models underwent reduction via the SMA plate (SMA group) or the cerclage cable plate (cable group) method in randomly selected pelvic limbs. An intraoperative evaluation was conducted to assess the surgical time and difficulty related to implant fitting. Clinical assessments, radiography, microcomputed tomography (micro-CT), histological analysis, positron emission tomography (PET)/CT, and galvanic corrosion analysis were conducted for 52 weeks to evaluate bone healing and blood perfusion. RESULTS: The results for bone healing and blood perfusion were not significantly different between the groups (p > 0.05). In addition, no evidence of galvanic corrosion was present in any of the implants. However, the median surgical time was 75 min (range, 53-82 min) for the SMA group and 126 min (range, 120-171 min) for the cable group, which was a statistically significant difference (p = 0.0286). CONCLUSIONS: This study assessed the ability of a newly developed shape memory alloy (SMA) to treat VB1 periprosthetic femoral fractures (PFFs) in canines for over a 52-week period and revealed outcomes comparable to those of traditional methods in terms of bone healing and mechanical stability. Despite the lower surgical complexity and potential time-saving benefits of this treatment, further research is needed to confirm its efficacy.

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.

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.

New clinical application of three-dimensional-printed polycaprolactone/ß-tricalcium phosphate scaffold as an alternative to allograft bone for limb-sparing surgery in a dog with distal radial osteosarcoma.

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/ß-tricalcium phosphate (PCL/ß-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/ß-TCP scaffold may be an effective alternative to cortical allograft in limb-sparing surgery for bone tumors.

Micro and ultrastructural changes monitoring during decellularization for the generation of a biocompatible liver.

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.

DNA aptamer immobilized hydroxyapatite for enhancing angiogenesis and bone regeneration.

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.

Clinical Application of Bone Morphogenetic Protein-2 Microcarriers Fabricated by the Cryopolymerization of Gelatin Methacrylate for the Treatment of Radial Fracture in Two Dogs.

Bone morphogenetic protein-2 (BMP-2) effectively induces bone healing. However, the efficacy of BMP-2 relies heavily on its delivery vehicle because of its short half-life. We utilized a microcarrier fabricated by the cryopolymerization of gelatin methacrylate (cryoGelMA) infused with bone morphogenetic protein-2 (cryoGelMA-BMP-2) for the sustained and localized release of growth factors. Two dogs with radius and ulnar fractures were treated with implanted cryoGelMA-BMP-2 to accelerate bone healing. The cases were followed up for 6 months and 2 months after surgery, respectively. Distinctive healing processes were observed. The operated limb regained its premorbid function, the fracture line disappeared, and the gait was functionally stable. Implantation of cryoGelMA-BMP-2 resulted in the successful healing of bone fractures.

Repair of rabbit radial bone defects using bone morphogenetic protein-2 combined with 3D porous silk fibroin/ß-tricalcium phosphate hybrid scaffolds.

Our study aimed to investigate the effect of bone morphogenetic protein-2 (BMP-2) bound to silk fibroin and ß-tricalcium phosphate (SF/ß-TCP) hybrid on the healing of critical-size radial defects in rabbits. A 15-mm critical-size defect was induced at mid-diaphysis in the left radius of 20 New Zealand white rabbits (average age, 3.5 months; weight, 2.5-3.0 kg). The animals were randomized into Group 1 (SF/ß-TCP combined with BMP-2), Group 2 (SF/ß-TCP alone), and Group 3 (nothing implanted). Radiographs were obtained every 2 weeks and euthanasia was performed after 8 weeks for visual, radiological, micro-computed tomography (micro-CT), and histological studies. Eight weeks after implantation (SF/ß-TCP combined with BMP-2), radiographs showed that new bone formed on the surface of the implant and had bridged the defect in Group 1. Micro-CT imaging also confirmed the formation of new bone around the implant, and the newly formed bone was quantified. Histological examination revealed newly formed bone in the implanted area. Meanwhile, there was no formation of new bone in Group 3. Among the groups, most active formation of new bones was found in Group 1, while there was no difference between Group 2 and Group 3. Based on these results, we concluded that BMP-2-SF/ß-TCP showed significant improvement in healing of critical-size defects. Therefore, the combination of BMP-2 and SF/ß-TCP would be useful in the field of bone tissue engineering.

Biocompatibility and hemocompatibility of efficiently decellularized whole porcine kidney for tissue engineering.

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.

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.

A Case of endoscopic retrieval of a long bamboo stick from a Humboldt penguin (Spheniscus humboldti).

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.

Umbilical-cord-blood-derived mesenchymal stem cells seeded onto fibronectin-immobilized polycaprolactone nanofiber improve cardiac function.

Stem cells seeded onto biofunctional materials have greater potency for therapeutic applications. We investigated whether umbilical-cord-blood-derived mesenchymal stem cell (UCB-MSC)-seeded fibronectin (FN)-immobilized polycaprolactone (PCL) nanofibers could improve cardiac function and inhibit left ventricle (LV) remodeling in a rat model of myocardial infarction (MI). Aligned nanofibers were uniformly coated with poly(glycidyl methacrylate) by initiated chemical vapor deposition followed by covalent immobilization of FN proteins. The degree of cell elongation and adhesion efficacy were improved by FN immobilization. Furthermore, genes related to angiogenesis and mesenchymal differentiations were up-regulated in the FN-immobilized PCL nanofibers in comparison to control PCL nanofibers in vitro. 4 weeks after the transplantation in the rat MI model, the echocardiogram showed that the UCB-MSC-seeded FN-immobilized PCL nanofiber group increased LV ejection fraction and fraction shortening as compared to the non-treated control and acellular FN-immobilized PCL nanofiber groups. Histological analysis indicated that the implantation of UCB-MSCs with FN-immobilized PCL nanofibers induced a decrease in MI size and fibrosis, and an increase in scar thickness. This study indicates that FN-immobilized biofunctional PCL nanofibers could be an effective carrier for UCB-MSC transplantation for the treatment of MI.

Comparing the osteogenic potential of canine mesenchymal stem cells derived from adipose tissues, bone marrow, umbilical cord blood, and Wharton's jelly for treating bone defects.

Alternative sources of mesenchymal stem cells (MSCs) for replacing bone marrow (BM) have been extensively investigated in the field of bone tissue engineering. The purpose of this study was to compare the osteogenic potential of canine MSCs derived from adipose tissue (AT), BM, umbilical cord blood (UCB), and Wharton's jelly (WJ) using in vitro culture techniques and in vivo orthotopic implantation assays. After canine MSCs were isolated from various tissues, the proliferation and osteogenic potential along with vascular endothelial growth factor (VEGF) production were measured and compared in vitro. For the in vivo assay, MSCs derived from each type of tissue were mixed with ß-tricalcium phosphate and implanted into segmental bone defects in dogs. Among the different types of MSCs, AT-MSCs had a higher proliferation potential and BM-MSCs produced the most VEGF. AT-MSCs and UCB-MSCs showed greater in vitro osteogenic potential compared to the other cells. Radiographic and histological analyses showed that all tested MSCs had similar osteogenic capacities, and the level of new bone formation was much higher with implants containing MSCs than cell-free implants. These results indicate that AT-MSCs, UCB-MSCs, and WJ-MSCs can potentially be used in place of BM-MSCs for clinical bone engineering procedures.