Cell-permeable bone morphogenetic protein 2 facilitates bone regeneration by promoting osteogenesis.

The use of the FDA-approved osteoinductive growth factor BMP2 is widespread for bone regeneration. However, its clinical application has been hindered by limitations in cell permeability and a short half-life in circulation. To address this issue, we have developed a modified version of BMP2, referred to as Cell Permeable (CP)-BMP2, which possesses improved cell permeability. CP-BMP2 incorporates an advanced macromolecular transduction domain (aMTD) to facilitate transfer across the plasma membrane, a solubilization domain, and recombinant human BMP2. Compared to traditional rhBMP2, CP-BMP2 exhibits enhanced cell permeability, solubility, and bioavailability, and activates Smad phosphorylation through binding to BMP receptor 2. The effectiveness of CP-BMP2 was evaluated in three animal studies focusing on bone regeneration. In the initial study, mice and rabbits with critical-size calvarial defects received subcutaneous (SC) injections of CP-BMP2 and rhBMP2 (7.5 mg/kg, 3 injections per week for 8 weeks).Following 8 weeks of administration, CP-BMP2 demonstrated a remarkable 65 % increase in bone formation in mice when compared to both the vehicle and rhBMP2. Moreover, rabbits exhibited faster bone formation, characterized by a filling pattern originating from the center. In a subsequent study involving injured horses, hind limb bones treated with CP-BMP2 exhibited an 85 % higher bone regeneration rate, as evidenced by Micro-CT results, in contrast to horses treated with the vehicle or rhBMP2 (administered at 150 µg/defect, subcutaneously, once a week for 8 weeks, without a scaffold). These results underscore the potential of CP-BMP2 to facilitate rapid and effective healing. No noticeable adverse effects, such as ectopic bone formation, were observed in any of the studies. Overall, our findings demonstrate that CP-BMP2 holds therapeutic potential as a novel and effective osteogenic agent.

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.

In vitro effects of monophosphoryl lipid A and Poly I:C combination on equine cells.

BACKGROUND: Toll-like receptor (TLR) agonists have been used as adjuvants to modulate immune responses in both animals and humans. OBJECTIVES: The objective of this study was to evaluate the combined effects of the TLR 4 agonist monophosphoryl lipid A (MPL) and the TLR 3 agonist polyinosinic:polycytidylic acid (Poly I:C) on equine peripheral blood mononuclear cells (PBMCs), monocyte-derived dendritic cells (MoDCs), and bone marrow-derived mesenchymal stromal cells (BM-MSCs). METHODS: The PBMCs, MoDCs, and BM-MSCs collected from three mixed breed horses were treated with MPL, Poly I:C, and their combination. The mRNA expression of interferon gamma (IFN-γ), interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-12p40, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1) was determined using real-time polymerase chain reaction. RESULTS: The combination of MPL and Poly I:C significantly upregulated immunomodulatory responses in equine cells/ without cytotoxicity. The combination induced greater mRNA expression of pro-inflammatory cytokines IFN-γ and IL-6 than MPL or Poly I:C stimulation alone in PBMCs. In addition, the combination induced significantly higher mRNA expression of IL-1ß, IL-6, and IL-12p40 in MoDCs, and IL-8, MCP-1, and VEGF in BM-MSCs compared to stimulation with a single TLR agonist. CONCLUSIONS: The combination of MPL and Poly I:C can be used as a potential adjuvant candidate for vaccines to aid in preventing infectious diseases in horses.

Impact of varying food hardness on mastication/swallowing.

The purpose of this study was to analyze mastication and swallowing in the elderly and confirm the association with dysphagia characteristics. A questionnaire was developed to evaluate the masticating and swallowing functions of the elderly. Mastication was analyzed using electromyography, and tongue/lip pressures were measured using Iowa Oral Performance Instrument. The results of the questionnaire showed that statistical difference in the number of teeth between the group without and with, decreased ability to swallow, and there was a correlation with lip pressure. Additionally, the higher number of teeth, the higher muscle activity, and there is a positive correlation between the number of chews and the lip pressure. Consequently, our findings suggested oral health parameters are closely associated with mastication/swallowing ability. Finally, based on the results obtained for different foods tested, we suggested that texture-modified foods are necessary to enhance swallowing ability.

Evaluation of concurrent vaccinations with recombinant canarypox equine influenza virus and inactivated equine herpesvirus vaccines.

Despite vaccination, equine influenza virus (EIV) and equine herpesvirus (EHV) infections still cause highly contagious respiratory diseases in horses. Recently, concurrent vaccination with EIV and EHV was suggested as a new approach; however, there have been no reports of concurrent vaccination with recombinant canarypox EIV and inactivated EHV vaccines. In this study, we aimed to compare the EIV-specific immune responses induced by concurrent administrations of a recombinant canarypox EIV vaccine and an inactivated bivalent EHV vaccine with those induced by a single recombinant canarypox EIV vaccine in experimental horse and mouse models. Serum and peripheral blood mononuclear cells (PBMCs) were collected from immunized animals after vaccination. EIV-specific serum antibody levels, serum hemagglutinin inhibition (HI) titers, and interferon-gamma (IFN-γ) levels were measured by enzyme-linked immunosorbent assay, HI assay, and quantitative polymerase chain reaction, respectively. Concurrent EIV and EHV vaccine administration significantly increased IFN-γ production, without compromising humoral responses. Our data demonstrate that concurrent vaccination with EIV and EHV vaccines can enhance EIV-specific cellular responses in horses.

Computed tomographic features of the temporomandibular joint in 10 Jeju horses.

BACKGROUND: The equine temporomandibular joint (TMJ) has a complex anatomical structure that makes diagnosis of TMJ disorders difficult. Computed tomography (CT) is now available in equine medicine; hence, TMJ evaluation has become more convenient. OBJECTIVES: The objectives of this study were to describe the CT features of the TMJ in Jeju horses and to compare these features with those of Thoroughbreds. METHODS: In this report, the TMJs of 10 Jeju horses (mean age: 4.5 ± 1.9 yr; mean body weight: 282.6 ± 40.3 kg) and 6 Thoroughbreds (mean age: 7.3 ± 1.6 yr; mean body weight: 479.7 ± 44.0 kg) were examined using CT. After CT scanning, the Hounsfield units (HU) and height to width ratio (H:W) of the mandibular condyle were measured. RESULTS: The mean H:W in Jeju horses was significantly lower than that in Thoroughbreds. The mean HU in Jeju horses was lower than that in Thoroughbreds; however, the difference was not significant. The most frequent CT finding was an irregular medial margin of the mandibular condyle in both breeds. CONCLUSIONS: In this study, the shape of the mandibular condyle in Jeju horses was flatter than that in Thoroughbreds. This report could be useful in evaluating the TMJ in Jeju horses. Moreover, CT could be a pragmatic choice for the examination of the TMJ in horses.

Therapeutic effects of platelet derived growth factor overexpressed-mesenchymal stromal cells and sheets in canine skin wound healing model.

Adipose-derived mesenchymal stromal cells (Ad-MSCs) have excellent potential for skin wound repair. Moreover, platelet-derived growth factor (PDGF) has strong wound healing properties. The purpose of the present study was to compare the healing effects of PDGF-overexpressing canine allogeneic Ad-MSCs (PDGF-MSCs) and their cell sheets (PDGF-CSs) as compared to unexpressed Ad-MSCs (U-MSCs) and their cell sheets (UCSs) in a cutaneous wound healing model induced upon dogs. In in vitro study, the expression of immunomodulatory and growth factors was assessed by qRT-PCR. In in vivo study, cells and sheets were transplanted into a square-shaped full-thickness (1.5×1.5 cm) skin defect model created in 12 dogs. After 5 and 10 days, wounds were harvested and evaluated macroscopically and histopathologically. The qRT-PCR results showed that the PDGF-B gene was significantly upregulated (p<0.05) in PDGF-CS and PDGF-MSCs groups. Upon gross analysis of the wound, all stromal cells and their sheet groups showed accelerated (p<0.05) cutaneous wound healing compared to the negative control groups. As compared to U-MSCs and UCSs, the PDGF-MSCs showed significant epithelization on days 5 and 10 of healing, whereas PDGF-CSs showed improved epithelization only on day 10. In the granulation tissue analysis, PDGF-CSs and UCSs promoted more formation (p<0.05) of upper granulation tissue, collagen, and activated fibroblasts than PDGF-MSCs, and U-MSCs. Especially, the PDGF-CSs presented the highest formation and maturation of granulation tissue among all groups. All considered, PDGF overexpressed stromal cells or cells sheets can improve cutaneous wound healing in a canine model.

Therapeutic Effects of Intravenous Injection of Fresh and Frozen Thawed HO-1-Overexpressed Ad-MSCs in Dogs with Acute Spinal Cord Injury.

Owing to the antioxidant and anti-inflammatory functions of hemeoxygenase-1 (HO-1), HO-1-expressing canine adipose-derived mesenchymal stem cells (Ad-MSCs) could be efficacious in treating spinal cord injury (SCI). Further, frozen thawed HO-1 Ad-MSCs could be instantly available as an emergency treatment for SCI. We compared the effects of intravenous treatment with freshly cultured HO-1 Ad-MSCs (HO-1 MSCs), only green fluorescent protein-expressing Ad-MSCs (GFP MSCs), and frozen thawed HO-1 Ad-MSCs (FT-HO-1 MSCs) in dogs with acute SCI. For four weeks, dogs were evaluated for improvement in hind limb locomotion using a canine Basso Beattie Bresnahan (cBBB) score. Upon completion of the study, injured spinal cord segments were harvested and used for western blot and histopathological analyses. All cell types had migrated to the injured spinal cord segment. The group that received HO-1 MSCs showed significant improvement in the cBBB score within four weeks. This group also showed significantly higher expression of NF-M and reduced astrogliosis. There was reduced expression of proinflammatory cytokines (IL6, TNF-α, and IL-1ß) and increased expression of anti-inflammatory markers (IL-10, HO-1) in the HO-1 MSC group. Histopathological assessment revealed decreased fibrosis at the epicenter of the lesion and increased myelination in the HO-1 MSC group. Together, these data suggest that HO-1 MSCs could improve hind limb function by increasing the anti-inflammatory reaction, leading to neural sparing. Further, we found similar results between GFP MSCs and FT-HO-1 MSCs, which suggest that FT-HO-1 MSCs could be used as an emergency treatment for SCI.

Comparison of the methods for platelet rich plasma preparation in horses.

Platelet rich plasma (PRP) is popularly used in the horse industry to enhance regeneration of tissue injury that has limitation of blood supply. This study aimed to compare the methods for platelet rich plasma preparation since they has not been established yet. Blood was collected from six horses and platelets were concentrated by three different methods (2-step centrifugation, separated centrifugation and separated centrifugation using histopaque). Concentrated blood was analyzed using Advia hematology systems. In the result, separated centrifugation with histopaque showed the significantly lower number of red blood cells than other groups. The 2-step centrifugation showed the significantly higher number of white blood cells than other groups, while it contained the highest concentration of red blood cells among three groups. In the 2-step centrifugation, separated centrifugation and separated centrifugation with histopaque, platelets were concentrated 4.5, 5.3 and 5.6 times, respectively. And no significant difference of the platelet concentration between the three groups was found. This study demonstrated that separated centrifugation using histopaque was the best method for platelet rich plasma preparation because of the proper amount of platelets and the separation of red blood cells from platelet rich plasma.

Synthetic anion transporters that bear a terminal ethynyl group.

Cl(-) transporters that bear a terminal ethynyl group were synthesized; they consist of non-pyrrolic hydrogen bond motifs such as phenolic OH, amide NH, and triazole CH. The ethynyl group of these non-pyrrolic analogs plays an important role in chloride efflux and they exhibit no significant cytotoxic activity.