Evaluation of canine adipose-derived mesenchymal stem cells for neurological functional recovery in a rat model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a common condition in veterinary medicine that is difficult to manage.Veterinary regenerative therapy based on adipose mesenchymal stem cells seem to be an effective strategy for the treatment of traumatic brain injury. In this study, we evaluated therapeutic efficacy of canine Adipose-derived mesenchymal stem cells (AD-MSCs)in a rat TBI model, in terms of improved nerve function and anti-neuroinflammation. RESULTS: Canine AD-MSCs promoted neural functional recovery, reduced neuronal apoptosis, and inhibited the activation of microglia and astrocytes in TBI rats. According to the results in vivo, we further investigated the regulatory mechanism of AD-MSCs on activated microglia by co-culture in vitro. Finally, we found that canine AD-MSCs promoted their polarization to the M2 phenotype, and inhibited their polarization to the M1 phenotype. What's more, AD-MSCs could reduce the migration, proliferation and Inflammatory cytokines of activated microglia, which is able to inhibit inflammation in the central system. CONCLUSIONS: Collectively, the present study demonstrates that transplantation of canine AD-MSCs can promote functional recovery in TBI rats via inhibition of neuronal apoptosis, glial cell activation and central system inflammation, thus providing a theoretical basis for canine AD-MSCs therapy for TBI in veterinary clinic.

Enhancement of the solubility and oral bioavailability of altrenogest through complexation with hydroxypropyl-ß-cyclodextrin.

Altrenogest (ALT), a synthetic progestogen, serves a critical role in estrus synchronization among animals like gilts and mares. However, its practical application in animal husbandry is hampered due to its poor solubility and limited oral bioavailability. To address this challenge, a solvent evaporation method was employed to create an inclusion complex of ALT with hydroxypropyl-ß-cyclodextrin (ALT/HP-ß-CD). The formation of this inclusion complex was confirmed by scanning electron microscopy, power X-ray diffraction, differential scanning calorimetry, Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and docking calculations. In addition, we further conducted pharmacokinetic investigation involving gilts, comparing ALT/HP-ß-CD inclusion complex to an ALT oral solution. The physicochemical characterization results unveiled a transformation of ALT's crystal morphology into an amorphous state, with ALT effectively entering the cavity of HP-ß-CD. Compared with ALT, the solubility of ALT/HP-ß-CD inclusion complex increased by 1026.51-fold, and its dissolution rate demonstrated significant improvement. Pharmacokinetic assessments further revealed that the oral bioavailability of ALT/HP-ß-CD inclusion complex surpassed that of the ALT oral solution, with a relative bioavailability of 114.08 %. In conclusion, complexation with HP-ß-CD represents a highly effective approach to improve both the solubility and oral bioavailability of ALT.

A Bioresorbable and Conductive Scaffold Integrating Silicon Membranes for Peripheral Nerve Regeneration.

Peripheral nerve injury represents one of the most common types of traumatic damage, severely impairing motor and sensory functions, and posttraumatic nerve regeneration remains a major challenge. Electrical cues are critical bioactive factors that promote nerve regrowth, and bioartificial scaffolds incorporating conductive materials to enhance the endogenous electrical field have been demonstrated to be effective. The utilization of fully biodegradable scaffolds can eliminate material residues, and circumvent the need for secondary retrieval procedures. Here, a fully bioresorbable and conductive nerve scaffold integrating N-type silicon (Si) membranes is proposed, which can deliver both structural guidance and electrical cues for the repair of nerve defects. The entire scaffold is fully biodegradable, and the introduction of N-type Si can significantly promote the proliferation and production of neurotrophic factors of Schwann cells and enhance the calcium activity of dorsal root ganglion (DRG) neurons. The conductive scaffolds enable accelerated nerve regeneration and motor functional recovery in rodents with sciatic nerve transection injuries. This work sheds light on the advancement of bioresorbable and electrically active materials to achieve desirable neural interfaces and improved therapeutic outcomes, offering essential strategies for regenerative medicine.

Deferoxamine Promotes Peripheral Nerve Regeneration by Enhancing Schwann Cell Function and Promoting Axon Regeneration of Dorsal Root Ganglion.

Deferoxamine (DFO) is a potent iron chelator for clinical treatment of various diseases. Recent studies have also shown its potential to promote vascular regeneration during peripheral nerve regeneration. However, the effect of DFO on the Schwann cell function and axon regeneration remains unclear. In this study, we investigated the effects of different concentrations of DFO on Schwann cell viability, proliferation, migration, expression of key functional genes, and axon regeneration of dorsal root ganglia (DRG) through a series of in vitro experiments. We found that DFO improves Schwann cell viability, proliferation, and migration in the early stages, with an optimal concentration of 25 µM. DFO also upregulates the expression of myelin-related genes and nerve growth-promoting factors in Schwann cells, while inhibiting the expression of Schwann cell dedifferentiation genes. Moreover, the appropriate concentration of DFO promotes axon regeneration in DRG. Our findings demonstrate that DFO, with suitable concentration and duration of action, can positively affect multiple stages of peripheral nerve regeneration, thereby improving the effectiveness of nerve injury repair. This study also enriches the theory of DFO promoting peripheral nerve regeneration and provides a basis for the design of sustained-release DFO nerve grafts.

Study on the preservation effects of the amputated forelimb by machine perfusion at physiological temperature.

PURPOSE: Ischemia and hypoxia are the main factors limiting limb replantation and transplantation. Static cold storage (SCS), a common preservation method for tissues and organs, can only prolong limb ischemia time to 4-6 h. The normothermic machine perfusion (NMP) is a promising method for the preservation of tissues and organs, which can extend the preservation time in vitro by providing continuous oxygen and nutrients. This study aimed to evaluate the difference in the efficacy of the 2 limb preservation methods. METHODS: The 6 forelimbs from beagle dogs were divided into 2 groups. In the SCS group (n = 3), the limbs were preserved in a sterile refrigerator at 4 °C for 24 h, and in the NMP group (n = 3), the perfusate prepared with autologous blood was used for the oxygenated machine perfusion at physiological temperature for 24 h, and the solution was changed every 6 h. The effects of limb storage were evaluated by weight gain, perfusate biochemical analysis, enzyme-linked immunosorbent assay (ELISA), and histological analysis. All statistical analyses and graphs were performed using GraphPad Prism 9.0 one-way or two-way analysis of variance (ANOVA). The p value of less than 0.05 was considered to indicate statistical significance. RESULTS: In the NMP group, the weight gained percentage was 11.72% ± 4.06%; the hypoxia-inducible factor-1α (HIF-1α) contents showed no significant changes; the shape of muscle fibers was normal; the gap between muscle fibers slightly increased, showing the intercellular distance of (30.19 ± 2.83) µm; and the vascular α-smooth muscle actin (α-SMA) contents were lower than those in the normal blood vessels. The creatine kinase level in the perfusate of the NMP group increased from the beginning of perfusion, decreased after each perfusate change, and remained stable at the end of perfusion showing a peak level of 4097.6 U/L. The lactate dehydrogenase level of the NMP group increased near the end of perfusion and reached the peak level of 374.4 U/L. In the SCS group, the percentage of weight gain was 0.18% ± 0.10%, and the contents of hypoxia-inducible factor-1α increased gradually and reached the maximum level of (164.85 ± 20.75) pg/mL at the end of the experiment. The muscle fibers lost their normal shape, and the gap between muscle fibers increased showing an intercellular distance of (41.66 ± 5.38) µm. The contents of vascular α-SMA were much lower in the SCS group as compared to normal blood vessels. CONCLUSIONS: NMP caused lesser muscle damage and contained more vascular α-SMA as compared to SCS. This study demonstrated that NMP of the amputated limb with perfusate solution based on autologous blood could maintain the physiological activities of the limb for at least 24 h.

Dual-bionic regenerative microenvironment for peripheral nerve repair.

Autologous nerve grafting serves is considered the gold standard treatment for peripheral nerve defects; however, limited availability and donor area destruction restrict its widespread clinical application. Although the performance of allogeneic decellularized nerve implants has been explored, challenges such as insufficient human donors have been a major drawback to its clinical use. Tissue-engineered neural regeneration materials have been developed over the years, and researchers have explored strategies to mimic the peripheral neural microenvironment during the design of nerve catheter grafts, namely the extracellular matrix (ECM), which includes mechanical, physical, and biochemical signals that support nerve regeneration. In this study, polycaprolactone/silk fibroin (PCL/SF)-aligned electrospun material was modified with ECM derived from human umbilical cord mesenchymal stem cells (hUMSCs), and a dual-bionic nerve regeneration material was successfully fabricated. The results indicated that the developed biomimetic material had excellent biological properties, providing sufficient anchorage for Schwann cells and subsequent axon regeneration and angiogenesis processes. Moreover, the dual-bionic material exerted a similar effect to that of autologous nerve transplantation in bridging peripheral nerve defects in rats. In conclusion, this study provides a new concept for designing neural regeneration materials, and the prepared dual-bionic repair materials have excellent auxiliary regenerative ability and further preclinical testing is warranted to evaluate its clinical application potential.

Remodeling Microenvironment for Endogenous Repair through Precise Modulation of Chondroitin Sulfate Proteoglycans Following Spinal Cord Injury.

The fluid-filled cystic cavity sealed by a dense scar developed following traumatic spinal cord injury (SCI) has been a major obstacle to neural regeneration and functional recovery. Here the transected lesion is bridged using a functional self-assembling peptide (F-SAP) hydrogel loaded with membrane-permeable intracellular sigma peptide (ISP) and intracellular LAR peptide (ILP), targeted at perturbing chondroitin sulfate proteoglycan (CSPG) inhibitory signaling. As compared to F-SAP hydrogel loaded with chondroitinase ABC, the F-SAP+ISP/ILP promotes a beneficial anti-inflammatory response via manipulation of microglia/macrophages infiltration and assembly of extracellular matrix (ECM) molecules into fibrotic matrix rather than scarring tissues. The remodeled ECM creates a permissive environment that supports axon regrowth and the formation of synaptic connections with neurons derived from endogenous neural stem cells. The remodeled networks contribute to functional recovery, as demonstrated by improved hind limb movements and electrophysiological properties. This work proposes a unique mechanism that ECM remodeling induced by CSPG-manipulation-based anti-inflammation can construct a permissive environment for neural regeneration, and shed light on the advancement of manipulation of cascading cellular and molecular events potential for endogenous repair of SCI.

Electrical stimulation accelerates Wallerian degeneration and promotes nerve regeneration after sciatic nerve injury.

Following peripheral nerve injury (PNI), Wallerian degeneration (WD) in the distal stump can generate a microenvironment favorable for nerve regeneration. Brief low-frequency electrical stimulation (ES) is an effective treatment for PNI, but the mechanism underlying its effect on WD remains unclear. Therefore, we hypothesized that ES could enhance nerve regeneration by accelerating WD. To verify this hypothesis, we used a rat model of sciatic nerve transection and provided ES at the distal stump of the injured nerve. The injured nerve was then evaluated after 1, 4, 7, 14 and 21 days post injury (dpi). The results showed that ES significantly promoted the degeneration and clearance of axons and myelin, and the dedifferentiation of Schwann cells. It upregulated the expression of BDNF and NGF and increased the number of monocytes and macrophages. Through transcriptome sequencing, we systematically investigated the effect of ES on the molecular processes involved in WD at 4 dpi. Evaluation of nerves bridged using silicone tubing after transection showed that ES accelerated early axonal and vascular regeneration while delaying gastrocnemius atrophy. These results demonstrate that ES promotes nerve regeneration by accelerating WD and upregulating the expression of neurotrophic factors.

Umbilical cord mesenchymal stem cells promote the repair of trochlear groove reconstruction in dogs.

Trochlear groove reconstruction (TGR) is a common treatment for patellar luxation (PL) in dogs. Nevertheless, the prognosis of TGR is poor due to the cartilage damage and secondary inflammation. To study the repair effect of canine umbilical cord mesenchymal stem cells (UC-MSCs) after TGR, 10 experimental dogs were given TGR surgery and then randomized into two groups: Treatment group (1 ml suspension allogeneic UC-MSCs (106 cells/kg) was injected into the cavum articulare on days 0, 7, and 14 after TGR); and the Model group (injected with 1 ml of physiological saline as negative control). The therapeutic effect of UC-MSCs was studied by blood routine examination, inflammatory factor index detection, double-blind knee score, histopathology, and computed tomography (CT) scans. The results showed that the total number of white blood cells and neutrophils in the model group were significantly higher than those in the treatment group on both 7 days and 21 days, postoperatively (P < 0.05); there were no significant changes in the levels of IL-6, MMP-13, and TGF-ß1 between the model group and the treatment group throughout the days of testing. The double-blind knee scores of the treatment group were significantly lower than the model group on 1st, 4th, and 5th days postoperatively (P < 0.05). The treatment group showed low-pain sensation, stable gait, and fast recovery of muscle strength in the knee score, and the wound healing of the treatment group returned to normal on the 5th day after surgery; CT scans and gross observation showed that the cartilage growth in the treatment group was faster than that in the model group. Histological observation of cases showed that fibro chondrocytes were predominantly found in the treatment group, and the distribution of chondrocytes was uneven, while the model group showed a large number of fibrous tissue hyperplasia, fissures, and unequal matrix staining. Intra-articular injection of UC-MSCs after TGR has the effect of relieving pain and promoting the repair of bone defects, making the operative limb recover function earlier, making up for the deficiency of TGR, and improving the effect of PL treatment. Future studies should furthermore explore the dose and frequency of therapy based on the multiple advantages of UC-MSCs and the mechanism of cartilage repair in dogs.

Immunomodulatory effects of mesenchymal stem cells in peripheral nerve injury.

Various immune cells and cytokines are present in the aftermath of peripheral nerve injuries (PNI), and coordination of the local inflammatory response is of great significance for the recovery of PNI. Mesenchymal stem cells (MSCs) exhibit immunosuppressive and anti-inflammatory abilities which can accelerate tissue regeneration and attenuate inflammation, but the role of MSCs in the regulation of the local inflammatory microenvironment after PNI has not been widely studied. Here, we summarize the known interactions between MSCs, immune cells, and inflammatory cytokines following PNI with a focus on the immunosuppressive role of MSCs. We also discuss the immunomodulatory potential of MSC-derived extracellular vesicles as a new cell-free treatment for PNI.

Protegrin-1 inhibits porcine ovarian granulosa cell apoptosis from H2O2-induced oxidative stress via the PERK/eIF2α/CHOP signaling pathway in vitro.

In mammals, oxidative stress-induced apoptosis of granulosa cells is one of the major causes of follicular atresia, affecting ovarian physiological function. Protegrin-1 (PG-1) is an antimicrobial peptide with effective antimicrobial activity, immunomodulatory function, and porcine growth-promoting effects. PG-1 has been detected in porcine ovaries follicles. This study aimed to investigate the effect of PG-1 on oxidative stress-induced apoptosis of porcine ovarian granulosa cells and the underlying molecular mechanism. Granulosa cells were obtained from porcine follicles and treated with H2O2 to establish the oxidative stress model, and then treated with or without PG-1 (10 µg/mL). PG-1 significantly suppressed H2O2-induced apoptosis in granulosa cells after 24 h of treatment. Furthermore, these results revealed that PG-1 increased the mRNA and protein expression of anti-apoptotic B cell lymphoma/leukemia 2 (BCL2) and the BCL2/Bcl-2-associated X protein (BAX) ratio while decreasing the expression of pro-apoptotic BAX and active caspase-3. Using Western blot analysis, it was found that PG-1 decreased the phosphorylation of RNA-like endoplasmic reticulum kinase (PERK) and the α-subunit of eukaryotic initiation factor 2 (eIF2α) as well as the protein expression level of CCAAT enhancer-binding protein homologous protein (CHOP), all of which were increased by H2O2. Moreover, inhibitors against PERK and phospho-eIF2ɑ both suppressed the H2O2-induced granulosa cells apoptosis and enhanced the anti-apoptosis effect of PG-1. Taken together, our findings demonstrated that PG-1 inhibited porcine ovarian granulosa cell apoptosis from oxidative stress via the PERK/eIF2α/CHOP signaling pathway in vitro, which suggests the novel regulatory function of the antimicrobial peptide in the ovary.

Isolation and identification of exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells.

BACKGROUND: Exosomes, internal proteins, lipids, and nucleic acids coated by phospholipid bilayer membranes, are one type of small extracellular vesicles, which can mediate cell-cell communication. In recent years, exosomes have gained considerable scientific interest due to their widely applied prospect in the diagnosis and therapeutics of human and animal diseases. In this study, we describe for the first time a feasible method designed to isolate and characterize exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells. RESULTS: Exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells were successfully isolated by differential centrifugation. Quantification and sizing of exosomes were assessed by transmission electron microscopy, flow nano analysis and western blotting. Detected particles showed the normal size (30-100 nm) and morphology described for exosomes, as well as presence of the transmembrane protein (TSG101, CD9, CD63, and CD81) known as exosomal marker. CONCLUSIONS: The results suggest that differential centrifugation is a feasible method for isolation of exosomes from different types of feline samples. Moreover, these exosomes can be used to further diagnosis and therapeutics in veterinary pre-clinical and clinical studies.

Manufacturing and banking canine adipose-derived mesenchymal stem cells for veterinary clinical application.

BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. RESULTS: After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. AD-MSCs presented normal karyotype, and did not form in vivo tumors. No adverse events were noted in the case treated with intravenously AD-MSCs. CONCLUSIONS: Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

Engineering Microenvironment for Endogenous Neural Regeneration after Spinal Cord Injury by Reassembling Extracellular Matrix.

The formation of a fluid-filled cystic cavity after spinal cord injury (SCI) is a major obstacle for neural regeneration. In this study, the post-SCI cavity was bridged by a functional self-assembling peptide (F-SAP) nanofiber hydrogel coupled with growth factor "cocktail". A sustained release of growth factors was achieved by carefully tailoring the physical hindrances and charge-induced interactions between the growth factors and the peptide nanofibers. Such an engineering microenvironment elicited axon regeneration, as determined by tracing of the descending pathway in the dorsal columns and immunochemical detection of regenerating axons beyond the lesion. Furthermore, the dynamic spatiotemporal activation line of endogenous NSCs (eNSCs) after severe SCI was thoroughly investigated. The results indicated that the growth factor-coupled F-SAP greatly facilitated eNSC proliferation, neuronal differentiation, maturation, myelination, and more importantly, the formation of interconnection with severed descending corticospinal tracts. The robust endogenous neurogenesis essentially led to the recovery of locomotion and electrophysiological properties. In conclusion, the growth factor-coupled F-SAP nanofiber hydrogel elucidated the therapeutic effect of eliciting endogenous neurogenesis by locally reassembling an extracellular matrix.

A Comparative Study of Biological Characteristics and Transcriptome Profiles of Mesenchymal Stem Cells from Different Canine Tissues.

Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy. Comparing the biological and transcriptome gene characteristics of MSCs from different sources provides an important basis for the screening of clinically used cells. The main purpose of this experiment was to establish methods for the isolation and culture of MSCs from five different canine sources, including adipose tissue, bone marrow, umbilical cord, amniotic membrane, and placenta, and compare biological and transcriptome characteristics of MSCs, in order to provide a basis for the clinical application of canine MSCs. MSCs were isolated from Chinese pastoral dogs, and the following experiments were performed: (1) the third, sixth, and ninth generations of cells were counted, respectively, and a growth curve was plotted to calculate the MSC population doubling time; (2) the expression of CD34 and CD44 surface markers was studied by immunofluorescence; (3) the third generation of cells were used for osteogenetic and adipogenic differentiation experiments; and (4) MSC transcriptome profiles were performed using RNA sequencing. All of the five types of MSCs showed fibroblast-like adherent growth. The cell surface expressed CD44 instead of CD34; the third-generation MSCs had the highest proliferative activity. The average population doubling time of adipose mesenchymal stem cells (AD-MSCs), placenta mesenchymal stem cells (P-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord mesenchymal stem cells (UC-MSCs), and amniotic mesenchymal stem cells (AM-MSCs) were 15.8 h, 21.2 h, 26.2 h, 35 h, and 41.9 h, respectively. All five types of MSCs could be induced to differentiate into adipocytes and osteoblasts in vitro, with lipid droplets appearing after 8 days and bone formation occurring 5 days after AD-MSC induction. However, the multilineage differentiation for the remaining of MSCs was longer compared to that of the AD-MSCs. The MSC transcriptome profiles showed that AD-MSC and BM-MSCs had the highest homology, while P-MSCs were significantly different compared to the other four types of MSCs. All the isolated MSCs had the main biological characteristics of MSCs. AD-MSCs had the shortest time for proliferation, adipogenesis, and osteogenic differentiation.

Advances in injectable self-healing biomedical hydrogels.

In recent years, implantable biomaterials have attracted significant interest owing to their potentials for use in the therapy of physical defects and traumas. Among the implantable biomaterials, hydrogels have received increasing attention for their tunable structures and good rheological behavior. However, the mechanical failures of traditional gel materials during normal operation remain a serious issue. To overcome this problem, hydrogel materials with self-healing and injectable abilities have been developed, with their potential for autonomous self-recovery and minimally invasive implantation. In this paper, the progress of injectable self-healing hydrogels is presented by combining developments in the fundamental knowledge of polymer designs and discussions on the practical biomedical applications of the materials. The mechanisms of different types of self-healing hydrogels are introduced first and their performances are then discussed, followed by a review of the self-healing hydrogels with injectability. The applications of the injectable self-healing hydrogels are discussed in the final section. STATEMENT OF SIGNIFICANCE: This paper provides an overview of the progress of a smart material, injectable self-healing hydrogel, during the past ten years and mainly focuses on its recent development. This paper presents developments in the fundamental knowledge in polymer designs and discussions on the practical biomedical application of the materials, which sheds more light on the advancement of injectable self-healing hydrogels. This paper should be of interest to the readers who are curious about the advances of injectable self-healing hydrogels.

Bionanotube/Poly(3,4-ethylenedioxythiophene) Nanohybrid as an Electrode for the Neural Interface and Dopamine Sensor.

Poly(3,4-ethylene dioxythiophene) (PEDOT) is a promising conductive material widely used for interfacing with tissues in biomedical fields because of its unique properties. However, obtaining high charge injection capability and high stability remains challenging. In this study, pristine carbon nanotubes (CNTs) modified by dopamine (DA) self-polymerization on the surface polydopamine (PDA@CNTs) were utilized as dopants of PEDOT to prepare hybrid films through electrochemical deposition on the indium tin oxide (ITO) electrode. The PDA@CNTs-PEDOT film of the nanotube network topography exhibited excellent stability and strong adhesion to the ITO substrate compared with PEDOT and PEDOT/ p-toulene sulfonate. The PDA@CNTs-PEDOT-coated ITO electrodes demonstrated lower impedance and enhanced charge storage capacity than the bare ITO. When applying exogenous electrical stimulation (ES), robust long neurites sprouted from the dorsal root ganglion (DRG) neurons cultured on the PDA@CNTs-PEDOT film. Moreover, ES promoted Schwann cell migration out from the DRG spheres and enhanced myelination. The PDA@CNTs-PEDOT film served as an excellent electrochemical sensor for the detection of DA in the presence of biomolecule interferences. Results would shed light into the advancement of conducting nanohybrids for applications in the multifunctional bioelectrode in neuroscience.

Assembly Pathway Selection of Designer Self-Assembling Peptide and Fabrication of Hierarchical Scaffolds for Neural Regeneration.

The self-assembling peptide (SAP) RADA 16-I has been modified with various functional motifs to improve its performances in biomedical applications. Nevertheless, the assembly mechanisms of designer functional RADA 16-I SAPs (F-SAPs) have not been clearly illustrated. The main problem is the difficulty in preparing a completely molecular aqueous solution of F-SAP. In the current study, we demonstrated that different procedures for preparing the F-SAP solution could result in the formation of different conformations and consequently micro/macroscopic morphologies. F-SAP was molecularly dissolved in an appropriate solvent, such as hexafluoroisopropanol (HFIP), as evidenced by random coil conformation characterized by circular dichroism spectroscopy and morphologies under transmission electron microscopy. The monomers were induced into monolayers when the F-SAP solution in HFIP was adsorbed on mica as observed by atomic force microscopy. However, nanoscaled filaments containing ß-sheets dominated in the F-SAP aqueous solution, in which case water acted as a poor solvent of F-SAP. Furthermore, the results of molecular dynamics simulation implicated that water facilitated F-SAP aggregation, whereas HFIP inhibited it. The ß-sheet assemblies formed in water exhibited a high kinetic stability and did not disassemble rapidly after the addition of HFIP. Our study indicated that selecting the right assembly pathway of F-SAP required for targeted functions, for example, delivery of hydrophobic drugs in aqueous conditions, could be achieved by optimizing the preparation protocol in addition to molecular design. Moreover, hierarchical scaffolds mimicking the natural extracellular matrix could be fabricated by the direct electrospinning of F-SAP molecular solution in HFIP and biodegradable polymer for applications in neural regeneration by promoting neural differentiation, neurite outgrowth, and synapse formation.

Evaluation of the Curative Effect of Umbilical Cord Mesenchymal Stem Cell Therapy for Knee Arthritis in Dogs Using Imaging Technology.

OBJECTIVE: The aim of this study was to assess the efficacy of canine umbilical cord mesenchymal stem cells (UC-MSCs) on the treatment of knee osteoarthritis in dogs. METHODS: Eight dogs were evenly assigned to two groups. The canine model of knee osteoarthritis was established by surgical manipulation of knee articular cartilage on these eight dogs. UC-MSCs were isolated from umbilical cord Wharton's jelly by 0.1% type collagenase I and identified by immunofluorescence staining and adipogenic and osteogenic differentiation in vitro. A suspension of allogeneic UC-MSCs (1 × 106) and an equal amount of physiological saline was injected into the cavitas articularis in the treated and untreated control groups, respectively, on days 1 and 3 posttreatment. The structure of the canine knee joint was observed by magnetic resonance imaging (MRI), B-mode ultrasonography, and X-ray imaging at the 3rd, 7th, 14th, and 28th days after treatment. Concurrently, the levels of IL-6, IL-7, and TNF-α in the blood of the examined dogs were measured. Moreover, the recovery of cartilage and patella surface in the treated group and untreated group was compared using a scanning electron microscope (SEM) after a 35-day treatment. RESULTS: Results revealed that the isolated cells were UC-MSCs, because they were positive for CD44 and negative for CD34 surface markers, and the cells were differentiated into adipocytes and osteoblasts. Imaging technology showed that as treatment time increased, the high signal in the MRI T2-weighted images decreased, the echo-free space in B ultrasonography images disappeared basically, and the continuous linear hypoechoic region at the trochlear sulcus thickened. On X-ray images, the serrate defect at the ventral cortex of the patella improved, and the low-density gap of the ventral patella and trochlear crest gradually increased in the treated group. On the contrary, the high signal in the MRI T2-weighted images and the echo-free space in B ultrasonography images still increased after a 14-day treatment in the untreated control group, and the linear hypoechoic region was discontinuous. On the X-ray images, there was no improvement in the serrate defect of the ventral cortex of the patella. Results for inflammatory factors showed that the blood levels of IL-6, IL-7, and TNF-α of the untreated control group were significantly higher than those of the treated group (P < 0.05) 7-14 days posttreatment. The result of SEM showed that the cartilage neogenesis in the treated group had visible neonatal tissue and more irregular arrangement of new tissue fibers than that of the untreated control group. Furthermore, more vacuoles but without collagen fibers were observed in the cartilage of the untreated control group, and the thickness of the neogenetic cartilage in the treated group (65.13 ± 5.29, 65.30 ± 5.83) and the untreated control group (34.27 ± 5.42) showed a significant difference (P < 0.01). CONCLUSION: Significantly higher improvement in cartilage neogenesis and recovery was observed in the treated group compared to the untreated control group. The joint fluid and the inflammatory response in the treated group decreased. Moreover, improved recovery in the neogenetic cartilage, damaged skin fascia, and muscle tissue around the joints was more significant in the treated group than in the untreated control group. In conclusion, canine UC-MSCs promote the repair of cartilage and patella injury in osteoarthritis, improve the healing of the surrounding tissues, and reduce the inflammatory response.