A Green Light-Activated Insulin Depot with Ultrafast In Vivo Efficiency in the Subcutaneous Space.

In this work, for the first time, we demonstrate light control of a therapeutic protein's release from a depot in the subcutaneous layer of the skin. The subcutaneous layer is a standard location for therapeutic protein depots due to its large size and ease of access, but prior attempts to utilize this space failed because insufficient light can reach this deeper layer. An analysis of existing biophysical literature suggested that an increase of photoactivation wavelength from 365 to 500 nm could allow an increase of depot irradiation in the subcutaneous by >100-fold. We therefore used a green light-activated thio-coumarin-based material and demonstrated robust release of a therapeutic, insulin, in response to skin illumination with an LED light source. We further demonstrated that this release is ultrafast, as fast or faster than any commercially used insulin, while maintaining the native insulin sequence. This release of insulin was then accompanied by a robust reduction in blood glucose, demonstrating the retention of bioactivity despite the synthetic processing required to generate the material. In addition, we observed that the material exhibits slow basal release of insulin, even in the absence of light, potentially through biochemical or photochemical unmasking of insulin. Thus, these materials can act much like the healthy pancreas does: releasing insulin at a slow basal rate and then, upon skin irradiation, releasing an ultrafast bolus of native insulin to reduce postprandial blood glucose excursions.

Hepatic enzymes and immunoinflammatory response to Bio-C Temp bioceramic intracanal medication implanted into the subcutaneous tissue of rats.

Our purpose was to evaluate the biocompatibility and hepatotoxicity of a new bioceramic intracanal medicament, Bio-C Temp (BIO). The biological properties of BIO were compared with calcium hydroxide-based intracanal medicament (Calen; CAL), used as gold pattern. Polyethylene tubes filled with BIO or CAL, and empty tubes (control group, CG) were implanted into subcutaneous tissue of rats. After 7, 15, 30 and 60 days, the samples were embedded in paraffin for morphological, quantitative and immunohistochemistry analyses. At 7 and 60 days, blood samples were collected for analysis of serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels. The data were submitted to two-way ANOVA and Tukey's test (p ≤ 0.05). No significant difference was detected in serum GOT and GPT levels among BIO, CAL and CG specimens. In all periods, BIO specimens exhibited lower number of inflammatory cells and immunoexpression of IL-6, a pro-inflammatory cytokine, than CAL specimens. The reduction of these parameters was accompanied by significant increase in the collagen content and in the immunoexpression of IL-10, a cytokine involved in the tissue repair, over time. Our findings indicate that Bio-C Temp is biocompatible and had no hepatotoxicity effect.

Graphene-Oxide Porous Biopolymer Hybrids Enhance In Vitro Osteogenic Differentiation and Promote Ectopic Osteogenesis In Vivo.

Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.

Weight-based cefuroxime dosing provides comparable orthopedic target tissue concentrations between weight groups - a microdialysis porcine study.

Antibiotic prophylaxis is a key element in prevention of surgical site infections. For the majority of orthopedic procedures, antibiotic administration follows fixed dosing regimens irrespective of weight. However, this may result in insufficient antibiotic target tissue concentrations and higher risk of surgical site infections in obese individuals. The aim of this study was to investigate the effect of weight-based cefuroxime dosing on plasma and target tissue concentrations. Eighteen female pigs were allocated into three groups differentiated by weight: 53-57 kg, 73-77 kg, and 93-97 kg. Microdialysis catheters were placed for continuous sampling in bone, muscle, and subcutaneous tissue during an 8h sampling interval. Blood samples were collected as reference. Cefuroxime was administered intravenously as a bolus according to weight (20 mg/kg). The primary endpoint was the time above the cefuroxime minimal inhibitory concentration for Staphylococcus aureus (T > MIC (4 µg/mL)). Comparable target tissue T > MICs (4 µg/mL) were found between weight groups. Mean T > MIC ranged between 116-137 min for plasma, 118-154 min for bone, 109-146 min for the skeletal muscle, and 117-165 min for subcutaneous tissue across the groups. Weight-based cefuroxime (20 mg/kg) dosing approach provides comparable perioperative plasma and target tissue T > MIC (4 µg/mL) in animals between 50-100 kg body weight, and thus a comparable prophylaxis of surgical site infections.

Beneficial Effects of High Intensity Interval Training and/or Linseed Oil Supplementation to Limit Obesity-Induced Oxidative Stress in High Fat Diet-Fed Rats.

High-intensity interval training (HIIT) and linseed oil (LO) supplementation are effective strategies to reduce obesity-induced oxidative stress. Our aim was to determine whether the HIIT + LO combination prevents obesity-induced oxidative stress in high fat diet (HFD)-fed rats. HFD-fed 8-week-old, male, Wistar rats were subdivided in four groups: HFD, LO (2% of sunflower oil replaced with 2% of LO in the HFD), HIIT (4 days/week for 12 weeks), and HIIT + LO. Wistar rats fed a low-fat diet (LFD) were used as controls. Epididymal and subcutaneous adipose tissue, gastrocnemius muscle, liver, and plasma samples were collected to measure oxidative stress markers (AOPP, oxLDL), antioxidant (SOD, CAT, and GPx activities) and pro-oxidant (NOx and XO) enzyme activities. Compared with the LFD, the HFD altered the pro/antioxidant status in different tissues (increase of AOPP, oxLDL, SOD and catalase activities in plasma, and SOD activity increase in liver and decrease in adipose tissues) but not in gastrocnemius. LO upregulated CAT activity and decreased NOx in liver. HIIT alleviated HFD negative effects in liver by reducing SOD and NOx activities. Moreover, the HIIT + LO combination potentiated SOD activity upregulation in subcutaneous tissue. HIIT and LO supplementation have independent beneficial effects on the pro/antioxidant balance. Their association promotes SOD activity in subcutaneous adipose tissue.

Imparting multi-functionality to covalent organic framework nanoparticles by the dual-ligand assistant encapsulation strategy.

The potential applications of covalent organic frameworks (COFs) can be further developed by encapsulating functional nanoparticles within the frameworks. However, the synthesis of monodispersed core@shell structured COF nanocomposites without agglomeration remains a significant challenge. Herein, we present a versatile dual-ligand assistant strategy for interfacial growth of COFs on the functional nanoparticles with abundant physicochemical properties. Regardless of the composition, geometry or surface properties of the core, the obtained core@shell structured nanocomposites with controllable shell-thickness are very uniform without agglomeration. The derived bowl-shape, yolk@shell, core@satellites@shell nanostructures can also be fabricated delicately. As a promising type of photosensitizer for photodynamic therapy (PDT), the porphyrin-based COFs were grown onto upconversion nanoparticles (UCNPs). With the assistance of the near-infrared (NIR) to visible optical property of UCNPs core and the intrinsic porosity of COF shell, the core@shell nanocomposites can be applied as a nanoplatform for NIR-activated PDT with deep tissue penetration and chemotherapeutic drug delivery.

Multiphysics Modeling and Simulation of Subcutaneous Injection and Absorption of Biotherapeutics: Sensitivity Analysis.

PURPOSE: A multiphysics simulation model was recently developed to capture major physical and mechanical processes of local drug transport and absorption kinetics of subcutaneously injected monoclonal antibody (mAb) solutions. To further explore the impact of individual drug attributes and tissue characteristics on the tissue biomechanical response and drug mass transport upon injection, sensitivity analysis was conducted and reported. METHOD: Various configurations of injection conditions, drug-associated attributes, and tissue properties were simulated with the developed multiphysics model. Simulation results were examined with regard to tissue deformation, porosity change, and spatiotemporal distributions of pressure, interstitial fluid flow, and drug concentration in the tissue. RESULTS: Injection conditions and tissue properties were found influential on the mechanical response of tissue and interstitial fluid velocity to various extents, leading to distinct drug concentration profiles. Intrinsic tissue porosity, lymphatic vessel density, and drug permeability through the lymphatic membrane were particularly essential in determining the local absorption rate of an mAb injection. CONCLUSION: The sensitivity analysis study may shed light on the product development of an mAb formulation, as well as on the future development of the simulation method.

Hybrid Nanoparticles Modified by Hyaluronic Acid Loading an HSP90 Inhibitor as a Novel Delivery System for Subcutaneous and Orthotopic Colon Cancer Therapy.

BACKGROUND: As a therapeutic target for cancer treatment, HSP90 has been explored extensively. However, the significant side effects of the HSP90 inhibitor 17AAG have limited its clinical use. METHODS: In this study, we used hyaluronic acid (HA)-decorated DOTAP-PLGA hybrid nanoparticles (HA-DOTAP-PLGA NPs) as 17AAG-delivery carriers for targeted colon cancer therapy. RESULTS: Different methods were used to characterize the successful fabrication of these hybrid PLGA NPs. Our results demonstrated that internalization of HA-NPs in colon cancer cells was governed by CD44receptor-mediated endocytosis. Annexin V-propidium iodide staining experiments revealed that cell apoptosis induced by HA-NPs-17AAG in colon cancer cells was more efficient than free 17AAG. In two animal models used to screen anticancer efficacy (Luc-HT29 subcutaneous xenograft and AOM/DSS-induced orthotopic tumor model), HA-NPs-17AAG significantly inhibited xenograft and orthotopic tumor growth, demonstrating HA-NPs-17AAG had much better therapeutic efficiency than free 17AAG. It is worth noting that great biocompatibility of HA-DOTAP-PLGA NPs was observed both in vitro and in vivo. CONCLUSION: Our research offers a preclinical proof of concept for colon cancer therapy with DOTAP-PLGA NPs as a creative drug-delivery system.

Specific Cytotoxic Effect of an Auristatin Nanoconjugate Towards CXCR4+ Diffuse Large B-Cell Lymphoma Cells.

BACKGROUND AND PURPOSE: Around 40-50% of diffuse large-B cell lymphoma (DLBCL) patients suffer from refractory disease or relapse after R-CHOP first-line treatment. Many ongoing clinical trials for DLBCL patients involve microtubule targeting agents (MTAs), however, their anticancer activity is limited by severe side effects. Therefore, we chose to improve the therapeutic window of the MTA monomethyl auristatin E developing a nanoconjugate, T22-AUR, that selectively targets the CXCR4 receptor, which is overexpressed in many DLBCL cells (CXCR4+) and associated with poor prognosis. METHODS: The T22-AUR specificity towards CXCR4 receptor was performed by flow cytometry in different DLBCL cell lines and running biodistribution assays in a subcutaneous mouse model bearing CXCR4+ DLBCL cells. Moreover, we determined T22-AUR cytotoxicity using cell viability assays, cell cycle analysis, DAPI staining and immunohistochemistry. Finally, the T22-AUR antineoplastic effect was evaluated in vivo in an extranodal CXCR4+ DLBCL mouse model whereas the toxicity analysis was assessed by histopathology in non-infiltrated mouse organs and by in vitro cytotoxic assays in human PBMCs. RESULTS: We demonstrate that the T22-AUR nanoconjugate displays CXCR4-dependent targeting and internalization in CXCR4+ DLBCL cells in vitro as well as in a subcutaneous DLBCL mouse model. Moreover, it shows high cytotoxic effect in CXCR4+ DLBCL cells, including induction of G2/M mitotic arrest, DNA damage, mitotic catastrophe and apoptosis. Furthermore, the nanoconjugate shows a potent reduction in lymphoma mouse dissemination without histopathological alterations in non-DLBCL infiltrated organs. Importantly, T22-AUR also exhibits lack of toxicity in human PBMCs. CONCLUSION: T22-AUR exerts in vitro and in vivo anticancer effect on CXCR4+ DLBCL cells without off-target toxicity. Thus, T22-AUR promises to become an effective therapy for CXCR4+ DLBCL patients.

Pro-angiogenic decellularized vessel matrix gel modified by silk fibroin for rapid vascularization of tissue engineering scaffold.

Current pro-angiogenic methods in the fields of tissue engineering always aim to enrich the vascular network but neglect to provide an appropriate environment for cells, which may lead to incomplete endothelium or thrombosis. Decellularized matrix gels derived from specific tissue are expected to be suitable for targeted tissue regeneration because they preserve the biochemical properties of the native tissue. Decellularized vascular matrix gel (DVMG) has shown promise for rapid vascularization. However, DVMG is difficult to directly apply due to its weak mechanical properties and rapid degradation. In this work, silk fibroin (SF) was introduced to the DVMG to improve the physical properties of the hybrid scaffolds. The performances of the SF/DVMG scaffolds were characterized, and the results showed that SF effectively improved the overall mechanical properties of the scaffold and decreased the degradation rate. SF/DVMG scaffolds also showed good cell growth promotion effects in vitro. After the scaffolds were subcutaneously implanted in the dorsa of rats, more CD34-positive endothelial cells were expressed in the DVMG-containing scaffolds, and the number of vascular loops significantly increased compared to that of the pure SF scaffold control. The development of DVMG creates more possibilities for the rapid vascular network generation of clinically engineered scaffolds.

Protective effect of the intravenous anesthetic propofol against a local inflammation in the mouse carrageenan-induced air pouch model.

Aim: 2,6-Di-isopropylphenol (propofol) is an intravenous general anesthetic widely used in the operating room for general anesthesia and in the intensive care unit for sedation. The mouse air pouch model is versatile in studying the anti-inflammatory effect of a drug on a local inflammation, which is induced by a variety of substances. In this study, using the carrageenan-induced air pouch inflammation model, we tested whether propofol mitigates inflammation occurring locally in the mouse air pouch. Methods: Carrageenan-induced air pouch inflammation model. Results: Propofol inhibited the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the pouch. Propofol also inhibited the production of neutrophil chemokines, KC and MIP-2, and decreased the number of both Ly-6G+/CD11b+ cells (assumed to be primarily neutrophils) and Ly-6G-/CD11b+ cells (assumed to be monocytes/macrophages), recruited into the pouch at 3 h after injection of carrageenan. Conclusion: Propofol has an anti-inflammatory property in the carrageenan-induced mouse air pouch local inflammation model, by inhibiting the production of pro-inflammatory cytokines (TNF-α and IL-6), as well as by inhibiting the production of chemokines (KC and MIP-2), which might be associated with the inhibition of intra-pouch recruitment of white blood cells.

Biocompatibility evaluation of heparin-conjugated poly(ε-caprolactone) scaffolds in a rat subcutaneous implantation model.

Vascular grafts prepared from synthetic polymers have serious shortcomings that can be resolved by surface modification, such as by immobilizing heparin. In this study, the mechanical properties, biocompatibility, anticoagulation property, and water contact angle of two heparin-conjugated poly(ε-caprolactone) scaffolds (PCL-hexamethylendiamine-heparin, PCL-HMD-H. PCL-lysine-heparin, PCL-LYS-H) were compared to identify a preferred heparin conjugation method. An evaluation of the subcutaneous tissue biocompatibility of the scaffolds demonstrated that PCL-HMD-H had better endothelial cell proliferation than the PCL-LYS-H and was therefore a promising scaffold candidate for use in vascular tissue-engineering.

Hydroxyapatite-Incorporated Composite Gels Improve Mechanical Properties and Bioactivity of Bone Scaffolds.

Reinforcing polymeric scaffolds with micro/nanoparticles improve their mechanical properties and render them bioactive. In this study, hydroxyapatite (HA) is incorporated into 5% (w/v) gelatin methacrylate (GelMA) hydrogels at 1, 5, and 20 mg mL-1 concentrations. The material properties of these composite gels are characterized through swelling, degradation, and compression tests. Using 3D cell encapsulation, the cytocompatibility and osteogenic differentiation of preosteoblasts are evaluated to assess the biological properties of the composite scaffolds. The in vitro assays demonstrate increasing cell proliferation and metabolic activity over the course of 14 d in culture. Furthermore, the scaffolds support osteogenic differentiation of the microencapsulated preosteoblasts. For the in vivo study, the composite scaffolds are subcutaneously implanted in rats for 14 d. The histological staining of the explanted in vivo samples exhibits the functional advantages of the scaffold's biocompatibility, biodegradability, and integration into the existing host tissue. This work demonstrates the enhanced mechanical and biological performance of HA-gelatin composite hydrogels for bone tissue engineering applications.

An evaluation of subcutaneous apomorphine for the treatment of Parkinson's disease.

INTRODUCTION: Heterogeneity of symptoms and individual variability of progression characterizes Parkinson's disease. Unmet therapeutic needs include a cure, disease modification, and improvement of available marketed dopamine-substituting compounds. Personalized treatment, tailored to the patients' needs and symptoms, aims to ameliorate impaired motor behavior and non-motor features. Injection or infusion of apomorphine is a therapeutic option for more advanced patients with severe levodopa associated motor complications. AREAS COVERED: This narrative review summarizes the subcutaneous administration, efficacy, and side effects of the non-ergot derivative dopamine agonist apomorphine following a non-systematic literature research. EXPERT OPINION: Subcutaneous apomorphine hydrochloride application rapidly terminates intervals with severe motor impairment with bolus injections. Oscillation of motor behavior well responds to continuous apomorphine infusions. Long-term application of the commercially available apomorphine hydrochloride solution sooner or later affects skin and oral mucosa. Onset of skin nodules associated with subcutaneous tissue inflammation probably results from the antioxidant preservative sodium metabisulfite in the apomorphine solution. Addition of another better tolerated and safer antioxidant instead of sodium metabisulphite or use of an already available concentrated apomorphine-free base formulation will enhance its future use, its tolerability, safety, and acceptance of subcutaneous and sublingual application.

Attenuation of subcutaneous insulin induced amyloid mass in vivo using Lumbrokinase and Serratiopeptidase.

The protein misfolded structure called amyloids is related with extensive range of pathologies like local amyloidosis and neurodegenerative diseases. Several studies have reported the potential of insulin to generate local amyloidosis under certain state. Reports also showed that fibrils of insulin generated local amyloid mass due to continuous subcutaneous injection in mouse as well as rat. The present study was designed to examine the consequence of insulin fibril injections in rats, as well as the ability of enzymes, Lumbrokinase (LK) and Serratiopeptidase (SP) in diminishing this amyloid mass progression. The results showed that insulin fibrils generated amyloid masses in rats after subcutaneous injection for two weeks which was significantly condensed in size for the groups injected with insulin fibrils combined with LK or SP. At higher doses of LK and SP, the absence of amyloid structure was observed in histopathological studies. Light microscopy, polarized microscopy as well as Lumia live in vivo imaging system was used to analyze the results. In conclusion, the overall outcome of this study showed the anti-amyloid potential of enzyme LK and SP in the attenuation of local amyloidosis.

In vivo biocompatibility of silicon dioxide nanofilm used as antimicrobial agent on acrylic surface.

the focus ofthis study was to testthe hypothesisthatthere would be no difference betweenthe biocompatibility of silicon dioxide nanofilms used as antimicrobial agents. Sixty male Wistar rats were divided into 4 groups (n=15): Group C (Control,Polyethylene), Group AR (Acrylic Resin), Group NP (Acrylic Resin coated with NP-Liquid), Group BG (Acrylic Resin coated with Bacterlon).the animals were sacrificed with 7,15 and 30 days and tissues analyzed as regardsthe events of inflammatory infiltrate, edema, necrosis, granulation tissue, mutinucleated giant cells, fibroblasts and collagen. Kruskal-Wallis and Dunn tests was used (P<0.05). Intense inflammatory infiltrate was shown mainly in Groups BG and AR, with significant difference from Control Group inthe time interval of 7days (P=0.004). Necrosis demonstrated significant difference between Group BG and Control Group (P<0.05) inthe time intervals of 7 days. For collagen fibers,there was significant difference betweenthe Control Group and Groups AR and BG inthe time interval of 7 days (P=0.006), and between BG and Control Groups inthe time intervals of 15 days (P=0.010).the hypothesis was rejected. Bacterlon demonstratedthe lowest level, and NP-Liquid Glassthe highest level of tissue compatibility, and best cell repair.the coating with NP-Liquid Glass was demonstrated to be highly promising for clinical use.

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Fibrin fragment E potentiates TGF-ß-induced myofibroblast activation and recruitment.

Fibrin is an essential constituent of the coagulation cascade, and the formation of hemostatic fibrin clots is central to wound healing. Fibrin clots are over time degraded into fibrin degradation products as the injured tissue is replaced by granulation tissue. Our goal was to study the role of the fibrin degradation product fragment E (FnE) in fibroblast activation and migration. We present evidence that FnE is a chemoattractant for fibroblasts and that FnE can potentiate TGF-ß-induced myofibroblast formation. FnE forms a stable complex with αVß3 integrin, and the integrin ß3 subunit is required both for FnE-induced fibroblast migration and for potentiation of TGF-ß-induced myofibroblast formation. Finally, subcutaneous infusion of FnE in mice results in a fibrotic response in the hypodermis. These results support a model where FnE released from clots in wounded tissue promote wound healing and fibrosis by both recruitment and activation of fibroblasts. Fibrin fragment E could thus represent a therapeutic target for treatment of pathological fibrosis.

Nanoclay-based 3D printed scaffolds promote vascular ingrowth ex vivo and generate bone mineral tissue in vitro and in vivo.

Acellular soft hydrogels are not ideal for hard tissue engineering given their poor mechanical stability, however, in combination with cellular components offer significant promise for tissue regeneration. Indeed, nanocomposite bioinks provide an attractive platform to deliver human bone marrow stromal cells (HBMSCs) in three dimensions producing cell-laden constructs that aim to facilitate bone repair and functionality. Here we present the in vitro, ex vivo and in vivo investigation of bioprinted HBMSCs encapsulated in a nanoclay-based bioink to produce viable and functional three-dimensional constructs. HBMSC-laden constructs remained viable over 21 d in vitro and immediately functional when conditioned with osteogenic media. 3D scaffolds seeded with human umbilical vein endothelial cells (HUVECs) and loaded with vascular endothelial growth factor (VEGF) implanted ex vivo into a chick chorioallantoic membrane (CAM) model showed integration and vascularisation after 7 d of incubation. In a pre-clinical in vivo application of a nanoclay-based bioink to regenerate skeletal tissue, we demonstrated bone morphogenetic protein-2 (BMP-2) absorbed scaffolds produced extensive mineralisation after 4 weeks (p < 0.0001) compared to the drug-free and alginate controls. In addition, HBMSC-laden 3D printed scaffolds were found to significantly (p < 0.0001) support bone tissue formation in vivo compared to acellular and cast scaffolds. These studies illustrate the potential of nanoclay-based bioink, to produce viable and functional constructs for clinically relevant skeletal tissue regeneration.

Subcutaneous priming of protein-functionalized chitosan scaffolds improves function following spinal cord injury.

Strategies using neural stem cells (NSCs) to aid regeneration following spinal cord injury (SCI) show much promise, but challenges remain regarding implementation and efficacy. In this work, we explored the use of an NSC-seeded scaffold consisting of covalently immobilized interferon-γ and rat NSCs within a hydrogel matrix (methacrylamide chitosan). We placed the scaffolds within the subcutaneous environment of rats, allowing them to incubate for 4 weeks in order to prime them for regeneration prior to being transplanted into a right lateral hemisection SCI model in the same animal. We found that subcutaneous priming reduced the lineage commitment of encapsulated NSCs, as observed by increased nestin expression and decreased NeuN expression. When combined with intracellular σ peptide administration (which reduces inhibition from the glial scar), subcutaneous maturation improved functional outcomes, which were assessed by BBB score and quantitative gait parameters (fore and hind limb duty factor imbalance, right and left paw placement accuracy). Although we did not observe any direct reconnection of the transplanted cells with the host tissue, we did observe neurofilament fibers extending from the host tissue into the scaffold. Importantly, the mechanism for improved functional outcomes is likely an increase in trophic support from subcutaneously maturing the scaffold, which is enhanced by the administration of ISP.