Enrichment of Nanofiber Hydrogel Composite with Fractionated Fat Promotes Regenerative Macrophage Polarization and Vascularization for Soft-Tissue Engineering.

BACKGROUND: Fractionated fat has been shown to promote dermal regeneration; however, the use of fat grafting for reconstruction of soft-tissue defects is limited because of volume loss over time. The authors have developed a novel approach for engineering of vascularized soft tissue using an injectable nanofiber hydrogel composite enriched with fractionated fat. METHODS: Fractionated fat was generated by emulsification of groin fat pads from rats and mixed in a 3:1 ratio with nanofiber hydrogel composite (nanofiber hydrogel composite with fractionated fat). Nanofiber hydrogel composite with fractionated fat or nanofiber hydrogel composite alone was placed into isolation chambers together with arteriovenous loops, which were subcutaneously implanted into the groin of rats (n = 8 per group). After 21 days, animals were euthanized and systemically perfused with ink, and tissue was explanted for histologic analysis. Immunofluorescent staining and confocal laser scanning microscopy were used to quantify CD34+ progenitor cell and macrophage subpopulations. RESULTS: Nanofiber hydrogel composite with fractionated fat tissue maintained its shape without shrinking and showed a significantly stronger functional vascularization compared to composite alone after 21 days of implantation (mean vessel count, 833.5 ± 206.1 versus 296.5 ± 114.1; p = 0.04). Tissue heterogeneity and cell count were greater in composite with fractionated fat (mean cell count, 49,707 ± 18,491 versus 9263 ± 3790; p = 0.005), with a significantly higher number of progenitor cells and regenerative CD163+ macrophages compared to composite alone. CONCLUSIONS: Fractionated fat-enriched nanofiber hydrogel composite transforms into highly vascularized soft tissue over 21 days without signs of shrinking and promotes macrophage polarization toward regenerative phenotypes. Enrichment of injectable nanofiber hydrogel composite with fractionated fat represents a promising approach for durable reconstruction of soft-tissue defects. CLINICAL RELEVANCE STATEMENT: The authors' approach for tissue engineering may ultimately lay the groundwork for clinically relevant applications with the goal of generating large volumes of vascularized soft tissue for defect reconstruction without donor site morbidity.

Tissue Engineering of Axially Vascularized Soft-Tissue Flaps with a Poly-(ɛ-Caprolactone) Nanofiber-Hydrogel Composite.

Objective: To develop a novel approach for tissue engineering of soft-tissue flaps suitable for free microsurgical transfer, using an injectable nanofiber hydrogel composite (NHC) vascularized by an arteriovenous (AV) loop. Approach: A rat AV loop model was used for tissue engineering of vascularized soft-tissue flaps. NHC or collagen-elastin (CE) scaffolds were implanted into isolation chambers together with an AV loop and explanted after 15 days. Saphenous veins were implanted into the scaffolds as controls. Neoangiogenesis, ultrastructure, and protein expression of SYNJ2BP, EPHA2, and FOXC1 were analyzed by immunohistochemistry and compared between the groups. Rheological properties were compared between the two scaffolds and native human adipose tissue. Results: A functional neovascularization was evident in NHC flaps with its amount being comparable with CE flaps. Scanning electron microscopy revealed a strong mononuclear cell infiltration along the nanofibers in NHC flaps and a trend toward higher fiber alignment compared with CE flaps. SYNJ2BP and EPHA2 expression in endothelial cells (ECs) was lower in NHC flaps compared with CE flaps, whereas FOXC1 expression was increased in NHC flaps. Compared with the stiffer CE flaps, the NHC flaps showed similar rheological properties to native human adipose tissue. Innovation: This is the first study to demonstrate the feasibility of tissue engineering of soft-tissue flaps with similar rheological properties as human fat, suitable for microsurgical transfer using an injectable nanofiber hydrogel composite. Conclusions: The injectable NHC scaffold is suitable for tissue engineering of axially vascularized soft-tissue flaps with a solid neovascularization, strong cellular infiltration, and biomechanical properties similar to human fat. Our data indicate that SYNJ2BP, EPHA2, and FOXC1 are involved in AV loop-associated angiogenesis and that the scaffold material has an impact on protein expression in ECs.

In Vivo Bioluminescence Imaging in a Rabbit Model of Orthopaedic Implant-Associated Infection to Monitor Efficacy of an Antibiotic-Releasing Coating.

BACKGROUND: In vivo bioluminescence imaging (BLI) provides noninvasive monitoring of bacterial burden in animal models of orthopaedic implant-associated infection (OIAI). However, technical limitations have limited its use to mouse and rat models of OIAI. The goal of this study was to develop a larger, rabbit model of OIAI using in vivo BLI to evaluate the efficacy of an antibiotic-releasing implant coating. METHODS: A nanofiber coating loaded with or without linezolid-rifampin was electrospun onto a surgical-grade locking peg. To model OIAI in rabbits, a medial parapatellar arthrotomy was performed to ream the femoral canal, and a bright bioluminescent methicillin-resistant Staphylococcus aureus (MRSA) strain was inoculated into the canal, followed by retrograde insertion of the coated implant flush with the articular surface. In vivo BLI signals were confirmed by ex vivo colony-forming units (CFUs) from tissue, bone, and implant specimens. RESULTS: In this rabbit model of OIAI (n = 6 rabbits per group), implants coated without antibiotics were associated with significantly increased knee width and in vivo BLI signals compared with implants coated with linezolid-rifampin (p < 0.001 and p < 0.05, respectively). On day 7, the implants without antibiotics were associated with significantly increased CFUs from tissue (mean [and standard error of the mean], 1.4 × 10 ± 2.1 × 10 CFUs; p < 0.001), bone (6.9 × 10 ± 3.1 × 10 CFUs; p < 0.05), and implant (5.1 × 10 ± 2.2 × 10 CFUs; p < 0.05) specimens compared with implants with linezolid-rifampin, which demonstrated no detectable CFUs from any source. CONCLUSIONS: By combining a bright bioluminescent MRSA strain with modified techniques, in vivo BLI in a rabbit model of OIAI demonstrated the efficacy of an antibiotic-releasing coating. CLINICAL RELEVANCE: The new capability of in vivo BLI for noninvasive monitoring of bacterial burden in larger-animal models of OIAI may have important preclinical relevance.

Local Release of Paclitaxel from Aligned, Electrospun Microfibers Promotes Axonal Extension.

Traumatic spinal cord injuries ultimately result in an inhibitory environment that prevents axonal regeneration from occurring. A low concentration administration of paclitaxel has been previously shown to promote axonal extension and attenuate the upregulation of inhibitory molecules after a spinal cord injury. In this study, paclitaxel is incorporated into electrospun poly(l-lactic acid) (PLA) microfibers, and it is established that a local release of paclitaxel from aligned, electrospun microfibers promotes neurite extension in a growth-conducive and inhibitory environment. Isolated dorsal root ganglion cells are cultured for 5 d directly on tissue culture polystyrene surface, PLA film, random, or aligned electrospun PLA microfibers (1.44 ± 0.03 µm) with paclitaxel incorporated at various concentrations (0%-5.0% w/w in reference to fiber weight). To determine the effect of a local release of paclitaxel, paclitaxel-loaded microfibers are placed in CellCrown inserts above cultured neurons. Average neurite extension rate is quantified for each sample. A local release of paclitaxel maintains neuronal survival and neurite extension in a concentration-dependent manner when coupled with aligned microfibers when cultured on laminin or an inhibitory surface of aggrecan. The findings provide a targeted approach to improve axonal extension across the inhibitory environment present after a traumatic injury in the spinal cord.

Formulation and stabilization of Francisella tularensis live vaccine strain.

Francisella tularensis live vaccine strain (F. tularensis LVS), a promising vaccine candidate for protection against F. tularensis exposure, is a particularly thermolabile vaccine and difficult to stabilize sufficiently for storage under refrigerated conditions. Our preliminary data show that F. tularensis LVS can be stabilized in the dried state using foam drying, a modified freeze drying method, with sugar-based formulations. The process was conducted under mild drying conditions, which resulted in a good titer retention following processing. The inclusion of osmolytes in the growth media resulted in an acceleration of growth kinetics, although no change in osmotolerance was observed. The optimized F. tularensis formulation, which contained trehalose, gelatin, and Pluronic F68 demonstrated stability for approximately 1.5 weeks at 37°C (i.e., time required for the vaccine to decrease in potency by 1 log(10) colony forming unit) and for 12 weeks at 25°C. At refrigerator storage condition (4°C), stabilized F. tularensis LVS vaccine exhibited no activity loss for at least 12 weeks. This stabilization method utilizes conventional freeze dryers and pharmaceutically approved stabilizers, and thus can be readily implemented at many manufacturing sites for large-scale production of stabilized vaccines. The improved heat stability of the F. tularensis LVS could mitigate risks of vaccine potency loss during long-term storage, shipping, and distribution.

Biophysical characterization of rotavirus serotypes G1, G3 and G4.

The stability of attenuated virus vaccines has traditionally been assessed by a plaque assay to measure the virus's loss of replication competency in response to a variety of environmental perturbations. Although this method provides information regarding the impact of the vaccine formulation, it involves an empirical approach to evaluate stability. Biophysical studies on the other hand have the potential to provide insight into the mechanisms of inactivation of a viral vaccine in response to a variety of stressed conditions. Herein, we have employed a variety of spectroscopic techniques (i.e., circular dichroism, fluorescence spectroscopy and dynamic light scattering) for a comprehensive examination of the thermal stability of three live-attenuated human-bovine reassortant rotavirus strains (G1, G3 and G4) in the 5-8 pH range. The spectroscopic methods employed are not specific and response changes reflect an average change over the entire virus structure. The present work, however, suggests the utility of these methods in early formulation of rotaviral vaccines due to their ability to identify regions of marginal stability over which high throughput excipient screening assays can be designed. We have further shown that these methods are sufficiently sensitive to differentiate the stability of the three homologous G-subtypes differing only in the composition of their surface antigenic proteins. The data from these spectroscopic methods are also compared to biological activity using a tissue culture viral infectivity assay. Partial correlation between the structural alterations and losses in activity are observed, further suggesting the utility of biophysical studies in early formulation studies of rotavirus vaccines.

Using spectroscopic and microscopic methods to probe the structural stability of human adenovirus type 4.

Adenovirus serotype 4 (Ad4) is a major cause of Ad-associated human diseases. Ad4 is also considered to be a potential delivery vector for gene therapy. In this study, multiple spectroscopic techniques together with transmission electron microscopy (TEM) were employed to probe viral stability and to improve pharmaceutical formulations of Ad4-based vaccines and DNA carriers. Perturbations of secondary, tertiary and quaternary structure of Ad4 proteins induced by elevated temperatures over a wide pH range (3-8) were analyzed using circular dichroism, UV absorption and intrinsic and extrinsic fluorescence spectroscopy as well as static and dynamic light scattering. The spectroscopic results obtained indicate a decrease in Ad4 stability as pH increases from 4 to 8, similar to the behavior reported previously for Ad2 and Ad5, although the Ad4 virion appears to possess slightly more tolerance to thermal stress. An empirical phase diagram (EPD) approach was used to summarize the data in the form of a colored map. In addition, the different physical states of Ad4 identified by the EPD were confirmed by TEM images. The results obtained in this study reveal both structural similarities among three commonly employed Ad subtypes (2, 4 and 5) as well as unique properties of Ad4.

Heat-stable measles vaccine produced by spray drying.

A combination of unique stabilizers and mild spray drying process conditions was employed to produce heat-stable measles vaccine powder. Live attenuated measles vaccine from Serum Institute of India was formulated with pharmaceutically approved stabilizers, including sugars, proteins, amino acids, polymers, surfactants, and plasticizers, as well as charged ions. In addition, the effects of buffer salt and pH on the storage stability of measles virus were examined. The potency of the dried vaccine stored at several temperatures was quantified by TCID(50) assay on Vero cells. As a comparison to other process methods, lead formulations were also subjected to freeze drying and foam drying. The optimized measles vaccine formulation tested at 37 degrees C was stable for approximately 8 weeks (i.e. time for 1 log TCID(50) loss). The measles titer decreased in a bi-phasic manner, with initial rapid loss within the first week but relative stability thereafter. Key stabilizers identified during the formulation screening processes were L-arginine, human serum albumin, and a combination of divalent cations. Spray drying was identified as the optimal processing method for the preparation of dried vaccine, as it generally resulted in negligible process loss and comparable, if not better storage stability, with respect to the other processes. Processing methods and formulation components were developed that produced a measles vaccine stable for up to 8 weeks at 37 degrees C, which surpassed the WHO requirement for heat stability of 1 week at that temperature.