Backscattered Scanning Electron Microscopy Approach for Assessment of Microvessels under Conditions of Normal Microanatomy and Pathological Neovascularization.

We evaluated the efficiency of an original method for studying of the microvascular bed under conditions of normal microanatomy and pathological neovascularization. The blood vessels, tissues surrounding the stent in the pulmonary artery and subcutaneously implanted titanium nickelide plate, atherosclerotic plaque, and vascular stent with restenosis were examined. The specimens were fixed in formalin and stained in OsO4, embedded into fresh epoxy resin, grinded, polished, and counterstained with uranyl acetate and lead citrate. Numerous vasa vasorum were found in all native vessels. Around the pulmonary artery stent and metal plates, numerous newly formed vessels of small diameter were seen. The intensity of neovascularization in atherosclerosis and carotid stent restenosis differed significantly. Our technique can be successfully used for evaluation of the microvascular bed.

Morphological study of human facial fascia and subcutaneous tissue structure by region through SEM observation.

Fascia of the facial area is contiguous between fat tissues of the subcutaneous and connective tissue layers and does not envelope the muscle surface like other parts of the human body. This structure is called the superficial musculoaponeurotic system (SMAS), which is accepted as an international anatomical terminology. This special structure is commonly used to pull facial muscles during plastic surgeries such as a face lift. Most reports regarding the facial subcutaneous tissue structure including SMAS are in the field of plastic surgery, and only a few studies from a morphological perspective has been reported. Since the facial fascia does not envelope the muscular surface layer which is different from the deep fascia found on the general skeletal muscle surface, a clear definition of this structure has not been established yet. The purpose of this study was to clearly identify the basic morphological structure of the subcutaneous tissue layer containing the SMAS three-dimensionally through a scanning electron microscope using dissected specimen rather than living subjects. Moreover, this study explores structural differences among seven aging facial areas; thereby further clarifying the properties of the structure and add clinical significance and considerations.

In Vitro and In Vivo Characterization of Biodegradable Reactive Isocyanate-Terminated Three-Armed- and Hyperbranched Block Copolymeric Tissue Adhesives.

Tissue adhesives are an attractive class of biomaterials, which can serve as a treatment for meniscus tears. In this study, physicochemical and adhesive properties of novel biodegradable three-armed- and hyperbranched block copolymeric adhesives are evaluated. Additionally, their degradation in vitro and in vivo, and the tissue reaction after subcutaneous injection in rats are assessed. The developed adhesives have sufficient adhesive strength to meniscus tissue after curing (66-88 kPa). Networks based on the three-armed adhesive have tensile properties that are in the same range as human meniscus. After 26 weeks, networks based on the hyperbranched adhesive show a faster mass loss (25.4%) compared to networks prepared from the three-armed ones (5.5%). Both adhesives induce an inflammatory reaction, however, no necrosis and only initial toxic effects on peripheral tissues are observed. The proposed materials are suitable candidates for the use as resorbable tissue adhesives for meniscus repair.

Vascularization Potential of Electrospun Poly(L-Lactide-co-Caprolactone) Scaffold: The Impact for Tissue Engineering.

BACKGROUND Electrospun nanofibers have widespread putative applications in the field of regenerative medicine and tissue engineering. When compared to naturally occurring collagen matrices, electrospun nanofiber scaffolds have two distinct advantages: they do not induce a foreign body reaction and they are not at risk for biological contamination. However, the exact substrate, structure, and production methods have yet to be defined. MATERIAL AND METHODS In the current study, tubular-shaped poly(L-lactide-co-caprolactone) (PLCL) constructs produced using electrospinning technology were evaluated for their potential application in the field of tissue regeneration in two separate anatomic locations: the skin and the abdomen. The constructs were designed to have an internal diameter of 3 mm and thickness of 200 µm. Using a rodent model, 20 PLCL tubular constructs were surgically implanted in the abdominal cavity and subcutaneously. The constructs were then evaluated histologically using electron microscopy at 6 weeks post-implantation. RESULTS Histological evaluation and analysis using scanning electron microscopy showed that pure scaffolds by themselves were able to induce angiogenesis after implantation in the rat model. Vascularization was observed in both tested groups; however, better results were obtained after intraperitoneal implantation. Formation of more and larger vessels that migrated inside the scaffold was observed after implantation into the peritoneum. In this group no evidence of inflammation and better integration of scaffold with host tissue were noticed. Subcutaneous implantation resulted in more fibrotic reaction, and differences in cell morphology were also observed between the two tested groups. CONCLUSIONS This study provides a standardized evaluation of a PLCL conduit structure in two different anatomic locations, demonstrating the excellent ability of the structure to achieve vascularization. Functional, histological, and mechanical data clearly indicate prospective clinical utilization of PLCL in critical size defect regeneration.

Histological evaluations of apatite-fiber scaffold cultured with mesenchymal stem cells by implantation at rat subcutaneous tissue.

BACKGROUND: There is a strong impetus for the development of alternative treatments for bone disease that avoid the complications associated with autografts and allografts. To address this, we previously developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional interconnected pores, and constructed tissue-engineered bone by culturing rat bone marrow cells (RBMCs) using AFSs in a radial-flow bioreactor (RFB). OBJECTIVE: To generate additional baseline data for the development of tissue-engineered bone constructed for clinical application using a RFB, we cultured RBMCs using AFSs under static conditions (hereafter, RBMC AFS culture), and monitored RBMC growth and differentiation characteristics in vitro, and two weeks after subcutaneous inoculation into recipient rats. METHODS: RBMCs were seeded to AFSs and growth, differentiation and calcification were monitored in vitro and in vivo by histological evaluation using hematoxylin eosin, alkaline phosphatase and alizarin red S stains. RESULTS: RBMCs in/on AFSs proliferated and differentiated normally in vitro and in vivo, and calcification was evident two weeks after subcutaneous AFS culture implantation. Histological assays revealed that AFSs and RBMC AFS cultures were biocompatible, and did not induce inflammation or immunological rejection in vivo. CONCLUSIONS: These findings suggest that AFSs are a conducive microenvironment for bone regeneration and are well tolerated in vivo. The results provide valuable baseline data for the design of implant studies using tissue-engineered bone constructed by RFB.

Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM).

Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland-related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d-glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2- to 3-µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in-solution ASEM for histology and to study vesicle secretion systems. Further, the high-throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine-related diseases.

In vitro and in vivo corrosion, cytocompatibility and mechanical properties of biodegradable Mg-Y-Ca-Zr alloys as implant materials.

This study introduces a class of biodegradable Mg-Y-Ca-Zr alloys novel to biological applications and presents evaluations for orthopedic and craniofacial implant applications. Mg-Y-Ca-Zr alloys were processed using conventional melting and casting techniques. The effects of increasing Y content from 1 to 4 wt.% as well as the effects of T4 solution treatment were assessed. Basic material phase characterization was conducted using X-ray diffraction, optical microscopy and scanning electron microscopy. Compressive and tensile tests allowed for the comparison of mechanical properties of the as-cast and T4-treated Mg-Y-Ca-Zr alloys to pure Mg and as-drawn AZ31. Potentiodynamic polarization tests and mass loss immersion tests were used to evaluate the corrosion behavior of the alloys. In vitro cytocompatibility tests on MC3T3-E1 pre-osteoblast cells were also conducted. Finally, alloy pellets were implanted into murine subcutaneous tissue to observe in vivo corrosion as well as local host response through H&E staining. SEM/EDS analysis showed that secondary phase intermetallics rich in yttrium were observed along the grain boundaries, with the T4 solution treatment diffusing the secondary phases into the matrix while increasing the grain size. The alloys demonstrated marked improvement in mechanical properties over pure Mg. Increasing the Y content contributed to improved corrosion resistance, while solution-treated alloys resulted in lower strength and compressive strain compared to as-cast alloys. The Mg-Y-Ca-Zr alloys demonstrated excellent in vitro cytocompatibility and normal in vivo host response. The mechanical, corrosion and biological evaluations performed in this study demonstrated that Mg-Y-Ca-Zr alloys, especially with the 4 wt.% Y content, would perform well as orthopedic and craniofacial implant biomaterials.

In vitro degradation and cytotoxicity response of Mg-4% Zn-0.5% Zr (ZK40) alloy as a potential biodegradable material.

Mg-4 wt.% Zn-0.5 wt.% Zr (ZK40) alloy was studied as a candidate material for biodegradable metallic implants in terms of its biocorrosion resistance, mechanical properties and cytocompatibility. The corrosion characteristics of ZK40 alloy were assessed by potentiodynamic polarization and immersion testing in DMEM+10% FBS solution. Analysis of the degradation characteristics by potentiodynamic polarization measurements shows the corrosion rates of ZK40 alloy in as-cast and solution treatment (T4) condition were slightly higher than those of pure Mg or as-drawn AZ31. Determination of the corrosion rate by the weight loss technique reveals that the as-cast ZK40 resulted in slower degradation than other alloy specimens after 7 days of immersion but exhibited accelerated degradation after 14 and 21 days, respectively. T4-treated ZK40 exhibited stable degradation rates compared to as-cast ZK40 and close to those of pure Mg and AZ31 during immersion testing for 14 and 21 days. In order to examine the in vitro cytocompatibility of ZK40 alloy, live/dead cell viability assay and indirect MTT assay were performed using a murine osteoblast-like cell line (MC3T3). After 3 days of direct culture of MC3T3 on ZK40 alloys the live/dead assay indicated favorable cell viability and attachment. The degradation product of ZK40 also showed minimal cytotoxicity when assessed in indirect MTT assay. The mechanical properties of the as-cast and T4-treated ZK40 alloy were superior to those of pure Mg and comparable to as-drawn AZ31. Solution treatment did not significantly enhance the cytocompatibility and mechanical properties of ZK40 alloy. Overall, the ZK40 alloy exhibited favorable cytocompatibility, biocorrosion, and mechanical properties rendering it a potential candidate for degradable implant applications.

Pulsed focused ultrasound exposures enhance locally administered gene therapy in a murine solid tumor model.

Gene therapy by intratumoral injection is a promising approach for treating solid tumors. However, this approach has limited success due to insufficient distribution of gene vectors used for gene delivery. Previous studies have shown that pulsed-focused ultrasound (pFUS) can enhance both systemic and local delivery of therapeutic agents in solid tumors and other disease models. Here, murine squamous cell carcinoma flank tumors were treated with single intratumoral injection of naked tumor necrosis factor-alpha (TNF-α) plasmid, either with or without a preceding pFUS exposure. The exposures were given at 1 MHz, at a spatial average, temporal peak intensity of 2660 W cm(-2), using 50 ms pulses, given at a pulse repetition frequency of 1 Hz. One hundred pulses were given at individual raster points, spaced evenly over the projected surface of the tumor at a distance of 2 mm. Exposures alone had no effect on tumor growth. Significant growth inhibition was observed with injection of TNF-α plasmid, and tumor growth was further inhibited with pFUS. Improved results with pFUS correlated with larger necrotic regions in histological sections and improved distribution and penetration of fluorescent surrogate nanoparticles. Electron microscopy demonstrated enlarged gaps between cells in exposed tissue, and remote acoustic palpation showed decreases in tissue stiffness after pFUS. Combined, these results suggest pFUS effects may be reducing barriers for tissue transport and additionally lowering interstitial fluid pressure to further improve delivery and distribution of injected plasmid for greater therapeutic effects. This suggests that pFUS could potentially be beneficial for improving local gene therapy treatment of human malignancies.

Subcutaneous tissue response to titanium, poly(ε-caprolactone), and carbonate-substituted hydroxyapatite-coated poly(ε-caprolactone) plates: a rabbit study.

The aim of this study was to evaluate the soft tissue response to poly(ε-caprolactone) (PCL) implants with and without carbonate-substituted hydroxyapatite (CHA) coating compared to the commonly used titanium alloy (Ti-6Al-4V)-machined surface. Experimental materials were implanted subcutaneously in New Zealand white rabbits for 5 weeks. The tissue attachment strength, as evaluated by a tissue peel test, histological and histomorphology analysis, as well as scanning electron microscopy were compared between groups. The peel test result revealed no statistically significant difference between groups. Histological analysis found fibrous capsule formation around all implant materials. The fibrous capsule around PCL implants with and without CHA coating was significantly thinner compared with the capsule thickness around the titanium implants. However, the inflammatory cells, as present at the fibrous capsule-implant interface, were found to be significantly lower in the Ti-group. In conclusion, the current data do not prove that PCL or PCL with a CHA coating results in a superior soft tissue response compared with a machined titanium implant.

Development of the platysma muscle and the superficial musculoaponeurotic system (human specimens at 8-17 weeks of development).

There is controversy regarding the description of the different regions of the face of the superficial musculoaponeurotic system (SMAS) and its relationship with the superficial mimetic muscles. The purpose of this study is to analyze the development of the platysma muscle and the SMAS in human specimens at 8-17 weeks of development using an optical microscope. Furthermore, we propose to study the relationship of the anlage of the SMAS and the neighbouring superficial mimetic muscles. The facial musculature derives from the mesenchyme of the second arch and migrates towards the different regions of the face while forming premuscular laminae. During the 8th week of development, the cervical, infraorbital, mandibular, and temporal laminae are observed to be on the same plane. The platysma muscle derives from the cervical lamina and its mandibular extension enclosing the lower part of the parotid region and the cheek, while the SMAS derives from the upper region. During the period of development analyzed in this study, we have observed no continuity between the anlage of the SMAS and that of the superficial layer of the temporal fascia and the zygomaticus major muscle. Nor have we observed any structure similar to the SMAS in the labial region.

Variability of the biological activity of oxidized titanium implants.

Oxidized titanium is a biologically inert material, but bioinertness reduces biomechanical characteristics of titanium implants. Modification of the structure of oxide surface layer of BT 5-1 titanium by increasing its thickness (by 1.7 times) and pore diameter (by 1.4 times) and by adding phosphorus, aluminum, and zinc oxides to its composition leads to radical modification of its biological characteristics. These implants acquire osteoinductive properties in in vivo systems not found in pure or oxidized BT 1-00 titanium and fairly well maintain in vitro growth of mesenchymal cells.

A simple way to reconstruct a human 3-d hypodermis: a useful tool for pharmacological functionality.

BACKGROUND: Adipose tissue engineering has been hampered by the inability to culture mature adipocytes. Adipose-derived stem cell (ASC) culture opens the way for the preparation of human 3-D hypodermis in large quantities. These models play a role in obesity-related active molecules and slimming agent screening. Moreover, they contribute to a better understanding of the mechanisms underpinning obesity. MATERIALS AND METHODS: Freshly extracted ASC from fat tissue were characterized by flow cytometry for CD73, CD90, CD105, HLA-ABC, CD14 and CD45 markers and by Western blot for pref-1. Their differentiation in mature adipocytes was followed by lipid and adiponectin secretion or by oil red O staining and radioimmunoassay. Neosynthesized extracellular matrix (ECM) of 3-D hypodermis was investigated by immunohistochemistry (collagen type I, V and VI) and transmission electron microscopy. RESULTS: Our results demonstrate that the culture of preadipocytes in proliferation medium for 15 days followed by 16 days of culture in differentiation medium allowed production of the thickest single-layer hypodermis in which preadipocytes and mature adipocytes coexist and synthesize adiponectin and ECM components. Functionality of our 3-D single-layer hypodermis was demonstrated both by a 3.5-fold glycerol production after its stimulation with norepinephrine (adrenergic agonist) and by its slimming after caffeine treatment versus the nontreated 3-D hypodermis. CONCLUSION: This economic 3-D model, easy to prepare and giving reproducible results after the treatment of actives, is useful for pharmacotoxicological trials as an alternative to animal experimentation.

Kinesin-1 and dynein at the nuclear envelope mediate the bidirectional migrations of nuclei.

Kinesin-1 and dynein are recruited to the nuclear envelope by the Caenorhabditis elegans klarsicht/ANC-1/Syne homology (KASH) protein UNC-83 to move nuclei. The mechanisms of how these motors are coordinated to mediate nuclear migration are unknown. Time-lapse differential interference contrast and fluorescence imaging of embryonic hypodermal nuclear migration events were used to characterize the kinetics of nuclear migration and determine microtubule dynamics and polarity. Wild-type nuclei display bidirectional movements during migration and are also able to roll past cytoplasmic granules. unc-83, unc-84, and kinesin-1 mutants have severe nuclear migration defects. Without dynein, nuclear migration initiates normally but lacks bidirectional movement and shows defects in nuclear rolling, implicating dynein in resolution of cytoplasmic roadblocks. Microtubules are highly dynamic during nuclear migration. EB1::green fluorescence protein imaging demonstrates that microtubules are polarized in the direction of nuclear migration. This organization of microtubules fits with our model that kinesin-1 moves nuclei forward and dynein functions to move nuclei backward for short stretches to bypass cellular roadblocks.

Vascularization around poly(tetrafluoroethylene) mesh with coating of gelatin hydrogel incorporating basic fibroblast growth factor.

For successful mesh hernia treatment with medical meshes, it is important to induce angiogenesis and fibroplasia around the site of the mesh implanted. The objective of this study is to combine a mesh with a gelatin hydrogel for basic fibroblast growth factor (bFGF) release and evaluate the angiogenic activity in vivo. The MotifMesh (MM) of poly(tetrafluoroethylene) was treated with corona discharge to make the surface hydrophilic. This corona discharge treatment increased the bonding strength between the gelatin hydrogel coated and the mesh surface. When implanted into the back subcutis of mice, the MM coated with the gelatin hydrogel incorporating bFGF showed significant angiogenesis around the implanted site, in contrast to the MM alone and that without gelatin hydrogel or bFGF incorporation. It is concluded that the coating of hydrogel incorporating bFGF is a promising technology to give the mesh angiogenic properties.

Degradation behavior of poly(glycerol sebacate).

Poly(glycerol sebacate) (PGS), a promising scaffold material for soft tissue engineering applications, is a soft, tough elastomer with excellent biocompatibility. However, the rapid in vivo degradation rate of PGS limits its use as a scaffold material. To determine the impact of crosslink density on degradation rate, a family of PGS materials was synthesized by incrementally increasing the curing time from 42 to 144 h, at 120 degrees C and 10 mTorr vacuum. As expected, PGS became a stiffer, tougher, and stronger elastomer with increasing curing time. PGS disks were subcutaneously implanted into rats and periodically harvested; only mild tissue responses were observed and the biocompatibility remained excellent. Regardless of crosslink density, surface erosion degradation was observed. The sample dimensions linearly decreased with implantation time, and the mass loss rates were constant after 1-week implantation. As surface erosion degradation frequently correlates with enzymatic digestion, parallel in vitro digestion studies were conducted in lipase solutions which hydrolyze ester bonds. Enzymatic digestion played a significant role in degrading PGS, and the mass loss rates were not a function of curing time. Alternative chemistry approaches will be required to decrease the enzymatic hydrolysis rate of the ester bonds in PGS polymers.

Biological evaluation of a novel chitosan-PVA-based local delivery system for treatment of periodontitis.

In the study, we intend to design a suitable localized drug delivery system (LDDS) with chitosan and poly vinyl alcohol (PVA) for treating serve periodontitis. For that, a novel formulation based on the incorporation of chitosan-based microspheres into PVA film was prepared. As the core parts of the novel formulation, chitosan-based microspheres were prepared form chitosan and/or carboxymethyl-chitosan (CM-chitosan) by using water-in-oil emulsification method. Then basic in vitro and in vivo experiments focusing on biocompatibility and biodegradability of the two chitosan-based microspheres were carried out to evaluate the feasibility of the novel LDDS. In vitro tests, besides having no hemolysis, chitosan microsphere (Cs1-Ms), and CM-chitosan microsphere (Cs2-Ms) have adsorbed little proteins on their surfaces. Moreover, plasma proteins adsorbed on Cs2-Ms, most of which can easily desorbed, are much less than that adsorbed on Cs1-Ms. This indicates that Cs2-Ms perhaps has better biocompatibility than Cs1-Ms. In vivo tests, Cs1-Ms and Cs2-Ms were subcutaneously implanted in rat to investigate the host tissue inflammatory response. Implantations of Cs1-Ms and Cs2-Ms induced a little more severe inflammation when compared with the implantation of PVA film. However, the difference on in vivo biocompatibility between Cs1-Ms and Cs2-Ms could not be confirmed by the implantation model of our experiments. Both Cs1-Ms and Cs2-Ms had suffered bioerosion when they were subcutaneously implanted. The hard and compact matrixes of Cs1-Ms were degraded very slowly, and only some trifling degradation had been found until 4 weeks of implantation. In contrast, Cs2-Ms is soft and more hydrophilic, and can be quickly degraded in a form of diffluence by the physiological circumstance. All these results suggested that Cs2-Ms had better potentials used as core parts of the novel designed LDDS in the future developments.

The role of skin and subcutaneous tissues in Dupuytren's contracture: an electron microscopic observation.

OBJECTIVE: To determine the relationship between the overlying tissues and recurrence of Dupuytren's contracture. METHODS: Forty-three patients (68 hands) who accepted surgical treatment were divided into two groups according to treatment methods: partial fasciectomy or dermofasciectomy and full-thickness skin graft. Diseased palmar fascia, subcutaneous tissues and skin obtained during surgery were then assessed by electron microscopy. RESULTS: All patients were followed up. None of the hands which accepted dermofasciectomy and full-thickness skin grafting recurred, while 46.4% of the hands which accepted partial fasciectomy recurred. Under electron microscopic observation, myofibroblasts were found in the skin and subcutaneous tissues. CONCLUSION: The overlying tissues play an important role in Dupuytren's contracture, which may be a reason for recurrence of this condition after surgical treatment.

High-resolution electron microscopy of multi-wall carbon nanotubes in the subcutaneous tissue of rats.

The atomic structure of multi-wall carbon nanotubes (MWCNTs) implanted in the subcutaneous tissue of rats was examined by means of high-resolution transmission electron microscopy (HRTEM). Clusters of the MWCNTs implanted in the subcutaneous tissue were well recognized by the TEM observations. It was indicated that some nanotubes were taken in phagocytes after the 1-year implantation. The deterioration of crystalline structure of the nanotubes in phagocytes was shown by the HRTEM observation. It was suggested that the deterioration of the nanotubes was due to the peeling of the outer graphene layers in the phagocytes.