Author Correction: Cardiovascular, hematological and neurosensory impact of COVID-19 and variants.

Correction to: European Review for Medical and Pharmacological Sciences 2021; 25 (8): 3350-3364-DOI: 10.26355/eurrev_202104_25747-PMID: 33928623, published online 30 April, 2021. After publication, the authors requested to correct the Acknowledgements of the above-mentioned article. There are amendments to this paper. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/25747.

Cardiovascular, hematological and neurosensory impact of COVID-19 and variants.

OBJECTIVE: The purpose of this article was to review our clinical experience with COVID-19 patients observed in the Cardiovascular Division of Pompidou Hospital (University of Paris, France) and the Department of Neurology of the Eastern Piedmont University (Novara, Italy), related to the impact on the cardiovascular, hematological, and neurologic systems and sense organs. PATIENTS AND METHODS: We sought to characterize cardiovascular, hematological, and neurosensory manifestations in patients with COVID-19 and variants. Special attention was given to initial signs and symptoms to facilitate early diagnosis and therapy. Indications of ECMO (extracorporeal membrane oxygenation) for cardiorespiratory support were evaluated. RESULTS: Preliminary neurosensorial symptoms, such as anosmia and dysgeusia, are useful for diagnosis, patient isolation, and treatment. Early angiohematological acro-ischemic syndrome includes hand and foot cyanosis, Raynaud digital ischemia phenomenon, skin bullae, and dry gangrene. This was associated with neoangiogenesis, vasculitis, and vessel thrombosis related to immune dysregulation, resulting from "cytokine storm syndrome". The most dangerous complication is disseminated intravascular coagulation, with mortality risks for both children and adults. CONCLUSIONS: COVID-19 is a prothrombotic disease with unique global lethality. A strong inflammatory response to viral infection severely affects cardiovascular and neurological systems, as well as respiratory, immune, and hematological systems. Rapid identification of acro-ischemic syndrome permits the treatment of disseminated intravascular coagulation complications. Early sensorial symptoms, such as gustatory and olfactory loss, are useful for COVID-19 diagnosis. New variants of SARS-CoV-2 are emerging, principally from United Kingdom, South Africa, and Brazil. These variants seem to spread more easily and quickly, which may lead to more cases of COVID.

Needleless electrospun and centrifugal spun poly-ε-caprolactone scaffolds as a carrier for platelets in tissue engineering applications: A comparative study with hMSCs.

The biofunctionalization of scaffolds for tissue engineering is crucial to improve the results of regenerative therapies. This study compared the effect of platelet-functionalization of 2D electrospun and 3D centrifugal spun scaffolds on the osteogenic potential of hMSCs. Scaffolds prepared from poly-ε-caprolactone, using electrospinning and centrifugal spinning technology, were functionalized using five different concentrations of platelets. Cell proliferation, metabolic activity and osteogenic differentiation were tested using hMSCs cultured in differential and non-differential medium. The porous 3D structure of the centrifugal spun fibers resulted in higher cell proliferation. Furthermore, the functionalization of the scaffolds with platelets resulted in a dose-dependent increase in cell metabolic activity, proliferation and production of an osteogenic marker - alkaline phosphatase. The effect was further promoted by culture in an osteogenic differential medium. The increase in combination of both platelets and osteogenic media shows an improved osteoinduction by platelets in environments rich in inorganic phosphate and ascorbate. Nevertheless, the results of the study showed that the optimal concentration of platelets for induction of hMSC osteogenesis is in the range of 900-3000 × 109 platelets/L. The study determines the potential of electrospun and centrifugal spun fibers with adhered platelets, for use in bone tissue engineering.

Electrospun nanofibers as support for the healing of intestinal anastomoses.

The breakdown of intestinal anastomosis is a serious postsurgical complication. The worst complication is anastomotic leakage, resulting in contaminated peritoneal cavity, sepsis, multi-organ failure and even death. In problematic locations like the rectum, the leakage rate has not yet fallen below 10 %. Such a life-threatening condition is the result of impaired healing in the anastomotic wound. It is still vital to find innovative strategies and techniques in order to support regeneration of the anastomotic wound. This paper reviews the surgical techniques and biomaterials used, tested or published. Electrospun nanofibers are introduced as a novel and potential material in gastrointestinal surgery. Nanofibers possess several, unique, physical and chemical properties, that may effectively stimulate cell proliferation and collagen production; a key requirement for the healed intestinal wound.

Biodegradation of porous calcium phosphate scaffolds in an ectopic bone formation model studied by X-ray computed microtomograph.

Three types of ceramic scaffolds with different composition and structure [namely synthetic 100% hydroxyapatite (HA; Engipore), synthetic calcium phosphate multiphase biomaterial containing 67% silicon stabilized tricalcium phosphate (Si-TCP; Skelite) and natural bone mineral derived scaffolds (Bio-oss)] were seeded with mesenchymal stem cells (MSC) and ectopically implanted for 8 and 16 weeks in immunodeficient mice. X-ray synchrotron radiation microtomography was used to derive 3D structural information on the same scaffolds both before and after implantation. Meaningful images and morphometric parameters such as scaffold and bone volume fraction, mean thickness and thickness distribution of the different phases as a function of the implantation time, were obtained. The used imaging algorithms allowed a direct comparison and registration of the 3D structure before and after implantation of the same sub-volume of a given scaffold. In this way it was possible to directly monitor the tissue engineered bone growth and the complete or partial degradation of the scaffold. Further, the detailed kinetics studies on Skelite scaffolds implanted for different length of times from 3 days to 24 weeks, revealed in the X-ray absorption histograms two separate peaks associated to HA and TCP. It was therefore possible to observe that the progressive degradation of the Skelite scaffolds was mainly due to the resorption of TCP. The different saturation times in the tissue engineered bone growth and in the TCP resorption confirmed that the bone growth was not limited the scaffold regions that were resorbed but continued in the inward direction with respect to the pore surface.

Kinetics of in vivo bone deposition by bone marrow stromal cells within a resorbable porous calcium phosphate scaffold: an X-ray computed microtomography study.

Resorbable ceramic scaffolds based on Silicon stabilized tricalcium phosphate (Si-TCP) were seeded with bone marrow stromal cells (BMSC) and ectopically implanted for 2, 4, and 6 months in immunodeficient mice. Qualitative and quantitative evaluation of the scaffold material was performed by X-ray synchrotron radiation computed microtomography (microCT) with a spatial resolution lower than 5 microm. Unique to these experiments was that microCT data were first collected on the scaffolds before implantation and then on the same scaffolds after they were seeded with BMSC, implanted in the mice and rescued after different times. Volume fraction, mean thickness and thickness distribution were evaluated for both new bone and scaffold phases as a function of the implantation time. New bone thickness increased from week 8 to week 16. Data for the implanted scaffolds were compared with those derived from the analysis of the same scaffolds prior to implantation and with data derived from 100% hydroxyapatite (HA) scaffold treated and analyzed in the same way. At variance with findings with the 100% HA scaffolds a significant variation in the density of the different Si-TCP scaffold regions in the pre- and post-implantation samples was observed. In particular a post-implantation decrease in the density of the scaffolds, together with major changes in the scaffold phase composition, was noticeable in areas adjacent to newly formed bone. Histology confirmed a better integration between new bone and scaffold in the Si-TCP composites in comparison to 100% HA composites where new bone and scaffold phases remained well distinct.

Bulk and interface investigations of scaffolds and tissue-engineered bones by X-ray microtomography and X-ray microdiffraction.

This review is presented of recent investigations concerning the structure of ceramic scaffolds and tissue-engineered bones and focused on two techniques based on X-ray radiation, namely microtomography (microCT) and microdiffraction. Bulk 3D information, with micro-resolution, is mainly obtained by microCT, whereas microdiffraction provides useful information on interfaces to the atomic scale, i.e. of the order of the nanometer. Since most of the reported results were obtained using synchrotron radiation, a brief description of the European Synchrotron Radiation Facility (ESRF) is presented, followed by a description of the two techniques. Then examples of microstructural investigations of scaffolds are reported together with studies on bone architecture. Finally, studies on ex vivo tissue-engineered bone and on bone microstructure in vivo are presented.

High-resolution X-ray microtomography for three-dimensional visualization of human stem cell muscle homing.

In the perspective of clinical translation of stem cell research, it would be advantageous to develop new techniques to detect donor cells after transplantation to track their fate and thus better understand their role in regeneration of damaged and diseased tissues. In this study we use X-ray computed microtomography for three-dimensional visualization of stem cells that were labeled with magnetic nanoparticles and transplanted via intra-arterial infusion. We show that X-ray computed microtomography offers the possibility to detect with high definition and resolution human cells after transplantation, and opens new possibilities for both experimental stem cell research.

Engineered bone from bone marrow stromal cells: a structural study by an advanced x-ray microdiffraction technique.

The mechanism of mineralized matrix deposition was studied in a tissue engineering approach in which bone tissue is formed when porous ceramic constructs are loaded with bone marrow stromal cells and implanted in vivo. We investigated the local interaction between the mineral crystals of the engineered bone and the biomaterial by means of microdiffraction, using a set-up based on an x-ray waveguide. We demonstrated that the newly formed bone is well organized inside the scaffold pore, following the growth model of natural bone. Combining wide angle (WAXS) and small angle (SAXS) x-ray scattering with high spatial resolution, we were able to determine the orientation of the crystallographic c-axis inside the bone crystals, and the orientation of the mineral crystals and collagen micro-fibrils with respect to the scaffold. In this work we analysed six samples and for each of them two pores were studied in detail. Similar results were obtained in all cases but we report here only the most significant sample.

Kinetics of in vivo bone deposition by bone marrow stromal cells into porous calcium phosphate scaffolds: an X-ray computed microtomography study.

In a typical bone tissue engineering application, osteogenic cells are harvested and seeded on a three-dimensional (3D) synthetic scaffold that acts as guide and stimulus for tissue growth, creating a tissue engineering construct or living biocomposite. Despite the large number of performed experiments in different laboratories, information on the kinetics of bone growth into the scaffolds is still scarce. Highly porous hydroxyapatite scaffolds were investigated before the implantation and after they were seeded with in vitro expanded bone marrow stromal cells (BMSC) and implanted for 8, 16, or 24 weeks in immunodeficient mice. Synchrotron x-ray computed microtomography (microCT) was used for qualitative and quantitative 3D characterization of the scaffold material and 3D evaluation of tissue engineered bone growth kinetics after in vivo implantation. Experiments were performed taking advantage of a dedicated set up at the European Synchrotron Radiation Facility (ESRF, Grenoble, France), which allowed quantitative imaging at a spatial resolution of about 5 microm. A peculiarity of these experiments was the fact that at first the data were obtained on the different pure scaffolds, then the same scaffolds were seeded by BMSC, implanted, and brought again to ESRF for investigating the formation of new bone. The volume fraction, average thickness, and distribution of the newly formed bone were evaluated as a function of the implantation time. New bone thickness increased from week 8 to week 16, but deposition of new bone was arrested from week 16 to week 24. Instead, mineralization of the newly deposited bone matrix continued up to week 24.

Tissue engineering of bone: search for a better scaffold.

BACKGROUND: Large bone defects still represent a major problem in orthopedics. Traditional bone-repair treatments can be divided into two groups: the bone transport (Ilizarov technology) and the graft transplant (autologous or allogeneic bone grafts). Thus far, none of these strategies have proven to be always resolving. As an alternative, a tissue engineering approach has been proposed where osteogenic cells, bioceramic scaffolds, growth factors and physical forces concur to the bone defect repair. Different sources of osteoprogenitor cells have been suggested, bone marrow stromal cells (BMSC) being in most cases the first choice. METHODS AND RESULTS: In association with mineral tridimensional scaffolds, BMSC form a primary bone tissue which is highly vascularized and colonized by host hemopoietic marrow. The chemical composition of the scaffold is crucial for the osteoconductive properties and the resorbability of the material. In addition, scaffolds should have an internal structure permissive for vascular invasion. Porous bioceramics [hydroxyapatite (HA) and tricalcium phosphate] are osteoconductive and are particularly advantageous for bone tissue engineering application as they induce neither an immune nor an inflammatory response in the implanted host. Earlier, we first reported a cell-based tissue engineering procedure to treat three patients with long bone segmental defects. Cells were loaded on a 100% HA porous ceramic. These scaffolds proved to have good osteoconductive properties resulting in a good functional recovery, but they have not been resorbed after more than 5 years from the implant. In addition, due to the high density of the mineral and the relatively low porosity (50-60%), it was very difficult to monitor the patient recovery during the post-surgery time using X-rays. CONCLUSIONS: We report here some pre-clinical testing of new scaffolds. To compare these second generation ceramic scaffolds more suitable for a tissue engineering approach we had to first establish animal models and analysis procedures including the use of X-ray-computed microtomography associated with X-rays synchroton radiation.

Phase-contrast microtomography of thin biomaterials.

Phase-contrast microtomography, performed at the beamline ID 22 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France), is demonstrated for high-resolution 3-D imaging of a hydroxyapatite sample. The technique, which relies on phase contrast imaging, gives the possibility to observe features inside samples with negligible absorption contrast. The positive results obtained suggest a possible future investigation of the influence of the distribution of pores and defects inside biomaterial coatings, on the growth of osteoblast cells.

Influence of fluorapatite minor additions on behavior of hydroxyapatite ceramics.

Fluorinated hydroxyapatite is known to be less soluble by body fluids, resulting in enhanced resistance to biodegradation in vivo conditions, as compared to the pure hydroxyapatite ceramics. The present work was aimed at the investigation of the effect of minor additions of ultrafine fluorapatite (up to 10 wt%) on the sintering behavior and mechanical properties of hydroxyapatite ceramics. In vitro testing for the osteoblast-like cells viability and proliferation was performed with the samples of varying fluorapatite content. It was found that the fluorapatite addition hinders the sintering shrinkage and lowers the strength, but does not generally affect negatively the viability of the cells.

Osteointegration of bioactive glass-coated and uncoated zirconia in osteopenic bone: an in vivo experimental study.

In elderly and osteoporotic patients an age-related loss of osteoinductivity could be the biological cause of implant failure regardless of the high quality of the implanted device. yttria stabilized tetragonal zirconia (YSTZ), either coated with the bioactive glass named RKKP bioglaze (RKKP) or uncoated, was implanted in the distal femurs of sham-operated and ovariectomized female rats. Animals were sacrificed at 30 and 60 days. Histomorphometry and microhardness tests were performed to assess osteointegration rate as well as bone quality around the implants. Significant decreases (p < 0.0005) in trabecular bone volume, BV/TV (41%), trabecular bone surface BS/TV (33%), trabecular thickness Tb.Th (20%), and trabecular number Tb.N (32%), together with a significant increase in trabecular separation Tb.Sp (184%), were found for the osteopenic rats compared with the sham-operated rats. At both experimental times the RKKP coating ensured a better osteointegration rate with higher AI values than the uncoated YSTZ, even when osteopenic rats were used (48% at 30 days and 12% at 60 days). No differences were observed at the bone-biomaterial interfaces for either material when comparing sham-operated with osteopenic rats. The present results demonstrate that the RKKP bioactive glass used as a coating ensures a high osteointegration rate even in osteoporotic bone, which is already visible from postoperative day 30 and is still apparent on day 60.

Synchrotron radiation microtomography of bone engineered from bone marrow stromal cells.

Osteoprogenitor cells expanded in vitro and associated with porous ceramic scaffolds have been proposed as bone substitutes. Animal models have been developed to test the efficacy of various cell populations and scaffolds in promoting bone repair. Qualitative analysis of the new bone formed within the ceramic scaffold is relatively easy by conventional histology. On the other hand, quantitative data are difficult to obtain. X-ray computed microtomography was used as a possible experimental technique to obtain quantitative data on the three-dimensional structure of newly formed bone and of remaining scaffold in implants after 8 weeks in vivo. Measurements were performed at the European Synchrotron Radiation Facility on beamline ID19 with a spatial resolution of about 5 microm. This study clearly indicates the possibility of nondestructive quantitative analysis of bone-engineered constructs. The technique appears suitable to compare different scaffolds (and possibly different cell populations) with regard to bone formation efficiency and reabsorbability of biomaterials in the immunodeficient mouse model.

X-ray micro-diffraction analysis of reconstructed bone at Zr prosthetic surface with sub-micrometre spatial resolution.

The purpose of the present investigation is to demonstrate the power of the x-ray micro-diffraction technique in biological studies. In particular the reported experiment concerns the study of the interface between a Zr prosthetic device implanted in a rat femur and the newly-formed bone, with a spatial resolution of 0.5 microm. The obtained results give interesting information on the Zr deformation and on the crystallographic phase, the grain size and the orientation of the new bone. Moreover the study reveals a marked difference in the structure of the reconstructed bone with respect to the native bone, which cannot be appreciated with other techniques.

Osteointegration of bioactive glass-coated zirconia in healthy bone: an in vivo evaluation.

Osteointegration of yttria stabilised tetragonal zirconia (YSTZ), either coated with bioactive glass named RKKP bioglaze (RKKP) or uncoated, was evaluated in an animal model. RKKP-coated and uncoated (controls) YSTZ cylinders were implanted in the distal femoral epiphyses of 14 Sprague Dawley rats under general anaesthesia. At the experimental times of 30 and 60 days after sacrifice, histomorphometry and SEM microanalysis were performed on methylmethacrylate-embedded undecalcified sections to determine the osteointegration rate. At 30 days, a significantly higher affinity index was demonstrated in vivo by histomorphometric evaluation in RKKP-coated versus uncoated YSTZ implants p < 0.05); at 60 days, the coated implants behaved better than controls (affinity index of + 32%), but the difference observed lay within the statistical uncertainty. SEM analysis demonstrated better bone adhesion to the material in RKKP-coated YSTZ at both 30 and 60 days. These findings suggest that YSTZ coated with the bioactive glass named RKKP enhances osteointegration of ceramics.

Improvement in zirconia osseointegration by means of a biological glass coating: An in vitro and in vivo investigation.

The biocompatibility and osseointegration of zirconia (ZrO(2)), either coated with RKKP bioglazeor uncoated, were evaluated in vitro and in vivo. The in vitro test was performed in human osteoblasts, whereas maximal sensitization was performed in 23 Dunkin Hurtley guinea pigs. RKKP bioglaze-coated and uncoated (controls) ZrO(2) cylinders were implanted in the distal femoral epiphyses of 14 Sprague-Dawley rats under general anesthesia, and animals were sacrificed at 30 and 60 days. Lactate dehydrogenase, alkaline phosphatase, and Thiazolyl Blue (MTT) were tested in vitro. A graded score was used for evaluating the results of the sensitization test. Histomorphometry and microhardness testing were performed to quantify the osseointegration rate, as well as bone quality around the implants. Neither in vitro cytotoxicity nor sensitization were observed. Histomorphometry demonstrated that at 30 days, the affinity index was significantly higher in coated implants than in uncoated ones (p < 0.05); at 60 days, the behavior of coated implants was better than that of uncoated ones, but differences were not significant. Significant increases in bone microhardness were found at 1000 microm from the interface area for both uncoated (p < 0.0005) and RKKP bioglaze-coated (p < 0.0005) ZrO(2), and also within 200 microm from the interface (p = 0.014) but only for coated ZrO(2.) These results suggest that RKKP bioglaze-coated ZrO(2) permits biocompatible devices with improved osseointegration properties to be manufactured.

Sugar-induced stabilization of the monoolein Pn3m bicontinuous cubic phase during dehydration.

To explore the molecular mechanism of the protective function of sugars on cubic lipidic systems, the mesomorphic properties of the monoolein-water system, dehydrated in the presence of a series of sugars, have been studied by osmotic stress experiments. Two bicontinuous inverse cubic structures (Pn3m and Ia3d) and a lamellar L(alpha) phase form under dehydration in pure water. In sugar solutions, the Pn3m phase shows an extraordinary stability: as a function of sugar concentration, the lattice parameter decreases to very low values, but no phase transitions occur. Instead, the Pn3m to Ia3d phase transition is obtained by equilibrating the lipid phase with aqueous polymer solutions of increasing osmotic pressure. As a result, the pressure at which the phase transition occurs strongly depends on sugar concentration. The free-energy curves obtained from the osmotic-pressure unit-cell data show that the sugar exerts an additional stabilization on both the cubic phases. The analysis of the structural parameters indicates that sugars alter the interface geometry. We suggest that a consequent release of stretching contributions in the chain packing or a reduction of the inhomogeneity in molecular splay mainly stabilize the Pn3m phase and prevent the transition to the Ia3d phase on dehydration.

Biological glass coating on ceramic materials: in vitro evaluation using primary osteoblast cultures from healthy and osteopenic rat bone.

ZrO2 and Al2O3 substrates were successfully coated by a double layer of a silica-based glass named RKKP, using a low-cost firing technique. RKKP is a glass well known for its bioactivity; therefore, a RKKP coating on Al2O3 or ZrO2, allows to combine the excellent mechanical properties of these strong ceramic substrates with its bioactivity. ZrO2 samples were easily coated using a double layer of RKKP by a simple enamelling technique. To accommodate the thermal expansion coefficient mismatch between Al2O3 and RK K P, this substrate was coated using a multilayered composite approach. All of the coatings were characterised from a morphological and compositional point of view, and an extensive biological evaluation was performed using fresh rat osteoblasts. Osteoblast primary cultures were derived from the trabecular bone of femoral condyles harvested from intact (NB) and osteopenic (OB) rats. After characterisation of their phenotype, osteoblasts were seeded on material samples of ZrO2 or Al2O3 coated with RKKP, and cultured for 7 days. Cell proliferation (MTT test) and cell differentiation (alkaline phosphatase activity) were evaluated at the end of the experiment, to assess osteoblast behaviour in the presence of biomaterials and determine if the results were related to the host bone quality. Results of both materials showed a good level of biocompatibility. In particular, MTT significant higher values were detected in NB cultures on ZrO2-RKKP samples; ALP activity significantly increased in NB cultures on Al2O3-RKKP and in OB cultures on both coated samples.