Roman V. Deev

Bioceramics composed of octacalcium phosphate demonstrate enhanced biological behavior.

Bioceramics are used to treat bone defects but in general do not induce formation of new bone, which is essential for regeneration process. Many aspects related to bioceramics synthesis, properties and biological response that are still unknown and, there is a great need for further development. In the most recent research efforts were aimed on creation of materials from biological precursors of apatite formation in humans. One possible precursor is octacalcium phosphate (OCP), which is believed to not only exhibit osteoconductivity but possess osteoinductive quality, the ability to induce bone formation. Here we propose a relatively simple route for OCP ceramics preparation with a specifically designed microstructure. Comprehensive study for OCP ceramics including biodegradation, osteogenic properties in ortopic and heterotopic models and limited clinical trials were performed that demonstrated enhanced biological behavior. Our results provide a possible new concept for the clinical applications of OCP ceramics.

pCMV-vegf165 Intramuscular Gene Transfer is an Effective Method of Treatment for Patients With Chronic Lower Limb Ischemia

Effective treatment of chronic lower limb ischemia is one of the most challenging issues confronting vascular surgeons. There are a number of choices available to the vascular surgeon. Open or endovascular revascularization is the treatment of choice when applicable. Current pharmacological therapies play an auxiliary role and cannot prevent disease progression. Therefore, new methods of treatment are needed. We conducted a phase 2b/3 multicenter randomized controlled clinical trial of the intramuscular transfer of a plasmid DNA encoding vascular endothelial growth factor (VEGF) 165 with cytomegalovirus promotor (CMV) in patients with atherosclerotic lower limb ischemia. A total of 100 patients were enrolled in the study, that is, 75 patients were randomized into the test group and received 2 intramuscular injections of 1.2 mg of pCMV-vegf165, 14 days apart together with standard pharmacological treatment. In all, 25 patients were randomized into the control group and received standard treatment only. The following end points were evaluated within the first 6 months of the study and during a 1.5-year additional follow-up period: pain-free walking distance (PWD), ankle–brachial index (ABI), and blood flow velocity (BFV). The pCMV-vegf165 therapy appeared to be significantly more effective than standard treatment. The PWD increased in the test group by 110.4%, 167.2%, and 190.8% at 6 months, 1 year, and 2 years after treatment, respectively. The pCMV-vegf165 intramuscular transfer caused a statistically significant increase in ABI and BFV. There were no positive results in the control group. Thus, pCMV-vegf165 intramuscular gene transfer is an effective method of treatment of moderate to severe claudication due to chronic lower limb ischemia.

3D Printing of Octacalcium Phosphate Bone Substitutes

Biocompatible calcium phosphate ceramic grafts are able of supporting new bone formation in appropriate environment. The major limitation of these materials usage for medical implants is the absence of accessible methods for their patient-specific fabrication. 3D printing methodology is an excellent approach to overcome the limitation supporting effective and fast fabrication of individual complex bone substitutes. Here, we proposed a relatively simple route for 3D printing of octacalcium phosphates (OCP) in complexly shaped structures by the combination of inkjet printing with post-treatment methodology. The printed OCP blocks were further implanted in the developed cranial bone defect followed by histological evaluation. The obtained result confirmed the potential of the developed OCP bone substitutes, which allowed 2.5-time reducing of defect’s diameter at 6.5 months in a region where native bone repair is extremely inefficient.

Ordinary and Activated Bone Grafts: Applied Classification and the Main Features

Bone grafts are medical devices that are in high demand in clinical practice for substitution of bone defects and recovery of atrophic bone regions. Based on the analysis of the modern groups of bone grafts, the particularities of their composition, the mechanisms of their biological effects, and their therapeutic indications, applicable classification was proposed that separates the bone substitutes into “ordinary” and “activated.” The main differential criterion is the presence of biologically active components in the material that are standardized by qualitative and quantitative parameters: growth factors, cells, or gene constructions encoding growth factors. The pronounced osteoinductive and (or) osteogenic properties of activated osteoplastic materials allow drawing upon their efficacy in the substitution of large bone defects.

Clinical-instrumental and morphological evaluation of the effect of autologous dermal fibroblasts administration

Basic molecular mechanisms, associated with the main cell population of the dermis – fibroblasts – are the basis of skin aging. The number of functionally active fibroblasts in the skin and their biosynthetic activity decreases with age, thus enhancement of their cell density with synthetically active cells is accepted as a one of the most effective methods. The objective of the present study was to evaluate the safety and effectiveness of intradermal administration of autologous dermal fibroblasts in a year after treatment of 17 patients, aged 45–65 years. Results obtained with modern instrumental skin diagnostic methods (vacuum cutometry, optical profilometry, VISIA photometric analysis, etc.) demonstrate the safety and clinical effectiveness of dermal autofibroblast therapy: after transplantation, cultured autofibroblasts keep their biosynthetic activity and produce extracellular matrix for at least 12 months. As a result, remodelling of the dermis microstructures is observed, accompanied by a progressive increase of collagen content and thickness of the dermis (up to 62.5 ±6.7% in 12 months). This is clinically expressed by increase of skin elasticity (24.0 ±4.3% in periorbital area) and thickness of the skin, and by decrease in the number and depth of wrinkles (46 ±7% by the end of observation period). Copyright

In vitro assessment of electrospun polyamide‐6 scaffolds for esophageal tissue engineering

Artificial tissue-engineered grafts offer a potential alternative to autologous tissue grafts for patients, which can be traumatic. After decellularizing Papio hamadryas esophagus and studying the morphology and physical properties of the extracellular matrix (ECM), we generated electrospun polyamide-6 based scaffolds to mimic it. The scaffolds supported a greater mechanical load than the native ECM and demonstrated similar 3D microstructure, with randomly aligned fibers, 90% porosity, 29 μm maximal pore size, and average fiber diameter of 2.87 ± 0.95 µm. Biocompatibility studies showed that human adipose- and bone marrow-derived mesenchymal stromal cells (AD-MSC and BMD-MSC) adhered to the scaffold surface and showed some proliferation: scaffold cell coverage was 25% after 72 h of incubation when seeded with 1000 cells/mm2 ; cells elongated processes along the polyamide-6, although they flattened 1.67-4 times less than on cell culture plastic. Human umbilical vein endothelial cells, however, showed poor adherence and proliferation. We thus provide in vitro evidence that polyamide-6 scaffolds approximating the esophageal biomechanics and 3D topography of nonhuman primates may provide a biocompatible substrate for both AD-MSC and BMD-MSCs, supporting their adhesion and survival to some degree.

186. Long-Term Results of pCMV-vegf165 Intramuscular Gene Transfer in Patients With Chronic Lower Limb Ischemia

Restriction of blood flow due to atherosclerosis leads to chronic lower limb ischemia (CLI) affected 3-8% of the general population. Current pharmacological therapies have limited effects. Therefore, developing new treatments is pivotal. Gene therapy is targeted at inducing angiogenesis in ischemic extremities via increased endogenous expression of vascular growth factors. Among many, plasmid DNA vectors play a significant role in gene therapy and are safe with low immunogenicity and no risk of insertional mutagenesis.A novel drug “Neovasculgen” became the first gene therapy for patients with CLI approved by regulatory authorities in Russia in 2011. It contains a plasmid DNA encoding vascular endothelial growth factor VEGF165 (pCMV-vegf165). There was conducted a multicentre randomised controlled clinical trial of the intramuscular transfer of this new gene product in 100 patients with CLI, stage 2a-3 disease according to Fontaine. The trial lasted 6 months. It was concluded that pCMV-vegf165 transfer significantly improved pain-free walking distance (PWD) and caused no adverse effects. Efficacy and safety of this therapy were evaluated and translated into practice. Most of patients enrolled in the trial gave their consent to participate in the long-term follow-up study. The primary endpoint was PWD, the secondary – ankle-brachial index (ABI) and transcutaneous oxygen tension (TcPO2).PWD in patients who received pCMV-vegf165 increased throughout the 3-year follow-up period: by the end of the 1st year the parameter increased by 167.2% as compared to the 135.3 m baseline (p<0.05). The tendency remained positive throughout the following two years of monitoring (Fig. 1Fig. 1). PWD decreased by 27% within a 3-year follow-up period in control patients who received conventional therapy alone (p<0.05).Fig. 1Pain-free walking distance in patients of test (pCMV-vegf165) and control groups.View Large Image | Download PowerPoint SlideChanges in secondary endpoints were less frequent and less pronounced. TcPO2 increased by 14% (10mm Hg) in the pCMV-vegf165 group by the end of the 1st year (p<0.05). The value remained stable during the next two years. TcPO2 slightly decreased in the control patients throughout the 3-year follow-up period. ABI significantly increased in the test group at each time point, but the difference comparing with control was not statistically significant.We conclude that gene therapy stably increases PWD and improves functional state of patients with CLI.

Results of 5-year follow-up study in patients with peripheral artery disease treated with PL-VEGF165 for intermittent claudication

Background: The effective treatment of chronic lower limb ischemia is one of the most challenging issues confronting vascular surgeons. Current pharmacological therapies play an auxiliary role and cannot prevent disease progression, and new treatment methods are needed. In 2011, a plasmid VEGF65-gene therapy drug was approved in Russia for the treatment of chronic lower limb ischemia (ClinicalTrials.gov identifier: NCT03068585). The objective of this follow-up study was to evaluate the long-term safety and efficacy of gene therapy in patients with limb ischemia of atherosclerotic genesis. Aims: To evaluate the long-term safety and efficacy of the therapeutic angiogenesis, 36 patients in the treatment group (pl-VEGF165) and 12 patients in the control group participated in a 5-year follow-up study. Planned examinations were carried out annually for 5 years after pl-VEGF165 administration. Results: Differences in the frequency of major cardiovascular events (pl-VEGF165 5/36 versus control 2/12; p = 0.85), malignancies (pl-VEGF165 1/36 versus control 0/12; p = 0.38) and impaired vision (there was none in either group) over the 5-year follow-up period did not achieve statistical significance. The target limb salvage was 95% (n = 36) and 67% (n = 12) in the pl-VEGF165 and control groups, respectively. The pain-free walking distance value increased by 288% from 105.7 ± 16.5 m to 384 ± 39 m in the treatment group by the end of the fifth year, with a peak of 410.6 ± 86.1 m achieved by the end of the third year. The ankle-brachial index (ABI) increased from 0.47 ± 0.01 to 0.56 ± 0.02 by the end of the first year, with a subsequent slight decrease to 0.51 ± 0.02 by the fifth year. The maximum increment of transcutaneous oximetry test (tcoO2) by 36%, from 66.6 ± 3.7 mm Hg to 90.7 ± 4.9 mm Hg, was observed by the end of the second year. Conclusion: The therapeutic effect of angiogenesis induction by gene therapy persists for 5 years.

Caroli syndrome: a clinical case with detailed histopathological analysis

Herein we present a clinical case of the Caroli syndrome caused by the compound heterozygous mutation in the PKHD1 gene. Histopathological assessment of liver detected biliary cirrhosis, numerous dilated bile ducts of various sizes, hyperplastic cholangiocytes containing a large amount of acid mucopolysaccharides, decreased ß-tubulin expression and increased proliferation of cholangiocytes. A significant proportion of hepatic tissue was composed of giant cysts lined with a single layer of cholangiocytes, containing pus and bile in its lumen and surrounded by granulation tissue. An accumulation of neutrophils in the lumen of the bile ducts was observed, as well as an infiltration of the ducts and cysts surrounding connective tissue by CD4+ and to a lesser extent CD8+ lymphocytes. This may be caused by the expression of HLA-DR by cholangiocytes. Atrophy and desquamation of the epithelium of collecting tubules with the formation of microcysts were detected in the kidneys without a clinically significant loss of renal function. Morphopathogenetic mechanisms of the Caroli syndrome can be targets for a potential pathogenetic therapy and prevention of its manifestations and complications.

Twenty-Year Clinical Progression of Dysferlinopathy in Patients from Dagestan

To date, over 30 genes with mutations causing limb-girdle muscle dystrophy have been described. Dysferlinopathies are a form of limb-girdle muscle dystrophy type 2B with an incidence ranging from 1:1,300 to 1:200,000 in different populations. In 1996, Dr. S. N. Illarioshkin described a family from the Botlikhsky district of Dagestan, where limb-girdle muscle dystrophy type 2B and Miyoshi myopathy were diagnosed in 12 members from three generations of a large Avar family. In 2000, a previously undescribed mutation in the DYSF gene (c.TG573/574AT; p. Val67Asp) was detected in the affected members of this family. Twenty years later, in this work, we re-examine five known and seven newly affected family members previously diagnosed with dysferlinopathy. We observed disease progression in family members who were previously diagnosed and noted obvious clinical polymorphism of the disease. A typical clinical case is provided.