Abstract:Treatment with leeches has been studied by researchers for centuries for rheumatic diseases, inflammatory diseases or post-surgical complications. There are over 100 bioactive substances with different properties in the salivary gland secretions of leeches.  These secretions have been shown to be effective in vasodilation, analgesia, suppression of inflammation, increase of anticoagulation in blood, inhibition of bacterial proliferation, resolution of edema, acceleration of microcirculation, increase of permeability and vitality in damaged vascular structures of organs and tissues, correction of hypoxia, reduction of blood pressure, reduction of pain and regulation of immunity (1).Food and Drug Administration of USA permitted the sale of leeches and their use in general purpose and plastic surgery and microsurgery in 2004.Ankylosing spondylitis (AS) is an inflammatory, chronic, progressive disease of unknown etiology. It often presents with insidious onset inflammatory low back pain. It is a disease that  progresses with sacroiliac joint inflammation in the early stages and axial and peripheral joint inflammation in the late stages, and can also involve the eye, lung and intestine. The HLA-B27 gene has a great effect on the etiology (2).The “European League Against Rheumatism (EULAR)” has developed evidence-based recommendations for the treatment and care of patients with AS. Nonsteroidal anti-inflammatory drugs (NSAIDs), local corticosteroid administration, disease-modifying antirheumatic drug therapy such as sulfasalazine (SLZ), methotrexate, and tumor necrosis factor inhibitors from biological treatments are among the treatment options as pharmacological treatments (3). All 3 of our patients have chronic low back pain and are being treated with the diagnosis of AS with axial involvement, which is an etiologically inflammatory disease. Patients regularly use NSAIDs and SLZ 2 gr/day.Patients received additional leech therapy while continuing standard medical therapy. The difficulty in doing activities at home and at work improved by 40% in the first patient, 25% in the second patient, and 37.5% in the third patient. One month after the leech application, morning stiffness, which lasted for 1 hour, disappeared completely in two of our patients, while it decreased to 10 minutes in the other patient. Fatigue scores of the patients decreased by 50%, 54% and 37.5%. All three of our patients stopped using NSAIDs and stated that they could tolerate their current pain with exercise. According to the VAS pain scale, three patients reported their pain before hirudotherapy as 7, after treatment as 4 in one patient and as 3 in the other two patients. It was observed that CRP values decreased to normal after 1 month in all three patients.Fatigue, morning stiffness and acute phase reactants decreased in all three patients. There were no side effects.In conclusion, although there are many studies with hirudotherapy in various diseases in the medical literature, research on inflammatory low back pain is limited. The results obtained from three patients show us that promising results can be obtained with hirudotherapy for AS patients.

Biocompatibility of ceramic materials in Ca2P2O7-Ca(PO3)2 system was investigated using different methods, including in vitro and in vivo tests. Ceramic materials in the Ca2P2O7-Ca(PO3)2 system were obtained by annealing cement-salt stone based on powder mixtures of calcium citrate tetrahydrate Ca3(C6H5O7)2·4H2O and monocalcium phosphate monohydrate (MCPM) Ca(H2PO4)2·H2O. The phase composition of cement-salt stone included brushite, monetite as a result of chemical reaction of starting components after adding of water. The presence of citric acid as by-product of chemical reaction, leads to increase the setting time of the cement-salt stone. Highly concentrated aqueous suspensions based on calcium citrate and MCPM powders providing content of calcium polyphosphate Ca(PO3)2 up to 20 wt % in ceramics were used for designing bioresorbable materials. The presence of an excess of monocalcium phosphate monohydrate makes it possible to reduce the annealing temperature of ceramics, which is associated with the formation of a lower melting phase of Ca(PO3)2. In vivo tests shown that obtained ceramic materials can be recommended for regenerative treatments for bone defects.Biocompatibility of ceramic materials in Ca2P2O7-Ca(PO3)2 system was investigated using different methods, including in vitro and in vivo tests. Ceramic materials in the Ca2P2O7-Ca(PO3)2 system were obtained by annealing cement-salt stone based on powder mixtures of calcium citrate tetrahydrate Ca3(C6H5O7)2·4H2O and monocalcium phosphate monohydrate (MCPM) Ca(H2PO4)2·H2O. The phase composition of cement-salt stone included brushite, monetite as a result of chemical reaction of starting components after adding of water. The presence of citric acid as by-product of chemical reaction, leads to increase the setting time of the cement-salt stone. Highly concentrated aqueous suspensions based on calcium citrate and MCPM powders providing content of calcium polyphosphate Ca(PO3)2 up to 20 wt % in ceramics were used for designing bioresorbable materials. The presence of an excess of monocalcium phosphate monohydrate makes it possible to reduce the annealing temperature of ceramics, which is associated with the formation of a lower melting phase of Ca(PO3)2. In vivo tests shown that obtained ceramic materials can be recommended for regenerative treatments for bone defects.

Introduction: Open fractures involving the distal femur are secondary to high velocity trauma. These patients often exhibit critical bone defects, which pose a serious therapeutic challenge. Case: A 19 years old male presented with an open fracture involving the right distal femur with substantial bone loss, involving near total metaphysis, mostly the medial column, part of the diaphysis and an articular fracture. The patient also had an ipsilateral patella and an extra articular proximal tibia fracture at the metaphysio-diaphysial junction. We planned for a two-staged intervention, where in the first stage we treated soft tissue injury defects along with definitive primary fixation of tibia and patella; we also stabilized the limb with a spanning external fixator. In the second stage we reconstructed the distal medial column with an appropriate size fibular strut graft, augmenting it with a reconstruction plate (novel technique). We followed up for two years, achieving osteosynthesis, perfect graft integration and consolidation, good functional outcome and an unaided painless ambulation. Conclusion: Preservation of biology with appropriate bone and soft tissue management, stabilization with a biomechanically superior construct of strut graft - plate combination to facilitate early mobilization, are key factors in completing the injury cycle and restoring the function of the damaged limb.

Taking in consideration the proved capability of stem/progenitor cells to differentiate to various directions depending of the respective environmental factors and conditions, the possibility about derivation of different mature types of cells from different sources in human and experimental animal models was tested. For this goal was investigated the capability of nonimmune cells from precursors with both hematopoietic and non-hematopoietic origin to acquire immune characteristics in appropriate circumstances. Human mesenchymal stem cells (MSCs), isolated from bone marrow material, as well as from adipose and cartilage tissues, were in vitro-incubated in appropriate conditions, necessary about differentiation to chondrocyte, osteoblast and osteoclast directions, respectively. SDS-Polyacrylamide Gel Electrophoresis (SDSPAGE) with subsequent label-free tandem mass spectrometry assay, combined with liquid chromatography (LC-MS/MS) were also performed. NK cells were isolated from spleen cells of mouse Balb/c and incubated in the presence of different cytokine combinations. Fibroblasts from 3T3 cell line, derived from embryos of experimental mice of the same breed, were pre-incubated in the presence of cultural fluid from different types of murine mature normal and malignant cells (both in the absence and in the presence of cells in the used suspensions). The chondrocyte, osteoblast and osteoclast morphology was proved by Alcian Blue, Alizarin Red and TRAP+Hematoxillin staining, respectively, as well as by Hematoxillin/Eosin (H/E) staining and Giemsa dye. These morphological characteristics were proved about the three types of mature cells, derived from the human MSCs, isolated from the three sources. These features were confirmed by the data from SDS-PAGE with subsequent label free LCMS/MS assay and were in agreement with the literature findings. When the derived from mouse spleen progenitors NK cells were pre-incubated in the presence of the cocktail IL-12/IL-15/IL-18, active production of Connexin 43 (Cx43) was proved by Western-blot assay. These results confirmed the literature data about the proved key role of this molecule in the regulation of certain immune functions, including of antibody-related immune disorders. In the sub-populations of mouse embryonic 3T3 cell line were pre-incubated with mouse malignant myeloma cells and in cultural fluid from these neo-plastic cells, was noted appearance of initial myeloid and lymphoid progenitors, respectively. When the so derived cells in the prepared mixed cultures were freezed in the presence of cryo-protector DMSO, thawed and re-incubated, appearance of availability of multi-nuclear osteoclast-like cells was observed. In pre-incubation of de novo-formed semiconfluent monolayers with the received osteoclastlike cells and of cultural fluid from them, differentiation to osteoblast-like and chondrocytelike directions was assessed. These changes could be explained with probable existence of able to differentiate to different lineages sub-populations of stem-like cells in the embryonic 3T3 fibroblast line, depending of the respective environmental factors. These data were in confirmation of the literature messages about the role of DMSO and drastic temperature changes in the activation of fusion processes.