Sanja Stojanović

Addition of blood to a phycogenic bone substitute leads to increased in vivo vascularization

The present study aimed to analyze the effects of the addition of blood to the phycogenic bone substitute Algipore(®) on the severity of in vivo tissue reaction. Initially, Fourier-transform infrared spectroscopy (FTIR) of the bone substitute was conducted to analyze its chemical composition. The subcutaneous implantation model in Balb/c mice was then applied for up to 30 d to analyze the tissue reactions on the basis of specialized histochemical, immunohistochemical, and histomorphometrical methods. The data of the FTIR analysis showed that the phycogenic bone substitute material is mainly composed of hydroxyapatite with some carbonate content. The in vivo analyses revealed that the addition of blood to Algipore(®) had a major impact on both angiogenesis and vessel maturation. The higher vascularization seemed to be based on significantly higher numbers of multinucleated TRAP-positive cells. However, mostly macrophages and a relatively low number of multinucleated giant cells were involved in the tissue reaction to Algipore(®). The presented data show that the addition of blood to a bone substitute impacts the tissue reaction to it. In particular, the immune response and the vascularization were influenced, and these are believed to have a major impact on the regenerative potential of the process of bone tissue regeneration.

Osteogenic potential of in vitro osteo-induced adipose-derived mesenchymal stem cells combined with platelet-rich plasma in an ectopic model

PurposeThe osteogenic potential of in vitro osteo-induced adipose-derived mesenchymal stem cells (ADSCs) combined with platelet-rich plasma (PRP) and implanted on bone mineral matrix (BMM) carrier was examined in a subcutaneous model in Balb/c mice.MethodsIn vitro osteogenic differentiation of ADSCs was monitored by relative bone-related gene expression and osteocalcin expression at the third, seventh and 15th day. Test implants consisting of in vitro osteo-induced ADSCs, PRP and BMM (OPC implants) and control implants consisting of PRP and BMM (PC implants) were examined. The relative expression of the bone-related genes encoding osterix, osteocalcin, collagen type I α1 and alkaline phosphatase was examined in implants extracted at one, two, four and eight weeks. Histochemical, immunohistochemical and histomorphometric analyses of implants extracted at two and eight weeks were performed.ResultsThe highest relative expression of bone-related genes and osteocalcin expression was found at the 15th day of in vitro osteogenic induction of the ADSCs. Permanent and continuous increased expression of bone-related genes was noticed in OPC implants at eight weeks. Expression peaks of bone-related genes in PC implants were at two and four weeks, but they significantly decreased at eight weeks. The signs of resorption, formation of callus-like tissue positive for osteocalcin and increased presence of bone cells were found in OPC implants compared with PC implants. A higher percentage of infiltrated tissue and vascularisation was found in OPC than in PC implants.ConclusionsThe combination of in vitro osteo-induced ADSCs and PRP on BMM carrier represents a promising approach for bone regeneration.

Instrumental methods and techniques for structural and physicochemical characterization of biomaterials and bone tissue: A review

A review of recent advances in instrumental methods and techniques for structural and physicochemical characterization of biomaterials and bone tissue is presented in this paper. In recent years, biomaterials attracted great attention primarily because of the wide range of biomedical applications. This paper focuses on the practical aspects of instrumental methods and techniques that were most often applied (X-ray methods, vibrational spectroscopy (IR and Raman), magnetic-resonance spectroscopy (NMR and ESR), mass spectrometry (MS), atomic absorption spectrometry (AAS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES), thermogravimetry (TG), differential thermal analysis (DTA) and differential scanning calorimetry (DSC), scanning electron microscopy (SEM), transmission electron microscopy (TEM)) in the structural investigation and physicochemical characterization of biomaterials and bone tissue. The application of some other physicochemical methods was also discussed. Hands-on information is provided about these valuable research tools, emphasizing practical aspects such as typical measurement conditions, their limitations and advantages, interpretation of results and practical applications.

Analysis of the in vitro degradation and the in vivo tissue response to bi-layered 3D-printed scaffolds combining PLA and biphasic PLA/bioglass components – Guidance of the inflammatory response as basis for osteochondral regeneration

The aim of the present study was the in vitro and in vivo analysis of a bi-layered 3D-printed scaffold combining a PLA layer and a biphasic PLA/bioglass G5 layer for regeneration of osteochondral defects in vivo Focus of the in vitro analysis was on the (molecular) weight loss and the morphological and mechanical variations after immersion in SBF. The in vivo study focused on analysis of the tissue reactions and differences in the implant bed vascularization using an established subcutaneous implantation model in CD-1 mice and established histological and histomorphometrical methods. Both scaffold parts kept their structural integrity, while changes in morphology were observed, especially for the PLA/G5 scaffold. Mechanical properties decreased with progressive degradation, while the PLA/G5 scaffolds presented higher compressive modulus than PLA scaffolds. The tissue reaction to PLA included low numbers of BMGCs and minimal vascularization of its implant beds, while the addition of G5 lead to higher numbers of BMGCs and a higher implant bed vascularization. Analysis revealed that the use of a bi-layered scaffold shows the ability to observe distinct in vivo response despite the physical proximity of PLA and PLA/G5 layers. Altogether, the results showed that the addition of G5 enables to reduce scaffold weight loss and to increase mechanical strength. Furthermore, the addition of G5 lead to a higher vascularization of the implant bed required as basis for bone tissue regeneration mediated by higher numbers of BMGCs, while within the PLA parts a significantly lower vascularization was found optimally for chondral regeneration. Thus, this data show that the analyzed bi-layered scaffold may serve as an ideal basis for the regeneration of osteochondral tissue defects. Additionally, the results show that it might be able to reduce the number of experimental animals required as it may be possible to analyze the tissue response to more than one implant in one experimental animal.

Blunt chest trauma--an audit of injuries diagnosed by the MDCT examination.

BACKGROUND/AIM Multidetector computed tomography (MDCT) characterised by speed and precision is increasingly accessible in emergency wards. The aim of our study was to determine the most common injuries to the chest region, as well as type associated extrathoracic injuries, and the treatment outcome. METHODS This prospective study included 61 patients with blunt trauma who were submitted to computed tomography (CT) of the thorax. The number of injuries was evaluated by organs and organ systems of the chest. The cause of the injury, the length and the outcome of the treatment, and the presence of injuries in other regions were assessed. RESULTS Chest injuries were associated with injuries to other regions in 80.3% cases, predominantly injuries to extremities or pelvic bones in 54.1% cases, followed by head injuries in 39.3% patients. Associated thoracic injuries were present in 90.9% of patients with lethal outcome. Lung parenchymal lesions, pleural effusions and rib fractures were the most common injuries affecting 77.1%, 65.6% and 63.9% of the cases, respectively. CONCLUSION Blunt chest trauma is a significant problem affecting predominantly males in their forties and it is usually caused by a motor vehicle accident. In case of pneumomediastinum or mediastinal haematoma, the use of 3D reconstructions is advised for diagnosing possible tracheobronchial ruptures and thoracic aorta injuries. Increased resolution of CT scanners yielded a large number of findings that are occult on radiography, especially in the event of lung parenchymal and pleural injuries. However, none imaging modality can replace surgical judgement.

SEM-EDX Analysis of Bio-Oss® Granules After Incubation In Cell Culture Medium

The aim of this study was to analyze changes in the surface and chemical composition of Bio-Oss® granules after incubation in cell culture medium. Appropriate amount of Bio-Oss® material was incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) at 37 °C for 3 days. After that, granules were dried, coated with gold and analyzed using scanning electron microscope (SEM) equipped with EDX (JEOL, JSM 5300). SEM analysis revealed that the surface of Bio-Oss® particles looks less grainy after incubation in DMEM. The impression is that an incubation in DMEM made erosion of cam bumps or that larger and wider reefs compared to the material before incubation are made by depositing of new material. Semi-quantitative analysis of calcium and phosphorous content in Bio-Oss® before and after incubation in DMEM was determined by EDX spectroscopy and results show that concentration of calcium and phosphorous ions increases after incubation in DMEM.

Crosstalk between possible cytostatic and antiinflammatory potential of ketoprofen in the treatment of culture of colon and cervix cancer cell lines.

OBJECTIVES The aim of this study was to test the cytostatic potential of ketoprofen in the in vitro treatment of cells derived from colon and cervix cancer. BACKGROUND NF-κB and cyclooxygenase can have a role in different stages of the development and progression of cancer. In recent years, special attention has been paid to the possible cytostatic potential of nonsteroidal anti-inflammatory drugs. There are no published data on the use of ketoprofen in pharmacotherapy of the colon and cervical carcinoma. METHODS We examined the effect of ketoprofen alone or in combination with cisplatin and 5-fluorouracil on proliferation of the two cell lines, HeLa (human cervical carcinoma cells) and Caco-2 (human colon cancer cells) by MTT test. Measurement of the level of NF-κB was also performed in the cells of both cell lines. RESULTS The results of present study have shown that at least one of the mechanisms of antiproliferating and/or cytostatic effects of different concentrations of ketoprofen on Caco-2 and HeLa cells could include the transcription factor NF-κB. CONCLUSIONS Since this transcription factor is controlled by the altered expression of COX-2, the inhibition of this enzyme by ketoprofen may represent a significant step in synergistic cascade of the therapy and prevention of colon and cervical cancer (Tab. 4, Ref. 31).

Locally applied cholecalciferol and alfacalcidol act differently on healing of femur defects filled with bone mineral matrix and platelet-rich plasma in ovariectomized rats

Vitamin D and its analogues that are often used in the treatment of osteoporosis have also been shown to affect the healing of bone fractures. To avoid the high-dose systemic treatments, needed to achieve faster healing of fractured bone, the approach of local application of these drugs has become very attractive. The aim of our study was to examine the influence of cholecalciferol and alfacalcidol, locally applied with bovine bone mineral matrix (Bio-Oss), supplemented with platelet-rich plasma on femur defect healing in ovariectomized rats. Experimental osteoporosis was induced by bilateral ovariectomy. To estimate the healing of defects, the formation of the new bone tissue and the resorption of Bio-Oss particles after two and eight weeks of defect healing, we performed biochemical, histological, immunohistochemical, histomorphometric and scanning electron microscopy analyses. Our results showed that locally applied cholecalciferol and alfacalcidol delay early bone healing of defect in ovariectomized rat model. Both of the examined vitamins, particularly alfacalcidol, decreased the resorption of Bio-Oss particles in defects. Only alfacalcidol affected the formation of histologically normal new bone tissue with a good integration into the surrounding osteoporotic bone. Cholecalciferol allowed callus mineralization and led to the formation of new bone with high quantity, with characteristics, similar to those of the control group. Our results suggested that alfacalcidol and cholecalciferol may be promising tools for the local treatment of osteoporotic fractures, but the goal of application should specify the choice of vitamin D form, as well as the composition of scaffolds, with which they are applied.

Synthesis, Swelling Properties and Evaluation of Genotoxicity of Hydrogels Based on (Meth)acrylates and Itaconic Acid

In this study we prepared hydrogels based on 2-hydroxyethyl methacrylate (HEMA): PHEMA homopolymer and two terpolymers of HEMA, itaconic acid (IA) and two poly(alkylene glycol) (meth) acrylates (PAGM): poly(ethylene glycol)6 acrylate (P(HEMA/IA/PAGM1)) and poly(propylene glycol)5 methacrylate (P(HEMA/IA/PAGM2)). Hydrogels were synthesized by gamma-irradiated radical polymerization and subjected to swelling measurements and genotoxicity evaluation. Swelling studies confirmed that these hydrogels deserve consideration as biomaterials due to their ability to swell in phosphate buffer but maintaining physical integrity for a prolonged contact time after equilibrium state has been reached. Comet assay showed certain genotoxic effect following cell exposure to extracts of hydrogels, which was dependent on the concentration of extracts, chemical composition of hydrogels and the degree of crosslinking. The influence of concentration on genotoxicity was the most pronounced. The synthesis of these novel HEMA-based hydrogels should be optimized so as to reduce their toxicity and enable the use in clinical practice.

Effects of bone tissue engineering triad components on vascularization process: comparative gene expression and histological evaluation in an ectopic bone-forming model

ABSTRACT Vascular development has a great significance in the osteogenic process and in bone tissue engineering (BTE). BTE is based on various combinations of three principal types of components: biomaterials as scaffolds, regulatory signals and cells. The aim of our study was to evaluate, at gene expression and histological level, the effect of BTE triad components on the vascularization process in an ectopic bone-forming model in mice. Bone mineral matrix (BMM) was used as a scaffold and a carrier, platelet-rich plasma (PRP) as a source of regulatory signals and adipose stem cells (ASCs) as a source of cells for endothelial differentiation, in order to show how and to what extent the biological enrichment of BMM influences the outcome of the osteogenic process and its key precondition, vascularization. Implants composed of BMM, PRP and ASCs in vitro induced into endothelial cells (EPB implants) and implants composed of BMM and PRP (PB implants) were compared with implants composed of BMM only (B implants). More pronounced endothelial-related gene expression and stronger VCAM-1 (vascular cell adhesion molecule-1) immunoexpression were observed in EPB implants in comparison with PB and B ones at later time points of the in vivo experimental period. Osteopontin gene expression and immunoexpression of osteopontin were significantly higher in EPB compared to PB and B implants. Therefore, addition of ASCs combined with PRP to BMM improved the vascularization process in the ectopic bone-forming model, which makes this BTE composition the most favourable among the examined types of implants for application in BTE.