Elif Bilgiç

Auricular cartilage repair using cryogel scaffolds loaded with BMP‐7‐expressing primary chondrocytes

The loss of cartilage tissue due to trauma, tumour surgery or congenital defects, such as microtia and anotia, is one of the major concerns in head and neck surgery. Recently tissue‐engineering approaches, including gene delivery, have been proposed for the regeneration of cartilage tissue. In this study, primary chondrocytes were genetically modified with plasmid‐encoding bone morphogenetic protein‐7 (BMP‐7) via the commercially available non‐viral Turbofect vector, with the aim of bringing ex vivo transfected chondrocytes to resynthesize BMP‐7 in vitro as they would in vivo. Genetically modified cells were implanted into gelatin–oxidized dextran scaffolds and cartilage tissue formation was investigated in 15 × 15 mm auricular cartilage defects in vivo in 48 New Zealand (NZ) white rabbits for 4 months. The results were evaluated via histology and early gene expression. Early gene expression results indicated a strong effect of exogenous BMP‐7 on matrix synthesis and chondrocyte growth. In addition, histological analysis results exhibited significantly better cartilage healing with BMP‐7‐modified (transfected) cells than in the non‐modified (non‐transfected) group and as well as the control. Copyright

The effect of poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx) and human mesenchymal stem cell (hMSC) on axonal regeneration in experimental sciatic nerve damage

This study is designed to evaluate the treatment effect of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and human mesenchymal stem cells (hMSC) on axonal regeneration in experimental rat sciatic nerve damage, and compare the results of this modality with autologous nerve grafting. In Spraque–Dawley albino rats, 10-mm-long experimental nerve gaps were created. Three groups were constituted, the gap was repaired with autologous nerve graft (autograft group), PHBHHx nerve graft alone (PHBHHx alone group), and PHBHHx nerve graft with hMSCs inside (PHBHHx with hMSC group), respectively. The results were evaluated with functional recovery, electrophysiological evaluation, and histological evaluation either with light microscopy and transmission electron microscopy for axonal regeneration and myelin formation. In functional evaluation, autograft and PHBHHx with hMSC groups showed functional improvement with time, whereas PHBHHx alone group did not. Electrophysiological evaluation showed better results in autograft and PHBHHx with hMSC groups when compared to PHBHHx alone group. There was no statistical difference between autograft and PHBHHx with hMSC groups. Histological evaluation showed regenerated axons in each group. Autograft group was better than the others, and PHBHHx with hMSC group was better than PHBHHx alone group both for axonal regeneration and myelin formation. This study showed that the nerve grafts which were prepared from PHBHHx with oriented nanofiber three-dimensional surfaces aided to nerve regeneration, either used alone or with hMSC. PHBHHx provided better nerve regeneration when used with hMSCs inside than alone, and reached the same statistical treatment effect in functional evaluation and electrophysiological evaluation when compared to autografting.

Cranial bone regeneration via BMP-2 encoding mesenchymal stem cells

Abstract Cranial bone repair and regeneration via tissue engineering principles has attracted a great deal of interest from researchers during last decade. Here, within this study, 6 mm critical-sized bone defect regeneration via genetically modified mesenchymal stem cells (MSC) were monitored up to 4 months. Cranial bone repair and new bone formations were evaluated by histological staining and real time PCR analysis in five different groups including autograft and bone morphogenetic protein-2 (BMP-2) transfected MSC groups. Results presented here indicate a proper cranial regeneration in autograft groups and a prospering regeneration for hBMP-2 encoding mesenchymal stem cells.

In vitro and in vivo evaluation of doxycycline-chondroitin sulfate/PCLmicrospheres for intraarticular treatment of osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease, which has no complete treatment with medication yet. Intraarticular hyaluronan (HA) injection can decrease pain and modify the natural course of OA. This study was designed to provide long term delivery of an MMP (matrix-metalloproteinase) inhibitor agent-doxycycline, together with matrix regenerative agent-chondroitin sulfate for treating OA which progress with matrix degenerations. Doxycycline (D) and doxycycline-chondroitin sulfate (D-CS) loaded poly-ɛ-caprolactone (PCL) microspheres (MS) were prepared as intraarticular delivery systems. Bio-effectiveness of developed microspheres was first evaluated with three-dimensional in vitro model of OA where both MS showed significant reduction in MMP-13 levels compared to untreated OA-chondrocytes at 15 and 24 days. Significant decrease was observed in GAG release into the media for both D MS and D-CS MS treated groups at 15 and 24 days. Second, the microspheres were injected to rabbit knee in hyaluronan (HA) to evaluate the effectiveness of the treatment. Radiographic scores of D MS and D-CS MS groups improved after 8 weeks when compared to OA group. Mankin-Pitzker histological scores similarly showed improvement with D MS and D-CSMS groups when compared to OA group. Ex vivo hardness tests of cartilages demonstrated superior hardness values with both doses of D-CSMS compared to OA group. D MS showed promising improvement of OA in histology results. Although, both MS groups had similar effects on cells in the in vitro model, D-CSMS had a positive contribution on all in vivo treatment outcomes and showed potential as a new strategy for treatment when applied to OA knee joints.

Apoptosis in chronic tonsillitis and tonsillar hypertrophy

OBJECTIVE Chronic tonsillitis is the persistent inflammation of the tonsillar tissue that occurs due to recurrent, acute or subclinical infection. The recurrent and chronic inflammation of palatine tonsils sometimes results in hypertrophy. Apoptosis provides an important balance between lymphocytes in tonsillar lymphoid tissue. The aim of this study is to investigate the apoptosis in tonsillar diseases. METHODS 43 patients with chronic tonsilitis and tonsillar hypertrophy underwent tonsillectomy. The specimens were examined immunohistochemically for apoptosis. Tonsils were assembled into groups according to their size. Specimens were compared for their apoptotic cell count. RESULTS The apoptosis difference between the tonsil size groups is not statistically significant (p>0.05). However, when the study group was divided into two at age 6, the difference was not statistically significant for patients at and below 6 years of age; but, the difference was statistically significant for patients above 6 years of age (p<0.05). The comparison of apoptosis in microcompartments of tonsil tissue (intrafollicular, interfollicular, subepithelial and intraepithelial) between tonsil size stages and between chronic tonsillitis and tonsillar hypertrophy groups revealed no statistical significance (p>0.05). There was a statistically significant positive correlation between intrafollicular and interfollicular, interfollicular and intraepithelial & subepithelial and intraepithelial areas (p<0.05). CONCLUSIONS In the light of these findings, it was concluded that apoptosis played a role in the tonsillar hypertrophy and atrophy. Apoptosis functioned to balance lymphocyte proliferation in tonsil tissue. The association of apoptosis with tonsillar hypertrophy seemed to be age-dependent.

Aligned bacterial PHBV nanofibrous conduit for peripheral nerve regeneration

Abstract The conventional method of peripheral nerve gap treatment is autografting. This method is limited. In this study, an aligned nanofibrous graft was formed using microbial polyester, Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The regenerative effect of the graft was compared with that of autografting in vivo. To determine the regenerative effect, rats were assessed with sciatic nerve functional index, electromyographic evaluation, and histological evaluation. Results found in this study include PHBV grafts stimulated progressive nerve regeneration, although regeneration was not comparable with that of autografting. We conclude that the study results were promising for aligned bacterial polymeric grafts for peripheral nerve regeneration.

Vancomycin containing PLLA/β-TCP controls experimental osteomyelitis in vivo.

BackgroundImplant-related osteomyelitis (IRO) is recently controlled with local antibiotic delivery systems to overcome conventional therapy disadvantages. In vivo evaluation of such systems is however too little.Questions/purposesWe asked whether vancomycin (V)-containing poly-l-lactic acid/β-tricalcium phosphate (PLLA/β-TCP) composites control experimental IRO and promote bone healing in vivo.MethodsFifty-six rats were distributed to five groups in this longitudinal controlled study. Experimental IRO was established at tibiae by injecting methicillin-resistant Staphylococcus aureus (MRSA) suspensions with titanium particles in 32 rats. Vancomycin-free PLLA/β-TCP composites were implanted into the normal and infected tibiae, whereas V-PLLA/β-TCP composites and coated (C)-V-PLLA/β-TCP composites were implanted into IRO sites. Sham-operated tibiae established the control group. Radiological and histological scores were quantified with microbiological findings on weeks 1 and 6.ResultsIRO is resolved in the CV- and the V-PLLA/β-TCP groups but not in the PLLA/β-TCP group. MRSA was not isolated in the CV- and the V-PLLA/β-TCP groups at all times whereas the bacteria were present in the PLLA/β-TCP group. Radiological signs secondary to infection are improved from 10.9 ± 0.9 to 3.0 ± 0.3 in the V-PLLA/β-TCP group but remained constant in the PLLA/β-TCP group. Histology scores are improved from 24.7 ± 6.5 to 17.6 ± 4.8 and from 27.6 ± 7.9 to 32.4 ± 8.9 in the CV-PLLA/β-TCP and the V-PLLA/β-TCP groups, respectively. New bone was formed in all the PLLA/β-TCP group at weeks 1 and 6.ConclusionsCV- and V-PLLA/β-TCP composites controlled experimental IRO and promoted bone healing.Clinical relevanceCV- and V-PLLA/β-TCP composites have the potential of controlling experimental IRO and promoting bone healing.

Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease

BACKGROUND Inflammatory activity originating from the epicardial adipose tissue (EAT) may have a role in coronary artery disease (CAD) pathogenesis. The relationship between macrophage infiltration, polarization in the EAT, and netrin-1 gene expression was investigated. METHODS Macrophage infiltration and polarization were examined by immunohistochemical methods and expression levels of netrin-1, Unc5b, and cytokines related with M1-macrophage subtype (IL-12 and IL-18) were determined by quantitative polymerase chain reaction in subcutaneous and epicardial adipose tissue obtained from patients undergoing coronary artery bypass grafting and non-coronary cardiac surgery. RESULTS CAD patients had higher CD68+ (p=0.005) and CD11c+ (p<0.001) macrophage count in EAT when compared to the controls. CD11c+/CD206+ macrophage ratio, which reflects dominancy of M1-macrophage phenotype, was significantly increased in EAT of CAD patients when compared to that of the controls (p=0.008). CAD patients had significantly higher netrin-1, Unc5b, and IL-18 gene expression in the EAT when compared to the control group (p<0.001, p<0.001, and p=0.006 respectively). Increased macrophage infiltration and polarization were associated with higher netrin-1, Unc5b, and IL-12 gene expression in EAT (p<0.05). CONCLUSIONS Findings suggest a link between enhanced netrin-1 expression in EAT and macrophage infiltration and polarization in patients with CAD.

Splenectomy-Induced Leukocytosis Promotes Intratumoral Accumulation of Myeloid-Derived Suppressor Cells, Angiogenesis and Metastasis

ABSTRACT Background: Enlargement of the spleen is commonly observed in animal models of cancer. Here, in a breast cancer model, it was aimed to determine the effect of splenectomy on circulating and tumor-infiltrating myeloid-derived suppressor cells (MDSCs), tumor angiogenesis, and metastasis. Methods: Mice were inoculated with 4T1 breast cancer cells and underwent splenectomy or sham laparotomy. Tumor growth and survival of animals were followed. Macroscopic and histopathological analyses were performed to determine splenomegaly and metastasis. Immunophenotyping of myeloid cells was performed with flow cytometric analysis of CD11b, Gr-1, F4/80, CD206, CD11c, and F4/80 markers. Suppressive function of MDSCs on T cell proliferation was studied in cocultures. Tumor angiogenesis and granulocytic myeloid cell infiltration in the metastatic foci were studied by CD31 and Ly6G immunohistochemistry, respectively. Results: The mice bearing breast tumors underwent total splenectomy at an early time point of tumorigenesis when only low levels of MDSCs had accumulated in the spleen. Circulating and tumor-infiltrating MDSCs, and tumor-associated macrophages (TAMs) were increased following splenectomy. Nevertheless, splenectomy could only lead to a temporary deceleration in tumor growth but favored lung metastasis and angiogenesis in the long run. Conclusion: Our data demonstrated a link among splenectomy-induced leukocytosis, accumulation of circulating and tumor-infiltrating MDSC, and enhanced angiogenesis and metastasis. Therefore, as a part of oncological surgery, favorable and unfavorable facets of the splenectomy must be considered to improve therapeutic efficacy.

Possible role of apoptosis in pathogenesis of adenoid hypertrophy and chronic adenoiditis: Prospective case-control study.

OBJECTIVE Apoptosis is a programmed cell death; it provides an important balance between lymphocytes in adenoid tissue. The aim of this study is to investigate the role of apoptosis in chronic adenoiditis and adenoid hypertrophy. This is the first study in the literature about apoptosis in adenoid hypertrophy and chronic adenoiditis. METHODS Prospective case-control study in a tertiary referral university hospital was conducted. 46 patients who had chronic adenoiditis and adenoid hypertrophy underwent adenoidectomy. Adenoids were evaluated for apoptosis and assembled into groups according to their size. Apoptotic cells were counted in three different microscopic fields and their average was taken for every microcompartment. As a result of immunohistochemical staining, specimens were compared for their apoptotic cell rate. RESULTS The difference in apoptosis of chronic adenoiditis and adenoid hypertrophy groups is statistically significant (p<0.05). The age 6 was used as a cut-off to compare apoptosis in adenoid tissue. The difference was not statistically significant for patients at and below 6 years of age; however, the difference was statistically significant for patients above 6 years of age. The comparison of apoptosis in microcompartments of adenoid tissue (intrafollicular, interfollicular, subepithelial and intraepithelial) between chronic adenoiditis and adenoid hypertrophy groups revealed significant differences for intrafollicular and intraepithelial areas, and insignificant differences for interfollicular and subepithelial areas. CONCLUSION Although apoptosis could not totally explain the pathogenesis of chronic adenoiditis and adenoid hypertrophy, it appeared to play an important role in it. Apoptosis functions to limit adenoid hypertrophy. Adenoid apoptosis appears to be age-dependent.