Y. Murat Elçin

Periodontal ligament cellular structures engineered with electrospun poly(DL-lactide-co-glycolide) nanofibrous membrane scaffolds

Periodontal tissue engineering is expected to overcome the limitations associated with the existing regenerative techniques for the treatment of periodontal defects involving alveolar bone, cementum, and periodontal ligament. Cell-based tissue engineering approaches involve the utilization of in vitro expanded cells with regenerative capacity and their delivery to the appropriate sites via biomaterial scaffolds. The aim of this study was to establish living periodontal ligament cell-containing structures on electrospun poly(DL-lactic-co-glycolic acid) (PLGA) nanofiber membrane scaffolds, assess their viability and characteristics, and engineer multilayered structures amenable to easy handling. Human periodontal ligament (hPDL) cells were expanded in explant culture and then characterized morphologically and immunohistochemically. PLGA nanofiber membranes were prepared by the electrospinning process; mechanical tensile properties were determined, surface topography, nanofiber size, and porosity status were investigated with SEM. Cells were seeded on the membranes at approximately 50,000 cell/cm(2) and cultured for 21 days either in expansion or in osteogenic induction medium. Cell adhesion and viability were demonstrated using SEM and MTT, respectively, and osteogenic differentiation was determined with IHC and immunohistomorphometric evaluation of osteopontin, osteocalcin, and bone sialoprotein marker expression. At days 3, 6, 9, and 12 additional cell/membrane layers were deposited on the existing ones and multilayered hybrid structures were established. Results indicate the feasibility of periodontal ligament cell-containing tissue-like structures engineering with PDL cells and electrospun nanofiber PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures amenable to macroscopic handling.

Neovascularization by bFGF releasing hyaluronic acid-gelatin microspheres: in vitro and in vivo studies.

Therapeutic angiogenesis with angiogenic growth factors has been described as a promising approach for tissue engineering, wound healing, and for treating ischemic tissues. Here, we assessed the merit of heparin-entrapped hyaluronic acid–gelatin (HA–G) microspheres for the sustained release of recombinant basic fibroblast growth factor (rbFGF) to promote localized neovascularization. HA–G microspheres were prepared by a water-in-oil emulsion method, and the in vitro release kinetics were first examined using three model proteins. Then, bFGF was incorporated into microspheres, and the bioactivity of the in vitro-released rbFGF was tested on human umbilical vein endothelial cell cultures. The ability to promote microvessel growth was assessed in vivo, at the subcutaneous groin fascia of Wistar rats after 3, 7, 14, and 21 days. Histological and morphometrical analysis indicated that heparin-entrapped HA–G microspheres have the capacity to release bioactive rbFGF, leading to localized neovascularization in the rat subcutaneous tissue.

Engineering of Rat Articular Cartilage on Porous Sponges : Effects of TGF-β1 and Microgravity Bioreactor Culture

The objective of this study was to develop an engineered rat hyaline cartilage by culturing articular chondrocytes on three-dimensional (3D) macroporous poly(DL-lactic-co-glycolic acid) (PLGA) sponges under chondrogenic induction and microgravity bioreactor conditions. Experimental groups consisted of 3D static and dynamic cultures, while a single cell monolayer (2D) served as the control. The effect of seeding conditions (static vs. dynamic) on cellularization of the scaffolds was investigated. MTT assay was used to evaluate the number of viable cells in each group at different time points. Formation of a hyaline-like cartilage was evaluated for up to 4 weeks in vitro. While 2D culture resulted in cell sheets with very poor matrix production, 3D culture was in the favor of tissue formation. A higher yield of cell attachment and spatially uniform cell distribution was achieved when dynamic seeding technique was used. Dynamic culture promoted cell growth and infiltration throughout the sponge structure and showed the formation of cartilage tissue, while chondrogenesis appeared attenuated more towards the outer region of the constructs in the static culture group. Medium supplemented with TGF-β 1 (5 ng/ml) had a positive impact on proteoglycan production as confirmed by histochemical analyses with Alcian blue and Safranin-O stainings. Formation of hyaline-like tissue was demonstrated by immunohistochemistry performed with antibodies against type II collagen and aggrecan. SEM confirmed higher level of cellularization and cartilage tissue formation in bioreactor cultures induced by TGF-β 1. The data suggest that PLGA sponge inside rotating bioreactor with chondrogenic medium provides an environment that mediates isolated rat chondrocytes to redifferentiate and form hyaline-like rat cartilage, in vitro.

Effect of Osteogenic Induction on the in Vitro Differentiation of Human Embryonic Stem Cells Cocultured With Periodontal Ligament Fibroblasts

Osteogenesis is one of the principal components of periodontal tissue development as well as regeneration. As pluripotent cells with unlimited proliferative potential and differentiation ability to all germ layer representatives, embryonic stem cells also hold the promise to become a cell source in bone tissue engineering. Our aim was to investigate osteogenic differentiation potential of human embryonic stem cells (hESCs) under the inductive influence of human periodontal ligament fibroblast (hPDLF) monolayers. After being expanded and characterized morphologically and immunohistochemically, hESCs (HUES-9) were cocultured with hPDLFs for 28 days. Two groups were established: (i) osteogenic induction group with ascorbic acid, beta-glycerophosphate, and dexamethasone containing hESC differentiation medium; and (ii) spontaneous differentiation group cultured in hESC differentiation medium. Morphological shift in cells was analyzed under an inverted microscope, and immunohistochemistry was performed on fixed specimens at days 1 and 28 using antibodies against alkaline phosphatase, osteonectin, osteopontin, bone sialoprotein (BSP), and osteocalcin (OSC). Reverse transcription-polymerase chain reaction was utilized for the detection of octameric binding protein-4, BSP, and OSC expression at mRNA level. Mineralization was assessed using alizarin red, and the surface topology shift in colonies was demonstrated with scanning electron microscopy. Results indicate the feasibility of osteogenic differentiation of hESCs in coculture, and suggest a role of periodontal ligament fibroblasts in their differentiation patterns. Advances in the field could allow for potential utilization of hESCs in periodontal tissue engineering applications involving regeneration of bone in periodontal compartment lost as a result of destructive periodontal diseases.

Proteome Analysis of Rat Bone Marrow Mesenchymal Stem Cell Differentiation

Bone marrow multipotent stromal cells (or mesenchymal stem cells; MSCs) have the capacity for renewal and the potential to differentiate in culture into several cell types including osteoblasts, chondrocytes, adipocytes, cardiomyocytes, and neurons. This study was designed to investigate the protein expression profiles of rat bone marrow MSCs during differentiation into adipogenic (by dexamethasone, isobutylmethylxanthine, insulin, and indomethacin), cardiomyogenic (by 5-azacytidine), chondrogenic (by ascorbic acid, insulin-transferrin-selenous acid, and transforming growth factor-β1), and osteogenic (by dexamethasone, β-glycerophosphate, and ascorbic acid) lineages by well-known differentiation inducers. Proteins extracted from differentiated MSCs were separated using two-dimensional gel electrophoresis (2-DE) and protein spots were detected using Sypro Ruby dye. Protein spots that were determined to be up- or down-regulated when the expression of corresponding spots (between weeks 1 and 2, 1 and 3, 1 and 4) showed an increase (≥2-fold) or decrease (≤0.5-fold) were successfully identified by MALDI-TOF-MS. In summary, 23 new proteins were identified either up- or down-regulated during differentiation experiments.

In Vitro Osteogenic Differentiation of Rat Mesenchymal Stem Cells in a Microgravity Bioreactor

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to differentiate into osteoblasts, chondroblasts, myocytes, and adipocytes. They have potential for bone tissue engineering by the utilization of in vitro expanded cells with osteogenic capacity and their delivery to the appropriate sites via biomaterial scaffolds. The objective was to evaluate the potential of rat bone marrow MSCs to form 3D bone-like tissue by the use of mineralized poly(DL-lactic-co-glycolic acid) (PLGA) foam and osteoinductive medium under rotating culture conditions. PLGA foams were prepared by solvent casting and particulate leaching, then mineralized by incubating in simulated body fluid. MSCs isolated from the bone marrow of young Wistar rats were expanded and seeded on the mineralized scaffolds. The cell-polymer constructs were then cultured in a slow turning lateral vessel-type rotating bioreactor for 4 weeks under the effect of osteogenic inducers, β-glycerophosphate, ascorbic acid and dexamethasone. Mineralization was evaluated using FT-IR and increases in dry mass; morphology changes of the mineralized foams and cell adhesion was characterized by SEM; cell viability was monitored by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Osteogenic differentiation was determined by using immunohistochemistry (anti-osteopontin). Results indicate the feasibility of bone tissue engineering with MSCs and mineralized PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures under appropriate bioreactor conditions.

Functional and Morphological Characteristics of Bovine Adrenal Chromaffin Cells on Macroporous Poly(D,L-lactide-co-glycolide) Scaffolds

Adrenal chromaffin cells (ACCs) secrete several neuroactive substances that are effective in influencing pain sensitivity in the central nervous system as well as enhancing the recovery of the intrinsic nigrostriatal dopaminergic system in patients with Parkinsons disease. ACC transplantation may be upregulated by the use of three-dimensional (3-D) scaffolds. In this study, we determined whether biodegradable poly(D,L-lactic-coglycolic acid) (PLGA) (85:15) sponges could be used as support for chromaffin cells. ACCs were isolated from bovine adrenal glands by standard perfusion (95% purity) followed by additional purification (>99.5% purity). ACC (approximately 5 x 10(5) cells) suspension in collagen (type I) was seeded on prewetted sponges and cultured in DMEM-F12 (1:1) medium (5% fetal bovine serum). The catecholamine and enkephalin levels of the samples were measured by high-performance liquid chromatography and radioimmunoassay. Cell morphology was examined by transmission electron microscopy. Morphological evidence showed prolonged viability of chromaffin cells on scaffolds having pores of 250-400 microm. Cell counts and scanning electron microscopy demonstrated that the majority of seeded cells were located within the scaffold. Chromaffin cells exhibited higher levels of enkephalins and catecholamines on PLGA scaffold compared with their monolayer cultures. By the use of 3-D PLGA as support for ACCs, it is possible to upregulate metabolic function and localize a high number of morphologically healthy-looking cells. Highly purified ACCs cultured on PLGA scaffold may have promise in transplantation studies, because these cells are less immunogenic and may be applied to in vivo settings by using short-term immunosuppression.

Translational applications of tissue engineering in cardiovascular medicine.

Cardiovascular diseases are the leading cause of global deaths. The current paradigm in medicine seeks novel approaches for the treatment of progressive or end-stage diseases. The organ transplantation option is limited in availability, and unfortunately, a significant number of patients are lost while waiting for donor organs. Animal studies have shown that upon myocardial infarction, it is possible to stop adverse remodeling in its tracks and reverse with tissue engineering methods. Regaining the myocardium function and avoiding further deterioration towards heart failure can benefit millions of people with a significantly lesser burden on healthcare systems worldwide. The advent of induced pluripotent stem cells brings the unique advantage of testing candidate drug molecules on organ-on-chip systems, which mimics human heart in vitro. Biomimetic three-dimensional constructs that contain disease-specific or normal cardiomyocytes derived from human induced pluripotent stem cells are a useful tool for screening drug molecules and studying dosage, mode of action and cardio-toxicity. Tissue engineering approach aims to develop the treatments for heart valve deficiency, ischemic heart disease and a wide range of vascular diseases. Translational research seeks to improve the patients quality of life, progressing towards developing cures, rather than treatments. To this end, researchers are working on tissue engineered heart valves, blood vessels, cardiac patches, and injectable biomaterials, hence developing new ways for engineering bio-artificial organs or tissue parts that the body will adopt as its own. In this review, we summarize translational methods for cardiovascular tissue engineering and present useful tables on pre-clinical and clinical applications.