Cell-nanofiber-based cartilage tissue engineering using improved cell seeding, growth factor, and bioreactor technologies

Biodegradable nanofibrous scaffolds serving as an extracellular matrix substitute have been shown to be applicable for cartilage tissue engineering. However, a key challenge in using nanofibrous scaffolds for tissue engineering is that the small pore size limits the infiltration of cells, which may result in uneven cell distribution throughout the scaffold. This study describes an effective method of chondrocyte loading into nanofibrous scaffolds, which combines cell seeding, mixing, and centrifugation to form homogeneous, packed cell-nanofiber composites (CNCs). When the effects of different growth factors are compared, CNCs cultured in medium containing a combination of insulin-like growth factor-1 and transforming growth factor-beta1 express the highest mRNA levels of collagen type II and aggrecan. Radiolabeling analyses confirm the effect on collagen and sulfated-glycosaminoglycans (sGAG) production. Histology reveals chondrocytes with typical morphology embedded in lacuna-like space throughout the entire structure of the CNC. Upon culturing using a rotary wall vessel bioreactor, CNCs develop into a smooth, glossy cartilage-like tissue, compared to a rough-surface tissue when maintained in a static environment. Bioreactor-grown cartilage constructs produce more total collagen and sGAG, resulting in greater gain in net tissue weight, as well as express cartilage-associated genes, including collagen types II and IX, cartilage oligomeric matrix protein, and aggrecan. In addition, dynamic culture enhances the mechanical property of the engineered cartilage. Taken together, these results indicate the applicability of nanofibrous scaffolds, combined with efficient cell loading and bioreactor technology, for cell-based cartilage tissue engineering.     

Epithelial cell rests of Malassez: tissue, cell, and molecular biology

This review presents an analysis of the current data on the structural and functional organization and biological significance of epithelial cell rests of Malassez (ERM), which are formed by persisting fragments of epithelial (Hertwig) sheath that participates in the tooth root development. ERM, found within the periodontal ligament, undergo intensive age-related involution and are traditionally described as small clusters of functionally inactive cells. Meanwhile, recent findings are indicative of high functional activity of ERM which are both producers of and targets for various cytokines, growth factors, adhesive substances, their receptors and other biologically active molecules. It is suggested that ERM participate in sustaining the optimal conditions for the normal functioning and regeneration of periodontium structural components. When periodontal tissue homeostasis is disturbed, ERM may grow up and acquire the capacity to destroy the surrounding tissues.     

Application of cell technologies to tissue defect closure in oncology

In vivo and in vitro experiments on the application of cell technologies to tissue defect closure were conducted; autologic mesenchymal stem cells on 3-demensional matrices were used. The authors analyze the results of the application of bioengineering tissue equivalents for the closure of soft tissue and upper airway defects after extensive resections performed in 52 oncological patients. Tissue equivalents with stem cells provide engraftment and long-term graft functioning; they also modify wound surface, thus stimulating wound epithelization. In this study the application of tissue equivalents led to wound healing and functional recovery in 87% of patients.     

Epithelial tissue chimerism after human hematopoietic cell transplantation is a real phenomenon

Bone marrow transplantation in animals has been shown to generate epithelial populations, a phenomenon that has also recently been suggested to take place after human hematopoietic cell transplantation. However, reports in humans are not conclusive because they still leave open the possibility that the identified donor-derived cells are not epithelial cells but intraepithelial lymphocytes. Here, we demonstrate that donor-derived CD45(+) hematopoietic cells in close contact with epithelial tissue may be falsely characterized as donor-derived epithelial cells if the three-dimensional structure of the tissue is not considered and the hematopoietic markers are not examined. By using a rigorous three-dimensional analysis on single sections of colon biopsies triple stained with donor-specific, epithelial-specific, and hematopoietic-specific markers we demonstrate that chimerism of colon epithelium is a real phenomenon occurring constantly after human hematopoietic cell transplantation. We exclude horizontal DNA transfer or cell fusion as the underlying mechanism of our findings. Tissue damage enhances the engraftment of the donor-derived epithelial cells. The physiological and therapeutical role of the donor-derived epithelial cells after human hematopoietic cell transplantation needs further investigation. However, their identification requires stringent and unequivocal detection systems.     

Epithelial and connective tissue cell CTGF/CCN2 expression in gingival fibrosis

Gingival overgrowth is a side effect of certain medications and occurs in non-drug-induced forms either as inherited (human gingival fibromatosis) or idiopathic gingival overgrowth. The most fibrotic drug-induced lesions develop in response to therapy with phenytoin; the least fibrotic lesions are caused by cyclosporin A; and intermediate fibrosis occurs in nifedipine-induced gingival overgrowth. Connective tissue growth factor (CTGF/CCN2) expression is positively related to the degree of fibrosis in these tissues. The present study has investigated the hypothesis that CTGF/CCN2 is expressed in human gingival fibromatosis tissues and contributes to this form of non-drug-induced gingival overgrowth. Histopathology/immunohistochemistry studies showed that human gingival fibromatosis lesions are highly fibrotic, similar to phenytoin-induced lesions. Connective tissue CTGF/CCN2 levels were equivalent to the expression in phenytoin-induced gingival overgrowth. The additional novel observation was made that CTGF/CCN2 is highly expressed in the epithelium of fibrotic gingival tissues. This finding was confirmed by in situ hybridization. Real-time polymerase chain reaction (PCR) analyses of RNA extracted from drug-induced gingival overgrowth tissues for CTGF/CCN2 were fully consistent with these findings. Finally, normal primary gingival epithelial cell cultures were analysed for basal and transforming growth factor beta1 (TGF-beta1) or lysophosphatidic acid-stimulated CTGF/CCN2 expression at protein and RNA levels. These data indicate that fibrotic human gingival tissues express CTGF/CCN2 in both the epithelium and connective tissues; that cultured gingival epithelial cells express CTGF/CCN2; and that lysophosphatidic acid further stimulates CTGF/CCN2 expression. These findings suggest that interactions between epithelial and connective tissues could contribute to gingival fibrosis.     

Non-invasive quantitative reconstruction of tissue elasticity using an iterative forward approach

A novel iterative approach is presented to estimate Young's modulus in homogeneous soft tissues using vibration sonoelastography. A low-frequency (below 100 Hz) external vibration is applied and three or more consecutive frames of B-scan image data are recorded. The internal vibrational motion of the soft tissue structures is calculated from 2D displacements between pairs of consecutive frames, which are estimated using a mesh-based speckle tracking method. An iterative forward finite element approach has been developed to reconstruct Young's modulus from the measured vibrational motion. This is accomplished by subdividing the 2D image domain into sample blocks in which Young's modulus is assumed to be constant. Because the finite element equations are internally consistent, boundary values other than displacement are not required. The sensitivity of the results to Poisson's ratio and the damping coefficient (viscosity) is investigated. The approach is verified using simulated displacement data and using data from tissue-mimicking phantoms.     

Postoperative irradiation following immediate breast reconstruction using a temporary tissue expander.

Ten consecutive patients at Howard University Hospital underwent modified radical mastectomy with immediate breast reconstruction using a temporary tissue expander. Postoperative irradiation was delivered to the breast mound encompassing the tissue expander. The effects of expansion on the delivery of postoperative irradiation was assessed. Dosimetric measurements with thermoluminescent dosimeters revealed that the saline-filled expander attenuated the photon beam 3% less than tissue-equivalent material of equal volume. This dose variation was negligible, so no adjustments were made. Postoperative treatment consisted of 5040 cGy to 5220 cGy delivered in 5 to 10 weeks using 4 mV photon tangentials. Cosmesis was assessed over a 2-year period. Six patients completed reconstruction and irradiation without complications. Cosmesis was good in five and fair in one. One patient developed a moist reaction secondary to postoperative irradiation; however, final cosmesis was good. Three patients developed complications leading to the loss of the reconstructed breast. Successful final reconstruction can be achieved with careful patient selection and close follow-up by the plastic surgeon and radiation oncologist.     

Epithelial cell apoptosis and lung remodeling

Lung epithelium is the primary site of lung damage in various lung diseases. Epithelial cell apoptosis has been considered to be initial event in various lung diseases. Apoptosis signaling is classically composed of two principle pathways. One is a direct pathway from death receptor ligation to caspase cascade activation and cell death. The other pathway triggered by stresses such as drugs, radiation, infectious agents and reactive oxygen species is mediated by mitochondria. Endoplasmic reticulum has also been shown to be the organeile to mediate apoptosis. Epithelial cell death is followed by remodeling processes, which consist of epithelial and fibroblast activation, cytokine production, activation of coagulation pathway, neoangiogenesis, re-epithelialization and fibrosis. Epithelial and mesenchymai interaction plays important roles in these processes. Further understanding of apoptosis signaling and its regulation by novel strategies may lead to effective treatments against various lung diseases. We review the recent advances in the understanding of apoptosis signaling and discuss the involvement of apoptosis in lung remodeling. Cellular ＆ Molecular Immunology.     

Bone cell transfection in tissue culture using hydroxyapatite microparticles

Coprecipitates of calcium phosphate and DNA have been used in vitro for several decades for cell transfection. We evaluated the efficiency of calcium phosphate ceramics associated to plasmid DNA in the transfection of bone cells in vitro when they are grown in tissue culture. Newborn rat calvariae and tibia epiphyses were grown on an agar surface for a period of 48 h to 30 days. The hydroxyapatite (HA)-particles were loaded with a plasmid bearing a galactosidase reporter gene by incubation of the plasmid solution in PBS with the particles. One milligram of HA-particles was then placed in contact with the bone explants for 8 and 30 days. Histological sections were then performed and the galactosidase activity was revealed using an X-gal solution. At eight days, very few cells expressing the galactosidase activity were detected. By 30 days, however, the explants appeared uniformly stained blue. The staining of sections showed that the osteoblasts, chondroblasts, perichondroblasts, and perisoteal cells all expressed the lacZ gene while the number of cells stained in the control was negligible. The time dependence of the transfection suggests that transfection using ceramics is linked to the degradation of the ceramic by the cells. Furthermore, the cells are stained remote from the particles suggesting that the particles induce a coprecipitate of DNA in the explant.     

Applications of MEMS technologies in tissue engineering

The success of therapeutic strategies within the fields of regenerative medicine, including tissue engineering, biomaterials engineering, and cell and tissue transplantation science, relies on researchers' understanding of the complex cellular microenvironments that occur within functional tissue. Microfabricated biomedical platforms provide tools for researchers to study cellular response to various stimuli with micro- and nanoscale spatial control. Initial studies utilizing relatively passive means of microenvironmental control have provided the fundamental knowledge required to begin to design microculture platforms that closely mimic these biological systems. In this review, we discuss second-generation cell and tissue culture platforms that utilize active components, borrowed from work in the development of microelectromechanical systems (MEMS). These microsystems offer the unprecedented opportunity to fabricate culture platforms designed to match tissue-specific growth parameters. In addition, the adoption of MEMS components opens up the door for future integration with the burgeoning field of microanalytical systems, providing analytical platforms that retain the sensitivity and resolution required within low-volume, microfluidic culture technologies.     

Results and outlooks of using cell technologies in the treatment of neurological diseases

An attempt was undertaken in the last decade of the 20th century to use a principally new approach to the treatment of neurological diseases--cell therapy. Main efforts were focused on developing a method related with replacement of neurons dying in neurodegenerative pathology, primarily, in Parkinson disease (PD). Outlined below are the key elements of the technology:--ensuring, in experiment, of a prolonged therapeutic effect in transplantation, to the affected part, first of embryonic neurons of the animal of the same species (allografting) and then of homologous embryonic neurons of man (heterografting);--obtaining, standardization and preparation (for transplantation) of embryonic nervous tissue of man; transplantation of embryonic nervous tissue of man to the brain of patient and evaluation, in situ, of the functional activity of its neurons; and evaluation of the therapeutic effect of grafting. Cell suspension of meseencephalon of 6-9 week human fetus containing around 10% of differentiating dopaminergic neurons was used for grafting in PD. Embryonic dopaminergic neurons, administered stereotactically into the striatum of patient, established synaptic links with neurons of the recipient, which was accompanied by the onset of synthesis and reverse uptake of dopamine (DA) as well as by the onset of spontaneous and stimulated release of DA. Neurografting ensured a temporary improvement of the condition in a part of PD patients but did not cure them. Moreover, such positive therapeutic effect was registered only in patients with the akineticorigid but not trembling variation of the disease. Hence, although there was a certain progress in clinical neurografting, the approach cannot be now recommended for introduction in neurology and neurosurgery. The limited therapeutic effect of the treatment method is primarily explained by a low rate of survival of transplanted dopaminergic neurons and, consequently, by the persisting DA deficit in patient's body. Therefore, the outlooks for perfecting the cell technology are related with increasing the survival rate of implanted dopaminergic neurons and with stimulating the innervation of target neurons in patient's striatum as well as with using the neural (glia) and non-neural (fibroblasts, myoblasts) cells with modified gene and stem cells. Finally, despite a certain progress of advancing the cell technology in neurology the approach still needs more research, which would enable further clinical trials.     

Bone tissue reconstruction using titanium fiber mesh combined with rat bone marrow stromal cells

The study aim was to evaluate the effect of bone marrow stromal cells (BMSCs) cultured in titanium fiber mesh and implanted in a rat cranial defect. A total of 24 titanium meshes were placed in a tube containing 10 ml BMSC suspension (3 x 10(6)cells/ml) and the tube was rotated on a rotation plate (2 rpm) during 3 h. Thereafter, meshes with cells were subcultured for 1 day under standard conditions. Cell-loaded implants and non-cell-loaded controls were placed in a 8 mm cranial defect and retrieved after 3, 15 and 30 days of implantation. Histology showed that after 3 days of implantation, the mesh porosity of both implant groups was mainly invaded with blood cells. On the other hand, at 15 days of implantation, the cell-loaded implants were filled for 15 +/- 10% of their volume with bone, while the controls showed 1.5 +/- 3.5% of bone. The 30-day cell-loaded implants showed 40 +/- 12.5% of bone and the 30-day control implants 17 +/- 14.5%. At both implantation times the differences were statistically significant. Therefore, we conclude that inoculation of titanium fiber mesh with BMSCs can improve the bone healing capacity of this material.     

In situ tissue engineering using magnetically guided three-dimensional cell patterning

Manipulation of cell patterns in three dimensions in a manner that mimics natural tissue organization and function is critical for cell biological studies and likely essential for successfully regenerating tissues--especially cells with high physiological demands, such as those of the heart, liver, lungs, and articular cartilage.(1, 2) In the present study, we report on the feasibility of arranging iron oxide-labeled cells in three-dimensional hydrogels using magnetic fields. By manipulating the strength, shape, and orientation of the magnetic field and using crosslinking gradients in hydrogels, multi-directional cell arrangements can be produced in vitro and even directly in situ. We show that these ferromagnetic particles are nontoxic between 0.1 and 10 mg/mL; certain species of particles can permit or even enhance tissue formation, and these particles can be tracked using magnetic resonance imaging. Taken together, this approach can be adapted for studying basic biological processes in vitro, for general tissue engineering approaches, and for producing organized repair tissues directly in situ.     

Gene therapy in soft tissue reconstruction

Gene therapy is defined as the introduction of a therapeutic gene into a cell, whose expression can lead to a cure of a disease or offer a transient advantage for tissue growth and regeneration. The delivery of genes can be undertaken for a number of purposes, usually it is attempted to enhance or add a function to a cell or a tissue or to delete or reduce another function. In this brief overview we describe various vehicles and techniques that have been developed to deliver therapeutic genes into cells, such as viral vectors and physical/chemical gene delivery methods including naked DNA and particle-mediated gene transfer, the microseeding technique and the application of lipids. Furthermore we review the potential utility of gene therapy from the perspective of a reconstructive surgeon. Several tissues will be discussed, particularly muscle, tendon, nerve, bone, skin and wounds.     

Silk fibroin in ocular tissue reconstruction

The silk structural protein fibroin displays potential for use in tissue engineering. We present here our opinion of its value as a biomaterial for reconstructing tissues of clinical significance within the human eye. We review the strengths and weaknesses of using fibroin in those parts of the eye that we believe are most amenable to cellular reconstruction, namely the corneoscleral limbus, corneal stroma, corneal endothelium and outer blood-retinal barrier (Ruysch's complex). In these areas we find that by employing the range of manufacturing products afforded by fibroin, relevant structural assemblies can be made for cells expanded ex vivo. Significant questions now need to be answered concerning the effect of this biomaterial on the phenotype of key cell types and the biocompatibility of fibroin within the eye. We conclude that fibroin's strength, structural versatility and potential for modification, combined with the relative simplicity of associated manufacturing processes, make fibroin a worthy candidate for further exploration.     

Sustained regeneration of high-volume adipose tissue for breast reconstruction using computer aided design and biomanufacturing

Adipose tissue engineering offers a promising alternative to the current breast reconstruction options. Here we investigated patient-specific breast scaffolds fabricated from poly(d,l)-lactide polymer with pore sizes >1 mm for their potential in long-term sustained regeneration of high volume adipose tissue. An optimised scaffold geometry was modelled in silico via a laser scanning data set from a patient who underwent breast reconstruction surgery. After the design process scaffolds were fabricated using an additive manufacturing technology termed fused deposition modelling. Breast-shaped scaffolds were seeded with human umbilical cord perivascular cells and cultured under static conditions for 4 weeks and subsequently 2 weeks in a biaxial rotating bioreactor. These in vitro engineered constructs were then seeded with human umbilical vein endothelial cells and implanted subcutaneously into athymic nude rats for 24 weeks. Angiogenesis and adipose tissue formation were observed throughout all constructs at all timepoints. The percentage of adipose tissue compared to overall tissue area increased from 37.17% to 62.30% between week 5 and week 15 (p < 0.01), and increased to 81.2% at week 24 (p < 0.01), while the seeded endothelial cells self-organised to form a functional capillary network. The presented approach of fabricating customised scaffolds using 3D scans represents a facile approach towards engineering clinically relevant volumes of adipose tissue for breast reconstruction.