Lentivirally administered glial cell line-derived neurotrophic factor promotes post-ischemic neurological recovery, brain remodeling and contralesional pyramidal tract plasticity by regulating axonal growth inhibitors and guidance proteins.

Owing to its potent longterm neuroprotective and neurorestorative properties, glial cell line-derived neurotrophic factor (GDNF) is currently studied in neurodegenerative disease clinical trials. However, little is known about the longterm effect of GDNF on neurological recovery, brain remodeling and neuroplasticity in the post-acute phase of ischemic stroke. In a comprehensive set of experiments, we examined the effects of lentiviral GDNF administration after ischemic stroke. GDNF reduced neurological deficits, neuronal injury, blood-brain barrier permeability in the acute phase in mice. As compared with control, enhanced motor-coordination and spontaneous locomotor activity were noted in GDNF-treated mice, which were associated with increased microvascular remodeling, increased neurogenesis and reduced glial scar formation in the peri-infarct tissue. We observed reduced brain atrophy and increased plasticity of contralesional pyramidal tract axons that crossed the midline in order to innervate denervated neurons in the ipsilesional red and facial nuclei. Contralesional axonal plasticity by GDNF was associated with decreased abundance of the axonal growth inhibitors brevican and versican in contralesional and ipsilesional brain tissue, reduced abundance of the growth repulsive guidance molecule ephrin b1 in contralesional brain tissue, increased abundance of the midline growth repulsive protein Slit1 in contralesional brain tissue and reduced abundance of Slit1's receptor Robo2 in ipsilesional brain tissue. These data indicate that GDNF potently induces longterm neurological recovery, peri-infarct brain remodeling and contralesional neuroplasticity, which are associated with the fine-tuned regulation of axonal growth inhibitors and guidance molecules that facilitate the growth of contralesional corticofugal axons in the direction to the ipsilesional hemisphere.

Influence of STRO-1 selection on osteogenic potential of human tooth germ derived mesenchymal stem cells.

Mesenchymal stem cells derived from the human tooth germ (hTGSCs) are a heterogeneous cell population that can differentiate into osteogenic, neurogenic, and adipogenic lineages. The aim of this study was to compare the osteogenic differentiation capacity of STRO-1 positive (STRO-1+) hTGSCs and unsorted heterogeneous hTGSCs and to establish if STRO-1+ cells are more committed to osteogenic differentiation. HTGSCs were isolated from impacted third molar tooth germ tissues of adolescents, and a subpopulation of STRO-1+ hTGSCs was obtained by fluorescence-activated cell sorting. STRO-1+, STRO-1 negative (STRO-1-), and unsorted cells were cultured in osteogenic and standard culture media to compare their capacity to differentiate towards osteoblastic lineage. Cells were tested for proliferation rates, alkaline phosphatase activity, and amounts of accumulated calcium. Gene expression levels of the RUNX2, osteocalcin, and osteonectin genes were analyzed with real time PCR. Mineralization and osteogenic protein expression were examined by using von Kossa staining and confocal microscopy. Our results indicated that osteogenically induced cell populations showed greater mineralization capacity than non-induced cells. However, expression levels of early and late osteogenic markers were not significantly different between STRO-1+ and unsorted cells. In conclusion, the selection by STRO-1 expression does not yield cells with osteogenic capacity higher than that of the heterogeneous hTGSC population. Cell sorting using osteogenic markers other than STRO-1 might be beneficial in obtaining a more sensitive osteogenic sub-population from unsorted heterogenous hTGSCs.

Biocompatibility of Accelerated Mineral Trioxide Aggregate on Stem Cells Derived from Human Dental Pulp.

The aim of this study was to evaluate the effects of several additives on the setting time and cytotoxicity of accelerated-set mineral trioxide aggregate (MTA) on stem cells of human dental pulp. ProRoot white MTA (WMTA) (Dentsply Tulsa Dental, Johnson City, TN) was mixed with various additives including distilled water, 2.5% disodium hydrogen phosphate (Na2HPO4) (Merck, Darmstadt, Germany), K-Y Jelly (Johnson & Johnson, Markham, ON, Canada), and 5% and 10% calcium chloride (CaCl2) (Merck). The setting times were evaluated using a Vicat apparatus (Alsa Lab, Istanbul, Turkey). Human dental pulp stem cells were isolated and seeded into 48-well plates at 2 × 10(3) cells per well and incubated with MTA samples for 24 hours, 3 days, and 7 days. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. MTA mixed with 10% CaCl2 showed the lowest setting time (P < .05). According to the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium results on the 1st, 3rd, and 7th days, a statistically significant difference was found (P < .05) between MTA groups and the control group. MTA mixed with K-Y Jelly in all groups showed the lowest cell viability at all time points (P < .05). The cell viability of MTA mixed with distilled water, 5% CaCl2, 10% CaCl2, and Na2HPO4 increased significantly through time (P < .05). This in vitro study found MTA mixed with 5% and 10% CaCl2 and Na2HPO4 is biocompatible with dental pulp stem cells in terms of cell viability. Further in vitro and in vivo investigations are required to prove the clinical applications of MTA mixed with various additives.

Incorporation of growth factor loaded microspheres into polymeric electrospun nanofibers for tissue engineering applications.

Nanofibrous double-layer matrices were prepared by electrospinning technique with the bottom layer formed from PCL (poly-ε-caprolactone)/PLLA (poly-l-lactic acid) nanofibers and the upper layer from PCL/Gelatin nanofibers. Bottom layer was designed to give mechanical strength to the system, whereas upper layer containing gelatin was optimized to improve the cell adhesion. Gelatin microspheres were incorporated in the middle of two layers for controlled growth factor delivery. Successful fabrication of the blend nanofibers were shown by spectroscopy. Scanning electron microscopy results demonstrated that bead-free nanofibers with uniform morphology could be obtained by 10% w/v concentrations of PCL/PLLA and PCL/Gelatin solutions. Microspheres prepared by 15% gelatin concentration and cross-linked with 7.5% glutaraldehyde solution were chosen after in vitro release studies for the incorporation to the double-layer matrices. The optimized conditions were used to prepare fibroblast growth factor-2 (FGF-2) loaded microspheres. Preliminary cell culture studies showed that the FGF-2 could be actively loaded into the microspheres and enhanced the cell attachment and proliferation. The complete system had a slow degradation rate in saline (18% weight loss in 2 months) and it could meanwhile preserve its integrity. This sandwich system prevented microsphere leakage from the scaffold, and the hydrophilic and bioactive nature of the fibers at the upper layer promoted cell attachment to the surface. PLLA/PCL layer, on the other hand, improved the mechanical properties of the system and enabled better handling.

Advanced cell therapies with and without scaffolds.

Tissue engineering and regenerative medicine aim to produce tissue substitutes to restore lost functions of tissues and organs. This includes cell therapies, induction of tissue/organ regeneration by biologically active molecules, or transplantation of in vitro grown tissues. This review article discusses advanced cell therapies that make use of scaffolds and scaffold-free approaches. The first part of this article covers the basic characteristics of scaffolds, including characteristics of scaffold material, fabrication and surface functionalization, and their applications in the construction of hard (bone and cartilage) and soft (nerve, skin, blood vessel, heart muscle) tissue substitutes. In addition, cell sources as well as bioreactive agents, such as growth factors, that guide cell functions are presented. The second part in turn, examines scaffold-free applications, with a focus on the recently discovered cell sheet engineering. This article serves as a good reference for all applications of advanced cell therapies and as well as advantages and limitations of scaffold-based and scaffold-free strategies.

A 3D aligned microfibrous myocardial tissue construct cultured under transient perfusion.

The goal of this study was to design and develop a myocardial patch to use in the repair of myocardial infarctions or to slow down tissue damage and improve long-term heart function. The basic 3D construct design involved two biodegradable macroporous tubes, to allow transport of growth media to the cells within the construct, and cell seeded, aligned fiber mats wrapped around them. The microfibrous mat housed mesenchymal stem cells (MSCs) from human umbilical cord matrix (Wharton's Jelly) aligned in parallel to each other in a similar way to cell organization in native myocardium. Aligned micron-sized fiber mats were obtained by electrospinning a polyester blend (PHBV (5% HV), P(L-D,L)LA (70:30) and poly(glycerol sebacate) (PGS)). The micron-sized electrospun parallel fibers were effective in Wharton's Jelly (WJ) MSCs alignment and the cells were able to retract the mat. The 3D construct was cultured in a microbioreactor by perfusing the growth media transiently through the macroporous tubing for two weeks and examined by fluorescence microscopy for cell distribution and preservation of alignment. The fluorescence images of thin sections of 3D constructs from static and perfused cultures confirmed enhanced cell viability, uniform cell distribution and alignment due to nutrient provision from inside the 3D structure.

Dynamic cell culturing and its application to micropatterned, elastin-like protein-modified poly(N-isopropylacrylamide) scaffolds.

In this study a tissue engineering scaffold was constructed from poly(N-isopropylacrylamide) (pNIPAM) to study the influence of strain on cell proliferation and differentiation. The effect of surface chemistry and topography on bone marrow mesenchymal stem cells was also investigated. Micropatterned pNIPAM films (channels with 10 microm groove width, 2 microm ridge width, 20 microm depth) were prepared by photopolymerization. The films were chemically modified by adsorption of a genetically engineered and temperature sensitive elastin-like protein (ELP). Dynamic conditions were generated by repeated temperature changes between 29 degrees C and 37 degrees C. ELP presence on the films enhanced initial cell attachment two fold (Day 1 cell number on films with ELP and without ELP were 27.6 x 10(4) and 13.2 x 10(4), respectively) but had no effect on proliferation in the long run. ELP was crucial for maintaining the cells attached on the surface in dynamic culturing (Day 7 cell numbers on the films with and without ELP were 81.4 x 10(4) and 12.1 x 10(4), respectively) and this enhanced the ability of pNIPAM films to transfer mechanical stress on the cells. Dynamic conditions improved cell proliferation (Day 21 cell numbers with dynamic and with static groups were 180.4 x 10(4) and 157.7 x 10(4), respectively) but decreased differentiation (Day 14 specific ALP values on the films of static and dynamic groups were 6.6 and 3.5 nmol/min/cell, respectively). Thus, a physically and chemically modified pNIPAM scaffold had a positive influence on the population of the scaffolds under dynamic culture conditions.

Sequential growth factor delivery from complexed microspheres for bone tissue engineering.

Aim of the study was to design a 3D tissue-engineering scaffold capable of sequentially delivering two bone morphogenetic proteins (BMP). The novel delivery system consisted of microspheres of polyelectrolyte complexes of poly(4-vinyl pyridine) (P(4)VN) and alginic acid loaded with the growth factors BMP-2 and BMP-7 which themselves were loaded into the scaffolds constructed of PLGA. Microspheres carrying the growth factors were prepared using polyelectrolyte solutions with different concentrations (4-10%) to control the growth factor release rate. Release kinetics was studied using albumin as the model drug and the populations that release their contents very early and very late in the release study were selected to carry BMP-2 and BMP-7, respectively. Foam porosity changed when the microspheres were loaded. Bone marrow derived stem cells (BMSC) from rats were seeded into these foams. Alkaline phosphatase (ALP) activities were found to be lowest and cell proliferation was highest at all time points with foams carrying both the microsphere populations, regardless of BMP presence. With the present doses used neither BMP-2 nor BMP-7 delivery had any direct effect on proliferation, however, they enhanced osteogenic differentiation. Co-administration of BMP enhanced osteogenic differentiation to a higher degree than with their single administration.