Suvi Haimi

Growth and Osteogenic Differentiation of Adipose Stem Cells on PLA/Bioactive Glass and PLA/β-TCP Scaffolds

The aim of this study was to compare the effects of novel three-dimensional composite scaffolds consisting of a bioactive phase (bioactive glass or beta-tricalcium phosphate [beta-TCP] 10 and 20 wt%) incorporated within a polylactic acid (PLA) matrix on viability, distribution, proliferation, and osteogenic differentiation of human adipose stem cells (ASCs). The viability and distribution of ASCs on the bioactive composite scaffolds was evaluated using Live/Dead fluorescence staining, environmental scanning electron microscopy, and scanning electron microscopy. There were no differences between the two concentrations of bioactive glass and beta-TCP in PLA scaffolds on proliferation and osteogenic differentiation of ASCs. After 2 weeks of culture, DNA content and alkaline phosphatase (ALP) activity of ASCs cultured on PLA/beta-TCP composite scaffolds were higher relative to other scaffold types. Interestingly, the cell number was significantly lower, but the relative ALP/DNA ratio of ASCs was significantly higher in PLA/bioactive glass scaffolds than in other three scaffold types. These results indicate that the PLA/beta-TCP composite scaffolds significantly enhance ASC proliferation and total ALP activity compared to other scaffold types. This supports the potential future use of PLA/beta-TCP composites as effective scaffolds for tissue engineering and as bone replacement materials.

Characterization of zinc-releasing three-dimensional bioactive glass scaffolds and their effect on human adipose stem cell proliferation and osteogenic differentiation

While the addition of zinc ions to bioactive ceramics has been shown to enhance the proliferation and osteogenic differentiation of osteoblast-like cells, contradictory results have been found. Therefore, the effect of zinc-releasing ceramics on cell proliferation and differentiation into osteogenic lineages requires further clarification. The aim of this study was to evaluate the effects of zinc addition on the degradation profile of three-dimensional bioactive glass scaffold, and on the proliferation and osteogenesis of human adipose stem cells (hASCs) in these scaffolds. Bioactive glass scaffolds containing Na(2)O, K(2)O, MgO, CaO, B(2)O(3), TiO(2), P(2)O(5) and SiO(2) were prepared. The degradation was evaluated by weight loss measurement, scanning electron microscopy and elemental analysis. The degradation profile of bioactive glass was shown to slow down with the addition of zinc. Qualitative live/dead staining showed that zinc addition to bioactive glass inhibits cell spreading and proliferation of hASCs. However, zinc addition had no significant effect on DNA content, alkaline phosphatase activity and osteopontin concentration of hASCs when measured quantitatively. Our results suggest that the possible stimulatory effect of addition of zinc on hASC proliferation and osteogenesis was not detected because addition of zinc slowed down the degradation rate of the studied bioactive glass scaffolds.

The effects of vibration loading on adipose stem cell number, viability and differentiation towards bone-forming cells

Mechanical stimulation is an essential factor affecting the metabolism of bone cells and their precursors. We hypothesized that vibration loading would stimulate differentiation of human adipose stem cells (hASCs) towards bone-forming cells and simultaneously inhibit differentiation towards fat tissue. We developed a vibration-loading device that produces 3g peak acceleration at frequencies of 50 and 100 Hz to cells cultured on well plates. hASCs were cultured using either basal medium (BM), osteogenic medium (OM) or adipogenic medium (AM), and subjected to vibration loading for 3 h d–1 for 1, 7 and 14 day. Osteogenesis, i.e. differentiation of hASCs towards bone-forming cells, was analysed using markers such as alkaline phosphatase (ALP) activity, collagen production and mineralization. Both 50 and 100 Hz vibration frequencies induced significantly increased ALP activity and collagen production of hASCs compared with the static control at 14 day in OM. A similar trend was detected for mineralization, but the increase was not statistically significant. Furthermore, vibration loading inhibited adipocyte differentiation of hASCs. Vibration did not affect cell number or viability. These findings suggest that osteogenic culture conditions amplify the stimulatory effect of vibration loading on differentiation of hASCs towards bone-forming cells.

Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro

IntroductionCurrently, human adipose stem cells (hASCs) are differentiated towards osteogenic lineages using culture medium supplemented with L-ascorbic acid 2-phosphate (AsA2-P), dexamethasone (Dex) and beta-glycerophosphate (β-GP). Because this osteogenic medium (OM1) was initially generated for the differentiation of bone marrow-derived mesenchymal stem cells, the component concentrations may not be optimal for the differentiation of hASCs. After preliminary screening, two efficient osteogenic media (OM2 and OM3) were chosen to be compared with the commonly used osteogenic medium (OM1). To further develop the culture conditions towards clinical usage, the osteo-inductive efficiencies of OM1, OM2 and OM3 were compared using human serum (HS)-based medium and a defined, xeno-free medium (RegES), with fetal bovine serum (FBS)-based medium serving as a control.MethodsTo compare the osteo-inductive efficiency of OM1, OM2 and OM3 in FBS-, HS- and RegES-based medium, the osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic marker genes (runx2A, DLX5, collagen type I, osteocalcin, and ALP).ResultsIn HS-based medium, the ALP activity increased significantly by OM3, and mineralization was enhanced by both OM2 and OM3, which have high AsA2-P and low Dex concentrations. ALP activity and mineralization of hASCs was the weakest in FBS-based medium, with no significant differences between the OM compositions due to donor variation. However, the qRT-PCR data demonstrated significant upregulation of runx2A mRNA under osteogenic differentiation in FBS- and HS-based medium, particularly by OM3 under FBS conditions. Further, the expression of DLX5 was greatly stimulated by OM1 to 3 on day 7 when compared to control. The regulation of collagen type I, ALP, and osteocalcin mRNA was modest under induction by OM1 to 3. The RegES medium was found to support the proliferation and osteogenic differentiation of hASCs, but the composition of the RegES medium hindered the comparison of OM1, OM2 and OM3.ConclusionsSerum conditions affect hASC proliferation and differentiation significantly. The ALP activity and mineralization was the weakest in FBS-based medium, although osteogenic markers were upregulated on mRNA level. When comparing the OM composition, the commonly used OM1 was least effective. Accordingly, higher concentration of AsA2-P and lower concentration of Dex, as in OM2 and OM3, should be used for the osteogenic differentiation of hASCs in vitro.

Hollow fibers of poly(lactide-co-glycolide) and poly(ε-caprolactone) blends for vascular tissue engineering applications.

At present the manufacture of small-diameter blood vessels is one of the main challenges in the field of vascular tissue engineering. Currently available vascular grafts rapidly fail due to development of intimal hyperplasia and thrombus formation. Poly(lactic-co-glycolic acid) (PLGA) hollow fiber (HF) membranes have previously been proposed for this application, but as we show in the present work, they have an inhibiting effect on cell proliferation and rather poor mechanical properties. To overcome this we prepared HF membranes via phase inversion using blends of PLGA with poly(ε-caprolactone) (PCL). The influence of polymer composition on the HF physicochemical properties (topography, water transport and mechanical properties) and cell attachment and proliferation were studied. Our results show that only the ratio PCL/PLGA of 85/15 (PCL/PLGA85/15) yielded a miscible blend after processing. A higher PLGA concentration in the blend led to immiscible PCL/PLGA phase-separated HFs with an inhomogeneous morphology and variation in the cell culture results. In fact, the PCL/PLGA85/15 blend, which had the most homogeneous morphology and suitable pore structure, showed better human adipose stem cell (hASC) attachment and proliferation compared with the homopolymers. This, combined with the good mechanical and transport properties, makes them potentially useful for the development of small-caliber vascular grafts.

Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation

Signals from the microenvironment around a cell are known to influence cell behavior. Material properties, such as biochemical composition and substrate stiffness, are today accepted as significant regulators of stem cell fate. The knowledge of how cell behavior is influenced by 3D geometric cues is, however, strongly limited despite its potential relevance for the understanding of tissue regenerative processes and the design of biomaterials. Here, the role of surface curvature on the migratory and differentiation behavior of human mesenchymal stem cells (hMSCs) has been investigated on 3D surfaces with well‐defined geometric features produced by stereolithography. Time lapse microscopy reveals a significant increase of cell migration speed on concave spherical compared to convex spherical structures and flat surfaces resulting from an upward‐lift of the cell body due to cytoskeletal forces. On convex surfaces, cytoskeletal forces lead to substantial nuclear deformation, increase lamin‐A levels and promote osteogenic differentiation. The findings of this study demonstrate a so far missing link between 3D surface curvature and hMSC behavior. This will not only help to better understand the role of extracellular matrix architecture in health and disease but also give new insights in how 3D geometries can be used as a cell‐instructive material parameter in the field of biomaterial‐guided tissue regeneration.

Autologous Adipose Stem Cells in Treatment of Female Stress Urinary Incontinence: Results of a Pilot Study

The purpose of our study was to find out whether transurethral injections of autologous adipose stem cells (ASCs) are an effective and a safe treatment for female stress urinary incontinence (SUI). We treated five SUI patients with ASCs combined with bovine collagen gel and saline. Prior to the treatment, the ASCs were isolated from subcutaneous fat and expanded for 3 weeks in a good manufacturing practice‐level laboratory. The mixture of ASCs and collagen was injected transurethrally via cystoscope. Additionally, viability, multipotency, and surface marker profile of ASCs were analyzed in vitro. We followed up with patients 3, 6, and 12 months after the injections. The primary endpoint was a cough test to measure objectively the effect of the treatment. Validated questionnaires were used to determine the subjective cure rate. After 6 months, 1 of 5 patients displayed a negative cough test with full bladder filled with 500 ml of saline. At 1 year, the cough test was negative with three patients; two of them were satisfied with the treatment and did not wish further treatment for SUI. Validated questionnaires showed some subjective improvement in all five patients. This is the first study describing the use of autologous ASCs in combination with collagen gel for female SUI treatments. Thus far, the treatment with autologous ASCs has proven safe and well tolerated. However, the feasibility and efficacy of the treatment were not optimal; therefore, additional research is needed to develop SUI injection therapies.

Porous polylactide/β-tricalcium phosphate composite scaffolds for tissue engineering applications

Porous polylactide/β‐tricalcium phosphate (PLA/β‐TCP) composite scaffolds were fabricated by freeze‐drying. The aim of this study was to characterize these graded porous composite scaffolds in two different PLA concentrations (2 and 3 wt%). Also, three different β‐TCP ratios (5, 10 and 20 wt%) were used to study the effect of β‐TCP on the properties of the polymer. The characterization was carried out by determining the pH, weight change, component ratios, thermal stability, inherent viscosity and microstructure of the scaffolds in 26 weeks of hydrolysis. This study indicated that no considerable change was noticed in the structure of the scaffolds when the β‐TCP filler was added. Also, the amount of β‐TCP did not affect the pore size or the pore distribution in the scaffolds. We observed that the fabrication method improved the thermal stability of the samples. Our results suggest that, from the structural point of view, these scaffolds could have potential for the treatment of osteochondral defects in tissue engineering applications. The porous bottom surface of the scaffold and the increased osteogenic differentiation potential achieved with β‐TCP particles may encourage the growth of bone cells. In addition, the dense surface skin of the scaffold may inhibit the ingrowth of osteoblasts and bone tissue, while simultaneously encouraging the ingrowth of chondrocytes. Copyright

Surface properties and interaction forces of biopolymer-doped conductive polypyrrole surfaces by atomic force microscopy

Surface properties and electrical charges are critical factors elucidating cell interactions on biomaterial surfaces. The surface potential distribution and the nanoscopic and microscopic surface elasticity of organic polypyrrole-hyaluronic acid (PPy-HA) were studied by atomic force microscopy (AFM) in a fluid environment in order to explain the observed enhancement in the attachment of human adipose stem cells on positively charged PPy-HA films. The electrostatic force between the AFM tip and a charged PPy-HA surface, the tip-sample adhesion force, and elastic moduli were estimated from the AFM force curves, and the data were fitted to electrostatic double-layer and elastic contact models. The surface potential of the charged and dried PPy-HA films was assessed with Kelvin probe force microscopy (KPFM), and the KPFM data were correlated to the fluid AFM data. The surface charge distribution and elasticity were both found to correlate well with the nodular morphology of PPy-HA and to be sensitive to the electrochemical charging conditions. Furthermore, a significant change in the adhesion was detected when the surface was electrochemically charged positive. The results highlight the potential of positively charged PPy-HA as a coating material to enhance the stem cell response in tissue-engineering scaffolds.

Characterizing and optimizing poly-l-lactide-co-ε-caprolactone membranes for urothelial tissue engineering

Different synthetic biomaterials such as polylactide (PLA), polycaprolactone and poly-l-lactide-co-ε-caprolactone (PLCL) have been studied for urothelial tissue engineering, with favourable results. The aim of this research was to further optimize the growth surface for human urothelial cells (hUCs) by comparing different PLCL-based membranes: smooth (s) and textured (t) PLCL and knitted PLA mesh with compression-moulded PLCL (cPLCL). The effects of topographical texturing on urothelial cell response and mechanical properties under hydrolysis were studied. The main finding was that both sPLCL and tPLCL supported hUC growth significantly better than cPLCL. Interestingly, tPLCL gave no significant advantage to hUC attachment or proliferation compared with sPLCL. However, during the 14 day assessment period, the majority of cells were viable and maintained phenotype on all the membranes studied. The material characterization exhibited potential mechanical characteristics of sPLCL and tPLCL for urothelial applications. Furthermore, the highest elongation of tPLCL supports the use of this kind of texturing. In conclusion, in light of our cell culture results and mechanical characterization, both sPLCL and tPLCL should be further studied for urothelial tissue engineering.