Comparison of bone marrow cell growth on 2D and 3D alginate hydrogels

Calcium cross-linked sodium alginate hydrogels have several advantageous features making them potentially suitable as tissue engineering scaffolds and this material has been previously used in many biomedical applications. 3D cell culture systems are often very different from 2D petri dish type cultures. in this study the effect of alginate hydrogel architecture was investigated by comparing rat bone marrow cell proliferation and differentiation on calcium cross linked sodium alginate discs and 1mm internal diameter tubes. It was found that bone marrow cell proliferation was diminished as the concentration of alginate in the 2D hydrogel substrates increased, yet proliferation was extensive on tubular alginate constructs with high alginate contents. Alginate gel thickness was found to be an important parameter in determining cell behaviour and the different geometries did not generate significant alterations in BMC differentiation profiles.     

Directed cell growth and alignment on protein-patterned 3D hydrogels with stereolithography

The stereolithography apparatus (SLA) is a computer-assisted, three-dimensional (3D) printing system that is gaining attention in the medical field for the fabrication of patient-specific prosthetics and implants. An attractive class of implantable biomaterials for the SLA is photopolymerisable hydrogels because of their resemblance to soft tissues and intrinsic support of living cells. However, most laser-based SLA machines lack the minimum feature size required to imitate cell growth and alignment patterns in complex tissue architecture. In this study, we demonstrate a simple method for aligning cells on 3D hydrogels by combining the micro-contact printing (µCP) technique with the stereolithographic process. Fibronectin modified with acrylate groups was printed on glass coverslips with unpatterned, 10, 50, and 100 µm wide line patterns, which were then transferred to hydrogels through chemical linkages during photopolymerisation. Fibroblasts cultured on protein-printed 3D hydrogels aligned in the direction of the patterns, as confirmed by fast Fourier transform and cell morphometrics.     

Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part 6: 3D hydrogels with positive and negative surface charges and polyelectrolyte complexes in spinal cord injury repair

Macroporous hydrogels are artificial biomaterials commonly used in tissue engineering, including central nervous system (CNS) repair. Their physical properties may be modified to improve their adhesion properties and promote tissue regeneration. We implanted four types of hydrogels based on 2-hydroxyethyl methacrylate (HEMA) with different surface charges inside a spinal cord hemisection cavity at the Th8 level in rats. The spinal cords were processed 1 and 6 months after implantation and histologically evaluated. Connective tissue deposition was most abundant in the hydrogels with positively-charged functional groups. Axonal regeneration was promoted in hydrogels carrying charged functional groups; hydrogels with positively charged functional groups showed increased axonal ingrowth into the central parts of the implant. Few astrocytes grew into the hydrogels. Our study shows that HEMA-based hydrogels carrying charged functional groups improve axonal ingrowth inside the implants compared to implants without any charge. Further, positively charged functional groups promote connective tissue infiltration and extended axonal regeneration inside a hydrogel bridge.     

Sequential assembly of 3D perfusable microfluidic hydrogels

Bottom-up tissue engineering provides a promising way to recreate complex structural organizations of native organs in artificial constructs by assembling functional repeating modules. However, it is challenging for current bottom-up strategies to simultaneously produce a controllable and immediately perfusable microfluidic network in modularly assembled 3D constructs. Here we presented a bottom-up strategy to produce perfusable microchannels in 3D hydrogels by sequentially assembling microfluidic modules. The effects of agarose–collagen composition on microchannel replication and 3D assembly of hydrogel modules were investigated. The unique property of predefined microchannels in transporting fluids within 3D assemblies was evaluated. Endothelial cells were incorporated into the microfluidic network of 3D hydrogels for dynamic culture in a house-made bioreactor system. The results indicated that the sequential assembly method could produce interconnected 3D predefined microfluidic networks in optimized agarose–collagen hydrogels, which were fully perfusable and successfully functioned as fluid pathways to facilitate the spreading of endothelial cells. We envision that the presented method could be potentially used to engineer 3D vascularized parenchymal constructs by encapsulating primary cells in bulk hydrogels and incorporating endothelial cells in predefined microchannels.     

Hyaluronic acid hydrogels incorporating platelet lysate enhance human pulp cell proliferation and differentiation

The restoration of dentine-pulp complex remains a challenge for dentists; nonetheless, it has been poorly addressed. An ideal system should modulate the host response, as well as enable the recruitment, proliferation and differentiation of relevant progenitor cells. Herein was proposed a photocrosslinkable hydrogel system based on hyaluronic acid (HA) and platelet lysate (PL). PL is a cocktail of growth factors (GFs) and cytokines involved in wound healing orchestration, obtained by the cryogenic processing of platelet concentrates, and was expected to provide the HA hydrogels specific biochemical cues to enhance pulp cells’ recruitment, proliferation and differentiation. Stable HA hydrogels incorporating PL (HAPL) were prepared after photocrosslinking of methacrylated HA (Met-HA) previously dissolved in PL, triggered by the Ultra Violet activated photoinitiator Irgacure 2959. Both the HAPL and plain HA hydrogels were shown to be able to recruit cells from a cell monolayer of human dental pulp stem cells (hDPSCs) isolated from permanent teeth. The hDPCs were also seeded directly over the hydrogels (5?×?10⁴ cells/hydrogel) and cultured in osteogenic conditions. Cell metabolism and DNA quantification were higher, in all time-points, for PL supplemented hydrogels (p?<?0,05). Alkaline phosphatase (ALPL) activity and calcium quantification peaks were observed for the HAPL group at 21 days (p?<?0,05). The gene expression for ALPL and COLIA1 was up-regulated at 21 days to HAPL, compared with HA group (p?<?0,05). Within the same time point, the gene expression for RUNX2 did not differ between the groups. Overall, data demonstrated that the HA hydrogels incorporating PL increased the cellular metabolism and stimulate the mineralized matrix deposition by hDPSCs, providing clear evidence of the potential of the proposed system for the repair of damaged pulp/dentin tissue and endodontics regeneration.     

Preparation and characterization of hydrophobically modified polyacrylamide hydrogels by grafting glycidyl methacrylate

A modified polyacrylamide (PAM-g-GMA) has been prepared by ring-opening reaction of glycidyl methacrylate (GMA) monomer grafted onto the –COO⁻ groups of partially hydrolytic polyacrylamide (PAM) chain. The modified polyacrylamide hydrogels were obtained via a free radical polymerization of PAM-g-GMA without adding any a crosslinker using potassium persulfate (KPS), as initiator, and triethanolamine (TEA), as a coupling agent in aqueous solution. The molecular structure of PAM-g-GMA was characterized by FT-IR, ¹H-NMR, and the thermal behaviors of hydrogels were studied by DSC. Furthermore, the swelling property and compressive properties of PAM-g-GMA hydrogels were investigated. The results show that the modified polyacrylamide hydrogels exhibit a remarkable hydration-dehydration change in response to pH in aqueous media and also undergo dramatic increase in volume with increasing temperature. So the modified polyacrylamide hydrogels will have promising and wide applications such as pharmaceutical use, water retention, electrophoretic media and so on.     

PEG-based bioresponsive hydrogels with redox-mediated formation and degradation

A hydrogel which will undergo macroscopic transition responding to redox stimuli is prepared. Mercapto precursors are prepared from 4-armed polyethylene glycol and after deprotection of thiolate anions, they can transform into disulfide crosslinked hydrogels within 3 min by responding to oxidant H₂O₂. Desirable elasticity is exhibited with a wide range of storage modulus from 50 Pa to 14 kPa through rheological investigation. In addition, the hydrogels are found to be hydrolytically stable but degrade within 75 days when exposed to reductant such as glutathione (GSH). So gelation time and degradation behavior can be regulated by concentrations of precursor, oxidant, reductant, temperature, and pH value. Notably, interest arises from the long-period degradation under low GSH concentration of 0.01 mM that is similar to extracellular level, but not the fast disintegration under high concentration intracellular, providing the possibility of “smart” degradation responding to those cell-secreted biomacromolecules during the process of tissue regeneration. Furthermore, both hydrogels and their degradation products show cell viability above 90% culturing with C2C12 cells, representing nontoxic properties. Such a stimuli-responsive degradation strategy will give promising application in tissue repair and regeneration; especially enable the achievement of matching the degradation kinetics with physiological environment.     

\upalpha 5\upbeta 1-integrin and MT1-MMP promote tumor cell migration in 2D but not in 3D fibronectin microenvironments

Cell migration is a crucial event for physiological processes, such as embryonic development and wound healing, as well as for pathological processes, such as cancer dissemination and metastasis formation. Cancer cell migration is a result of the concerted action of matrix metalloproteinases (MMPs), expressed by cancer cells to degrade the surrounding matrix, and integrins, the transmembrane receptors responsible for cell binding to matrix proteins. While it is known that cell-microenvironment interactions are essential for migration, the role of the physical state of such interactions remains still unclear. In this study we investigated human fibrosarcoma cell migration in two-dimensional (2D) and three-dimensional (3D) fibronectin (FN) microenvironments. By using antibody blocking approach and cell-binding site mutation, we determined that $\upalpha _{5}\upbeta _{1}$ -integrin is the main mediator of fibrosarcoma cell migration in 2D FN, whereas in 3D fibrillar FN, the binding of $\upalpha _{5}\upbeta _{1}$ - and $\upalpha _\mathrm{v}\upbeta _{3}$ -integrins is not necessary for cell movement in the fibrillar network. Furthermore, while the general inhibition of MMPs with GM6001 has no effect on cell migration in both 2D and 3D FN matrices, we observed opposing effect after targeted silencing of a membrane-bound MMP, namely MT1-MMP. In 2D fibronectin, silencing of MT1-MMP results in decreased migration speed and loss of directionality, whereas in 3D FN matrices, cell migration speed is increased and integrin-mediated signaling for actin dynamics is promoted. Our results suggest that the fibrillar nature of the matrix governs the migratory behavior of fibrosarcoma cells. Therefore, to hinder migration and dissemination of diseased cells, matrix molecules should be directly targeted, rather than specific subtypes of receptors at the cell membrane.     

Evaluating cell proliferation based on internal pore size and 3D scaffold architecture fabricated using solid freeform fabrication technology

The scaffold, as a medical component to regenerate tissues or organs in humans, plays an important role in tissue engineering. Recently, solid freeform fabrication (SFF) technology using computer-assisted methods was applied to address the problems of conventional fabrication methods in which the internal/outer architectures cannot be controlled. In this report, we propose suitable scaffolds for bone tissue regeneration considering the internal pore size and scaffold architecture. Poly(propylene fumarate) was used as the biodegradable photopolymer, and scaffolds were fabricated using microstereolithography (MSTL). We observed the relationship between the internal pores and architecture, and the proliferation of pre-osteoblast cells. To demonstrate the superiority of MSTL, we fabricated conventional and SFF scaffolds, and measured the cell proliferation rates for each. The results showed that cell proliferation on the MSTL scaffold was clearly superior and indicated that MSTL would be a good replacement for current conventional methods.     

Fibrillized peptide microgels for cell encapsulation and 3D cell culture

One of the advantages of materials produced by self-assembly is that in principle they can be formed in any given container to produce materials of predetermined shapes and sizes. Here, we developed a method for triggering peptide self-assembly within the aqueous phase of water-in-oil emulsions to produce spherical microgels composed of fibrillized peptides. Size control over the microgels was achieved by specification of blade type, speed, and additional shear steps in the emulsion process. Microgels constructed in this way could then be embedded within other self-assembled peptide matrices by mixing pre-formed microgels with un-assembled peptides and inducing gelation of the entire composite, offering a route towards multi-peptide materials with micron-scale domains of different peptide formulations. The gels themselves were cytocompatible, as was the microgel fabrication procedure, enabling the encapsulation of NIH 3T3 fibroblasts and C3H10T-1/2 mouse pluripotent stem cells with good viability.     

Evaluation of cell proliferation and differentiation on a poly(propylene fumarate) 3D scaffold treated with functional peptides

Synthetic polymers were used to fabricate a three-dimensional (3D) porous scaffold of poly(propylene fumarate)/diethyl fumarate (PPF/DEF). PPF-based materials are good candidates for bone regeneration, because of their non-toxic, biodegradable byproducts, and excellent mechanical properties. However, they exhibit hydrophobic surface properties that have negative effects on cell adhesion. To change the surface properties of a PPF/DEF scaffold, the authors used three peptide modifications (RGD, cyclo RGD, and RGD-KRSR mixture) to the scaffold and tested the effects on MC3T3-E1 pre-osteoblast adhesion, proliferation, and differentiation. The results indicated that peptide modification (particularly the RGD-KRDR mixture) altered the hydrophobic surface properties of the PPF/DEF scaffold, and promoted cell adhesion. Thus, it was suggest that peptide modification is a useful method for changing the properties of the PPF/DEF scaffold surface and may be applicable in bone tissue engineering.     

3D cell growth and proliferation on a RGD functionalized nanofibrillar hydrogel based on a conformationally restricted residue containing dipeptide

Three-dimensional (3D) hydrogels incorporating a compendium of bioactive molecules can allow efficient proliferation and differentiation of cells and can thus act as successful tissue engineering scaffolds. Self-assembled peptide-based hydrogels can be worthy candidates for such applications as peptides are biocompatible, biodegradable and can be easily functionalized with desired moieties. Here, we report 3D growth and proliferation of mammalian cells (HeLa and L929) on a dipeptide hydrogel chemically functionalized with a pentapeptide containing Arg-Gly-Asp (RGD) motif. The method of functionalization is simple, direct and can be adapted to other functional moieties as well. The functionalized gel was noncytotoxic, exhibited enhanced cell growth promoting properties, and promoted 3D growth and proliferation of cells for almost 2 weeks, with simultaneous preservation of their metabolic activities. The presence of effective cell growth supporting properties in a simple and easy to functionalize dipeptide hydrogel is unique and makes it a promising candidate for tissue engineering and cell biological applications.     

Controlling fibroblast adhesion and proliferation by 1D Al2 O3 nanostructures

The fibrotic encapsulation, which is mainly accompanied by an excessive proliferation of fibroblasts, is an undesired phenomenon after the implantation of various medical devices. Beside the surface chemistry, the topography plays also a major role in the fibroblast–surface interaction. In the present study, one‐dimensional aluminium oxide (1D Al₂ O₃) nanostructures with different distribution densities were prepared to reveal the response of human fibroblasts to the surface topography. The cell size, the cell number and the ability to form well‐defined actin fibres and focal adhesions were significantly impaired with increasing distribution density of the 1D Al₂ O₃ nanostructures on the substratum.Inspec keywords: biomechanics, adhesion, surface chemistry, biomedical materials, cellular biophysics, surface topography, nanostructured materials, alumina, nanomedicineOther keywords: fibrotic encapsulation, medical devices, surface chemistry, human fibroblasts, surface topography, cell size, cell number, well‐defined actin fibres, focal adhesions, distribution density, fibroblast adhesion, 1D nanostructures, distribution densities, fibroblast proliferation, fibroblast‐surface interaction, one‐dimensional aluminium oxide nanostructures, Al₂ O₃      

细胞培养用温度敏感凝胶的合成与性能研究

用丙烯酸（ARc）对壳聚糖（CS）进行化学改性，合成反应中问体壳聚糖衍生物CS-ARc，进一步合成不同配比的CS-ARc与N-异丙基丙烯酰胺（NIPA）的共聚凝胶P（CS-AAc-NIPA），通过红外光谱和元素分析等表征了产物的结构和组成，并研究了P（CS—ARc-NIPA）凝胶在水中和细胞培养基中的溶胀性能．结果表明共聚凝胶在水中和培养基中均显示较好的温度敏感性．对P（CS-ARc-NIPA）共聚凝胶进行细胞培养研究发现，其表面可成功种植成纤维细胞（L929），细胞贴附生长情况良好，表明材料具有很好的细胞相容性．当环境温度降低后，共聚凝胶发生疏水．亲水变化，导致其表面细胞自动脱附，从而避免了使用酶解法脱附细胞造成的细胞功能损伤．’     

The formation and characterization of freon 22 cluster beams

Freon 22 (CF₂HCl) clusters have been found upon gasdynamic cooling of these molecules in a pulsed supersonic beam. A method for diagnostics of the cluster beams of freon 22 has been developed based on the phenomenon of UV multiphoton ionization in combination with the time-of-flight mass spectrometry and the IR photodissociation of clusters. The velocities of directed motion and the longitudinal and transverse velocity components of the thermal motion of (CF₂HCl)_n clusters in a beam of freon 22 were measured for various stagnation pressures P ₀. The degree of beam clusterization was estimated.     

Micro- and nanoscale modification of poly(2-hydroxyethyl methacrylate) hydrogels by AFM lithography and nanoparticle incorporation

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels are widely used as biomaterials. Due to their unique combination of biocompatibility and good mechanical properties, they have potential as scaffolds for tissue engineering applications. To this purpose, topographic and chemical patterning at the nano- to the mesoscale is crucial in order to favor and to characterize cell adhesion and proliferation. Here we report the characterization of as-prepared and patterned PHEMA hydrogels, produced by conventional radical polymerization in water and dimethylformamide. We have obtained chemical and morphological micro- and nanoscale patterning by atomic force microscopy based lithography. We also demonstrate that it is possible to incorporate carbon nanoparticles in the hydrogel matrix by supersonic cluster beam deposition.     

Gland cell cultures into 3D hyaluronan-based scaffolds

In this study we report a preliminary investigation of the feasibility of non-woven/sponge fabrics of a hyaluronan derived biomaterials (benzyl ester of HA (HYAFF-11 FAB, Abano Terme, Italy) for the in vitro culture of rat hepatocytes and rat beta cells. Cell growth on hyaluronan derived biomaterials were tested in the presence of complete medium and in the presence of ECM (extracellular matrix) secreted by fibroblasts previously cultured into the scaffold. Hepatocytes and beta cells were extracted from rat liver/pancreas and seeded either on the HYAFF-11 scaffold alone, or on HYAFF-11 scaffold containing ECM. Direct assay of cell proliferation was performed with MTT test. For morphological observations samples were stained with hematoxylin and eosin. The results obtained by MTT test showed that hepatocytes cultivated in both the above described conditions were able to proliferate up to 14 days and Langerhans islet up to 21 days. After this time, cells started to undergo apoptosis. The morphological analyses showed cell aggregation in three-dimensional structures promoted by the fibers of the biomaterial. Our results confirmed that HYAFF-11 meshes represent a suitable scaffold for hepatocyte adhesion/Langerhans islet organization and proliferation. In particular, the presence of a fibroblast secreted extracellular matrix improves the biological property of the scaffold.