Tissue engineering: strategies, stem cells and scaffolds

Tissue engineering scaffolds are designed to influence the physical, chemical and biological environment surrounding a cell population. In this review we focus on our own work and introduce a range of strategies and materials used for tissue engineering, including the sources of cells suitable for tissue engineering: embryonic stem cells, bone marrow-derived mesenchymal stem cells and cord-derived mesenchymal stem cells. Furthermore, we emphasize the developments in custom scaffold design and manufacture, highlighting laser sintering, supercritical carbon dioxide processing, growth factor incorporation and zoning, plasma modification of scaffold surfaces, and novel multi-use temperature-sensitive injectable materials.     

Attachment and growth of fibroblasts on poly(L-lactide/epsilon-caprolactone) scaffolds prepared in supercritical CO2 and modified by polyethylene imine grafting with ethylene diamine-plasma in a glow-discharge apparatus

In this study, a copolymer of L-lactide and epsilon-caprolactone (Mn: 73,523, Mw: 127,990 and PI: 1.74) was synthesized by ring-opening polymerization by using stannous octoate as the catalyst. FTIR, 1H-NMR and DSC confirmed the copolymer formation. The copolymer films were prepared and a novel method was developed to produce highly porous sponges for potential use in tissue engineering. Films were subjected to supercritical CO2 at 3300 psi and 70 degrees C to create porous structures for production of possible tissue engineering scaffolds. The pore sizes were in the range of 40-80 microm. The copolymer films were pre-wetted with polyethylene imine (PEI) and then treated with ethylene diamine (EDA)-plasma in glow-discharge apparatus. Gas plasma surface modification of three-dimensional scaffolds fabricated by supercritical carbon dioxide technique was demonstrated to enhance cell adhesion, proliferation, and differentiation over 6 days in culture using L929 fibroblast cell line. Alkaline phosphatase (ALP) activity and glucose uptake in cell culture medium were followed in the cell culture experiments. Fibroblastic cell attachment and growth on the EDA-plasma treated scaffolds were rather low. However, both cell attachment and growth were significantly increased by PEI pre-treatment before EDA-plasma. The changes in ALP activity and glucose uptake also supported the cell growth behavior on these PEI and EDA-plasma treated scaffolds.     

心脏组织工程中细胞和生物支架的研究热点

文题释义：心脏组织工程：是基于组织工程的技术和原理,利用合适来源的细胞和生物支架制造心脏移植物,用于代替受损心脏组织或促进心肌细胞增殖,以恢复或改善心脏功能的技术。 生物支架：是组织工程技术中用于对细胞成分起支撑作用的移植物,其构成成分类似于细胞外基质成分,部分支架具有孔隙等允许血液中氧气及营养物质通过。 背景：心脏组织工程技术的出现和发展,为心血管疾病尤其是心肌梗死的治疗提供了新的选择。 目的：通过对心脏组织工程的2个核心要素即细胞和生物支架的研究进展进行综述,以期为心脏组织工程技术应用于心血管疾病治疗提供参考及依据。 方法：通过检索PubMed 数据库及中国知网数据库2010至2019年期间心脏组织工程相关文章,以“cardiac tissue engineering,cardiomyocytes differentiation,cardiac tissue engineering,cardiomyocytes differentiation,bone marrow derived stem cells,human embryonic stem cells,induced pluripotent stem cells,menstrual blood stem cells,biological scaffolds”为英文检索词,以“心脏组织工程,心肌细胞分化,干细胞,生物支架”等为中文检索词,最终选择78篇英文文献纳入研究。 结果与结论：多种来源的细胞(包括心肌细胞、骨骼肌细胞、心脏成纤维细胞、骨髓来源干细胞、胚胎干细胞、诱导多能干细胞、月经血干细胞)和生物支架(包括水凝胶、脱细胞支架、细胞片及心脏芯片)都可应用于心脏组织工程,但心脏组织工程仍然存在诸多需要解决的问题,如合适的细胞来源、新型支架材料的研发、诱导分化技术的优化,植入时机及途径的优化。 ORCID:0000-0003-2763-5535(王萍) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Solid freeform fabrication of designer scaffolds of hyaluronic acid for nerve tissue engineering

The field of tissue engineering and regenerative medicine will tremendously benefit from the development of three dimensional scaffolds with defined micro- and macro-architecture that replicate the geometry and chemical composition of native tissues. The current report describes a freeform fabrication technique that permits the development of nerve regeneration scaffolds with precisely engineered architecture that mimics that of native nerve, using the native extracellular matrix component hyaluronic acid (HA). To demonstrate the flexibility of the fabrication system, scaffolds exhibiting different geometries with varying pore shapes, sizes and controlled degradability were fabricated in a layer-by-layer fashion. To promote cell adhesion, scaffolds were covalently functionalized with laminin. This approach offers tremendous spatio-temporal flexibility to create architecturally complex structures such as scaffolds with branched tubes to mimic branched nerves at a plexus. We further demonstrate the ability to create bidirectional gradients within the microfabricated nerve conduits. We believe that combining the biological properties of HA with precise three dimensional micro-architecture could offer a useful platform for the development of a wide range of bioartificial organs.     

Electrospun bio-composite P(LLA-CL)/collagen I/collagen III scaffolds for nerve tissue engineering

One of the biggest challenges in peripheral nerve tissue engineering is to create an artificial nerve graft that could mimic the extracellular matrix (ECM) and assist in nerve regeneration. Bio-composite nanofibrous scaffolds made from synthetic and natural polymeric blends provide suitable substrate for tissue engineering and it can be used as nerve guides eliminating the need of autologous nerve grafts. Nanotopography or orientation of the fibers within the scaffolds greatly influences the nerve cell morphology and outgrowth, and the alignment of the fibers ensures better contact guidance of the cells. In this study, poly (L-lactic acid)-co-poly(ε-caprolactone) or P(LLA-CL), collagen I and collagen III are utilized for the fabrication of nanofibers of different compositions and orientations (random and aligned) by electrospinning. The morphology, mechanical, physical, and chemical properties of the electrospun scaffolds along with their biocompatibility using C17.2 nerve stem cells are studied to identify the suitable material compositions and topography of the electrospun scaffolds required for peripheral nerve regeneration. Aligned P(LLA-CL)/collagen I/collagen III nanofibrous scaffolds with average diameter of 253 ± 102 nm were fabricated and characterized with a tensile strength of 11.59 ± 1.68 MPa. Cell proliferation studies showed 22% increase in cell proliferation on aligned P(LLA-CL)/collagen I/collagen III scaffolds compared with aligned pure P(LLA-CL) scaffolds. Results of our in vitro cell proliferation, cell-scaffold interaction, and neurofilament protein expression studies demonstrated that the electrospun aligned P(LLA-CL)/collagen I/collagen III nanofibrous scaffolds mimic more closely towards the ECM of nerve and have great potential as a substrate for accelerated regeneration of the nerve.     

Phase separation,pore structure,and properties of nanofibrous gelatin scaffolds

The development of three-dimensional (3D) biomimetic scaffolds which provide an optimal environment for cells adhesion, proliferation and differentiation, and guide new tissue formation has been one of the major goals in tissue engineering. In this work, a processing technique has been developed to create 3D nanofibrous gelatin (NF-gelatin) scaffolds, which mimic both the physical architecture and the chemical composition of natural collagen. Gelatin matrices with nanofibrous architecture were first created by using a thermally induced phase separation (TIPS) technique. Macroporous NF-gelatin scaffolds were fabricated by combining the TIPS technique with a porogen-leaching process. The processing parameters were systematically investigated in relation to the fiber diameter, fiber length, surface area, porosity, pore size, interpore connectivity, pore wall architecture, and mechanical properties of the NF-gelatin scaffolds. The resulting NF-gelatin scaffolds possess high surface areas (>32 m²/g), high porosities (>96%), well-connected macropores, and nanofibrous pore wall structures. The technique advantageously controls macropore shape and size by paraffin spheres, interpore connectivity by assembly conditions (time and temperature of heat treatment), pore wall morphology by phase separation and post-treatment parameters, and mechanical properties by polymer concentration and crosslinking density. Compared to commercial gelatin foam (Gelfoam^®), the NF-gelatin scaffold showed much better dimensional stability in a tissue culture environment. The NF-gelatin scaffolds, therefore, are excellent scaffolds for tissue engineering.     

Osteoblast response to PLGA tissue engineering scaffolds with PEO modified surface chemistries and demonstration of patterned cell response

Because tissues are characterized by a well-defined three-dimensional arrangement of cells, tissue engineering scaffolds that facilitate the organization and differentiation of new tissue will have improved performance in comparison to scaffolds that only provide surfaces for cell attachment and growth. We hypothesize that instructions for cells can be incorporated into tissue engineering scaffolds by patterning the scaffold's architecture and surface chemistry. Our goals for the presented work were to collect data about cell response to three-dimensional, porous scaffolds with uniformly modified surfaces chemistries, and to demonstrate patterning of cell response by patterning surface chemistry. Our system was osteoblast response to poly(l-lactide-co-glycolide) scaffolds modified with poly(ethylene oxide) (PEO). Scaffolds were fabricated using the Three-Dimensional Printing (3DP) process which has control over scaffolds properties to a resolution of approximately 100 microm in all three dimensions. At higher PEO concentrations, adhesion, growth rates, and migration of rat osteoblasts were reduced; alkaline phosphate activity was increased, and cells were less spread and had microvilli. Patterned regions of low and high cell adhesion were demonstrated on scaffolds fabricated with 1 mm thick stripes of PEO and non-PEO regions.     

Manufacturing of Multi-Layered Nanofibrous Structures Composed of Polyurethane and Poly(ethylene oxide) as Potential Blood Vessel Scaffolds

One of the current limitations in using electrospun nanofibrous materials for tissue engineering is that cells have difficulty penetrating into the materials. For this, multi-layered electrospun structures composed of polyurethane (PU) and poly(ethylene oxide) (PEO) were fabricated and tested in vitro. A 20% (w/v) PU solution was electrospun for 30 min, while a 20% (w/v) PEO solution was electrospun for 5, 15 or 30 min, alternatively. Then, the PEO was extracted by immersing the structure in distilled water to make multi-layered structure. The characteristics of fabricated structures were examined by SEM, FT-IR spectroscopy, mechanical tests and cell penetration test. The bioactivities of smooth muscle cells (SMCs) on these scaffolds were assessed by quantifying DNA, collagen and glycosaminoglycan (GAG) levels. Although hybrid PEO-extracted scaffolds had a little of residual PEO, they were more penetrable than PU alone scaffolds. Also, they showed higher bioactivity than PU-alone scaffolds. The results of this study provided potential of this structure in the application not only to the development of artificial blood vessels but also to other types for tissue engineering.     

Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts

Nanotechnology has enabled the engineering of nanostructured materials to meet current challenges in bone replacement therapies. Biocomposite nanofibrous scaffolds of poly(l-lactic acid)-co-poly(?-caprolactone), gelatin and hydroxyapatite (HA) were fabricated by combining the electrospinning and electrospraying techniques in order to create a better osteophilic environment for the growth and mineralization of osteoblasts. Electrospraying of HA nanoparticles on electrospun nanofibers helped to attain rough surface morphology ideal for cell attachment and proliferation and also achieve improved mechanical properties than HA blended nanofibers. Nanofibrous scaffolds showed high pore size and porosity up to 90% with fiber diameter in the range of 200–700 nm. Nanofibrous scaffolds were characterized for their functional groups and chemical structure by FTIR and XRD analysis. Studies on cell–scaffold interaction were carried out by culturing human fetal osteoblast cells (hFOB) on both HA blended and sprayed PLACL/Gel scaffolds and assessing their growth, proliferation, mineralization and enzyme activity. The results of MTS, ALP, SEM and ARS studies confirmed, not only did HA sprayed biocomposite scaffolds showed better cell proliferation but also enhanced mineralization and alkaline phosphatase activity (ALP) proving that electrospraying in combination with electrospinning produced superior and more suitable biocomposite nanofibrous scaffolds for bone tissue regeneration.     

Engineered pullulan-collagen composite dermal hydrogels improve early cutaneous wound healing

New strategies for skin regeneration are needed to address the significant medical burden caused by cutaneous wounds and disease. In this study, pullulan-collagen composite hydrogel matrices were fabricated using a salt-induced phase inversion technique, resulting in a structured yet soft scaffold for skin engineering. Salt crystallization induced interconnected pore formation, and modification of collagen concentration permitted regulation of scaffold pore size. Hydrogel architecture recapitulated the reticular distribution of human dermal matrix while maintaining flexible properties essential for skin applications. In vitro, collagen hydrogel scaffolds retained their open porous architecture and viably sustained human fibroblasts and murine mesenchymal stem cells and endothelial cells. In vivo, hydrogel-treated murine excisional wounds demonstrated improved wound closure, which was associated with increased recruitment of stromal cells and formation of vascularized granulation tissue. In conclusion, salt-induced phase inversion techniques can be used to create modifiable pullulan-collagen composite dermal scaffolds that augment early wound healing. These novel biomatrices can potentially serve as a structured delivery template for cells and biomolecules in regenerative skin applications.     

Layer-by-layer tissue microfabrication supports cell proliferation in vitro and in vivo

Layer-by-layer biofabrication represents a novel strategy to create three-dimensional living structures with a controlled internal architecture, using cell micromanipulation technologies. Laser assisted bioprinting (LAB) is an effective printing method for patterning cells, biomolecules, and biomaterials in two dimensions. "Biopapers," made of thin polymer scaffolds, may be appropriate to achieve three-dimensional constructs and to reinforce mechanical properties of printed materials. The aim of this work was to evaluate the effect of the tridimensional organization of cells and biomaterials on cell proliferation in vitro and in vivo. The experimental LAB setup was comprised of an infrared laser, focused onto a glass ribbon coated with an absorbing layer of gold. The cell bioink was made of MG63 cells (50 millions cells/mL in culture medium and 1% alginate), transduced with Luciferase gene for tracking and quantification. The printing substrate was a 100-μm-thick polycaprolacton (PCL) electrospun scaffold. The building sequence comprised sequential layers of cells and PCL scaffolds stacked using two different tridimensional arrangements, which were compared in this study (layer-by-layer vs. seeding on a single locus of the scaffolds). Then the cell-seeded materials were cultured in vitro or implanted in vivo in NOD-SCID mice. The qualitative follow-up involved scanning electron microscopy (SEM) observations, live-dead assays, and histology. The cell amount was quantified by photon imager during 21 days in vitro and 2 months in vivo. Live- dead assay and SEM revealed that the cells survived after printing and spread onto PCL membranes. Circle-shaped patterns were maintained in vitro during the first week but they were no longer observable after 2 weeks, due to cell proliferation. Luciferase tracking displayed that the cell amount was increased in vitro and in vivo when the materials and the cells where stacked layer by layer. Histological sections of the in vivo samples revealed a thicker fibrous tissue in the layer-by-layer samples. We have demonstrated in this study that PCL electrospun biopapers can act as a shock-absorbing mattress for cell printing and could further support cell proliferation. The layer-by-layer printing provided an appropriate 3D environment for cell survival and enhanced cell proliferation in vitro and in vivo.     

Precipitation of nanohydroxyapatite on PLLA/PBLG/Collagen nanofibrous structures for the differentiation of adipose derived stem cells to osteogenic lineage

Tissue engineering and nanotechnology have enabled engineering of nanostructured materials to meet the current challenges in bone treatment owing to rising occurrence of bone diseases, accidental damages and defects. Poly(l-lactic acid)/Poly-benzyl-l-glutamate/Collagen (PLLA/PBLG/Col) scaffolds were fabricated by electrospinning and nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering (BTE). The abundance and accessibility of adipose derived stem cells (ADSC) may prove to be novel cell therapeutics for bone repair and regeneration. ADSCs were cultured on these scaffolds and were induced to undergo osteogenic differentiation in the presence of PBLG/n-HA for BTE. The cell-biomaterial interactions were analyzed using cell proliferation, SEM and CMFDA dye extraction techniques. Osteogenic differentiation of ADSC was confirmed using alkaline phosphatase activity (ALP), mineralization (ARS) and dual immunofluorescent staining using both ADSC marker protein and Osteocalcin, which is a bone specific protein. The utmost significance of this study is the bioactive PBLG/n-HA biomolecule introduced on the polymeric nanofibers to regulate and improve specific biological functions like adhesion, proliferation and differentiation of ADSC into osteogenic lineage. This was evident from the immunostaining and CMFDA images of ADSCs showing cuboidal morphology, characteristic of osteogenic lineage. The observed results proved that the PLLA/PBLG/Col/n-HA scaffolds promoted greater osteogenic differentiation of ADSC as evident from the enzyme activity and mineralization profiles for bone tissue engineering.     

Property of Polyvinyl Alcohol-Calcium Alginate as Composite Scaffolds for Tissue Engineering

Objective:A novel PVA-CaAIg composite material by polyvinyl alcohol (PVA) and sodium alginate(SA) was fabricated to investigate the feasibility serving as a scaffold for tissue engineering and to find out the most ideal proportion according to their properties.Methods: Film,graininess and sponge scaffolds of PVA-CaAlg were fabricated by three different methods.Water content and swelling ratio were tested.SEM was used to observe the configuration of the cross section.Results:Different proportional scaffolds could be obtained with different PVA molecular weight,alcoholysis degree and different SA dosages.The water content of different scaffolds ranged from 48% to 93% and showed different inner configuration with swelling ratio between 120% and 470%.SEM proved that different composite materials had different porous structures.Conclusion:A scaffold for tissue engineering with high water content and proper swelling ratio can be fabricated using PVA and SA.The porous structure shows potential in tissue engineering and cell culture.     

Evaluation of biodegradable, three-dimensional matrices for tissue engineering of heart valves

A crucial factor in tissue engineering of heart valves is the type of scaffold material. In the following study, we tested three different biodegradable scaffold materials, polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), and poly-4-hydroxybutyrate (P4HB), as scaffolds for tissue engineering of heart valves. We modified PHA and P4HB by a salt leaching technique to create a porous matrix. We constructed trileaflet heart valve scaffolds from each polymer and tested them in a pulsatile flow bioreactor. In addition, we evaluated the cell attachment to our polymers by creating four tubes of each material (length equals 4 cm; inner diameter, 0.5 cm), seeding each sample with 8,000,000 ovine vascular cells, and incubating the cell-polymer construct for 8 days (37 degrees C and 5% CO2). The seeded vascular constructs were exposed to continuous flow for 1 hour. Analysis of samples included DNA assay before and after flow exposure, 4-hydroxyproline assay, and environmental scanning electron microscopy (ESEM). We fabricated trileaflet heart valve scaffolds from porous PHA and porous P4HB, which opened and closed synchronously in a pulsatile bioreactor. It was not possible to create a functional trileaflet heart valve scaffold from PGA. After seeding and incubating the PGA-, PHA-, and P4HB-tubes, there were significantly (p < 0.001) more cells on PGA compared with PHA and P4HB. There were no significant differences among the materials after flow exposure, but there was a significantly higher collagen content (p < 0.017) on the PGA samples compared with P4HB and PHA. Cell attachment and collagen content was significantly higher on PGA samples compared with PHA and P4HB. However, PHA and P4HB also demonstrate a considerable amount of cell attachment and collagen development and share the major advantage that both materials are thermoplastic, making it possible to mold them into the shape of a functional scaffold for tissue engineering of heart valves.     

Synergic effects of nanofiber alignment and electroactivity on myoblast differentiation

The interactions between cells and materials play critical roles in the success of new scaffolds for tissue engineering, since chemical and physical properties of biomaterials regulate cell adhesion, proliferation, migration, and differentiation. We have developed nanofibrous substrates that possess both topographical cues and electroactivity. The nanofiber scaffolds were fabricated through the electrospinning of polycaprolactone (PCL, a biodegradable polymer) and polyaniline (PANi, a conducting polymer) blends. We investigated the ways in which those properties influenced myoblast behaviors. Neither nanofiber alignment nor PANi concentration influenced cell growth and proliferation, but cell morphology changed significantly from multipolar to bipolar with the anisotropy of nanofibers. According to our analyses of myosin heavy chain expression, multinucleate myotube formation, and the expression of differentiation-specific genes (myogenin, troponin T, MHC), the differentiation of myoblasts on PCL/PANi nanofibers was strongly dependent on both nanofiber alignment and PANi concentration. Our results suggest that topographical cues and the electroactivity of nanofibers synergistically stimulate muscle cell differentiation to make PCL/PANi nanofibers a suitable scaffold material for skeletal tissue engineering.     

Porogen-based solid freeform fabrication of polycaprolactone-calcium phosphate scaffolds for tissue engineering

Drop on demand printing (DDP) is a solid freeform fabrication (SFF) technique capable of generating microscale physical features required for tissue engineering scaffolds. Here, we report results toward the development of a reproducible manufacturing process for tissue engineering scaffolds based on injectable porogens fabricated by DDP. Thermoplastic porogens were designed using Pro/Engineer and fabricated with a commercially available DDP machine. Scaffolds composed of either pure polycaprolactone (PCL) or homogeneous composites of PCL and calcium phosphate (CaP, 10% or 20% w/w) were subsequently fabricated by injection molding of molten polymer-ceramic composites, followed by porogen dissolution with ethanol. Scaffold pore sizes, as small as 200 microm, were attainable using the indirect (porogen-based) method. Scaffold structure and porosity were analyzed by scanning electron microscopy (SEM) and microcomputed tomography, respectively. We characterized the compressive strength of 90:10 and 80:20 PCL-CaP composite materials (19.5+/-1.4 and 24.8+/-1.3 Mpa, respectively) according to ASTM standards, as well as pure PCL scaffolds (2.77+/-0.26 MPa) fabricated using our process. Human embryonic palatal mesenchymal (HEPM) cells attached and proliferated on all scaffolds, as evidenced by fluorescent nuclear staining with Hoechst 33258 and the Alamar Blue assay, with increased proliferation observed on 80:20 PCL-CaP scaffolds. SEM revealed multilayer assembly of HEPM cells on 80:20 PCL-CaP composite, but not pure PCL, scaffolds. In summary, we have developed an SFF-based injection molding process for the fabrication of PCL and PCL-CaP scaffolds that display in vitro cytocompatibility and suitable mechanical properties for hard tissue repair.     

皮肤组织工程-细胞支架的构筑及其生物相容性评价

皮肤组织工程的发展提供了一种无损伤修复创伤和功能重建的皮肤治疗模式.作为组织工程的三要素之一,细胞支架发挥着重要的作用.为满足组织工程中对细胞支架在力学性能、物理结构及生物相容性等方面的要求,我们首先制备了聚乳酸(PDLLA)、聚乳酸-己内酯(PLACL)多孔支架,并以生物相容性较好的猪的无细胞真皮(acellular dermis matrix,ADM)为参比,分别把三种材料植入大鼠背部肌层,术后定期取大鼠皮下埋藏组织进行组织学检测.结果发现PDLLA与PLACL多孔支架的降解周期、力学性能、孔隙率及其孔径都可以根据皮肤组织工程中的要求进行调控.组织学检查,移植物内无明显炎性细胞,21天后,均完全血管化且分布较均匀.说明PDLLA与PLACL的生物相容性较ADM差,但并未出现明显的异物排斥反应,两者的生物相容性基本上可以满足组织工程中对支架的要求,这为聚乳酸类人工皮肤的进一步研究提供了有意义的实验依据.     

Three-dimensional nanocomposite scaffolds fabricated via selective laser sintering for bone tissue engineering

Bionanocomposites formed by combining biodegradable polymers and nanosized osteoconductive inorganic solids have been regarded as promising biomimetic systems which possess much improved structural and functional properties for bone tissue regeneration. In this study three-dimensional nanocomposite scaffolds based on calcium phosphate (Ca-P)/poly(hydroxybutyrate–co-hydroxyvalerate) (PHBV) and carbonated hydroxyapatite (CHAp)/poly(l-lactic acid) (PLLA) nanocomposite microspheres were successfully fabricated using selective laser sintering, which is a rapid prototyping technology. The sintered scaffolds had controlled material microstructure, totally interconnected porous structure and high porosity. The morphology and mechanical properties of Ca-P/PHBV and CHAp/PLLA nanocomposite scaffolds as well as PHBV and PLLA polymer scaffolds were studied. In vitro biological evaluation showed that SaOS-2 cells had high cell viability and normal morphology and phenotype after 3 and 7 days culture on all scaffolds. The incorporation of Ca-P nanoparticles significantly improved cell proliferation and alkaline phosphatase activity for Ca-P/PHBV scaffolds, whereas CHAp/PLLA nanocomposite scaffolds exhibited a similar level of cell response compared with PLLA polymer scaffolds. The nanocomposite scaffolds provide a biomimetic environment for osteoblastic cell attachment, proliferation and differentiation and have great potential for bone tissue engineering applications.     

Preparation and characterization of highly porous, biodegradable polyurethane scaffolds for soft tissue applications

In the engineering of soft tissues, scaffolds with high elastance and strength coupled with controllable biodegradable properties are necessary. To fulfill such design criteria we have previously synthesized two kinds of biodegradable polyurethaneureas, namely poly(ester urethane)urea (PEUU) and poly(ether ester urethane)urea (PEEUU) from polycaprolactone, polycaprolactone-b-polyethylene glycol-b-polycaprolactone, 1,4-diisocyanatobutane and putrescine. PEUU and PEEUU were further fabricated into scaffolds by thermally induced phase separation using dimethyl sulfoxide (DMSO) as a solvent. The effect of polymer solution concentration, quenching temperature and polymer type on pore morphology and porosity was investigated. Scaffolds were obtained with open and interconnected pores having sizes ranging from several mum to more than 150 microm and porosities of 80-97%. By changing the polymer solution concentration or quenching temperature, scaffolds with random or oriented tubular pores could be obtained. The PEUU scaffolds were flexible with breaking strains of 214% and higher, and tensile strengths of approximately 1.0 MPa, whereas the PEEUU scaffolds generally had lower strengths and breaking strains. Scaffold degradation in aqueous buffer was related to the porosity and polymer hydrophilicity. Smooth muscle cells were filtration seeded in the scaffolds and it was shown that both scaffolds supported cell adhesion and growth, with smooth muscle cells growing more extensively in the PEEUU scaffold. These biodegradable and flexible scaffolds demonstrate potential for future application as cell scaffolds in cardiovascular tissue engineering or other soft tissue applications.     

Optimization of PAM scaffolds for neural tissue engineering: preliminary study on an SH-SY5Y cell line

Engineering neural tissue is one of the most challenging goals of tissue engineering. Neural tissue is highly complex and possesses an organized three-dimensional (3D) distribution that is essential for tissue function. An optimal scaffold for tissue engineering has to provide this distribution until the cells are able to activate their normal functions and develop neural connections with the host tissue. Different strategies such as gene therapy and cell transplantation particularly in retinal tissue have been tested, but so far they have only induced retinal degeneration in animals. The objective of this work was to study neural cell assembly as a function of scaffold features and surface chemistry for application in retinal tissue engineering using microfabricated patterns with a well-defined geometry. Because retinal neurons are known to be arranged in hexagonal arrays, hexagonal scaffolds of poly(DL-lactide-co-glycolide) acid were fabricated using a pressure-assisted microsyringe (PAM) system. The behavior of a model cell, neuroblastoma originating from human retina (SH-SY5Y), was analyzed after seeding on the scaffolds, measuring cell density as a function of line width and length of the scaffold to identify the optimal hexagonal geometry. We also evaluated the influence of scaffold on cell metabolism using the methyl thiazolyl tetrazolium assay and on neurite extension. As far as two-dimensional scaffolds are concerned, the results show that although metabolic activity per cell remains constant, hexagons with sides of 500 microm and line widths of 20 +/- 5 microm are optimum for neural cell adhesion in terms of cell density. On 3D scaffolds, cell metabolism is about three times higher than controls, and the optimum number of layers in the scaffold is three or four.