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.     

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.     

Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs

Most tissue engineering (TE) strategies for creating functional replacement tissues or organs rely on the application of temporary three-dimensional scaffolds to guide the proliferation and spread of seeded cells in vitro and in vivo. The characteristics of TE scaffolds are major concerns in the quest to fabricate ideal scaffolds. This paper identifies essential structural characteristics and the pre-requisites for fabrication techniques that can yield scaffolds that are capable of directing healthy and homogeneous tissue development. Emphasis is given to solid freeform (SFF), also known as rapid prototyping, technologies which are fast becoming the techniques of choice for scaffold fabrication with the potential to overcome the limitations of conventional manual-based fabrication techniques. SFF-fabricated scaffolds have been found to be able to address most, if not all the macro- and micro-architectural requirements for TE applications. This paper reviews the application/potential application of state-of-the-art SFF fabrication techniques in creating TE scaffolds. The advantages and limitations of the SFF techniques are compared. Related research carried out worldwide by different institutions, including the authors' research are discussed.     

Solid freeform fabrication and in-vitro response of osteoblast cells of mPEG-PCL-mPEG bone scaffolds

Bone tissue engineering is an emerging approach to provide viable substitutes for bone regeneration. Poly(ethylene glycol) (PEG) is a good candidate of bone scaffold because of several advantages such as hydrophilicity, biocompatibility, and intrinsic resistance to protein adsorption and cell adhesion. However, its low compressive strength limits application for bone regeneration. Poly(ε-caprolactone) (PCL), a hydrophobic nonionic polymer, is adopted to enhance the compressive strength of PEG alone.We aimed to investigate the in-vitro response of osteoblast-like cells cultured with porous scaffolds of triblock PEG-PCL-PEG copolymer fabricated by an air pressure-aided deposition system. A desktop air pressure-aided deposition system that involves melting and plotting PEG-PCL-PEG was used to fabricate three-dimensional scaffolds having rectangular pores. The experimental results showed that PEG-PCL-PEG with a molecular weight of 25,000 can be melted and stably deposited through a heating nozzle at an air pressure of 0.3 MPa and no crack occurs after it solidifies. The scaffolds with pre-determined pore size of 400× 420 μm and a porosity of 79 % were fabricated, and their average compressive strength was found to be 18.2 MPa. Osteoblast-like cells, MC3T3-E1, were seeded on fabricated scaffolds to investigate the in-vitro response of cells including toxicity and cellular locomotion. In a culture period of 28 days, the neutral-red stained osteoblasts were found to well distributed in the interior of the scaffold. Furthermore, the cellular attachment and movement in the first 10 h of cell culture were observed with time-lapse microscopy indicating that the porous PEG-PCL-PEG scaffolds fabricated by air pressure-aided deposition system is non-toxicity for osteoblast-like cells.     

Solid lipid templating of macroporous tissue engineering scaffolds

Macroporous biodegradable cell carriers (scaffolds) provide the three-dimensional matrix for tissue formation in vitro. In this study, we present the fabrication of macroporous scaffolds with high inter-pore connectivity from different biodegradable polymers using the recently developed solid lipid templating technique. Starting from a polymer solution and solid lipid microparticles, a dispersion is prepared and subsequently transferred into molds, which are finally submerged in warm hexane to precipitate the polymer and extract the porogens. The study shows how to control pore structure, pore size and porosity of the scaffold using this technique. The process parameters dispersion viscosity, porogen size and type of polymer are considered. Limits of viscosity are examined by macroscopic and microstructure evaluation of the scaffolds prepared at different viscosities. An approach to rationalize these data by oscillation rheometry is shown. Pore size can be controlled by porogen particle size and adaptation of the viscosity of the polymer solution. Porosity can be modified by changing the ratio of porogen to polymer. The suitability of the resulting scaffolds was shown using an established cartilage cell culture model.     

Nanomechanical analysis of bone tissue engineering scaffolds

Copolymers of (2-hydroxyethyl methacrylate) (HEMA) and methacrylamide monomers conjugated with amino acids were synthesized and crosslinked with ethylene glycol dimethacrylate. The resulting library of copolymers was mineralized in vitro using two distinct methods. In the first mineralization method, the copolymers were polymerized in the presence of a sub-micron hydroxyapatite (HA) suspension. In the second method, copolymers were mineralized with HA using a urea-mediated process. The mechanical properties of all of the copolymers, both mineralized and not, were determined using nanoindentation under both load and displacement control. A power law fit to the initial unloading curve was used to determine a reduced elastic modulus for each material. Between 30 and 300 indentations were performed on each material, and ANOVA analysis was run to determine the statistical significance of differences in modulus between samples. Using nanoindentation, the 22 different samples had reduced modulus values ranging from 840 MPa to 4.14 GPa. Aspartic acid-methacrylate (Asp-MA) copolymers were not distinguishable from the pHEMA control material. Polymerization in the presence of HA created a more uniform material than the urea method of mineralization. Several challenges and solutions encountered in the nanomechanical testing of soft, heterogeneous materials are discussed. These results demonstrate that with proper experimental design, the mechanical properties of tissue engineering scaffold materials based on polymer-ceramic composite materials can be determined using small samples and nanoindentation techniques.     

Electrospun nanostructured scaffolds for bone tissue engineering

The current challenge in bone tissue engineering is to fabricate a bioartificial bone graft mimicking the extracellular matrix (ECM) with effective bone mineralization, resulting in the regeneration of fractured or diseased bones. Biocomposite polymeric nanofibers containing nanohydroxyapatite (HA) fabricated by electrospinning could be promising scaffolds for bone tissue engineering. Nanofibrous scaffolds of poly-l-lactide (PLLA, 860 ± 110 nm), PLLA/HA (845 ± 140 nm) and PLLA/collagen/HA (310 ± 125 nm) were fabricated, and the morphology, chemical and mechanical characterization of the nanofibers were evaluated using scanning electron microscopy, Fourier transform infrared spectroscopy and tensile testing, respectively. The in vitro biocompatibility of different nanofibrous scaffolds was also assessed by growing human fetal osteoblasts (hFOB), and investigating the proliferation, alkaline phosphatase activity (ALP) and mineralization of cells on different nanofibrous scaffolds. Osteoblasts were found to adhere and grow actively on PLLA/collagen/HA nanofibers with enhanced mineral deposition of 57% higher than the PLLA/HA nanofibers. The synergistic effect of the presence of an ECM protein, collagen and HA in PLLA/collagen/HA nanofibers provided cell recognition sites together with apatite for cell proliferation and osteoconduction necessary for mineralization and bone formation. The results of our study showed that the biocomposite PLLA/collagen/HA nanofibrous scaffold could be a potential substrate for the proliferation and mineralization of osteoblasts, enhancing bone regeneration.     

Biomimetic nanofibrous scaffolds for bone tissue engineering

Bone tissue engineering is a highly interdisciplinary field that seeks to tackle the most challenging bone-related clinical issues. The major components of bone tissue engineering are the scaffold, cells, and growth factors. This review will focus on the scaffold and recent advancements in developing scaffolds that can mimic the natural extracellular matrix of bone. Specifically, these novel scaffolds mirror the nanofibrous collagen network that comprises the majority of the non-mineral portion of bone matrix. Using two main fabrication techniques, electrospinning and thermally-induced phase separation, and incorporating bone-like minerals, such as hydroxyapatite, composite nanofibrous scaffolds can improve cell adhesion, stem cell differentiation, and tissue formation. This review will cover the two main processing techniques and how they are being applied to fabricate scaffolds for bone tissue engineering. It will then cover how these scaffolds can enhance the osteogenic capabilities of a variety of cell types and survey the ability of the constructs to support the growth of clinically relevant bone tissue.     

Chitosan-alginate hybrid scaffolds for bone tissue engineering

A biodegradable scaffold in tissue engineering serves as a temporary skeleton to accommodate and stimulate new tissue growth. Here we report on the development of a biodegradable porous scaffold made from naturally derived chitosan and alginate polymers with significantly improved mechanical and biological properties as compared to its chitosan counterpart. Enhanced mechanical properties were attributable to the formation of a complex structure of chitosan and alginate. Bone-forming osteoblasts readily attached to the chitosan-alginate scaffold, proliferated well, and deposited calcified matrix. The in vivo study showed that the hybrid scaffold had a high degree of tissue compatibility. Calcium deposition occurred as early as the fourth week after implantation. The chitosan-alginate scaffold can be prepared from solutions of physiological pH, which may provide a favorable environment for incorporating proteins with less risk of denaturation. Coacervation of chitosan and alginate combined with liquid-solid separation provides a scaffold with high porosity, and mechanical and biological properties suitable for rapid advancement into clinical trials.     

Design of oligolactone-based scaffolds for bone tissue engineering

Novel difunctional oligolactone macromers have been synthesized by ring-opening oligomerization of various lactones (L-lactide, glycolide, p-dioxanone) in the presence of suitable diols (propane-1,2-diol, dianhydro-D-glucitol) and subsequent endcapping of the formed oligolactones with methacrylate moieties. Based on these macromers, two fabrication procedures were developed to fabricate highly porous scaffolds and the material properties including in vitro biodegradation behaviour of the resulting polymeric scaffolds were investigated. Preliminary in vitro studies on the cytocompatibility of the fabricated scaffolds and on osteoblast cultivation on the optimized polymeric materials demonstrated that the oligolactide based polymer networks possess an excellent biocompatibility and that they are promising candidates as scaffolds in bone tissue engineering.     

3D打印骨组织工程支架的研究与应用

背景：虽然应用传统方法制作骨组织工程支架取得一定成就,但在支架的三维结构、力学强度、支架个性化方面不太满意,通过3D打印技术制作支架的方法有望改变这些不足。 目的：对3D打印技术制作骨组织工程支架作一综述,对支架的未来优化进行展望。 方法：应用计算机检索PubMed和谷歌学术数据库中,2008至2015年关于3D打印技术制作骨组织工程支架的文章。纳入包含骨组织工程支架结构设计、材料及通过不同3D打印技术制作的支架性能研究文章,排除观点重复和陈旧的文章,最后对37篇文献进行归纳总结。 结果与结论：目前可用作骨组织工程支架制作的3D打印技术有熔融层积成型、立体平版印刷、选区激光烧结及3DP技术。3D打印技术制作的骨组织工程支架在力学、结构、个性化方面有其独特优势,但该技术仍有很多问题需要解决,比如原材料的问题、不同3D打印技术的不足问题及3D打印机器的改进问题等。相信在未来多学科的共同合作下,可以制作出适合于临床的骨组织工程支架,造福于人类。  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     

Versatile fabrication of vascularizable scaffolds for large tissue engineering in bioreactor

Despite significant progresses were achieved in tissue engineering over the last 20 years, a number of unsolved problems still remain. One of the most relevant issues is the lack of a proper vascularization that is limiting the size of the engineered tissues to smaller than clinically relevant dimensions. Sacrificial molding holds great promise to engineered construct with perfusable vascular architectures, but there is still the need to develop more versatile approaches able to be independent of the nature and dimensions of the construct. In this work we developed a versatile sacrificial molding technique for fabricating bulk, cell-laden and porous scaffolds with embedded vascular fluidic networks. These branched fluidic architectures are created by highly resistant thermoplastic sacrificial templates, made of poly(vinyl alcohol), representing a remarkable progress in manufacturability and scalability. The obtained architecture, when perfused in bioreactor, has shown to prevent the formation of a necrotic core in thick cell-laden constructs and enabled the rapid fabrication of hierarchically branched endothelium. In conclusion we demonstrate a novel strategy towards the engineering of vascularized thick tissues through the integration of the PVA-based microfabrication sacrificial approach and perfusion bioreactors. This approach may be able to scale current engineered tissues to clinically relevant dimensions, opening the way to their widespread clinical applications.     

Optimising bioactive glass scaffolds for bone tissue engineering

A 3D scaffold has been developed that has the potential to fulfil the criteria for an ideal scaffold for bone tissue engineering. Sol-gel derived bioactive glasses of the 70S30C (70 mol% SiO2, 30 mol% CaO) composition have been foamed to produce 3D bioactive scaffolds with hierarchical interconnected pore morphologies similar to trabecular bone. The scaffolds consist of a hierarchical pore network with macropores in excess of 500 microm connected by pore windows with diameters in excess of 100 microm, which is thought to be the minimum pore diameter required for tissue ingrowth and vasularisation in the human body. The scaffolds also have textural porosity in the mesopore range (10-20 nm). The scaffolds were sintered at 600, 700, 800 and 1000 degrees C. As sintering temperature was increased to 800 degrees C the compressive strength increased from 0.34 to 2.26 MPa due to a thickening of the pore walls and a reduction in the textural porosity. The compressive strength is in the range of that of trabecular bone (2-12 MPa). Importantly, the modal interconnected pore diameter (98 microm) was still suitable for tissue engineering applications and bioactivity is maintained. Bioactive glass foam scaffolds sintered at 800 degrees C for 2 h fulfill the criteria for an ideal scaffold for tissue engineering applications.     

Calcium phosphate-chitosan composite scaffolds for bone tissue engineering

Macroporous calcium phosphate-chitosan composite scaffolds were fabricated and evaluated for use in bone tissue engineering. Human osteoblast-like MG63 cells were cultured on the composite scaffolds, and their response to the materials was studied. Cell morphology, total protein content, and expression of classic markers for osteoblast differentiation were characterized. MG63 cells on the hydroxyapatite scaffolds nesting chitosan sponges (HC1) showed significantly higher alkaline phosphatase (ALP) level and osteocalcin (OC) production during the 11-day culture period, compared with the control culture on tissue culture plates. Cells on the chitosan scaffolds incorporated with hydroxyapatite powders (HC2) exhibited lower ALP activity during the 11-day culture period and OC secretion during the first 7 days, in comparison with that on HC1. The addition of calcium phosphate glass as in HC3 scaffolds increased the ALP and OC levels of MG63 cells. Our study indicated that the hydroxyapatite-matrix composite scaffolds might enhance the phenotype expression of MG63 cells, in comparison with chitosan-matrix scaffolds. Soluble calcium phosphate glasses should be added to the scaffolds to prevent chitosan from fast degradation that may affect the differentiation of osteoblast cells.     

Microporous nanofibrous fibrin-based scaffolds for bone tissue engineering

The fibrotic response of the body to synthetic polymers limits their success in tissue engineering and other applications. Though porous polymers have demonstrated improved healing, difficulty in controlling their pore sizes and pore interconnections has clouded the understanding of this phenomenon. In this study, a novel method to fabricate natural polymer/calcium phosphate composite scaffolds with tightly controllable pore size, pore interconnection, and calcium phosphate deposition was developed. Microporous, nanofibrous fibrin scaffolds were fabricated using sphere-templating methods. Composite scaffolds were created by solution deposition of calcium phosphate on fibrin surfaces or by direct incorporation of nanocrystalline hydroxyapatite (nHA). The SEM results showed that fibrin scaffolds exhibited a highly porous and interconnected structure. Osteoblast-like cells, obtained from murine calvaria, attached, spread and showed a polygonal morphology on the surface of the biomaterial. Multiple cell layers and fibrillar matrix deposition were observed. Moreover, cells seeded on mineralized fibrin scaffolds exhibited significantly higher alkaline phosphatase activity as well as osteoblast marker gene expression compared to fibrin scaffolds and nHA incorporated fibrin scaffolds (0.25 and 0.5g). All types of scaffolds were degraded both in vitro and in vivo. Furthermore, these scaffolds promoted bone formation in a mouse calvarial defect model and the bone formation was enhanced by addition of rhBMP-2.     

Freeze casting of hydroxyapatite scaffolds for bone tissue engineering

Although extensive efforts have been put into the development of porous scaffolds for bone regeneration, with encouraging results, all porous materials have a common limitation: the inherent lack of strength associated with porosity. Hence, the development of porous hydroxyapatite scaffolds has been hindered to non-load bearing applications. We report here how freeze casting can be applied to synthesize porous scaffolds exhibiting unusually high compressive strength, e.g. up to 145 MPa for 47% porosity and 65 MPa for 56% porosity. The materials are characterized by well-defined pore connectivity along with directional and completely open porosity. Various parameters affecting the porosity and compressive strength have been investigated, including initial slurry concentration, freezing rate, and sintering conditions. The implications and potential application as bone substitute are discussed. These results might open the way for hydroxyapatite-based materials designed for load-bearing applications. The biological response of these materials is yet to be tested.     

Synthesis of macroporous hydroxyapatite scaffolds for bone tissue engineering

A novel method of preparing macroporous hydroxyapatite (HA) by dual-phase mixing was developed: HA slurry and Polymethylmethacrylate (PMMA) resin were mixed together at the volume ratio of 1:1. After pyrolytic removal of the PMMA phase, HA with an open porous structure was obtained. In this way, the porosity of the ceramic was limited to 50%. Attempts to increase the porosity by adding more PMMA resin were confronted with the technical hurdle of sample collapse during the pyrolysis process. To increase the porosity and to improve pore interconnection, an extra foaming step was introduced before the polymerization of PMMA resin. Three foaming agent systems were tried, based on the reactions of citric acid and (bi)carbonate salts: sodium bicarbonate, calcium carbonate, and ammonium bicarbonate. Although all the three foaming agents were able to increase the porosity up to 70%, keeping all the pores interconnected throughout, only ammonium bicarbonate system turned out to be applicable to make HA scaffolds or implants, because both NaHCO(3) and CaCO(3) systems caused alkalic residues in the final ceramic. The porous HA samples were fully characterized by FTIR, XRD, ESEM (EDX), and optical microscopy.     

Solvent-free polymer/bioceramic scaffolds for bone tissue engineering: fabrication,analysis, and cell growth

This study examines the potential use of porous polycaprolactone (PCL) and polycaprolocatone/hydroxyapatite (PCL/HA) scaffolds fabricated through melt molding and porogen leaching for bone tissue engineering. While eliminating organic solvents is desirable, the process steps proposed in this study for uniformly dispersing HA particles (~5?μm in size) within the scaffold can also contribute to homogeneous properties for these porous composites. Poly(ethylene oxide) (PEO) was chosen as a porogen due to its similar density and melting point as PCL. Pore size of the scaffold was controlled by limiting the size of PCL and PEO particles used in fabrication. The percent of HA in the fabricated scaffolds was quantified by thermogravimetric analysis (TGA). Mechanical testing was used to compare the modulus of the scaffolds to that of bone, and the pore size distribution was examined with microcomputed tomography (μCT). Scanning electron microscopy (SEM) was used to examine the effect on scaffold morphology caused by the addition of HA particles. Both μCT and SEM results showed that HA could be incorporated into PCL scaffolds without negatively affecting scaffold morphology or pore formation. Energy-dispersive X-ray spectroscopy (EDS) and elemental mapping demonstrated a uniform distribution of HA within PCL/HA scaffolds. Murine calvaria-derived MC3T3-E1 cells were used to determine whether cells could attach on scaffolds and grow for up to 21 days. SEM images revealed an increase in cell attachment with the incorporation of HA into the scaffolds. Similarly, DNA content analysis showed a higher cell adhesion to PCL/HA scaffolds.