Rapid Mineralization of Electrospun Scaffolds for Bone Tissue Engineering

We investigated different techniques to enhance calcium phosphate mineral precipitation onto electrospun poly(_L-lactide) (PLLA) scaffolds when incubated in concentrated simulated body fluid (SBF), 10×SBF. The techniques included the use of vacuum, pre-treatment with 0.1 M NaOH and electrospinning gelatin/PLLA blends as means to increase overall mineral precipitation and distribution throughout the scaffolds. Mineral precipitation was evaluated using environmental scanning electron microscopy, energy dispersive spectroscopy mapping and the determination of the mineral weight percents. In addition we evaluated the effect of the techniques on mechanical properties, cellular attachment and cellular proliferation on scaffolds. Two treatments, pre-treatment with NaOH and incorporation of 10% gelatin into PLLA solution, both in combination with vacuum, resulted in significantly higher degrees of mineralization (16.55 and 15.14%, respectively) and better mineral distribution on surfaces and through the cross-sections after 2 h of exposure to 10×SBF. While both scaffold groups supported cell attachment and proliferation, 10% gelatin/PLLA scaffolds had significantly higher yield stress (1.73 vs 0.56 MPa) and elastic modulus (107 vs 44 MPa) than NaOH-pre-treated scaffolds.     

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.     

Preparation,in vitro degradability,cytotoxicity, and in vivo biocompatibility of porous hydroxyapatite whisker-reinforced poly(L-lactide) biocomposite scaffolds

Biodegradable and bioactive scaffolds with interconnected macroporous structures, suitable biodegradability, adequate mechanical property, and excellent biocompatibility have drawn increasing attention in bone tissue engineering. Hence, in this work, porous hydroxyapatite whisker-reinforced poly(L-lactide) (HA-w/PLLA) composite scaffolds with different ratios of HA and PLLA were successfully developed through compression molding and particle leaching. The microstructure, in vitro mineralization, cytocompatibility, hemocompatibility, and in vivo biocompatibility of the porous HA-w/PLLA were investigated for the first time. The SEM results revealed that these HA-w/PLLA scaffolds possessed interconnected pore structures. Compared with porous HA powder-reinforced PLLA (HA-p/PLLA) scaffolds, HA-w/PLLA scaffolds exhibited better mechanical property and in vitro bioactivity, as more formation of bone-like apatite layers were induced on these scaffolds after mineralization in SBF. Importantly, in vitro cytotoxicity displayed that porous HA-w/PLLA scaffold with HA/PLLA ratio of 1:1 (HA-w₁/PLLA₁) produced no deleterious effect on human mesenchymal stem cells (hMSCs), and cells performed elevated cell proliferation, indicating a good cytocompatibility. Simultaneously, well-behaved hemocompatibility and favorable in vivo biocompatibility determined from acute toxicity test and histological evaluation were also found in the porous HA-w₁/PLLA₁ scaffold. These findings may provide new prospects for utilizing the porous HA whisker-based biodegradable scaffolds in bone repair, replacement, and augmentation applications.     

Mineralization of hydroxyapatite in electrospun nanofibrous poly(L-lactic acid) scaffolds

A highly porous electrospun poly(L-lactic acid) (PLLA) nanofibrous scaffold was used as a matrix for mineralization of hydroxyapatite. The mineralization process could be initiated by immersing the electrospun scaffold in the simulated body fluids (SBF) at 37 degrees C for varying periods of time. Scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), wide-angle X-ray diffraction (WAXD), Fourier transform infrared (FTIR), and Raman spectroscopy were used to characterize the composition and the structure of the deposited mineral on the nanofiber surface. Results indicated that the mineral phase was a carbonated apatite with thin flake-like nanostructures. The effects of functional groups on the scaffold surface and anionic additives in the incubation fluids on the nucleation and growth of the mineral were investigated. It was found that a minuscule amount of anionic additives (e.g., citric acid and poly-L-aspartic acid) in the SBF could effectively inhibit the mineral growth. Surface modification of the scaffold was carried out by hydrolysis of PLLA scaffold in NaOH aqueous solution, where carboxylic acid groups were produced without compromising the scaffold integrity. The mineralization process from modified PLLA electrospun scaffolds was significantly enhanced because the calcium ions can bind to the carboxylate groups on the fiber surface.     

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.     

A functional agarose-hydroxyapatite scaffold for osteochondral interface regeneration

Regeneration of the osteochondral interface is critical for integrative and functional cartilage repair. This study focuses on the design and optimization of a hydrogel-ceramic composite scaffold of agarose and hydroxyapatite (HA) for calcified cartilage formation. The first study objective was to compare the effects of HA on non-hypertrophic and hypertrophic chondrocytes cultured in the composite scaffold. Specifically, cell growth, biosynthesis, hypertrophy, and scaffold mechanical properties were evaluated. Next, the ceramic phase of the scaffold was optimized in terms of particle size (200 nm vs. 25 μm) and dose (0-6 w/v%). It was observed that while deep zone chondrocyte (DZC) biosynthesis and hypertrophy remained unaffected, hypertrophic chondrocytes measured higher matrix deposition and mineralization potential with the addition of HA. Most importantly, higher matrix content translated into significant increases in both compressive and shear mechanical properties. While cell hypertrophy was independent of ceramic size, matrix deposition was higher only with the addition of micron-sized ceramic particles. In addition, the highest matrix content, mechanical properties and mineralization potential were found in scaffolds with 3% micro-HA, which approximates both the mineral aggregate size and content of the native interface. These results demonstrate that the biomimetic hydrogel-ceramic composite is optimal for calcified cartilage formation and is a promising design strategy for osteochondral interface regeneration.     

In vitro cell performance on hydroxyapatite particles/poly(L-lactic acid) nanofibrous scaffolds with an excellent particle along nanofiber orientation

Highly porous hydroxyapatite (HA)/poly(L-lactide) (PLLA) nanofibrous scaffolds were prepared by incorporating needle-shaped nano- or micro-sized HA particles into PLLA nanofibers using electrospinning. The scaffolds had random or aligned fibrous assemblies and both types of HA particles were perfectly oriented along the fiber long axes. The biocompatibility and cell signaling properties of these scaffolds were evaluated by in vitro culture of rat osteosarcoma ROS17/2.8 cells on the scaffold surface. Cell morphology, viability and alkaline phosphatase (ALP) activity on each scaffold were examined at different time points. The HA/PLLA scaffolds exhibited higher cell viability and ALP activity than a pure PLLA scaffold. In addition, micro-sized HA particles supported cell proliferation and differentiation better than nano-sized ones in random scaffolds through a 10 day culture period and in aligned scaffolds at an early culture stage. The fibrous assembly of the scaffold had a pronounced impact on the morphology of the cells in direct contact with the scaffold surface, but not on cell proliferation and differentiation. Thus, HA/PLLA nanofibrous scaffolds could be good candidates for bone tissue engineering.     

基于三维打印的磷酸三钙骨组织工程支架烧结工艺研究

磷酸三钙(TCP)是构建骨组织工程支架常用的生物陶瓷材料。三维(3D)打印的TCP支架具有精确可控的孔隙结构,但存在力学性能不足的问题。由于烧结工艺对生物陶瓷支架力学性能的影响至关重要,本文详细探讨了不同烧结温度对3D打印TCP支架的力学性能的影响,测试了不同烧结温度制备的支架的表观形貌、质量和体积收缩率、孔隙率、力学性能以及降解性能。结果表明,当烧结温度为1150℃时,晶粒生长充分、气孔最少,支架具有最大的体积收缩率、最小的孔隙率以及最优的力学性能,压缩模量和抗压强度可以分别达到(100.08±18.6)MPa和(6.52±0.84)MPa,能够满足人体松质骨力学强度的要求。此外,与其他烧结温度下制备的支架相比,1150℃下烧结制备的支架在酸性环境中降解最慢,进一步说明其在长期植入时具有更佳的力学稳定性。该支架可支持骨髓间充质干细胞(BMSCs)黏附和快速增殖,具有良好的生物相容性。综上,本文优化了3D打印TCP支架的烧结工艺,提高了其力学性能,为其作为承重骨的应用奠定了基础。     

Simultaneous electrospin–electrosprayed biocomposite nanofibrous scaffolds for bone tissue regeneration

Currently, the application of nanotechnology in bone tissue regeneration is a challenge for the fabrication of novel bioartificial bone grafts. These nanostructures are capable of mimicking natural extracellular matrix with effective mineralization for successful regeneration of damaged tissues. The simultaneous electrospraying of nanohydroxyapatite (HA) on electrospun polymeric nanofibrous scaffolds might be more promising for bone tissue regeneration. In the current study, nanofibrous scaffolds of gelatin (Gel), Gel/HA (4:1 blend), Gel/HA (2:1 blend) and Gel/HA (electrospin–electrospray) were fabricated for this purpose. The morphology, chemical and mechanical stability of nanofibres were evaluated by means of field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy and with a universal tensile machine, respectively. The in vitro biocompatibility of different nanofibrous scaffolds was determined by culturing human foetal osteoblasts and investigating the proliferation, alkaline phosphatase (ALP) activity and mineralization of cells. The results of cell proliferation, ALP activity and FESEM studies revealed that the combination of electrospinning of gelatin and electrospraying of HA yielded biocomposite nanofibrous scaffolds with enhanced performances in terms of better cell proliferation, increased ALP activity and enhanced mineralization, making them potential substrates for bone tissue regeneration.     

Micromechanical finite-element modeling and experimental characterization of the compressive mechanical properties of polycaprolactone-hydroxyapatite composite scaffolds prepared by selective laser sintering for bone tissue engineering

Bioresorbable scaffolds with mechanical properties suitable for bone tissue engineering were fabricated from polycaprolactone (PCL) and hydroxyapatite (HA) by selective laser sintering (SLS) and modeled by finite-element analysis (FEA). Both solid gage parts and scaffolds having 1-D, 2-D and 3-D orthogonal, periodic porous architectures were made with 0, 10, 20 and 30 vol.% HA. PCL:HA scaffolds manufactured by SLS had nearly full density (99%) in the designed solid regions and had excellent geometric and dimensional control. Through optimization of the SLS process, the compressive moduli for our solid gage parts and scaffolds are the highest reported in the literature for additive manufacturing. The compressive moduli of solid gage parts were 299.3, 311.2, 415.5 and 498.3 MPa for PCL:HA loading at 100:0, 90:10, 80:20 and 70:30, respectively. The compressive effective stiffness tended to increase as the loading of HA was increased and the designed porosity was lowered. In the case of the most 3-D porous scaffold, the compressive modulus more than doubled from 14.9 to 36.2 MPa when changing the material from 100:0 to 70:30 PCL:HA. A micromechanical FEA model was developed to investigate the reinforcement effect of HA loading on the compressive modulus of the bulk material. Using a first-principles based approach, the random distribution of HA particles in a solidified PCL matrix was modeled for any HA loading to predict the bulk mechanical properties of the composites. The bulk mechanical properties were also used for FEA of the scaffold geometries. The results of the FEA were found to be in good agreement with experimental mechanical testing. The development of patient- and site-specific composite tissue-engineering constructs with tailored properties can be seen as a direct extension of this work on computational design, a priori modeling of mechanical properties and direct digital manufacturing.     

In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres

Biomimetic composites consisting of polymer and mineral components, resembling bone in structure and composition, were produced using a rapid prototyping technique for bone tissue engineering applications. Solid freeform fabrication, known as rapid prototyping (RP) technology, allows scaffolds to be designed with pre-defined and controlled external and internal architecture. Using the indirect RP technique, a three-component scaffold with a woodpile structure, consisting of poly-l-lactic acid (PLLA), chitosan and hydroxyapatite (HA) microspheres, was produced that had a macroporosity of more than 50% together with micropores induced by lyophilization. X-ray diffraction analysis indicated that the preparation and construction of the composite scaffold did not affect the phase composition of the HA. The compressive strength and elastic modulus (E) for the PLLA composites are 0.42 and 1.46 MPa, respectively, which are much higher than those of chitosan/HA composites and resemble the properties of cellular structure. These scaffolds showed excellent biocompatibility and ability for three-dimensional tissue growth of MC3T3-E1 pre-osteoblastic cells. The pre-osteoblastic cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores, as seen from the histological analysis and electron microscopy. The proliferating cells formed an extracellular matrix network and also differentiated into mature osteoblasts, as indicated by alkaline phosphatase enzyme activity. The properties of these scaffolds indicate that they can be used for non-load-bearing applications.     

Modification and cytocompatibility of biocomposited porous PLLA/HA-microspheres scaffolds

Poly(L-lactic acid) and hydroxyapatie (PLLA/HA) composite scaffolds have good properties and suit to use as bone tissue engineering. In this work, hollow HA microspheres (HAM) with poor crystallinity were fabricated by a flame-drying method. The HAM has the potential to be used to release drugs or proteins in addition to improve osteoconductivity. Different ratios of PLLA/HAM were used to prepare porous composite scaffolds using the thermally induced phase separation technique. The HAMs were randomly incorporated into the PLLA porous scaffolds. As the HAMs ratio was increased, the porous composite scaffolds changed from ladder-like into isotropic structure. In addition, the compressive strength of PLLA/HAMs composite scaffolds improved first and declined with the increasing of HAMs ratio in the scaffolds. In vitro experiment showed that PLLA/HAMs composite scaffolds improved the attachment, migration, and differentiation of osteoblastic cells. These results demonstrated that the PLLA/HAMs composite scaffolds were superior to plain PLLA scaffold for bone tissue engineering.     

牙周膜细胞在网格型与无纺型聚乳酸纳米纤维支架材料上体外培养的研究比较

研究对比牙周膜细胞在无纺型和网格型聚乳酸纳米纤维膜上的生长行为,探讨支架结构对细胞生长的影响.采用静电纺丝技术,用金属平板或金属网分别接收,得到无纺型和网格型聚乳酸纳米纤维膜;通过SEM观察两种支架形貌差异,并测试比较它们的力学性能.通过MTT测试和SEM观察,比较无纺型和网格型纳米纤维膜对细胞生长的影响.实验结果:网格型膜的纤维直径平均为500～600 nm;无纺型膜的纤维直径大于网格型膜,平均直径约为700 nm,但网格型膜的拉伸断裂应变略大.牙周细胞与支架联合培养的MTT结果显示,与在聚苯乙烯(TCPS) 培养板上的培养比较,两种纳米纤维膜都显示出促进细胞增殖的效果,其中网格膜的促进效果比无纺膜更加明显.SEM观察的结果显示,细胞无法进入无纺型膜内部生长,而网格型膜中由疏松纤维堆积形成的大孔结构则非常有利于细胞进入支架内部,细胞在后者上生长良好.因此,网格型纳米纤维支架是一种优于纤维为完全无纺排布的支架,更适用于组织工程研究.     

Pre-osteoblast infiltration and differentiation in highly porous apatite-coated PLLA electrospun scaffolds

Electrospun polymer/apatite composite scaffolds are promising candidates as functional bone substitutes because of their ability to allow pre-osteoblast attachment, proliferation, and differentiation. However these structures usually lack an adequate pore size to permit sufficient cell migration and colonization of the scaffold. To overcome this limitation, we developed an apatite-coated electrospun PLLA scaffold with varying pore size and porosity by utilizing a three-step water-soluble PEO fiber inclusion, dissolution, and mineralization process. The temporal and spatial dynamics of cell migration into the scaffolds were quantified to determine the effects of enhanced pore size and porosity on cell infiltration. MC3T3-E1 pre-osteoblast migration into the scaffolds was found to be a function of both initial PEO content and time. Scaffolds with greater initial PEO content (50% and 75% PEO) had drastically accelerated cell infiltration in addition to enhanced cell distribution throughout the scaffold when compared to scaffolds with lower PEO content (0% and 25% PEO). Furthermore, scaffolds with an apatite substrate significantly upregulated MC3T3-E1 alkaline phosphatase activity, osteocalcin content, and cell-mediated mineralization as compared to PLLA alone. These findings suggest that such a scaffold enhances pre-osteoblast infiltration, colonization, and maturation in vitro and may lead to overall improved bone formation when implanted in vivo.     

Novel mesoporous silica-based antibiotic releasing scaffold for bone repair

Tissue engineering scaffolds with a micro- or nanoporous structure and able to deliver special drugs have already been confirmed to be effective in bone repair. In this paper, we first evaluated the biomineralization properties and drug release properties of a novel mesoporous silica–hydroxyapatite composite material (HMS–HA) which was used as drug vehicle and filler for polymer matrices. Biomineralization can offer a credible prediction of bioactivity for the synthetic bone regeneration materials. We found HMS–HA exhibited good apatite deposition properties after being soaked in simulated body fluid (SBF) for 7 days. Drug delivery from HMS–HA particle was in line with Fick’s law, and the release process lasted 12 h after an initial burst release with 60% drug release. A novel tissue engineering scaffold with the function of controlled drug delivery was developed, which was based on HMS–HA particles, poly(lactide-co-glycolide) (PLGA) and microspheres sintering techniques. Mechanical testing on compression, degradation behavior, pH-compensation effect and drug delivery behavior of PLGA/HMS–HA microspheres sintered scaffolds were analyzed. Cell toxicity and cell proliferation on the scaffolds was also evaluated. The results indicated that the PLGA/HMS–HA scaffolds could effectively compensate the increased pH values caused by the acidic degradation product of PLGA. The compressive strength and modulus of PLGA/HMS–HA scaffolds were remarkably high compared to pure PLGA scaffold. Drug delivery testing of the PLGA/HMS–HA scaffolds indicated that PLGA slowed gentamycin sulfate (GS) release from HMS–HA particles, and the release lasted for nearly one month. Adding HMS–HA to PLGA scaffolds improved cytocompatibility. The scaffolds demonstrated low cytotoxicity, and supported mesenchymal stem cells growth more effectively than pure PLGA scaffolds. To summarize, the data supports the development of PLGA/HMS–HA scaffolds as potential degradable and drug delivery materials for bone replacement.     

Improvement of dual-leached polycaprolactone porous scaffolds by incorporating with hydroxyapatite for bone tissue regeneration

Polycaprolactone (PCL)/hydroxyapatite (HA) composite scaffolds were prepared by combining solvent casting and salt particulate leaching with a polymer leaching technique. The hydrophilicity of the dual-leached scaffold was improved by alkaline (NaOH) treatment. Well-defined interconnected pores were detected by scanning electron microscopy. The water absorption capacity of the NaOH-treated PCL/HA dual-leached scaffold increased greatly, confirming that the hydrophilicity of the scaffold was improved by NaOH treatment. The compressive modulus of the PCL/HA dual-leached scaffold was greatly increased by the addition of HA particles. An indirect evaluation of the cytotoxicity of all PCL dual-leached scaffolds with mouse fibroblastic cells (L929) and mouse calvaria-derived pre-osteoblastic cells (MC3T3-E1) indicated that the PCL dual-leached scaffolds are non-toxic to cells. The ability of the scaffolds to support mouse calvaria-derived pre-osteoblastic cell (MC3T3-E1) attachment, proliferation, differentiation, and mineralization was also evaluated. Although the viability of cells was lower on the PCL/HA dual-leached scaffold than on the tissue-culture polystyrene plates (TCPS) and on the other substrates at early time points, both the PCL and NaOH-treated PCL/HA dual-leached scaffolds supported the attachment of MC3T3-E1 at significantly higher levels than TCPS. During the proliferation period (days 1–3), all of the PCL dual-leached scaffolds were able to support the proliferation of MC3T3-E1 at higher levels than the TCPS; in addition, the cells grown on NaOH-treated PCL/HA dual-leached scaffolds proliferated more rapidly. The cells cultured on the surfaces of NaOH-treated PCL/HA dual-leached scaffolds had the highest rate of mineral deposition.     

bFGF-containing electrospun gelatin scaffolds with controlled nano-architectural features for directed angiogenesis

Current therapeutic angiogenesis strategies are focused on the development of biologically responsive scaffolds that can deliver multiple angiogenic cytokines and/or cells in ischemic regions. Herein, we report on a novel electrospinning approach to fabricate cytokine-containing nanofibrous scaffolds with tunable architecture to promote angiogenesis. Fiber diameter and uniformity were controlled by varying the concentration of the polymeric (i.e. gelatin) solution, the feed rate, needle to collector distance, and electric field potential between the collector plate and injection needle. Scaffold fiber orientation (random vs. aligned) was achieved by alternating the polarity of two parallel electrodes placed on the collector plate thus dictating fiber deposition patterns. Basic fibroblast growth factor (bFGF) was physically immobilized within the gelatin scaffolds at variable concentrations and human umbilical vein endothelial cells (HUVEC) were seeded on the top of the scaffolds. Cell proliferation and migration was assessed as a function of growth factor loading and scaffold architecture. HUVECs successfully adhered onto gelatin B scaffolds and cell proliferation was directly proportional to the loading concentrations of the growth factor (0-100 bFGF ng/mL). Fiber orientation had a pronounced effect on cell morphology and orientation. Cells were spread along the fibers of the electrospun scaffolds with the aligned orientation and developed a spindle-like morphology parallel to the scaffold's fibers. In contrast, cells seeded onto the scaffolds with random fiber orientation, did not demonstrate any directionality and appeared to have a rounder shape. Capillary formation (i.e. sprouts length and number of sprouts per bead), assessed in a 3-D in vitro angiogenesis assay, was a function of bFGF loading concentration (0 ng, 50 ng and 100 ng per scaffold) for both types of electrospun scaffolds (i.e. with aligned or random fiber orientation).     

Characterization of mineralized collagen-glycosaminoglycan scaffolds for bone regeneration

Mineralized collagen–glycosaminoglycan scaffolds designed for bone regeneration have been synthesized via triple co-precipitation in the absence of a titrant phase. Here, we characterize the microstructural and mechanical properties of these newly developed scaffolds with 50 and 75 wt.% mineral content. The 50 wt.% scaffold had an equiaxed pore structure with isotropic mechanical properties and a Ca–P-rich mineral phase comprised of brushite; the 75 wt.% scaffold had a bilayer structure with a pore size varying in the through-thickness direction and a mineral phase comprised of 67% brushite and 33 wt.% monetite. The compressive stress–strain response of the scaffolds was characteristic of low-density open-cell foams with distinct linear elastic, collapse plateau and densification regimes. The elastic modulus and strength of individual struts within the scaffolds were measured using an atomic force microscopy cantilevered beam-bending technique and compared with the composite response under indentation and unconfined compression. Cellular solids models, using the measured strut properties, overestimated the overall mechanical properties for the scaffolds; the discrepancy arises from defects such as disconnected pore walls within the scaffold. As the scaffold stiffness and strength decreased with increasing overall mineral content and were less than that of natural, mineralized collagen scaffolds, these microstructural/mechanical relations will be used to further improve scaffold design for bone regeneration applications.     

A simple method for fabricating 3-D multilayered composite scaffolds

One limitation of electrospinning stems from the charge build-up that occurs during processing, preventing further fibre deposition and limiting the scaffold overall thickness and hence their end-use in tissue engineering applications targeting large tissue defect repair. To overcome this, we have developed a technique in which thermally induced phase separation (TIPS) and electrospinning are combined. Thick three-dimensional, multilayered composite scaffolds were produced by simply stacking individual polycaprolactone (PCL) microfibrous electrospun discs into a cylindrical holder that was filled with a 3% poly(lactic-co-glycolic acid) (PLGA) solution in dimethylsulfoxide (a good solvent for PLGA but a poor one for PCL). The construct was quenched in liquid nitrogen and the solvent removed by leaching out in cold water. This technique enables the fabrication of scaffolds composed principally of electrospun membranes that have no limit to their thickness. The mechanical properties of these scaffolds were assessed under both quasi-static and dynamic conditions. The multilayered composite scaffolds had similar compressive properties to 5% PCL scaffolds fabricated solely by the TIPS methodology. However, tensile tests demonstrated that the multilayered construct outperformed a scaffold made purely by TIPS, highlighting the contribution of the electrospun component of the composite scaffold to enhancing the overall mechanical property slate. Cell studies revealed cell infiltration principally from the scaffold edges under static seeding conditions. This fabrication methodology permits the rapid construction of thick, strong scaffolds from a range of biodegradable polymers often used in tissue engineering, and will be particularly useful when large dimension electrospun scaffolds are required.