We investigated the fabrication of highly porous scaffolds made of three different materials [poly(propylene fumarate (PPF) polymer, an ultra-short single-walled carbon nanotube (US-tube) nanocomposite, and a dodecylated US-tube (F-US-tube) nanocomposite] in order to evaluate the effects of material composition and porosity on scaffold pore structure, mechanical properties, and marrow stromal cell culture. All scaffolds were produced by a thermal-crosslinking particulate-leaching technique at specific porogen contents of 75, 80, 85, and 90 vol%. Scanning electron microcopy, microcomputed tomography, and mercury intrusion porosimetry were used to analyze the pore structures of scaffolds. The porogen content was found to dictate the porosity of scaffolds. There was no significant difference in porosity, pore size, and interconnectivity among the different materials for the same porogen fraction. Nearly 100% of the pore volume was interconnected through 20 μm or larger connections for all scaffolds. While interconnectivity through larger connections improved with higher porosity, compressive mechanical properties of scaffolds declined at the same time. However, the compressive modulus, offset yield strength, and compressive strength of F-US-tube nanocomposites were higher than or similar to the corresponding properties for the PPF polymer and US-tube nanocomposites for all the porosities examined. As for in vitro osteoconductivity, marrow stromal cells demonstrated equally good cell attachment and proliferation on all scaffolds made of different materials at each porosity. These results indicate that functionalized ultra-short single-walled carbon nanotube nanocomposite scaffolds with tunable porosity and mechanical properties hold great promise for bone tissue engineering applications.
We have developed a new fabrication technique to create three-dimensional (3D) porous poly(ε-caprolactone fumarate) (PCLF) scaffolds using hydrogel microparticle porogens, as an alternative to overcome certain limitations of traditional scaffold fabrication techniques such as a salt leaching method. Both natural hydrogel, gelatin, and synthetic hydrogel, poly(ethylene glycol) sebacic acid diacrylate, were used as porogens to fabricate 3D porous PCLF scaffolds. Hydrogel microparticles were prepared by a single emulsion technique with the particle size in the range of 100–500 μm after equilibrium in water. The pore size distribution, porosity, pore interconnectivity, and spatial pore heterogeneity of the 3D PCLF scaffolds were assessed using micro-computed tomography and imaging analysis. Scaffolds fabricated with the hydrogel porogens had higher porosity and pore interconnectivity as well as more homogeneous spatial pore distribution, compared to the scaffolds made from the salt leaching process. Compressive moduli of the scaffolds were also measured and showed that lower porosity yielded greater modulus of the scaffolds. Overall, the new fabrication technology using hydrogel porogens may be beneficial for certain tissue engineering applications.
The objective of this study was to determine how the incorporation of surface-modified alumoxane nanoparticles into a biodegradable fumarate-based polymer affects in vivo bone biocompatibility (characterized by direct bone contact and bone ingrowth) and in vivo degradability. Porous scaffolds were fabricated from four materials: poly(propylene fumarate)/propylene fumarate-diacrylate (PPF/PF-DA) polymer alone; a macrocomposite consisting of PPF/PF-DA polymer with boehmite microparticles; a nanocomposite composed of PPF/PF-DA polymer and mechanically-reinforcing surface-modified alumoxane nanoparticles; and a low molecular weight PPF polymer alone (tested as a degradation control). Scaffolds were implanted in the lateral femoral condyle of adult goats for 12 weeks and evaluated by micro-computed tomography and histological analysis. For all material groups, small amounts of bone, some soft tissue, and a few inflammatory elements were observed within the pores of scaffolds, though many pores remained empty or filled with fluid only. Direct contact between scaffolds and surrounding bone tissue was also observed in all scaffold types, though less commonly. Minimal in vivo degradation occurred during the 12 weeks of implantation in all materials. These results demonstrate that the incorporation of alumoxane nanoparticles into porous PPF/PF-DA scaffolds does not significantly alter in vivo bone biocompatibility or degradation.
Bone tissue engineering; Nanocomposite; Biocompatibility; Nanobiomaterials; Micro-computed tomography
A series of crosslinkable nanocomposites has been developed using hydroxyapatite (HA) nanoparticles and poly(propylene fumarate) (PPF). PPF/HA nanocomposites with four different weight fractions of HA nanoparticles have been characterized in terms of thermal and mechanical properties. To assess surface chemistry of crosslinked PPF/HA nanocomposites, their hydrophilicity and capability of adsorbing proteins have been determined using static contact angle measurement and MicroBCA protein assay kit after incubation with 10% fetal bovine serum (FBS), respectively. In vitro cell studies have been performed using MC3T3-E1 mouse pre-osteoblast cells to investigate the ability of PPF/HA nanocomposites to support cell attachment, spreading, and proliferation after 1, 4, and 7 days. By adding HA nanoparticles to PPF, the mechanical properties of crosslinked PPF/HA nanocomposites have not been increased due to the initially high modulus of crosslinked PPF. However, hydrophilicity and serum protein adsorption on the surface of nanocomposites have been significantly increased, resulting in enhanced cell attachment, spreading, and proliferation after 4 days of cell seeding. These results indicate that crosslinkable PPF/HA nanocomposites are useful for hard tissue replacement because of excellent mechanical strength and osteoconductivity.
Poly(propylene fumarate) (PPF); Hydroxyapatite (HA); Nanocomposite; Protein adsorption; Osteoblast response
Tissue engineering of the small intestine remains experimental despite worldwide attempts to develop a functional substitute for short bowel syndrome. Most published studies have reported predominant use of PLLA (poly-L-lactide acid)/PGA (polyglycolic acid) copolymer as the scaffold material, and studies have been limited by in vivo experiments. This lack of progress has inspired a fresh perspective and provoked further investigation and development in this field of tissue engineering. In the present paper, we exploit a relatively new nanocomposite of POSS (polyhedral oligomeric silsesquioxane) and PCL [poly(caprolactone-urea)urethane] as a material to develop porous scaffolds using a solvent casting/particulate leaching technique to fabricate porous scaffolds in different pore sizes and porosities. Scaffolds were characterized for pore morphology and porosity using scanning electron microscopy and micro-computed tomography. Rat intestinal epithelial cells were then seeded on to the polymer scaffolds for an in vitro study of cell compatibility and proliferation, which was assessed by Alamar Blue™ and lactate dehydrogenase assays performed for 21 days post-seeding. The results obtained demonstrate that POSS–PCL nanocomposite was produced as a macroporous scaffold with porosity over the range of 40–80% and pore size over the range of 150–250 μm. This scaffold was shown to support epithelial cell proliferation and growth. In conclusion, as a further step in investigating small intestinal tissue engineering, the nanocomposite employed in this study may prove to be a useful alternative to poly(lactic-co-glycolic acid) in the future.
intestinal epithelial cell (IEC); nanocomposite; poly(caprolactone-urea)urethane (PCL); scaffold; tissue engineering; DMAC, dimethylacetamide; FTIR, Fourier-transform infrared; IEC, intestinal epithelial cell; LDH, lactate dehydrogenase; micro-CT, micro-computed tomography; PCL, poly(caprolactone-urea)urethane; PCU, poly(carbonate-urea)urethane; PGA, polyglycolic acid; PLGA, poly(lactic-co-glycolic acid); PN, parenteral; POSS, polyhedral oligomeric silsesquioxane; SBS, short bowel syndrome; SEM, scanning electron microscopy
It is now recognized that geometric structures of scaffolds at several size levels have profound influences on cell adhesion, viability, proliferation and differentiation. This study aims to develop an integrated process to fabricate scaffolds with controllable geometric structures at nano-, micro- and macro-scales. A phase separation method is used to prepare interconnected poly(l-lactide) (PLLA) nanofibrous (NF) scaffolds. The pore size of the NF scaffold at the scale of several hundred micrometers is controlled by the size of porogen, paraffin spheres. At millimeter scale and above, the overall shape of the scaffold is defined by a wax mold produced using a three-dimensional printer. The printer utilizes a stereo lithographic file generated from computed tomographic files retrieved from the National Library of Medicine's Visual Human Project. NF PLLA scaffolds with a human digit shape are successfully prepared using this process. Osteoblast cell line MC3T3-E1 cells are then seeded and cultured in the prepared scaffolds. Cell proliferation, differentiation and biomineralization are characterized to demonstrate the suitability of the scaffolds for the digit bone tissue engineering application.
Integrating an advanced manufacturing technique, nanocomposite material and controlled delivery of growth factor to form multifunctional tissue engineering scaffolds was investigated in this study. Based on calcium phosphate (Ca–P)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) nanocomposite microspheres, three-dimensional Ca–P/PHBV nanocomposite scaffolds with customized architecture, controlled porosity and totally interconnected porous structure were successfully fabricated using selective laser sintering (SLS), one of the rapid prototyping technologies. The cytocompatibility of sintered Ca–P/PHBV nanocomposite scaffolds, as well as PHBV polymer scaffolds, was studied. For surface modification of nanocomposite scaffolds, gelatin was firstly physically entrapped onto the scaffold surface and heparin was subsequently immobilized on entrapped gelatin. The surface-modification improved the wettability of scaffolds and provided specific binding site between conjugated heparin and the growth factor recombinant human bone morphogenetic protein-2 (rhBMP-2). The surface-modified Ca–P/PHBV nanocomposite scaffolds loaded with rhBMP-2 significantly enhanced the alkaline phosphatase activity and osteogenic differentiation markers in gene expression of C3H10T1/2 mesenchymal stem cells. Together with osteoconductive nanocomposite material and controlled growth factor delivery strategies, the use of SLS technique to form complex scaffolds will provide a promising route towards individualized bone tissue regeneration.
nanocomposite; scaffold; selective laser sintering; surface modification; bone tissue engineering
Tissue engineering scaffolds require a controlled pore size and interconnected pore structures to support the host tissue growth. In the present study, three dimensional (3D) hybrid scaffolds of poly lactic acid (PLA) and poly glycolic acid (PGA) were fabricated using solvent casting/particulate leaching. In this case, partially fused NaCl particles were used as porogen (200-300µ) to improve the overall porosity (≥90%) and internal texture of scaffolds. Differential scanning calorimeter (DSC) analysis of these porous scaffolds revealed a gradual reduction in glass transition temperature (Tg) (from 48°C to 42.5°C) with increase in hydrophilic PGA content. The potential applications of these scaffolds as implants were further tested for their biocompatibility and biodegradability in four simulated body fluid (SBF) types in vitro. Whereas, simulated body fluid (SBF) Type1 with the optimal amount of HCO3− ions was found to be more appropriate and sensible for testing the bioactivity of scaffolds. Among three combinations of polymer scaffolds, sample B with a ratio of 75:25 of PLA: PGA showed greater stability in body fluids (pH 7.2) with an optimum degradation rate (9% to 12% approx). X-ray diffractogram also confirmed a thin layer of hydroxyapatite deposition over sample B with all SBF types in vitro.
poly (lactic-co-glycolic acid) scaffolds; simulated body fluid; solvent immersion; polymer degradation; hydroxyapatite
In this study, new nano-fluor-hydroxyapatite (nFHA)/polyurethane composite scaffolds were fabricated for potential use in bone tissue engineering. Polyester urethane samples were synthesized from polycaprolactone, hexamethylene diisocyanate, and 1,4-butanediol as chain extender. Nano fluor-hydroxyapatite (nFHA) was successfully synthesized by sol-gel method. The solid–liquid phase separation and solvent sublimation methods were used for preparation of the porous composites. Mechanical properties, chemical structure, and morphological characteristics of the samples were investigated by compressive test, Fourier transform infrared, and scanning electron microscopy (SEM) techniques, respectively. The effect of nFHA powder content on porosity and pore morphology was investigated. SEM images demonstrated that the scaffolds were constituted of interconnected and homogeneously distributed pores. The pore size of the scaffolds was in the range 50–250 μm. The result obtained in this research revealed that the porosity and pore average size decreased and compressive modulus increased with nFHA percentage. Considering morphological, physical, and mechanical properties, the scaffold with a higher ratio of nFHA has suitable potential use in tissue regeneration.
polyester urethane; composite; fluor-hydroxyapatite; scaffold
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 g and 0.5 g). 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.
Macroporous scaffolds with controllable pore structure and mechanical properties were fabricated by a porogen fusion technique. Biodegradable material poly (d, l-lactide) (PDLLA) was used as the scaffold matrix. The effects of porogen size, PDLLA concentration and hydroxyapatite (HA) content on the scaffold morphology, porosity and mechanical properties were investigated. High porosity (90% and above) and highly interconnected structures were easily obtained and the pore size could be adjusted by varying the porogen size. With the increasing porogen size and PDLLA concentration, the porosity of scaffolds decreases, while its mechanical properties increase. The introduction of HA greatly increases the impact on pore structure, mechanical properties and water absorption ability of scaffolds, while it has comparatively little influence on its porosity under low HA contents. These results show that by adjusting processing parameters, scaffolds could afford a controllable pore size, exhibit suitable pore structure and high porosity, as well as good mechanical properties, and may serve as an excellent substrate for bone tissue engineering.
tissue engineering; composite scaffolds; mechanical property; porogen fusion technique
Nanohydroxyapatite (n-HA)/nylon 6,6 composite scaffolds were produced by means of the salt-leaching/solvent casting technique. NaCl with a distinct range size was used with the aim of optimizing the pore network. Composite powders with different n-HA contents (40%, 60%) for scaffold fabrication were synthesized and tested. The composite scaffolds thus obtained were characterized for their microstructure, mechanical stability and strength, and bioactivity. The microstructure of the composite scaffolds possessed a well-developed interconnected porosity with approximate optimal pore size ranging from 200 to 500 μm, ideal for bone regeneration and vascularization. The mechanical properties of the composite scaffolds were evaluated by compressive strength and modulus tests, and the results confirmed their similarity to cortical bone. To characterize bioactivity, the composite scaffolds were immersed in simulated body fluid for different lengths of time and results monitored by scanning electron microscopy and energy dispersive X-ray microanalysis to determine formation of an apatite layer on the scaffold surface.
scaffold; nanohydroxyapatite; nylon 6,6; salt-leaching/solvent casting; bioactivity
To validate the importance of uniformity in pore size and structure of a scaffold for tissue engineering, we fabricated two types of scaffolds with uniform (inverse opal scaffolds) and non-uniform pore sizes and structures, and then evaluated their properties in terms of diffusion of macromolecules, spatial distribution of fibroblasts, and differentiation of preosteoblasts. Our results confirmed the superior performance of the inverse opal scaffolds due to the uniform pore size, homogeneous environment, and high interconnectivity: a higher diffusion rate, a uniform distribution of cells, and a higher degree of differentiation. In addition, we found that both the differentiation of cells and secretion of extracellular matrix were dependent on the properties of the individual pore to which the cells were attached to, rather than the bulk properties of a scaffold. Our results clearly indicate that inverse opal scaffolds could provide a better microenvironment for cells in comparison to a scaffold with non-uniform size and structure.
Inverse opal scaffold; pore uniformity; tissue engineering
Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.
In vitro human mesenchymal stem cell (hMSC) proliferation and differentiation is dependent on scaffold design parameters and specific culture conditions. In this study, we investigate how scaffold microstructure influences hMSC behavior in a perfusion bioreactor system. Poly-l-lactic acid (PLLA) scaffolds are fabricated using supercritical carbon dioxide (SC-CO2) gel drying. This production method results in scaffolds fabricated with nanostructure. To introduce a microporous structure, porogen leaching was used in addition to this technique to produce scaffolds of average pore size of 100, 250, and 500 µm. These scaffolds were then cultured in static culture in well plates or dynamic culture in the tubular perfusion system (TPS) bioreactor. Results indicated that hMSCs were able to attach and maintain viability on all scaffolds with higher proliferation in the 250 µm and 500 µm pore sizes of bioreactor cultured scaffolds and 100 µm pore size of statically cultured scaffolds. Osteoblastic differentiation was enhanced in TPS culture as compared to static culture with the highest alkaline phosphatase expression observed in the 250 µm pore size group. Bone morphogenetic protein-2 was also analyzed and expression levels were highest in the 250 µm and 500 µm pore size bioreactor cultured samples. These results demonstrate cellular response to pore size as well as the ability of dynamic culture to enhance these effects.
supercritical fluids; scaffold; PLLA; human mesenchymal stem cells; tissue engineering; bioreactor
A novel scaffold fabrication method utilizing both polymer blend extrusion and gas foaming techniques to control pore size distribution is presented. Seventy five per cent of all pores produced using polymer blend extrusion alone were less than 50 μm. Introducing a gas technique provided better control of pore size distribution, expanding the range from 0-50 to 0-350 μm. Varying sintering time, annealing temperature and foaming pressure also helped reduced the percentage of pore sizes below 50 μm. Scaffolds chosen for in vitro cellular studies had a pore size distribution of 0-300 μm, average pore size 66 ± 17 μm, 0.54 ± 0.02% porosity and 98% interconnectivity, measured by micro computed tomography (microCT) analysis. The ability of the scaffolds to support osteogenic differentiation and cranial defect repair was evaluated by static and dynamic (0.035 ± 0.006 m s-1 terminal velocity) cultivation with dura mater stem cells (DSCs). In vitro studies showed minimal increases in proliferation over 28 days in culture in osteogenic media. Alkaline phosphatase expression remained constant throughout the study. Moderate increases in matrix deposition, as assessed by histochemical staining and microCT analysis, occurred at later time points, days 21 and 28. Although constructs cultured dynamically showed greater mineralization than static conditions, these trends were not significant. It remains unclear whether bioreactor culture of DSCs is advantageous for bone tissue engineering applications. However, these studies show that polycaprolactone (PCL) scaffolds alone, without the addition of other co-polymers or ceramics, support long-term attachment and mineralization of DSCs throughout the entire porous scaffold.
polycaprolactone; polymer blend; gas foaming; bioreactor culture; dura; bone repair
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.
A biomimetic approach involving the self-assembly of mineral within the pores of three-dimensional porous polymer scaffolds is a promising strategy to integrate advantages of inorganic and organic phases into a single material for hard tissue engineering. Such a material enhances the ability of progenitor cells to differentiate down an osteoblast lineage in vitro and in vivo, compared with polymer scaffolds. The mechanisms regulating mineral formation in this one-step process, however, are poorly understood, especially the effects of ionic activity products (IP) of the mineralizing solution and incubation time. The aims of this study were to define the structure and composition of mineral formed within the pores of biodegradable polymer scaffolds as a function of IP and time. Three-dimensional poly(lactide-co-glycolide) scaffolds were fabricated by solvent casting/particulate leaching and incubated for 4–16 days in six variants of simulated body fluid whose IPs were varied by adjusting ionic concentrations. Scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy demonstrated the formation of carbonated apatite with sub-micrometer sized crystals that grew into spherical globules extending out of the scaffold pore surfaces. As IP increased, more mineral grew on the scaffold pore surfaces, but the apatite became less crystalline and the Ca/P molar ratio decreased from 1.63 ± 0.005 to 1.51 ± 0.002. Since morphology, composition, and structure of mineral are factors that affect cell function, this study demonstrates that the IP of the mineralizing solution is an important modulator of material properties, potentially leading to enhanced control of cell function.
biomimetic; mineralization; apatite; ionic activity products; scaffold
Free form fabrication and high resolution imaging techniques enable the creation of biomimetic tissue engineering scaffolds. A 3D CAD model of canine trabecular bone was produced via micro CT and exported to a fused deposition modeler, to produce polybutylene terephthalate (PBT) trabeculated scaffolds and four other scaffold groups of varying pore structures. The five scaffold groups were divided into subgroups (n=6) and compression tested at two load rates (49 N/s and 294 N/s). Two groups were soaked in a 25 °C saline solution for 7 days before compression testing. Micro CT was used to compare porosity, connectivity density, and trabecular separation of each scaffold type to a canine trabecular bone sample. At 49 N/s the dry trabecular scaffolds had a compressive stiffness of 4.94±1.19 MPa, similar to the simple linear small pore scaffolds and significantly more stiff (p<0.05) than either of the complex interconnected pore scaffolds. At 294 N/s, the compressive stiffness values for all five groups roughly doubled. Soaking in saline had an insignificant effect on stiffness. The trabecular scaffolds matched bone samples in porosity; however, achieving physiologic connectivity density and trabecular separation will require further refining of scaffold processing.
Scaffolds; Rapid prototyping; Fused deposition modeling; Micro CT; Bone
Bone-mimetic electrospun scaffolds consisting of polycaprolactone (PCL), collagen I and nanoparticulate hydroxyapatite (HA) have previously been shown to support the adhesion, integrin-related signaling and proliferation of mesenchymal stem cells (MSCs), suggesting these matrices serve as promising degradable substrates for osteoregeneration. However, the small pore sizes in electrospun scaffolds hinder cell infiltration in vitro and tissue-ingrowth into the scaffold in vivo, limiting their clinical potential. In this study, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col I/nanoHA scaffolds: limited protease digestion, decreasing the fiber packing density during electro-spinning, and inclusion of sacrificial fibers of the water-soluble polymer PEO. The PEO sacrificial fiber approach was found to be the most effective in increasing scaffold pore size. Furthermore, the use of sacrificial fibers promoted increased MSC infiltration into the scaffolds, as well as greater infiltration of endogenous cells within bone upon placement of scaffolds within calvarial organ cultures. These collective findings support the use of sacrificial PEO fibers as a means to increase the porosity of complex, bone-mimicking electrospun scaffolds, thereby enhancing tissue regenerative processes that depend upon cell infiltration, such as vascularization and replacement of the scaffold with native bone tissue.
Bone tissue engineering; Cellular infiltration; Porosity; Scaffold; Biomimetic material; Organ culture
Porous collagen scaffold is integrated with surface activated PLLA nanoparticles fabricated by lyophilizing and crosslinking via EDC treatment. In order to prepare surface-modified PLLA nanoparticles, PLLA was firstly grafted with poly (acrylic acid) (PAA) through surface-initiated polymerization of acrylic acid. Nanoparticles of average diameter 316 nm and zeta potential −39.88 mV were obtained from the such-treated PLLA by dialysis method. Porous collagen scaffold were fabricated by mixing PLLA nanoparticles with collagen solution, freeze drying, and crosslinking with EDC. SEM observation revealed that nanoparticles were homogeneously dispersed in collagen matrix, forming interconnected porous structure with pore size ranging from 150 to 200 μm, irrespective of the amount of nanoparticles. The porosity of the scaffolds kept almost unchanged with the increment of the nanoparticles, whereas the mechanical property was obviously improved, and the degradation was effectively retarded. In vitro L929 mouse fibroblast cells seeding and culture studies revealed that cells infiltrated into the scaffolds and were distributed homogeneously. Compared with the pure collagen sponge, the number of cells in hybrid scaffolds greatly increased with the increment of incorporated nanoparticles. These results manifested that the surface-activated PLLA nanoparticles effectively reinforced the porous collagen scaffold and promoted the cells penetrating into the scaffold, and proliferation.
We have developed a biodegradable composite scaffold for bone tissue engineering applications with a pore size and interconnecting macroporosity similar to those of human trabecular bone. The scaffold is fabricated by a process of particle leaching and phase inversion from poly(lactideco-glycolide) (PLGA) and two calcium phosphate (CaP) phases both of which are resorbable by osteoclasts; the first a particulate within the polymer structure and the second a thin ubiquitous coating. The 3–5 μm thick osteoconductive surface CaP abrogates the putative foreign body giant cell response to the underlying polymer, while the internal CaP phase provides dimensional stability in an otherwise highly compliant structure. The scaffold may be used as a biomaterial alone, as a carrier for cells or a three-phase drug delivery device. Due to the highly interconnected macroporosity ranging from 81% to 91%, with macropores of 0.8∼1.8 mm, and an ability to wick up blood, the scaffold acts as both a clot-retention device and an osteoconductive support for host bone growth. As a cell delivery vehicle, the scaffold can be first seeded with human mesenchymal cells which can then contribute to bone formation in orthotopic implantation sites, as we show in immune-compromised animal hosts. We have also employed this scaffold in both lithomorph and particulate forms in human patients to maintain alveolar bone height following tooth extraction, and augment alveolar bone height through standard sinus lift approaches. We provide a clinical case report of both of these applications; and we show that the scaffold served to regenerate sufficient bone tissue in the wound site to provide a sound foundation for dental implant placement. At the time of writing, such implants have been in occlusal function for periods of up to 3 years in sites regenerated through the use of the scaffold.
bone regeneration; scaffold; composite; biodegradable; clot retention; osteoconduction; cell delivery; extraction socket; sinus lift; clinical
Alkaline phosphatase (ALP) promotes bone formation by degrading inorganic pyrophosphate (PPi), an inhibitor of hydroxyapatite formation, and generating inorganic phosphate (Pi), an inducer of hydroxyapatite formation. Pi is a crucial molecule in differentiation and mineralization of osteoblasts. In this study, a method to immobilize ALP on fibrin scaffolds with tightly controllable pore size and pore interconnection was developed, and the biological properties of these scaffolds were characterized both in vitro and in vivo. Microporous, nanofibrous fibrin scaffolds (FS) were fabricated using a sphere-templating method. ALP was covalently immobilized on the fibrin scaffolds using 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC). Scanning electron microscopic observation (SEM) showed that mineral was deposited on immobilized alkaline phosphatase fibrin scaffolds (immobilized ALP/FS) when incubated in medium supplemented with β-glycerophosphate, suggesting that the immobilized ALP was active. Primary calvarial cells attached, spread and formed multiple layers on the surface of the scaffolds. Mineral deposition was also observed when calvarial cells were seeded on immobilized ALP/FS. Furthermore, cells seeded on immobilized ALP/FS exhibited higher osteoblast marker gene expression compared to control FS. Upon implantation in mouse calvarial defects, both the immobilized ALP/FS and FS alone treated group had higher bone volume in the defect compared to the empty defect control. Furthermore, bone formation in the immobilized ALP/FS treated group was statistically significant compared to FS alone group. However, the response was not robust.
Alkaline phosphatase; Fibrin; Bone tissue engineering; Phosphate; Pyrophosphate
Solid Free-Form Fabrication (SFF) technologies allow the fabrication of anatomical 3D scaffolds from computer tomography (CT) or magnetic resonance imaging (MRI) patients’ dataset. These structures can be designed and fabricated with a variable, interconnected and accessible porous network, resulting in modulable mechanical properties, permeability, and architecture that can be tailored to mimic a specific tissue to replace or regenerate. In this study, we evaluated whether anatomical meniscal 3D scaffolds with matching mechanical properties and architecture are beneficial for meniscus replacement as compared to meniscectomy. After acquiring CT and MRI of porcine menisci, 3D fiber-deposited (3DF) scaffolds were fabricated with different architectures by varying the deposition pattern of the fibers comprising the final structure. The mechanical behaviour of 3DF scaffolds with different architectures and of porcine menisci was measured by static and dynamic mechanical analysis and the effect of these tissue engineering templates on articular cartilage was assessed by finite element analysis (FEA) and compared to healthy conditions or to meniscectomy. Results show that 3DF anatomical menisci scaffolds can be fabricated with pore different architectures and with mechanical properties matching those of natural menisci. FEA predicted a beneficial effect of meniscus replacement with 3D scaffolds in different mechanical loading conditions as compared to meniscectomy. No influence of the internal scaffold architecture was found on articular cartilage damage. Although FEA predictions should be further confirmed by in vitro and in vivo experiments, this study highlights meniscus replacement by SFF anatomical scaffolds as a potential alternative to meniscectomy.
Rapid prototyping; Mechanical analysis; Finite element analysis; Scaffolds; Meniscus; Tissue engineering.
We demonstrate high-resolution photocross-linking of biodegradable poly(propylene fumarate) (PPF) and diethyl fumarate (DEF) using UV excimer laser photocuring at 308 nm. The curing depth can be tuned in a micrometre range by adjusting the total energy dose (total fluence). Young's moduli of the scaffolds are found to be a few gigapascal, high enough to support bone formation. The results presented here demonstrate that the proposed technique is an excellent tool for the fabrication of stiff and biocompatible structures on a micrometre scale with defined patterns of high resolution in all three spatial dimensions. Using UV laser photocuring at 308 nm will significantly improve the speed of rapid prototyping of biocompatible and biodegradable polymer scaffolds and enables its production in a few seconds, providing high lateral and horizontal resolution. This short timescale is indeed a tremendous asset that will enable a more efficient translation of technology to clinical applications. Preliminary cell tests proved that PPF : DEF scaffolds produced by excimer laser photocuring are biocompatible and, therefore, are promising candidates to be applied in tissue engineering and regenerative medicine.
laser photocuring; scaffolds; tissue engineering; biocompatibility; poly(propylene fumarate)