Biodegradable polylactide/hydroxyapatite nanocomposite foam scaffolds for bone tissue engineering applications

Supercritical carbon dioxide processing of poly-L-lactide (PLLA)/hydroxyapatite (nHA) nanocomposites was investigated as a means to prepare foams suitable as scaffolds in bone tissue engineering applications. For given foaming parameters, addition of nHA to the PLLA gave reduced cell sizes and improved homogeneity in the size distribution, but did not significantly affect the degree of crystallinity, which remained of the order of 50 wt% in all the foams. The compressive modulus and strength were primarily influenced by the porosity and there was no significant reinforcement of the matrix by the nHA. The mechanical properties of the foams were nevertheless comparable with those of trabecular bone, and by adjusting the saturation pressure and depressurization rate it was possible to generate porosities of about 85 %, an interconnected morphology and cell diameters in the range 200-400 μm from PLLA containing 4.17 vol% nHA, satisfying established geometrical requirements for bone replacement scaffolds.     

Hierarchically engineered fibrous scaffolds for bone regeneration

Surface properties of biomaterials play a major role in the governing of cell functionalities. It is well known that mechanical, chemical and nanotopographic cues, for example, influence cell proliferation and differentiation. Here, we present a novel coating protocol to produce hierarchically engineered fibrous scaffolds with tailorable surface characteristics, which mimic bone extracellular matrix. Based on the sol–gel method and a succession of surface treatments, hollow electrospun polylactic acid fibres were coated with a silicon–calcium–phosphate bioactive organic–inorganic glass. Compared with pure polymeric fibres that showed a completely smooth surface, the coated fibres exhibited a nanostructured topography and greater roughness. They also showed improved hydrophilic properties and a Young''s modulus sixfold higher than non-coated ones, while remaining fully flexible and easy to handle. Rat mesenchymal stem cells cultured on these fibres showed great cellular spreading and interactions with the material. This protocol can be transferred to other structures and glasses, allowing the fabrication of various materials with well-defined features. This novel approach represents therefore a valuable improvement in the production of artificial matrices able to direct stem cell fate through physical and chemical interactions.     

Preparation and characterization of polycaprolactone/forsterite nanocomposite porous scaffolds designed for bone tissue regeneration

Biocomposite scaffolds made from polymers and bioceramics can provide the mechanical structure necessary for osteoinductivity in the growth of new bone. The aim of this research was to investigate the properties of a novel nanocomposite scaffold made from a combination of polycaprolactone (PCL) and forsterite nanopowder which could find use in bone tissue engineering applications. The scaffold itself was fabricated by a method of solvent casting and particle leaching. The effect of forsterite content on the mechanical properties, bioactivity, biodegradability, and cytotoxicity of the scaffolds was investigated. Significant improvement in the mechanical properties was observed in the nanocomposite scaffolds as compared to that seen in the pure PCL scaffolds. Bioactivity was also observed in the nanocomposite scaffolds, a trait which was not present in the pure PCL scaffolds. Biodegradation assay indicated that the addition of forsterite nanopowder could modulate the degradation rate of PCL. In vitro tests of cytotoxicity and osteoblast proliferation showed that the nanocomposite scaffolds were non-cytotoxic, thereby allowing cells to adhere, grow, and proliferate on the surface of these scaffolds. The results obtained in this experiment suggest that the combination of PCL with forsterite nanopowder can be used to form scaffolds suitable for use in bone tissue engineering. The exact material behavior required can be adjusted through variation of the ratio between PCL and forsterite nanopowder used to form the scaffold.     

Injectable silk/hydroxyapatite nanocomposite hydrogels with vascularization capacity for bone regeneration

Localized and sustained osteogenic-angiogenic stimulation to bone defects is critical for effective bone repair.Here,desferrioxamine(DFO)was loaded on silk fibroin nanofibers and blended with hydroxyapatite nanorods(HA),forming injectable DFO-loaded silk fibroin-HA nanocomposite hydrogels.The composite hydrogels remained homogeneous distribution of HA with high ratio(60%)and also higher stiffness than that of pure silk fibroin nanofiber hydrogels,which provided stable osteogenic stimulation niches for tissue regeneration.Without the scarify of injectability,the hydrogels achieved slow delivery of DFO for above 60 days,resulting in suitable angiogenesis in vitro and in vivo and better osteogenesis than DFO-free systems.Compared to previous injectable silk fibroin-HA hydrogels,the introduction of vascularization capacity further stimulated the osteogenic differentiation of stem cells and accelerated new bone formation.Quicker and better bone healing were detected at defect sites after the injection of DFO-loaded nanocomposite hydrogels,indicating the effective synergistic effect of osteogenic and angiogenic cues.This work provides a simple and effective strategy of introducing angiogenic cues to bone matrices.We believe that the injectable nanocomposite hydrogels are suitable for the regeneration of bone tissues.     

Porous ceramic bone scaffolds for vascularized bone tissue regeneration

Hydroxyapatite scaffolds with a multi modal porosity designed for use in tissue engineering of vascularized bone graft substitutes were prepared by three dimensional printing. Depending on the ratio of coarse (mean particle size 50 microm) to fine powder (mean particle size 4 microm) in the powder granulate and the sintering temperature total porosity was varied from 30% to 64%. While macroscopic pore channels with a diameter of 1 mm were created by CAD design, porosity structure in the sintered solid phase was governed by the granulate structure of the printing powder. Scaffolds sintered at 1,250 degrees C were characterized by a bimodal pore structure with intragranular pores of 0.3-0.4 microm and intergranular pores of 20 microm whereas scaffolds sintered at 1,400 degrees C exhibit a monomodal porosity with a maximum of pore size distribution at 10-20 microm. For in-vivo testing, matrices were implanted subcutaneously in four male Lewis rats. Scaffolds with 50% porosity and an average pore size of approximately 18 microm were successfully transferred to rats and vascularized within 4 weeks.     

In vivo performance of bilayer hydroxyapatite scaffolds for bone tissue regeneration in the rabbit radius

The objective of this study was to investigate the in vivo biomechanical performance of bone defects implanted with novel bilayer hydroxyapatite (HAp) scaffolds that mimic the cortical and cancellous organization of bone. The scaffolds maintained architectural continuity in a rabbit radius segmental defect model and were compared to an untreated defect group (negative control) and autologous bone grafts (positive control). Micro-CT evaluations indicated total bone and scaffold volume in the experimental group was significantly greater than the defect group but lesser than the autologous bone graft treatment. The flexural toughness of the scaffold and the autograft groups was significantly greater than the flexural toughness of the defect group. Interestingly, the absolute density of the bone mineral as well as calcium to phosphorus (Ca/P) ratio in that mineral for the scaffold and autograft contralateral bones was significantly higher than those for the defect contralaterals suggesting that the scaffolds contributed to calcium homeostasis. It was concluded from this study that new bone regenerated in the bilayer HAp scaffolds was comparable to the empty defects and while the HAp scaffolds provided significant increase in modulus when compared to empty defect and their flexural toughness was comparable to autografts after 8 weeks of implantation.     

Microsphere-based scaffolds encapsulating tricalcium phosphate and hydroxyapatite for bone regeneration

Bioceramic mixtures of tricalcium phosphate (TCP) and hydroxyapatite (HAp) are widely used for bone regeneration because of their excellent cytocompatibility, osteoconduction, and osteoinduction. Therefore, we hypothesized that incorporation of a mixture of TCP and HAp in microsphere-based scaffolds would enhance osteogenesis of rat bone marrow stromal cells (rBMSCs) compared to a positive control of scaffolds with encapsulated bone-morphogenic protein-2 (BMP-2). Poly(d,l-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds encapsulating TCP and HAp mixtures in two different ratios (7:3 and 1:1) were fabricated with the same net ceramic content (30 wt%) to evaluate how incorporation of these ceramic mixtures would affect the osteogenesis in rBMSCs. Encapsulation of TCP/HAp mixtures impacted microsphere morphologies and the compressive moduli of the scaffolds. Additionally, TCP/HAp mixtures enhanced the end-point secretion of extracellular matrix components relevant to bone tissue compared to the “blank” (PLGA-only) microsphere-based scaffolds as evidenced by the biochemical, gene expression, histology, and immunohistochemical characterization. Moreover, the TCP/HAp mixture groups even surpassed the BMP-2 positive control group in some instances in terms of matrix synthesis and gene expression. Lastly, gene expression data suggested that the rBMSCs responded differently to different TCP/HAp ratios presented to them. Altogether, it can be concluded that TCP/HAp mixtures stimulated the differentiation of rBMSCs toward an osteoblastic phenotype, and therefore may be beneficial in gradient microsphere-based scaffolds for osteochondral regeneration.     

Composite scaffolds of nano calcium deficient hydroxyapatite/multi-(amino acid) copolymer for bone tissue regeneration

In this study, nano calcium deficient hydroxyapatite (n-DA)/multi-(amino acid) copolymer composite scaffolds were prepared by injection molding foaming method using calcium sulphate dihydrate as a foaming agent. The composite scaffolds showed well interconnected macropores with the pore size of ranging from 100 to 600 μm, porosity of 81 % and compressive strength of 12 MPa, and the compressive strength obviously affected by the porosity. The composite scaffolds could be slowly degraded in phosphate buffered solution (PBS), which lost its initial weight of 61 w % after immersion into PBS for 12 weeks, and the porosity significantly affected the degradability of the scaffolds. Moreover, it was found that the composite scaffolds could promote the MG-63 cells growth and proliferation, and enhance its alkaline phosphatase activity. The implantation of the scaffolds into the femoral bone of rabbits confirmed that the composite scaffolds were biocompatibitive, degradable, and osteoconductive in vivo.     

Poly(vinyl alcohol)–hydroxyapatite biomimetic scaffold for tissue regeneration

Hydroxyapatite–poly(vinyl alcohol) composite blocks were produced by freeze–thawing in situ synthesized hybrid suspension. Matrix mediated precipitation of hydroxyapatite particles in the polymer, controlled the particle size in nanometer range (< 100 nm) and hydrogelation induced an ordered three-dimensional assembly of the particles. Morphological and crystallographic characterization revealed the formation of a macro-porous hydroxyapatite–poly(vinyl alcohol) nanocomposite block. Initial results indicate compositional dependence of modulus of elasticity of the composite.     

Heparinized hydroxyapatite/collagen three-dimensional scaffolds for tissue engineering

Currently, in bone tissue engineering research, the development of appropriate biomaterials for the regeneration of bony tissues is a major concern. Bone tissue is composed of a structural protein, collagen type I, on which calcium phosphate crystals are enclosed. For tissue engineering, one of the most applied strategies consists on the development and application of three dimensional porous scaffolds with similar composition to the bone. In this way, they can provide a physical support for cell attachment, proliferation, nutrient transport and new bone tissue infiltration. Hydroxyapatite is a calcium phosphate with a similar composition of bone and widely applied in several medical/dentistry fields. Therefore, in this study, hydroxyapatite three dimensional porous scaffolds were produced using the polymer replication method. Next, the porous scaffolds were homogeneously coated with a film of collagen type I by applying vacuum force. Yet, due to collagen degradability properties, it was necessary to perform an adequate crosslinking method. As a result, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was employed as an efficient and non-toxic crosslinking method in this research. The composites were characterized by means of SEM, DSC and TNBS. Furthermore, heparin was incorporated in order to accomplish sustained delivery of a growth factor of interest namely, bone morphogenetic proteins (BMP-2). BMP-2 binding and release of non-heparinized and heparinized scaffolds was evaluated at specific time points. The incorporation of heparin leads to a reduced initial burst phase when compared to the non heparinized materials. The results show a beneficial effect with the incorporation of heparin and its potential as a localized drug delivery system for the sustained release of growth factors.     

In vitro and in vivo evaluation of electrospun PCL/PMMA fibrous scaffolds for bone regeneration

Abstract

Scaffolds were fabricated by electrospinning using polycaprolactone (PCL) blended with poly(methyl methacrylate) (PMMA) in ratios of 10/0, 7/3, 5/5 and 3/7. The PCL/PMMA ratio affected the fiber diameter, contact angle, tensile strength and biological in vitro and in vivo properties of the scaffolds, and the 7/3 ratio resulted in a higher mechanical strength than 5/5 and 3/7. In vitro cytotoxicity and proliferation of MG-63 osteoblast cells on these blended scaffolds were examined by MTT assay, and it was found that PCL/PMMA blends are suitable for osteoblast cell proliferation. Confocal images and expression of proliferating cell nuclear antigen confirmed the good proliferation and expression of cells on the 7/3 PCL/PMMA fibrous scaffolds. In vivo bone formation was examined using rat models, and bone formation was observed on the 7/3 PCL/PMMA scaffold within 2 months. In vitro and in vivo results suggest that 7/3 PCL/PMMA scaffolds can be used for bone tissue regeneration.     

Calcium silicate ceramic scaffolds toughened with hydroxyapatite whiskers for bone tissue engineering

Calcium silicate possessed excellent biocompatibility, bioactivity and degradability, while the high brittleness limited its application in load-bearing sites. Hydroxyapatite whiskers ranging from 0 to 30 wt.% were incorporated into the calcium silicate matrix to improve the strength and fracture resistance. Porous scaffolds were fabricated by selective laser sintering. The effects of hydroxyapatite whiskers on the mechanical properties and toughening mechanisms were investigated. The results showed that the scaffolds had a uniform and continuous inner network with the pore size ranging between 0.5 mm and 0.8 mm. The mechanical properties were enhanced with increasing hydroxyapatite whiskers, reached a maximum at 20 wt.% (compressive strength: 27.28 MPa, compressive Young's modulus: 156.2 MPa, flexural strength: 15.64 MPa and fracture toughness: 1.43 MPa·m^1/2) and then decreased by addition of more hydroxyapatite whiskers. The improvement of mechanical properties was due to whisker pull-out, crack deflection and crack bridging. Moreover, the degradation rate decreased with the increase of hydroxyapatite whisker content. A layer of bone-like apatite was formed on the scaffold surfaces after being soaked in simulated body fluid. Human osteoblast-like MG-63 cells spread well on the scaffolds and proliferated with increasing culture time. These findings suggested that the calcium silicate scaffolds reinforced with hydroxyapatite whiskers showed great potential for bone regeneration and tissue engineering applications.     

Hybrid bilayered chitosan-xanthan/PCL scaffolds as artificial periosteum substitutes for bone tissue regeneration

Journal of Materials Science - The periosteum, a bilayered membrane that covers bone surfaces, acts as a source of bone-forming cells and plays a pivotal role in bone homeostasis and defect...     

一种新型仿生骨组织工程支架的制备与表征

利用改性生物玻璃粉体和胶原、透明质酸钠、磷酸丝氨酸等天然生物分子复合制备仿生型三维多孔骨组织工程支架材料,利用体外模拟实验结合SEM、FTIR、XRD 等测试方法对材料的显微结构、生物矿化性能进行了综合研究,研究表明该材料具有良好的孔隙结构,在模拟生理溶液(SBF)中反应24h即可在支架表面形成碳酸羟基磷灰石(HCA).     

Electrospun bioactive nanocomposite scaffolds of polycaprolactone and nanohydroxyapatite for bone tissue engineering

Nanocomposite scaffolds based on nanofibrous poly(epsilon-caprolactone) (PCL) and nanohydroxyapatite (nanoHA) with different compositions (wt%) were prepared by electrostatic co-spinning to mimic the nano-features of the natural extracellular matrix (ECM). NanoHA was found to be well dispersed in polymers up to the addition of 20 wt%, after ultrasonication. The composite scaffolds were characterized for structure and morphology using XRD, EDX, SEM, and DSC. The scaffolds have a porous nanofibrous morphology with fibers (majority) having diameters in the range of 450-650 nm, depending on composition, and interconnected pore structures. SEM, EDX, and XRD analyses have confirmed the presence of nanoHA in the fibers. As the nanoHA content in the fibers increases, the surface of fibers becomes rougher. The mechanical (tensile) property measurement of the electrospun composites reveals that as the nanoHA content increases, the ultimate strength increases from 1.68 MPa for pure PCL to 2.17, 2.65, 3.91, and 5.49 MPa for PCL/nanoHA composites with the addition of 5, 10, 15, and 20 wt% nanoHA, respectively. Similarly the tensile modulus also increases gradually from 6.12 MPa to 21.05 MPa with the increase of nanoHA content in the PCL/nanoHA fibers, revealing an increase in stiffness of the fibers due to the presence of HA. DSC analysis reveals that as nanoHA in the composite scaffolds increases, the melting point slightly increases due to the good dispersion and interface bonding between PCL and nanoHA.     

Carbon nanofibers produced from modified electrospun PAN/hydroxyapatite precursors as scaffolds for bone tissue engineering

In the current study we have proposed a method to obtain a carbon/HAp bioactive nanofibrous scaffold. The modified carbon nanofibrous nonwoven' fabrics were obtained by the use of electrospinning and subsequent stabilization and carbonization processes. The modified with HAp powder nanofibrous PAN nonwovens were thermally stabilized using a multi-stage process in the temperature ranging from 100 °C to 300 °C in an oxidative environment and then carbonized at 1000 °C in argon atmosphere. The changes of properties of composite precursor membranes taking place during stabilization and carbonization processes were investigated using the methods of: DSC, TGA, FTIR, SEM, EDX, WAXD and mechanical tests. Bioactivity was determined by assessing the formation of crystalline apatite on the surface of membranes upon immersion in Simulated Body Fluid (SBF). The FTIR, SEM and WAXD investigation clearly prove that hydroxyapatite added to the electrospinning solution was present also in composites nanofibrous nonwovens after stabilization and carbonization process. It was found that due to HAp addition: the significant decrease of fibers average diameter occurs and that the average pore size for modified membranes is smaller than for the unmodified one. On the other hand it was shown that the ceramic additive protects fibers from mass reduction during the stabilization treatment. Finally a drastic increase of mineralization activity of nCF/HAp scaffolds as compared to their nCF counterparts has been proved.     

Chitosan/gelatin scaffolds support bone regeneration

Chitosan/Gelatin (CS:Gel) scaffolds were fabricated by chemical crosslinking with glutaraldehyde or genipin by freeze drying. Both crosslinked CS:Gel scaffold types with a mass ratio of 40:60% form a gel-like structure with interconnected pores. Dynamic rheological measurements provided similar values for the storage modulus and the loss modulus of the CS:Gel scaffolds when crosslinked with the same concentration of glutaraldehyde vs. genipin. Compared to genipin, the glutaraldehyde-crosslinked scaffolds supported strong adhesion and infiltration of pre-osteoblasts within the pores as well as survival and proliferation of both MC3T3-E1 pre-osteoblastic cells after 7 days in culture, and human bone marrow mesenchymal stem cells (BM-MSCs) after 14 days in culture. The levels of collagen secreted into the extracellular matrix by the pre-osteoblasts cultured for 4 and 7 days on the CS:Gel scaffolds, significantly increased when compared to the tissue culture polystyrene (TCPS) control surface. Human BM-MSCs attached and infiltrated within the pores of the CS:Gel scaffolds allowing for a significant increase of the osteogenic gene expression of RUNX2, ALP, and OSC. Histological data following implantation of a CS:Gel scaffold into a mouse femur demonstrated that the scaffolds support the formation of extracellular matrix, while fibroblasts surrounding the porous scaffold produce collagen with minimal inflammatory reaction. These results show the potential of CS:Gel scaffolds to support new tissue formation and thus provide a promising strategy for bone tissue engineering.     

A method to design biomimetic scaffolds for bone tissue engineering based on Voronoi lattices

In regenerative medicine, 3D scaffolds are used to sustain the regeneration of tissues in removed or damaged parts of the human body. As such practices are being widely experimented in clinical applications, the design, the materials and the manufacturing process to obtain efficient 3D biocompatible lattices are being significantly investigated. Nevertheless, most of the proposed designs are based on regular 3D shapes obtained from the repetition of unit cells disposed in a three-dimensional array. This approach does not exploit the whole potential of computer-aided design tools coupled with manufacturing capabilities for freeform shapes. In this paper, we propose a method to model biomimetic lattices controlling the porosity and the pores size of scaffolds to be integrated with the anatomical shape of the defect. The method has been implemented in bone tissue case study and implements a generative design approach based on Voronoi diagrams.