Effect of chitosan scaffold microstructure on mesenchymal stem cell chondrogenesis

Although numerous biomaterials have been investigated as scaffolds for cartilage tissue engineering, the effect of their microstructure on final construct characteristics remains unclear. The biocompatibility of chitosan and its similarity with glycosaminoglycans make it attractive as a scaffold for cartilage engineering. Our objective was to evaluate the effect of chitosan scaffold structure on mesenchymal stem cell proliferation and chondrogenesis. Chitosan fibrous scaffolds and chitosan sponges were seeded with mesenchymal stem cells in a chondrogenic medium. Constructs were analyzed 72 h after seeding via scanning electron microscopy (SEM), weight measurements and DNA quantification. Constructs were cultured for 10 or 21 days prior to confocal microscopy, SEM, histology, quantitative analysis (weight, DNA and glycosaminoglycan (GAG)), and quantitative real-time polymerase chain reaction. Mesenchymal stem cells maintained a viability above 90% on all chitosan scaffolds. The cell numbers in the constructs were similar at 72 h, 10 days and 21 days. However, matrix production was improved in chitosan fibrous constructs based on the GAG quantification and collagen II mRNA expression. Chondrogenesis on chitosan scaffolds is superior on microfibers compared to macroporous sponges.     

Chemical modification of collagen improves glycosaminoglycan retention of their co-precipitates

Being prevalent extracellular matrix components, collagen and glycosaminoglycan (GAG) are co-precipitated as scaffolds for tissue regeneration. However, the amount of GAG incorporated and its long-term retention present a persistent problem. In this study, chemical modifications, namely deamination, methylation and amination, were used to alter the net charge of collagen prior to fabrication of collagen–GAG co-precipitate. While most GAGs were lost in the untreated group and the deaminated group within 1 day, methylation and amination of collagen retained over 20% and 40% GAG after 6 days, respectively. Moreover, over 60% of GAG retention was achieved in the aminated group after cell seeding for 8 days. Furthermore, amination of collagen increased the GAG/hydroxyproline ratio in the co-precipitate to >4.5, approaching that of native nucleus pulposus. Ultrastructural analysis showed that the aminated group contains abundant granular substances resembling the extracellular matrix of native nucleus pulposus. Despite lower initial cell adhesion than untreated, all modified scaffolds promoted proliferation of human mesenchymal stem cells (hMSCs) and showed >95% cell viability at all time points. Cell morphology was distinct among the different groups, being round in the untreated control and methylated groups but elongated in deaminated and aminated groups. hMSCs adhered to scaffolds via collagen receptor integrin α2β1 in all groups, while all but the aminated group showed extensive expression of the general matrix receptor integrin αv. This work reports an effective method, namely amination of collagen, to improve GAG incorporation and retention in collagen–GAG co-precipitates, facilitating the fabrication of GAG-rich collagenous scaffold for intervertebral disc tissue engineering.     

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.     

Density–property relationships in collagen–glycosaminoglycan scaffolds

Collagen–glycosaminoglycan scaffolds for the regeneration of skin have previously been fabricated by freeze-drying a slurry containing a co-precipitate of collagen and glycosaminoglycan. The mechanical properties of the scaffold are low (e.g. the dry compressive Young’s modulus is roughly 30 kPa and the dry compressive strength is roughly 5 kPa). There is interest in using these scaffolds for tendon and ligament regeneration where there is a need for improved mechanical properties. Previous attempts to increase the mechanical properties of the scaffold by increasing the solid volume fraction of the scaffolds were limited by the increasing viscosity of the slurry, making it more difficult to mix and giving inhomogeneous scaffolds. Our recent work on mineralized collagen–glycosaminoglycan scaffolds used a vacuum filtration technique to increase the volume fraction of solids in the slurry, thereby increasing the density and mechanical properties of the scaffolds. In this work, we used this technique to fabricate collagen–glycosaminoglycan scaffolds with dry densities between 0.0076 and 0.0311 g cm^?3 and pore sizes between 250 and 350 μm, values appropriate for soft tissue growth. The compressive Young’s modulus and strength in the dry state increased from 32 to 127 kPa and from 5 to 19 kPa, respectively, with increasing density. The tensile Young’s modulus in the dry state increased from 295 to 3.1 MPa with increasing density. Finally, we showed that the attachment of cells onto the scaffold was directly proportional to the specific surface area of the scaffold, which defines the total internal surface area per volume of scaffold.     

Evaluation of extracellular matrix formation in polycaprolactone and starch-compounded polycaprolactone nanofiber meshes when seeded with bovine articular chondrocytes

Cartilage defects are a major health problem. Tissue engineering has developed different strategies and several biomaterial morphologies, including natural-based ones, for repairing these defects. We used electrospun polycaprolactone (PCL) and starch-compounded PCL (SPCL) nanofiber meshes to evaluate extracellular matrix (ECM) formation by bovine articular chondrocytes (BACs). The main aim of this work was to evaluate the suitability of PCL and SPCL nanofiber meshes in chondrocyte cultures, and their capability to produce ECM when seeded onto these nanostructured materials. The effect of culture conditions (static vs dynamic) on ECM formation was also assessed. BACs were seeded onto PCL and SPCL nanofiber meshes using a dynamic cell-seeding procedure and cultured under static or dynamic conditions for 4 weeks. Constructs were characterized using scanning electron microscopy, histology, immunolocalization of collagen types I and II, and glycosaminoglycan (GAG) quantification. Results show an extensive cell colonization of the entire nanofiber mesh, for both materials, and that chondrocytes presented typical spherical morphology. Some degree of cell infiltration inside the nanofiber meshes was noticeable for both materials. ECM formation and GAG were detected throughout the materials, evidencing typical construct maturation. PCL and SPCL nanofiber meshes are suitable as supports for ECM formation and therefore are adequate for cartilage tissue-engineering approaches.     

Influence of hydration on fiber geometry in electrospun scaffolds

Finite element models of tissue engineering scaffolds are powerful tools to understand scaffold function, including how external mechanical signals deform the scaffold at the meso- and microscales. Fiber geometry is needed to inform finite element models of fiber-based tissue engineering scaffolds; however, the accuracy and utility of these models may be limited if they are informed by non-hydrated geometries. Scanning electron microscopy and confocal microscopy, coupled with Fourier analysis of the resulting images, were used to quantify how hydration alters fiber geometry in electrospun collagen and polycaprolactone (PCL) scaffolds. The results also quantify how image size affects fiber geometry. Hydration is demonstrated to increase fiber tortuosity, defined as the ratio of actual fiber length:end-to-end fiber length. For collagen scaffolds, hydration increased the mean tortuosity from 1.05 to 1.21, primarily from large ～2- to 10-fold) increases in smaller (<40 μm) wavelength amplitudes. For PCL fibers, the mean tortuosity increased from 1.01 to only 1.04, primarily from modest ～2-fold) increases in larger (>100 μm) wavelength amplitudes. The results demonstrate that mechanical simulations of electrospun scaffolds should be informed with hydrated scaffold geometries of at least 200 μm scale, in order to capture geometrical effects associated with fiber straightening.     

Incorporation of bioactive polyvinylpyrrolidone–iodine within bilayered collagen scaffolds enhances the differentiation and subchondral osteogenesis of mesenchymal stem cells

Polyvinylpyrrolidone–iodine (Povidone-iodine, PVP-I) is widely used as an antiseptic agent for lavation during joint surgery; however, the biological effects of PVP–I on cells from joint tissue are unknown. This study examined the biocompatibility and biological effects of PVP–I on cells from joint tissue, with the aim of optimizing cell-scaffold based joint repair. Cells from joint tissue, including cartilage derived progenitor cells (CPC), subchondral bone derived osteoblast and bone marrow derived mesenchymal stem cells (BM-MSC) were isolated. The concentration-dependent effects of PVP–I on cell proliferation, migration and differentiation were evaluated. Additionally, the efficacy and mechanism of a PVP–I loaded bilayer collagen scaffold for osteochondral defect repair was investigated in a rabbit model. A micromolar concentration of PVP–I was found not to affect cell proliferation, CPC migration or extracellular matrix production. Interestingly, micromolar concentrations of PVP–I promote osteogenic differentiation of BM-MSC, as evidenced by up-regulation of RUNX2 and Osteocalcin gene expression, as well as increased mineralization on the three-dimensional scaffold. PVP–I treatment of collagen scaffolds significantly increased fibronectin binding onto the scaffold surface and collagen type I protein synthesis of cultured BM-MSC. Implantation of PVP–I treated collagen scaffolds into rabbit osteochondral defect significantly enhanced subchondral bone regeneration at 6 weeks post-surgery compared with the scaffold alone (subchondral bone histological score of 8.80 ± 1.64 vs. 3.8 ± 2.19, p < 0.05). The biocompatibility and pro-osteogenic activity of PVP–I on the cells from joint tissue and the enhanced subchondral bone formation in PVP–I treated scaffolds would thus indicate the potential of PVP–I for osteochondral defect repair.     

Substrate stiffness and contractile behaviour modulate the functional maturation of osteoblasts on a collagen–GAG scaffold

Anchorage-dependent cells respond to the mechanical and physical properties of biomaterials. One such cue is the mechanical stiffness of a material. We compared the osteogenic potential of collagen–glycosaminoglycan (CG) scaffolds with varying stiffness for up to 6 weeks in culture. The mechanical stiffness of CG scaffolds were varied by cross-linking by physical (dehydrothermal (DHT)) and chemical (1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDAC) and glutaraldehyde (GLUT)) methods. The results showed that all CG substrates allowed cellular attachment, infiltration and osteogenic differentiation. CG scaffolds treated with EDAC and GLUT were mechanically stiffer, retained their original scaffold structure and resisted cellular contraction. Consequently, they facilitated a 2-fold greater cell number, probably due to the pore architecture being maintained, allowing improved diffusion of nutrients. On the other hand, the less stiff substrates cross-linked with DHT allowed increased cell-mediated scaffold contraction, contracting by 70% following 6 weeks (P < 0.01) of culture. This reduction in scaffold area resulted in cells reaching the centre of the scaffold quicker up to 4 weeks; however, at 6 weeks all scaffolds showed similar levels of cellular infiltration, with higher cell numbers found on the stiffer EDAC- and GLUT-treated scaffolds. Analysis of osteogenesis showed that scaffolds cross-linked with DHT expressed higher levels of the late stage bone formation markers osteopontin and osteocalcin (P < 0.01) and increased levels of mineralisation. In conclusion, the more compliant CG scaffolds allowed cell-mediated contraction and supported a greater level of osteogenic maturation of MC3T3 cells, while the stiffer, non-contractible scaffolds resulted in lower levels of cell maturation, but higher cell numbers on the scaffold. Therefore, we found scaffold stiffness had different effects on differentiation and cell number whereby the increased cell-mediated contraction facilitated by the less stiff scaffolds positively modulated osteoblast differentiation while reducing cell numbers.     

Composite chondroitin-6-sulfate/dermatan sulfate/chitosan scaffolds for cartilage tissue engineering

Conjugating a single glycosaminoglycan (GAG) species such as chondroitin-6-sulfate (CSC) to chitosan is beneficial to chondrocyte culture and extracellular matrix (ECM) production, but whether fabrication of 3D chitosan scaffolds with additional minor GAG species such as dermatan sulfate (DS) further improves the ECM production is unknown. In this study, Response Surface Methodology (RSM) was employed to design CSC/DS/chitosan scaffolds of various formulations for cartilage engineering and to investigate the roles of individual GAG species in cartilage formation. The CSC/DS formulation affected neither the physical properties of scaffolds nor cell adhesion, but influenced cell morphology, GAGs and collagen production and chondrocytic gene expression. The linear effects elucidated by RSM analysis suggested that within the level range higher CSC levels favored GAGs and collagen production, whereas lower DS levels were desired for these responses. Nonetheless, the quadratic effects of DS and two-way interactions between CSC and DS also contributed to the GAGs and collagen production. Accordingly, the optimal formulation, as predicted by RSM and validated by experiments, comprised 2.8 mg CSC and 0.01 mg DS per scaffold. This study confirmed the importance of DS in cartilage tissue engineering and implicated the feasibility of rational CSC/DS/chitosan scaffold design with the aid of RSM.     

Tissue-engineered cartilage constructs using composite hyaluronic acid/collagen I hydrogels and designed poly(propylene fumarate) scaffolds

Our approach to cartilage tissue-engineering scaffolds combines image-based design and solid free-form (SFF) fabrication to create load-bearing constructs with user-defined parameters. In this study, 3-dimensional scaffolds with cubic and ellipsoidal pore architecture were fabricated using poly(propylene fumarate) (PPF). To increase seeding efficiency and cellular retention, hydrogels were used to deliver cells into the scaffolds. The first objective of this study was to evaluate the concentrations of composite hyaluronic acid (HyA) and collagen I hydrogels best able to stimulate proteoglycan synthesis in porcine chondrocytes in vitro and in vivo. The second objective was to evaluate the differences in extracellular matrix production due to pore geometry and scaffold design. For the in vitro assessment, chondrocytes were encapsulated in collagen I hydrogels with varying concentrations of HyA. Hydrogels were cultured for 1 and 2 weeks, and then the sulfated glycosaminoglycan (sGAG) content was quantified using a dimethyl-methylene blue assay. The concentration of HyA best able to increase ECM synthesis was 5% HyA/collagen I, or 0.23 mg/mL HyA. The results from the in vitro experiment were used as culture parameters for the in vivo analysis. Composite 5% HyA/collagen I or collagen I-only hydrogels were used to seed chondrocytes into SFF-fabricated scaffolds made of PPF with designed cubic or ellipsoidal pore geometry. The scaffolds were implanted subcutaneously in immunocompromised mice for 4 weeks. Histomorphometric analyses of sections stained with Safranin O were used to quantify the amount of ECM deposited by cells in the scaffolds. Scaffolds seeded with 5% HyA/collagen hydrogels had significantly greater areas of positive Safranin O staining (approximately 60%, compared with 30% for scaffolds with collagen I hydrogels only), indicating that greater numbers of chondrocytes retained their metabolic activity in the ectopic environment. These scaffolds also had greater stain intensities (corresponding to greater amounts of sGAG in the ECM) than their counterparts seeded with collagen I hydrogels alone. Significant differences in matrix production were not found between the scaffold pore designs. Overall, these results indicate that a combination of composite HyA hydrogels and designed SFF scaffolds could provide a functional tissue-engineered construct for cartilage repair with enhanced tissue regeneration in a load-bearing scaffold.     

Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.     

Density–property relationships in mineralized collagen–glycosaminoglycan scaffolds

Mineralized collagen–glycosminoglycan scaffolds have previously been fabricated by freeze-drying a slurry containing a co-precipitate of calcium phosphate, collagen and glycosaminoglycan. The mechanical properties of the scaffold are low (e.g. the dry Young’s modulus for a 50 wt.% mineralized scaffold is roughly 780 kPa). Our previous attempt to increase the mechanical properties of the scaffold by increasing the mineralization (from 50 to 75 wt.%) was unsuccessful due to defects in the more mineralized scaffold. In this paper, we describe a new technique to improve the mechanical properties by increasing the relative density of the scaffolds. The volume fraction of solids in the slurry was increased by vacuum-filtration. The slurry was then freeze-dried in the conventional manner to produce scaffolds with relative densities between 0.045 and 0.187 and pore sizes of about 100–350 μm, values appropriate for bone growth. The uniaxial compressive stress–strain curves of the scaffolds indicated that the Young’s modulus in the dry state increased from 780 to 6500 kPa and that the crushing strength increased from 39 to 275 kPa with increasing relative density. In the hydrated state, the Young’s modulus increased from 6.44 to 34.8 kPa and the crushing strength increased from 0.55 to 2.12 kPa; the properties were further increased by cross-linking. The modulus and strength were well described by models for cellular solids.     

Biomimetic poly(lactide) based fibrous scaffolds for ligament tissue engineering

The aim of this study was to fabricate a fibrous scaffold that closely resembled the micro-structural architecture and mechanical properties of collagen fibres found in the anterior cruciate ligament (ACL). To achieve this aim, fibrous scaffolds were made by electrospinning l-lactide based polymers. l-Lactide was chosen primarily due to its demonstrated biocompatibility, biodegradability and high modulus. The electrospun fibres were collected in tension on a rotating wire mandrel. Upon treating these fibres in a heated aqueous environment, they possessed a crimp-like pattern having a wavelength and amplitude similar to that of native ACL collagen. Of the polymer fibre scaffolds studied, those made from poly(l-lactide-co-d,l-lactide) PLDLA exhibited the highest modulus and were also the most resilient to in vitro hydrolytic degradation, undergoing a slight decrease in modulus compared to the other polymeric fibres over a 6 month period. Bovine fibroblasts seeded on the wavy, crimp-like PLDLA fibres attached, proliferated and deposited extracellular matrix (ECM) molecules on the surface of the fibrous scaffold. In addition, the deposited ECM exhibited bundle formation that resembled the fascicles found in native ACL. These findings demonstrate the importance of replicating the geometric microenvironment in developing effective tissue engineering scaffolds.     

The modification of scaffold material in building artificial dermis

Type X collagen is principal extracellular matrix (ECM) in natural dermis. To prepare artificial dermis, collagen is traditional, and most superior biomaterial. But beside collagen, the dermis also contains many other ECM. Among them, glycosaminoglycan (GAG) is another important substance. To imitate the natural dermis, and modificate the scaffold materials, two types of scaffolds were prepared: one is traditional type X collagen spongy scaffold, the other is collagen-chondroitin sulfate (CS) spongy scaffold. Collagen was blended with CS, one kind of GAG, and cross-linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Dermis fibroblast was isolated from neonate prepuce, and dermis fibroblasts were cultured on the scaffolds. The physical and chemical properties of the scaffolds were tested, including SEM, DSC, H&E staining, immunohistochemical staining and CS content analysis and so on. The results indicated that EDC is an effective and non-cytotoxic cross-link reagent, and attaching CS into collagen scaffold could improve the stability and histocompatibility of scaffold.     

Influence of the fiber diameter and surface roughness of electrospun vascular grafts on blood activation

Electrospun grafts have been widely investigated for vascular graft replacement due to their ease and compatibility with many natural and synthetic polymers. Here, the effect of the processing parameters on the scaffold’s architecture and subsequent reactions of partially heparinized blood triggered by contacting these topographies were studied. Degrapol® (DP) and poly(lactic-co-glycolic acid) (PLGA) electrospun fibrous scaffolds were characterized with regard to fiber diameter, pore area and scaffold roughness. The study showed that electrospinning parameters greatly affect fiber diameter together with pore dimension and overall scaffold roughness. Coagulation cascade activation, early platelet adhesion and activation were analyzed after 2 h of exposure of blood to the biomaterials. While no differences were found between DP and PLGA with similar topographies, the blood reactions were observed to be dependent on the fiber diameter and scaffold roughness. Scaffolds composed of thin fibers (diameter <1 μm) triggered very low coagulation and almost no platelets adhered. On the other hand, scaffolds with a bigger fiber diameter (2–3 μm) triggered higher thrombin formation and more platelets adhered. The highest platelet adhesion and activations rates as well as coagulation cascade activation were found in blood incubated in contact with the scaffolds produced with the biggest fiber diameter (5 μm). These findings indicate that electrospun grafts with small fiber diameter (<1 μm) could perform better with reduced early thrombogenicity due to lower platelet adhesion and lower activation of platelets and coagulation cascade.     

Synergistic effect of surface modification and scaffold design of bioplotted 3-D poly-ε-caprolactone scaffolds in osteogenic tissue engineering

The hydrophobic nature and the regular scaffold architecture of bioplotted poly(ε-caprolactone) (PCL) scaffolds present some hurdles for homogeneous tissue formation and differentiation. The current hypothesis is that a synergistic effect of applied surface modification and scaffold design enhances colonization and osteogenic differentiation. First, PCL scaffolds with a 0/90° lay-down pattern (0/90) were plotted and subjected to an oxygen plasma (O₂) or multistep surface modification, including post-argon 2-amino-ethylmethacrylate grafting (AEMA), followed by immobilization of gelatin type B (gelB) and physisorption of fibronectin (gelB Fn). Secondly, scaffolds of different designs were plotted (0/90° shift (0/90 S), 0/45° and 0/90° with narrow pores (0/90 NP)) and subjected to the double protein coating. Preosteoblasts were cultured on the scaffolds and the seeding efficiency, colonization and differentiation were studied. The data revealed that a biomimetic surface modification improved colonization (gelB Fn > gelB > AEMA > O₂). Compact scaffold architectures (0/90 NP, 0/45, 0/90 S > 0/90) positively influenced the seeding efficiency and differentiation. Interestingly, the applied surface modification had a greater impact on colonization than the scaffold design. In conclusion, the combination of a double protein coating with a compact design enhances tissue formation in the plotted PCL scaffolds.     

Shape-memory porous alginate scaffolds for regeneration of the annulus fibrosus: Effect of TGF-β3 supplementation and oxygen culture conditions

Disc herniation as a result of degenerative or traumatic injury is believed to be the primary instigator of low back pain. At present there is a lack of viable treatment options to repair damaged annulus fibrosus (AF) tissue. Developing alternative strategies to fill and repair ruptured AF tissue is a key challenge. In this work we developed a porous alginate scaffold with shape-memory properties which can be delivered using minimally invasive approaches and recover its original geometry once hydrated. Covalently cross-linked alginate hydrogels were created using carbodiimide chemistry, followed by a freeze-drying step to impart porosity and create porous scaffolds. Results showed that porous alginate scaffolds exhibited shape-memory recovery and mechanical behaviour that could be modulated depending on the cross-linker concentrations. The scaffold can be repeatedly compressed and expanded, which provides the potential to deliver the biomaterial directly to the damaged area of the AF tissue. In vitro experiments demonstrated that scaffolds were cytocompatible and supported cell seeding, penetration and proliferation under intervertebral-disc-like microenvironmental conditions (low glucose media and low oxygen concentration). Extracellular matrix (ECM) was secreted by AF cells with TGF-β3 stimulation and after 21 days had filled the porous scaffold network. This biological matrix was rich in sulfated glycosaminoglycan and collagen type I, which are the main compounds of native AF tissue. Successful ECM deposition was also confirmed by the increase in the peak stress of the scaffold. However, the immaturity of the matrix network after only 21 days of in vitro culture was not sufficient to attain native AF tissue mechanical properties. The ability to deliver porous scaffolds using minimal invasive approaches that can potentially promote the regeneration of AF defects provides an exciting new avenue for disc repair.     

Fabrication and characterization of biomimetic collagen–apatite scaffolds with tunable structures for bone tissue engineering

The objective of the current study is to prepare a biomimetic collagen–apatite scaffold for improved bone repair and regeneration. A novel bottom–up approach has been developed, which combines a biomimetic self-assembly method with a controllable freeze-casting technology. In this study, the mineralized collagen fibers were generated using a simple one-step co-precipitation method which involved collagen self-assembly and in situ apatite precipitation in a collagen-containing modified simulated body fluid (m-SBF). The precipitates were then subjected to controllable freeze casting, forming scaffolds with either an isotropic equiaxed structure or a unidirectional lamellar structure. These scaffolds were comprised of collagen fibers and poorly crystalline bone-like carbonated apatite nanoparticles. The mineral content in the scaffold could be tailored in the range 0–54 wt.% by simply adjusting the collagen content in the m-SBF. Further, the mechanisms of the formation of both the equiaxed and the lamellar scaffolds were investigated, and freezing regimes for equiaxed and lamellar solidification were established. Finally, the bone-forming capability of such prepared scaffolds was evaluated in vivo in a mouse calvarial defect model. It was confirmed that the scaffolds well support new bone formation.     

Effects of the architecture of tissue engineering scaffolds on cell seeding and culturing

The advance of rapid prototyping techniques has significantly improved control over the pore network architecture of tissue engineering scaffolds. In this work, we have assessed the influence of scaffold pore architecture on cell seeding and static culturing, by comparing a computer designed gyroid architecture fabricated by stereolithography with a random pore architecture resulting from salt leaching. The scaffold types showed comparable porosity and pore size values, but the gyroid type showed a more than 10-fold higher permeability due to the absence of size-limiting pore interconnections. The higher permeability significantly improved the wetting properties of the hydrophobic scaffolds and increased the settling speed of cells upon static seeding of immortalised mesenchymal stem cells. After dynamic seeding followed by 5 days of static culture gyroid scaffolds showed large cell populations in the centre of the scaffold, while salt-leached scaffolds were covered with a cell sheet on the outside and no cells were found in the scaffold centre. It was shown that interconnectivity of the pores and permeability of the scaffold prolonged the time of static culture before overgrowth of cells at the scaffold periphery occurred. Furthermore, novel scaffold designs are proposed to further improve the transport of oxygen and nutrients throughout the scaffolds and to create tissue engineering grafts with a designed, pre-fabricated vasculature.