Highlights of 2015

Surface improvement of implants is essential for achieving a fast osseo-integration. Technically, the creation of a precise pattern on a titanium alloy surface is challenging. Here, the femtosecond laser was chosen as an innovative technology for texturing with accuracy a nano-micro topography. By adjusting the laser parameters, three biomimetic textures were fabricated on the titanium surface: micropits with nano-ripples in the pits, micropits with nano-ripples around the pits, and a texture with only nano-ripples.

Mesenchymal stem cells (MSCs, C3H10T1/2) grown on these surfaces displayed altered morphometric parameters, and modified their focal adhesions in term of number, size, and distribution depending on surface type. These results indicate that the MSCs perceived subtle differences in topography. Dynamic analyses of early cellular events showed a higher speed of spreading on all the textured surfaces as opposed to the polished titanium.

Concerning commitment, all the laser-treated surfaces strongly inhibited the expression of adipogenic-related genes (PPARϒ2, C/EBPα) and up-regulated the expression of osteoblastic-related genes (RUNX2, osteocalcin). Interestingly, the combination of micropits to nano-ripples enhanced their osteogenic potential as seen by a twofold increase in osteocalcin mRNA. Alkaline phosphatase activity was increased on all the textured surfaces, and lipid production was down-regulated.

The functionalization of metallic surfaces by this high-resolution process will help us understand the MSCs' interactions with substrates for the development of textured implants with predictable tissue integrative properties.

Hybrid scaffolds made of xanthan and magnetite nanoparticles (XCA/mag) were prepared by dipping xanthan membranes (XCA) into dispersions of magnetic nanoparticles for different periods of time. The resulting hybrid scaffolds presented magnetization values ranging from 0.25 emu g⁻¹ to 1.80 emu g⁻¹ at 70 kOe and corresponding iron contents ranging from 0.25% to 2.3%, respectively. They were applied as matrices for in vitro embryoid body adhesion and neuronal differentiation of embryonic stem cells; for comparison, neat XCA and commercial plastic plates were also used. Adhesion rates were more pronounced when cells were seeded on XCA/mag than on neat XCA or plastic dishes; however, proliferation levels were independent from those of the scaffold type. Embryonic stem cells showed similar differentiation rates on XCA/mag scaffolds with magnetization of 0.25 and 0.60 emu g⁻¹, but did not survive on scaffolds with 1.80 emu g⁻¹. Differentiation rates, expressed as the number of neurons obtained on the chosen scaffolds, were the largest on neat XCA, which has a high density of negative charge, and were smallest on the commercial plastic dishes. The local magnetic field inherent of magnetite particles present on the surface of XCA/mag facilitates synapse formation, because synaptophysin expression and electrical transmission were increased when compared to the other scaffolds used. We conclude that XCA/mag and XCA hydrogels are scaffolds with distinguishable performance for adhesion and differentiation of ESCs into neurons.

Functionalization of a biomaterial surface with adhesive ligands is an effective way to promote specific cell adhesion. Ideally, biomaterial for applications in biomedical implants should simultaneously promote host cell adhesion and inhibit bacterial adhesion. Currently, little attention has been paid to the design of antimicrobial biomaterial with selective adhesiveness towards only targeted cells or tissues. In this study, the role of two typical adhesive ligands on the bioadhesion functions of a model antimicrobial film was elucidated. First, an adhesive ligand including an RGD peptide or collagen (CL) was chemically coupled to an antimicrobial polymeric multilayer composed of dextran sulfate (DS) and chitosan (CS). It was demonstrated that the density of RGD and CL immobilized on the DS/CS multilayer ranges between 4 to 137 ng cm⁻² and 100 to 1000 ng cm⁻², respectively. Then the effect of immobilized RGD or CL on both bacterial and fibroblast adhesion was investigated. By determining the density and morphology of adherent fibroblast on a DS/CS multilayer with or without an adhesive ligand, it was shown that RGD or CL effectively promoted fibroblast adhesion and proliferation in a concentration-dependent manner. Interestingly, the type of adhesive ligands imposed distinct effects in bacterial adhesion. Immobilized RGD did not enhance Staphylococcus aureus and Escherichia coli adhesion on DS/CS multilayers under all concentrations. In contrast, CL triggered significant S. aureus adhesion on DS/CS multilayers even at low surface concentration and when fibroblast adhesion was absent. Moreover, the detachment forces of individual S. aureus on CL coated DS/CS multilayers probed by atomic force microscopy (AFM) was 3 times and 20 times higher than that on the control substrate and on unmodified DS/CS multilayers, respectively. Interestingly, the lowest detachment force of E. coli was found on the CL coated DS/CS multilayers. This study demonstrated the possibility of engineering an antimicrobial multilayer coating with tailored adhesive properties towards specific cell types for potential applications in biomedical implants.

Bone scaffolds for regenerative medicine applications should have the ability to promote adhesion, proliferation and differentiation of osteoblast cells. Osteoconductive, osteoinductive and osteopromotive properties of the material are essential for rapid bone regeneration and new bone formation. In this study, the osteogenic potential of two novel tri-component scaffolds composed of krill chitosan, bacterial β-1,3-glucan and bioceramics (HAp or a mix of HAp/β-TCP granules) was investigated. The typical markers of the first (type I collagen), second (bone alkaline phosphatase) and third stages (osteocalcin) of the osteoblast differentiation process were evaluated during in vitro experimentation. The study was carried out using three various osteoblastic cell lines (normal human fetal osteoblast cells hFOB 1.19, human osteoblast-like cells derived from osteosarcoma Saos-2 and mouse calvarial preosteoblast cells MC3T3-E1 Subclone 4). The bone alkaline phosphatase (bALP) and osteocalcin (OC) were determined quantitatively using enzyme-linked immunosorbent assays, and type I collagen (Col I) was evaluated qualitatively using the direct immunofluorescence (DIF) method. The data obtained clearly prove that novel scaffolds have the ability to increase bALP activity, to enhance extracellular matrix synthesis (Col I and OC) and to induce mineralized nodule formation during osteogenic differentiation. In conclusion, novel tri-component materials have osteoconductive and osteopromotive properties, and thus are promising materials in bone tissue engineering applications to accelerate the bone regeneration process.

Elastic fibers are essential for the proper function of organs including cardiovascular tissues such as heart valves and blood vessels. Although (tropo)elastin production in a tissue-engineered construct has previously been described, the assembly to functional elastic fibers in vitro using human cells has been highly challenging. In the present study, we seeded primary isolated human vascular smooth muscle cells (VSMCs) onto 3D electrospun scaffolds and exposed them to defined laminar shear stress using a customized bioreactor system. Increased elastin expression followed by elastin deposition onto the electrospun scaffolds, as well as on newly formed fibers, was observed after six days. Most interestingly, we identified the successful deposition of elastogenesis-associated proteins, including fibrillin-1 and -2, fibulin-4 and -5, fibronectin, elastin microfibril interface located protein 1 (EMILIN-1) and lysyl oxidase (LOX) within our engineered constructs. Ultrastructural analyses revealed a developing extracellular matrix (ECM) similar to native human fetal tissue, which is composed of collagens, microfibrils and elastin. To conclude, the combination of a novel dynamic flow bioreactor and an electrospun hybrid polymer scaffold allowed the production and assembly of an elastic fiber-containing ECM.

Central nervous system neurons in adult mammals display limited regeneration after injury, and functional recovery is poor following complete transection (>4 mm gap) of a rat spinal cord. A novel combination scaffold composed of 3D nanofibrous hydrogel PuraMatrix and a honeycomb collagen sponge was used to promote spinal repair and locomotor functional recovery following complete transection of the spinal cord in rats. We transplanted this scaffold into 5 mm spinal cord gaps and assessed spinal repair and functional recovery using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. The BBB score of the scaffold-transplanted group was significantly higher than that of the PBS-injected control group from 24 d to 4 months after the operation (P < 0.001–0.01), reaching 6.0  ±  0.75 (mean ± SEM) in the transplant and 0.70  ±  0.46 in the control groups. Neuronal regeneration and spinal repair were examined histologically using Pan Neuronal Marker, glial fibrillary acidic protein, growth-associated protein 43, and DAPI. The scaffolds were well integrated into the spinal cords, filling the 5 mm gaps with higher numbers of regenerated and migrated neurons, astrocytes, and other cells than in the control group. Mature and immature neurons and astrocytes in the scaffolds became colocalized and aligned longitudinally over >2 mm, suggesting their differentiation, maturation, and function. The spinal cord NF200 content of the transplant group, analyzed by western blot, was more than twice that of the control group, supporting the histological results. Transplantation of this novel scaffold promoted functional recovery, spinal repair, and neuronal regeneration.

We report on the design and fabrication of a frame-supported nanofibrous membrane for the transplantation of retinal pigment epithelial (RPE) cells, which is a promising therapeutic option for the treatment of degenerative retinal disorders. The membranous cell carrier prepared from 640 nm-thick poly(DL-lactide) fibres uniquely combines high porosity, large pore size and low thickness, to maximize the nutrient supply to the transplanted cells in the subretinal space and thus to enhance the therapeutic effect of the transplantation. The carrier was prepared by electrospinning, which made it easy to embed a 95 μm-thick circular supporting frame 2 mm in diameter. Implantations into enucleated porcine eyes showed that the frame enabled the ultrathin membrane to be handled without irreversible folding, and allowed the membrane to regain its flat shape when inserted into the subretinal space. We further demonstrated that the minimum membrane thickness compatible with the surgical procedure and instrumentation employed here was as low as 4 μm. Primary porcine RPE cells cultivated on the membranes formed a confluent monolayer, expressed RPE-specific differentiation markers and showed transepithelial resistance close to that of the native RPE. Most importantly, the majority of the RPE cells transplanted into the subretinal space remained viable. The ultrathin, highly porous, and surgically convenient cell carrier presented here has the potential to improve the integration and the functionality of transplanted RPE cells.

Corneal transplantation has become a common procedure to improve visual acuity by replacing the opaque or distorted host tissue with clear healthy donor corneal tissue. However, globally its wide spread clinical utility is limited due to a lack of supply of high quality corneas. Bioengineered neo-corneas using discarded human corneas to isolate corneal endothelial and epithelial cells, as well as corneal stroma as a scaffolding material, could help address this shortage. The objective of this study was to fabricate multilayered corneal equivalents that could be suitable for full thickness cornea transplantation. To achieve this goal human corneal endothelial cells (hCEC) and human limbal epithelial cells (hLEC) were isolated from discarded human corneas and expanded in vitro, maintaining their phenotype for at least 3 passages. We used our previously described process of human cornea decellularization to create corneal scaffolds that preserve the native extracellular matrix of the corneal stroma. The corneal scaffolds were seeded with hCEC and hLEC, using a special apparatus that enabled seeding both sides of the scaffold. The cell-seeded corneal constructs supported hCEC and hLEC growth and multi-cellular organization for 2 weeks in vitro. Immunohistochemical analysis showed expression of typical hCEC and hLEC markers on their corresponding sides. Importantly, the cell-seeded corneal constructs were more transparent than non-seeded corneal scaffolds. Taken together, this study demonstrates the feasibility of creating multilayered cornea equivalents, exclusively from human donor-derived materials. These constructs may be suitable for corneal transplantation, and as a short-term application, may serve for ophthalmological drug testing purposes.

Degradation behavior is very important in the field of silk-based biomaterials. Mulberry and nonmulberry silk fibroins are structurally and functionally distinguishable; however, no studies have examined the differences in the degradation behaviors of silk materials from various silkworm species. In this study, Ca(NO₃)₂ was used as a uniform solvent to obtain regenerated mulberry and nonmulberry (Antheraea pernyi and Antheraea yamamai) silk fibroin (SF) solutions, and the degradation behaviors of various SF scaffolds were examined. In vitro and in vivo results demonstrated that regenerated mulberry SF scaffolds exhibited significantly higher mass loss and free amino acid content release than did nonmulberry SF scaffolds. The differences in the primary structures and condensed structures between mulberry and nonmulberry SF contributed to the significant difference in degradation rates, in which the characteristic (–Ala–)_n repeats, compact crystal structure and high α-helix and β-sheet contents make nonmulberry SF more resistant than mulberry SF to enzymatic degradation. Moreover, the Antheraea pernyi and Antheraea yamamai SFs possess similar primary structures and condensed structures, although a slight difference in degradation was observed; this difference might depend on the differences in molecular weight following the regeneration process. The results indicate that the original sources of SF significantly influence the degradation rates of SF-based materials; therefore, the original sources of SF should be fully considered for preparing tissue engineering scaffolds with matched degradation rates.

Gelatin hydrogels have been designed and prepared for the controlled release of the transforming growth factor (TGF-b1) and the platelet-derived growth factor (PDGF-BB). PRP (Platelet rich plasma) contains many growth factors including the PDGF and TGF-b1. The objective of this study was to evaluate the regeneration of periodontal tissue following the controlled release of growth factors in PRP. For the periodontal ligament cells and osteoblast, PRP of different concentrations was added. The assessment of DNA, mitochondrial activity and ALP activity were measured. To evaluate the TGF-β1 release from PRP incorporated gelatin sponge, amounts of TGF-β1 in each supernatant sample were determined by the ELISA. Transplantation experiments to prepare a bone defect in a rat alveolar bone were an implanted gelatin sponge incorporated with different concentration PRP. In DNA assay and MTT assay, after the addition of PRP to the periodontal ligament cells and osteoblast, the cell count and mitochondrial activity had increased the most in the group with the addition of 5  ×  PRP. In the ALP assay, after the addition of PRP to the periodontal ligament cells, the cell activity had increased the most in the group with the addition of 3  ×  PRP. In the transplantation, the size of the bone regenerated in the defect with 3  ×  PRP incorporated gelatin sponge was larger than that of the other group.

The goal of this study was to evaluate the effects of urethral reconstruction with a three-dimensional (3D) porous bacterial cellulose (BC) scaffold seeded with lingual keratinocytes in a rabbit model. A novel 3D porous BC scaffold was prepared by gelatin sponge interfering in the BC fermentation process. Rabbit lingual keratinocytes were isolated, expanded, and seeded onto 3D porous BC. BC alone (group 1, N  =  10), 3D porous BC alone (group 2, N  =  10), and 3D porous BC seeded with lingual keratinocytes (group 3, N  =  10) were used to repair rabbit ventral urethral defects (2.0   ×   0.8 cm). Scanning electron microscopy revealed that BC consisted of a compact laminate while 3D porous BC was composed of a porous sheet buttressed by a dense outer layer. The average pore diameter and porosity of the 3D porous BC were 4.23   ±   1.14 μm and 67.00   ±   6.80%, respectively. At 3 months postoperatively, macroscopic examinations and retrograde urethrograms of urethras revealed that all urethras maintained wide calibers in group 3. Strictures were found in all rabbits in groups 1 and 2. Histologically, at 1 month postoperatively, intact epithelium occurred in group 3, and discontinued epithelium was found in groups 1 and 2. However, groups 2 and 3 exhibited similar epithelial regeneration, which was superior to that of group 1 at 3 months (p  <  0.05). Comparisons of smooth muscle content and endothelia density among the three groups revealed a significant increase at each time point (p  <  0.05). Our results demonstrated that 3D porous BC seeded with lingual keratinocytes enhanced urethral tissue regeneration. 3D porous BC could potentially be used as an optimized scaffold for urethral reconstruction.