Susan Liao

The fabrication of nano-hydroxyapatite on PLGA and PLGA/collagen nanofibrous composite scaffolds and their effects in osteoblastic behavior for bone tissue engineering

Bone is a nanocomposite consisting of two main components, nano-hydroxyapatite (n-HA) and Type I collagen (Col). The aim is to exploit the nano-scale functional and material characteristics of natural bone in order to modulate cellular functions for optimal bone repair in bone graft systems. Here, we present an effective and novel technique in obtaining n-HA in cognate with native apatite on electrospun nanofibers within minutes without any pre-treatment. Using an alternate calcium and phosphate (Ca-P) solution dipping method, n-HA was formed on poly(lactide-co-glycolide) acid (PLGA) and blended PLGA/Col nanofibers. The presence of the functional groups of collagen significantly hastened n-HA deposition closed to nine-fold. The quantity of n-HA impinged upon the specific surface area, whereby mineralized PLGA/Col had a greater surface area than non-mineralized PLGA/Col, whereas n-HA did not significantly improve the specific surface area of mineralized PLGA compared to pure PLGA. The novelty of the process was that n-HA on PLGA had a positive modulation on early osteoblast capture (within minutes) compared to pure PLGA. Contrary, cell capture on mineralized PLGA/Col was comparable to pure PLGA/Col. Interestingly, although n-HA impeded proliferation during the culture period (days 1, 4 and 7), the cell functionality such as alkaline phosphatase (ALP) and protein expressions were ameliorated on mineralized nanofibers. The amount of n-HA appeared to have a greater effect on the early stages of osteoblast behavior (cell attachment and proliferation) rather than the immediate/late stages (proliferation and differentiation).

Biomimetic electrospun nanofibers for tissue regeneration

Nanofibers exist widely in human tissue with different patterns. Electrospinning nanotechnology has recently gained a new impetus due to the introduction of the concept of biomimetic nanofibers for tissue regeneration. The advanced electrospinning technique is a promising method to fabricate a controllable continuous nanofiber scaffold similar to the natural extracellular matrix. Thus, the biomedical field has become a significant possible application field of electrospun fibers. Although electrospinning has developed rapidly over the past few years, electrospun nanofibers are still at a premature research stage. Further comprehensive and deep studies on electrospun nanofibers are essential for promoting their biomedical applications. Current electrospun fiber materials include natural polymers, synthetic polymers and inorganic substances. This review briefly describes several typically electrospun nanofiber materials or composites that have great potential for tissue regeneration, and describes their fabrication, advantages, drawbacks and future prospects.

Synergistic effects of electrospun PLLA fiber dimension and pattern on neonatal mouse cerebellum C17.2 stem cells.

Topographical features, including fiber dimensions and pattern, are important aspects in developing fibrous scaffolds for tissue engineering. In this study aligned poly(l-lactide) (PLLA) fibers with diameters of 307+/-47, 500+/-53, 679+/-72 and 917+/-84 nm and random fibers with diameters of 327+/-40, 545+/-54, 746+/-82 and 1150+/-109 nm were obtained by optimizing the electrospinning parameters. We cultured neonatal mouse cerebellum C17.2 cells on the PLLA fibers. These neural stem cells (NSCs) exhibited significantly different growth and differentiation depending upon fiber dimension and pattern. On aligned fibers cell viability and proliferation was best on 500 nm fibers, and reduced on smaller or larger fibers. However, on random fibers cell viability and proliferation was best with the smallest (350 nm) and largest (1150 nm) diameter fibers. Polarized and elongated cells were orientated along the fiber direction on the aligned fibers, with focal contacts bridging the cell body and aligned fibers. Cells of spindle and polygonal morphologies were randomly distributed on the random fibers, with no focal contacts observed. Moreover, longer neurites were obtained on the aligned fibers than random fibers within the same diameter range. Thus, the surface topographic morphologies of fibrous scaffolds, including fiber pattern, dimensions and mesh size, play roles in regulating the viability, proliferation and neurite outgrowth of NSCs. Nevertheless, our results indicated that aligned 500 nm fiber are most promising for fine tuning the design of a nerve scaffold.

Processing nanoengineered scaffolds through electrospinning and mineralization suitable for biomimetic bone tissue engineering.

Processing scaffolds that mimic the extracellular matrix (ECM) of natural bone in structure and chemical composition is a potential promising option for engineering physiologically functional bone tissue. In this article, we report a novel method, by combining electrospinning and mineralization, to process a series of nano-fibrous scaffolding systems with desirable characteristics suitable for biomimetic bone tissue engineering. We have chosen two types of polymers, namely collagen and poly (lactic-co-glycolic acid) (PLGA), natural and synthetic of its kind, respectively, to electrospin into nano-fibrous scaffolds. The electrospun scaffolds have high surface area, high porosity and well connected open pore network. In order to mimic the chemical composition of native bone ECM, the electrospun scaffolds were subjected to mineralization under optimal conditions. From the experimental results, we observed that the formation of bone-like apatite into collagen was relatively abundant and significantly more uniform than PLGA. The major finding of this study has suggested that the surface functional groups of the scaffolding material, such as carboxyl and carbonyl groups of collagen, are important for the mineralization in vitro. In addition, this study revealed that the mineralization process predominantly induce the formation of nanosize carbonated hydroxyapatite (CHA) during collagen mineralization, whilst nanosize hydroxyapatite (HA) is formed during PLGA mineralization. These findings are critically important while selecting the material for processing bone scaffolding system.

Osteoblasts Adherence and Migration through Three-dimensional Porous Mineralized Collagen Based Composite: nHAC/PLA

Osteoblast cells were separated from the neonatal rat calvaria and co-cultured on a novel mineralized hydroxyapatite/collagen/poly(lactic acid) composite scaffold. By using this static cell culture, a three-dimensional osteoblasts/composite bone-like was constructed in vitro. The culture process was observed by scanning electron microscopy, fluorescence microscopy, confocal laser scanning microscopy, and histological analysis. Cells were observed to spread and proliferate throughout the inner-pores of the scaffold material. After a 12-day culture, the cells had grown into the interior scaffold about 200–400 μm depth of the composite by histological section observation. This mobile behavior of osteoblasts appeared to be similar to the composition and hierarchical structure of bone tissue. The adherence and migration of osteoblast cells in this three-dimensional composite is clinically important for large bone defect repair based on tissue engineering.

Fabrication of Mineralized Polymeric Nanofibrous Composites for Bone Graft Materials

Poly-L-lactic acid (PLLA) and PLLA/collagen (50% PLLA+50% collagen; PLLA/Col) nanofibers were fabricated using electrospinning. Mineralization of these nanofibers was processed using a modified alternating soaking method. The structural properties and morphologies of mineralized PLLA and PLLA/Col nanofibers were investigated using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and contact angle measurements. Human bone-derived osteoblasts were cultured on the materials for up to 1 week to assess the biological properties of the nanofibrous composites. Cell attachment on these nanocomposites was also tested within 1 h of culture at room temperature. The mechanical properties of the cell-nanocomposite constructs were determined using tensile testing. From our results, the bone-like nano-hydroxyapatite (n-HA) was successfully deposited on the PLLA and PLLA/Col nanofibers. We observed that the formation of n-HA on PLLA/Col nanofibers was faster and significantly more uniform than on pure PLLA nanofibers. The n-HA significantly improved the hydrophilicity of PLLA/Col nanofibers. From the results of cell attachment studies, n-HA deposition enhanced the cell capture efficacy at the 20-minute time point for PLLA nanofibers. The E-modulus values for PLLA+n-HA with cells (day 1 and day 4) were significantly higher than for PLLA+n-HA without cells. Based on these observations, we have demonstrated that n-HA deposition on nanofibers is a promising strategy for early cell capture.

Degradation of Electrospun Nanofiber Scaffold by Short Wave Length Ultraviolet Radiation Treatment and Its Potential Applications in Tissue Engineering

Development in the field of tissue engineering has brought much attention in the fabrication and preparation of scaffold with biodegradable synthetic polymer nanofibers. Electrospun biodegradable polymeric nanofibers are increasingly being used to fabricate scaffolds for tissue engineering applications as they provide high surface area-to-volume ratio and possess high porosity. One common way to sterilize polymeric nanofiber scaffolds is 254-nm ultraviolet (UV) irradiation. In this study, we aim to evaluate the effects of UV radiation on the degradation in polymeric nanofibers, and then capitalize on UV-induced degradation and UV photolithography in polymeric nanofiber scaffolds for tissue engineering applications. Poly(D,L-lactic-co-glycolic) acid (PLGA, 75:25) and poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL), 70:30] nanofibrous meshes were produced by electrospinning. The nanofibers were irradiated by commercial germicide UV (lambda=254 nm) lamp for different intervals. We found that UV sterilization induced significant degradation of nanofiber. At 1 h UV irradiation, the average molecular weight of PLGA and P(LLA-CL) nanofibers were reduced by 46% and 35%, respectively, with corresponding reduction in the tensile strength of 26% for PLGA and 28% for P(LLA-CL). Hence, precautions may have to be taken into consideration when sterilizing polymeric nanofibers by UV treatment. UV-induced degradation on nanofibers was applied to fabrication of a three-dimensional (3D) tissue engineering scaffold by UV photolithography. Masked exposure to UV could generate patterned holes (d=100 microm) on the nanofibrous mesh. Cell culture study showed that smooth muscle cells were able to migrate into the holes. This method can be used to fabricate a 3D nanofibrous scaffold with micropores.

Biomimetic nanocomposites for bone graft applications.

Allograft bone, dematerialized bone matrix and calcium-based synthetic materials have long been used as bone graft substitutes. First-generation bone graft substitutes as stand-alone graft substitutes have not developed as hoped. It remains a great challenge to design an ideal bone graft that emulates natures own structures or functions. To further improve the performance of such bone graft substitutes, scientists are investigating biomimetic processes to incorporate the desirable nano-features into the next generation of biomaterials. In this regard, nanostructured biomaterials less than 100 nm in at least one dimension, in particular nanocomposites, are perceived to be beneficial and potentially ideal for bone applications, owing to their nanoscale functional characteristics that facilitate bone cell growth and subsequent tissue formation. In fact, bone itself is a nanocomposite system with a complex hierarchical structure. This review reports the impact of biomimetically derived nanocomposite biomaterials for use in bone applications and provides possible suggestions for future research and development.

Effects of nanotopography on stem cell phenotypes

Stem cells are unspecialized cells that can self renew indefinitely and differentiate into several somatic cells given the correct environmental cues. In the stem cell niche, stem cell-extracellular matrix (ECM) interactions are crucial for different cellular functions, such as adhesion, proliferation, and differentiation. Recently, in addition to chemical surface modifications, the importance of nanometric scale surface topography and roughness of biomaterials has increasingly becoming recognized as a crucial factor for cell survival and host tissue acceptance in synthetic ECMs. This review describes the influence of nanotopography on stem cell phenotypes.

Manufacture of PLGA Multiple-Channel Conduits with Precise Hierarchical Pore Architectures and In Vitro/Vivo Evaluation for Spinal Cord Injury

By the method of injection molding combined with thermally induced phase separation (TIPS), a novel nerve conduit with a plurality of channels and macro-/microporous architecture was fabricated using poly (lactide-co-glycolide) (PLGA, 75:25; Mn=1.22x10(5)). The diameter of the conduits and the number of channels could be regulated by changing the parameters of the mold, and the porosity of the conduit was as high as 95.4%. Meanwhile, the hierarchical pore architecture of the walls could be controlled through varying the solution concentration and the contents of porogen. The degradation study in vitro showed that 7-channel conduit could hold its apparent geometry for about 12 weeks in phosphate buffer solution (PBS) at 37degreesC, and the pH values of the degradation solution were detected in the range 4.1-4.5. The influences of the conduit architecture on the cell attachment, spreading, and proliferation were evaluated by culturing rat mesenchymal stem cells alone or together with Schwann cells in vitro. The implantation of the PLGA conduit in the spinal cord showed that it had good biocompatibility, and no obvious inflammatory response was detected. Therefore, the results implied that these PLGA multiple-channel nerve conduits have the potential use for spinal cord injury.