Fabrication of nano-hydroxyapatite/collagen/osteonectin composites for bone graft applications.

Mineralized type I collagen (collagen I) nanofibers and their nanofibril bundles make up the microstructure of natural bone tissue, which range from nanometers to micrometers. However, attempts to achieve this hierarchically assembled structure in vitro have been unsuccessful. In this study, we added osteonectin into the collagen I solution, either at a high or low weight ratio (osteonectin: collagen I = 1:30 or 1:90) before co-precipitation. Results indicated that spindle-like nano-hydroxyapatite (nano-HA) was deposited on collagen/osteonectin and pure osteonectin (control) groups. Furthermore, transmission electron microscope (TEM) and scanning electron microscope (SEM) results showed that the assembled mineralized fiber bundles were formed randomly at different levels from 50 nm, 250 nm to 1100 nm. However, when we replaced collagen I with collagen II, osteonectin addition did not induce the formation of mineralized fiber bundles. The participation of osteonectin in the assembly of the mineralized fibers could provide new insights into the novel mineralization function of osteonectin for bone development in vivo and advancing new biomimetic methods for bone graft applications.

Degradation behaviors of electrospun resorbable polyester nanofibers.

Biodegradable materials are widely used in the biomedical field because there is no postoperative surgery after implantation. Widely used synthetic biodegradable materials are polyesters, especially those used in tissue engineering. Advances in the tissue engineering field have brought much attention in terms of scaffold fabrication, such as with biodegradable polyester nanofibers. The rationale for using nanofibers for tissue engineering is that the nonwoven polymeric meshwork is a close representation of the nanoscale protein fiber meshwork in native extracellular matrix (ECM). Electrospinning technique is a promising way to fabricate controllable continuous nanofiber scaffold mimicking the ECM structure. Electrospun nanofibers provide high surface-to-volume ratio and high porosity as a promising scaffold for tissue engineering. Because the degradation behaviors of scaffolds significantly affect new tissue regeneration, the degradation of the material becomes one of the crucial factors when considering using polyester nanofibers as scaffolds in tissue engineering. In this review paper, we focus on the degradation studies of several bioresorbable polyester nanofibrous scaffolds used in tissue engineering. The degradable properties of nanofibers were compared with the corresponding degradable materials in macroscale. The factors that might affect the degradation behaviors were analyzed.

The influence of laminin-derived peptides conjugated to Lys-capped PLLA on neonatal mouse cerebellum C17.2 stem cells.

Chemical guiding cues are being exploited to stimulate neuron adhesion and neurite outgrowth. In this study, an amino-functioned PLLA, lysine-capped PLLA [K-(CH(2))(n)-PLLA (n=2, 5, 8)], was synthesized with different length of linking spaces between lysine molecule and PLLA backbone. Drop-cast films were fabricated from K-(CH(2))(n)-PLLA/PLLA blends (10/90, w/w) and amino groups were detected on the surfaces of the resultant films. More amine groups were detected on the surface and the hydrophilicity of the films was obviously improved by annealing the films in water. The representative atomic force microscopy (AFM) images indicated that incorporation of lysine-capped PLLA into PLLA matrix increased the roughness of the films and resulted in a phase separation with distinct two nano-domains which may correspond to the hydrophilic and hydrophobic domains. Furthermore, the laminin-derived peptides, CYIGSR (Cys-Tyr-Ile-Gly-Ser-Arg) and CSIKVAV (Cys-Ser-Ile-Lys-Val-Ala-Val), were jointly tethered to the amine groups of lysine-capped PLLA by a linking reagent sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC). The neonatal mouse cerebellum C17.2 stem cells were seeded on the peptides-grafted K-(CH(2))(n)-PLLA/PLLA (n=2, 5, 8) films and pure PLLA films were used as controls. Improved viability and longer neurites were obtained on the peptide-grafted films than PLLA film over the cultivation period, especially for K-(CH(2))(5)-PLLA/PLLA, which had the highest peptide density of 0.28+/-0.03 microg/cm(2). This study highlights the potential of using the lysine-capped PLLA with laminin-derived peptides for promoting nerve regeneration.

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.

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.

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).

Systematic fabrication of nano-carbonated hydroxyapatite/collagen composites for biomimetic bone grafts.

A novel biomimetic self-assembly method was designed to create nano-carbonated hydroxyapatite/collagen (nCHAC) composites by means of incorporating various collagen and carbonate concentrations using solutions such as CaCl(2), H(3)PO(4), and Na(2)CO(3). At a given range of collagen and carbonate content, the nanosized inorganic phase of the newly synthesized material has a low degree of crystallinity which resembles that of natural bone. By manipulating the concentrations of collagen and carbonates, various morphologies of the nCHAC can be obtained. The crystal size of nCHAC is dependent on the concentration of carbonate and collagen present in the composites. For instance, higher collagen concentration results in smaller crystal nCHAC crystal size. Conversely, the higher the carbonate content, the smaller are the crystal size and the collagen fibril assembly. As the carbonate content increased, the plate-like crystals first became needle-like structures, subsequently short needle-like crystals and eventually became spherical particles. From this study, our method showcased the flexibility of fabricating various types of nCHAC composites which can be designed for different bone applications.