Study On Porous Nano-hydroxyapatite/polyamide66 Composite Scaffolds For Bone Tissue Engineering

The development of bone tissue engineering brings forward a promisingalternative for the clinic treatment on bone defects, and attracts gradually increasinginterest from the scientific researchers. In bone tissue engineering, scaffold served asthe matrices of tissue formation plays a pivotal role, and has to fulfill a few basicrequirements, that is, high porosity and proper pore size, required surface propertiespermitting cell adhesion, differentiation and proliferation, desirable mechanicalintegrity to maintain the predesigned tissue structure, non-cytotoxicity andosteoconductivity.The selection of the most appreciate material to produce a scaffold to be used inbone tissue engineering application is a critically important step towards theconstruction of a tissue engineered product. Hydroxyapatite (HA) has beenconsidered to be the ideal material to build bone tissue engineering scaffold due toits osteoconductivity and osteoinductivity. But its brittleness and poor performanceof mechanical stability limit its use for the regeneration of load-bearing bone defects.On the other hand, biocompatible polymers have also been regarded as the propercandidates for tissue-engineered scaffolds. However, a number of tough practicalproblems still persist, that is, difficulty in controlling the in vivo degradation ofbioresorbable polymers, low efficiency of cell seeding, cytotoxicity of thebreakdown products produced during scaffold degradation, and more commonlyhappened, poor mechanical properties incomparable with natural hard tissues.To overcome these shortages presented by bioceramics and biopolymers,ceramic/polymer composite scaffolds, and prepare three-dimensionally porous structure with adequate mechanical properties, we fabricated a novel compositetissue-engineered scaffold using n-HA/PA66 composite biomaterials which hasalready been proven to possess both good bioactivity and comparative mechanicalstrength to that of cortical bone. In this study, we employed phase inversion andphase inversion in combination with particle leaching processing methods to prepareporous scaffold. Then the morphologies and properties of the scaffolds werecharacterized by scanning electronic microscope (SEM), X-ray diffraction (XRD),infrared spectroscopy (IR) and mechanical testing. The results indicated thatn-HA/PA66 composite scaffold possessed ideal pore size (100～500μm) andinterconnectivity suitable for new bone ingrowth, and the porosity can be controlledby changing the parameters of the processing method. Moreover, the scaffoldexhibited good mechanical strength that was similar to the cancellous bone(2～10MPa). Because of its non-biodegradability, this composite scaffold is able toprovide a long-term stable support for the regeneration of new bone.On the other hand, the other important aspect of bone tissue engineering is theintroduction of bioactive cells into the three-dimensionally porous scaffold.Mesenchymal stem cells (MSCs) are present in many human tissues and can bedirectly derived from marrow, easily isolated, easily cultured and rapidlyproliferated in the laboratory setting. More importantly, MSCs serve as a readilyavailable source of undifferentiated cells that are capable to give rise to diversetissues, including bone, cartilage, tendon, muscle and so on. The advantages ofMSCs encourage us to introduce these cells into n-HA/PA composite scaffolds forbone tissue engineering application. Therefore, in this study, we investigate thebiocompatibility and cytotoxicity of the n-HA/PA composite scaffolds in directcontact with MSCs by in vitro tests, and study the in vivo biocompatibility andosteogenesis of the MSCs hybridized n-HA/PA scaffold in the animal experiments.The MSCs/scaffold constructs were cultured for up to 7 days and the adhesion,proliferation and differentiation of MSCs into osteoblastic phenotype weredetermined using MTT assay, alkaline phosphatase (ALP) activity and collagen typeⅠ(COLⅠ) immunohistochemical staining and scanning electronic microscopy (SEM). The results confirm that n-HA/PA scaffolds are biocompatible and have no negativeeffects on tile MSCs in vitro. To investigate the in vivo biocompatibility andosteogenesis of the composite scaffolds, both pure n-HA/PA scaffolds andMSCs/scaffold constructs were implanted in rabbit mandibles and studiedhistologically and microradiographically. The results show that n-HA/PA compositescaffolds exhibit good biocompatibility and extensive osteoconductivity with hostbone. Moreover, the introduction of MSCs to the scaffolds dramatically enhancesthe efficiency of new bone formation, especially at the initial stage afterimplantation. In long term (more than 12 weeks implantation), however, the purescaffolds show as good biocompatibility and osteogenesis as the hybrid ones.Nowadays, another common problem encountered when using such scaffolds fortissue engineering is the rapid formation of tissue on the outer edge, which leads tothe development of a necrotic core due to the limitations of cell penetration andnutrient exchange. Most tissue-engineered scaffolds with irregular and isotropicporous architecture normally possess relatively low interconnectivity, which makesit difficult for the cells and newly formed tissue to penetrate into the center ofscaffold and influences bone reconstruction. To solve this problem and prepare ascaffold with similar anisotropic property in both structure and physical propertieslike bone, which improve the ingrowth of cells and tissues into the interior part ofthe scaffold and enhance the nutrient exchange, we design and prepare n-HA/PA66scaffold with anisotropic structure and mechanical strength using a modified phaseinversion processing method. Then the processing method, physical and chemicalproperties, macro-morphology and mechanical property of the anisotropic scaffoldwere investigated, and the biocompatibility was evaluated using in vitro cells culture.Consequently, this n-HA/PA66 scaffold presents anisotropy in both morphology andmechanical activity, that is, the compressive strength of the scaffold in the directionparallel to the pore direction is much larger than that perpendicular to the poredirection. Additionally, the result of cells culture indicates that both MSCs andosteoblasts have the tendency to grow, migrate and proliferate along the orientationparallel to the pore direction. After all, these results indicate that n-HA/PA66 composite scaffolds fulfill thebasic requirements of bone tissue engineering scaffold, and have the potential to beapplied in orthopedic, reconstructive and maxillofacial surgery.