Yanfei Tan

Synthesis and evaluation of collagen–chitosan–hydroxyapatite nanocomposites for bone grafting

Incorporation of hydroxyapatite (HA) into the matrix of collagen (Col) and chitosan (Chi) by in situ synthesis was introduced to prepare nanocomposites. Structural investigations of the pure Col-Chi mixture validated the influence of Chi on Col assembly, but the molecular interactions between Col and Chi was partially depressed during the intervention of in situ HA synthesis, as revealed by FTIR and DSC analyses. A series of Col-Chi-HA (CCHA) nanocomposites with varying HA content were thereby prepared by a sequential method, involving in situ synthesis in the Col-Chi system, then gelling at 25 degrees C and subsequently washing the resultant elastic gel followed by dehydration consolidation. The structural characteristics and biological properties of the dehydrated CCHA nanocomposites were further evaluated by using XRD, FTIR, TG, and SEM analyses and the osteoblast culture experiment. Formation of a well integrated microstructure of organic fibers (ca. 90 nm in size) and dense matrix including inorganic aggregates (less than 30 nm in size) was found in these nanocomposites. Rat Ros 17/2.8 Osteoblasts proliferated and attached well on the surface of both CCHA nanocomposite and Col-Chi mixture. These results indicated that in situ HA synthesis in the Col-Chi system provided a feasible route for bone grafting nanocomposites.

Enhanced effect of β-tricalcium phosphate phase on neovascularization of porous calcium phosphate ceramics: In vitro and in vivo evidence

Neovascularization plays a key role in bone repair and regeneration. In the present study, four types of porous calcium phosphate (CaP) ceramics, namely hydroxyapatite (HA), biphasic calcium phosphates (BCP-1 and BCP-2) and β-tricalcium phosphate (β-TCP), with HA to β-TCP ratios of 100/0, 70/30, 30/70 and 2/98, respectively, were investigated in terms of their angiogenic induction. The in vitro cell culture revealed that the ceramics could promote proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs). This result could be achieved by stimulating CCD-18Co human fibroblasts to secrete angiogenic factors (vascular endothelial growth factor, basic fibroblast growth factor and transforming growth factor-β) as a paracrine effect, as well as by up-regulating HUVECs to express these angiogenic factors and their receptors (KDR, FGFR1 and ACVRL1) and the downstream eNOS as an autocrine effect. These effects were more significant in β-TCP and BCP-2, which had a higher content of β-TCP phase. In the in vivo implantation into the thigh muscles of mice, the process of neovascularization of the ceramics was initiated at 2 weeks and the mature vascular networks were formed at 4 weeks as visualized by hematoxylin and eosin staining and scanning electron microscopy. Microvessel density count confirmed that β-TCP and BCP-2 induced more microvessels to form than HA or BCP-1. This phenomenon was further confirmed by the significantly up-regulated expressions of angiogenesis-related genes in the ingrowth of cells into the inner pores of the two ceramics. All the results confirmed the angiogenic induction of porous CaP ceramics, and a higher content of β-TCP phase had an enhanced effect on the neovascularization of the ceramics.

Effect of phase composition on protein adsorption and osteoinduction of porous calcium phosphate ceramics in mice

The purpose of this study was to investigate the effect of phase compositions of porous calcium phosphate (CaP) ceramics on their protein adsorption behaviors in vitro and osteoinductive potentials in vivo in mice. Under competitive conditions, a high adsorption of bone morphogenetic protein 2 (BMP-2) was observed at a high initial concentration of BMP-2 in the multi-protein solution on all the four types of ceramics, indicating their strong affinity for BMP-2. No significant difference in BMP-2 adsorption between the ceramics was noted, indicating that phase composition could have little influence on BMP-2 adsorption. After implantation into the thigh muscles of mice for 45 and 90 days, the histological and histomorphometric analyses showed that porous biphasic calcium phosphate (BCP) ceramic consisting of 30% hydroxyapatite HA and 70% tricalcium phosphate (β-TCP), i.e. BCP-2 had stronger osteoinductive ability than the other three groups of ceramics. The immunohistochemical staining showed the highest expression of BMP-2 and osteocalcin (OCN) in BCP-2 group. Osteoinduction of porous CaP ceramics might be influenced by the amount of BMP-2 present in the local microenvironment in the implant, which was regulated by the phase composition of the ceramics. BCP-2 promoted the highest expression of BMP-2 and then showed the strongest osteoinduction in mice.

Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics

Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway.

Biomimetic fabrication of a three-level hierarchical calcium phosphate/collagen/hydroxyapatite scaffold for bone tissue engineering

A three-level hierarchical calcium phosphate/collagen/hydroxyapatite (CaP/Col/HAp) scaffold for bone tissue engineering was developed using biomimetic synthesis. Porous CaP ceramics were first prepared as substrate materials to mimic the porous bone structure. A second-level Col network was then composited into porous CaP ceramics by vacuum infusion. Finally, a third-level HAp layer was achieved by biomimetic mineralization. The three-level hierarchical biomimetic scaffold was characterized using scanning electron microscopy, energy-dispersive x-ray spectra, x-ray diffraction and Fourier transform infrared spectroscopy, and the mechanical properties of the scaffold were evaluated using dynamic mechanical analysis. The results show that this scaffold exhibits a similar structure and composition to natural bone tissues. Furthermore, this three-level hierarchical biomimetic scaffold showed enhanced mechanical strength compared with pure porous CaP scaffolds. The biocompatibility and osteoinductivity of the biomimetic scaffolds were evaluated using in vitro and in vivo tests. Cell culture results indicated the good biocompatibility of this biomimetic scaffold. Faster and increased bone formation was observed in these scaffolds following a six-month implantation in the dorsal muscles of rabbits, indicating that this biomimetic scaffold exhibits better osteoinductivity than common CaP scaffolds.

Comparison of ectopic bone formation process induced by four calcium phosphate ceramics in mice

Phase composition played a key role in the biodegradation of calcium phosphate ceramics (CaP), which in turn influences the osteoinductive ability. The in vivo biological mechanism is still poorly understood. In this study, four types of porous CaP ceramics were investigated, namely, hydroxyapatite (HA), β-tricalcium phosphate (TCP), and biphasic calcium phosphates BCP1 and BCP2, with HA to β-TCP ratios of 70/30 and 30/70, respectively. The four types of ceramics were implanted into thigh muscle of mice for 16weeks. Longitudinal ectopic bone formation process at gene, protein, and tissue level induced by the material was assessed. Histological analysis revealed that BCP2 was the only group that had promoted new bone formation after 16weeks. In micro-CT analysis of biodegradation, the BCP2 group had the least increment of porosity due to the new bone formation, resulting in a significant elevation in material density. Instead of a steady increase, multiple peaks were observed in most of the temporal gene expression patterns. The gene expression results were further confirmed by immunohistochemical staining of the corresponding proteins. Among the target genes, Osterix and type I collagen were activated successively. The osteoinductive BCP2 group showed earlier and significantly higher peaks in BMP2, BMPR1A, and OPG expressions than non-bone forming groups. These findings revealed that the occurrence time and magnitude of these osteogenetic gene expression peaks can be crucial in the osteoinduction process.

The material and biological characteristics of osteoinductive calcium phosphate ceramics

Abstract The discovery of osteoinductivity of calcium phosphate (Ca-P) ceramics has set an enduring paradigm of conferring biological regenerative activity to materials with carefully designed structural characteristics. The unique phase composition and porous structural features of osteoinductive Ca-P ceramics allow it to interact with signaling molecules and extracellular matrices in the host system, creating a local environment conducive to new bone formation. Mounting evidence now indicate that the osteoinductive activity of Ca-P ceramics is linked to their physicochemical and three-dimensional structural properties. Inspired by this conceptual breakthrough, many laboratories have shown that other materials can be also enticed to join the rank of tissue-inducing biomaterials, and besides the bones, other tissues such as cartilage, nerves and blood vessels were also regenerated with the assistance of biomaterials. Here, we give a brief historical recount about the discovery of the osteoinductivity of Ca-P ceramics, summarize the underlying material factors and biological characteristics, and discuss the mechanism of osteoinduction concerning protein adsorption, and the interaction with different types of cells, and the involvement of the vascular and immune systems.

DOX-encapsulated intelligent PAA-g-PEG/PEG–Fa polymeric micelles for intensifying antitumor therapeutic effect via active-targeted tumor accumulation

Stimuli-responsive targeted polymeric micelles as drug delivery systems have recently attracted significant attention for the treatment of various cancers, which could improve delivery efficiency by tumor-specific recognition via active targeting strategies. In this research, DOX-incorporated bioreducible polymeric micelles based on PAA-g-PEG/PEG-Fa conjugated polymers were prepared and characterized as nanocarriers for promoting intracellular anti-tumor drug delivery efficiency via folate receptor-mediated endocytosis. The anti-proliferative activity on cancer cells, biodistribution, active-/passive-targeting efficiency, tumor growth inhibition efficiency, and biological toxicity were evaluated in vitro and in vivo. The MTT assay demonstrated that the DOX-encapsulated active-targeting PAA-g-PEG/PEG-Fa micelles had much greater growth inhibition effect against 4T1 and KB than passive-targeting micelles. These active-targeting micelles showed superior tumor accumulation and excellent tumor growth inhibition effect as revealed by the fluorescence optical imaging technique and tumor volume change investigation, as well as the survival study of the tumor-bearing Balb/c mice. Furthermore, the active targeting micelles greatly decreased the toxicity of DOX on the heart and other organs. These potential results encouraged us to further optimize the molecular structure to achieve more excellent targeted therapeutic effect.

In Vivo Evaluation of Nano-HA/PDLLA Composite

The purpose of this study was to evaluate the behavior of nano-hydroxyapatite/ poly(D,L)lactide (n-HA/PDLLA) composite in vivo. The composite rods containing about 40wt% n-HA and control HA rods with a diameter of 2mm and a length of 6mm were implanted into the femora of 16 New Zealand rabbits. Composite wafers with a diameter of 5mm and a thickness of 1mm were implanted into the dorsal subcutis of 18 Wistar Albino rats. After definite intervals, the histological analysis was completed by light microscopy and the degradation behavior was observed by scanning electron microscopy. The histological analysis showed no obvious difference between n-HA /PDLLA composite and pure HA that had good biocompatibility and osteoconductivity. SEM analysis of the surface and cross section of the samples showed that the degradation of the composite started from surface, then into the inner gradually and formed multiple pores at surface. The pore size and porosity gradually increased along with time and a porous network may be formed.

Methacrylamide-modified Collagen Hydrogel with Improved Anti-actin-mediated Matrix Contraction Behavior

For an ideal biomimetic microenvironment to realize reliable cartilage regeneration, the ability to induce mesenchymal stem cell (MSCs) differentiation along the chondrogenic lineage and prevent further dedifferentiation is expected. With native bioactivity, collagen has been proved to be preferential for inducing the chondrogenic differentiation of MSCs. However, the phenotypic maintenance of differentiated chondrocytes in a collagen matrix is still a challenge. Actin traction, which causes drastic contraction of the collagen matrix, is frequently observed and might be an important factor that affects cell fates including chondrogenic differentiation and phenotypic maintenance. In this study, photochemical modification was applied to acquire collagen hydrogels with improved mechanical strength and creep behavior. Accompanied by inherited bioactivity, the photo-crosslinked collagen hydrogel well supported the actin cytoskeleton functionalization while resisting the actin-mediated matrix contraction. Benefitting from this, the hydrogel system promoted MSCs proliferation and chondrogenic differentiation, and more importantly, prevented further dedifferentiation. By exploring the mesenchymal development-related signal transduction markers, it was revealed that the promoted chondrogenesis was achieved through inhibiting the over-expression of MAPK and Wnt/β-catenin signaling pathways that up-regulated dedifferentiated gene expression. The strategy of applying the hydrogel system to cartilage regeneration is foreseeable based on the positive heterotopic and orthotopic chondrogenic differentiation.