Yifan Ma

β-Tricalcium phosphate/poly(glycerol sebacate) scaffolds with robust mechanical property for bone tissue engineering.

Despite good biocompatibility and osteoconductivity, porous β-TCP scaffolds still lack the structural stability and mechanical robustness, which greatly limit their application in the field of bone regeneration. The hybridization of β-TCP with conventional synthetic biodegradable PLA and PCL only produced a limited toughening effect due to the plasticity of the polymers in nature. In this study, a β-TCP/poly(glycerol sebacate) scaffold (β-TCP/PGS) with well interconnected porous structure and robust mechanical property was prepared. Porous β-TCP scaffold was first prepared with polyurethane sponge as template and then impregnated into PGS pre-polymer solution with moderate viscosity, followed by in situ heat crosslinking and freezing-drying process. The results indicated that the freezing-drying under vacuum process could further facilitate crosslinking of PGS and formation of Ca(2+)-COO(-) ionic complexing and thus synergistically improved the mechanical strength of the β-TCP/PGS with in situ heat crosslinking. Particularly, the β-TCP/PGS with 15% PGS content after heat crosslinking at 130°C and freezing-drying at -50°C under vacuum exhibited an elongation at break of 375±25% and a compressive strength of 1.73MPa, 3.7-fold and 200-fold enhancement compared to the β-TCP, respectively. After the abrupt drop of compressive load, the β-TCP/PGS scaffolds exhibited a full recovery of their original shape. More importantly, the PGS polymer in the β-TCP/PGS scaffolds could direct the biomineralization of Ca/P from particulate shape into a nanofiber-interweaved structure. Furthermore, the β-TCP/PGS scaffolds allowed for cell penetration and proliferation, indicating a good cytobiocompatibility. It is believed that β-TCP/PGS scaffolds have great potential application in rigid tissue regeneration.

Strontium attenuates rhBMP-2-induced osteogenic differentiation via formation of Sr-rhBMP-2 complex and suppression of Smad-dependent signaling pathway

UNLABELLED Strontium (Sr(2+)) has pronounced effects on stimulating bone formation and inhibiting bone resorption in bone regeneration. In this current study, the effect and the underlying mechanism involved of Sr(2+) on the biological activity of bone morphogenetic protein-2 (BMP-2) were studied in detail with pluripotent skeletal muscle myogenic progenitor C2C12 model cell line. The results indicated that Sr(2+) could bind recombinant human BMP-2 (rhBMP-2) rapidly, even in the presence of Ca(2+) and Mg(2+), and inhibited rhBMP-2-induced osteogenic differentiation in vitro and osteogenetic efficiency in vivo. Further studies demonstrated that Sr(2+) treatment undermined the binding capacity of rhBMP-2 with its receptor BMPRIA and thus attenuated Smad 1/5/8 phosphorylation without affecting their dephosphorylation in C2C12 cells. Furthermore, circular dichroism spectroscopy, fluorescence spectroscopy and X-ray photoelectron spectroscopy all revealed that the inhibitory effect of Sr(2+) on the rhBMP-2 osteogenic activity was associated with the formation of Sr-rhBMP-2 complex and ensuing enhancement of β-sheet structure. Our work suggests the activity of rhBMP-2 to induce osteogenic differentiation was decreased by directly interaction with free Sr ions in solution, which should provide guide and assist for development of BMP-2-based materials for bone regeneration. STATEMENT OF SIGNIFICANCE Due to easy denaturation and ensuing the reduced activity of rhBMP-2, preserving/enhancing the capacity of rhBMP-2 to induce osteogenic differentiation is of critical importance in developing the protein-based therapy. Cations as effective elements influence the conformation and thereby the bioactivity of protein. Strontium (Sr(2+)), stimulating bone formation and inhibiting bone resorption, has been incorporated into biomaterials/scaffold to improve the bioactivity for bone-regeneration applications. However, Sr(2+)-induced changes in the conformation and bioactivity of BMP-2 have never been investigated. In this study, the formation of Sr-rhBMP-2 complex inhibited the osteogenic differentiation in vitro and osteogenetic efficiency in vivo through the inhibition of BMP/Smad signaling pathway, providing guidance for development of Sr-containing BMP-2-based bone scaffold/matrice and other Sr-dopped protein therapy.

Poly(glycerol sebacate)-modified polylactic acid scaffolds with improved hydrophilicity, mechanical strength and bioactivity for bone tissue regeneration

Polylactic acid (PLA) has been extensively researched in biomedical engineering applications due to its superior mechanical strength and biocompatibility in vivo. But the inherent brittleness, slow degradability and inferior hydrophilicity greatly hamper its successful application. Here, a biodegradable crosslinked elastomer poly(glycerol sebacate) (PGS) was adapted to modify PLA scaffold for bone tissue engineering in this study. A highly interconnected and large porous, three-dimensional (3D) PLA-based scaffold was prepared by a NaCl particulate-leaching method and the PGS prepolymer (pre-PGS) was introduced either by pre-molding binary blend (B.B) or by surface coating (S.C) of a homogeneous PGS onto PLA-based scaffolds with and without oxygen plasma pretreatment (O.P and D.C). After curing at 130 °C, the resulting PLA/PGS scaffolds all exhibited well interconnected open-cell structures. The incorporation of PGS to PLA both by B.B and S.C could effectively improve the hydrophilicity, degradation, toughness and ductility, and the best efficacy was observed for the S.C with the oxygen plasma pretreatment. Specifically, at the ratio of PLA/PGS 9:1 and 7:3, the fracture strain of the PLA/PGS scaffolds by O.P were improved from 8% (pure PLA) to 13% and 24%, respectively. Further studies indicated that enhanced hydrophilicity and increased surface roughness were the main contributors to the above positive effect of oxygen-based plasma treatment. Additionally, these hybrid PLA/PGS scaffolds exhibited good mineralization, high cell biocompatibility, and enhanced cell adhesion and osteogenic differentiation for bone mesenchymal stem cells (BMSCs), especially for scaffolds by S.C. The present results suggest that the surface coating of PGS with oxygen-based plasma pretreatment is an effective strategy to modify the properties of PLA and the hybrid PLA/PGS scaffold represents a promising candidate in the formulation of bone tissue regeneration.

MBG-Modified β-TCP Scaffold Promotes Mesenchymal Stem Cells Adhesion and Osteogenic Differentiation via a FAK/MAPK Signaling Pathway

The β-TCP scaffold has been widely used as a bone graft substitute, but the traditional PMMA molding method-induced undesirable mechanical strength and poor interconnectivity still have not been addressed until now. In this study, a MBG-based PU foam templating method was developed to fabricate β-TCP scaffolds with desirable microtopography. The MBG gel, as both binder and modifier, prepared by a modified sol-gel method with controlled viscosity is incorporated with β-TCP powder and thereafter is impregnated into PU foam. The resultant hybrid scaffolds exhibited interconnected macropores (200-500 μm) and distinctive micropores (0.2-1.5 μm), especially for the TCP/25MBG (with 25 wt % content MBG). As expected, the compression strength of β-TCP/MBG composite scaffolds was enhanced with increasing MBG content, and TCP/50MBG (with 50 wt % content MBG) exhibited almost 100-fold enhancement compared to the pure β-TCP. Intriguingly, the cell affinity and osteogenic capacity of rBMSCs were also dramatically improved the best on TCP/25MBG. Further investigation found that the subtle, grainy-like microtopography, not the chemical composition, of the TCP/25MBG favored the adsorption of Fn and expression of integrin α5β1 and further facilitated FA formation and the expression of p-FAK, following activation of the MAPK/ERK signaling pathway and ultimately upregulated expression of osteogenic genes. Further in vivo experiments confirmed the promoted osteogenesis of TCP/25MBG in vivo. The results suggest that such a novel MBG-based PU foam templating method offers new guidance to construct hierarchically porous scaffolds, and the prepared MBG-modified β-TCP scaffold will have great potential for future use in bone tissue regeneration.

PEGylated poly(glycerol sebacate)-modified calcium phosphate scaffolds with desirable mechanical behavior and enhanced osteogenic capacity.

UNLABELLED Calcium phosphate (CaP) scaffolds have been widely used as bone graft substitutes, but undesirable mechanical robustness and bioactivity greatly hamper its availability in clinic application. To address these issues, PEGylated poly (glycerol sebacate) (PEGS), a hydrophilic elastomer, was used to modify a model calcium phosphate cement (CPC) scaffold for bone regeneration in this study. The PEGS pre-polymer with PEG content from 0% to 40% was synthesized and was subsequently coated onto the pre-fabricated CPC scaffolds by facile infiltration and thermal-crosslink process. Compression strength and toughness of the CPC/PEGS composite scaffold (defined as CPX/Y, X referred to the PEG content in PEGS and Y referred to PEGS amount in final scaffold) were effectively tailored with increasing coating amount and PEG content, and CPX/Y exhibited an optimal compressive strength of 3.82MPa and elongation at break of 13.20%, around 5-fold and 3-fold enhancement compared to the CPC. In vitro cell experiment with BMSCs as model indicated that coating and PEG-modified synchronously facilitated cell attachment and proliferation in a dose-dependent manner. Particularly, osteogenic differentiation of BMSCs on PEGS/CPC scaffold was strongly enhanced, especially for CP20/18. Further in vivo experiments confirmed that PEGS/CPC induced promoted osteogenesis in striking contrast to CPC and PGS/CPC. Collectively, hybrids scaffolds (around 18% coating amount and PEG content from 20% to 40%) with the combination of enhanced mechanical behavior and up-regulated cellular response were optimized and PEGS/CaP scaffolds can be deemed as a desirable option for bone tissue engineering. STATEMENT OF SIGNIFICANCE Insufficient mechanical robustness and bioactivity still limit the availability of calcium phosphate (CaP) scaffolds in clinic application. Herein, calcium phosphate cement (CPC) scaffold, as a model CaP-matrix material, was modified with PEGylated PGS (PEGS) polymers by facile infiltration and thermal-crosslink process. Such biomimetic combination of PEGS and CaP-matrix porous scaffold was first explored, without affecting its porous structure. In this study, CPC scaffold was endowed with robust mechanical behavior and promoted bioactivity by simultaneously optimizing the amount of polymer-coating and the PEG content in PGS. In rat critical-sized calvarial defects repairing, osteogenic efficacy of PEGS/CPC further demonstrated the potential for application in bone tissue regeneration. The design concept proposed in this study might provide new insights into the development of future tissue engineering materials.

RhBMP-2 loaded MBG/PEGylated poly(glycerol sebacate) composite scaffolds for rapid bone regeneration

With the worldwide rising need of severe bone defect treatment, the development of available bone substitutes with optimal mechanical strength, sustained drug release, cell affinity and osteoinductivity remains a great challenge. In this study, an rhBMP-2 loaded polymer-coated mesoporous bioactive glass (MBG) composite scaffold was developed. The uncrosslinked poly(glycerol sebacate) (PGS) or PEGylated PGS (PEGS) coating modification had enhanced the mechanical strength of the composite scaffold, solved the brittleness problem of the MBG matrix, increased the cell affinity of the material surface, and diminished the initial burst release of rhBMP-2 from mesopores of MBG. The results indicated that the PGS coating promoted the proliferation of rat bone marrow stem cells (rBMSCs), while the PEGS coating exhibited an enhancement in the osteogenic differentiation of rBMSCs. The in vivo ectopic bone formation results provide strong evidence that the rhBMP-2-loaded MBG/PEGS composite scaffolds exhibited a rapid bone forming capacity and might yield extraordinary achievements in the field of bone tissue engineering. The design considerations can be extended to other artificial scaffolds and are expected to provide new thoughts on the development of future tissue engineering materials.

Strontium doping promotes bioactivity of rhBMP-2 upon calcium phosphate cement via elevated recognition and expression of BMPR-IA

Preserving and improving osteogenic activity of bone morphogenetic protein-2 (BMP-2) upon implants remains one of the key limitations in bone regeneration. With calcium phosphate cement (CPC) as model, we have developed a series of strontium (Sr)-doped CPC (SCPC) to address this issue. The effects of fixed Sr on the bioactivity of recombinant human BMP-2 (rhBMP-2) as well as the underlying mechanism were investigated. The results suggested that the rhBMP-2-induced osteogenic activity was significantly promoted upon SCPCs, especially with a low amount of fixed Sr (SrCO3 content <10wt%). Further studies demonstrated that the Sr-induced enhancement of bioactivity of rhBMP-2 was related to an elevated recognition of bone morphogenetic protein receptor-IA (BMPR-IA) to rhBMP-2 and an increased expression of BMPR-IA in C2C12 model cells. As a result, the activations of BMP-induced signaling pathways were different in C2C12 cells incubated upon CPC/rhBMP-2 and SCPCs/rhBMP-2. These findings explicitly decipher the mechanism of SCPCs promoting osteogenic bioactivity of rhBMP-2 and signify the promising application of the SCPCs/rhBMP-2 matrix in bone regeneration implants.

Microporous density-mediated response of MSCs on 3D trimodal macro/micro/nano-porous scaffolds via fibronectin/integrin and FAK/MAPK signaling pathways

The microporous architecture of biomaterials/scaffolds plays a critical role in cellular behaviors of marrow stromal cells in the field of tissue regeneration, but the role of microporous density in this process and its underlying molecular mechanism are poorly understood. In the present work, a series of three-dimensional (3D) trimodal macro/micro/nano-porous MBG scaffolds (TMSs) with different microporous densities were developed to investigate the influence of microporous density on the attachment, proliferation and osteogenic differentiation of rat bone marrow stromal cells (rBMSCs), and the fundamental molecular mechanism was explored. The results demonstrated that scaffolds with micropores significantly promoted initial cell adhesion, ALP activity and osteogenesis-related gene/protein expressions, especially the one with 20% microporous density (TMS 20). We found that the appropriate microporous density modulated the adsorption of fibronectin (Fn), and in turn facilitated integrin receptor binding affinity, focal adhesion complex formation and subsequent FAK/MAPK signaling pathway activation. Based on these studies, it can be confirmed that microporous density contributes to the regulation of cellular response, which can provide a new insight into the design of future bone substitutes in a 3D environment.

Controlled synthesis and transformation of nano-hydroxyapatite with tailored morphologies for biomedical applications

Hydroxyapatites (HAps) with nano-sized structures are promising materials in various biomedical areas, but the synthesis of high quality particles is still challenged by the insufficient precision of size and morphology, as well as the presence of severe agglomeration. An inadequate knowledge of the early nucleation, growth and transformation might limit our exploration and application of HAp. Here, we report a novel oil/water microemulsion-hydrothermal hybrid strategy for the preparation of highly dispersive HAps with tailored morphologies and controlled size. Through the synergetic effect of the oleic acid and microemulsion system, a well-dispersed HAp nucleus was first generated at 2 h. By tuning the ensuing hydrothermal conditions from room temperature to 140 °C, the nucleus would grow from spherical to needle-like nanoparticles. The size of the particles could be regulated by the alteration of the hydrothermal temperature. In addition, we experimentally demonstrated the complete evolution of HAp growth and transformation at a critical temperature of 90 °C by quenching the reaction at various intervals. The obtained particles were explored as potential cellular delivery carriers and polymer fillers.

Bioactivation of Calcium Phosphate Cement by Growth Factors and Their Applications

Calcium phosphate cement (CPC) scaffold has been widely used as bone graft substitutes. In order to deal with formidable defects in clinic, such as critical-sized bone defects or elderly patients with low regeneration capacity, a recombinant human bone morphogenetic protein-2 (rhBMP-2) was further loaded into CPC scaffold currently. In this chapter, effects of the surface properties, microstructure, and chemical composition on the bioactivity of rhBMP-2 were carried out. The osteogenic activity of CPC/rhBMP-2 in vitro and in vivo and the underlying mechanism were reported. Additionally, the clinical application of this active CPC/rhBMP-2 scaffold was also presented. These findings will provide insightful guide for the design and fabrication of rhBMP-2-based scaffolds/implants and further promote the clinical translation of growth factor-loaded porous scaffolds for bone regeneration.