Preparation And Characterization Of Nano-Biomimetic Materials Modified With The Peptide Derived From Bone Morphogenetic Protein-2

Bone defects and fracture non-union are common problems in China, affecting as many as several millions patients. Previously, these cases have been treated by surgery, using techniques such as autologous bone grafting or artificial bone grafting. However, autologous bone grafts have some problems including donor-site problems, the limitations of harvested bone or the weak strength of graft-bone, while artificial bone grafts also have associated problems caused by the use of biomaterials, including immunogenicity, biodegradation or strength limitations. To overcome these problems, further development in new types of biomaterials and new cytokines which can enhance bone regeneration has been pursued.Natural bone is a complex biomineralized system with an intricate hierarchical structure. It is a typical example of an"organic matrix-mediated"self-assembling and biomineralization process which was constituted of nano-size plate-like crystals of carbonated hydroxyapatite(HA) orderly grown in intimate contact with collagen fibers. Meanwhile, investigations showed that this biomineralization process was affected by the content and space distribution of anionic functional groups. Mineralization during vertebrate bone growth is a classic example in which hydrophobic collagen fibrils are organized into parallel sheets with periodically staggered"hole zones". These spaces are rich in phospho- and glycoproteins, creating local charge accumulation. The anionic nature of the hole zone, along with structural and stereochemical interactions, are thought to lead to attraction of calcium-rich mineral nuclei and initiation of mineral growth. It means that the best biomineralization effect could be attained only when the anionic functional groups density on the surface of the material is suitable.Until now, there are no artificial materials with such ingenious nano-structure and excellent biologic activity, especially the bioactivity of osteoinduction. Poly (lactic acid) (PLA), poly (glycolic acid) (PGA), and their copolymers (PLGA) have been widely used in the fields of tissue engineering. They have favorable biocompatibility and biodegradation, but they have week mechanic strength, high hydrophobicity and low cell adhesiveness. Most importantly, they are short of activity of bone conduction and bone induction, thereby limiting their application.In recent years, biologically inspired self-assembling and biomineralization approach is always used to prepare organic-/ inorganic nano-composite and modify materials with peptide and growth factors. Biomimetic"intelligence"materials with specific structure and function are prepared to induce definite and controllable bio-response at nano-level. This belongs to leading edge topics in the field of biomaterials and tissue engineering.Using a hierarchical approach that mimics natural material formation processes, we developed a method to produce materials with controlled physical structures at both the micrometer- and nanometer scale. We synthesized bone morphogenetic protein-2(BMP-2) derived peptide P24 and combined the peptide with PLGA-[ASP-PEG]n materials. A new biomimetic bone tissue engineering scaffold material, BMP-2-derived peptide/nano- HA/PLGA-[ASP-PEG] composite, was synthesized by a biologically inspired self- assembling and biomineralization approach.The whole experiment was divided into two parts as follows: 1. The bioactivity of osteoinduction of BMP-2-derived peptideObjective: To investigate the activity of osteoinduction of BMP-2-derived peptide.Methods: The BMP-2-derived peptide, P24, was synthesized by FMOC/tBu solid-phase peptide synthesis (SPSS). HPLC and mass spectrometer (MS) were used to evaluate synthesis of the peptide. BMP-2-derived peptide solution was absorbed by pieces of absorbable collagen sponge in different concentration and then the material was freezed. The experimental material was put into one side of the musculus sacrospinalis of each rat in the form of 0.4mg peptide/ collagen sponge in one group or 0.1 mg peptide/ collagen sponge in the other group, while a piece of pure collagen sponge in similar size was put into the other side of the muscle. After 3, 6 and 8 weeks, four rats were sacrificed and assessed with X rays, CT and evaluated by histology. The time-effect relationship and dose-effect relationship of ectopic bone formation were observed.Results: Both groups which contained the peptide showed ectopic bone formation by X ray, CT and histological evaluation. In addition, its dose and time dependence effects can also be observed. The group of 0.4mg peptide/ collagen sponge demonstrated evident new bone formation in contrast with moderate new bone formation in the group of 0.1 mg peptide/ collagen sponge. Only some fibrous tissue was seen in the group of pure collagen sponge without any bone formation.Conclusion: The BMP-2-derived peptide has excellent activity of osteoinduction and the capability of ectopic bone formation similar to properties of BMP-2. It can be applied widely in the domains of osteoanagenesis and bone tissue engineering.2. Preparation and characterization of nano- biomimetic materials modified with the peptide derived from BMP-2(1) Self-assembling synthesis of bone-like HA/ PLGA- (PEG-ASP)n compositeObjective: To synthesize biomimetic bone tissue engineering scaffold material by a biologically inspired self-assembling biomineralization approach. The biomineralization potential of a novel biodegradable PLGA-(PEG-ASP) n copolymer with abundant pendant amine which can act as functional groups for nucleation of calcium was also investigated.Methods: The PLGA-(PEG-ASP)n porous scaffolds were incubated in modified simulated body fluid(mSBF), with PLGA scaffolds as the control group. Growth of HA nanocrystals is confirmed by calcium ion binding analyses, SEM, mass increase measurements and quantification of phosphate content within scaffolds.Results: SEM analysis demonstrated the nucleation and growth of a continuous bonelike, low crystalline HA nanocrystals on the inner pore surfaces of the PLGA-(PEG-ASP)n scaffolds. The amount of calcium binding, total mass and the mass of phosphate on experimental PLGA-(PEG-ASP)n scaffolds at different incubation times in mSBF was significantly greater than that of control PLGA scaffolds.Conclusion: The ASP-PEG alt-prepolymer modified PLGA copolymer provide a controllable high surface density and distribution of anionic functional groups which would enhance nucleation and growth of bonelike mineral following exposure to mSBF. This biomimetic treatment provides a simple method for surface functionalization and subsequent mineral nucleation and self-assembling on biodegradable polymer scaffolds for tissue engineering. This nano-HA/PLGA-(PEG-ASP)n composite shows some features of natural bone both in main composition and hierarchical microstructure.(2) Preparation of peptide modified PLGA-[ASP-PEG]n biomimetic scaffoldsObjective: To prepare a new biomimetic bone matrix material for tissue engineering by integrating peptide, which has excellent osteoinduction, with modified PLGA scaffold using cross-linkers.Methods: We devised and synthesized the BMP-2-derived peptide, and modificated PLGA-[ASP-PEG] with peptide by methods of physical entrapment and chemical coupling, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N- hydroxysuccinimide (NHS) as cross-linkers. We used scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS) and ion binding experiment to investigate the products.Results: The contents of sulphur element on the surfaces of coupled composition and entrapped composition were 1.5%and 0.09%. The amounts of ions attach to the coupled composition were 0.126mg at 12h and 0.231mg at 24h, while amounts of ions attach to the entrapped composition were 0.053mg at 12h and 0.102mg at 24h. The coupled composition showed greater ability to attach to ions than that of the entrapped composition.Conclusion: The BMP-2 derived peptide P24 was successfully coupled with PLGA-(PEG-ASP)n by EDC and NHS. The coupled material established the basement of preparing nano-HA/PLGA-(PEG-ASP)n materials.(3) Bioinspired growth of hydroxyapatite nanocrystals on PLGA- (PEG-ASP)n scaffolds modified with peptide derived from BMP-2Objective: In this study, PLGA-(PEG-ASP)n scaffolds were modified with P24 and a new biomimetic bone tissue engineering scaffold material with enhanced bioactivity was synthesized by a biologically inspired mineralization approach.Methods: Peptide P24 was introduced into PLGA-(PEG-ASP)n scaffolds using cross-linkers. Then the P24 modified scaffolds and the simple PLGA-(PEG-ASP)n scaffolds were incubated in modified simulated body fluid (mSBF) for 10 days. Growth of HA nanocrystals on the materials was confirmed by observation SEM and measurements EDS and XRD.Results: SEM analysis demonstrated the well growth of bonelike HA nanocrystals on P24 modified PLGA-(PEG-ASP)n scaffolds than that of the control scaffolds. The main component of mineral of the P24 modified scaffolds was hydroxyapatite containing low crystalline nanocrystals, and the Ca/P ratio was nearly 1.60, similar to that of natural bone, while that of the control scaffolds was 1.52.Conclusion: The resulted peptide P24 modified- HA/PLGA- (PEG- ASP)n composite showed some features of natural bone both in main composition and hierarchical microstructure. Meanwhile, the introduction of peptide P24 into PLGA-(PEG-ASP)n copolymer provided excellent osteoinduction to the materials.