The Preparation Of Macromolecule Nano-materials And Primary Study Of Its Application In Bone Tissue Engineering

To repair bone defect derived from a variety of causes (trauma, infection, tumorresection, congenital diseases, etc) is always the dilemma in such the fields as clinicalorthopaedics,biomaterial science and tissue engineering. But traditional bonesubstitute materials including autograft, allograft and synthetic materials have theirintrinsic disadvantages to a certain extent, consequently, people are obliged to study insearch for ideal bone substitute materials. Bone tissue engineering emerge as the timesrequire in 1980s of the 20th century.One of its characteristics is that seed cells areplanted on scaffolds materials and the tissue engineering bone is planted into thebone defect area once it form. Nowadays,scaffolds research is the hotspot in bonetissue engineering field. An ideal bone tissue engineering material should meet thedemands as follows: (1)right biocompatibility; (2)right biodegradability orresorbablity; (3)its degradation speed corresponds with the bone growth capability;(4)porous fabrication with aperture from 200μm to 400μm; (5)very strong penetrationpower; (6)exact lacuna size for growth of seed cells; (7)mechanical strength providingmicro-stress circumstance for cells; (8)appropriate surface structure for cellsadhesion; (9)improvement upon the secretion capability of extracellular matrix;(10)capability of being the carriers of signal molecules such as growth factors.Scaffold materials in existence involve in natural and synthetic materials. Thescaffold materials which received considerable investigation includecollagen, PLA, PGA, PMMA, HA, TCP and coral. Synthetic macromolecule materialshave advantages such as controllable capability, no immunogenicity and rightbiocompatibility. PLA, PGA and PLGA are the biodegradable materials applied mostbroadly. Their disadvantages include as following:low mechanical strength, aciddegradation outcomes unfavorable to cells growth, not corresponding biodegradatonspeed with bone growth, toxic organic solvents remained.In this study, literature in abundance were collected and analysed with a view to theshortcomings currently existed in bone tissue engineering scaffoldings materials.L-lysine ethyl ester was consequently chosen as raw material to synthesizelysine diisocyanate which was subsequently polymerized with glycerol for lysinediisocyanate-glycerol polymer. The obtained macromolecule material—LDIG polymerwas purified and nanometerized by means of SAS and ultrasound disperse. Amacromolecule Scaffold material with certain aperture and lacuna rate was acquired via solvent moulding/grain strain process.In the article,stress was laid on preparationmethods,physical and chemical characteristics, degradation features andbiocompatibility of the macromolecule material.The polymer was commingled withn-HA to obtain a bionic composite which was to be applied to repair bone defect inrabbits.The experimental study was divided into 5 portions as follows:1. Preparation and characteristics of macromolecule nano-material(1)L-lysine ethyl ester and triphosgene were applied as raw material to synthesizelysine diisocyanate (LDI) by improved traditional process, then macromoleculematerial was obtained by polymerization of LDI and glycerol in this experiment.(2) Aqueous macromolecule or solid nano-particles were obtained by ultrasonicdispersion,emulsification/solvent diffusion and SAS, respectively. The size of aqueousmacromolecule nano-particles was 80.0～200.0 nm (average 140.0±56.3 nm) incomparative uniformity. The size of crystal fibres acquired by SAS was 100.0～350.0nm (average 156.0±67.5 nm). The crystal fibres in nanometer were interwoven intomicropores fabrication, the pore diameter and the porosity rate, which weremeasured by scanning electron microscope, was 100.0～400.0μm (average 276.0±87.2μm), 75.6ï¼…, respectively.(3) The macromolecule material was revealed as a potential tissue engineeringmaterial by a series of characterization in physical capability: The glass-transformingtemperature (Tg) was 88.6℃, tangency angle was 67.3°, average originalcompression intensity 4.26±0.78 Mpa and anti-rupture intensity 11.63±2.30 Mpa.(4) The obtained macromolecule material was pure and nontoxic due to innoxiousraw material and process which accorded with the viewpoint of 'green chemistry' andelimination of organic solvent in the course of preparation.2. Degradation of macromolecule nano-material in vitro(1) The dynamic changes of pH value, hydrophilicity, weight, mechanical strength anddegradation outcome were examined to evaluate the degradability of lysinediisocyanate-glycerol polymer in vitro, accordingly, the material was proved to be anideal, nontoxic and biodegradable macromolecule material.(2) The weight loss of the material showed a tendency from slowness torapidness: the weight of LDIG decreased slowly in 6 weeks and decreased rapidlyafter 6 weeks, half of original weight was left at the time of 10 weeks.(3) The pH value of LDIG lixivium kept stable in the course of degradation for 12 weeks and was 7.30 in the end of 12 weeks indicating that degradability of thepolymer did not alter pH value of medium.(4) Compression intensity of LDIG decreased slowly in 6 weeks and rapidly after 6weeks and was equal to half of original one in the end of 8 weeks indicating that thedynamic changes of mechanical strength,weight and hydrophilicity accorded withits degradation.(5) The degradation of LDIG in vitro was hydrolyzation of ester binding of poly(urethane), The outcome of degradation was some micromoleculesubstances: lysine, glycerol, ethanol and CO₂.3. Biological evaluation of macromolecule nano-material(1) According to the correlative regulations of Technical Evaluation Standards ofBiomedical Materials and Medical Instruments promulgated by National Ministry ofHealth,the experiments of acute toxicity, pyrogen, hemolysis and implantation intomuscles were investigated to evaluate the biocompatibility of the polymer. The resultsof experiments on acute toxicity, pyrogen, hemolysis, implantation into muscles met theregulatons mentioned above.(2) The macromolecule material (LDIG) had fight biological characteristics whichmet the basic conditions demanded in bone tissue engineering.4. Preparation and characterization of n-HA/n-LDIG composites for bone repair(1) The examined results showed that n-HA/n-LDIG prepared in this experimentobviously excels common u-HA/n-LDIG in mechanical property. By comparison,n-HA/n-LDIG we prepared was better than those reported before on crystal degreeand shape and sizes, which was more similar to the natural bone.(2) The composite (n-HA/n-LDIG) had a closer combination and a smaller crackthan before.n-HA was equally distributed in n-LDIG with the sameshape. Consequently, it has better bioactivity, biocompatibility and mechanicalcapability, which are very important for its clinical application and making up thosevarious disadvantages of bone materials reported before.(3) The composite(n-HA/n-LDIG)was a greater improvement than the beforeresearch work.The newly man-made bone material was believed to have moreapplication value and a much wider prospect.5. Experimental study of macromolecule bone substitute material (n-HA/n-LDIG)on reconstruction of rabbit thighbone defect(1) Macromolecule bone substitute nano-material (n-HA/n-LDIG) was prepared by emultion commingling and TIPS methods and applied in repairing rabbit thighbonedefect successfully, displaying nice reconstruction function of the composite.(2) Gradual biodegradaton of n-HA/n-LDIG accorded with bone growth in bonedefect area.(3) The experimental study showed that n-HA/n-LDIG composite was endowed withsatisfactory biocompatibility and osteoconduction and capability of repairingpenetrating defect in long rabbit bone effectively.