Preparation And Evaluation Of A BMP-2 Loaded PPLA Microspheres

The Bone defect is a common clinical disease,that is,the structural integrity of the bone is destroyed.The main causes of bone defects are trauma,infection,cancer,osteomyelitis surgery debridement,and a variety of congenital diseases.The comminuted fracture caused by a large number of bone defects brings about many difficulties in practical treatment.At present,the autologous bone graft is used to treat patients in clinical.However,autologous bone transplantation in the source is limited while there are still some problems such as bone pain,bleeding,and other issues.In recent years,bone tissue engineering has been developed to assist osteogenesis by investigating the development of bioavailability material that induces osteogenic activity.For now,there have been many studies trying to enhance bone repair rate by adding some specific osteogenic growth factors or cytokines such as bone morphogenetic protein,vascular endothelial growth factor,insulin-like growth factor,and platelet-derived growth factor.Bone morphogenetic protein(BMP)belongs to the transforming growth factor-beta superfamily family.BMP-2 plays a strong role in the field of osteogenesis,it is a bone-induced growth factor of the BMP family which was studied early and widely.Plenty of studies have shown that as a promoter of the initial stage of fracture,BMP-2 can induce mesenchymal cells to differentiate into osteocytes,promote osteoblast differentiation and maturation,participate in bone and cartilage growth and reconstruction process,and thus accelerate bone defect repair.Nevertheless,BMP-2 also has some common troubles like poor biological stability,short half-life,rapid diffusion in vivo,easy to be degraded etc.Hence it can be restricted in the clinic for its poor efficacy in the expected part.On that account,this paper intends to develop a suitable sustained-release system for BMP-2 to improve its metabolic process in vivo and increase its half-life to play a better role in osteogenesis.In recent years,the biodegradable polymer materials prepared into microspheres for carrying protein have gradually become one of the hot spots.Biodegradable polymer materials refer to macromolecular polymeric materials that are naturally or artificially synthesized and can be degraded in the human body by enzymatic or non-enzymatic hydrolysis.Typically,polylactic acid(PLA)has become one of the extensive polymeric materials because of its good biocompatibility,biodegradability,and non-toxicity.In clinical orthopedics and surgical sutures,the use of PLA has also been approved by FDA.But the direct use of PLA usually can't meet the requirements of carrying different drugs for its numerous ester bonds which lead to its poor hydrophilicity,so the modification of PLA is particularly vital.The most common modification method is to add a hydrophilic substance into the PLA chain,such as polyethylene glycol(PEG).PEG has excellent biocompatibility and good safety.The insert of PEG has the following advantages:(1)improving the hydrophilicity of PLA;(2)reducing the adsorption of the protein on the surface of the protein and the adhesion of the cells,avoiding being recognized by the immune system in vivo,and protect the modified PLA from the immune system;(3)synthetic amphiphilic copolymer can be modified further,such as inserting some active groups in the terminal group.In this study,BMP-2 was used as the model drug,and the biodegradable polymer polylactic acid-polyethylene glycol amphiphilic block copolymer(PPLA)was used as carrier material.BMP-2/PPLA sustained-release microspheres for the treatment of bone defects were successfully prepared by the double emulsion solvent evaporation method.Relevant evaluation of BMP-2/PPLA microspheres has been done including the design of prescription,the technical study,the physical and chemical properties,and the in vitro release.1.The prescription and techology of BMP-2/PPLA MicrospheresThe encapsulation efficiency of BMP-2 was determined by the dissolution-ultracentrifugation method and methodology was inspected.The effects on encapsulation efficiency(EE)of the polyvinyl alcohol(PVA)concentration,the PPLA concentration,the ratio of internal water phase to oil phase,the ratio of oil phase to the external water phase,stirring time and stirring speed were tested.On the basis of single-factor experiments,the optimum parameters were achieved using orthogonal test.The result showed that the free BMP-2 and BMP-2/PPLA microspheres can be completely separated by the centrifugation-filtration method.The data of the drug concentration displayed a good linearity within the range of 5.5?49.5?g/mL.The recovery and the RSDs(intra-day and inter-day)meet the requirement.The optimal prescription was obtained by the single factor and orthogonal test.The highest EE(%)of BMP-2 was achieved with a PVA concentration of 4%,a PPLA concentration of 90 mg/ml,the ratio of internal water phase to oil phase is 1:5,the ratio of oil phase to external water phase is 1:10,and a stirring time of 1.5 min at 15000 r/min.2.The physicochemical properties of BMP-2/PPLA MicrospheresThree batches of microspheres were prepared using the optimized formulation.The morphology was observed by optical microscope and super-depth microscope.The particle size and Zeta potential were conducted by dynamic light scattering.Encapsulation efficiency and drug loading were measured by Ultraviolet spectrophotometry(UV).The appearance of the microspheres was spherical and the distribution of the microspheres was homogeneous.Mean diameter and Zeta potential of microspheres were(3.48±0.51)?m and(-23.27±0.76)mv respectively.The microspheres prepared according to the optimal technique parameters exhibit excellent appearance,high encapsulation efficiency,and good reproducibility.3.In vitro release of BMP-2/PPLA MicrospheresIn vitro release property of BMP-2 from microspheres was performed by dynamic dialysis method.The release behavior of BMP-2/PPLA microspheresin vitrowas investigated in pH 4.0 acetate buffer solution containing 0.02%NaN3.Free BMP-2 released fast and its cumulative release percentagereached to 89.34%in 50 minutes.BMP-2/PPLA microsphere is relatively slow while its cumulative release percentage is 27.91%in one day,40%in three days,and 85%in 12 days,whichdemonstrated significant sustained release effect.