Study Of PDGF-BB Releasing N-Nacre/PDLLA/FG Composite Scaffold

BackgroundBone defect results from many clinical problem, such as trauma tumor resection and infection. Autografts and allografts have been used to heal bone defects, but these treatments have limitations. With the rapid development of bone tissue engineering, it provides a new idea for bone defect reconstruction.Nacre is the inner layer of the shell of mollusc pearl oyster or bivalve. It is a hard tissue consisting of more than95%calcium carbonate crystals and about5%of organic matrix. Nacre organic matter containing macromolecules such as proteins andpolysaccharides. Studies have shown that nacre has the incomparable advantage of other inorganic materials as a biological substitute materials, it is a material from organisms rich source of low-cost, easy processing, physical and chemical quality and biological characteristics of the animal and human bones veryclose. The osteoinductive function of nacre was firstly found by French scientist Lopez. Since then, lots of researches had proved that this kind of biomaterial had satisfactory biocompatibility, osteoinduction and osteoconduction. Recently, much attention has been paid to nacre as a new bone substitute biomaterial.With the requirements to further improve the performance of scaffolds, single-phase biological scaffolds have been difficult to meet clinical requirements, and thus the different nature of the material compound in order to obtain excellent clinical performance biomaterials has become the hotspot of current materials research in the field. Poly-lactide acid (PLA) is one of the most commonly used biodegradable polymers in the field of tissue engineering because of its outstanding biocompatibility, variable degradability, good mechanical properties, thermal stability, nontoxic degraded products, as well as easy processing. Poly-D,L-lactide acid (PDLLA) has a more appropriate degradability and can be chose as a bone substitute. Although PLA has many advantages, but it is the presence of high hydrophobicity, poor cell affinity, insufficient mechanical strength and the product is easy to cause inflammation and other shortcomings so that the application is limited. The main component of the nacre is CaCO3crystals, with basic properties, good mechanical properties and incorporate nacre into the PDLLA to improve the bioactivity and mechanical properties of the scaffolds. Therefore, our group use "Pressure-enhanced Technology" patented technology to prepared nacre/PDLLA composite artificial bone in early stage. After a series of in vitro and vivo studies have confirmed that this composite scaffold has good biocompatibility and osteogenic capacity. At the same time, we found that the nacre degradation is very slow speed of degradation and new bone growth rate is not proportional.In recent years, nano-materials have made extensive progress. Therefore, we attempt to explore the application of n-Nacre artificial bone to improve the nacre degradation and maintain its biological activity. We used nacre nanoparticles, which were prepared by using a planetary ball mill, to generate a novel porous PDLLA/nacre nanocomposite scaffold though the salt leaching processing technique. Preliminary studies indicated that small particle size of PDLLA/nacre nanocomposite scaffold is easily to degradation and absorption, and has good biocompatibility and biological activity. However, PDLLA/nacre nanocomposite scaffold incorporate PDLLA, then reduces the content of the nacre, which reduces the potential osteoinductivity of the scaffold. Our studies have demonstrated that platelet-derived growth factor BB (PDGF-BB) can accelerate bone formation and also promote osteoclast resorption, which play an important role in the regulation of bone reconstruction. Thus we try to improve pore size and porosity of PDLLA/nacre nanocomposite scaffold and loaded PDGF-BB, thereby enhancing osteoinductivity and promote bone formation.We fabricated a PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold by solvent casting/particulate leaching and Freeze-drying technology. The physical and chemical properties, mechanical properties, biocompatibility and osteogenesis of the scaffold were investigated, it provides a experimental basis for clinical applications.Objectives:1. To investigate the preparation and characterization of a PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold.2. To investigate the MC3T3-E1cell biocompatibility of a PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold.3. To investigate the biocompatibility and biological safety of a PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold in vivo.4. To investigate the repair of rabbit radius segmental bone defects(15mm in length) after the implantation of PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold. Methods1. Preparation and characterization of PDGF-BB releasing n-Nacre/PDLLA/FGcomposite scaffold(1) Charatherization of nacre nanoparticlesNacre nanoparticles were prepared by using a planetary ball mill. The crystalline phase of the obtained nacre nanoparticles was analyzed with an XRD. The morphology of the nacre nanoparticles was examined by a TEM.(2) Preparation of PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffoldPDLLA scaffold (S1) and n-Nacre/PDLLA scaffold (S2) were fabricated by a simple method of solvent casting and salt leaching, and fibrin glue (FG) mixed with PDGF-BB was infused into the S2and lyophilized to develop a PDGF-BB releasing nano-nacre/PDLLA/FG composite scaffold (S3). The morphology and structure properties of the scaffolds were investigated by scanning electron microscope (SME) and mechanical property testing. The porosity of the scaffold was measured by the Archimedes method. We detected the PDGF-BB releasing amount during the degradation of S3by ELISA.2. Evaluation of the MC3T3-E1cell biocompatibility of PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffoldMC3T3-E1cells cultured on PDLLA scaffold (S1), n-Nacre/PDLLA scaffold (S2), PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold (S3) and on the empty wells were classified as control group, respectively. At the indicated time, cell morphology was observed by inverted phase contrast microscope and scanning electron microscope. In addition, MTT assay and ALP activity test were used to evaluate the cells abilities of proliferation and osteogenetic-differentiation respectively. The amount and area of calcium nodules in each group were observed by alizarin red staining. 3. In vivo study of biocompatibility of PDGF-BB releasing n-Nacre/PDLLA/FGcomposite scaffold(1) PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold was immersed into normal saline inorder to obtain extracts. Then the extracts were subjected to acute systemic toxicity test, intradermal stimulation test and pyrogen test.(2) Implant test:PDLLA scaffold (S1), n-Nacre/PDLLA scaffold (S2) and PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold (S3) were implanted in back subcutaneous tissue in12New Zealand rabbits. Respectively1,4,8,12weeks after the X-ray examination, the scaffolds underwent hematoxylin-eosin(HE) staining and optical microscopy to observe the tissue response to the situation.4. Osteogenesis of PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold(1) Group settingsA total of36adult male New Zealand rabbits (2-3kg body weight) were randomly divided into4groups. A segmental bone defect (15mm in length) was created at bilateral radial in each rabbit. The defects were implanted with various grafts:PDLLA scaffold (S1), n-Nacre/PDLLA scaffold (S2) and PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold (S3), no implant as the control group.(2) Establishment of rabbit model of15mm radial bone defectSurgical procedure is listed below:After anesthesia, the experimental rabbit was skin prepared, sterilized and towel draped. An incision was made along the long axis of the radial shaft, and middle of the radial was exposed by successive dissection then a segmental bone defect (15mm in length) was created at bilateral radial in each rabbit.. Finally, a cylindrical scaffold was grafted into the defect respectively.(3) Acquirement of the specimens and observation indicatorsAnimals were sacrificed at each time point. Gross observation, X-ray examination, bone mineral density (BMD) examination, Micro-CT3D reconstruction and histological examination were performed on the specimens.Results1. The diffractogram from XRD analysis of the nacre nanoparticles was indicated that the samples were phase-pure and well crystallized. TEM micrographs of the nacre nanoparticles were shown that nacre crystals were spherical shape, with a diameter range from45to95nm. PDLLA scaffold (S1), n-Nacre/PDLLA scaffold (S2) and PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold (S3) were prepared, they are all cylinder shape (3.5mm×3.5mm×15mm and8mm×8mm×15mm).2. These scaffolds possessed porous structure, the pore sizes of the these scaffolds were (330.0±55.6)μm,(322.5±50.8)μm and (303.3±47.0)μm. There was no significant difference between them(P=0.141). The porosities of the these scaffolds were81.0±1.1%,82.5±0.8%and80.2±0.7%. There was no significant difference between them(P=0.211). The compressive strength of the3scaffolds were (3.6±0.12) MPa,(5.0±0.35) MPa and (5.1±0.26) MPa. S1was significant difference between S2and Ss(P<0.001). In vitro experiments showed that PDGF-BB released from the S3scaffold sustained30days.3. The MC3T3-E1cells adhered and grew well on the surface of the S3scaffold, it was significantly better than S1and S2scaffolds. After4h and24h culture the efficiency of cell adhesion in the S3scaffold was higher the the S1and S2scaffolds(P<0.001). At the same time point compared S3group with other groups, MTT detection and the expression of the ALP in S3group was the highest at the same time point, there was significant difference (P<0.001). The number of mineralized nodules and area of Group S3were more obvious than Group S1, Group S2and blank control group. 4. Experiments of acute systemic toxicity test, intradermal stimulation test, pyrogen test and implant in back subcutaneous tissue were investigated to evaluate the biocompatibility of the PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold. The results showed that the scaffold was non-toxie, non-stimulation, no pyrogen and possessed good biocompatibility in vivo and met the needs of bone tissue engineering.5. General observation:all animals healed by first intention after operation, no fracture occurred. The activities of eating and mental state of the animals were normal basically. Results of radiographic examination: at4weeks after surgery, the floccose low-dense imaging manifestation of callus was observed in S3scaffolds and a little callus formed at the ends of bone. A lot of new bone filled in the defects, bone bridge formed between the ends of bone, the medullary cavity achieved partial recanalisation. At12weeks after surgery, the medullary cavity achieved full recanalisation, and bone defects were repaired completely. According to Lane-Sandhu X-ray criteria, Lane-Sandhu scores of S3scaffolds were much higher than S2scaffolds at4,8and12weeks(P<0.05). Lane-Sandhu scores of S2scaffolds were much higher than S1scaffolds and control group at4,8and12weeks(P<0.05). Results of BMD examination:the BMD values of S3scaffolds were much higher than S1and S2scaffolds at4,8and12weeks(P<0.001), and BMD values of S3scaffolds did not differ from that of normal radii(P>0.05). Gross speciments observation:at12weeks after surgery, the bone defects were repaired completely in S3scaffolds, the bone defect achieved partial repaired in S2scaffolds, the bone defect achieved a little repaired in S1scaffolds and nothing filled in the bone defect in blank control group. Micro-CT scan reconstruction: the results showed regular styloid bone structure had formed in Group S3. The formation of new bone structure were irregular, some bone defects were partly repaired in Group S2. There had been a little bone formation in Group S2and no bone formation in control group. Results of histological examination:at4weeks after surgery, the junction of the bone ends and S3scaffolds’inner showed a few new cartilages, immature bone tissues, fibrous tissue ingrowth and a small amout of blood vessels. At8weeks after surgery, the new bone increased and the mature osteocytes and woven bones appeared, S3scaffolds partially degraded, vascular generated in the S3scaffolds. The new bone tissues rebuilt gradually till12weeks post-operation, and abundant of woven bones or mature lamellar bones connected between the bone ends and S3scaffolds, the medullary cavity achieved recanalisation.Conclusions1. PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold has an appropriate structure, suitable compressive strength and slow release of PDGF-BB.2. PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold is non-cytotoxic biological materials, which has good biocompatibility with MC3T3-E1cells, and it promoted MC3T3-E1cell differentiation and minerlization.3. PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold which was non-toxie, non-stimulation, no pyrogen and possessed good biocompatibility in vivo and met the needs of bone tissue engineering.4. PDGF-BB releasing n-Nacre/PDLLA/FG composite scaffold possessed the ability of osteoinduction and it had good osteogenesis in vivo and in vitro test.