Biocompatibility Of New Biomaterials Electrospun With PLA,Ⅰ Collagen And HA Mimicking The BECM

Reconstruction of bone defects caused by trauma or surgical resection is a major issue in orthopedic surgery. The shortage of donor organs, the high cost and possible complications of transplant surgery have created the need for an alternate source of tissue. In the last couple of decades a new approach to repairing tissues has evolved that employs engineering tissues by using cells and scaffolds. Tissue engineering aims at restoring, maintaining, or improving tissue function, but tissue engineering does still suffer from various limitations to address clinical demands in treating tissue defects. One of the problems is the lack of appropriate biomimetic scaffolds. The ideal tissue repair material should be natural polymers possessing the mechanical and biological functions of the nature bone extracellular matrix(BECM), so mimicking the BME'S contents and architecture has attached more and more attention, popularity and funds. The recent understanding the BME is a nature 3-D cell supporter with complicated ultrastructure determines the direction of scaffold design development. Over the last decade electrospinning has been recognized as a simple and versatile process to fabricate various polymeric and inorganic fiber mats with the high surface area-to-volume ratio and porosity. Moreover, electrospun fibrous matrices with nanoscale diameters mimic morphological nanofeatures of native ECM.Bone extracelluar matrix is an organic-inorganic hybrid with nanofibrous collagen and nanohydroxyapatite. Therefore, biocomposite scaffolds of nanofibrous collagen and hydroxyapatite nanoparticles could be more promisings scaffolds for bone tissue engineeringm, because collagen and hydroxyapatite are bioactive and osteoconductive, and the cell recognition sites present in this matrices. Novel nanofibrous scaffolds of native polymer such as collagen o(rand) hydroxyapatite(HA) have been reported for tissue engineering constructs. But the present study reports the electrospinning of collagen (typeⅠ) and nanosize hydroxyapatite in different conditions and we report the different hydrophilicity between the electrospinning materials and non-electrospinnig materials for the first time. And we researched how the different contents with the nano-structure affected the hMSCs'adhesion and proliferation, the osteoblasts'functions for the first time. Our research included five parts from the construction of biomaterials to the cytobiology.1. Electrospinning the PLA,Collagen,collagen and HA construct the the biomaterials mimicking the BEMC's ultrastructure and contentAims: PLA/Collagen/HA were electrospun at different conditions in order to find the better condition; analyzing the surface characterization of different scaffolds including the diameter/density/porositycomparing the hydrophilicity and degradation in different scaffolds.1.2 Methods: we electrospun PLA/collagen/HA at different conditions, and find the better condition through checking the surface characterization. The morphology of collagen typeⅠand nanoHA scaffolds were observed using scanning electron microscope (SEM, applied by State key laboratory of supramolecular structure and materials, Jilin University). The diameters of the fibers and the thickness of the scaffolds were measured from SEM image with a custom image analysis program. Water contact angle of the electrospun scaffolds and the non-electrospun scaffold were measured to compare their hydrophilicity. Water contact angle was measured with the sessile drop method and a drop shape analysis at 20-25℃. The weight losing were recorded after the different scaffolds were soaked in buffer mimicking the vivo conditions.1.3 Results: The electrospun nanocomposite fibers on the collector formed a randomly oriented nonwoven fabric structure having nanofibers in the 50-500 nm diameter range, depending on the composition and concentration. The present nanofibrous biocomposite scaffolds have well interconnected pore network structure and large surface area necessary for cellular attachment, vascularization, and resulting cellular in-growth. The porosity calculation from apparent density measurements of the matrices showed 80-95% porosity. the porosity is close to the native bone matrix porosity about 80% . The SEM images showed that electrospun fabrics of both collagen and HA obtained less beads under the optimized spinning conditions (electric potential =12kV, and distance between needle and collector=8cm). Measuring the water contact angle and rate of degradation improve that the electrospun scaffolds was better than non- electrospun scaffolds(P<0.001).1.4 Conclusions: As a conclusion of this study, electrospinning is a potential single-step method for producing 3D scaffolds. in this way we harvest a satisfied bioscaffold whose average diameter is 220.7±22.8nm and contents are collagen and HA. the scaffold had high porosity 88.6%. the datas is so close to the parameters of the nature BECM. we enhance the hydrophilicity of the scaffold through electrospinning. We change the rate of degradation of the scaffolds by the way.2. Isolation,cultivation and assessment the characterization of hMSCs and inducing the hMSCs to osteogenesis2.1 Aims: used bone marrow collection sets were evaluated as a valuable source of hMSCs. We want to find an effective way to Isolate and cultivate hMSCs and identify the characterization of the harvest cells. inducing the hMSCs to Adipogenesis and osteogenesis2.2 Methods: we isolate the hMSCs from bone marrow in plastic plate by 70% percoll according the rule that the hMSCs only exit in the low concentration lay of the total bone marrow and the hMSCs easily adhere on the surface of the plastic. Cell morphology was inspected and documented continuously with light inverted microscope; The measurements of the cell surface molecule pattern were performed using ?ow cytometer; Adipogenesis was induced by a-MEM supplemented with10% FBS,1% penicillin,0.5 mM dexamethasone,50 mM indomethacin and 0.5 mM isobu-thylmethylxanthine Differentiation was demonstrated by Oil Red O staining of intracellular lipid droplets after 3-week culture;Osteogenesis was induced by a-MEM supplemented with dexamethasone,ascorbic acid,and b-glycerophosphate。Differentiation was evaluated by Alizarin Red staining,ALP staining andⅠcollagen staining after a 2-week culture.2.3 Results: After the initial 3 days of primary culture, human MSCs adhered to a plastic surface and presented spindle shape. hMSCs displayed blue smooth nuclear and red cytoplasm. The cells were considered normal on the basis of typical morphology. The hMSCs were positive for CD29, CD90. Oil Red-O staining proved that most of the cells had actively deposited intracellular lipids; The osteogenic differentiation was proved by Alizarin Red staining,ALP staining andⅠcollagen staining after a 2-week culture.2.4 Conclusions: we control an effective way to isolate and cultivate hMSCs in vitro. We successfully induced the hMSCs to Adipogenesis and osteogenesis which were proved by staining,flowing cytometer and immunochemistry.3. Researching the biocompatibility of the new mimicking biomaterials3.1 Aims: To investigate the cytotoxicity of electrospun bioscaffolds with PLA/collagen/HA that mimic the nature structure and contents of bone extracellular matrix in vitro.3.2 Methods: The Human bone mesenchymal cells were exposed to different concentrations of cell culture medium which new scaffolds had been soaked for 48h. Finally cell viability and growth were assessed by MTT assay at 1d,3d,5d,7d.3.3 Results: No significant cytotoxicity was observed among different concentrations and negative control group(P<0.05)3.4 Conclusions: Electrospun bioscaffolds with PLA/collagen/HA that mimic the nature structure and contents of nature bone extracellular matrix doesn't demonstrate any significant toxic effects to human mesenchymal cells in vitro.4. The adhesion and proliferation of the hMSCs on the different new mimicking biomaterials4.1 Aims: we want know whether the ultrasturcture of the mimicking bioscaffolds influence the adhesion and proliferation of the hMSCs on the surface of the different materials. And we want to know whether the mimicking the ultra-structure and content of BECM can influence the ALP/Ca2+/osteocalcin (OCN)of osteoblast induced by hMSCs.4.2 Methods: the hMSCs were cultured on the surface of the different scaffolds including electrospinning and non- electrospinning. We count the alive cells on the surface of scaffolds by cytometer at 4h,12h,24h,48h. we check the proliferation of different scaffolds by MTT. we cultured the hMSCs on the surface of the six kinds of scaffolds including electrospinning and non- electrospinning. After 1 week, the cells was induced by a-MEM supplemented with dexamethasone,ascorbic acid,and b-glycerophosphate. Cell morphology was inspected and documented continuously with light inverted microscope and SEM; The osteogenic differentiation was proved by Alizarin Red staining ;The total protein and ALP were detected after 2 weeks by Coomassie brilliant blue and kit. The mRNA of OCN were checked by RT-PCR after2 weeks.4.3 Results: the adhesion and proliferation of the hMSCs on the surface of electrospun bioscaffolds are better than non-electrospun bioscaffolds(P<0.05); The function of osteogenesis had been found that the mimicking the structure's team is better than the control group(P<0.05)and the mimicking the structure and content's group is obviously better than the mimicking the structure's team(P<0.05).4.4 Conclusions: The new electrospun bioscaffolds can enhance the adhesion of the adhesion and proliferation of the hMSCs on the surface of the different materials. The osteogenic differentiation was proved by Alizarin Red staining after a 2-week inducing culture. The cell is activity and osteogenesis; The new electrospun bioscaffolds can enhance the the function of osteogenesis of the hMSCs too, they are promising materials for bone tissue engineer.As a conclusion of this study,electrospinning is a potential single-step method for producing 3D scaffolds. And in this way we mimic the architecture and content of the bone extracellular matrix in four ways: first, contents of the electrospun composition are the native contents of the native bone extracellular matrix which can supply attachment sites, and proteins of promoting cell proliferate, And the HA can regulates cell function and promotes osteogenesis and mineralization of bone. second, the diameter of the fiber resembles the extracellular mineral protein fiber diameter, thus allowing cellular attachment and facilitating cellular migration. Third, the porosity simulates that of the native BCM such that microvascular capillary tube formation is enhanced . Finally, we enhance the hydrophilicity of the scaffold through electrospinning. So a combination of nanofibrous collagen and nanohydroxyapatite that mimics the features of the native bone extracellular matrix could be promising scaffold materials for bone tissue engineering.