Creation And Research On MMP Enzyme Degradable Novel Silk Fibroin Materials Releasing BMP-2

Native silk fibroin(SF)from Bombyx mori is a natural polymer renowned for its impressive properties and has attracted attentions as a biomaterial for bone tissue engineering.However,silk fibroin materials may not meet some specific demands for bone tissue engineering materials such as the lack of bone-related growth factors and appropriate degradation rate.Bone Morphogenetic Protein-2(BMP-2)is a growth factor indispensably required for bone tissue regeneration and has been explored in the field of silk biomaterials.Up to date,different conjugation schemes were adopted to functionalize and enable the release of BMP-2 from SF-based materials formats such as porous 3D sponges,films,microparticles and electrospun mats.The combination of silk fibroin biomaterials with BMP-2 has mostly applied non-covalent conjugation strategies that are associated with a burst release phenomenon during the first days.Therefore,developing SF materials with new proteins or growth factors and enable their release in controlled manner i.e.minimizing the extent of burst release is an interesting topic in the research of silk materials applications for bone tissue engineering.Although,new desirable functions can be implemented at multilevel modification including feeding,chemical modification,mesoscale assembly and macroscale mixing,however,these strategies apply complex modification experiments every time they are used,and it is difficult to ensure the distribution consistency of each batch of materials.Silkworm germline transformation has attracted much attention as a strategy for the functionalization of silk fibroin with therapeutic proteins.This approach consists of modifying silk by inserting the exogenous gene into the silkworm genome to acquire the desired properties that can be stably maintained in silkworm genome and inherited in the offspring.We hypothesized that silkworm germline transformation can be a functionalization approach suitable to alleviate the above-mentioned issues.Recently,particular emphasis is placed on smart drug delivery systems that respond to tissue environmental signals to release growth factor according to cellular demand,so-called ‘release on demand’.Release on demand can be designed by the introduction of stimuli-responsive components into delivery systems.The most commonly used triggering mechanisms involve enzymes.If enzyme cleavable linker is used to immobilize growth factor to delivery carrier,this approach would offer a distinct advantage in controlling the degradation rate of silk fibroin scaffold materials but also in the release of the encapsulated growth factor in a controlled manner.Based on the above considerations,this study combines silkworm germline transformation technology,high-efficiency expression system of silk gland and on demand release strategy to create a new functional silk material with advantageous features for bone tissue engineering.We firstly designed fusion genes that specifically expressed in the posterior silk gland of the silkworm.The structure of the fusion gene consists of BMP-2 with MMP-2 linkers at both sides and respectively connected with the N-terminal and C-terminal of silk fibroin heavy chain;secondly,silkworm germline transformation technology was applied to create transgenic silkworm strains that specifically expressing the designed fusion protein in the posterior silk gland of the silkworm;finally,the new silk fiber spun out by transgenic silkworm strains was characterized.The results showed that our strategy achieved functional silk that can effectively overcome the shortcomings of the conventional approaches for silk functionalization,and also provide a new approach to control the degradation of silk materials.The main results of this study are summarized as follows: 1.Design of on demand release BMP-2 fusion protein structure and constructionof silk gland expression system vectorsThe sequence of human Bone Morphogenetic Protein BMP-2 was first optimized to the codon bias of the silkworm and commercially synthesized.To design h BMP-2 gene flanked with MMP linkers,specific oligonucleotides encoding for MMP-2 cleavable linkers presenting high(PAGLWA),moderate(PLGLWA)and low(VLGLWA)catalytic activity were used to direct the insertion.DNA sequencing results confirmed that MMP-2 cleavable linkers were successfully incorporated at both terminus of h BMP-2 gene yielding three different target genes briefly denoted in the dissertation as B1,B2 and B3.The construction of fusion genes was based on silk fibroin heavy chain expression system(R3)previously reported in our research group for producing foreign proteins in the posterior silk gland cells of transgenic silkworms(Zhao et al,2010).The fragment of EGFP was first released out from R3 and separately replaced with that of B1,B2 or B3 genes.We constructed heavy-chain-B1,heavy-chain-B2 and heavychain-B3 fusion genes carrying 2304 nucleotides of Fib-H P3 that consists of 5’ upstream sequences,the exon 1,the intron and the 5’ end of exon 2 of heavy chains;1206 nucleotides of B1,B2 or B3 coding sequence and 333 nucleotides of LBS consisted of the 3’ end of exon 2 and poly-A sequences.The obtained fusion genes were separately cloned into piggy Bac transposon-derived vector to yield p Bac[3x P3-Ds Red]-B1,p Bac[3x P3-Ds Red]-B2 and p Bac[3x P3-Ds Red]-B3.DNA sequencing results showed that three transformation vectors were constructed correctly.2.Creation and analysis of Transgenic silkwormThree recombinant piggy Bac vectors were transformed into G0 B.mori embryos via microinjection-based germline transformation method.Transgenic individuals were screened for the expression of ocular Ds Red expression from G1 broods using fluorescence stereomicroscope equipped with a filter set for Ds Red.According to the screening results,the rate of Ds Red-positive G1 broods was 13.4%,19% and 6.3% for p Bac[3x P3-Ds Red]-B1,p Bac[3x P3-Ds Red]-B2 and p Bac[3x P3-Ds Red]-B3,respectively.PCR analysis of genomic DNA extracted from Ds Red-positive adult moths confirmed the genomic integration of the transgenes into silkworm genome.Total proteins were extracted from cocoons harvested from wild type and G1 transgenic silkworm cocoons subjected to SDS-PAGE and immunoblotted with h BMP-2 antibody.A visible single band was detected from total proteins of the three different types of transgenic cocoons.Total proteins from wild type cocoons did not show a signal for BMP-2 antibody,confirming that BMP-2 was successfully expressed in each type of transgenic cocoons.The estimation of BMP-2 content in transgenic silkworm cocoons was about 7.2%,10.05% and 5.87% for transgenic lines expressing B1,B2 and B3 proteins,respectively.These results suggest that heavy-chain-B1,heavy-chain-B2 and heavy-chain-B3 fusion proteins were successfully secreted into the lumen of posterior silk gland together with fibroin,and further expelled into cocoons.3.The incorporation of MMP cleavable linkers into silk fibroin is a promisingstrategy to control the degradation rateThe processing of silk fibroin into different formats has been extensively applied for tissue engineering.It was therefore of high importance to prior evaluate the effects of these processes on the content of the exogenous protein.Native silk fibroin in the form of slices and regenerated formats such as hydrogels,films and lyophilized powder were fabricated and reversibly dissolved in lithium thiocyanate.The obtained solution was used for Western blotting analysis,and the results showed that the initial content of BMP-2 was significantly decreased by 40%,52% and 60% after regeneration processes required for fabricating hydrogel,film and lyophilized powder,respectively.However,the remaining content of BMP-2(80%)into native silk fibroin slices was still sufficient for subsequent experiment.Hence,only silk fibroin cocoon slices served for subsequent experiments.We subcutaneously implanted silk fibroin cocoon slices into rabbits and H&E staining results demonstrated cellular infiltration during 4 weeks after implantation.The observed cells were expected to be inflammatory cells.Therefore,we tested the presence and the activity of MMP-2 enzymes from inflammatory cells including monocytes,unpolarized and polarized macrophages M1 using gelatin zymography method.Results revealed that all monocytes and macrophage cells exhibit a time dependent secretion of MMP-2 enzyme.Two bands of apparent molecular weight of 62 and 72 k Da were detected.The 72 k Da band represented a pro-form of MMP-2,whereas the 62 k Da band represented an active form.Monocyte cells THP1 and unpolarized macrophages only secrete MMP-2 in pro-form states.Slight amount of active MMP-2 was detected on day 5 after macrophage polarization and the large amounts of active MMP-2 was found in conditioned media from macrophage M1 at day 7.It is known that different phases of bone healing are regulated by different type of cells mainly macrophages,osteoblasts and osteoclasts.Of particular interest,macrophages have been shown to be the first responder after the implantation of SFbased materials.Our results showed that macrophages M1 can serve as an affordable source of MMP-2 enzymes and in vitro model for MMP-mediated degradation.Sterile silk fibroin slices were therefore co-cultured with or without inflammatory macrophages M1 and incubated over a period of 4 weeks.Mass weight was measured to monitor quantitative changes during the course of degradation.Slices were observed under Scanning Electron Microscopy to visualize morphological changes.Upon the exposure of silk fibroin cocoon slices to macrophage-secreted MMP-2,the degradation rate was not significantly different in all experimental groups within the first three days.The possible reason was that MMP-2 was still in its pro-form state.The quantity and the activity of MMP-2 in M1-conditioned culture media was sufficient to cause the difference in the degradation of silk fibroin slices after 7 days.After 28 days,silk fibroin incorporated with high catalytic activity showed a great mass loss(B1,38.07%)compared with silk fibroin slices incorporated with moderate(B2,23.40%)and low catalytic activity(B3,11.93%).The reduction in dry weight was slight even none when silk fibroin cocoon slices were cultured in cell-free media implying that degradation was likely mediated by MMP-2 secreted by macrophages.Morphological analysis using Scanning Electron Microscopy showed that the incorporation of MMP-2 cleavable linkers into silk fibroin heavy chain promoted the degradation at certain level.Wild type silk fibroin slices retained their original structure while slices from transgenic groups TSL-B1,TSL-B2 and TSL-B3showed particulate debris on the surface.4.The release rate of BMP-2 followed similar trends with the catalytic activity ofthe inserted MMP-2 linkers peptide.Referred to the design of the target protein B1,B2,and B3,the embedded BMP-2 specifically necessitate the cleavage of MMP-2 linker to be released.We set in vitro MMP-triggered release system in which macrophages were used as a source of MMP-2 enzymes.Soluble aqueous solution was prior used to ascertain the susceptibility of cleavable linkers PAGLWA,PLGLWA,VLGLWA and the freeing of BMP-2 protein in the presence of MMP-2 enzymes secreted by M1 macrophage cells.Silk fibroin solution was reacted with M1 macrophage conditioned media that served as a source of MMP-2 enzymes.After reaction,aliquots were withdrawn and fractionated on 12% polyacrylamide gel.The change in the molecular weight was verified by Western blotting using BMP-2 antibody.Results showed that in enzyme conditions,the band corresponding to the exogenous protein was reduced in molecular weight.Hence,it could be verified that the cleavable linkers were recognized and cleaved by active MMP-2 enzyme.The cleavage of sensitive sites separated BMP-2 protein from the rest silk fibroin sequence.The molecular weight of BMP-2 fusion protein was not declined in cell-free culture media,suggesting that the release of BMP-2 growth factor was specifically triggered by the presence of active MMP-2.Macrophage-mediated release was adapted to verify whether the variation in catalytic activity of MMP-2 linkers may affect the release kinetics of BMP-2.Silk fibroin slices from SF-B1,SF-B2 and SF-B3 with identical content of BMP-2 were subjected to release analyses in presence of M1 macrophages.At each time points,the content of BMP-2 was quantified in the release media using ELISA kit and in the remained insoluble fibers using Western blotting analysis.The obtained results demonstrated that after 4 weeks of culture with macrophage cells,the release kinetic significantly differed between three groups.SF-B1 exhibited the fastest release kinetics(52.92% of BMP-2),followed by SF-B2(32.24% of BMP-2)and SF-B1(11.74% of BMP-2)suggesting that the kinetics of drug release(BMP-2)followed similar trends as the catalytic activity(MMP-2).A decrease in the values of catalytic activity was related to a reduction in the BMP-2 release and as the kinetic activity increases,so does the BMP-2 release rate.No BMP-2 release was detected in any of the samples when the culture was cell-free even after long-term incubation.We have successfully obtained a new degradable silk material that responsively release bone growth factor BMP-2 in the presence of MMP-2.The effect of released BMP-2 on the proliferation of mesenchymal stem cell(MSC)was assessed through DNA quantification analysis.After 1 week,in vitro results showed the DNA content of the MSCs co-cultured with cocoon slices from WT was significantly lower than TSL-B1,TSL-B2,and TSL-B3,implying that the released BMP-2 from transgenic silk fibroin cocoon slices presented biological capacity to enhance the proliferation of osteo-progenitor cells,a preliminary step for bone regeneration process.All together,we created new silk fibroin materials responsive to MMP-2 enzymes as biological stimuli secreted in the process of bone regeneration.Compared to conventional strategies,our developed silk materials can sustainably release BMP-2 when exposed to matrix metalloproteinases secreted by macrophages.At the same time,our study provides a new strategy for controlling the degradation of silk scaffolds in bone regeneration engineering...