Studies On Tissue-engineered Bone Modified By Anchorage Of Bio-peptides Via Ammonia Plasma Treatment

BackgroundMassive bone defects can lead to delayed union or nonunion of fractures. It is common in traumatic cause of disability. The family, economic and social problems caused by disability should not be ignored. Although autogenous bone grafting is still the gold standard in repair of bony defects, it is also limited in clinical application. We have a new choice in treatment of bony defects with the development of tissue-engineered bone. Tissue-engineered scaffolds grafted into defects as temporary substitutes should have the biological characteristics similarly to autogenous bone. Therefore, seeking for appropriate bone substitutes becomes a principle task in bone tissue engineering.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. It is approved by-the FDA for clinical use in the 1970s. Poly-D,L-lactide acid (PDLLA) has a more appropriate degradability and can be chose as a bone substitute. However, low hydrophilicity/ surface energy and lack of bioactive sites have been shown as two negative factors to affect cell adhesion, proliferation and osteogenic differentiation in three-D biomaterial scaffolds. To solve these problems, many researches have attempted to modify the PDLLA surface. Surface modification methods, such as surface coating, surface chemical modification and plasma treatment, etc. were applied.Coating the surface with extracellular matrix (ECM) proteins provides an adhesive interface between the polymer scaffold surface and cells that resemble the native cellular milieu. However, it is often time-consuming and expensive. Moreover, passive adsorption could induce the competitive adsorption of other materials in the system and change the configuration of the adsorbed protein molecules. As a result, its cell binding activity would be influenced and reduced. An improved method of the chemical modification for promotion of the anchorage is the covalently conjugating the bioactive molecules to biomaterials. The covalent couple promotes effective and stable conjugation and has been recommended by several researchers. However, surfaces of organic polymers, such as PDLLA, customarily are short of bioactive groups. Moreover, excess use of chemical reagents might lead to complicated reaction, over side reaction, and difficulty to dispose excess reagents.Low-temperature plasma technique was demonstrated to be an efficient method for modifying the surface of biomaterials without changing the bulk properties. It improves the hydrophilicity/surface energy and roughness of the polymer. The plasma technique easily induces the desired groups or chains onto the surface of the polymer, such as amine group, hydroxyl group, etc. The active groups on the modified polymer surface will be a chance to further conjugate the bioactive molecules (collagen, bio-peptides, etc.). Compared with chemical modification, the plasma treatment has merits in simple process, easy management, non-pollution, no impact on bulk properties, no limit to surface shape and high sterilization ability. However, the active radicals on polymer surface will decrease with time.So-called'biomimetic'bone, the artificial bioactive scaffold may mimics many roles of ECM in vitro. Investigators used to utilize ECM proteins, including collagen, laminin and fibronectin, etc., as coatings on polymer surfaces. Later, the native ECM protein was found tending to be randomly folded upon such that the receptor binding domains are not always sterically available. However, the short peptide sequences are relatively more stable during the modification process than long chain proteins. In addition, short peptide sequences can be massively synthesized in laboratories more economically. Therefore, numerous biomaterials have been anchored by bio-peptides for academic studies and clinical applications. The most commonly used peptide for surface modification is Arg-Gly-Asp (RGD), the signaling domain derived from ECM proteins. It is one of the most extensively investigated ligand for integrins. Once the RGD sequence is recognized by and binds to integrins, it will initiate an integrin-mediated cell adhesion process and activate signal transduction between the cell and ECM, thus influencing cell behavior on the substrate including proliferation, differentiation, apoptosis, survival and migration.In the study, we explore the convenient and effective method to anchor the surface of PDLLA with RGD-containing bio-peptides, and further investigate the osteogeneic ability of the bio-modified PDLLA in vitro and in vivo. In order to promote amide linkage-anchorage to the surface of the PDLLA scaffolds, we explored the variable quantities of N-containing groups conjugated to PDLLA scaffolds in different treatment parameters of NH3 plasma, and consequently looked for reaction between-NH2 and carboxyl (-COOH) end group of GRGDS peptides. The resultant PDLLA scaffolds have acquired the stable bioactivity. Meanwhile, the drawback of instability of groups on plasma-treated PDLLA surfaces was overcome.Objectives1. To study the feasibility of preparation of the poly-L), L-lactide acid (PDLLA) scaffolds treated by ammonia plasma and subsequent conjugation of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide linkage formation.2. To establish a method of the isolation, culture and identification of Sprague-Dawley (SD) rats derived bone marrow mesenchymal stem cells (BMSCs) in vitro, and culture and identify the BMSCs transfected with red fluorescent protein by lentivirus (RFP-BMSCs).3. To study the adhesion, proliferation, metabolism and mineralization of RFP-BMSCs in ammonia plasma treated and GRGDS anchored PDLLA scaffolds, and investigate the effects of the bio-modification on osteogenic abilities in vitro.4. To establish a SD rat model of 8-mm femoral defect, and study the ability of the new type of bio-modified PDLLA scaffold to repair the bone defect in vivo.Methods1. Preparation and detection of PDLLA scaffolds modified by ammonia plasma treated and GRGDS anchored(1) Fabrication of three-D PDLLA scaffoldsPrepare the three-D PDLLA scaffolds with partial modification to our previous patent of "Pressure-enhanced Technology". The finished products were cylindrical sponge-like scaffolds (8mm×8mm×10mm). The scaffolds were sliced into uniform wafers with 1 mm of thickness prior to use.(2) Preparation and detection of aminated PDLLA (A/PDLLA) scaffoldsThe wafers of PDLLA scaffolds were placed in the plasma reactor chamber. The chamber was evacuated to less than 10 Pa before filling with the NH3. After the pressure of the chamber was stabilized at 30Pa, plasma treatment was initiated for 2, 5,10,20 and 30 min using a power of 50 W and pulsed frequency of 13.56 MHz. Observation of surface topography and measurement of aperture, porosity and surface contact angle (water) were performed on PDLLA scaffolds before and after plasma treatment.(3) Preparation and detection of peptides conjugated A/PDLLA (PA/PDLLA) scaffoldsThe A/PDLLA scaffolds were immersed into the sterile FITC-GRGDS solution which contained EDC.HCl and NHS with the molar ratio of peptides, EDC.HCl and NHS was 1:1.5:1.5. The solution was brachytely sloshed in room temperature for 24hs. X-ray photoelectron spectroscopy (XPS) was performed on PDLLA scaffolds before and after plasma treatment and following conjugation of FITC-GRGDS in order to determine the changes of surface chemistry using a Ultra DLD spectrometer. The amounts of FITC-GRGDS conjugated to scaffolds were appraised by confocal laser scanning microscope and HPLC. 2. Isolation, culture and identification of SD rats derived BMSCs(1) Primary culture and identification of SD rats derived BMSCsPrimary BMSCs were harvested from the long bones of young adult SD rats by whole bone marrow adherence method. The isolated marrow was centrifuged, re-suspended in the growth medium, and seeded in 25 ml culture flask. The flasks were incubated in a humidified 5% CO2 incubator at 37℃. The cells were subcultured when they were 90% confluent. The cultured cells were observed through an inverted phase-contrast microscope. The cell growth curve was investigated by MTT detection. Expressed proteins of BMSCs (P3) include CD29, CD34, CD44 and CD45 were analyzed by flow cytometry. After 21 days of osteogenic induction, cells were identified by alkaline phosphatase (ALP) staining and alizarin red staining.(2) Culture and identification of SD rats derived RFP-BMSCs.The frozen RFP-BMSCs were resuscitated, and incubated in a humidified 5% CO2 incubator at 37℃. The cells were subcultured when they were 90% confluent. The cultured cells were observed through an inverted phase-contrast microscope. The cell growth curve was investigated by MTT detection. After 21 days of osteogenic induction, RFP-BMSCs (P4) were identified by alkaline phosphatase (ALP) staining and alizarin red staining. Moreover, cells were adipogenic induced until the appearance of more and larger lipid droplets, and identified by oil-red O staining.3. Co-culture of GRGDS-modified PDLLA scaffolds with seed cells in vitro(1) Group settingsPDLLA scaffolds were prepared in a diameter of 8mm-circle with 1mm-thickness and divided into 3 groups, Group P (untreated) as control group, Group A (PDLLA pre-treated with 20 min of NH3 plasma) and Group PA (PDLLA pre-treated with 20 min of NH3 plasma and conjugated GRGDS peptides).(2) Co-culture with RFP-BMSCsScaffolds in groups were seeded with osteogenic-induced RFP-BMSCs.①After seeding and continued culture for 1,2,4,6,8,10 and 12 days, cell proliferation and metabolism in scaffolds were detected by CyQuant NF and AlamarBlue reagents, respectively.②After seeding and continued culture for 7,14 and 21 days, cell adhesion and proliferation were observed through an inverted phase-contrast microscope. Meanwhile, staining of the calcification in vitro was studied by calcein fluorescent dye as well.(3) Co-culture with BMSCsScaffolds in groups were seeded with osteogenic-induced BMSCs and cultured for 3,7 and 14 days. Real time quantitative PCR (RT-qPCR) was used to detect the osteocalcin (OCN), collagenⅠ(Col-Ⅰ), ALP, bone morphogenetic protein 2 (BMP-2) and osteopontin (OPN) mRNA expression of BMSCs in scaffolds in each group. After seeding and continued culture for 7,14 and 21 days, ALP activities were measured. Scaffolds seeded with non-induced BMSCs were observed by scanning electron microscope (SEM) after continued cultured for 4 and 8 days.4. Repair of rat femoral defects using GRGDS-modified PDLLA scaffolds(1) Group settingsA total of 45 SD adult male rats (350-500 g body weight) were randomly divided into 3 groups, the femoral defect with no engineered scaffold as blank control group, with unmodified PDLLA/BMSCs as control group (Group PDLLA) and with peptides-modified PDLLA/BMSCs (Group PA/PDLLA) as experimental group. After 4,8 and 12 weeks of operation, five rats in each group were sacrificed, and the specimens were acquired.(2) Establishment of SD rat model of 8-mm femoral defectCylindrical sponge-like scaffolds were seeded with cell suspension by multi-point injection. The suspension was adjusted to a density of 2.0×107-3.0×107 cells/mL, and 100μL of the suspension was seeded into each scaffold. Surgical procedure is listed below:After anesthesia, the experimental rat was skin prepared, sterilized and towel draped. An incision was made along the long axis of the femoral shaft, and proximal and distal metaphysises were exposed by successive dissection. Shaft of femur was drilled and laterally fixed with a steel plate by 4 screws. The proximal and distal sides of femur were tightened up by 2 screws, respectively. After remove of the plate and screws, the shaft of the femur was truncated by a circular saw and an 8-mm femoral defect was established. The defect was fixed by steel plate and screws, and enhanced by stainless steel wires. Finally, a cylindrical scaffold seeded with cell suspension was grafted into the defect.(3) Acquirement of the specimens and observation indicatorsAnimals were sacrificed at each time point. Gross observation, X-ray, histological examination and real-time quantitative polymerase chain reaction were performed on the specimens.Results1. Results of the fabricated scaffolds(1) The three-D porous and interconnected architecture of the scaffolds' inner was observed by SEM with no difference among the 3 groups. Before and after ammonia plasma treatment, no porous and porosity difference of PDLLA was observed (P>0.05). With increase of ammonia plasma process time, water contact angle of A/PDLLA scaffolds'surface was decreased. The comparison between groups of 20-min and 30-min, P-value was 0.088. The remaining groups compared with each other, P-values were all lower than 0.001.(2) XPS survey spectra show the presence of nitrogen and sulfur in addition to carbon and oxygen after plasma treatment and conjugation. The peak of N 1s in the spectra increased with increasing the treatment time except at the 30 min. During each treatment, N 1s peak has accordingly further increased after conjugation of peptides. C 1s in the spectra from pristine samples was deconvoluted into four peaks. NH3 plasma treatment and FITC-GRGDS conjugation produced two new peaks with binding energies of 285.7 and 288.3 eV, which were attributed to-C-NH-(amine) and -C=O-NH- (amide) groups, respectively. NH3 plasma treatment brings about N-containing radicals, which are primarily amine groups.-Further conjugation results in the decline of amine groups and raise of amide groups.(3) Qualitative conjugation of the peptides to the scaffolds was visually exhibited by the fluorescent confocal images. The fluorescent intensities have obviously strengthened with the time of plasma treatment except the 30 min of plasma pre-treatment and peptides conjugation. Quantitative results of peptides anchored to the scaffolds were determined by HPLC. Conjugation was undetectable in the scaffolds with 0 (untreated),2 and 5 min of NH3 plasma pre-treatment and FITC-GRGDS conjugation. The peak of the anchored peptides appeared at 20 min of the NH3 plasma pre-treatment. It is significantly elevated from 10 min and reduced at 30min(P<0.01).2. Isolation, culture and identification of SD rats derived BMSCs(1) SD rats derived BMSCs and RFP-BMSCs were observed spindle and 2 or 3 processes in shape under the optical microscope. Additionally, RFP-BMSCs were exhibited intense red fluoresence under the fluorescent microscope. Both of the growth curves of BMSCs and RFP-BMSCs were observed as S-shapes.(2) The flow cytometry research has suggested that BMSCs were positive for CD29 and CD44, and negative for CD34 and CD45. Furthermore, both of BMSCs and RFP-BMSCs were positive for ALP staining and alizarin red staining. Oil-red O staining of RFP-BMSCs was also demonstrated a positive result.3. Co-culture of GRGDS-modified PDLLA scaffolds with seed cells in vitro(1) The RFP-BMSCs seeded on the 3 scaffolds all showed proliferative activity at different time points after cell seeding, and the cell numbers decreased significantly in the order of PA>A>P (P<0.001). The cell number was significantly greater in group PA than in group A at all the time points except for days 10 (P=0.077) and 12 (P=0.491), and gradually became similar with the passage of time. The metabolic changes of the cells follow a similar pattern of cell proliferation. RFP-BMSCs showed more active proliferation in group A and group PA than in group P. On days 14 and 21, the intensity of green fluorescence decreased in the order of group PA, A and P.(2) The results from qPCR showed that bone-related gene expression in Group PA was higher than Group P at 3 days. Also, it was higher than Group A except OCN mRNA on the same day. The fold changes of gene expression of OCN (13.13±1.28), Col-Ⅰ(23.71±6.51) and OPN (27.4±7.17) in Group A were the highest among the three groups at 7 days, and it was take second place in Group PA. Most of the gene expressions were elevated in all groups at 14 days. The fold changes of gene expression in Group A and Group PA were higher than the control. OCN (51.54±7.09), ALP (24.26±3.41) and BMP-2 (11.82±2.38) mRNA in Group PA was significantly higher than Group A (P<0.05). ALP activity rose continuously in groups A and PA. It was slightly declined in Group P at 21 days. There were no statistical significances in ALP between groups A and PA (P>0.05). But, both were higher than the controls (P<0.01). In groups P, A, and PA, there were statistical significances in ALP between each pair of groups at 14 and 21 days. ALP activity in Group PA was the highest among the three groups. Group P was the lowest one. The BMSCs showed better adhesion in group PA than in group A, and the cells in group P appeared more scattered under SEM.4. Repair of the rat femoral defects using GRGDS-modified PDLLA scaffolds(1) At 4 weeks after surgery, a few new cartilages and immature bone tissues were observed in bone ends and scaffolds'inner in Group PA/PDLLA. Eight weeks post-operation, the new bone increased and abundant of the mature osteocytes and woven bones appeared. The new bone tissues rebuilt gradually till 12 weeks post-operation, and abundant of woven bones or mature lamellar bones connected between the bone ends and the scaffolds. The boundary became indistinct. X-ray examination confirmed that the floccose high-dense imaging manifestation of callus was observed in Group PA/PDLLA at 4 weeks post-operation. The manifestations of the bone repair were better in Group PA/PDLLA than that in Group PDLLA at all of the time points post-operation. At 12 weeks after surgery, a great of amount of callus connected between the bone ends and the scaffolds. The density of the new bone was homogeneous and compact, and the bone defects became obscured.(2) At 12 weeks after surgery, there were statistical significances in fold changes of gene expression of OCN, Col-Ⅰ, ALP, BMP2 and OPN among the three groups of blank control, Group PDLLA and Group PA/PDLLA (P<0.001). Furthermore,95% confidence intervals for means in Group PDLLA and Group PA/PDLLA were not include "1". Gene expression in Group PA/PDLLA was the highest among the three groups. The blank control group was the lowest one. Conclusions1. NH3 plasma treatment promotes the anchorage of GRGDS peptides to the PDLLA scaffolds via the formation of amide linkage.2. The method of "whole bone marrow adherence" can effectively isolates and amplifies the SD derived BMSCs. The cultured cells express a combination of surface markers characteristic for BMSCs, and can be able to differentiate into osteoblasts. RFP-BMSCs have the ability to differentiate into osteoblasts or lipocytes. Both of BMSCs and RFP-BMSCs are appropriate for uses in the field of bone tissue engineering.3. Bioactive modification of PDLLA by ammonia treatment and conjugation with GRGDS peptides promotes the adhesion, proliferation, metabolism and mineralization of RFP-BMSCs seeded on PDLLA scaffolds. The treatment of ammonia plasma will promote early osteo-differentiation of BMSCs in PDLLA scaffolds, and bioactive modification of PDLLA may have better ossification in vitro.4. Bioactive modification of PDLLA by ammonia treatment and conjugation with GRGDS peptides may accelerates the repair of the rat femoral defects via up-regulation of the expression of osteogenesis-related genes.