Biological Evaluation Of Human Degenerated Nucleus Pulposus Cells In Functionalized Self-assembling Peptide Nanofiber Hydrogel Scaffolds

BackgroundLow back pain (LBP) is a common disease that affects both the quality of life and the labor capacity of patients and results in a large economic burden due to the use of health services. Although the causes of LBP are thought to be multifactorial, intervertebral disc (IVD) degeneration is a significant cause. Current clinical therapies for IVD degeneration typically start with nonoperative treatments including physiotherapy or pain medication. Surgeries such as disc decompression, fusion or disc arthroplasty are employed as the last resort and may cause many complications such as adjacent segment degeneration and loss of spinal mobility. Furthermore, these treatments eliminate symptoms rather than repair IVD. Therefore, IVD degeneration therapies should not be focused on minimizing the symptoms but instead pay more attention to the restoration of the IVD.Many studies have demonstrated that the decrease of the extracellular matrix (ECM), in particular collagen II and aggrecan, secreted by nucleus pulposus cells (NPCs) plays important roles in the process of IVD degeneration. Over the past few years, many biological approaches have been used to promote the biological activity of NPCs and to stimulate the regeneration of the IVD, such as the injection of growth factors, biomaterials and cells. Although tremendous progress has been accomplished, therapeutic limits still exist when these approaches are used alone. The growth factors always degenerate too quickly to result in long-time bioactivity. The biomaterials usually do not have sufficient biological behavior and generate spacious injuries during the process of implantation. In addition, the transplanted cells have low survival rates due to the harsh internal environment of the IVD. Thus, one promising therapeutic strategy should be to combine growth factors, biomaterials and cells together.Recently, self-assembling peptide nanofiber scaffolds have been successfully used as promising biomaterials in tissue engineering and regenerative medicine. These scaffolds exhibit excellent biocompatibility and bioactivity. As one of the most commonly used self-assembling nanofiber peptides, RADA16-Ⅰ (AcN-RADARADARADARADA-CONH2) can undergo spontaneous assembly into well-ordered interwoven nanofibers through alternating hydrophobic and hydrophilic amino acids under physiological conditions and further form an ECM-like hydrogel both rapidly and orderly. These properties enable the cells encapsulated in the self-assembling peptide to be used in a three-dimension (3D) culture. Additionally, unlike other natural hydrogels derived from animals, RADA16is composed of synthetic amino acids and can be tailor-made according to specific requirements. Various bioactive short peptide motifs that act as growth factors or ECM macromolecules have been incorporated into the C-terminus of RADA16to promote cell migration, proliferation and differentiation. These functionalized self-assembling peptide nanofiber scaffolds have exhibited extensive potential in bone, vascular and neural regeneration.The bone morphogenetic protein-7(BMP7) is an important member of the transforming growth factor-β superfamily. It has been observed that BMP7can not only stimulate the synthesis of proteoglycans and collagen type II in vitro, but also increase the expression of ECM and the disc height of the degenerated disc in rabbits. However, the clinical application of BMP7is limited due to its short half-life. As the BMP7has approximately400peptides, it is difficult to conjugate the whole BMP7to nanomaterials to prolong its half-life. More recently, the study had observed that the short functional peptide, SNVILKKYRN, KPSSAPTQLN and KAISVLYFDDS, were very important short bioactive motifs of the BMP7. More importantly, these short motifs could be easily chemically functionalized onto nanoscaled biomaterials.Therefore, in the present study, we designed three new functionalized peptides that conjugated the short functional motifs onto the C-terminus of RADA16-I and then further mixed the functionalized peptides solution with the RAD A16-1solution at a volume ratio of1:1to form the novel functionalized peptide nanofiber scaffolds. Then, the biocompatibilities and bioactivities of these three functionalized self-assembling peptides for human degenerated nucleus pulposus cells (NPCs) were further observed in vitro. We expected that these functionalized self-assembling peptide nanofiber hydrogel scaffolds could not only provide ECM-like three-dimensional microenvironments for hNPCs, but also have BMP7-like bioactivities and have long half-life. Finally, the bioactivities of human degenerated NPCs could be improved by the functionalized self-assembling peptides, and the intervertebral disc degeneration ultimately repaired.Objective1. To construct three functionalized self-assembling peptides based on the self-assembling peptide RADA16-I and the C-terminus of RAD A16-1were extended by three short functional motifs of BMP7, respectively.2. To observe the hydrogel properties and microstructural characteristics of RADA16-I and functionalized self-assembling peptides.3. To study the biocompatibilities and bioactivities of these designer self-assembling peptides for hNPCs, and then to choose the most suitable functionalized self-assembling peptide for potential application in NP tissue regeneration.4. To compare the bioactivities of functionalized self-assembling peptides and BMP7for hNPCs.5. To observe the feasibility that the IVD degeneration is repaired by functionalized self-assembling peptides nanofiber hydrogel scaffolds in vitro.Methods1. Preparation of self-assembling peptide solutionsRADA16-I(AcN-RADARADARADARADA-CONH2), RSNV(AcN-RADARADARADARADA-GG-SNVILKKYRN-CONH2), RKPS (AcN-RADARADARADARADA-GG-KPSSAPTQLN-CONH2) and RKAI(AcN-RADARADARADARADA-GG-KAISVLYFDDS-CONH2) were solid-phase synthesized. These peptide powders were dissolved in MilliQ water at a final concentration of1%(w/v,10mg/mL), sonicated for30minutes and further filter-sterilized for future use. Then, these functionalized self-assembling peptide solutions were mixed at a volume ratio of1:1with a1%RADA16-I solution to obtain1%functionalized RAD/SNV, RAD/KPS and RAD/KAI peptide mixtures, respectively.2. Preparation of self-assembling peptide nanofiber hydrogel scaffoldsThe transwell inserts were placed into a24-well culture plate with400μL of the culture medium placed in each well. Then,100μL of the peptide solution (RADA16-I, RAD/SNV, RAD/KPS or RAD/KAI) was added directly into each of the inserts and then incubated at37℃for more than1hour for gelation. Next,400μL of the culture medium was gently loaded onto the gel and then incubated overnight at37℃. After gelatinization, the medium was carefully removed and changed at least twice to equilibrate the growth environment to physiological pH.3. Detection of the structural characteristics of self-assembling peptidesThe ability of self-assembling nanofibers, secondary structures and microstructures of the designer self-assembling peptides were detected by atomic force microscopy (AFM), circular dichorism (CD), scanning electron microscopy (SEM), respectively.4. Isolation and culture of human degenerated NPCsHuman degenerated IVD tissue was obtained from the disc level L5-S1of a46-year-old woman, who had undergone a fusion surgery due to lumbar disc herniation. Human degenerated NPCs were isolated via collagenase digestion method, and cultured and passaged to the third passage for further using.5. Three-dimensional (3D) culture of hNPCsTranswell inserts were placed into a24-well culture plate with400μL of the culture medium in each well, and the hNPCs were suspended in10%sucrose before seeding. Then,5×104hNPCs in20μL of the cell suspension were rapidly mixed with100μL of the peptide solution, and then, the cell/peptide mixture was immediately moved into the insert. Then,400μL of the culture medium was very slowly added onto the hydrogel for gelation at37℃for10min, the medium was changed for another30min. The medium was further changed at least twice to enhance self-assembly and to equilibrate the physiological pH.6. Biocompatibilities of designer self-assembling peptide nanofiber hydrogel scaffolds for hNPCsHuman degenerated NPCs were3D cultured in designer self-assembling peptide nanofiber hydrogel scaffolds. After culturing for7days and14days, the adhesion of hNPCs in designer self-assembling peptide nanofiber hydrogel scaffolds were observed by SEM. After culturing for1day,3days and7days, the viabilities/cytotoxicities of hNPCs were detected by live/dead assays.7. Bioactivities of designer self-assembling peptide nanofiber hydrogel scaffolds for hNPCsAfter hNPCs cultured on the surfaces of designer self-assembling peptide nanofiber hydrogel scaffolds for1day,3days and7days, the cell adhesion and morphology on the surface of these hydrogel scaffolds and the migrations were observed by laser confoncal microscopy (LCM). Human degenerated NPCs were3D cultured in the peptide hydrogels, and hNPCs were cultured in culture plates without or with10ng/mL BMP7that were used as the negative control group (NCG) and positive control group (PCG). After culturing for1day,3days,5days and7days, the cell proliferation were detected by CCK-8assay. After culturing for7days,14days and28days, the secretions of collagen I, collagen II and aggrecan were detected via Elisa assays, and the mRNA expression of collagen I, collagen II, collagen X, aggrecan, Sox-9and versican were analyzed by qRT-PCR.Results1. Synthesis and self-assembly of peptidesThe designed SNVI, KPSS and KAIS peptides were synthesized via direct attachment to the C-terminal of the self-assembling peptide RADA16-I using solid phase synthesis. After the peptide powders were dissolved and cultured in transwells, all the peptides (RADA16-I, RAD/SNV, RAD/KPS and RAD/KAI) could self-assemble and form transparent viscous hydrogels under physiological conditions.2. AFM assayUniform and interweaved nanofibers were observed in all designer self-assembling peptides. However, the density and diameters of the nanofibers in these self-assembling peptides were different. The average diameters of the nanofibers in RAD/SNV, RAD/KPS and RAD/KAI were larger than these in RAD A16-1. Moreover, the length of these nanofibers ranged from several hundreds of nanometers to a few micrometers.3. CD assayTypical spectrums for β-sheet structures with a negative maximum mole residue ellipticity at216nm and a positive maximum at195nm were observed in all the designer self-assembling peptides RADA16-I, RAD/SNV, RAD/KPS and RAD/KAI. However, compared with the RADA16-I solutions, the intensities of the mole residue ellipticity at216nm and195nm of the RAD/SNV and RAD/KPS solutions decreased, while these in RAD/KAI increased.4. SEM assayThe self-assembling nanofibers were formed in all the designer self-assembling peptides and that the nanofibers interweaved into porous structures with diameters of10-40nm and pores between5-200nm. These structures were similar to ECM and allowed the cells to attach tightly and nutrition to be exchanged easily. After the hNPCs3D cultured in the peptide hydrogel scaffolds, the hNPCs could be truly embedded in the hydrogel scaffolds. The cell bodies exhibited a3D spindle-shaped morphology and were tightly adhered to the surrounding nanofibers in the scaffolds by stretching a large number of pseudopodia after14days.5. Viability of hNPCs cultured in the designer self-assembling peptide hydrogel scaffoldsAll the designer self-assembling peptide hydrogel scaffolds had extremely low toxicity for the hNPCs and with time the cytotoxicities were not significantly increased (P>0.05). There was no significance difference between RADA16-Ⅰ and the functionalized self-assembling peptide hydrogel scaffolds during the7days in terms of the survival rates of the hNPCs (p>0.05). All of the cell survival rates were greater than86%after culturing for7days.6. Migration of hNPCs cultured in the designer self-assembling peptide hydrogel scaffoldsAfter culturing on the surface of the self-assembling peptide hydrogel scaffolds for1day, the hNPCs had attached and exhibited typical spindle-shaped morphology. Cell clusters were only observed on RAD/SNV and RAD/KPS after culturing for7days.The reconstructed3D images further demonstrated that the hNPCs seeded on the surface of all the hydrogel scaffolds could spontaneously migrate several hundreds of micrometers in depth and had a positive correlation with the cultural time. After culturing for7days, the migration distance of RAD/SNV(290μm) and RAD/KPS (295μm) were significantly longer than that of RAD/KAI (169μm), and RADA16-I had the shortest migration distance (128μm).7. Proliferation of hNPCs cultured in the designer self-assembling peptide hydrogel scaffoldsThe hNPCs could proliferate both in the peptide hydrogel scaffolds and the control groups. After3D culturing for7days, the cells seeded in the hydrogel scaffolds exhibited a significantly higher proliferation speed than that in the control groups (P<0.05), and there was no obviously difference between the NCG and PCG (P>0.05). Moreover, the cell number in both RAD/SNV and RAD/KPS were significantly higher than these in RADA16-I and RAD/KAI (P<0.05), and there was no obviously difference between RAD/SNV andRAD/KPS (P>0.05).8. ECM secretion of hNPCs cultured in the designer self-assembling peptide hydrogel scaffoldsAfter culturing for28days, compared to NCG, the mount of aggrecan and collagen II secreted by hNPCs in designer peptide hydrogel scaffolds were significantly increased and collagen I were obviously decreased (P<0.05), while all the change were lower than these in PCG (P<0.05). Among the three functionalized peptide hydrogel scaffolds, the abilities of promoting collagen II and aggrecan secretion and inhibiting collagen I secretion were significantly superior than those in RADA16-I and RAD/KAI RAD/KAI (P<0.05), and the secretion of aggrecan in RAD/KPS was higher than that in RAD/SNV (P<0.05), while there were no significantly differences in secretion of collagen I and collagen II between RAD/SNV and RAD/KPS (P>0.05). Furthermore, the ECM were mainly secreted by hNPCs within the first14days, especially the first7days, and could maintain at least for28days.9. Genes expression of hNPCs cultured in the designer self-assembling peptide hydrogel scaffoldsAfter culturing for28days, compared to NCG, the mRNA of collagen II, aggrecan and Sox-9expressed by hNPCs in designer peptide hydrogel scaffolds were significantly increased and the mRNA of collagen I and versican expressed were obviously decreased (P<0.05), while all the change were lower than these in PCG (P<0.05). There was no significant statistically differences of the collagen X mRNA expression among all the groups. Among the three functionalized peptide hydrogel scaffolds, although the abilities of promoting collagen Ⅱ, aggrecan and Sox-9mRNA expression and inhibiting collagen I and versican mRNA expression were significantly superior than those in RADA16-I and RAD/KAI (P<0.05), the expression of aggrecan and Sox-9were obviously higher than those in RAD/SNV (P<0.05), and there were no significantly differences in expression of collagen I, collagen Ⅱ and versican between RAD/SNV and RAD/KPS (P>0.05). Furthermore, the ECM were mainly secreted by hNPCs within the first14days, especially the first 7days, and could maintain at least for28days.Conclusion1. Functionalized self-assembling peptides can be constructed via conjugating short functional motifs of BMP7onto the C-terminus of RAD Al6-1.2. All the designer self-assembling peptides RADA16-I, RAD/SNV, RAD/KPS and RAD/KAI can self-assemble into nanofiber hydrogel scaffolds in physiological conditons in vitro.3. All the designer self-assembling peptides RADA16-I, RAD/SNV, RAD/KPS and RAD/KAI have excellent biocompatibilities for human degenerated NPCs.4. All the designer self-assembling peptides RADA16-I, RAD/SNV, RAD/KPS and RAD/KAI have excellent bioactivities for human degenerated NPCs, but less than BMP7, and BMP7> RAD/KPS≥RAD/SNV>RAD/KAI≥RADA16-I.5. RAD/KPS has the BMP7-like bioactivities for human degenerated NPCs, and the bioactivities can be maintained at least more than28days.Thence, as a novel injectable functionalized self-assembling peptide nanofiber hydrogel scaffold, RAD/KPS can effectively integrate BMP7into nanoscale biomaterial. It can not only provide ECM-like microstructure for human degenerated NPCs, but also have the BMP7-like bioactivities for hNPCs, and can prolong the half-life of BMP7. Therefore, the functional self-assembling peptide RAD-KPS exhibit great potential for IVD degeneration biological treatment.