Biomechanics And Biocompatibility Evaluation Of A Functional Self-assembling Peptide Nanofiber Hydrogel Scaffold RADKPS

Degenerative disc disease is frequently occurring in clinical, which is closely related to genetic factors, age, sex, occupation, smoking and other factors. On the basis of cellular number decrease or cellular activity decrease in nucleus pulposus(NP), and the following decrease of extracellular matrix components such as type II collagen and proteoglycans, the water content and biomechanical condition of intervertebral disc(IVD) get changed finally. This process is an important pathological basis of degenerative disc disease. IVD is the largest avascular and nerveless tissue in human body, whose inner microenvironment is relatively maintaining a hypoxia, hypertonic and low PH condition different from other tissues and organs. And the microenvironment will get even harsher when the IVD gets degenerated. Hence, in this harsh microenvironment, degenerative disc couldn’t c local progenitor cells like bone, muscle, liver and other tissues and organs to endogenously repair degenerated intervertebral disc, although a lot of independent researches from several laboratories have confirmed the exist of mesenchymal stem cells in degenerative IVD just like bone, muscle, liver and other tissues and organs. Restricted by the harsh IVD microenvironment and relatively insufficient growth factors in degenerated IVD, the degenerated IVD couldn’t recruit native stem cells and activate local progenitor cells well. This must be an important reason why degenerated IVD is limited in endogenously repair. Conservative treatments such as hormone stoss therapy, anti-inflammatory and analgesic, traction therapy and operative treatment such as removal of NP, intervertebral decompression and fusion are all symptomatic treatment which couldn’t fundamentally cure degenerative disc disease by block the course of IVD degeneration from its pathological basis.Numerous researches in vitro and in vivo reveal that the growth factor bone morphogenetic protein-7(BMP-7) can not only significantly reduce the apoptosis of NP cells in vitro culture conditions, but also effectively increase the secretion of proteoglycans, type II collagen, water content of the NP and the intervertebral height in the degenerated IVD of rabbits, dogs. AS that were reported by many researches, the self-assembling nanofiber peptides RADA16-I(AcN-RADARADARADARADA-CONH2) possess the properties of classic hydrogel scaffolds such as high water content similar to human NP, can be injected into IVD, and can self-assemble into a gel-like nanofiber scaffold for three-dimensional attachment of cells with the high ionic salt concentrations of IVD. The C- terminal of RADA16-I is capable to incorporate short bioactive peptide motifs(no more than 15 amino acids) that act as growth factors or ECM macromolecules. And then the material turn to functionalized self-assembling peptide nanofiber scaffolds, of which the biological activity is effectively promoted and the function is specific for a certain tissue. Functionalized self-assembling peptide nanofiber scaffolds, RADA16-I with short bioactive peptide motifs incorporated in the C- terminal, fail to gelatinize well, but when it is mixed with RADA16-I in equal proportions, the material return to gelatinize well. Till now, Functionalized self-assembling peptide nanofiber scaffolds compounded based on this biological properties of RADA16-I have been widely used in many areas such as bone, nerves, heart and blood vessel regeneration. And since 2007 mesenchymal stem cells(MSCs) having similar capabilities to human marrow-derived MSCs from human degenerated IVD, NP, annulus fibrosus(AF) and cartilaginous endplate were consecutively demonstrated by Risbud, Feng, Blanco and Liu.So the methods of recruiting and activating IVD MSVs with functionalized self-assembling peptide nanofiber scaffolds through modifying RADA16-I with short bioactive peptide motifs of BMP-7 may bring new ideas for the biological treatment for degenerative disc disease.Firstly, our research team has successfully incorporated three very important short bioactive motif of the BMP7 into the C-terminal of RADA16-I, so that three functionalized peptide nanofiber scaffold, RADA-SNVI, RADA-KPSS and RADA-KAIS,were originally compounded. And then after mixing each of them with RADA16-I in equal proportions, three novel functionalized peptide nanofiber scaffold, RADSNV, RADKPS and RADKAI, were finally got. Through previous in vitro experiments, we found that RADKPS out of the three novel functionalized peptide nanofiber scaffold is more excellent in recruit human degenerative NP cells to move in, and effectively improve cellular proliferation, significantly increase proteoglycan, type II collagen and other important extracellular matrix gene expression and secretion. Whether the novel functionalized peptide nanofiber scaffold RADKPS is able to help recruit and activate surrounding MSCs, improve cellular number and activity, enhance cellular proliferation and important extracellular matrix gene expression and secretion, which will be directly related to the possibility of promote endogenous repair of degenerated IVD through block the pathological basis of degenerative disc disease.Normal NP is a naturally highly hydrated gelatinous, viscoelastic tissue of high moisture content of 70%-85%. So it is necessary for tissue engineering materials of NP to possess similar biomechanical characteristic. Meanwhile the biocompatibility of implantable biomaterials must be well tested before in vivo experiments to ensure the safety of its application in vivo.In order to learn more about the biomechanics feature of the novel functionalized peptide nanofiber scaffold RADKPS, and to provide in vivo experimental evidence for the use of novel functionalized peptide nanofiber scaffold RADKPS in animals and even in human degenerated IVD, this paper designed biomechanical and biocompatibility test for the novel functionalized peptide nanofiber scaffold RADKPS.Objective:To test the biomechanical properties of RADKPS through a rheometer and Atomic force microscope(AFM).To evaluation the biocompatibility of RADKPS, through which providing experimental basis for the use of RADKPS in vivo studies in the field of regeneration of degenerated NP.Methods:1. The young’s modulus of RADKPS was detected by AFM, and the rheological property(viscoelasticity) of RADKPS was detected through by a rheometer.2. Cell toxicity test, in-vitro IVD organs of rabbits implantation test, hemolysis test and subcutaneous injection of mice were conducted to detected the cytocompatibility, blood compatibility and tissue compatibility.Results:1. The AFM detection revealed that the young’s modulus of RADKPS was 0.519±0.244 KPa. And the rheological property test demonstrated that the storage modulus(G’) of 1%RADKPS was always bigger than its loss modulus(G’’), which well showed the good viscoelasticity of the novel functionalized peptide nanofiber scaffold RADKPS. And the rheological property test also showed that G’ and G’’ had nothing to do with Shear Frequency, which convince the reticulate structure of 1%RADKPS. RADKPS is similar with NP tissue in biomechanical characteristic.2. The functional self-assembling peptide solution RADKPS presented a homogeneous transparent hydrogel like state as long as in touch with L-DMEM. After BMSCs cultured in RADKPS for 7 days, scanning electron microscopy(SEM) showed reliable attachment between the cells and the scaffolds. And cell viability was maintained over 90 percent. MTT assay revealed that RADKPS of 0.1% 、 0.05% 、 0.025% concentrations dependently increased the proliferation of BMSCs. The result of hemolytic assay revealed that the hemolysis ratios of all the RADKPS solutions with different concentrations were less than 5%, which meet the requirement well of hemolytic assay standard for medical biomaterials. Hypodermic injection assay in KM mouse showed no vesicle, erythema and eschar formation around the injection area 28 days later. Meanwhile, HE staining showed inflammatory cells infiltration, such as lymphocytes, and the hydrogel scaffold had already been replaced by fibrous connective tissue. All these phenomenon revealed good tissue compatibility.Conclusion1.The results showed that the young’s modulus of the novel functionalized peptide nanofiber scaffold RADKPS was 0.519 ± 0.244 KPa, in which partial initial mechanical support could be offered by RADKPS after being injected into degenerated IVD. And RADKPS was able to self-assemble into viscoelastic Hydrogel with a reticulate structure.2. The novel functional self-assembling peptide nanofiber hydrogel scaffold RADKPS showed satisfactory biocompatibility and biological reliability, which would be suitable for tissue engineering repair and regeneration of nucleus pulposus tissue.