| For the first time, we report an experimental design, development and evaluation of in situ nanomechanics of cell-biomaterial composites for tissue engineering applications. A blend of two biopolymers (Chitosan and Polygalacturonic acid) was chosen with hydroxyapatite nanoparticles to mimic the natural bone (Chi-PgA-HAP). These substrates swell in presence of cell culture media as found by our in situ topographical, chemical and mechanical analyses for 48 days. Biocompatibility experiments were performed using human osteoblasts (CRL 11732) and results indicate that these substrates favor cell adhesion and proliferation. Over cell culture duration of 22 days, osteoblasts generated bone-like nodules onto Chi-PgA-HAP substrates in absence of any stimulants for osteogenesis. In vitro generated bone nodule mimics the structure, chemistry and nanomechanical properties of natural bone as revealed by Atomic Force Microscopy (AFM), and Fourier Transform Infrared (FTIR) analyses on bone nodule. Hierarchically organized extracellular matrix of bone nodule consisting of mineralized collagen fibers, fibrils and mineral deposits was revealed by high resolution AFM images. FTIR analyses on bone nodule suggests that bone nodule is chemically similar to human bone due to the presence of major bands of collagen (Amide I, II, and III) and biological apatite (CO32- and HPO 43). Live cell and cell-substrate nanoindentation experiments on cell seeded Chi-PgA-HAP nanocomposites were conducted under the physiological conditions (cell culture Name: Rohit Khanna medium; 37°C) for culture duration of 1, 4, 8, and 22 days, respectively. Dynamic mechanical responses of cells are indicated by stiffer elastic responses of flat cells as compared to round cells. Dynamic mechanical behavior of cell-degrading substrate is indicated by their corresponding elastic moduli: ECell-Chi-PgA-HAP, 1 day, 2000 nm= 10.3-20.2 MPa, ECell-Chi-PgA-HAP, 4 days, 2000 nm = 5.2-8.4 MPa and ECell-Chi-PgA-HAP. 8 days, 2000 nm= 6.2-16.7 MPa. The spread in elastic moduli indicate the dynamic mechanical responses from varying depths of cell-substrate composites. Substrate dependent differences in mechanical properties of cells were found by performing similar experiments on tissue culture polystyrene. The major conclusion from this research is that microenvironment plays a key role in modulating the mechanical behavior of cell-substrate interactions. |
