(805e) Controllable Effects of Mechanical Moduli On Osteoblast Differentiation of Mesenchymal Stem Cells On Polyurethane Substrates

Statement of Purpose: Cell therapy, which is the transplantation of autologous or allogeneic cells to restore the viability or function of deficient tissues, has attracted much attention in regenerative medicine. In order to design cell carriers for the application of cell therapy in tissue repair, there is a compelling need to understand the mechanisms by which regenerative stem cells sense and respond to the physical properties, such as elastic modulus and pore size, of 3D scaffolds. Hydrogels have been extensively investigated as carriers for stem cells, and matrix rigidity has been shown to regulate stem cell differentiation for elastic moduli up to 1 MPa[1]. However, the effects of matrix rigidity on osteogenic differentiation in 3D scaffolds with moduli exceeding 1 MPa, which is representative of the mineralized extracellular matrix in bone, has been investigated in only a limited number of studies. Polyurethane scaffolds, which are porous, biodegradable, and biocompatible, have been reported to support the migration of cells and ingrowth of new tissue in vitro and as well as in bone models, with non-toxic degradation product[2]. Moreover, the mechanical properties can be modified by changing the structures of hard and soft segments, which can be easily achieved by controlling the chain length of polyol and isocyanate in the reaction[3].

Method: In this study, we have synthesized both 2D films and 3D scaffolds from polyester triol, COSCAT® 83 catalyst, and hexamethylene diisocyanate trimer (HDIt) to ensure the same surface chemistry. To precisely control the porosity, morphology, and pore size of 3D polyurethane scaffolds, we used 3D molds from 3D-Biotek to form scaffolds with completely interconnected pores. Different chain lengths of polyester triol (e.g., from 300 Da to 3000 Da) were used to regulate the elastic moduli of the synthesized polyurethanes. Longer chain (higher molecular weight) of polyester triol yielded scaffolds with lower mechanical moduli (noted as “compliant”), while lower molecular weight polyesters yielded “rigid” scaffolds. Stem cell osteogenic differentiation was then studied on the developed polyurethane materials in vitro. Bone-marrow derived mesenchymal stem cells from mice were utilized in this study, and alkaline phosphatase (ALP) activity from cell lysate was selected as the early marker of osteoblast differentiation while secreted osteocalcin was selected as the late marker of osteogenesis. Metabolism level (MTS assay) of cultured stem cells and total protein from cell lysate were used as representative of cell proliferation in this study. Real-time PCR of bone differentiation markers including ALP, osteocalcin and Runx2 were also measured to further compare the differentiation level of stem cells on polyurethane materials.

Results: The elastic moduli at the cellular interface of the material of all scaffolds were analyzed by the Agilent G200 nanoindenter, yielded a moduli range from 10 – 3800 MPa. MSCs were seeded on polyurethane materials by pre-coating the surface with fibronectin based on calculated values of the specific surface of the 3D scaffold. Osteogenic induction medium was added after attachment of cells to the surface and changed daily to study osteoblast differentiation. Both cell metabolism level and total protein from harvested cell lysate indicated that MSCs were able to attach to and proliferate on the polyurethane materials for up to 2 weeks. Time-course ALP measurements in vitro showed that ALP activity of stem cells cultured in osteoinductive medium peaked between D7 and D10 on the rigid polyurethane. Similarly, both ALP and osteocalcin measurements showed that ALP activity from lysed MSCs and osteocalcin from the culture medium were significantly higher on the rigid polyurethane. Furthermore, real-time PCR of the bone differentiation markers also proved higher osteogenesis level of inducted stem cells on rigid polyurethane.

Conclusions: Polyurethane scaffolds with tunable mechanical properties (10 – 3800 MPa) were investigated as a potential polymer carrier for cell therapy in regeneration of bone defects. Bone-marrow derived mesenchymal stem cells responded to the mechanical properties of substrates at elastic moduli >10 MPa. Future work will focus on the study of the effects of other physical properties of this polyurethane carrier (i.e. pore size and surface chemistry) on stem cell behavior and cell fate, as well as identifying the underlying mechanisms of the cell-substrate interactions and the application of this tunable polymer carrier in cell therapy.