(496h) Tissue Origami for Biomineralization

Gulden Camci-Unal

University of Massachusetts Lowell, Department of Chemical Engineering, 1 University Avenue, Lowell, MA 01854, USA

Due to disease, degeneration, trauma, or aging, loss or damage to bone occurs in the body. Although there have been significant improvements in development of bone scaffolds, it remains difficult to fabricate porous and biocompatible constructs in physiologically relevant sizes in cm-scale. Thus, there is an unmet need for development of inexpensive, easy-to-use, and widely accessible biomaterials that are capable of supporting biomineralization for bone repair. In this work, we developed biomineralized origami-inspired paper scaffolds in three-dimensions (3D). This work is the first demonstration that paper can be used as a 3D construct to induce template-guided mineralization by osteoblasts.

We used the principles of origami to fabricate free-standing paper scaffolds in cm-scale. Because paper is an extremely flexible material that can easily be cut, creased, and folded to form 3D constructs, the scaffolds were easily fabricated in a variety of different geometries. This feature can be highly useful in fabricating constructs for patient-specific applications especially for patient who have defects of irregular sizes and shapes. Whatman brand filter paper was employed due to its large pore size (25-30 micron) and thickness (190 micron) for cellular migration. After sterilizing the constructs, they were seeded with mouse osteoblasts at a density of 20 million cells/mL in a collagen matrix (2 mg/mL). The samples were cultured up to 21 days and mineralization was evaluated using various assays including colorimetric assays, immunocytochemistry, high-resolution imaging (SEM), and micro-computed tomography (micro-CT) at different time points (days 0, 3, 7, 14, 21). We also performed in vivo experiments in a rat model.

In this work, for the first time, we performed tissue origami using paper for template-guided mineralization. We generated paper scaffolds in different shapes, sizes, and configurations. Due to its porous structure, paper allowed for transport of oxygen and nutrients across its thickness. Paper scaffolds supported a uniform distribution of cells within their 3D structures. In our experiments, proliferation of osteoblasts increased until day 3 and then decreased. Because cells proliferate first and then differentiate, a decrease in proliferation is expected to correlate with the initialization of the mineralization process. Hydroxyapatite content of the samples indicated that there was a progressive increase in the amount of hydroxyapatite in the paper scaffolds over 21 day of culture period. We found that the expression of osteocalcin increased until day 14 and decreased. This result can be attributed to increasing mineralization and consequent transformation of osteoblasts to osteocytes after day 14. We used SEM to visualize the deposition of mineral clusters, and EDAX to calculate the ratio of calcium to phosphate, which was found to be 1.5. Our in vivo experiments demonstrated that paper substrate did not cause inflammation. The paper implant integrated with the existing tissue well and rapidly vascularized.

In summary, we have demonstrated origami-inspired tissue engineering for mineralized tissues. We successfully obtained partially mineralized scaffolds in various 3D geometries. Mouse osteoblasts deposited calcium phosphate in these scaffolds and induced template-guided mineralization. Our approach used paper, a readily available material, as the cell culture scaffold. Paper has great potential to tackle the limitations of traditional scaffolds including cost, availability, accessibility, porosity, flexibility, rigidity, and ease of fabrication. In the future, paper-based scaffolds could potentially guide and accelerate bone repair using patient specific cells.