Biodegradable Visible Light-cured Thiol-acrylate Hydrogels for Craniofacial Bone Tissue Engineering

Arbi M Emmakah, Purdue University

Abstract

A craniofacial skeleton is a complex biological construction containing varieties of soft and hard tissues. For several decades, cranial bone defects owing to congenital malformations, severe trauma, and tumor surgery, have been a major challenge to restore and to reconstruct functions of lost bone tissues. Thus, successful regeneration of cranial bony defects is an integral step to restore craniofacial function. However, the restoration of craniofacial structure has been challenging due to its complex geometry, limited donor site availability, and poor graft integration. Current advances in tissue engineering and regenerative medicine have the promise of increasingly innovative techniques to improve bone restoration. Natural bone grafting methods, such as autografts, allografts, and xenografts, have been regularly used in craniofacial defects. Nevertheless, these methods do not yield completely satisfactory outcomes. In contrast, advances in synthetic biomaterials used in craniofacial repair, e.g. alloplastic bone grafting, tolerate the biological and mechanical functions in craniofacial bone defects. To address these problems, we investigated the use of a thiol-acrylate hydrogel as a cell carrier to facilitate cranial regeneration. Thiol-acrylate hydrogels were formulated with 5–15 wt% poly(ethylene glycol)-diacrylate (PEGDA) and 1–9 mM dithiothreitol (DTT). The degradation rate, percent of weight change, and shear modulus of the resulting hydrogel were first characterized. Then, pre-osteoblast-like cells (MC3T3-E1) were encapsulated in the hydrogel and cultured for up to 21 days. Our results demonstrate that compared to samples formulated from 15wt% PEGDA, 5wt% PEGDA samples showed lower storage modulus at day 10 (0.7kPa vs. 8.3kPa), 62.7% higher in weight change after soaking for 10 days. While 5wt% PEGDA group showed an 85% weight loss between day 10 and 21, 15 wt% PEGDA group showed a 5% weight gain in the same time period. Cell viability with 15wt% PEGDA and 5 mM DTT hydrogel decreased 41.3% compared to 5wt% PEGDA and 5mM DTT gel at day 7. However, histological analysis of cells after 21 days in culture revealed that they had pericellular mineral deposition indicating that the cells were differentiating into osteoblasts lineage in all experimental groups. Furthermore, the effects of photoencapsulating Bone Morphogenetic Protein-2 (BMP2) in biodegradable thiol-acrylate hydrogels were investigated. Immortalized mouse bone marrow stromal cells (MSCs) were Photoencapsulated with or without BMP2 in 10wt% PEGDA hydrogels. The rheometry indicated that the addition of BMP2 into prepolymer solutions did not significantly affect the mechanical stiffness and degradation behavior of the resultant gels compared to the control group of 10 wt% PEGDA hydrogels. However, the immortalized photoencapsulated-MSCs with BMP2 (BMBMP2) demonstrate 54.61% higher metabolic activity than photoencapsulated-MSCs alone (BMSC) on day 3. Furthermore, confocal microscopy images showed almost no dead cells captured over the 21-dayculture in basal medium for both groups (BMSC and BMBMP2). Additionally, BMBMP2 shows significantly increased ALP activity (by 11.84%) on day 3 compared to BMSCs. These results were confirmed by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) data. The expression of ALP upregulated significantly (** p < 0.01) on day 1 in BMBMP2 (8.52 fold change) compared to BMSC. Additionally, the expression of cFOS on day 3 in the BMBMP2 group increased significantly by 3.86-fold compared to BMSC’s 2.52-fold change at day 7. Additionally, the effect of visible light-cured thiol-acrylate hydrogels was also examined on craniofacial bone defects. Two formulations of PEGDA (5 and 15 wt%) hydrogels were used. Bone Marrow and Dental Pulp Stromal Cells (BMSCs and DPSCs) were photoencapsulated in 5 or 15 wt% PEGDA hydrogels and incubated in Basal (BSL) or Osteogenic (OST) medium. AlamarBlue cell viability assay revealed significantly higher levels of cell viability in 5wt% PEGDA hydrogel compared to 15 wt% PEGDA hydrogel in both types of MSCs. Conversely, 15wt% PEGDA groups demonstrated significant increases in ALP activity at day 7 compared to 5wt% PEGDA hydrogel groups in both types of MSCs. (Abstract shortened by ProQuest.)

Degree

Ph.D.

Advisors

Chu, Purdue University.

Subject Area

Histology|Biomedical engineering|Chemical engineering

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