Engineering 3D constructs of human bone matrix in a mechanically-active environment
Background: A biocompatible, osteoconductive and osteoinductive framework for ingrowth of host cells that can mimic the remodelling behaviour of normal bone matrix would be of significant benefit to the tissue engineering field. A solution would be to create a mineralised matrix, an engineered ‘off the shelf’ bone replacement material, containing the multitude of growth factors that would facilitate its integration into bone and induce bone regeneration. Mechanical loading is an important determinant of bone mass and architecture, and has been shown to promote osteogenic differentiation in vitro. A suitable scaffold for culturing osteoblasts must therefore have the strength to withstand mechanical stimulation over extended periods of times. The present study explores the suitability of semi-synthetic hyaluronan-gelatin PEGDA cross-linked hydrogel for culturing of calvarial and femoral osteoblasts. This semi-synthetic material has a close resemblance to the extracellular matrix, and offers the opportunity to optimise its bio-mechanical characteristics through modification of hydrogel cross-linking. Aims: 1) To develop a 3-D hydrogel cell-culture model for engineering artificial mineralised bone matrix in vitro; 2) Validate assays for the measurement of osteoblast proliferation and hydroxyapatite deposition; 3) Examine suitability of the model for the study of mRNA expression under mechanical strain. Methods: Human foetal calvarial osteoblasts (HCO) and femoral osteoblasts (HFO) were cultured in thiol-modified hyaluronan-gelatin-PEGDA cross-linked hydrogel. Two degrees of cross-linking were used and cell proliferation and hydroxyapatite deposition was quantified over a 21-day culture period. Confocal microscopy was also used to assess the penetration of the hydrogel by the assay dyes. An enhanced cross-linked hydrogel was used for mechanical strain experiments. Quantitative polymerase chain reaction (qPCR) was used to measure mRNA expression of alkaline phosphatase (ALP), osteocalcin (OC) and bone morphogenetic protein 2 (BMP2) genes. Results: Cell proliferation and hydroxyapatite deposition increased for each cell line over 21 days. The highly cross-linked hydrogel was found to be a better scaffold for osteoblast attachment. Confocal microscopy showed assay stains had limited hydrogel penetration. A cyclic compressive mechanical load did not affect ALP expression in either the HCO (p=0.243) or the HFO (p=0.132) but it caused a 53.9-fold increase in BMP2 in the HFO group (p=0.0043). No OC mRNA was detected in either group. Conclusion: Thiol-modified hyaluronan-gelatin-PEGDA cross-linked hydrogel is a suitable scaffold for the study of osteoblast proliferation and hydroxyapatite deposition in vitro. Further experiments are required to assess the differences in the responses of calvarial and femoral osteoblasts to mechanical strain.
Advisor: Meikle, Murray; Milne, Trudy; Farella, Mauro; Cannon, Richard
Degree Name: Doctor of Clinical Dentistry
Degree Discipline: Oral Sciences
Publisher: University of Otago
Keywords: bone; scaffold; tissue-engineering; osteoblast; 3D-tissue-culture; hydrogel; mechanobiology; mechanically-active-environment
Research Type: Thesis