In vivo and in vitro degradation of tissue engineered collagen and mineralised collagen scaffolds used in bone tissue engineering
A clinical need exists to provide alternatives to autologous bone grafting for the reconstruction of head and neck bone defects. This thesis examines the utility and success of a tissue engineering approach to this problem using novel collagen based scaffolds combined with mesenchymal stem cells.
The objective of this work was to determine how the addition of a calcium phosphate mineral phase to a collagen-based scaffold, designed for use in bone tissue engineering, affects the in-vitro and in-vivo scaffold degradation characteristics in addition to the rate of tissue healing as assessed by new bone formation and host immune response in a rat calvarial model.
In vitro analysis of the collagen calcium phosphate (CCP) scaffolds revealed minimal degradation and loss of mechanical properties over time in a non enzymatic degradation media, these results were similar to those of pure collagen (collagen) and collagen glycosaminogylcan (CollGAG) scaffolds. However, in a bacterial collagenases media the mineralised CCP scaffolds were relatively resistant to degradation compared to the collagen and CollGAG scaffolds.
The CollGAG and CCP scaffolds were then implanted into a 7mm trans-osseouscritically sized defect created in the calvarium of Wistar rats. Half of each group were pre-cultured with mesenchymal stem cells (MSC). Animals were sacrificed at 4 and 8 weeks post implantation. Quantitative histomorphometry identified significantly better rates of new bone formation in non MSC seeded scaffolds, with superior results for the mineralised collagen scaffold at 8 weeks (37.24%V13.15%, p<0.05). Scaffolds pre cultured with MSCs showed an accumulation of fibrous tissue at the periphery of the scaffold.
In the knowledge that macrophages play an important role in fracture healing and that this fibrous tissue surrounding the MSC seeded scaffolds appeared inflammatory in nature, immunohistochemical staining was performed to confirm the presence of macrophages (CD68) and to phenotype the macrophage response (CD163, CCR7). A marked macrophage response to the MSC seeded scaffolds, with only a moderate response to non seeded irnplants.was seen. Whilst all scaffold types demonstrated an M2 (immunomodulatory and tissue remodelling) macrophage phenotype response the location of this response was confined to the scaffold periphery in the MSC seeded group as opposed to areas of new bone formation in the non seeded group.
In conclusion this thesis demonstrates quantitatively superior new bone formation in non MSC seeded mineralised (CCP) collagen scaffolds. Aside from increasing the scaffolds mechanical properties the addition of a mineral phase also retards scaffold degradation. Furthermore, an appropriate macrophage response is necessary for successful bone deposition in collagen scaffolds and appears hindered by current tissue engineering approaches.
First SupervisorProfessor Fergal O'Brien
Second SupervisorProfessor Michael Walsh
CommentsSubmitted for the degree of Master of Surgery (MCh) to the Royal College of Surgeons in Ireland, 2010
Published CitationKieran SM. In vivo and in vitro degradation of tissue engineered collagen and mineralised collagen scaffolds used in bone tissue engineering. [MCh Thesis]. Dublin: Royal College of Surgeons in Ireland; 2010.
- Master of Surgery (MCh)