Formation of human bone via tissue engineering technique with the addition of growth factors and telomerase gene transfection

Abstract

Tissue engineering aims to create an autologous or universal tissue or organ in order to restore and enhance biological function by combining the principles of engineering and life sciences. This study is an endeavour to devise a strategy for human bone tissue engineering that encompasses the process of sample harvesting, cell expansion, three-dimensional construct formation in vitro and the implantation of the autologous engineered bone construct to repair critical-sized bone defect in an animal model. There were five distinct phases in the study. In Phase I, four cell sources: periosteum, cancellous bone, cortical bone and bone marrow aspirate were harvested and expanded in culture. The four cell sources exhibited osteogenic differentiation capabilities and hence can be pooled for generating three-dimensional bone construct. Periosteum proved to be superior in terms of growth profile and the degree of osteogenic advancement. However, bone marrow mesenchymal stem cells (BMSCs), a potential candidate for future clinical applications had been further characterized for surface markers (CD markers) and telomerase activity. Due to the decreased proliferation capacity and telomerase activity in BMSCs of aged individuals, it was hypothesized that the ectopic expression of human telomerase reverse transcriptase (hTERT), the key component of telomerase activity, in BMSCs will increase cellular proliferation and life span. The effect of BMSC transfection with hTERT gene was hence being studied. Phase II of the study then explored the use of growth factors to expedite the proliferation and differentiation of BMSCs in vitro. The effect of transforming growth factor-beta 1 (TGF-B1), insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP-2) in different dosages on the monolayer cultures were evaluated. bFGF addition effected a significant increase in BMSCs proliferation in parallel with increasing dosage. Phase III embarked on the formation of three-dimensional bone constructs. Ceramic hydroxyapatite (HA) and a composite of tricalcium phosphate and hydroxyapatite (TCP/HA) were selected for use as the scaffolding material. Continuous seeding is the method of choice for cell seeding when compared to static seeding and human plasma derivative, upon polymerization, acted as a cell carrier and growth factor delivery. In Phase IV, the two ceramic materials; HA and TCP/HA were compared in parallel. Evaluations of in vitro and in vivo constructs supported the hypothesis that TCP/HA, due to its higher biodegradability accelerated new bone formation and is the preferred scaffold material for bone tissue engineering. Finally, for proof of concept, one- centimeter tubular bone was removed from the right tibia of 21 three-month old New Zealand White Rabbits and was replaced with autologous tissue engineered bone constructs, allografts, fresh bone marrow loaded scaffolds or were left blank as controls. New bone developed in the tissue engineered bone constructs, which fused onto adjacent native bone. Greater biomechanical strength and physiological function were achieved in tissue engineered bones when compared to other approaches. In conclusion, we have proved that bone repair using the tissue engineering approach is feasible.