The Study Of Tissue Engineering Bone Cultured By Perfusion For Repair Of Segmental Bone Defects

Segmental bone defects resulting from trauma,excision of bone tumor or pathology represent a common and significant clinical problem. Limb amputation was historically the principal treatment option for these defects as they typically do not heal spontaneously. With advances in medicine and science, alternative treatment options have developed such as the use of bone-grafting techniques including autologous,allogeneic and tissue engineering bone. Autologous bone grafts are preferred as they possess inherent osteoconductivity, osteogenicity and osteoinductivity. However, there is often limited supply of suitable bone for autologous grafting, and its collection is frequently associated with donor-site morbidity. Aonther alternative is to use allogeneic bone grafts from donors or cadavers. These circumvent some of the limitations associated with harvesting autologous grafts, but allogeneic bone grafts lack osteogenicity, have limited osteoinductivity and present a risk of disease transmission. These limitations necessitate the pursuit of alternatives for the management of segmental bone defects, with the latest approach being to use tissue-engineering techniques. At present, the feasibility of segmental bone defects repaired by the scaffolds combined with BMSC are being studied in some institution. In the current paper, we find out a novel method of investigating lively cells on great scaffold material , through observing the combination of BMSC labeled by DiI andβ-TCP scaffold ;And we present a tissue-engineering strategy for the culture of tissue engineering bone by perfusion culture bioreactor designed by ourselves.β-TCP scaffolds combined with auto-BMSC were cultured by perfusion culture bioreactor,and implanted into 30mm-sized defects in an established goat model. Defects and scaffolds were stabilized with a load-sharing device (forcing plate). The aim was to investigate the effect of tissue engineering bone cultured by dynamic perfusion on segmental defect repair in the goat tibia.1. Observation Of The DiI Labeled Bone Mesenchymal Stem Cells Combined With The Great Scaffold by Fluorescent MicroscopeObjective To find out a novel method of investigating lively cells on great scaffold material , through observing the combination of BMSC labeled by DiI andβ-TCP scaffold. To offer a new feasible method to study the tissue engineering bone. Methods The third passage BMSC of goats digested were divided into two groups: the experiment group was labeled by DiI, and the normal group not. They were planted into cell culture plates with 12 holes at the same level ,each group with 6 holes per plate. From then on , we took one plate each day digest, count cells for 7 days continuously and draw the growth curve. Otherwise, we made two group cells of the same number combine with theβ-TCP scaffold, then cultured and evaluated their adhesion rate. Digesting and counting each group cells on the day of 3,7,14 , to observe the increasing state with theβ-TCP scaffold. And to investigate the growth state of the DiI labeled cells by fluorescent microscope. Results The growth curve of each group is not significant on the whole, and so are the cell numbers on different time(P>0.05). The increasing rates of the two groups on theβ-TCP scaffold are not significant on the day of 3,7,14(P>0.05). BMSC labeled by DiI can be observed clearly by fluorescent microscope. Conclusions Compared with the normal cells, the proliferation and appearance of BMSC labeled by DiI are not significant. The BMSC labeled by DiI can be well compatible withβ-TCP scaffold and be observed continuously in living state. The novel method of observation the cells on the opaque scaffold is feasible to investigate the interaction between the cells and the opaque great scaffold. The method can be utilized extensively in the tissue engineering bone.2. The study of tissue engineering bone cultured by using the dynamic perfusion bioreactorObjective To investigate the feasibility of using perfusion culture bioreactor for bone mesenchymal stem cell proliferation in large scaleβ-tricalcium phosphate (β-TCP) scaffold. Methods In the dynamic perfusion culture group, the porousβ-TCP cylindrical scaffolds combined with the sheep mesenchymal stem cells were continuously perfused with the complete DMEM medium by a peristaltic pump for 2 weeks. While in the static culture group, the hybrid constructs were immersed in the medium without perfusion for 2 weeks. The cell proliferation and distribution were examined by the daily glucose consumption, the cell viability and undecalcified histological study. Results The daily glucose consumption increased with time. The increase was much more evident in the first-10 days than in the last-4 days. The daily glucose consumption was higher in the dynamic culture group than in the static culture group. The cell viability was also higher in the dynamic culture group(P < 0.05). Under dynamic perfusion culture, the mesenchymal stem cells survived and proliferated through the scaffolds. However, the mesenchymal stem cells survived and proliferated only in the peripheral pores of the scaffolds under static culture. Histomorphometrical study indicated that there were much more cells in dynamic culture group than in the static group. Conclusion Perfusion culture permitted the persistent nutrition supply and gas exchange into the centre of large scaffold. This perfusion bioreactor makes the mesencymal stem cells survive and proliferate through a large three-dimensional scaffold.3. The study of segmental bone defects repaired by tissue engineering bone cultured by dynamic perfusionObjective To study the effect of bioactive bone ,which is made up ofβ-TCP combined with self-BMSC and is cultured by dynamic perfusion,repairs the segmental bone defects. Methods Isolate and culture the goat BMSC in vitro. The test group wasβ-TCP scaffolds combined with self-BMSC which were cultured by perfusion culture bioreactor made by ourselves. The control group wasβ-TCP scaffolds combined with self-BMSC which were cultured in static. After two weeks the scaffolds in test and control groups were respectively transplanted into the 30mm-defects of the goat left and right middle tibias. The X-ray of goats tibias were gathered in 1 week,1,3,6 months after operation. The test and control bioactive bones of the goats tibia were analyzed by Histology and Micro-CT. Results Osteogenesis in the test groups outweighs in the control groups according to X-ray analysis. The rate of the test groups osteogenesis is higher than the control groups (p<0.05). By micro-CT analysis, BMD and TMD in the test groups are vastly different of the control groups (p<0.05). Conclusions The osteogenesis capacity and quantity of the bioactive bone, which is cultured by perfusion culture bioreactor, outweighs that cultured in static. So does the bioactive bone repair for the segmental defect of bone. The technology of the bioactive bones cultured by perfusion culture bioreactor will become a clinical method to repair the segmental defects of bone.