In vitro and in vivo evaluation of osteogenesis of human umbilical cord blood derived mesenchymal stem cells on partially demineraliz

In vitro and in vivo evaluation of osteogenesis of human umbilical cord blood derived mesenchymal stem cells on partially demineralized bone matrix

GUANGPENG LIU, M.D., Ph.D., YULIN LI, B.S., JIAN SUN, B.S., HENG ZHOU, M.S., WENJIE ZHANG, M.D., Ph.D., LEI CUI, M.D., Ph.D., and YILIN CAO, M.D., Ph.D.

Summary:

When regeneration of bone defects is needed, bone grafts taken from the patient are usually used. However, when the bone defect is rather large, grafts cannot be done because not enough material can be extracted for the grant. One solution to this problem is using tissue engineering to develop a degradable scaffold that contains osteogenically differentiated mesenchymal stem cells to regrow the bone. These scaffolds are called partially demineralized bone matrix (pDBM). The purpose of this lab was to determine whether such a scaffold can support the osteogenic differentiation of mesenchymal stem cells obtained from umbilical cord blood (UCB-MSCs), which are easier to obtain than those from bone marrow (BMSC), both in vitro and in vivo.

First, human umbilical cord blood samples were taken and processed in heparnized tubes to avoid coagulation. The blood sample was diluted, and mononuclear cells were isolated using density gradient centrifugation. These cells were then seeded in dishes and grown in basic culture media. After the culture was fairly confluent, single separated colonies of fibroblast like cells were detached and replated (essentially picking out the cells most thought to be MSCs. The cells were then passaged up to 10 times for use. Cell quantification was done through the use of a DNA assay to determine the doubling time, which was about 33 hours for the UCB-MSCs. Further analysis of the MSC’s seeded were done using flow cytometry with various cell surface antigen markers to determine when the MSC cultures were most pure. MSC s were determined to be the populations that determined a homogenous, spindle-like population, and tested positive for the CD29, CD105, CD106, CD 166, and Stro-1 antigen markers.



The differentiation potential of the UCB-MSCs was determined by inducing osteogenic, adipogenic, and chrondrogeinc differentiation through various methods. Staining was used to determine whether differentiation occurred or not, and it was found that the UCB-MSCs readily differentiated in all 3 ways when induced, but did not differentiate spontaneously when not induced.



Meanwhile, the pDBM scaffolds were prepared from porcine trabecular bone. The bone samples were extracted in absolute ethanol, and then decullarized in TritonX-100, and demineralized in an HCl solution. The scaffolds were then dried and sterilized so that they could be seeded. Then, UCB-MSC samples were added onto the scaffold and incubated for 2 hours, after which the whole cell/scaffold composite was transferred to a separate well Some of these composites served as a control, with no induced differentiation, while others were cultured in osteogenic medium to induce osteogenesis. Sputter coating and SEM were used to visualize the culture growth on the scaffold, and DNA assays were used to determine the cell number of the culture. It was found that both induced and non induced cells spread and attached well to the pdBM scaffold, but the osteogenically induced cells grew for longer and to a higher total cell number than the non-induced ones.

To assess the osteogenesis of the cells on the scaffolds, multiple tests were then done. First, tests for alkaline phosphatase (via phosphate substrate solution and absorbance readings) and osteocalcin (via ELISA) were performed, and it was found that the induced group of cells had increased alkaline phosphatase activity and osteocalcin levels when compared to the control. After 14 days of culture, RT-PCR was performed, and four genes related to osteoblasts, alkaline phosphatase, osteocalcin, osteopontin, and collagen type I were amplified. The induced cells were shown to be expressing all 4 of these genes, while the control cells did not express osteocalicn or osteopontin transcripts, and expressed the alkaline phosphatase at a severely reduced level.



After this analysis, the in vitro part of the experiment was begun. A critical-sized bone defect was created in the rat’s skull, and the defect was then filled with an induced UCB-MSC/pDBM composite, with a non-induced composite, with just a pDBM scaffold, or nothing at all. Micro-CT scans, BMD readings, and histological tests were done of the inserted area 6 and 12 weeks after surgery. After 12 weeks, it was found that the defects filled with the induced complexes had started to form new bone tissue, and the pDBM scaffold was completely reabsorbed. Meanwhile, the other groups did not display much evidence of bone growth or union, and the scaffold (if present) was still present in the defect.


The BMD and histological results confirmed what the micro-CT scans showed. The bone formed from the induced complexes had a very high BMD, comparable to that of normal bone. The other two groups that had a scaffold inserted (but did not contain induced cells) had very low BMDs that actually decreased through the 12 weeks, most likely due to scaffold degeneration. When examined histologically, these results were further confirmed. After 6 weeks, the induced complex group formed new osteoid-like tissue in between the fragments of scaffold still left, while the other groups formed only thick fibrous tissue if a scaffold was present, or very thin fibrous tissue if no scaffold was present. After 12 weeks, the induced group displayed no residual scaffold, and the defect was almost completely bridged by bone, while the other groups showed only minimal bone growth.

Discussion and Critique:

In general, the differentiation of UCB-MSCs was successful, indicating that they could be a possible future cell source for bone tissue engineering. The results of this study indicated that the pDBM scaffold could support UCB-MSC cell growth. Additionally, UCB-MSC osteogenic differentiation proved to be successful on this scaffold, as confirmed by SEM, RT-PCR, and the alkaline phosphatase and osteocalcin assays performed. In vivo studies indicated that these differentiated scaffolds were very successful in bone regeneration as shown by the Micro-CT scans, BMD readings, and histological examinations.

Overall, this paper did a fair job in covering its bases and using multiple ways to confirm its results, or explaining any limitations or unexpected finding in the study, such as how the scaffold and bone densities were hard to distinguish from each other in micro-CT scans, or how non-induced cells still had alkaline phosphatase activity. In both cases, because multiple tests were done, such setbacks could be countered by plausible explanations. However, there were a few points that I, as a reader, was concerned about. First, I felt that the cell proliferation should be measured not only by a DNA fluorescence assay, but supplanted with actual cell counting/live dead assay. The DNA measurements could be affected easily by any contamination or the presence of DNA from dead cells. Both of these limitations could be picked up by the cell counting methods. Additionally, I think that RT-PCR and such assays should have also been done on the various differentiated UCB-MSC’s which were first tested on just the culture plate. That way, the effect of the scaffold on levels of gene expression of the osteogenically-induced cells could be compared. Lastly, I was somewhat skeptical by the lack of any immunological response that was reported by the study. During the characterization of the UCM-MSCs, mouse antibodies easily attached to the cells and were used in testing. I found it difficult to believe that a mouse antibody would stick so readily to the cell culture, but a rat would not produce any significant immunological response.