Regeneration Of Bone Through The Use Of Adult Mesenchymal Stem Cells

Introduction

Bone degeneration occurs naturally with age, it has been estimated that 64-67% of people over the age of 70 have abnormal bone mass and with osteoarthritis currently being the most common form of arthritis (affecting approximately a third of people over the age of 45) treatment for bone regeneration is at an all-time high [2].

[image: Figure 1]Adult mesenchymal stem cells (MSCs) have great medicinal outcomes at sites of inflammation, tissue injury and disease, they can be supplied in-vivo or in-vitro and reside in the bone marrow stroma. They provide microenvironmental support for hematopoietic stem cells and are able to differentiate into a range of mesodermal lineages (see figure 1) [1][4]. MSCs have the capacity to secrete either trophic and/or immunomodulatory mediators and are equipped to assist the natural regenerative capability of almost every tissue in the body due to their multipotency [1]. The medical ability of MSCs is determined by where, when and by what tools or mechanism they are delivered [1].

Figure 1- The potential lineages MSCs can divide into and their requirements for in-vitro culture (boxed). Dotted arrows indicate the potential for some events to be reversed. Italics indicate the signalling components and pathways involved in lineage-specific differentiation [5].

The role of MSCs in bone regeneration

The general effect of switched-on, locally situated MSCs is to help manage tissue regeneration by hindering the quick-fix apparatus of the formation of scar tissue [1]. At the site of bone and tissue injury there is an instant trigger to the severe inflammatory response which aids to immerse the site with cells and molecules to prevent invasion of foreign bodies and toxic molecules [1]. This acute inflammation conditions the site for either scarring, regeneration or repair [1]. MSCs prevent the invasion of immune probing cells protecting the site from toxic molecules. Connective tissue cells are inhibited by activated MSCs to prevent them from secreting large quantities of collagen that would generate scar tissue, therefore serving as protection from degenerative events and allowing regenerative repairs to be commenced [1]. They are able to do this due to their low expression of histocompatibility class I and II and the absence of co-stimulatory molecules such as CD40 which is required for activation of antigen-presenting cells combined with the observation that MSCs do not prompt a proliferative response from allogeneic lymphocytes [4].

In-vitro manipulation of BM MSCs

Bone marrow is typically taken from the iliac crest of a donor using a needle and is diluted using Dulbeccos modified Eagles medium (DMEM), the marrow is centrifuged at 1800 r.p.m for 10 minutes to remove anticoagulants and washed with DMEM having removed the supernatant. Mononuclear cells are layered onto a Ficoll density gradient to be isolated and washed with DMEM [3]. The cells are then plated and cultured in DMEM medium and supplemented with substrates such as pen-strep (to protect against bacterial contamination), 10% fetal bovine serum (FBS – rich in proteins and low in antibodies therefore aiding in growth) and human plasma to increase osteoblast differentiation [3]. Culture surface substrates such as unmodified polystyrene is added to the medium to increase cell adherence, any cells that haven’t adhered after 48 hours are then removed. The medium is replenished every 3 days to maintain growth factors and once a confluency of approximately 90% is reached the cells are dissociated using 0.25% trypsin EDTA (which breaks down the proteins enabling the cells to stick to the medium and one another) and sub-cultured for propagation [3][7]. After 3 weeks the cells are resuspended and washed once more using phosphate-buffered saline (PBS), PBS is used to maintain the pH of the cells. Other factors affecting MSC proliferation include oxygen percentage, pH and plate density [3][7].

Growth factors should be added to the media immediately after culture before the cells enter into interphase (G1, S and G2) as this is the point in which the cell will grow and the nuclear DNA will be duplicated, this phase lasts for approximately 23-25 hours. As cells are multiplying more media will be needed to supply the growth of the recently proliferated cells, hence the need to change and add new media and growth factors every 3 days.

Stem cells divide and multiply meaning a lag phase is experienced before exponential growth occurs, cell growth will then plateau when a carrying capacity is reached, for example when there is no longer space or culture medium available (see figure 2).

To determine the presence of stem cells an immune positive criterion is used to identify clusters of differentiation (CD) such as CD73 (produces anti-inflammatory substances) and ensure the absence of clusters such as CD40, these act as cell markers by determining the function of the cell at hand [3].

Delivery of MSCs

MSCs should be delivered to the site of damage or inflammation by systemic delivery however data has proven that exogenous MSCs do not circulate well, are very fragile and can be abolished very quickly so the likelihood of the MSCs reaching the target area is slim [1]. Direct injections of MSCs are often used for this reason, particularly when targeting joints or intervertebral discs [1]. Research into encapsulating exogenous MSCs so they are not susceptible to damage when inserted into the bloodstream has also been successfully attempted, MSCs have been injecting into Adipocytes and introduced to a culture, the MSCs crawled out onto the plate after 4-7 days [1]. Chemo-attractants are used to draw the MSCs to the site of inflammation within the body, thus proving to be an extremely useful treatment for diseases such as osteoarthritis [1].

Conclusion

The future for BM MSCs in the regeneration of bone is promising, with the current treatments for bone regeneration being fairly limited, the increase in demand for new and improved treatments is at its peak. Stem cells have the potential to develop into lineages, such as the osteoblastic lineage, which can replicate and recreate the function of tissues and organs, these cells occur naturally in-vivo but can be cultured in-vitro in order to be used at sites of damage, therefore meaning they are readily available, making them a favorable candidate for treatments to come.

References

1. Caplan, A.I., (2015) “Adult Mesenchymal Stem Cells: When, Where, and How.” Stem cells international vol.
2. Glyn-Jones S et al. Osteoarthritis. Lancet. 2015;386(9991):376-87
3. Pal, R., Hanwate, M., Jan, M., Totey, S., (2009). “Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow-derived mesenchymal stem cells”. Journal of tissue engineering and regenerative medicine research, article 3: 163 – 174.
4. Pontikoglou, C., Deschaseaux, F., Sensebé, L., & Papadaki, H. A. (2011). “Bone marrow mesenchymal stem cells: Biological properties and their role in hematopoiesis and hematopoietic stem cell transplantation”. Stem Cell Reviews and Reports, 7(3), 569-89
5. Tuan, R.S., Boland, G., Tulli, R. (2003). “Adult mesenchymal stem cells and cell-based tissue engineering”. Arthritis research and therapy.
6. Yang, J. D., Cheng-Huang, Wang, J. C., Feng, X. M., Li, Y. N., & Xiao, H. X. (2014). The isolation and cultivation of bone marrow stem cells and evaluation of differences for neural-like cells differentiation under the induction with neurotrophic factors. Cytotechnology, 66(6), 1007–1019.
7. Zhang, L., Peng, L-P., Wu, N., Li, L-P., (2012). “Development of bone marrow mesenchymal stem cell culture in-vitro”. Chinese Medical Journal: 2012 – Volume 125 – Issue 9 – p 1650–1655.