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Abhijit S. Dighe, PhD, examines morphological characteristics of stem cells after they are co-cultured with allogeneic or autologous T-cells.

AAOS Now

Published 9/1/2016
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Mark Crawford

Stimulating Bone Growth

OREF grant recipient looks at the potential of allogeneic MSCs
In the United States, fractures cost approximately $20 billion annually to treat and are associated with 100 million days of restricted activity. The surgical treatment for many fractures includes bone grafting. However, performing bone graft harvests has been associated with additional morbidity.

Mesenchymal stem cells (MSCs) have been used to treat a variety of musculoskeletal diseases, and researchers are investigating their potential to treat fractures. There are various types of MSCs, but autologous MSCs are the least likely to be rejected by the patient's immune system. Unfortunately, autologous MSCs exhibit less osteogenic function in key patient groups, such as the elderly, females, and individuals taking certain prescription medications.

On the other hand, allogeneic MSCs derived from young healthy males have the best osteogenic potential. However, the previous conclusion that these allogeneic MSCs are immune privileged has come under scrutiny. Some studies show that the patient's T-cells will destroy allogeneic MSCs.  More recently, other research has shown that suppressing T-cell function enables allogeneic MSCs to induce osteogenesis in syngeneic (ie, genetically similar or identical) mice.

MSCs in bone repair
To learn more about how T-cells impact the effectiveness of allogeneic stem cells, Abhijit S. Dighe, PhD, a research scientist for the University of Virginia's department of orthopaedic surgery in Char­lottesville, is applying funding from a 2014 Orthopaedic Research and Education Foundation (OREF)/Musculoskeletal Transplant Foundation (MTF) Research Grant to investigate the interaction between MSCs and T-cells during osteogenesis.

"The claim that allogeneic MSCs are immune privileged is based on insufficient evidence," said Dr. Dighe. "Our prior work has shown that allogeneic MSCs fail to induce successful osteogenesis in vivo. This study will identify the cellular and noncellular targets that retard osteogenesis of allogeneic MSCs. Hopefully these targets can then be modulated to allow successful use of allogeneic MSCs for bone repair and various other musculoskeletal conditions."

Allogeneic MSC therapy has huge potential for many people whose autologous MSCs cannot induce osteogenesis. For these populations, receiving allogeneic MSCs that have been given immune privilege status would be a tremendous breakthrough in bone repair. "Our project will hopefully lead to the establishment of allogeneic stem cell storage banks where the MSCs are obtained from young healthy males," said Dr. Dighe. "It would be immensely useful to have such allogeneic MSCs available for repairing fractures in those populations for which autologous MSCs don't work well." (Fig. 1).

MSCs and immune response
Dr. Dighe and his research team want to determine how various subtypes of T-cells modulate osteogenic differentiation of MSCs.

One goal of Dr. Dighe's research is to test whether syngeneic MSCs can induce T-regulatory and Th2 phenotypes in naïve T-cells, inhibit Th1 and Th17 phenotypes, escape cytolysis by CD8+ T-cells, and differentiate into osteoblasts in presence of the T-cells in vitro.

Dr. Dighe and his research team cultured the MSCs and naïve T-cells isolated from mice and human bone marrow in allogeneic and syngeneic settings for 15 days. The researchers assessed the polarization of naïve T-cells into different phenotypes and the osteoblastic differentiation of MSCs using flow cytometry, mineralization assays, enzyme-linked immunosorbent assays, and real-time polymerase chain reaction (PCR). They used Annexin V and propidium iodide staining in flow cytometry to determine the survival of MSCs.

Abhijit S. Dighe, PhD, examines morphological characteristics of stem cells after they are co-cultured with allogeneic or autologous T-cells.
Fig. 1 Potential uses for allogeneic MSCs derived from young healthy males.
Courtesy of Abhijit S. Dighe, PhD

Dr. Dighe and his research team also wanted to determine if selective elimination of CD4+ or CD8+ T-cells is sufficient to allow osteogenesis induced by MSCs in an allogeneic environment—a relationship that has yet to be investigated in the field.

The researchers implanted bone-marrow-derived stem cells from Balb/c mice in syngeneic Balb/c, allogeneic B6, CD4 -/- B6, and CD8 -/- B6 mice. The researchers determined if osteogenesis was present using radiography, micro-CT, histology, and mRNA expression of osteogenic markers. They studied the host immune response using flow cytometry and mRNA expression of cytokines and determined MSC survival using PKH126 staining.

T-cells vs. stem cells
The researchers are still evaluating the data, but preliminary results show that in the genetically controlled mice, stem cells are not immune privileged—they cannot escape the T-cells of the host's immune system. Dr. Dighe and his research team hope that further analysis will reveal more about how T-cells modulate osteogenic differentiation of MSCs.

For example, Dr. Dighe expects the data will provide information on comparative abilities of allogeneic and syngeneic MSCs to escape the cytotoxic T-cells derived from CD8+ T-cells—"something that has not been reported earlier in the literature," he said. "We also want to know if specific removal of CD4+ T-cells or CD8+ T-cells is sufficient to allow osteogenesis induced by allogeneic MSCs."

Dr. Dighe is also hopeful he can discover what cell types (Th1, Th17, C8+ T, CTLs) in allogeneic and syngeneic implants predominantly express cytokines that inhibit osteogenesis. These specific targets can then potentially be modulated to enhance osteogenesis induced by MSCs in an allogeneic environment.

"Our ultimate goal is to identify the culprit in the T-cell population that suppresses the ability of allogeneic stem cells to form bone," said Dr. Dighe. This is a critical step in advancing the implantation of allogeneic MSCs as an effective method of bone repair.

OREF supports every career stage
Dr. Dighe said that one of the reasons he applied for funding through OREF is that he knew some of his seniors and fellow faculty had gone on to obtain funding from the National Institutes of Health and Department of Defense after receiving their OREF grants.

"I was well aware that OREF can be a stepping stone. I think OREF has a very good structure. It funds researchers at every stage in their research careers. They fund residents. They fund people who have never had a grant," he said.

"This OREF/MTF research grant started my career. Pursuing ideas that are not in agreement within the scientific world is very difficult. A lot of preliminary data is needed to push that idea forward and apply for bigger funding," he said.

Mark Crawford is a contributing writer for OREF. He can be reached at communications@oref.org