Immuno-Protective and Immuno-Suppressive Behavior Displayed by a Novel Viable Cell Bone Matrix
+1Murray H; 2Patel S; 3Javier C; 1D’Antonio P; 3Ponticiello M; 2Rameshwar P
+1Biomet, Parsippany, NJ; 2University of Medicine and Dentistry of New Jersey; 3Biomet, Irvine, CA
[email protected]
INTRODUCTION
Autograft is considered the gold standard for bone grafting materials
because it contains osteoinductive growth factors, osteogenic cells, and
an osteoconductive scaffold onto which bone can grow. However, there
is a limited supply of autograft tissue that can be used for grafting
purposes and harvesting autograft tissue can result in donor site
morbidity. Many currently available bone grafting materials have some
of the features necessary for successful bone healing, but few have the
osteogenic component of the bone healing triad. This study evaluated the
safety profile of a new allogeneic viable cell bone matrix (VCBM)
confirming the safe delivery of osteoinductive growth factors and
osteogenic cells within an osteoconductive scaffold for bone healing
applications.
METHODS
VCBM Fabrication: Donor ilium tissue meeting the America
Association of Tissue Banks donor suitability requirements was
procured and processed. Briefly, cancellous tissue was harvested from
the ilium and ground into 1-4 mm diameter granules. Tissue was treated
with a proprietary process to remove the marrow fraction and then
soaked in an antibiotic solution to eliminate potential bioburden.
Concurrently, cortical bone was harvested and ground into a 125-850
µm diameter powder. Ground cortical powder was demineralized in
HCl and rinsed. Cancellous tissue and demineralized cortical bone
powder were combined in a 50:50 ratio and frozen in CryoStor CS10
(StemCell Technologies, Vancouver, Canada) at -80oC (Figure 1).
Mixed Lymphocyte Reaction (MLR): 106 activated or unactivated
peripheral blood mononuclear cells (PBMCs) were plated in co-culture
in 12-well plates with gamma-irradiated mesenchymal stem cells
(MSCs) expressing major histocompatibility class II antigens that can
elicit allogeneic responses (positive controls). Frozen VCBM was
defrosted in a 37oC water bath. The cryoprotectant on the bone matrix
was discarded and the tissue was rinsed with phosphate buffered saline
(PBS). The rinsed bone matrix was added to the wells with plated
PBMCs and incubated. After 4 days each well was pulsed with 2 µCi of
tritiated thymidine. On day 5 the cells were harvested on glass fiber
filters and counted in a scintillation counter for the number of activated
cells. Changes in proliferation are presented as stimulation indices as
determined by the tritiated thymidine incorporation into the PBMCs.
Flow Cytometry: Frozen VCBM was defrosted in a 37oC water bath.
The cryoprotectant on the bone matrix was discarded and the tissue was
rinsed with PBS. The bone tissue was then digested on an orbital shaker
for 30 minutes with 1 mg/ml collagenase (Sigma, St. Louis, MO). Cells
were collected and stained with antibodies for CD2, CD3, and CD8.
Cells were run through a FACScan (Becton Dickinson, Franklin Lakes,
NJ) flow cytometer to determine the % of cells expressing the T-cell
markers.
Osteogenic Differentiation: Frozen VCBM was defrosted in a 37oC
water bath. The cryoprotectant on the bone matrix was discarded and the
tissue was rinsed with PBS. The bone matrix was plated in tissue culture
to allow cells to grow out. Cells were culture expanded in StemPro
MSC medium (Invitrogen, Carlsbad, CA) and then plated in StemPro
Osteogenesis Differentiation Medium (Invitrogen) for 9 days prior to
alkaline phosphatase staining.
RESULTS
When introduced to unactivated PBMCs, the VCBM did not elicit an
immune response. As opposed to PBMCs cultured with gammairradiated MSCs alone, there was minimal stimulation of the PBMCs
when cultured with the VCBM (Figure 2a). Furthermore, when exposed
to activated PBMCs, the VCBM had an immuno-suppressive response.
Cell viability of the activated PBMCs was not affected, indicating that
the VCBM was not toxic to the PBMCs. However, there was significant
decrease (p<0.05) in the stimulation indices of activated PBMCs when
exposed to the VCBM (Figure 2b).
Flow cytometry analysis indicated that cells harvested from the VCBM
had minimal expression of CD2, CD3, and CD8 indicating that these
immune-reactive T-cells are not present in the VCBM (Table1). Further
characterization of the cells from the VCBM indicated the cells are
capable of differentiating into bone forming cells. Only in the presence
of osteogenic differentiation medium did the cells stain positively for
bone formation activity (Figure 3). Taken together with the MLR data,
this suggests that there are MSCs present in the VCBM.
Figure 2: A. MLR performed with unactivated PBMCs. PBMCs were
not activated in the presence of the VCBM. B. MLR performed with
activated PBMCs. The VCBM was immuno-suppressive when exposed
to the activated PBMCs.
Markers
CD2
CD3
CD8
% Shift
1.16
2.30
1.35
Table 1: Flow cytometry results for cell harvested from the VCBM.
Cells did not express markers for CD2, CD3, and CD8.
Figure 3: A. Cells growing out from the VCBM. B, C. Alkaline
phosphatase staining of cells harvested from the VCBM after 9 days in
differentiation medium and MSC medium (negative control),
respectively. Microscope magnification: 10x.
DISCUSSION
A novel VCBM was designed for use in bone grafting applications and
evaluated for its ability to safely deliver cells capable of osteogenic
differentiation. Previous research has shown that MSCs can be immunoprotective1. A similar assay was used to confirm that the VCBM is also
immuno-protective. Interestingly, when the assay was repeated using
activated PBMCs harvested from a donor with a viral infection, the
VCBM decreased the activity of the PBMCs by10% suggesting that the
VCBM is immuno-suppressive as well as immuno-protective. Cells
harvested from the VCBM were capable of osteogenic differentiation in
the presence of osteogenic differentiation medium. It is possible that the
VCBM exhibited immuno-protective behavior in the MLR assay due to
MSCs present within the bone matrix. The results from this study
suggest that this bone grafting material is a safe and effective delivery
system of osteogenic cells. Future research will aim to characterize the
osteoinductivity of the VCBM and evaluate its bone forming activity in
a posterolateral spine fusion model and ectopic bone formation model.
SIGNIFICANCE
This study is the first investigation to describe the immuno-protective
and immuno-suppressive properties of a novel osteoinductive,
osteogenic, and osteoconductive bone grafting material.
REFERENCES
1. Potian JA et al. J Immunology. 171:3426-3434 (2003).
Poster No. 0641 • ORS 2012 Annual Meeting