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Xenogeneic Implantation of Human Mesenchymal Stem cells to Promote
Fracture Healing in Atrophic Non-Union Model
Tulyapruek Tawonsawatruk, MD1,2, Robert J. Wallace, PhD1, Antonio M. Spadaccino, Bsc. Hons, MRes1, Hamish RW Simpson,
DMed, ChB, MB, FRCS, MA1.
1
The university of Edinburgh, Edinburgh, United Kingdom, 2Ramathibodi Hospital, Bangkok, Thailand.
Disclosures:
T. Tawonsawatruk: None. R.J. Wallace: None. A.M. Spadaccino: None. H.R. Simpson: 1; N/A. 2; Orthofix, Inc.. 3A; N/A. 3B; N/A.
3C; N/A. 4; N/A. 5; Stryker, Pfizer. 6; N/A. 7; Saunders/Mosby-Elsevier.
Introduction: Recently cellular therapy using Mesenchymal stem cells (MSCs) has become a promising strategy to improve
fracture healing in established non-union cases. MSCs are derived from bone marrow and are identified by their ability to
proliferate and undergo mutilineage differentiation (1). MSCs are believed to represent bone precursors and their ability to
undergo osteogenesic differentiation is desirable for bone repair and regeneration. However, several conditions may impair the
therapeutic potential of MSCs such as aging (2), smoking (3) and excessive alcohol ingestion (4). Therefore as MSCs are
considered not to trigger immune response (5), it would be advantageous for MSCs to be derived from healthy hosts and then
used as universal donor cells. A xenogeneic model has been reported to represent the most extreme immune response (6). Thus,
if there are any benefits in using human MSCs to promote fracture healing in an immunocompetent animal model, the results
should also reflect the outcomes of using universal donor cells in a clinical setting. The objectives of this study were 1) to
compare the bone regeneration potential of xenotransplantation and allogeneic MSC implantation in atrophic non-union 2) to
demonstrate the immune response after MSC implantation in vivo.
Methods: This study was conducted using male adult Wistar rats. An atrophic non-union was created at the tibial mid shaft by
stripping the periosteum and endosteum as well as creating a small (1.0 mm) non-critical gap. This procedure was validated and
characterised in 11 animals. To study the therapeutic effect of MSC implantation, atrophic non-union was created in 24 animals
which were then randomly allocated into three groups according to treatment; phosphate buffered saline (PBS) injection (n = 8),
rat MSCs injection (n = 8) and human MSC injection (n = 8). Either cells (five million) or PBS was administered using percutaneous
injection under fluoroscopy three weeks after the atrophic non-union procedure. Fracture healing was determined using
radiography, histology, biomechanical testing and micro-CT at 4 and 8 weeks after treatment. Implanted cells were traced using
CM-Dil and anti-human Nuclei Antibody. The panel of inflammatory cytokines from the rat serum were assessed using a
commercially available kit; Rat Inflammatory Cytokines Multi-Analyte ELISArray Kit every two weeks. Popliteal lymph node
histomorphology including the size of lymph node, the follicle number, the presence of infiltrated cells at the capsular sinus and
histocytes at medullar sinus was evaluated at the 4 and 8 week time points. The healing time was compared using Kaplan-Meier
survival analysis. All fracture healing parameters for each treatment and time point were analysed using a two-way ANOVA with
a Bonferroni post hoc test for multiple comparisons. A p-value less than 0.05 was considered statistically significant.
Results: Non-critical size defect non-unions were successfully induced in all animals (n=11). The typical characteristics of
atrophic non-unions were demonstrated by radiography, micro-CT and histology (Figure 1). The mean healing time of animals in
the rMSC injection group was 4.3 weeks (SD = 2.1) and in the hMSC injection group was 5.4 weeks (SD = 1.9). The survival curves
of the rMSC injection group and the hMSC injection group were significantly different from the control (P = 0.003 and P = 0.008,
respectively) (Figure 2). However, there was no significant difference between the rMSC injection group and the hMSC injection
group (P = 0.36). Examination of the radiographs showed the progression of fracture healing over the 8 week period after cell
injection. In contrast, the control group demonstrated progression of the fracture site to full characteristics of an atrophic nonunion. Micro-CT analysis showed that the percentage bone volume (BV/TV) and bone mineral density (BMD) in the rMSC and
hMSC injection groups to be significantly greater at both 4 and 8 weeks after injection compared to the control group (ANOVA, P
< 0.01). There were no significant differences in ultimate load, ultimate stress, young’s modulus and toughness between
implantation with rat MSC and human MSC at 4 weeks and 8 weeks after injection (Figure 3). Samples in the PBS group were
unsuitable for the bending tests as they deformed freely under their own weight; clearly indicating that insufficient healing had
occurred. The amount of mineralised tissue in the interfragmetary gap, as assessed from histological sections with either the rat
or human MSC implantation was significantly more than in the control specimens at both 4 and 8 weeks (ANOVA, P<0.01)
(Figure 4). There were no hMSCs detected at the fracture gap at 4 weeks and 8 weeks, while rMSCs were still found at 4 weeks,
but not at 8 weeks. In the hMSC treatment group, there was an increase in the GM-CSF level at both 4 and 8 weeks; however
histomorphological analysis showed no significant immune reaction from the lymph node in the rat and human MSC groups.
Discussion: Our results suggest that hMSC and rMSC are comparable and they both lead to an improvement in fracture healing
in an early atrophic non-union model. There were no clinical adverse effects detected with xenogeneic transplantation. Although
there was an increase in GM-CSF level 8 weeks after hMSC injection, there were no significant changes in the size of the lymph
node, a number of secondary follicles, presences of infiltrated cells at the capsular sinus and macrophages at the medullary
sinus. The cell tracing results suggested that the hMSCs did not persist at the fracture site. Therefore, the xenogeneic MSCs
contributed to the fracture healing process, but the cells disappeared when fracture progressed to union, suggesting that
exogenous MSCs may improve fracture healing via their paracrine effect but not as progenitor cells. Although there was some
immune response, the therapeutic effect of MSCs in bone repair was not significantly inhibited. This study demonstrated that
xenogeneic cells can improve bone healing, supporting the feasibility of using universal donor cells for bone repair. However,
further studies on the mechanisms of immune response and the interactions of MSCs in an allogeneic or xenogeneic
environment should be performed before MSCs from a universal donor are used to improve fracture healing in the clinical
setting.
Significance: The present findings have demonstrated the therapeutic effects of MSC to improve fracture healing in a
xenogeneic atrophic non-union and represent an excellent initial step toward the concept of using MSCs as universal donor cells
in fracture repair patients.
Acknowledgments: We would like to thank: Professor Sarah Howie who gave us much valuable advice in the early stages of this
work, Mr. James Baily who guided us in the methods to evaluate the histological sections and Professor Bruno Péault for his
collaboration with our department.
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3. Gullihorn L, Karpman R, Lippiello L. Differential effects of nicotine and smoke condensate on bone cell metabolic activity. J
Orthop Trauma. 2005 Jan;19(1):17-22.
4. Chakkalakal DA. Alcohol-induced bone loss and deficient bone repair. Alcohol Clin Exp Res. 2005 Dec;29(12):2077-90.
5. Jones BJ, McTaggart SJ. Immunosuppression by mesenchymal stromal cells: from culture to clinic. Exp Hematol. 2008
Jun;36(6):733-41.
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9.
ORS 2014 Annual Meeting
Poster No: 0649