Abstract
In the setting of bone defects, the injured vasculature and loss of hemodynamic inflow
leads to hematoma formation and low oxygen tension which stimulates vascular expansion
through the HIf-1α pathway. Most importantly, this pathway upregulates sprouting of
type H vessels (CD31hiEmcnhi vessels). H vessels engage in direct interaction with
perivascular osteoprogenitor cells (OPCs), osteoblasts, and preosteoclasts of bone
formation and remodeling. This angiogenic-osteogenic coupling leads to synchronous
propagation of vascular and bony tissue for regenerative healing. A growing body of
literature demonstrates that H vessels constitute a large portion of bone's innate
capacity for osteogenic healing. We believe that CD31hiEmcnhi vessels play a role
in bone healing during distraction osteogenesis (DO). DO is a procedure that utilizes
traction forces to facilitate induction of endogenous bone formation and regeneration
of surrounding soft tissues such as skin, muscle, tendon, and neurovascular structures.
While the H vessel response to mechanical injury is adequate to facilitate healing
in normal healthy tissue, it remains inadequate to overcome the devastation of radiation.
We posit that the destruction of CD31hiEmcnhi vessels plays a role in precluding DO's
effectiveness in irradiated bone defect healing. We aim, therefore, to recapitulate
the normal pathway of bony healing by utilizing the regenerative capacity of H vessels.
We hypothesize that using localized application of deferoxamine (DFO) will enhance
the H vessel-mediated vasculogenic response to radiation damage and ultimately enable
osteogenic healing during DO. This discovery could potentially be exploited by developing
translational therapeutics to hopefully accelerate bone formation and shorten the
DO consolidation period, thereby potentially expanding DO's utilization in irradiated
bone healing.
Sprague–Dawley rats were divided into three groups: DO, radiation with DO (xDO), and
radiation with DO and DFO implantation (xDODFO). Experimental groups received 35 Gy
of radiation. All groups underwent DO. The treatment group received injections into
the osteotomy site, every other day, beginning on postoperative day (POD) 4 of DFO.
Animals were sacrificed on POD 40. For immunohistochemical analysis, mandibles were
dissected and fixed in 4% paraformaldehyde for 48 hours, decalcified in Cal-Ex II
for 2 days, dehydrated through graded ethanol of increasing concentration, and then
embedded in paraffin. Samples were cut into 7-μm thick longitudinally oriented sections
including the metaphysis and diaphysis. CD31 and Emcn double immunofluorescent staining
were performed to evaluate the extent of CD31hiEmcnhi vessel formation. Bone sections
were then stained with conjugated antibodies overnight at 4°C. Nuclei were stained
with Hoechst. Slides were also double stained with Osterix and CD31 to study the quantity
of H vessel-mediated recruitment of OPCs to accelerate bone healing. Images were acquired
with a Nikon Ti2 widefield microscope and analyzed in NIS- Elements Advanced Research
5.41.02 software. The abundance of type H vessels is represented by the area fraction
of CD31 + Emcn+ vessel area inside the regenerate sample. OPC concomitant proliferation
into the distraction gap is represented by the area fraction of Osterix+ cell area
inside of the regenerate sample.
There were 6× more type H vessels in DO groups than in xDO groups. Localized DFO significantly
increased the abundance of type H vessels of irradiated DO animals compared to xDO
by 15× (p = 0.00133531). Moreover, the DO and xDODFO groups with higher abundance of type H
vessels also demonstrated better angiogenesis and osteogenesis outcomes. Interestingly,
xDODFO groups doubled the quantity of H vessel formation compared to DO, indicating
a supraphysiologic response (p = 0.044655055). Furthermore, H vessel-mediated recruitment of OPCs mimicked the described
H vessel formation trend in our study groups. Irradiated DO groups contained 3× less
OPCs compared to DO controls. DFO treatment to xDO animals remediated irradiation
damage by containing 12× Osterix+ cells. Finally, DFO treatment of irradiated animals
quadrupled osteoprogenitor recruitment into the distraction gap compared to DO controls.
In this study, we developed a novel approach to visualize CD31hiEmcnhi in paraffin
sections to study DO regeneration. Normal DO demonstrated a significant upregulation
of H vessel formation and associated angiogenic-osteogenic coupling. Radiation severely
decreased H vessel formation along with an associated significant diminution of new
bone formation and nonunion. DFO administration, however, resulted in vascular replenishment
and the restoration of high quantities of CD31hiEmcnhi and OPCs, recapitulating the
normal process of bony regeneration and repair. DFO treatment remediated new bone
formation and bony union in irradiated fields associated with increased H vessel angiogenic-osteogenic
coupling. While further studies are required to optimize this approach, the results
of this study are incredibly promising for the long-awaited translation of localized
DFO into the clinical arena.
Keywords
distraction osteogenesis (DO) - deferoxamine (DFO) - radiation - bone regeneration
