New emerging concepts in the medical management of local radiation injury.

Abstract

Treatment of severe radiation burns remains a difficult medical challenge. The response of the skin to ionizing radiation results in a range of clinical manifestations. The most severe manifestations are highly invalidating. Although several therapeutic strategies (excision, skin grafting, skin or muscle flaps) have been used with some success, none have proven entirely satisfying. The concept that stem cell injections could be used for reducing normal tissue injury has been discussed for a number of years. Mesenchymal stem cells therapy may be a promising therapeutic approach for improving radiation-induced skin and muscle damages. Pre-clinical and clinical benefit of mesenchymal stem cell injection for ulcerated skin and muscle restoration after high dose radiation exposure has been successfully demonstrated. Three first patients suffering from severe radiological syndrome were successfully treated in France based on autologous human grade mesenchymal stem cell injection combined to plastic surgery or skin graft. Stem cell therapy has to be improved to the point that hospitals can put safe, efficient, and reliable clinical protocols into practice.

Full text

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New emerging concepts in the medical management of local radiation
injury

Emerging concept of local radiation treatment

Marc Benderittera, Jean Jacques Latailladeb, Eric Beyc, Marie Pratb and Patrick
Gourmelona

a Institut de Radioprotection et de Sûreté Nucléaire, Laboratoire de radiopathologie et de
thérapie cellulaire, BP 17, 92262 Fontenay-aux-Roses, France.

b Centre de transfusion sanguine des armées, Département de recherche et de thérapie
cellulaire, 92141 Clamart, France.

c Service de Chirurgie plastique, Hôpital d'Instruction des Armées Percy, 92141 Clamart,
France.



Abstract
Treatment of severe radiation burns remains a difficult medical challenge. The response of the skin to ionizing
radiation results in a range of clinical manifestations. The most severe manifestations are highly invalidating.
Although several therapeutic strategies (excision, skin grafting, skin or muscle flaps) have been used with
some success, none have proven entirely satisfying. The concept that stem cell injections could be used for
reducing normal tissue injury has been discussed for a number of years. Mesenchymal Stem Cells (MSC)
therapy may be a promising therapeutic approach to improve radiation-induced skin and muscle damages. Pre-
clinical and clinical benefit of MSC injection for ulcerated skin and muscle restoration after high dose
radiation exposure has been successfully demonstrated. Three first patients suffering from severe radiological
syndrome were successfully treated in France based on combined autologuous human grade MSC injection to
plastic surgery. Stem cell therapy must be now improved to the point that hospitals can put safe, efficient,
reliable and inexpensive clinical protocols into practice.


KEYWORDS: Stem cell therapy, Mesenchymal stem cell, radiological burn

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1. Scientific and clinical background

The medical management of severe radiation burns after accidental overexposure to ionizing
radiation is still a major therapeutic challenge [1] unresolved with the classical therapeutic
approach derived from the management of thermal or electrical burns.
There are marked differences between radiation and thermal burns in terms of patho-physiological
mechanisms, clinical aspects and evolution. The main feature of severe radiation burns is the
occurrence of unpredictable successive inflammatory waves leading to the extension, in surface
and in depth, of the necrotic process. After an initial period marked by a clinical picture limited to a
rash and itching, subsequent ulceration and necrosis develop, which may extend to the deep dermal
and underlying muscle structures. Moreover, inflammatory waves are associated with
uncontrollable pain highly resistant to morphinics. The patho-physiological process, which
involves a cascade of inflammatory mediators and a continuous activation of target cells
(endothelial cells and fibroblasts), is not totally elucidated [2-6].
The surgical management of severe necrotic radiation burns is theoretically easy to perform. The
conventional main strategy is the excision of the necrotic tissues followed by a rotation flap or a
good quality skin autograft. In practice, the planning of such a surgical approach often encounters
insurmountable technical difficulties due to the occurrence of successive and unpredictable
inflammatory waves associated with a progressive extension of the necrotic process. Then, the
evolution of the radiation lesion often becomes uncontrolled and the final option is a last surgical
act leading to a very high morbidity and disability. Thus, in two highly irradiated Peruvian and
Georgian victims, previously treated in 1999 and 2002, with the classical surgical approach
combining excision and skin graft, it was not possible, in the Peruvian case, even after amputation
of the irradiated leg, to manage the huge extension, at the perineal level, of the radionecrotic
process. Concerning the Georgian case, the conventional treatment was a failure since four
successive excisions followed by skin autografts were always inefficient 440 days post-exposure
and only an autonomous, vascularized tissue (omentum flap) covered by skin allograft allowed
healing at 500 days PI [7-8].
Thus, to date, the best therapeutic approach for severe radiation burns remains unknown.

2. New emerging concept

Irradiation kills normal cells directly or indirectly and the basic issue is to replace them.
Replenishment of the depleted stem cell compartment and/or stem cell plasticity should allow better
regeneration of irradiated tissues. Adult stem cell therapy was postulated to favour also the
radiation burn healing process through the secretion of trophic factors (growth factors, cytokines)
that may counteract the local inflammatory wave processes and favour angiogenesis. Stem cell
therapy using bone marrow mesenchymal stem cells (MSC) may be a promising therapeutic
approach to improve radiation-induced normal tissue damage.
The first challenge in MSC transplantation is that the cultured cells retain their quality and their
differentiation potential during the expansion process. In order to treat tissue injury using cell
therapy, the number of cells required can be very high. Stem cells are a small percentage of the total
cellularity, so their pool has to be expanded ex vivo and injected taking into account the need for
immature cells – stem cells and progenitors – or differentiated cells. To be of therapeutic use, the
produced cells must retain normal function, differentiation pattern and regulation during culture.

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Mesenchymal stem cells (MSC) have been described in the bone marrow as multipotent progenitor
cells that differentiate into endothelial cells, epithelial cells, stromal cells but also osteocytes,
chondrocytes and adipocytes. Their ability to differentiate according to multiple lineage
characteristics is preserved along the expansion process. It has been demonstrated that MSCs can
be easily recovered from bone marrow or adipose tissue and enriched through their property of
adhering to tissue culture surfaces. Several groups have recently expanded MSCs up to a million
fold in vitro for hematologically and orthopedically relevant applications.


Moreover, MSCs are able to migrate towards injured tissular lesions where they deliver a high
number of growth factors that are required for immunoregulation and repair processes [9, 13].
Their positive effect in promoting the healing of radiation burn lesions in a preclinical
immunodeficient non obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model
was demonstrated [10-13]. In this model, the intravenous administration of human MSCs strongly
improved the healing of burn lesions induced by a 30 Gy irradiation [13]. The presence of hMSC in
the mouse skin 21 days after transplant suggest that incorporation of transplanted cells in skin
structure can only be seen 3 weeks after wounding. Once implanted in the injured area, bone
marrow cells could promote the migration, proliferation and differentiation of epidermal cells. The
presence of hMSC was evidenced in the irradiated epidermis. These results are the first evidence,
using human cells, of the possible use of human MSC for the treatment of the acute cutaneous
radiation syndrome [13].


The mechanisms leading to the observed positive effects of hMSC therapy on skin repair remain to
be studied. It has been reported that bone marrow cells could become perifollicular cells, blood
vessel cells or perisebaceous gland cells during the healing process [14]. Bone marrow cells could
differentiate into myofibroblasts and play an important role in the formation of granulation tissue
during the wound healing process when combined with an occlusive dressing. Injected bone
marrow MSC could give rise to functional skin cells at a high frequency and regenerate skin tissue.
Furthermore, in a skin defect model, MSC associated with Fibroblast Growth Factor (FGF) could
accelerate cutaneous wound healing as MSC differentiate into the epithelium [15]. The production
and secretion of beta-TGF by hMSC could be involved, suggesting a possible effect of hMSC on
the skin lesion through paracrine mediator release. Indeed, it has been reported that beta-TGF may
induce the growth of stem cells at the level of the skin and may stimulate the repair process by
enhancing extra-cellular matrix synthesis [16-18].

The successful transplant of stem cells and subsequent reduction in radiation-induced
complications may open the road to completely new strategies in cutaneous radiation syndrome
therapy. All together, these results are of clinical significance, as a drastic reduction of skin
necrosis may be a major advance in the treatment of acute cutaneous radiation reactions. This work
supports the use of hMSC infusion to repair skin injuries in patients after accidental irradiation.

3. Medical breakthrough in the treatment of radiological burn : 2 case reports

Clinical case report 1: The accident of Conception (Chili-2005) [20-21]
A 27-year-old Chilean man was overexposed, on 15 December 2005, to a gammagraphy radio-
active source (192Ir, 3.3 TBq). He picked up the source with his left hand and put it in the back left
pocket of his trousers. Following a multifocal localized irradiation, he rapidly exhibited severe
radiation burns located to the hand and the buttock. The early occurrence of skin lesions (ringed
permanent erythema with a central atonic area) at the buttock level within the first days after
irradiation strongly suggested a very high-dose exposure. The buttock skin lesion evolved into
moist epidermitis (4–5 cm in diameter), then quickly worsened and progressed to ulceration. These
radiation skin lesions were accompanied by classical intense pain, which was only partially

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alleviated by morphine. The early development of the buttock lesion without any latency phase, its
fast evolution toward ulceration and the uncontrolled pain were characteristic of a very severe
radiation burn with poor prognosis.
The strategy adopted to reconstruct the accidental dose distribution delivered to the patient was
based on numerical simulations. The numerical dosimetric reconstruction of the radiation accident
requires simulating the source, its environment and the patient body using a numerical anthropo-
morphic phantom. The doses absorbed by the tissues were then calculated using a computer code.
The dose absorbed at the skin lesion center was very high (almost 2000 Gy), but dropped rapidly
due to the combined effect of distance and tissue attenuation. Based on the dose reconstruction
mapping, a wide resection in apparently healthy tissues was performed on day 21 PI. All tissues
exposed to a dose over 20 Gy that were situated between the center of the lesion and the 20 Gy
isodose were excised according to hemisphere of 10 cm in diameter and then covered with a
cryopreserved allograft.
A new therapeutic strategy combining this classical surgery procedure (excision plus skin
autograft) and a local MSC therapy was designed. For MSC production, an autologous bone
marrow collection was performed, allowing a two-step MSC expansion producing 182 × 106 cells
at the first passage (P1) and 227 × 106 cells at the second passage (P2). Expanded cells exhibited
antigenic characteristics of MSCs: they did not express CD45, but expressed CD90, CD105 and
CD73 antigens. MSC purity was up to 97% and their clonogenic efficiency obtained at P1 was 225.
No telomerase activity was found in the expanded MSCs. The pluripotentiality of expanded MSC
was controlled by in vitro osteogenic, adipogenic and chondrogenic assays. Local administrations
of 168 × 106 MSC on day 90 PI and 226 × 106 on day 99 PI were performed in circle around the
lesion at the cutaneous and muscular levels, and in the wound bed of the lesion under the skin graft.
The lesion was further dressed with an artificial derma (Integra®). Following this combined therapy,
the healing of the lesion proceeded smoothly. There was no side effect. Optimal healing persists 2
years after the procedure.
Clinical case report 2: The accident of Dakar (Senegal-2006) [19]
A second experience of therapeutic management of a radiation accident victim combining stem cell
therapy using autologous mesenchymal stem cells and surgery is reported. On 3 June 2006, in
Dakar, during a gammagraphy operation, whilst inserting the source in the storage container, the
end of the remote control broke off for unknown reasons. Due to this technical failure, the 192Ir
radioactive source was lodged unexpectedly in the source ejection duct instead of being returned to
its shielded storage container, without this malfunction being detected. The remote control and the
ejection duct (holding the source) were stored temporarily under the stairs in an entrance hall. Four
patients were irradiated and send to the Percy hospital in France. The most severe irradiated patient
presented a very severe arm radiation burn which was treated by several surgical times/ iterative
excision, skin graft, latissimus muscle dorsi flap and forearm radial flap. Local autologous MSC
were administered as an adjuvant to improve the surgical approach. The clinical evolution,
radiation pain and healing progression was favourable and no recurrence of radiation inflammatory
waves was observed during the eight month patient’s follow-up suggesting that MSC act as ‘cell
drug” in modulating radiation inflammatory processes.

4. Conclusion
In conclusion, these data and first clinical application open new prospects in the medical management of
severe radiation burns. If confirmed in further radiation accidents, it would bring a major therapeutic
improvement. Stem cell therapy must be improved to the point that hospitals can put safe, efficient and
reliable and inexpensive clinical protocols into practice. The Institute of Radioprotection and Nuclear Safety
(IRSN) develops procedures that should achieved tissue repair in the long term to the benefice of a great
number of patients with skin complications after high dose radiation exposure. Furthermore, this novel
multidisciplinary therapeutic approach using physical techniques, surgical procedures and cellular therapy
with adult stem cells may open new prospects in the field of radiotherapy complications.

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