regenerative facial reconstruction of terminal stage osteoradionecrosis and other advanced...

7/31/2019 Regenerative Facial Reconstruction of Terminal Stage Osteoradionecrosis and Other Advanced Craniofacial Disease

1/12

PEDIATRIC/CRANIOFACIA

Regenerative Facial Reconstruction of TerminalStage Osteoradionecrosis and Other Advanced

Craniofacial Diseases with Adult Cultured Stemand Progenitor Cells Jose J. Mendonca, M.D.,

D.M.D., Ph.D.Pedro Juiz-Lopez, M.D.

Lugo, Spain

Background: Treatment options in cases of severe craniofacial disorders withbone loss and tissue damage are usually limited to vascularized and nonvascu-larized tissue transfers, allografts, mechanical devices and, more recently, facialtransplantation.Despite the therapies available, the demand for new approachesis realized in cases where current therapies are unable to resume form andfunction. This study presents the feasibility of alternative treatments based oncultured bone marrow cells that yield mixed populations of mesenchymal,hematopoietic, and endothelial lineages at very early stages implemented as part of a novel regenerative procedure.Methods: One hundred milliliters of a bone marrow aspirate was inoculatedinto the automated single-pass perfusion technology system, AastromReplicell,for the development of the cellular product, tissue repair cells. After 12 days of incubation, cells were exposed to a specially designed osteogenic environment in an autogenous fibrin-rich and platelet-rich clot and membrane with a mineralbase of -tricalcium phosphate and hydroxyapatite.Results: A case of maxillary and mandibular radionecrosis (stage IIIR) withpathologic fracture presented early osteogenesis, total recovery from alveolarnerve anesthesia, facial nerve reinnervation, and skin regeneration. Anothercase with nonhealed fracture, bone loss, and bilateral paresthesia demonstratedcallus formation, bone regeneration, and nerve recovery.Finally, maxillaryboneregenerated after massive deficiency. Oral functional restoration with implantsand fixed prosthesis was accomplished in all cases.Conclusion: After nerve, bone, skin, and vessel formation in three patients withsevere abnormality, bone marrowderived mixed cultured stem cell lineagescould be considered a new paradigmatic approach to advanced disease. ( Plast.Reconstr. Surg. 126: 1, 2010.)

Treatment options in cases of severe facialbone and tissue loss are usually accom-plished by autografts, 1 allografts, cytokines,mechanical devices and, more recently, facial

transplantation. Occasionally, the disease out- weighs the balance between destruction and re-generation, mainly when tissues other than boneare involved. 2,3 Many severe conditions remainchallenging, without a clear curative solution, andpatients are left with palliative or limited thera-pies. Autogenous grafts have been considered the

standard treatment in all osseous regenerativeprocedures. Bonesubstitutes, cytokines,and othersubstances are designed to enhance conventionalgrafting techniques or as an alternative to bone

harvesting, potentially improving grafts or avoid-ing donor-site surgery and morbidity. The iliaccrest has passed the test of time, 1 proving to be themost prolific donor site of the body, with the great-est variety of bony structures and cell populations.The most remarkable quality is the relatively largepopulations of stem and progenitor cells in the

From the Head and Neck Surgery Unit, POLUSA Hospital.Received for publication January 22, 2010; accepted May 10, 2010.Copyright 2010 by theAmerican Society of Plastic Surgeons

DOI: 10.1097/PRS.0b013e3181f24164

Disclosure: The authors have no financial interest to declare in relation to the content of this article.

www.PRSJournal.com 1

ot


2/12

marrow and adjacent areas. These cell popula-tions are occasionally able to survive transplanta-tion and hypoxia, revitalizing grafts through che-motaxis, mitosis, and differentiation.

The use of whole or subsets of bone marrow isnot new; hematopoietic recovery in myeloprolifera-tive disorders followed by high-dose radiochemo-therapy was successfully accomplished years ago us-ing this technique. More recently, patient-specifictherapies have overcome the limitations of progen-itor pools by ex vivo culture of certain lineages. 4 In vitro differentiation of marrow-derived stem cells isable to yield basic tissue components, such as neu-rons, hepatocytes, adipocytes, cardiomyoblasts,endothelial cells, Langerhans islets, osteoblasts,and others. 59 The concept and attributed poten-tial of these cells has changed dramatically in re-cent years, and they are currently considered aspossible pluripotent cells. 10,11 The acquisition of local phenotypes caused by native factors and pos-sibly cell fusion reveals an unexpected plasticity and behavior. Animal experimentation is quiteadvanced, demonstrating dramatic results in un-treatable conditions 12; nevertheless, strictly con-trolled legal clinical experience in humans isscarce, with a paucity of reports available in theliterature. 1315 There are not only many ethical andlegal limitations to human investigation but alsoenormous difficulties in translating basic researchor animal investigation.

Aging maxillary bone loss is a common findingin humans and is frequently related to tooth lossand periodontal disease; trauma, radiotherapy,and tumors also account for severe osseous tissuedestruction. Despite the variety of therapies avail-able, some cases are beyond the scope of moderntreatments or are performed with important risksand morbidity.

Osteoradionecrosis occurs when the normalbone turnover is exceeded by the degradative pro-cess within an irradiated field. It was first describedin the mandible by Marx 3 as a process related to

endarteritis and hypocellularity, leading to tissuehypoxia, apoptosis, fibrosis, and hypovascularity.

Final stages are often attributable to iatrogenicintervention, mainly tooth extraction leading tointraoral bone exposure and eventually a patho-logic fracture and fistula. Osteoradionecrosis isnot limited to the mandible and skin; mucosa,fat, periosteum, and muscle are devastated as well, initiating a vicious cycle that results ingreater instability.

In this article, we report the use of a new autologous cell therapy in which a small volume of iliac marrow aspirate cultured ex vivo generateslarge amounts of early-stage mesenchymal, endo-thelial, hematopoietic stem, and progenitor cells(tissue repair cells) appropriate for use in bonegrafting. 16 Tissue repair cells have been previously used to substitute for bone marrow transplants incancer patients to restore hematopoiesis. 4

PATIENTS AND METHODSOne hundred milliliters of heparinized bone

marrow aspirate was obtained from the poste-rior ileum under conscious sedation. A specifictechnique based on multiple-point minimal vol-ume smooth aspiration provided a rich nucle-ated cell aspirate with a maximum of viable cells( see Video, Supplemental Digital Content 1, which demonstrates bone marrow aspiration,http://links.lww.com/PRS/A217 ) that was sent in aspecial transport to a granulocyte-macrophageprogenitor processing unit and inoculated intothe closed, automated, computer-controlled Aas-tromReplicell System (Aastrom Biosciences, Inc., Ann Arbor, Mich.) and cultured for 12 days at 37C in Iscoves Modified Dulbeccos Mediumsupplemented with 10% fetal bovine serum, 10%horse serum, hydrocortisone, gentamicin sulfate(5 g/ml), L-glutamine (4 mM), and vancomycin(20 g/ml). To confirm nondetectable levels of

Supplemental digital content is available forthis article. Direct URL citations appear in theprinted text; simply type the URL address intoany Web browser to access this content. Click-able links to the material are provided in theHTML text of this article on the Journal s Website (www.PRSJournal.com). Video 1. Supplemental Digital Content 1 demonstrates bone

marrow aspiration, http://links.lww.com/PRS/A217 .

Plastic and Reconstructive Surgery November 2010

2

Q: 1

rich3/zpr-prs/zpr-prs/zpr01110/zpr3966-10z xppws S 1 8/23/10 7:03 4/Color Figure(s): F1-4,F6,F9-13 Art: PRS202603 Input-nlm
http://links.lww.com/PRS/A217http://links.lww.com/PRS/A217http://links.lww.com/PRS/A217http://links.lww.com/PRS/A217http://links.lww.com/PRS/A217


3/12

bacterial and fungal contaminants and endotox-ins, the culture medium was sampled 48 hoursbefore harvest. Cells were harvested on day 12.Reagents added during culture were below thedetectable limits of sensitive enzyme-linked immu-nosorbent assays. Flow cytometry, cell viability,and clonogenic assays confirmed tissue repair cellintegrity. Tissue repair cells were suspended in 150mlofNormosol (Hospira, Inc.,LakeForest, Ill.) with0.5% humanserum albumin andtransported at 4Cfor use within 8 hours. Cell samples for viability,quality, and characterization were taken before in-oculation and after production (Tables 1 thro-ugh 4). ( See Document, Supplemental Digital Con-tent 2, which demonstrates cell culture proceduresand release criteria, http://links.lww.com/PRS/A218 .)

Fifty milliliters of citrated venous blood drawnduring surgery was processed in approved systemsfor platelet concentration. Platelet-rich plasmaand platelet-poor plasma were clotted using au-tologous thrombin resulting from blood clot closedcentrifugation.Cellswere mixed with platelet-rich and platelet-poor plasma 17,18 and -tricalciumphosphate/hydroxyapatite, forming a bioactive ma-trix (Fig. 1). ( See Video, Supplemental Digital Con-tent 3, demonstrating injecting cells, temporaryscaf-fold, http://links.lww.com/PRS/A219 .)

A membrane created by compressing theplatelet-rich plasma 19 was injected with cells and

used as another temporary scaffold. Three pa-tients with severe disease were selected after meet-ing the following special criteria:

1. Severe facial tissue loss previously treated un-successfully.

2. Routine procedures for surgery.3. Extensive informed consent, with a witness,obtained 15 days before surgery.

4. Hospital ethics committee approval.5. Spanish ministry of health approval, Euro-

pean regulations for compassionate use of au-tologous stem cells.

6. All Conformite Europeenne/U.S. Food andDrug Administrationcertified materials. Ex-clusion of organic substances.

7. Extensive imaging diagnostics.8. Exclusion criteria were as follows:

Active or recent cancer, major health dis-orders. Smoking. Substance abuse. Metabolic bone or collagen disease. Poor oral hygiene. Pregnant or nursing women, women not

using contraceptives. Inadequate medications, allergy to com-

mon drugs.

Table 1. Bone Marrow Aspirate

Patient Volume

(ml)

BM CellConcentration

( 106/ml)

TNCsCollected

( 106) Viability

(%)1 98 20.76 2035.4 83.32 98 20.31 1991 85.13 96 20.68 1986 84.4BM, bone marrow; TNCs, total nuclear cells.

Table 2. Tissue Repair Cell Product (Output)

Patient Volume (ml)TNCs Released

( 106)Cell Concentration

( 106/ml) Viability (%) TNC Fold Expansion1 10.4 500.24 48.1 84.1 3.82 11.7 518.31 44.3 82.5 4.33 12.1 488.84 40.4 88.2 3.5TNCs, total nuclear cells.

Table 3. Tissue Repair Cell Product Phenotypes (Output)

Patient Thy-1 FoldExpansion CD90 (%) CD45 (%)

CD105 /CD166 (%)

CD144 /CD146 (%)

CXCR4 / VEGFR1 (%)

CD34 /lin (%)

1 29.8 35.88 66.77 14.1 3.3 11.8 0.522 31.5 25.07 62.81 13.8 4.2 12.1 0.853 38.7 33.78 73.83 14.4 3.5 12.6 0.67Thy-1, thymocyte differentiation antigen; CD, cluster of differentiation; CXCR4, chemokine receptor 4; VEGFR1, vascular endothelial growth

factor receptor 1; lin, lineage.

Table 4. Tissue Repair Cell Product ColonyForming(Output)

Patient CFU-F

(%)CFU-F FoldExpansion

CFU-GM(%)

CFU-GM FoldExpansion

1 3.9 76.9 0.8 1.12 3.2 86.2 0.6 13 3.6 78.7 0.6 0.9CFU-F, colony forming unit, fibroblast; CFU-GM, colony formingunit, granulocyte, monocyte.

Volume 126, Number 6 Regenerative Facial Reconstruction

3

1-4

,F11

Q: 3

Q: 4

http://links.lww.com/PRS/A218http://links.lww.com/PRS/A218http://links.lww.com/PRS/A219http://links.lww.com/PRS/A219http://links.lww.com/PRS/A219http://links.lww.com/PRS/A218


4/12

9. Patient permission: screening for viruses andcommon tumor markers.

10. Consistently the same surgeons and anesthetist.11. Healthy psychological background.12. Agreement to publish outcomes in scientific

journals regardless of poor or negative results.13. Following the guidelines of the International

Society for Stem Cell Research.

Case TechniquesFor radionecrosis, necrotic tissue was excised

excluding most of the cortical plates. The remain-ing bone was trimmed and carved to harbor thegraft and thoroughly perforated until bleedingfrom adjacent tissue appeared. A damaged inac-tive alveolar nerve was debrided with a micro-scope, followed by cell microinjections with apolycarbonate syringe and wrapping in the plate-let-poor plasma cell membrane. The platelet-poorplasma cell matrix was embedded carefully intothe defect and injected again (Fig. 1). A mandib-ular reconstruction plate was placed, and the wound was closed after cell flushing and inocula-tion into the skin, muscle, and areas adjacent tothe facial nerve and the necrosed vascular stumps.Intravenous administration of tissue repair cells was avoided. (See Video, Supplemental Digital Con-tent 4, which demonstrates procedure animation,http://links.lww.com/PRS/A220 ; and Video, Supple-mental Digital Content 5, which demonstrates the sur-gical procedure, http://links.lww.com/PRS/A221 .)

Posterior maxillary bone loss required sinuslift and platelet-poor plasma cell scaffold; muco-periosteal flaps and elevated sinus floors were mi-croinjected with tissue repair cells. Anterior max-illary atrophy was reconstructed with poly( L-lacticacid) plates that fixed and stabilized the grafts.

Other TechniquesOther techniques included lateralization of

the alveolar nerve with cell injection in the canaland nerve sheath (Fig. 2) ( see Video, Supplemen-tal Digital Content 6, which demonstrates nervecell injection, http://links.lww.com/PRS/A222 .)and 5-mm periodontal pocket debridement andgrafting. Perioperative antibiotics, dexametha-sone, and dexketoprofen trometamol were deliv-ered intravenously, and dexketoprofen trometa-

Fig. 1. Surgical site preparation and grafting of platelet-poorplasma plus mineral scaffold plus cells.

Video 2. Supplemental Digital Content 3 demonstrates inject-ing cells, temporary scaffold, http://links.lww.com/PRS/A219 .

Video 3. Supplemental Digital Content 4 demonstrates proce-dure animation, http://links.lww.com/PRS/A220 .


4

http://links.lww.com/PRS/A220http://links.lww.com/PRS/A220http://links.lww.com/PRS/A221http://links.lww.com/PRS/A221http://links.lww.com/PRS/A222http://links.lww.com/PRS/A222http://links.lww.com/PRS/A219http://links.lww.com/PRS/A219http://links.lww.com/PRS/A220http://links.lww.com/PRS/A220http://links.lww.com/PRS/A220http://links.lww.com/PRS/A219http://links.lww.com/PRS/A222http://links.lww.com/PRS/A221http://links.lww.com/PRS/A220


5/12

mol and amoxicillin clavulanate were prescribedorally for 7 days together with a mouth rinse. Allpatients were discharged from the hospital on theday after surgery following two episodes of generalanesthesia and one episode of conscious sedation.

Four months postoperatively, in all cases, arough-surface, high-quality implant was insertedin grafted and nongrafted areas. Two monthslater, fixed screwretained porcelain prostheses were placed on the implants.

RESULTSPatient 1, a 63-year-old man with a history of

radiation therapy for tonsillar cancer, reportedpain in the right hemimandible. Computed to-mographic scans (Fig. 3) disclosed a cystic lesionin the molar area. Diagnosed with limited man-

dibular and maxillary radionecrosis, the patient was treated elsewhere, returning 1 year later witha history of tooth extraction, pathologic fracture,life-threatening infection, prolonged hospitaliza-tion, and severe chronic pain with dysfunction.Several treatment strategies included surgical de-bridement, 45 dives in hyperbaric oxygen, antibi-otic therapy, and others. Despite these efforts, thedisease worsened, and a new computed tomo-graphic scan disclosed a severely fractured man-dible (Fig. 3). ( See Figure, Supplemental DigitalContent 7, which demonstrates a new computed

tomographic scan disclosing a severely fracturedmandible, http://links.lww.com/PRS/A223 .)Symptoms now included intraoral and ex-

traoral suppuration from two fistulas, severe tris-mus, total lip anesthesia from alveolar nerve dam-

Fig. 2. Nerve injection.

Video 5. Supplemental Digital Content 6 demonstrates nervecell injection, http://links.lww.com/PRS/A222 .

Fig. 3. Three-dimensional computed tomographic scans show evolution of radionecrosis to pathologicfracture after dental extractions (stage IIIR).


5

3

http://links.lww.com/PRS/A223http://links.lww.com/PRS/A223http://links.lww.com/PRS/A222http://links.lww.com/PRS/A222http://links.lww.com/PRS/A222http://links.lww.com/PRS/A223


6/12

age, xerostomia, and intense chronic pain.Neurophysiologic tests revealed partial dener- vation of the marginal mandibular nerve andabsence of right masseter activity. The diseasehad been the cause of early retirement. Freefibula grafting was rejected by the patient andevaluated as a high-risk procedure because of the damage to local vessels and the complexaccess to distant arteries.

Bone marrow aspirate, cell procedures, andsurgery took place as described previously without complications. A satisfactory recovery led to early hospital discharge 24 hours after surgery. Routineantibiotics and antiinflammatory drugs were pre-scribed for 1 week. Mild pain and swelling sub-sided completely just after surgery, whereas lipnumbness recovered progressively with an elec-tric tingling sensation 4 to 6 weeks postopera-tively. Thermoalgesic and tactile functionsarecur-rently near normal values.

Osteogenesis occurred within the first 3 monthsand continues to date (Figs. 4 and 5). ( See Figures,Supplemental Digital Content 8, which shows a com-puted tomographic scan obtained 3 months post-operatively, http://links.lww.com/PRS/A224 ; Sup- plemental Digital Content 9, which shows acomputed tomographic scan of the lower border,http://links.lww.com/PRS/A225 ; and SupplementalDigital Content 10, which shows a computed tomo-graphic scan of the coronal section demonstrating

bone formation, http://links.lww.com/PRS/A226 .)Two high standard dental implants were placed ingrafted areas of the maxilla and mandible for res-toration with fixed prostheses 2 months later. Bone

biopsy specimens during implant surgery demon-stratedanunusual purelycortical morphologyin themandible, with active osteocytes and osteoblasts, os-teoid formation, and partially resorbed biomaterial(Fig. 6). ( See Figure, Supplemental Digital Content 11, which shows histology of the grafted maxillary sinus, http://links.lww.com/PRS/A227 .) Dramaticchanges in dermal and epidermal morphology, in-cluding angiogenesis,appearedina skinbiopsyspec-imen taken during suture removal (Fig. 6).

Gadolinium magnetic resonance imaging at 9months showed high levels of blood supply to thegraft, with arteriogenesis of the facial and lingual vessels and profuse angiogenesis in the form of atortuous bundle from both of these arteries(Fig. 7). ( See Figures, Supplemental Digital Con-tent 12, which demonstrates a three-dimensional

magnetic resonance imaging scanof angiogenesis,http://links.lww.com/PRS/A228 ; and SupplementalDigital Content 13, which shows gadolinium up-take in th e grafted area demonstrating v ast angio-genesis, http://links.lww.com/PRS/A229 .) Neuro-physiologic testing disclosed changes in the latent period and right marginal mandibular nerve re-innervation pulses. Electrical and functional ac-tivity was also detected in the masseter muscle.

Twenty months after surgery, all improve-ments continued and the patient had returnedto a normal life without complications or se-

quelae. ( See Figure, Supplemental Digital Con-tent 14, which shows a view of the treated area,almost invisible scar, and high blood supply,http://links.lww.com/PRS/A230 .) Very recently, sia-

Fig. 4. Three-dimensional computed tomographic scans demonstrate critical state of necrosis and mas-sive osteogenesis 4 months later. Necrotic and grafted areas are delimited by arrows and circles .


6

4-5

http://links.lww.com/PRS/A224http://links.lww.com/PRS/A224http://links.lww.com/PRS/A225http://links.lww.com/PRS/A225http://links.lww.com/PRS/A226http://links.lww.com/PRS/A226http://links.lww.com/PRS/A227http://links.lww.com/PRS/A227http://links.lww.com/PRS/A228http://links.lww.com/PRS/A228http://links.lww.com/PRS/A229http://links.lww.com/PRS/A229http://links.lww.com/PRS/A229http://links.lww.com/PRS/A230http://links.lww.com/PRS/A230http://links.lww.com/PRS/A230http://links.lww.com/PRS/A229http://links.lww.com/PRS/A228http://links.lww.com/PRS/A227http://links.lww.com/PRS/A226http://links.lww.com/PRS/A225http://links.lww.com/PRS/A224


7/12

lography confirmed inexplicable reactivation of aradiotherapy-devastated parotid gland (Fig. 8).

Patient 2 was a 56-year-old man with chronic

pain, masticatory dysfunction and paresthesia of both lower lips, and a history of severe craniofacialtrauma with maxillomandibular fractures 12 yearspreviously. After several operations, the patient developed oral fistulas, chronic pain, functionallimitations, and bilateral alveolar nerve impair-ment. Diagnostic imaging revealed poor consoli-dation, bone loss, and invasion of the mandibularcanal with a screw.

Cell application procedures were similar, ex-cept for bilateral nerve lateralization, sheath dis-section, and cell injection (Fig. 2). Both sinuses

were grafted, and the anterior maxilla was recon-structed with two poly( L-lactic acid) plates andgrafts. Plates were exposed slightly 4 weeks later without infection but were not removed. Fourmonths later, thesame implants were inserted intoboth jaws, except in a small area of exposed plates where the bone was considered inappropriate.One year after surgery, the patient had resumednormal oral function with a fixed prosthesis. Bothmental nerves recovered despite mild paresthesiaat the screw-perforated site. Pain had subsided.

Patient 3 was a 52-year-old man with mastica-

tory dysfunction and joint pain resulting from the

absence of maxillary molars, with severe bone lossin the posterior maxilla, residual bone height ar-eas of 1 mm, and perforations to the sinus. Lower

limb and hip pathologic conditions contraindi-cated iliac harvesting. Calvarial harvesting was dis-carded because of unpredictable results with great volume defects, and allografts or xenografts alone were also rejected.

Cell processing followed the same techniquesdescribedabove; sinus lifts were grafted with plate-let-poor plasma cells and platelet-rich plasma cellscontralaterally. One implant was placed simulta-neously, and the rest were placed at 4 months. A fixed prosthesis was inserted 2 months later.

All patients led a normal life, with implant-

supported fixed prostheses (Fig. 9). ( See Figure,Supplemental Digital Content 15, which showsprosthetic reh abilitation in the area of prev iousradionecrosis, http://links.lww.com/PRS/A231 .) Allimplants survived, and bone density and functioncontinue to increase over time. More than 1 yearlater, a slight plate exposure remains the only complication to be reported.

DISCUSSIONThe therapeutic use of bone marrowderived

stem and progenitor cells for human disease is not

new. Native bone marrow, stimulation with gran-

Fig. 5. Preoperative and postoperative computed tomographic scans demonstrate significant bone formation.


7

8

http://links.lww.com/PRS/A231http://links.lww.com/PRS/A231http://links.lww.com/PRS/A231http://links.lww.com/PRS/A231


8/12

Fig. 6. Photomicrographs show changes in thehistology of bone andskin.Osteocytes, osteoblasts, andosteoid formation

are found at the time of implant insertion and4 monthsafter grafting (note unresorbed allograft on the right). Skin samplebefore and after surgery.

Fig. 7. Gadolinium-enhanced magnetic resonance imagingscan at 7 months demonstrates arteriogenesis of the facial andlingual vessels. Note theprofuse angiogenesis at thelevel of thegraft and the closed circuit with the maxillary artery.

Fig. 8. Sialographic image demonstrates parotid gland reacti-vation years after intense radiotherapy.


8



9/12

ulocyte colony-stimulating factor, and bloodapheresis and expanded bone marrow in autolo-gous and homologous procedures have been usedfor years in hematopoietic replacement after iat-rogenic destruction of blood-generative organs by radiotherapy, chemotherapy, or both. Hemato-poietic stem cell therapy is not only a consolidated

procedure but also a successful one. Adult stemcells have been the object of innumerable in vitroand animal experiments, demonstrating signifi-cant potential for the treatment of numerous dis-eases. Nevertheless, there is a paucity of reports inthe literature on therapies in humans with otheradvanced conditions.

In thisarticle,wereport the first use of a mixedpopulation of autologous cultured bone marrowderived stem andprogenitor cells in the treatment of severe craniofacial disorders, where an unex-pected outcome demonstrated the regeneration

of nervous, vascular, and dermal structures andbone. Osteoradionecrosis of the mandible devel-ops over a period ranging from months to yearsafter intense exposure to radiotherapy. Early stages are treated conservatively with routine den-tal care, oral hygiene, antibiotics, and extremecaution with extractions. Marx 20 hypothesized theprophylactic and therapeutic use of hyperbaricoxygen, assuming revascularization and cell in-duction by high partial pressure of oxygen. Crit-icism and controversy 20 followed, given the expen-sive and exclusive setup required and the

uncertain results, mainly in advanced cases. The

mechanisms involved are controversial and arebased not only on vascular deprivation but alsoon fibrous tissue formation and deficiencies inbone turnover. Furthermore, double-blind, pla-cebo-controlled studies that found no benefit from hyperbaric oxygen for advanced osteora-dionecrosis of the mandible 21 have led to the

emergence of new treatments. Restoration of the blood supply to the affected area continuesto be critical. One of the first images of humanangiogenesis and arteriogenesis after stem celltherapy in a case of radionecrosis is presented inthis article (Fig. 7). ( See Figures, SupplementalDigital Content 12, which showsa three-dimensionalmagnetic resonance imaging sc an of angiogenesis,http://links.lww.com/PRS/A228 ; and SupplementalDigital Content 13, which shows high gadoliniumuptake in t he grafted area demonstrating vast an-giogenesis, http://links.lww.com/PRS/A229 .) Endo-

thelial precursor cells and a wide variety and highlevels of angiogenic cytokines (mainly vascular en-dothelial growth factor) may be responsible forthe microvascular and macrovascular vessel devel-opment. It can be hypothesized that early vasculargraft ingrowth is the basis of regeneration of alltissues, delivering oxygen, nutrients, chemical sig-naling, migrating cells, and immunity. Osteogen-esis requires a copious blood supply but also anadequate cell pool.

Other effects, such as marginal mandibularnerve function, the recovery of three completely

inactive alveolar nerves, new skin formation

Fig. 9. Final radiographs obtained just after prosthesis placement.


9

Q: 6

http://links.lww.com/PRS/A228http://links.lww.com/PRS/A228http://links.lww.com/PRS/A228http://links.lww.com/PRS/A229http://links.lww.com/PRS/A229http://links.lww.com/PRS/A229http://links.lww.com/PRS/A229http://links.lww.com/PRS/A228


10/12

(Fig. 6), or masseter recovery, are more difficult toexplain. The human peripheral nervous systemshows a recognized ability to support limited oc-casional axonal regeneration. Although all nerves were inactive and recovered almost 100 percent of their functionality, it is possible that microinjec-tions of cells into the neural sheath led to immuneup-regulation, elimination of toxins, addition of nerve growth factors, and increase in blood sup-ply, or even influenced Schwann cells. The sameprocess could apply to the skin, but the possibility of real histogenesis cannot be completely discarded inthese cases. The therapy presented in this articleprovided clear evidence of the restoration of most lost facial tissues and functions, but the mechanismsinvolved remain to be understood.

Basic research has demonstrated that somephenotypes (CD105, CD166, CD105/166, CD146,CD90, and others) have a high osteogenic poten-tial; furthermore, mixed lineages, including non-specific fibroblast colony-forming unit and hema-topoietic CD34, CD13, CD14, and Gly-A, seem tobe more effective bone generators with additionalin vitro angiogenic capacities. The design of amixed population of stem and progenitor cells, 16as present in the culture used in this study, isconsistent with this principle; a tissue begins andends with different populations and depends onthe synergistic processing actions of multiple celllineages. The concept of a selected mixed popu-

lation versus a single population is a revolutionary point of view of histogenesis (Tables 2 through 4). Another critical concern in stem cell trans-

plantation is the delivery process. Some clinicalaspects of stem cell therapy may have been over-looked or underestimated because the resultshave not yet met the expectations and promises of a new age in medicine. One of the most challeng-ing aspects of the grafting procedure is cell scaf-folding and signaling. The design of a temporary (seeding the fibrin) and half-term scaffold has thepurpose of retaining cells until adhesion, and al-

lowing the development of a microenvironment.Fibrinogen and thrombin solutions must be care-fully considered for cell delivery because they af-fect the three-dimensional fibrin clot structureand cell proliferation. 18 The combination of acommercial blend of -tricalcium phosphate 22and hydroxyapatite (well-known in vitro nestinggrounds) was also added and trapped in the clot.Finally, an osteogenic environment would encour-age cells to adhere, divide, and differentiate (onexposure to growth factors and other factors).Contaminated areas were removed, but most of

the cortical bone remained after thorough stim-

ulation by deep trimming, becoming another scaf-fold with additional osteogenic potential.Multipleperforations expose underlying tissues, healthy bone marrow, and blood vessels to the graft, as-sisting notonly incoming chemoattracted cells but also dramatically increasing local distress bio-chemical signaling 19 (Fig. 1).

Another major concern in stem cell therapy issafety. Samples are processed and transported inspecial containers by certified agencies and aretested for contamination, disease, viruses, cell vi-ability, andDNA stability (clonogenic assays). Foldexpansions of the different cell populations arelimited and extend to only 12 days because of therisk of DNA transcription errors in an excessivenumber of mitoses. At this stage, it is possible toadvance the opinion that adult stem cells usedunder exceptionally controlled conditions, withclearance and approval from international healthcare government agencies, are very safe. To ourknowledge, there have been no reports of adversereactions or complications. Patient protocols in-clude multiple viral and tumor tests and generalmedical, surgical, and psychological evaluation. All patients were discharged from the hospital onthe day after surgery; this is unusual, especially inthe case of radionecrosis.

Laboratory experimentation seems to provideevidence of the great potential of embryonic stemcells. Nevertheless, insurmountable obstacles

such as immunogenicity,23

tumorigenicity,24

andothersstill represent serious impediments andrisks to clinical applications. Adult stem cell ther-apies have the potential to be sound, socially ac-ceptable techniques in future regenerative proce-dures in craniofacial reconstruction and otherdisciplines.

Jose J. Mendonca, M.D., D.M.D., Ph.D.Salvador de Madariaga, 1-1A

Lugo 27002, Spain [email protected]

ACKNOWLEDGMENTSThe authors thank Maria Jesus Lopez, neurophys-

iologist, and Paco Vidal, bioengineer, for enthusiastic and unselfish collaboration on this research.

REFERENCES1. Sullivan WG, Szwajkun PR. Revascularization of cranial ver-

sus iliac crest bone grafts in the rat. Plast Reconstr Surg. 1991;87:11051109.

2. Teng MS, Futran ND. Osteoradionecrosis of the mandible.Curr Opin Otolaryngol Head Neck Surg. 2005;13:217221.

3. Marx RE. Osteoradionecrosis: A new concept of its patho-

physiology. J Oral Maxillofac Surg. 1983;41:283288.


10



11/12

4. Stiff P, Chen B, FranklinW, et al. Autologous transplantationof ex vivo expanded bone marrow cells grown from smallaliquots after high-dose chemotherapy for breast cancer.Blood 2000;95:21692174.

5. Stiff P, Chen B, FranklinW, et al. Autologous transplantationof ex vivo expanded bone marrow cells grown from smallaliquots after high-dose chemotherapy for breast cancer.

Blood 2000;95:21692174.6. Munoz-El as G, Woodbury D, Black IB. Marrow stromal cells,

mitosis, and neuronal differentiation: Stem cell and precur-sor functions. Stem Cells 2003;21:437448.

7. Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bone marrow differentiate into func-tional hepatocyte-like cells. J Clin Invest. 2002;1091:12911302.

8. Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endoder-mal, and mesodermal genes prior to neurogenesis. J Neurosci Res. 2002;69:908917.

9. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can begenerated from marrow stromal cells in vitro. J Clin Invest.1999;103:697705.

10. PhinneyDG, Isakova I. Plasticityand therapeutic potential of mesenchymal stem cells in the nervous system. Curr Pharm Des . 2005;11:12551265.

11. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:4149. Erratum in: Nature 2007;447:879880.

12. MankaniMH, KuznetsovSA, Wolfe RM, Marshall GW, Robey PG. In vivo bone formation by human bone marrow stromalcells: Reconstruction of the mouse calvarium and mandible.Stem Cells 2006;24:21402149.

13. Quarto R, Mastrogiacomo M, Cancedda R, et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med. 2001;344:385386.

14. Jimenez ML, Lyon T, Nowinski G, et al. Stem and progenitor

cell therapy for management of refractory long bone non-unions: A multicenter clinical feasibility study (abstract).Paper presented at: 74th Annual Meeting of the American

Academy of Orthopaedic Surgeons Annual Meeting; Febru-ary 14-18, 2007; San Diego, Calif.

15. Morishita T, HonokiK, Ohgushi H, Kotobuki N, Matsushima A, Takakura Y. Tissue engineering approach to the treatment of bone tumors: Three cases of cultured bone grafts derivedfrom patients mesenchymal stem cells. Artif Organs 2006;30:115118.

16. Dennis JE, Esterly K, Awadallah A, Parrish CR, Poynter GM,Goltry KL. Clinical-scale expansion of a mixed population of bone-marrow-derived stem and progenitor cells for potentialuse in bone-tissue regeneration. Stem Cells 2007;25:25752582.

17. Bensad W, Triffitt JT, Blanchat C, Oudina K, Sedel L, PetiteH. A biodegradable fibrin scaffold formesenchymal stem celltransplantation. Biomaterials 2003;24:24972502.

18. Ho W, Tawil B, Dunn JC, Wu BM. The behavior of humanmesenchymal stem cells in 3D fibrin clots: Dependence onfibrinogen concentration and clot structure. Tissue Eng.2006;12:15871595.

19. Mendonca-Caridad JJ, Juiz-Lopez P, Rubio-Rodriguez JP.Frontal sinus obliteration and craniofacial reconstruction with platelet rich plasma in a patient with fibrous dysplasia.

Int J Oral Maxillofac Surg. 2006;35:8891.20. Schwartz HC. Is the use of hyperbaric oxygen necessary?

J Oral Maxillofac Surg. 1982;40:412420.21. Annane D, Depondt J, Aubert P, et al. Hyperbaric oxygen

therapy for radionecrosis of the jaw: A randomized, placebo-controlled, double-blind trial from the ORN96 study group. J Clin Oncol. 2004;22:48934900.

22. Goshima J, Goldberg VM, Caplan AI. The origin of boneformed in composite grafts of porous calcium phosphateceramic loaded withmarrow cells. Clin Orthop Relat Res. 1991;269:274283.

23. Swijnenburg RJ, Tanaka M, Vogel H, et al. Embryonic stemcell immunogenicity increases upon differentiation aftertransplantation into ischemic myocardium. Circulation 2005;

112(9 Suppl):I166I172.24. Blum B, Benvenisty N. The tumorigenicity of human em-bryonic stem cells. Adv Cancer Res. 2008;100:133158.


11



12/12

BNAME: AUTHOR QUERIES PAGE: 1 SESS: 3 OUTPUT: Mon Aug 23 07:04:08 2010h3/zpr prs/zpr prs/zpr01110/zpr3966 10z

AQ1: AUTHORThere were two reference 5s on the ref list; they were renumbered as 5 and 6,and subsequent references 6 through 19 were renumbered as refs 7 through 20. (The originalreference list did not have a ref. 20, so the original numbering resumed with ref. 21.) Correctas edited? If not, please revise reference citations and reference list as needed.

AQ2: AUTHORGMP spelled out correctly?

AQ3: AUTHORTable 1, column 3: Please confirm that the multiplication symbol (x) is correct inthe column heading (x10(6)/ml) or revise as needed.

AQ4: AUTHORTable 2, column 4: Please confirm that the multiplication symbol (x) is correct inthe column heading (x10(6)/ml) or revise as needed.

AQ5: AUTHORDescription of Supplemental Digital Content 12 correct as edited (three-dimensional magnetic resonance imaging scan of angiogenesis)? If not, please revise asneeded.

AQ6: AUTHORRenumbered reference 20 is by Schwartz et al., not Marx. Please reconcile.

AQ7: AUTHORRef. 14: Meeting information correct? If not, please revise as needed.

AUTHOR QUERIES

AUTHOR PLEASE ANSWER ALL QUERIES

regenerative facial reconstruction of terminal stage osteoradionecrosis and other advanced...

Documents