PEDIATRIC/CRANIOFACIAL

Regenerative Facial Reconstruction of Terminal Stage Osteoradionecrosis and Other Advanced Craniofacial Diseases with Adult Cultured Stem and Progenitor Cells
Foot

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 with bone loss and tissue damage are usually limited to vascularized and nonvascularized tissue transfers, allografts, mechanical devices and, more recently, facial transplantation. Despite the therapies available, the demand for new approaches is realized in cases where current therapies are unable to resume form and function. This study presents the feasibility of alternative treatments based on cultured 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 inoculated into 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 mineral base of -tricalcium phosphate and hydroxyapatite. Results: A case of maxillary and mandibular radionecrosis (stage IIIR) with pathologic fracture presented early osteogenesis, total recovery from alveolar nerve anesthesia, facial nerve reinnervation, and skin regeneration. Another case with nonhealed fracture, bone loss, and bilateral paresthesia demonstrated callus formation, bone regeneration, and nerve recovery. Finally, maxillary bone regenerated after massive deficiency. Oral functional restoration with implants and fixed prosthesis was accomplished in all cases. Conclusion: After nerve, bone, skin, and vessel formation in three patients with severe abnormality, bone marrow derived mixed cultured stem cell lineages could be considered a new paradigmatic approach to advanced disease. (Plast. Reconstr. Surg. 126: 1, 2010.)

reatment options in cases of severe facial bone and tissue loss are usually accomplished by autografts,1 allografts, cytokines, mechanical devices and, more recently, facial transplantation. Occasionally, the disease outweighs the balance between destruction and regeneration, mainly when tissues other than bone are involved.2,3 Many severe conditions remain challenging, without a clear curative solution, and patients are left with palliative or limited therapies. Autogenous grafts have been considered the
From the Head and Neck Surgery Unit, POLUSA Hospital. Received for publication January 22, 2010; accepted May 10, 2010. Copyright 2010 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0b013e3181f24164

standard treatment in all osseous regenerative procedures. Bone substitutes, cytokines, and other substances are designed to enhance conventional grafting techniques or as an alternative to bone harvesting, potentially improving grafts or avoiding donor-site surgery and morbidity. The iliac crest has passed the test of time,1 proving to be the most prolific donor site of the body, with the greatest variety of bony structures and cell populations. The most remarkable quality is the relatively large populations of stem and progenitor cells in the

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

www.PRSJournal.com

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marrow and adjacent areas. These cell populations are occasionally able to survive transplantation and hypoxia, revitalizing grafts through chemotaxis, mitosis, and differentiation. The use of whole or subsets of bone marrow is not new; hematopoietic recovery in myeloproliferative disorders followed by high-dose radiochemotherapy was successfully accomplished years ago using this technique. More recently, patient-specific therapies have overcome the limitations of progenitor pools by ex vivo culture of certain lineages.4 In vitro differentiation of marrow-derived stem cells is able to yield basic tissue components, such as neurons, hepatocytes, adipocytes, cardiomyoblasts, endothelial cells, Langerhans islets, osteoblasts, and others.59 The concept and attributed potential of these cells has changed dramatically in recent years, and they are currently considered as possible pluripotent cells.10,11 The acquisition of local phenotypes caused by native factors and possibly cell fusion reveals an unexpected plasticity and behavior. Animal experimentation is quite advanced, demonstrating dramatic results in untreatable conditions12; nevertheless, strictly controlled legal clinical experience in humans is scarce, with a paucity of reports available in the literature.1315 There are not only many ethical and legal limitations to human investigation but also enormous difficulties in translating basic research or animal investigation. Aging maxillary bone loss is a common finding in humans and is frequently related to tooth loss and periodontal disease; trauma, radiotherapy, and tumors also account for severe osseous tissue destruction. Despite the variety of therapies available, some cases are beyond the scope of modern treatments or are performed with important risks and morbidity. Osteoradionecrosis occurs when the normal bone turnover is exceeded by the degradative process within an irradiated field. It was first described in the mandible by Marx3 as a process related to endarteritis and hypocellularity, leading to tissue hypoxia, apoptosis, fibrosis, and hypovascularity. Final stages are often attributable to iatrogenic intervention, mainly tooth extraction leading to intraoral bone exposure and eventually a pathologic fracture and fistula. Osteoradionecrosis is not limited to the mandible and skin; mucosa, fat, periosteum, and muscle are devastated as well, initiating a vicious cycle that results in greater 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 generates large amounts of early-stage mesenchymal, endothelial, hematopoietic stem, and progenitor cells (tissue repair cells) appropriate for use in bone grafting.16 Tissue repair cells have been previously used to substitute for bone marrow transplants in cancer patients to restore hematopoiesis.4

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PATIENTS AND METHODS

One hundred milliliters of heparinized bone marrow aspirate was obtained from the posterior ileum under conscious sedation. A specific technique based on multiple-point minimal volume smooth aspiration provided a rich nucleated 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 a special transport to a granulocyte-macrophage progenitor processing unit and inoculated into the closed, automated, computer-controlled AastromReplicell System (Aastrom Biosciences, Inc., Ann Arbor, Mich.) and cultured for 12 days at 37C in Iscoves Modified Dulbeccos Medium supplemented 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

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Supplemental digital content is available for this article. Direct URL citations appear in the printed text; simply type the URL address into any Web browser to access this content. Clickable links to the material are provided in the HTML text of this article on the Journals Web site (www.PRSJournal.com).

Video 1. Supplemental Digital Content 1 demonstrates bone marrow aspiration, http://links.lww.com/PRS/A217.

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bacterial and fungal contaminants and endotoxins, the culture medium was sampled 48 hours before harvest. Cells were harvested on day 12. Reagents added during culture were below the detectable limits of sensitive enzyme-linked immunosorbent assays. Flow cytometry, cell viability, and clonogenic assays confirmed tissue repair cell integrity. Tissue repair cells were suspended in 150 ml of Normosol (Hospira, Inc., Lake Forest, Ill.) with 0.5% human serum albumin and transported at 4C for use within 8 hours. Cell samples for viability, quality, and characterization were taken before inoculation and after production (Tables 1 through 4). (See Document, Supplemental Digital Content 2, which demonstrates cell culture procedures and release criteria, http://links.lww.com/PRS/A218.) Fifty milliliters of citrated venous blood drawn during surgery was processed in approved systems for platelet concentration. Platelet-rich plasma and platelet-poor plasma were clotted using autologous thrombin resulting from blood clot closed centrifugation. Cells were mixed with plateletrich and platelet-poor plasma17,18 and -tricalcium phosphate/hydroxyapatite, forming a bioactive matrix (Fig. 1). (See Video, Supplemental Digital Content 3, demonstrating injecting cells, temporary scaffold, http://links.lww.com/PRS/A219.) A membrane created by compressing the platelet-rich plasma19 was injected with cells and
Table 1. Bone Marrow Aspirate
Volume (ml) 98 98 96 BM Cell Concentration ( 106/ml) 20.76 20.31 20.68 TNCs Collected ( 106) 2035.4 1991 1986 Viability (%) 83.3 85.1 84.4

used as another temporary scaffold. Three patients with severe disease were selected after meeting the following special criteria: 1. Severe facial tissue loss previously treated unsuccessfully. 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, European regulations for compassionate use of autologous stem cells. 6. All Conformite Europeenne/U.S. Food and Drug Administrationcertified materials. Exclusion of organic substances. 7. Extensive imaging diagnostics. 8. Exclusion criteria were as follows: Active or recent cancer, major health disorders. Smoking. Substance abuse. Metabolic bone or collagen disease. Poor oral hygiene. Pregnant or nursing women, women not using contraceptives. Inadequate medications, allergy to common drugs.

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Table 4. Tissue Repair Cell Product Colony Forming (Output)

Patient 1 2 3 CFU-F (%) 3.9 3.2 3.6 CFU-F Fold Expansion 76.9 86.2 78.7 CFU-GM (%) 0.8 0.6 0.6 CFU-GM Fold Expansion 1.1 1 0.9

Patient 1 2 3

BM, bone marrow; TNCs, total nuclear cells.

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CFU-F, colony forming unit, fibroblast; CFU-GM, colony forming unit, granulocyte, monocyte.

Table 2. Tissue Repair Cell Product (Output)

Patient 1 2 3 Volume (ml) 10.4 11.7 12.1 TNCs Released ( 106) 500.24 518.31 488.84 Cell Concentration ( 106/ml) 48.1 44.3 40.4 Viability (%) 84.1 82.5 88.2 TNC Fold Expansion 3.8 4.3 3.5

TNCs, total nuclear cells.

Table 3. Tissue Repair Cell Product Phenotypes (Output)

Patient 1 2 3 Thy-1 Fold Expansion 29.8 31.5 38.7 CD90 (%) 35.88 25.07 33.78 CD45 (%) 66.77 62.81 73.83 CD105 / CD166 (%) 14.1 13.8 14.4 CD144 / CD146 (%) 3.3 4.2 3.5 CXCR4 / VEGFR1 (%) 11.8 12.1 12.6 CD34 / lin (%) 0.52 0.85 0.67

Thy-1, thymocyte differentiation antigen; CD, cluster of differentiation; CXCR4, chemokine receptor 4; VEGFR1, vascular endothelial growth factor receptor 1; lin, lineage.

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Case Techniques For radionecrosis, necrotic tissue was excised excluding most of the cortical plates. The remaining bone was trimmed and carved to harbor the graft and thoroughly perforated until bleeding from adjacent tissue appeared. A damaged inactive alveolar nerve was debrided with a micro scope, followed by cell microinjections with a polycarbonate syringe and wrapping in the platelet-poor plasma cell membrane. The platelet-poor plasma cell matrix was embedded carefully into the defect and injected again (Fig. 1). A mandibular reconstruction plate was placed, and the wound was closed after cell flushing and inoculation into the skin, muscle, and areas adjacent to the facial nerve and the necrosed vascular stumps. Intravenous administration of tissue repair cells was avoided. (See Video, Supplemental Digital Content 4, which demonstrates procedure animation, http://links.lww.com/PRS/A220; and Video, Supplemental Digital Content 5, which demonstrates the surgical procedure, http://links.lww.com/PRS/A221.) Posterior maxillary bone loss required sinus lift and platelet-poor plasma cell scaffold; mucoperiosteal flaps and elevated sinus floors were microinjected with tissue repair cells. Anterior maxillary atrophy was reconstructed with poly(L-lactic acid) plates that fixed and stabilized the grafts. Other Techniques Other techniques included lateralization of the alveolar nerve with cell injection in the canal and nerve sheath (Fig. 2) (see Video, Supplemental Digital Content 6, which demonstrates nerve cell injection, http://links.lww.com/PRS/A222.) and 5-mm periodontal pocket debridement and grafting. Perioperative antibiotics, dexamethasone, and dexketoprofen trometamol were delivered intravenously, and dexketoprofen trometa-

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

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

9. Patient permission: screening for viruses and common 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.

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

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mol and amoxicillin clavulanate were prescribed orally for 7 days together with a mouth rinse. All patients were discharged from the hospital on the day after surgery following two episodes of general anesthesia and one episode of conscious sedation. Four months postoperatively, in all cases, a rough-surface, high-quality implant was inserted in grafted and nongrafted areas. Two months later, fixed screwretained porcelain prostheses were placed on the implants.

RESULTS
Patient 1, a 63-year-old man with a history of radiation therapy for tonsillar cancer, reported pain in the right hemimandible. Computed tomographic scans (Fig. 3) disclosed a cystic lesion in the molar area. Diagnosed with limited mandibular and maxillary radionecrosis, the patient was treated elsewhere, returning 1 year later with a history of tooth extraction, pathologic fracture, life-threatening infection, prolonged hospitalization, and severe chronic pain with dysfunction. Several treatment strategies included surgical de bridement, 45 dives in hyperbaric oxygen, antibiotic therapy, and others. Despite these efforts, the disease worsened, and a new computed tomographic scan disclosed a severely fractured mandible (Fig. 3). (See Figure, Supplemental Digital Content 7, which demonstrates a new computed tomographic scan disclosing a severely fractured mandible, http://links.lww.com/PRS/A223.) Symptoms now included intraoral and extraoral suppuration from two fistulas, severe trismus, total lip anesthesia from alveolar nerve dam-

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Fig. 2. Nerve injection.

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

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

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age, xerostomia, and intense chronic pain. Neurophysiologic tests revealed partial denervation of the marginal mandibular nerve and absence of right masseter activity. The disease had been the cause of early retirement. Free fibula grafting was rejected by the patient and evaluated as a high-risk procedure because of the damage to local vessels and the complex access to distant arteries. Bone marrow aspirate, cell procedures, and surgery took place as described previously without complications. A satisfactory recovery led to early hospital discharge 24 hours after surgery. Routine antibiotics and antiinflammatory drugs were prescribed for 1 week. Mild pain and swelling subsided completely just after surgery, whereas lip numbness recovered progressively with an electric tingling sensation 4 to 6 weeks postoperatively. Thermoalgesic and tactile functions are currently near normal values. Osteogenesis occurred within the first 3 months and continues to date (Figs. 4 and 5). (See Figures, Supplemental Digital Content 8, which shows a computed tomographic scan obtained 3 months postoperatively, http://links.lww.com/PRS/A224; Supplemental Digital Content 9, which shows a computed tomographic scan of the lower border, http://links.lww.com/PRS/A225; and Supplemental Digital Content 10, which shows a computed tomographic scan of the coronal section demonstrating bone formation, http://links.lww.com/PRS/A226.) Two high standard dental implants were placed in grafted areas of the maxilla and mandible for restoration with fixed prostheses 2 months later. Bone biopsy specimens during implant surgery demonstrated an unusual purely cortical morphology in the mandible, with active osteocytes and osteoblasts, osteoid 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.) Dramatic changes in dermal and epidermal morphology, including angiogenesis, appeared in a skin biopsy specimen taken during suture removal (Fig. 6). Gadolinium magnetic resonance imaging at 9 months showed high levels of blood supply to the graft, with arteriogenesis of the facial and lingual vessels and profuse angiogenesis in the form of a tortuous bundle from both of these arteries (Fig. 7). (See Figures, Supplemental Digital Content 12, which demonstrates a three-dimensional magnetic resonance imaging scan of angiogenesis, http://links.lww.com/PRS/A228; and Supplemental Digital Content 13, which shows gadolinium uptake in the grafted area demonstrating vast angiogenesis, http://links.lww.com/PRS/A229.) Neurophysiologic testing disclosed changes in the latent period and right marginal mandibular nerve reinnervation pulses. Electrical and functional activity was also detected in the masseter muscle. Twenty months after surgery, all improvements continued and the patient had returned to a normal life without complications or sequelae. (See Figure, Supplemental Digital Content 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-

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

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

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lography confirmed inexplicable reactivation of a radiotherapy-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 craniofacial trauma with maxillomandibular fractures 12 years previously. After several operations, the patient developed oral fistulas, chronic pain, functional limitations, and bilateral alveolar nerve impairment. Diagnostic imaging revealed poor consolidation, bone loss, and invasion of the mandibular canal with a screw. Cell application procedures were similar, except for bilateral nerve lateralization, sheath dissection, and cell injection (Fig. 2). Both sinuses were grafted, and the anterior maxilla was reconstructed with two poly(L-lactic acid) plates and grafts. Plates were exposed slightly 4 weeks later without infection but were not removed. Four months later, the same implants were inserted into both jaws, except in a small area of exposed plates where the bone was considered inappropriate. One year after surgery, the patient had resumed normal oral function with a fixed prosthesis. Both mental nerves recovered despite mild paresthesia at the screw-perforated site. Pain had subsided. Patient 3 was a 52-year-old man with masticatory dysfunction and joint pain resulting from the

absence of maxillary molars, with severe bone loss in the posterior maxilla, residual bone height areas of 1 mm, and perforations to the sinus. Lower limb and hip pathologic conditions contraindicated iliac harvesting. Calvarial harvesting was discarded because of unpredictable results with great volume defects, and allografts or xenografts alone were also rejected. Cell processing followed the same techniques described above; sinus lifts were grafted with platelet-poor plasma cells and platelet-rich plasma cells contralaterally. One implant was placed simultaneously, and the rest were placed at 4 months. A fixed prosthesis was inserted 2 months later. All patients led a normal life, with implantsupported fixed prostheses (Fig. 9). (See Figure, Supplemental Digital Content 15, which shows prosthetic rehabilitation in the area of previous radionecrosis, http://links.lww.com/PRS/A231.) All implants survived, and bone density and function continue to increase over time. More than 1 year later, a slight plate exposure remains the only complication to be reported.

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DISCUSSION
The therapeutic use of bone marrow derived stem and progenitor cells for human disease is not new. Native bone marrow, stimulation with gran-

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Fig. 6. Photomicrographs show changes in the histology of bone and skin. Osteocytes, osteoblasts, and osteoid formation are found at the time of implant insertion and 4 months after grafting (note unresorbed allograft on the right). Skin sample before and after surgery.

Fig. 7. Gadolinium-enhanced magnetic resonance imaging scan at 7 months demonstrates arteriogenesis of the facial and lingual vessels. Note the profuse angiogenesis at the level of the graft and the closed circuit with the maxillary artery.

Fig. 8. Sialographic image demonstrates parotid gland reactivation years after intense radiotherapy.

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

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ulocyte colony-stimulating factor, and blood apheresis and expanded bone marrow in autologous and homologous procedures have been used for years in hematopoietic replacement after iatrogenic destruction of blood-generative organs by radiotherapy, chemotherapy, or both. Hematopoietic stem cell therapy is not only a consolidated procedure but also a successful one. Adult stem cells have been the object of innumerable in vitro and animal experiments, demonstrating significant potential for the treatment of numerous diseases. Nevertheless, there is a paucity of reports in the literature on therapies in humans with other advanced conditions. In this article, we report the first use of a mixed population of autologous cultured bone marrow derived stem and progenitor cells in the treatment of severe craniofacial disorders, where an unexpected outcome demonstrated the regeneration of nervous, vascular, and dermal structures and bone. Osteoradionecrosis of the mandible develops over a period ranging from months to years after intense exposure to radiotherapy. Early stages are treated conservatively with routine dental care, oral hygiene, antibiotics, and extreme caution with extractions. Marx20 hypothesized the prophylactic and therapeutic use of hyperbaric oxygen, assuming revascularization and cell induction by high partial pressure of oxygen. Criticism and controversy20 followed, given the expensive and exclusive setup required and the uncertain results, mainly in advanced cases. The

mechanisms involved are controversial and are based not only on vascular deprivation but also on fibrous tissue formation and deficiencies in bone turnover. Furthermore, double-blind, placebo-controlled studies that found no benefit from hyperbaric oxygen for advanced osteoradionecrosis of the mandible21 have led to the emergence of new treatments. Restoration of the blood supply to the affected area continues to be critical. One of the first images of human angiogenesis and arteriogenesis after stem cell therapy in a case of radionecrosis is presented in this article (Fig. 7). (See Figures, Supplemental Digital Content 12, which shows a three-dimensional magnetic resonance imaging scan of angiogenesis, http://links.lww.com/PRS/A228; and Supplemental Digital Content 13, which shows high gadolinium uptake in the grafted area demonstrating vast angiogenesis, http://links.lww.com/PRS/A229.) Endothelial precursor cells and a wide variety and high levels of angiogenic cytokines (mainly vascular endothelial growth factor) may be responsible for the microvascular and macrovascular vessel development. It can be hypothesized that early vascular graft ingrowth is the basis of regeneration of all tissues, delivering oxygen, nutrients, chemical signaling, migrating cells, and immunity. Osteogenesis requires a copious blood supply but also an adequate cell pool. Other effects, such as marginal mandibular nerve function, the recovery of three completely inactive alveolar nerves, new skin formation

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(Fig. 6), or masseter recovery, are more difficult to explain. The human peripheral nervous system shows a recognized ability to support limited occasional axonal regeneration. Although all nerves were inactive and recovered almost 100 percent of their functionality, it is possible that microinjections of cells into the neural sheath led to immune up-regulation, elimination of toxins, addition of nerve growth factors, and increase in blood supply, or even influenced Schwann cells. The same process could apply to the skin, but the possibility of real histogenesis cannot be completely discarded in these cases. The therapy presented in this article provided clear evidence of the restoration of most lost facial tissues and functions, but the mechanisms involved remain to be understood. Basic research has demonstrated that some phenotypes (CD105, CD166, CD105/166, CD146, CD90, and others) have a high osteogenic potential; furthermore, mixed lineages, including nonspecific fibroblast colony-forming unit and hematopoietic CD34, CD13, CD14, and Gly-A, seem to be more effective bone generators with additional in vitro angiogenic capacities. The design of a mixed population of stem and progenitor cells,16 as present in the culture used in this study, is consistent with this principle; a tissue begins and ends with different populations and depends on the synergistic processing actions of multiple cell lineages. The concept of a selected mixed population versus a single population is a revolutionary point of view of histogenesis (Tables 2 through 4). Another critical concern in stem cell transplantation is the delivery process. Some clinical aspects of stem cell therapy may have been overlooked or underestimated because the results have not yet met the expectations and promises of a new age in medicine. One of the most challenging aspects of the grafting procedure is cell scaffolding and signaling. The design of a temporary (seeding the fibrin) and half-term scaffold has the purpose of retaining cells until adhesion, and allowing the development of a microenvironment. Fibrinogen and thrombin solutions must be carefully considered for cell delivery because they affect the three-dimensional fibrin clot structure and cell proliferation.18 The combination of a commercial blend of -tricalcium phosphate22 and hydroxyapatite (well-known in vitro nesting grounds) was also added and trapped in the clot. Finally, an osteogenic environment would encourage cells to adhere, divide, and differentiate (on exposure to growth factors and other factors). Contaminated areas were removed, but most of the cortical bone remained after thorough stimulation by deep trimming, becoming another scaffold with additional osteogenic potential. Multiple perforations expose underlying tissues, healthy bone marrow, and blood vessels to the graft, assisting not only incoming chemoattracted cells but also dramatically increasing local distress biochemical signaling19 (Fig. 1). Another major concern in stem cell therapy is safety. Samples are processed and transported in special containers by certified agencies and are tested for contamination, disease, viruses, cell viability, and DNA stability (clonogenic assays). Fold expansions of the different cell populations are limited and extend to only 12 days because of the risk of DNA transcription errors in an excessive number of mitoses. At this stage, it is possible to advance the opinion that adult stem cells used under exceptionally controlled conditions, with clearance and approval from international health care government agencies, are very safe. To our knowledge, there have been no reports of adverse reactions or complications. Patient protocols include multiple viral and tumor tests and general medical, surgical, and psychological evaluation. All patients were discharged from the hospital on the day after surgery; this is unusual, especially in the case of radionecrosis. Laboratory experimentation seems to provide evidence of the great potential of embryonic stem cells. Nevertheless, insurmountable obstacles such as immunogenicity,23 tumorigenicity,24 and othersstill represent serious impediments and risks to clinical applications. Adult stem cell therapies have the potential to be sound, socially acceptable techniques in future regenerative procedures in craniofacial reconstruction and other disciplines.
Jose J. Mendonca, M.D., D.M.D., Ph.D. Salvador de Madariaga, 1-1A Lugo 27002, Spain joaquin@cirujanosdelmundo.com

ACKNOWLEDGMENTS

The authors thank Maria Jesus Lopez, neurophysiologist, and Paco Vidal, bioengineer, for enthusiastic and unselfish collaboration on this research.
REFERENCES
1. Sullivan WG, Szwajkun PR. Revascularization of cranial versus 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 pathophysiology. J Oral Maxillofac Surg. 1983;41:283288.

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Volume 126, Number 6 Regenerative Facial Reconstruction

4. Stiff P, Chen B, Franklin W, et al. Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer. Blood 2000;95:21692174. 5. Stiff P, Chen B, Franklin W, et al. Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer. Blood 2000;95:21692174. 6. Munoz-Elas G, Woodbury D, Black IB. Marrow stromal cells, mitosis, and neuronal differentiation: Stem cell and precursor functions. Stem Cells 2003;21:437448. 7. Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest. 2002;1091:1291 1302. 8. Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res. 2002;69:908917. 9. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest. 1999;103:697705. 10. Phinney DG, Isakova I. Plasticity and 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. Mankani MH, Kuznetsov SA, Wolfe RM, Marshall GW, Robey PG. In vivo bone formation by human bone marrow stromal cells: 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 nonunions: A multicenter clinical feasibility study (abstract). Paper presented at: 74th Annual Meeting of the American Academy of Orthopaedic Surgeons Annual Meeting; February 14-18, 2007; San Diego, Calif. Morishita T, Honoki K, Ohgushi H, Kotobuki N, Matsushima A, Takakura Y. Tissue engineering approach to the treatment of bone tumors: Three cases of cultured bone grafts derived from patients mesenchymal stem cells. Artif Organs 2006; 30:115118. 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 potential use in bone-tissue regeneration. Stem Cells 2007;25:25752582. Bensad W, Triffitt JT, Blanchat C, Oudina K, Sedel L, Petite H. A biodegradable fibrin scaffold for mesenchymal stem cell transplantation. Biomaterials 2003;24:24972502. Ho W, Tawil B, Dunn JC, Wu BM. The behavior of human mesenchymal stem cells in 3D fibrin clots: Dependence on fibrinogen concentration and clot structure. Tissue Eng. 2006;12:15871595. 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. Schwartz HC. Is the use of hyperbaric oxygen necessary? J Oral Maxillofac Surg. 1982;40:412420. Annane D, Depondt J, Aubert P, et al. Hyperbaric oxygen therapy for radionecrosis of the jaw: A randomized, placebocontrolled, double-blind trial from the ORN96 study group. J Clin Oncol. 2004;22:48934900. Goshima J, Goldberg VM, Caplan AI. The origin of bone formed in composite grafts of porous calcium phosphate ceramic loaded with marrow cells. Clin Orthop Relat Res. 1991; 269:274283. Swijnenburg RJ, Tanaka M, Vogel H, et al. Embryonic stem cell immunogenicity increases upon differentiation after transplantation into ischemic myocardium. Circulation 2005; 112(9 Suppl):I166I172. Blum B, Benvenisty N. The tumorigenicity of human embryonic stem cells. Adv Cancer Res. 2008;100:133158.

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AUTHOR QUERIES
AUTHOR PLEASE ANSWER ALL QUERIES
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 original reference list did not have a ref. 20, so the original numbering resumed with ref. 21.) Correct as 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 in the column heading (x10(6)/ml) or revise as needed. AQ4: AUTHORTable 2, column 4: Please confirm that the multiplication symbol (x) is correct in the column heading (x10(6)/ml) or revise as needed. AQ5: AUTHORDescription of Supplemental Digital Content 12 correct as edited (threedimensional magnetic resonance imaging scan of angiogenesis)? If not, please revise as needed. 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. 1