Autologous stem cell therapy of large bone defects

TREATMENT OF SEVERE BONE DEFECTS WITH AUTOLOGOUS STEM CELLS:

FUNCTIONAL RECOVERY AND BONE REMODELING

VULCANO E, MD

; MURENA L, MD

; CHERUBINO P, MD

; FALVO DA, MD

; ROSSI A, MD

; BAJ A, MD, PhD

; TONIOLO A, MD

Department of Orthopedics and Traumatology

and Laboratory of Medical Microbiology

, University of Insubria Medical School & Ospedale di Circolo e Fondazione Macchi, Varese, Italy

Corresponding author:

Ettore Vulcano, MD

Dipartimento di Scienze Chirurgiche Ricostruttive e Tecnologie Avanzate Università dell’Insubria & Ospedale di Circolo e Fondazione Macchi Viale Luigi Borri 57

21100 Varese, ITALY

Tel: +39 (0)332 278 826 Fax: +39 (0)332 278 825

Email: ettorevulcano@hotmail.com

Keywords:

bone marrow; bone marrow concentrate; mesenchymal stem cells; bone defects; bone cyst; non

unions; pseudoarthosis; virus infections

Abstract

Introduction: Mesenchymal stem cells may differentiate into angiogenic and osteoprogenitor cells.

The effectiveness of autologous pluripotent mesenchymal cells for treating bone defects has not been investigated in humans. The present study aimed at evaluating the merit of nucleated cells from autologous bone marrow aspirates in the treatment of severe bone defects that failed to respond to traditional treatments.

Materials and Methods: Fourteen adult patients (mean age 58.4 years) with severe bone defects were investigated. Lower limb bone defects were ≥5 cm

, upper limb defects were ≥2 cm

. Before surgery, patients were tested for antibodies to common pathogens. Treatment consisted of bone allogeneic scaffold enriched with bone marrow nucleated cells harvested from the iliac crest and concentrated using an FDA-cleared device. Clinical and radiographic follow-up was performed at 1, 3, 6, and 12 months after surgery. To assess viability, morphology, and the immunophenotype, bone marrow nucleated cells were cultured in vitro, then tested for sterility and evaluated for the possible replication of adventitious viruses.

Results: In 12 of 14 patients, both clinical and radiographic healing of the bone defect together with bone graft integration was observed at the mean time of 5 months. Two patients failed to respond.

No post-operative complications were observed. Bone marrow nucleated cells were enriched 4.25- fold by a single concentration step. Enriched cells were free of microbial contamination. The immunophenotype of adherent cells was compatible with that of mesenchymal stem cells. Among investigated cases, replication of Epstein-Barr virus was detected in 2/14 bone marrow cell cultures.

Reactivation of hepatitis B virus, cytomegalovirus, parvovirus B19, and endogenous retrovirus HERV-K was not detected. From 470 to 1,150 million nucleated cells were grafted into each patient.

Discussion: The method was safe and easy to perform. Compared to treatment with autologous bone graft from the iliac crest, the procedure was not associated with complications at the donor site and equally effective.

Conclusion: This preliminary series of patients, with a mean follow-up of almost 2 years,

demonstrates that allogeneic bone scaffold enriched with concentrated autologous bone marrow

cells obtained from the iliac crest provides orthopaedic surgeons with a novel option for treating

important bone defects unresponsive to traditional therapies.

Introduction

Bone defects that do not respond to conventional medical and surgical treatments represent a major challenge for orthopaedic surgeons. Cell-based therapies aim at regenerating damaged tissues that fail to heal upon traditional treatment. In orthopaedic surgery, platelet-derived growth factors and bone morphogenetic proteins (BMPs) have been used to stimulate bone regeneration in a variety of clinical conditions, including long bone non-unions, spinal fusion surgery, repair of symptomatic posterolateral lumbar spine non-unions

. In contrast to cardiology where stem cells of different ori- gins (e.g., bone marrow, peripheral blood, skeletal muscle myoblasts) have been used in attempts to repair the infarcted heart

, cell therapy has received little attention in orthopaedic surgery

.

In small animals, bone marrow stem cells have been shown to favor fracture healing and tendons`

repair

. Bone marrow mesenchymal stem cells (MSCs) are multipotent cells that reside in the bone marrow

close to haematopoietic stem cell niches, and are involved in bone marrow home- ostasis and in regulating the maturation of haematopoietic and non-haematopoietic cells. MSCs have the potential to proliferate and differentiate into osteoblasts, chondroblasts, odontoblasts, adipocytes

. MSCs are of interest for clinical applications, since they can be easily obtained from bone marrow aspirates in adults, and either directly implanted in vivo, or expanded in vitro before implantation. Thus, when matrix and osteoprogenitors are scarce - as in large bone defects – cell therapy may provide novel ways of treatment

. Bone healing requires that implanted cells differenti- ate into osteoblasts, that these bone precursors proliferate, secrete matrix constituents and, finally, differentiate into osteocytes. Differentiation processes require that several genes are turned on and off in a growth- and differentiation-specific manner. For instance, in mice it has been shown that Notch signaling inhibits osteoblast differentiation through the Hes/Hey proteins that reduce Runx2 transcriptional activity

. These studies suggest that MSCs could be expanded in vitro by activating the Notch pathway, whereas in vivo differentiation and bone formation would require suppressing this pathway. In the same line, overexpression of the dentin matrix protein 1 in MSCs was shown to induce differentiation into odontoblast-like cells

. The process requires the regulated activation of transcription factors (e.g., core binding factor 1), expression of BMP2 and BMP4, of genes associ- ated with extracellular matrix deposition (alkaline phosphatase, osteopontin, osteonectin, osteocal- cin) and of late genes like DMP2 and dentin sialoprotein.

Though experimental knowledge is abundant, applications of adult MSCs for treating large bone de-

fects are still in their infancy

.Differentiation of stem cell into bone tissue is influenced by en-

vironmental factors, including the interaction of cells with an adequate extracellular scaffold, the

supply of oxygen and growth factors, the mechanical properties of the microenvironment

. These

studies indicate that substantial numbers of cells and an adequate scaffold are essential for allowing

bone formation. In principle, a cellular therapy protocol for bone regeneration should include isolat- ing, expanding in vitro and differentiating progenitor cells before re-implantation within an ade- quate extracellular matrix. Ethical and legislative considerations, however, regulate these proce- dures

. In this context, autologous sourcing of sufficient numbers of MSCs together with imme- diate implantation is an attractive approach. In particular, this approach would eliminate culture-de- rived problems, including cell aging, cell reprogramming, contamination/infection by microbes and/or non-autologous culture components

. Recently, the US Pharmacopeia addressed the safety aspects of cell therapy recommending simple procedures whenever applicable

.

The current study will present a novel approach based on the use of autologous MSCs and allogeneic bone graft as a scaffold for treating large bone defects that failed to respond to traditional medical and surgical therapies.

Patients, Materials and Methods

From November 2008 to September 2011, 14 patients affected by severe bone defects were recruited in the study. Inclusion criteria comprised: 1) bone defects and altered bone-consolidations unresponsive to previous surgical treatments; 2) minimum defect size ≥5 cm

(lower limb) or ≥2 cm

(upper limb); 3) in the case of non-union or delayed consolidation, a minimum of 2 months was required from the last surgery. Exclusion criteria included: 1) local or systemic infection; 2) Radiographic evidence of epiphyseal growth cartilage; 3) severe soft tissue alterations that could not allow proper bone covering at the implantation site; 4) pathologic fractures or neoplastic lesions; 5) severe metabolic and autoimmune disease; 6) antineoplastic therapy; 7) pregnancy.

Patients were treated at the Department of Orthopaedic Surgery and Traumatology, University of Insubria, Varese, Italy. Approval of the Hospital Ethics Committee and the written informed consent of patients were obtained for all cases. The mean age of patients was 62.5 years (range, 17- 84 years). Different pathologies were treated: 5 periprosthetic osteolysis secondary to hip acetabular cup mobilisation, 6 femur non-unions, 1 humerus non-union, 1 very severe bone loss secondary to complex exposed leg fracture, 1 proximal humerus cyst which had determined 3 humeral shaft fractures treated conservatively. Patient characteristics are summarized in Table 5. As shown in Table 1, prior to surgery patients were tested for serum antibodies to common pathogens (Treponema pallidum, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and parvovirus B19).

Harvest and processing of bone marrow cells

The SmartPReP-2 Bone Marrow Aspirate Concentrate System (Harvest Technologies GmbH,

Munich, Germany) was used for concentrating nucleated bone marrow cells. SmartPReP-2 is a dedicated, FDA-cleared and CE-marked device consisting of a centrifuge system for producing autologous bone marrow aspirate concentrate (BMAC). Fresh bone marrow aspirate (BMA; 60-120 ml) is introduced in a disposable dual chamber of the table-top centrifuge system. The first chamber contains a floating layer of a specific density. During the centrifugation phase, red blood cells (RBCs) are separated from nucleated cells, platelets and plasma. Cellular elements and plasma are automatically decanted into the second chamber and concentrated by centrifugation. A portion of plasma is removed, and cellular elements are resuspended in about 12 ml of plasma. The concentration step is accomplished in 15 minutes. BMAC contains unchanged proportions of myelocytes, granulocytes, lymphocytes, monocytes, proerythroblasts and erythroblasts as compared to the initial bone marrow aspirate

. BMA is obtained from the posterior iliac crest using a 6 lumen Jamshidi type trocar needle and 20 ml syringes pre-flushed with Na-heparin (1,000 units/ml). The trocar is initially directed at 30° to the horizontal, parallel to the plane of the crest. It is then inserted 5 cm toward the anterior cortex. At least 60 ml of BMA are extracted while rotating and slowly withdrawing the needle toward the cortex. BMA is then concentrated using the SmartPReP-2 system in the operating suite. BMAC (≥10 ml) is then transferred, under sterile conditions, to the surgical field, together with an appropriate amount of homologous bone graft obtained from the local bone bank.

Surgical methods

The surgical technique requires exposure of the bone defect, swabs to be sent for microbiological cultures, debridement of the bony surface until viable tissue is visible, followed by grafting of allogeneic bone (Bone Bank; Istituto Ortopedico G. Pini, Milan, Italy) enriched with BMAC. In the case of revision surgery for mobilized acetabular cup, the latter was substituted with a new cup.

Mechanical stability was always thoroughly evaluated and accounted for. At the end of the surgical procedure, 2 ml of BMA and approximately 0.5 ml of BMAC were submitted to the Microbiology laboratory for the following procedures: 1) pre- and post-concentration cell count; 2) sterility tests;

3) culture of nucleated cells to assess cell morphology, replication, and differentiation; 4) assessment of virus-induced CPE and of adventitious viruses.

Pre-operatory serological status of patients

Serum specimens taken from patients before surgery were tested for multiple pathogens. Antibodies

to HIV, HBV, HCV, CMV, EBV, and parvovirus B19 were tested using enzyme-linked

immunosorbent assays (ELISA; Diasorin, Saluggia, Italy; Biotrin, Dublin, Ireland). Antibodies to

T. pallidum were assayed against recombinant antigens (Diasorin, Saluggia, Italy).

Bone marrow cell cultures

Two samples of bone marrow (pre- and post-concentration) were obtained in heparin tubes from each patient. Blood cell counts were obtained using routine analyzers (Sysmex; Norderstedt, Germany). Nucleated cells were isolated by centrifugation on two different Ficoll-Histopaque gradients (1.077 and 1.199 g/ml). Cell viability was evaluated by the trypan blue exclusion method.

Cell suspensions were plated in 75 cm

tissue culture flasks using Mesenchymal Stem Cell Growth Medium (Lonza; Basel, Switzerland) plus basic fibroblast growth factor (bFGF; 5 ng/ml) and incubated at 37°C in air with 5% CO

. Stromal cells adhered to plastic within 48 hrs from plating;

then cell replication started. Upon reaching confluency in 5-7 days, adherent cells were harvested using trypsin-EDTA and replated for subsequent passages. Supernatant was collected from each culture at different times, clarified by low-speed centrifugation, aliquoted and frozen at –70°C before processing.

Cultures were observed at 3-day intervals using a phase-contrast microscope with a digital camera in order to evaluate cell morphology and the possible development of CPE. Images were acquired with a 10X, 20X, and 40X objective and recorded using a computerized image analysis system (Im- age DB; Amplimedical, Mira, IT).

Sterility tests and Mycoplasma detection

Aliquots of BMA, BMAC, and bone marrow cells cultured for 2-4 weeks were inoculated into aerobic and anaerobic blood culture vials (BACTEC™ Plus Aerobic/F* and Plus Anaerobic/F*

Vials; Becton Dickinson, Franklin Lakes, New Jersey) and incubated for 14 days. Cell culture supernatants were also tested for Mycoplasma spp. using a commercial kit (MycoAlert; Lonza Rockland, ME) based on enzymatic ATP-conversion combined with luminescence measurement.

The test can detect a wide range of Mycoplasma species.

Detection of adventitious viruses in cultured bone marrow cells

At the end of the 4-week culture period, cultures were tested for adventitious viruses by polymerase

chain reaction (PCR). DNA was extracted from cell pellets (10

cells) using QIAamp DNA Blood

Midi Kit (QIAGEN GmbH, Hilden, Germany); the QIAamp DNA mini kit (QIAGEN GmbH,

Hilden, Germany) was used for supernatants. RNA was extracted from supernatants using the

QIamp Viral RNA minikit (Qiagen, Hilden, Germany). Both DNA and RNA were eluted in 50 μL

of buffer. RNA was extracted from cultured cells using the acid guanidinium thiocyanate and

phenol chloroform method (Trizol; Invitrogen, Carlsbad, California). Samples were mixed with 2 vols RNAzol and extracted with 200 μl chloroform. The RNA-containing aqueous phase was precipitated with 500 μl isopropanol. The RNA pellet was washed with 75% ethanol and resuspended in 50 μl sterile water. Viral genomes were detected using amplification methods specific to HBV, CMV, EBV, herpes simplex virus-1/2 (HSV-1/2), parvovirus B19 (diagnostic kits from Qiagen-Artus, Hilden, Germany). Viral RNA was retrotranscribed with the SuperScript VILO cDNA synthesis kit (Invitrogen, Grand Island, New York) and random hexamer primers. The cDNA product was then amplified using methods specific for HCV and HIV (Qiagen-Artus, Hilden, Germany), as well as in-house tests for the endogenous retrovirus K

(HERV-K; Jeong et al., 2010).

Characterization of cultured bone marrow cells

The surface expression of differentiation markers was evaluated by flow cytometry (FACSCalibur;

Becton Dickinson Biosciences, Franklin Lakes, New Jersey) and immunofluorescence. The following mouse monoclonal antibodies were used: CD105, CD73, CD90 (expected expression by MSCs), CD45, CD34, CD14 (expected lack of expression by MSCs). Cultured adherent cells were washed with PBS, detached by scraping, and counted. Cell suspensions (approximately 10

/ml in FACS buffer) were dispensed in sterile tubes (0.3 ml/tube) and incubated on ice with antibodies directed against different markers. As secondary antibody, FITC-labeled goat anti-mouse was used.

Two negative controls were stained with FITC secondary antibody: unstained cells and cells incubated with a non-relevant mAb (anti-HIV p24).

Follow-up of patients

According to the routine protocol used at our Department of Orthopaedics and Traumatology, each patient was evaluated clinically and radiographically (2 plane x-rays) at five different times: 1) pre- operatory, 2) 1 month post-op, 3) 3 months post-op, 4) 6 months post-op, 5) 12 months post-op.

Adverse events experienced by patients during the study were recorded and reported. Results were analyzed using observational statistics.

Results

Serological evaluation before surgery

As shown in Table 1, no positivity against T.pallidum, HIV, and HCV was detected. Anti-HBc

antibody was found in 6 of 14 patients, no one of which was HBsAg-positive. IgG to parvovirus

B19 was detected in 10 of 12 patients; IgG against EBV and CMV were present in all patients.

These data substantiate the risk for surgeons of accidental infections by HBV, EBV, CMV, and parvovirus B19.

Ability of the SmartPReP-2 to concentrate nucleated cells in bone marrow aspirates

As shown in Table 2, cell counts performed before and after concentration showed that the procedure strongly reduced RBC numbers and concentrated nucleated cells by 4.25 fold on the average. Thus, the concentration step allowed administering to each patient cell suspensions containing 470->1,000 million nucleated cells plus 2-10 billion platelets.

Cell culture of bone marrow aspirates: sterility tests and phenotypic characterization of adherent cells

Nucleated cells of BMA were cultured for 4 weeks. Two days after plating, a mixture of fibroblast- like and hemispherical adherent cells started growing and formed distinct colonies that progressively transformed into monolayers of adherent fibroblast-like cells with rare hemispherical adherent cells (Figure 1 a-d).

Sterility tests performed on cultures incubated for 4 weeks showed that no bacterial or fungal contamination could be detected; mycoplasma contamination was also absent (Table 3). Cell viability was 93-99%. Cytofluorimetry and immunofluorescence showed that adherent bone marrow cells from all patients were negative for the CD45 haematopoietic marker, negative for CD34 (endothelial marker), and substantially negative for CD14 (monocyte marker). Cultured cells from most patients expressed the surface markers CD73, CD90, and CD105. The results are in agreement with the criteria of the International Society for Cellular Therapy for defining multipotent mesenchymal stromal cells

. Thus, considerable numbers of mesenchymal stromal cells were present in all concentrated bone marrow aspirates from adult patients.

Detection of adventitious viruses in bone marrow cultures

Adventitious viruses were evaluated by observing the development of CPE in cell cultures and by

amplification of viral genomes (Table 4). After 2-4 weeks incubation, 2 of 11 bone marrow cultures

developed CPE in fibroblast-like cells that were characterized by multiple cytoplasmic vacuoles and

cell fusion (Figure 1e,f). Cells of both patients continued to grow, but showed reduced viability (88-

93%). These cultures were positive for EBV by PCR. Cultures of patient PM contained 1,600

genome copies/10

cells, whereas patient PA had only 10 genome copies/10

cells. While the latter

value is normal for an EBV-positive individual, the first one indicates that EBV did replicate in

cultured cells. Given the oncogenic potential of EBV, cell therapy using bone marrow cells that had

been expanded in vitro from this individual might involve biological risk. This case supports the indications of the US Pharmacopeia

that recently made virology controls mandatory for cells expanded in culture for therapeutic use. It is interesting to notice that CMV and parvovirus B19 (agents against which the majority of patients was seropositive) did not reactivate in cultured bone marrow cells. Similarly, no evidence was obtained for replication of the endogenous retrovirus HERV-K.

Clinical outcome

Clinical data is summarized in Table 5. As evaluated using standard 2 plane radiographs (7, 26), the mean volume of bone defects was 50.8 cm

(range, 33-85 cm

). Unfortunately, the precision of the method is only ± 30%

. The mean number of previous surgical treatments was 2.57 (range, 1-15 treatments) with a mean of 11.5 month duration (range, 4-68 months). In two patients affected by non-union, rupture of previously positioned plates was observed. The mean follow-up time was 23.5 months (range, 16-34 months). During surgery, three patients underwent the substitution of plates either for plate failure or inadequate size. The clinical characteristics of patients are summarized in Table 5, which also presents the size of the bone defect and the number of implanted cells.

On the average, 5 months after surgical intervention (range 2-12 months) 12 of 14 cases showed full integration of the bone graft and complete healing of the bone defect, as observed on radiographs (Fig.2-4). In two patients, neither clinical nor radiographic healing was achieved. The first failure concerned a patient affected by aseptic loosening of the acetabular cup in a total hip replacement, complicated by a voluminous haematoma of the anterior region of the thigh. One month after acetabular cup substitution, and treatment with bone graft and bone marrow concentrate, the patient presented with a recurrence of the thigh haematoma and partial resorption of the bone graft. Clinical tests, radiographs, and microbiological assays over the following months failed to identify local or systemic infection. Furthermore, osteointegration of the new acetabular cup failed to occur.

Suspecting subclinical infection, the patient underwent repeated surgery nine months after the last intervention. The hip prosthesis was removed and substituted with an antibiotic spacer.

Microbiological and histological examinations of the haematoma and periprosthetic soft tissues were performed, yet no active infection was detected.

The second failure occurred in a patient affected by an open leg fracture with significant loss of skin

and bone, secondary to a penetrating trauma. After initial surgery of soft tissues and osteosynthesis

with an external fixator, local infection by Pseudomonas aeruginosa was detected. The patient was

treated accordingly. Upon negativisation of repeated microbiological tests, the patient underwent

plastic surgery to cover the soft-tissue defect. Four months later, in the absence of clinical and laboratory signs of infection, the patient was treated with allogeneic bone graft and BMAC. One month after surgery, a pus-secreting fistula appeared at the affected site, later confirmed as a reactivation of the P. aeruginosa infection. Despite the infection, four months after surgery, radiographic examination documented integration of about 50% of the bone graft at the proximal portion of the bone defect. The graft, however, was insufficient to guarantee mechanical stability.

Discussion

The key to bone restoration and regeneration was described by Giannoudis et al in 2007, when the

“diamond concept” was presented

. The authors provided evidence that in order to achieve bone formation and overcome impaired fracture healing, four parameters had to be respected: utilisation of growth factors, scaffolds, mesenchymal stem cells, and stable mechanics.

Experimental and clinical studies have shown that autologous mononuclear cells from bone marrow in combination with biocompatible scaffolds can support osteogenesis in animal models

40

and in humans

. Differentiation of osteoprogenitor cells is significantly dependent on the microenvironment, making the use of BMAC an option for bone regeneration

. The present study reports complete healing of bone defects refractory to traditional surgery within 3-12 months post- treatment in 12 of 14 patients. Two failures, however, need to be addressed in view of the great complexity of cases. One patient with a severe leg injury was admitted to the experimental procedure as an extreme measure to avoid limb amputation. A second case represents a problematic clinical scenario that has not yet been clarified.

The investigated cases were heterogeneous and clinically complex. However, all of them had been unresponsive to conventional medical and surgical therapies. Hence, the complete healing of 12 pa- tients can be viewed as encouraging.

Nonetheless, our study presents three major limitations: the small number of cases, the lack of a control group, and the heterogeneity of clinical diagnoses. Despite clinical and radiographic success in most cases, our conclusions cannot necessarily guarantee comparable results for different diag- noses leading to bone defects or for larger defects. Beyond any reasonable doubt, a larger random- ized controlled prospective study could provide more certain answers in regards of the use of the hereby presented technique.

Despite symptomatic improvement and significant bone formation observed in 12 of 14 patients,

treatment with autologous BMAC in combination with allogenic bone graft failed to cure all bone

defects. In this limited series, however, the method proved to be safe in both the short and long-run,

and had no significant impact on the total operating time. Bone marrow samples (before and after

concentration) were consistently sterile. The cost of the procedure was relatively cheap, especially if compared to alternative methods and to other more expensive drugs (e.g., bone morphogenetic proteins) that are used to treat large bone defects. In addition, the morbidity associated with autolo- gous bone graft transplantation (infections, seromas, hematomas, herniation of abdominal contents, vascular injuries, neurologic injuries, and iliac wing fractures)

was abolished

, and no significant side effect was observed. A further advantage of the one-stage isolation procedure in comparison to the ex-vivo expansion of autologous cells from bone marrow is the reduced cost and the absence of infections attributable to the complex procedures required for cellular expansion

.

Analysis of cell differentiation markers and virological studies show that considerable numbers of mesenchymal stem cells are easily obtainable from bone marrow aspirates of adult patients, and that virus reactivation is rarely detected in bone marrow cells. Furthermore, another major advantage is the elevated platelet concentration (about 380%) reported in our study. When activated, platelets release granules rich in tissue (including bone) growth factors, cytokines and chemochines: platelet- derived growth factor (PDGF-αα, ββ and αβ isomers), transforming growth factor (TGF-β, β1 and β2 isomers), platelet factor 4 (PF4), interleukin-1 (IL-1), platelet-derived angiogenesis factor (PDAF), VEGF, epidermal growth factor (EGF), platelet-derived endothelial growth factor (PDEGF), epithelial cell growth factor (ECGF), insulin-like growth factor (IGF), osteocalcin, osteonectin, fibrinogen, vitronectin, fibronectin, and thrombospondin

. Adult mesenchymal stem cells, osteoblasts, fibroblasts, endothelial cells and epidermal cells all respond to the abovementioned proteins

, thus promoting tissue/bone formation.

Conclusions

We present a new technique to try overcome bone healing impairment in patients affected by large bone defects unresponsive to traditional therapies. Despite the promising results, we strongly encourage further multicenter, prospective, controlled studies to better evaluate and understand the effectiveness of BMAC grafts in treating complex bone defects. It is expected that the procedure will provide clinical outcomes comparable to those of current autologous bone graft transplantation, yet with significantly less morbidity related to the procedure.

Overall, BMAC supplemented with allogeneic bone can locally deliver platelet-derived growth factors, mesenchymal stem cells, and mechanical stability conferred by the bone scaffold;

the above elements meet the “Diamond criteria” of Giannoudis et al

. Given the safety, efficacy,

cost-effectivenes, and simplicity of the presented technique, we encourage the use of allogeneic

bone graft enriched with autologous concentrated bone marrow cells obtained from the iliac crest

for treating large bone defects unresponsive to traditional therapies. Future investigations need to

consider that the organismal state of the patient does influence the mechanisms regulating the production and functions of bone marrow precursor stem cells

.

ACKNOWLEDGEMENTS

This work has been supported by Regione Lombardia (grant “Piano Sangue-2009”). We are grateful to Franco Locatelli (Pavia, Italy) and Gianni Zatti (Monza, Italy) for their biological and clinical contributions.

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Legends to Figures

Figure 1. A and B. cultures of adherent bone marrow cells 5 days post-plating. In both cases, most cells show fibroblast-like morphology (case 04AG, A; case 10BA, B. Original magnification 100x).

C and D: cultures of adherent bone marrow cells 12 days post-plating. In both cases, mixtures of fi- broblast-like and hemispherical adherent cells are present (case 01ZL, C; case 11MLD, D. Original magnification 200x).

E and F: culture of adherent bone marrow cells 12 days post-plating. Cytopathic effect associated to Epstein-Barr virus infection is see as multiple cytoplasmic vacuoles in fibroblast-like cells (case 05PM. Original magnification: 200x, E; 400x, F ).

Figure 2. 84 year-old female, distal femur fracture treated with plate in May 2008. Failure of the plate in May 2009, with evident non-union. In May 2009 the patient underwent surgery: removal of hardware, implantation of an intramedullary retrograde nail, and BMAC plus allogeneic bone grafting. Bone healing at 6 months.

Figure 3. 17 year-old male affected by a proximal humerus bone cyst that caused 3 fractures.

Treated with BMAC plus allogeneic bone grafting. Bone healing at 6 months.

Figure 4. 71 year-old male, mid humerus shaft fracture treated conservatively, developing non-

union. Treated with plate and BMAC plus allogeneic bone grafting. Bone healing at 3 months.