Positive early clinical outcomes of bone marrow aspirate concentrate for osteoarthritis using a novel fenestrated trocar.

BACKGROUND: This study sought to assess early clinical outcomes for knee osteoarthritis (OA) patients undergoing bone marrow aspirate concentrate (BMAC) treatment using a novel closed-end, fenestrated trocar (FT) that does not require centrifugation. METHODS: A prospective cohort of 17 knee OA patients undergoing BMAC treatment with the FT system from March 2018 to March 2019 was retrospectively evaluated. Approximately 10 mL of BMAC was harvested, no centrifugation was performed, and the BMAC was injected into the affected knee. Clinical outcomes were assessed at baseline, six weeks, and 12 weeks. This study has no affiliation with/vested-interest in the FT system. RESULTS: There were significant improvements in nearly all outcomes from baseline to 12 weeks. Specific improvements included Knee Injury and OA Outcome Score (KOOS) activities-of-daily-living (61.1 ± 9.2 [mean ± 95% confidence interval] to 89.3 ± 6, p = 0.001), quality-of-life (32.7 ± 9.3 to 66.1 ± 17.9, p = 0.003), sports/recreation (36.9 ± 10.6 to 72.6 ± 26.3, p = 0.006), and pain (53.8 ± 9.3 to 83 ± 10.2, p = 0.001); Lysholm scores (55.5 ± 8.4 to 77.3 ± 10.5, p = 0.009); and visual analog pain scores (5.68 ± 1.14 to 2.07 ± 1.86, p = 0.003). Individually, at least 75% of patients exhibited improvement in all KOOS categories at six weeks and at least 85% at 12 weeks. CONCLUSIONS: BMAC treatment with an FT system that does not require centrifugation resulted in significant improvements in early pain and function scores for knee OA. The symptomatic improvements in this study were similar to or greater than what has been reported using traditional needles. These data may provide clinicians with comfort in using an FT system and provide motivation for future randomized-controlled trials comparing aspiration techniques.

Isolation, Differentiation, and Characterization of Human Bone Marrow Stem Cells In Vitro and In Vivo.

In this chapter, we describe techniques for the isolation and characterisation of skeletal stem cells from human bone marrow. The methods for enrichment of STRO-1+ and STRO-4+ cells using magnetic activated cell sorting are described and we also detail techniques for establishing and characterizing osteogenic, adipogenic, and chondrogenic cultures from these cells. Finally, we present methods for studying the ability of these cells to produce bone in vivo using diffusion chambers which have been implanted subcutaneously into mice.

A Review of Commercially Available Point-of-Care Devices to Concentrate Bone Marrow for the Treatment of Osteoarthritis and Focal Cartilage Lesions.

OBJECTIVE: Mesenchymal stem cells (MSCs) are a promising cell-based therapy treatment option for several orthopedic indications. Because culture expansion of MSC is time and cost intensive, a bedside concentration of bone marrow (BM) aspirate is used as an alternative. Many commercial systems are available but the available literature and knowledge regarding these systems is limited. We compared different point-of-care devices that concentrate BM (BMC) by focusing on technical features and quality parameters to help surgeons make informed decisions while selecting the appropriate device. METHODS: We compared published data on the BMC devices of Arteriocyte, Arthrex, Celling Biosciences, EmCyte, Exactech, ISTO Tech, Harvest Tech/Terumo BCT, and Zimmer/BIOMET regarding technical features (centrifugation speed/time, input/output volume, kit components, type of aspiration syringes, filter usage) and quality parameters of their final BMC product (hematocrit, concentration of platelets and total nucleated cells, concentration of MSC and connective tissue progenitor cells). RESULTS: The systems differ significantly in their technical features and centrifugation parameters. Only the fully automated systems use universal kits, which allow processing different volumes of BM. Only the Arthrex system allows selection of final hematocrit. There was no standardized reporting method to describe biologic potency. CONCLUSIONS: Based on the data obtained in this review, recommending a single device is not possible because the reported data could not be compared between devices. A standardized reporting method is needed for valid comparisons. Furthermore, clinical outcomes are required to establish the true efficacy of these systems. We are conducting additional studies for more careful comparison among the devices.

Percutaneous Ultrasound-Guided Musculoskeletal Applications of Autologous Bone Marrow Aspirate Concentrate: Preliminary Experience From a Single Institution.

Emerging musculoskeletal applications for local administration of autologous bone marrow aspirate concentrate (BMAC) include treatment of fractures, osteonecrosis, osteochondral injuries, osteoarthritis, ligament injury, tendon injury, and tendonopathies. Ultrasound-guided technique for various BMAC injection sites is detailed in this technical report and our preliminary clinical experience outlined.Five patients, 1 woman and 4 men, were treated with 6 peri/intratendinous (n = 4) or intraarticular (n = 2) BMAC injections between July 5, 2015 and December 31, 2016 for the clinical indications of common hamstrings origin tendinosis (n = 4), hip labral tear (n = 1), and osteochondral lesion of the talus (n = 1).All procedures were technically successful, with BMAC locally administered to the therapeutic target and no procedural complications observed. Clinical follow-up was available for 5 of 6 procedures. Four of 5 injections resulted in self-reported symptomatic improvement (clinical follow-up range, 2-12 months). One 72-year-old man with right common hamstrings origin tendinosis reported no improvement after BMAC injection.The technology is now available to support ultrasound-guided, autologous BMAC administration by the musculoskeletal interventionalist for common indications. Our initial clinical experience is consistent with early reports in the literature. This technique is well tolerated by symptomatic patients on an outpatient basis, and rates of self-reported symptomatic relief are high. Mechanism of action, long-term safety, and long-term clinical efficacy remain largely undefined.

Combined Therapy with Shock Wave and Retrograde Bone Marrow-Derived Cell Transplantation for Osteochondral Lesions of the Talus.

Multiple treatment strategies have been developed for osteochondral lesions (OCLs) of the talus. The purpose of this retrospective study was to assess retrograde autologous bone marrow cell (BMC) transplantation via core drilling (CD) combined with focused extracorporeal shock wave treatment (ESWT) in undisplaced OCL of the talus. A total of 69 patients with unilateral osteochondral lesions of the talus (Hepple grade I-III) were divided into two groups: 41 patients received combined therapy of ESWT and BMC transplantation (group A), while 28 were administered BMC transplantation alone (group B). The patients were followed up clinically and radiographically for a minimum of 2 years. Mean follow-up was 4.1 ± 2.8 years. AOFAS scores increased more significantly while pain intensity levels decreased in group A after treatment, compared with group B values (P < 0.001). In MRI follow-up, a more remarkable improvement of OCLs of the talus was observed in group A compared with group B (P = 0.040). Therefore, the combined technique reported here is a highly effective therapeutic option in OCLs of the talus with intact cartilage. It promotes patient recovery with pain control, and improves clinical outcome for more than 2 years after surgery.

Trasplante de médula ósea en pacientes con anemia falciforme. Experiencia en un centro / Bone marrow transplant in patients with sickle cell anaemia. Experience in one centre

INTRODUCCIÓN: La enfermedad de células falciformes (ECF), pese a la mejora en el manejo médico, persiste asociada a morbilidad y a menor supervivencia. El alotrasplante de progenitores hematopoyéticos (alo-TPH) es actualmente la única opción curativa. Describir la evolución clínico-analítica de los pacientes trasplantados en nuestro centro. MATERIAL Y MÉTODO: Estudio unicéntrico descriptivo, incluye a pacientes con ECF en los que se realiza alo-TPH de médula ósea de hermano HLA-idéntico desde enero del 2010 hasta diciembre del 2014. Se recogen datos epidemiológicos, clínicos y analíticos con tiempo de seguimiento hasta diciembre del 2015. Los datos se presentan como frecuencias, porcentajes y medianas (rango). RESULTADOS: Se recluta a 11 pacientes (8 varones), mediana de edad: 7 años (2-13), todos ellos con comorbilidad previa al TPH. Se consigue injerto estable en 10/11 pacientes, quimerismo completo en 9/11 y quimerismo mixto estable tras un año del TPH en 1/11. Un paciente presenta fallo secundario de injerto con reaparición de clínica el día +180. Complicaciones post-TPH: complicaciones neurológicas 4/11 pacientes (hemorragia subaracnoidea, crisis), HTA 7/11, fallo renal agudo 3/11, reactivación CMV 9/11, EICHa cutáneo 6/11, uno de ellos desarrolla EICH intestinal grado IV causando su fallecimiento (día +51). Ningún paciente desarrolla EICH crónico. Supervivencia global y libre de eventos a los 3,1 años de seguimiento: 90,9 y 81,9%, respectivamente. CONCLUSIONES: El alo-TPH, única opción curativa, no está exento de morbimortalidad, encontramos un riesgo de muerte similar a otras series (1/11), siendo su primera causa el EICH agudo. Otros problemas son fallo de injerto (1/11) y complicaciones neurológicas (4/11), aunque las secuelas permanentes son leves

INTRODUCTION: Sickle cell disease (SCD), despite the improvement in the medical management, is still associated with severe morbidity and decreased survival. Allogenic hematopoietic stem cell transplantation (Allo-HSCT) currently provides the only curative therapy. A report is presented on our experience in children with SCD, who underwent Allo-HSCT in a single centre. Material and method. A single centre descriptive study was conducted on patients with SCD who underwent a bone marrow transplant from an HLA-identical sibling donor between January 2010 and December 2014. Epidemiological, clinical and analytical parameters were collected with a follow-up to December 2015. Data are presented as frequencies, percentages, and medians (range). RESULTS: Allo-HCST was performed in 11 patients (8 males) with a median age of 7 years (2-13), all of them with comorbidity prior to the HCST. A stable graft was achieved in 10 out of 11 patients, 9 of them with complete donor chimerism, and one patient with stable mixed chimerism after 1 year of allo-HSCT. One patient has secondary graft failure with re-appearance of symptoms associated with SCD on day 180. Complications of Allo-HSCT are: arterial hypertension 7/11, acute renal failure 3/11, CMV reactivation 9/11, neurological complications 4/11 (subarachnoid haemorrhage, seizure), and acute graft versus host disease (aGVHD) of the skin 6/11, one of whom developed grade IV intestinal aGVHD, causing his death (day 51). None of the patients developed chronic GVHD. The overall survival and event-free survival was 90.9% and 81.9%, respectively, with a median follow-up of 3.1 (1-5.7) years. CONCLUSIONS: Allo-HSCT, the only curative therapy, remains associated with morbidity. There was a transplant related mortality in our study, consistent with multicentre studies (1/11), and with aGVHD being the main cause. Other problems still include graft failure (1/11), and neurological complications (4/11), although the permanent sequelae are mild

bFGF binding cardiac extracellular matrix promotes the repair potential of bone marrow mesenchymal stem cells in a rabbit model for acute myocardial infarction.

To assess the effect of basic fibroblast growth factor-binding extracellular matrix (bFGF-ECM) combined with bone marrow mesenchymal stem cells (BMSCs) transplantation on acute myocardial infarction (AMI) and explore the underlying mechenisms. Rabbit hearts were processed by decellularization with sodium dodecyl sulfate (SDS) perfusion, heparin immobilization, bFGF-binding and homogenization, for preparation of bFGF-binding cardiac ECM suspension (bFGF-ECM). Thereafter, the characteristics of bFGF release were analyzed in vitro. Following ligation of the mid-third of the left anterior descending artery, the rabbits were divided into a control group (no treatment), BMSCs group (BMSCs transplantation), bFGF-ECM group (bFGF-ECM implantation), and BMSCs + bFGF-ECM group (BMSCs and bFGF-ECM implantation). Apoptosis and differentiation of implanted BMSCs, and the left ventricular (LV) remodeling and function were assessed. The ex vivo proliferation, apoptosis, migration and differentiation of BMSCs were determined after exposure to bFGF and/or ECM. The ECM could sustainably release bFGF. 24 h and 6 weeks after the operation, improved viability and differentiation of the implanted BMSCs, as well as inhibited dilatation and preserved function of the left ventricle (LV), were significant in the BMSCs + bFGF-ECM group compared with other groups (P < 0.05), although BMSCs and ECM-bFGF groups also showed better results than control group (P < 0.05). Additionally, ECM and bFGF showed a synergistic effect on BMSCs proliferation, viability, migration and differentiation. The combination of bFGF-binding ECM and BMSCs implantation may promote myocardial regeneration and LV function, and become a new strategy for the treatment of AMI.

Análisis de la supervivencia de los niños con inmunodeficiencias primarias que han recibido un trasplante de progenitores hematopoyéticos en Espana / Survival analysis of hematopoietic stem cell transplantation in children with primary immunodeficiency in Spain

INTRODUCCIÓN: Los niños afectados de inmunodeficiencias primarias presentan infecciones graves y mayor prevalencia de manifestaciones autoinmunitarias, alergias y enfermedad linfoproliferativa. El trasplante alogénico de precursores hematopoyéticos ha sido el único tratamiento curativo durante décadas. PACIENTES Y MÉTODOS: Pacientes con inmunodeficiencias primarias que recibieron trasplante alogénico de precursores hematopoyéticos desde 1985 hasta 2011, recogidos en el Registro Nacional del Grupo Español para Trasplante de Médula Ósea en Niños. RESULTADOS: Ciento cincuenta y nueve niños recibieron un total de 173 trasplantes, 97 por inmunodeficiencia combinada grave, 30 por enfermedades de disregulación inmunitaria, 25 por síndrome de Wiskott-Aldrich y 21 por defectos de número y/o función de los fagocitos. La mediana de edad al diagnóstico fue de 6 meses (17 días-168 meses) y de 12 meses (1 mes-189 meses) al trasplante. Los donantes fueron hermano HLA idéntico en 30 (19%), donante familiar alternativo en 40 (25%) y donante no emparentado en 89 (56%). La fuente de progenitores fue médula ósea en 68 (43%), sangre de cordón umbilical en 52 (33%) y sangre periférica en 39 (24%). Permanecen vivos 98 niños (61,6%), 57 (35,9%) fallecieron. La supervivencia libre de enfermedad a los 10 años fue del 63, el 90% para los pacientes trasplantados de hermano HLA idéntico, el 36% para los trasplantados de un donante familiar alternativo y el 66 para los trasplantados de donante no emparentado

INTRODUCTION: Children with primary immunodeficiency have severe life-threatening infections and a higher prevalence of autoimmune problems, allergy and lymphoproliferative disorders. Allogenic hematopoietic stem cell transplantation has been the only potentially curative option. PATIENTS AND METHODS: Patients with primary immunodeficiency underwent allogenic stem cell transplantation in the period 1985-2011, and registered in the Spanish Working Party for Bone Marrow Transplantation in Children. RESULTS: One hundred and fifty nine patients underwent 173 allogenic stem cell transplantations, of whom 97 had severe combined immunodeficiency, 30 with immune dysregulation disorders, 25 Wiskott-Aldrich syndrome, and 21 phagocyte disorders. The median patient age at diagnosis was 6 months (range: 17 days - 168 months) and the median patient age at transplant was 12 months (range: 1 month - 189 months). The donors were 30 (19%) identical siblings, 40 (25%) alternative family donors, and 89 (56%) unrelated donors. The source of stem cells was bone marrow in 68 (43%), cord blood in 52 (33%), and peripheral blood in 39 (24%). Ninety eight (61.6%) are alive, 57 (35.9%) died. Event-free survival at 10 years was 63%, with 90% for children transplanted from identical siblings, 36% for those transplanted from alternative family donors, and 66% for those transplanted from unrelated donors. CONCLUSIONS: The best results have been obtained with identical siblings, but other options may be considered

Comparison of free fibular flaps with reamer-irrigator-aspirator bone grafts for the reconstruction of critical-sized mandibular defects.

OBJECTIVE: Vascularized bone flaps are currently indicated for reconstructing mandibular defects 6 cm or larger. This technique can result in donor-site morbidity and requires microsurgery. To explore alternative methods of mandibular reconstruction, we sought to compare bone graft obtained with the reamer-irrigator-aspirator (RIA) device with the free fibula (FF) flap for the reconstruction of a critical-sized mandibular defect. METHODS: Sixteen 3-month-old Yorkshire pigs underwent 6-cm full-thickness resection of the left mandible. For the FF group (n = 8), an osseous FF flap was raised from the left leg and placed into the defect. For the RIA group (n = 8), a RIA Instrument Set was used on the ipsilateral femur to ream the femoral canal and harvest RIA putty. This putty containing medullary bone marrow contents and cortical bone was packed into the defect. At the study end point, volumetric, biomechanical, and histologic analyses were performed. RESULTS: Operative times were significantly shorter in the RIA group (RIA,126 [30] min; FF, 346 [50] min; P < 0.05). Biomechanical testing of reconstructed sites showed no significant difference in maximum fracture loads between both groups (RIA, 468 [97] N; FF, 689 [262] N; P = 0.11). Mean (SD) volume ratio of bone growth at the reconstructed sites was comparable between both groups (RIA, 71% [4.5%]; FF, 72% [3.3%]; P = 0.60). Equal bone quality was confirmed histologically. CONCLUSIONS: The RIA technique significantly reduces operative time and provides bone of equal strength and histologic quality to FF flap reconstruction in a large animal model. The RIA method may represent an efficient technique for the reconstruction of craniomaxillofacial defects.

A prospective comparison of 3 approved systems for autologous bone marrow concentration demonstrated nonequivalency in progenitor cell number and concentration.

OBJECTIVES: To evaluate the efficacy of 3 commercially available systems: the Harvest SmartPReP 2 BMAC, Biomet BioCUE, and Arteriocyte Magellan systems. We compared the number and concentration of progenitor cells achieved both before and after centrifugation and the percentage of progenitor cells salvaged after centrifugation. METHODS: Forty patients, mean age 47 ± 18 years (range: 18-92 years, 19 male/21 female) were prospectively consented for bilateral iliac crest aspiration. The first 20 aspirations compared the Harvest and Biomet systems, and based on those results, the second 20 compared the Harvest and Arteriocyte systems. One system was randomly assigned to each iliac crest. Each system's unique marrow acquisition process and centrifugation mechanism was followed. Samples for analysis were taken both immediately before the marrow was put into the centrifugation system (after acquisition), and after centrifugation. The number of progenitor cells in each sample was estimated by counting the connective tissue progenitors (CTPs). RESULTS: The Harvest system achieved a significantly greater number and concentration of CTPs both before and after centrifugation when compared to the Biomet system. There was no difference in the percent yield of CTPs after centrifugation. There was no significant difference in the number and concentration of CTPs between the Harvest and Arteriocyte systems before centrifugation, but the Harvest system had a significantly greater number and concentration of CTPs after centrifugation. The Harvest system also had a significantly higher percent yield of CTPs after centrifugation compared with the Arteriocyte system. CONCLUSIONS: The Harvest system resulted in a greater CTP number and concentration after centrifugation when compared with the Biomet and Arteriocyte systems and may thus provide increased osteogenic and chondrogenic capacity.

Comparing peripheral blood stem cell collection using the COBE Spectra, Haemonetics MCS+, and Baxter Amicus.

Peripheral blood stem cells (PBSC) have become the most common source of hematopoietic cells for allogeneic or autologous blood and marrow transplantation (BMT). We performed an evaluation of PBSC collections using three different apheresis systems in two major transplantation centers in Singapore. Patients undergoing autologous BMT and donors collecting for allogeneic BMT were harvested using the COBE Spectra, Haemonetics MCS+, or Baxter Amicus. There were 99 Spectra collections (61 were autologous), 81 MCS+ collections (35 were autologous) and 38 Amicus collections (33 were autologous). Our data shows that the Amicus not only processed larger peripheral blood volumes but also yielded larger PBSC volume (P-value<0.05). In terms of PBSC products, the Spectra produced more WBC, WBC/liter blood processed, and WBC/kg (P-value<0.05). The Spectra and MCS+ produced comparable amount of CD34+ cells. Amicus collected 50% less platelets compared to Spectra and MCS+. The total CD34+ cells in the PBSC products was linearly correlated to the circulating CD34+ cells using Spectra, MCS+, and Amicus. Our results suggest that, compared to MCS+ and Amicus, collecting PBSC using the COBE Spectra can produce more WBC with a similar number of CD34+ cells. With a linear correlation of circulating CD34+ cells to the total CD34+ cells in the products, the availability of an automated procedure, no rotating seal, and a small extracorporeal volume, the Spectra appears to be the preferred machine for PBSC collection.

Treatment of severe post-traumatic bone defects with autologous stem cells loaded on allogeneic scaffolds.

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. We present a case series to evaluate the rationale of using nucleated cells from autologous bone marrow aspirates in the treatment of severe bone defects that failed to respond to traditional treatments. Ten adult patients (mean age, 49.6-years-old) with severe bone defects were included in this study. Lower limb bone defects were >or=5 cm3 in size, and upper limb defects .or=2 cm3. 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-approved device. Postsurgery clinical and radiographic follow-up was performed at 1, 3, 6, and 12 months. To assess viability, morphology, and immunophenotype, bone marrow nucleated cells were cultured in vitro, tested for sterility, and assayed for the possible replication of adventitious (contaminating) viruses. In 9 of 10 patients, both clinical and radiographic healing of the bone defect along with bone graft integration were observed (mean time, 5.6 months); one patient failed to respond. No post-operative complications were observed. Bone marrow nucleated cells were enriched 4.49-fold by a single concentration step, and these enriched cells were free of microbial contamination. The immunophenotype of adherent cells was compatible with that of mesenchymal stem cells. We detected the replication of Epstein-Barr virus in 2/10 bone marrow cell cultures tested. Hepatitis B virus, cytomegalovirus, parvovirus B19, and endogenous retrovirus HERV-K replication were not detected. Overall, 470 to 1,150 million nucleated cells were grafted into each patient. This case series, with a mean follow-up of almost 2 years, demonstrates that an allogeneic bone scaffold enriched with concentrated autologous bone marrow cells obtained from the iliac crest provides orthopedic surgeons a novel option for treating important bone defects that are unresponsive to traditional therapies.

Transendocardial autologous bone marrow in myocardial infarction induced heart failure, two-year follow-up in an open-label phase I safety study (the TABMMI study).

AIMS: To assess the hypothesis that fluoroscopically-guided helical needle transendocardial delivery of autologous bone marrow (ABM) mononuclear cells (MNCs) in chronic post myocardial infarction patients is safe and improves ejection fraction (EF). METHODS AND RESULTS: Twenty ischaemic heart failure patients with an EF ≤40% were enrolled. ABMMNCs were prepared, counted for CD34+ and CD133+ content, and delivered percutaneously to the heart at 5 to 10 peri-infarct sites. Two-dimensional (2D) transthoracic echocardiography, EF measurements, Holter, and exercise tolerance time (ETT) were performed at baseline, one week (wk), and 6, 12, and 24 months (mo). 96±29 million ABMMNCs were injected into 8.5±2.6 peri-infarct sites over 42±17 minutes (n=20). There were no adverse events associated with the catheter-based cell transplantation procedure or significant increases in ventricular events on Holter. EF improved over baseline from 34.9±4.3% to 41.9±5.1% at 12 mo to 42.2±7.1% (p=0.00005) at 24 mo. ETT improvements were statistically significant from 246±113 sec to 373±183 sec at 12 mo and 371±181 sec at 24 mo (p=0.006). CONCLUSIONS: ABMMNCs delivered with the helical needle transendocardial catheter was safe in this uncontrolled open label study. Increased EF and ETT support the safety of the procedure and technologies involved and warrant additional investigation.

The subchondral bone in articular cartilage repair: current problems in the surgical management.

As the understanding of interactions between articular cartilage and subchondral bone continues to evolve, increased attention is being directed at treatment options for the entire osteochondral unit, rather than focusing on the articular surface only. It is becoming apparent that without support from an intact subchondral bed, any treatment of the surface chondral lesion is likely to fail. This article reviews issues affecting the entire osteochondral unit, such as subchondral changes after marrow-stimulation techniques and meniscectomy or large osteochondral defects created by prosthetic resurfacing techniques. Also discussed are surgical techniques designed to address these issues, including the use of osteochondral allografts, autologous bone grafting, next generation cell-based implants, as well as strategies after failed subchondral repair and problems specific to the ankle joint. Lastly, since this area remains in constant evolution, the requirements for prospective studies needed to evaluate these emerging technologies will be reviewed.

Intraoperative adjunctive stem cell treatment in patients with critical limb ischemia using a novel point-of-care device.

INTRODUCTION: In a prospective trial we tested whether adjunctive intraoperative stem cell treatment in patients with critical limb ischemia (CLI) can be performed safely in combination with bypass surgery and/or interventional treatment. The end point of our study was the safety and integrity of a novel point-of-care system used in patients with CLI. METHODS: We included only patients with CLI and tissue loss according to Rutherford categories 4-6. The Harvest Bone Marrow Aspirate Concentrate System consists of an automated, microprocessor-controlled dedicated centrifuge with decanting capability and the accessory BMAC Pack for processing a patient's bone marrow aspirate (BMA). The centrifuge is portable and enables BMA to be rapidly processed in the operating room to provide an autologous concentrate of nucleated cells for immediate injection. The surgeon aspirated 120 ml BMA from the iliac crest. RESULTS: Eight consecutive patients were treated according to the study protocol. The mean follow-up period was 9.2 months (range 2-18). Stem cells were always injected during the final revascularization attempt. One minor amputation and two major amputations were required. In five of eight patients there was a discrete increase in the ankle-brachial index post-stem cell treatment. The dose of stem cells after centrifugation was 17.2 (range 13.8-54.2)x10E6 CD34-positive cells and 7.8 (range 1.8-35.9)x10E6 CD133-positive cells. The injected dose of VEGFR-2-coexpressing stem cells was 0.5-5.7x10E4. CONCLUSION: We were able to show that the buffy coat preparation using a point-of-care system is a simple and fast method to enrich stem cells from BMAs. This automated system gives high recovery rates and good reproducibility.

Natural bone collagen scaffold combined with autologous enriched bone marrow cells for induction of osteogenesis in an ovine spinal fusion model.

Autologous bone graft, the standard of bone grafting in achieving spinal fusion, is associated with several limitations and complications. The use of bone marrow cells (BMCs) as a potential cell source for spinal fusion, combined with a suitable scaffold to promote bone formation, may be a better choice. The aims of this study were to evaluate the efficacy of natural bone collagen scaffold (NBCS) combined with autologous-enriched BMCs for induction of osteogenesis in vitro and in vivo. Ovine-enriched BMCs were co-cultured with NBCS for 1, 2, 3, and 4 weeks to investigate whether NBCS would support the population expansion and differentiation of enriched BMCs. Using an ovine interbody fusion model, NBCS seeded with autologous enriched BMCs was implanted into the lumbar disc space. Fusion outcomes were compared with the use of the autograft, NBCS without BMCs, and BMCs without NBCS. In vitro results demonstrated that NBCS facilitated the population expansion and differentiation of ovine-enriched BMCs, promoting the expression of collagen type I and the formation of a mineralized matrix. The use of NBCS combined with enriched BMCs in vivo enhanced the spinal fusion rate (6 of 6 at 10 week) (p < 0.05), the biomechanical stiffness of fusion masses, and bone volume at the fusion site (p < 0.05). Histological findings also revealed that a combination of NBCS and BMCs induced new bone formation that integrated well with host bone tissue. In conclusion, NBCS is an effective scaffold that supports ovine-enriched BMCs. The combination of NBCS and BMCs may be a useful alternative for autograft in induction of spinal fusion.

Autologous bone marrow graft and treatment of delayed and non-unions of long bones: technical aspects.

This article reports the technique of percutaneous autologous bone marrow injection as a minimally invasive method alternative to open grafting techniques in the treatment of delayed unions and non-unions. Despite continuous advances in the treatment of long bone fractures, disturbances of healing processes remain a difficult challenge for orthopaedic surgeons. Percutaneous administration of substances with osteoinductive and osteogenic properties offers the advantage of decreased morbidity associated with the classic open grafting techniques. This makes it worth exploring before embarking on more extensive open surgery. The authors present the main technical stages of the percutaneous bone marrow grafting (bone marrow aspiration, concentration, intra-osseous re-injection and post-operative protocol) with a short literature review about this topic.

Overlap between in vitro donor antihost and in vivo posttransplantation TCR Vbeta use: a new paradigm for designer allogeneic blood and marrow transplantation.

Following allogeneic blood and marrow transplantation (BMT), mature donor T cells can enhance engraftment, counteract opportunistic infections, and mount graft-versus-tumor (GVT) responses, but at the risk of developing graft-versus-host disease (GVHD). With the aim of separating the beneficial effects of donor T cells from GVHD, one approach would be to selectively deplete subsets of alloreactive T cells in the hematopoietic cell inoculum. In this regard, TCR Vbeta repertoire analysis by CDR3-size spectratyping can be a powerful tool for the characterization of alloreactive T-cell responses. We investigated the potential of this spectratype approach by comparing the donor T-cell alloresponses generated in vitro against patient peripheral blood lymphocytes (PBLs) with those detected in vivo posttransplantation. The results indicated that for most Vbeta families that exhibited alloreactive CDR3-size skewing, there was a robust overlap between the in vitro antipatient and in vivo spectratype histograms. Thus, in vitro spectratype analysis may be useful for determining the alloreactive T-cell response involved in GVHD development and, thereby, could serve to guide select Vbeta family depletion for designer transplants to improve outcomes.