Radovan Borojevic

Transendocardial, Autologous Bone Marrow Cell Transplantation for Severe, Chronic Ischemic Heart Failure

Background—This study evaluated the hypothesis that transendocardial injections of autologous mononuclear bone marrow cells in patients with end-stage ischemic heart disease could safely promote neovascularization and improve perfusion and myocardial contractility. Methods and Results—Twenty-one patients were enrolled in this prospective, nonrandomized, open-label study (first 14 patients, treatment; last 7 patients, control). Baseline evaluations included complete clinical and laboratory evaluations, exercise stress (ramp treadmill), 2D Doppler echocardiogram, single-photon emission computed tomography perfusion scan, and 24-hour Holter monitoring. Bone marrow mononuclear cells were harvested, isolated, washed, and resuspended in saline for injection by NOGA catheter (15 injections of 0.2 cc). Electromechanical mapping was used to identify viable myocardium (unipolar voltage ≥6.9 mV) for treatment. Treated and control patients underwent 2-month noninvasive follow-up, and treated patients alone underwent a 4-month invasive follow-up according to standard protocols and with the same procedures used as at baseline. Patient population demographics and exercise test variables did not differ significantly between the treatment and control groups; only serum creatinine and brain natriuretic peptide levels varied in laboratory evaluations at follow-up, being relatively higher in control patients. At 2 months, there was a significant reduction in total reversible defect and improvement in global left ventricular function within the treatment group and between the treatment and control groups (P =0.02) on quantitative single-photon emission computed tomography analysis. At 4 months, there was improvement in ejection fraction from a baseline of 20% to 29% (P =0.003) and a reduction in end-systolic volume (P =0.03) in the treated patients. Electromechanical mapping revealed significant mechanical improvement of the injected segments (P <0.0005) at 4 months after treatment. Conclusions—Thus, the present study demonstrates the relative safety of intramyocardial injections of bone marrow–derived stem cells in humans with severe heart failure and the potential for improving myocardial blood flow with associated enhancement of regional and global left ventricular function.

Improved Exercise Capacity and Ischemia 6 and 12 Months After Transendocardial Injection of Autologous Bone Marrow Mononuclear Cells for Ischemic Cardiomyopathy

Background—We recently reported the safety and feasibility of autologous bone marrow mononuclear cell (ABMMNC) injection into areas of ischemic myocardium in patients with end-stage ischemic cardiomyopathy. The present study evaluated the safety and efficacy of this therapy at 6- and 12-month follow-up. Methods and Results—Twenty patients with 6- and 12-month follow-up (11 treated subjects; 9 controls) were enrolled in this prospective, nonrandomized, open-label study. Complete clinical and laboratory evaluations as well as exercise stress (ramp treadmill), 2-dimensional Doppler echocardiography, single-photon emission computed tomography (SPECT) perfusion scanning, and 24-hour Holter monitoring were performed at baseline and follow-up. Transendocardial delivery of ABMMNCs was performed with the aid of electromechanical mapping to identify viable myocardium. Each patient received 15 ABMMNC injections of 0.2 mL each. At 6 and 12 months, total reversible defect, as measured by SPECT perfusion scanning, was significantly reduced in the treatment group as compared with the control group. At 12 months, exercise capacity was significantly improved in the treatment group. This improvement correlated well with monocyte, B-cell, hematopoietic progenitor cell, and early hemapoietic progenitor cell phenotypes. Conclusions—The 6- and 12-month follow-up data in this study suggest that transendocardial injection of ABMMNCs in patients with end-stage ischemic heart disease may produce a durable therapeutic effect and improve myocardial perfusion and exercise capacity.

Characterization of a bovine collagen–hydroxyapatite composite scaffold for bone tissue engineering

Different biomaterials have been used as scaffolds for bone tissue engineering. Here we characterize a biomaterial composed of sintered (1100 degrees C) and powdered hydroxyapatite (HA) and type I collagen (Coll), both of bovine origin, designed for osteoconductive and osteoinductive scaffolds. Coll/HA proportions were 1/2.6 and 1/1 (wet weight), and particles sizes varied from 200 to 400 microm. Vv (volume density) and Sv (surface to volume density) for the HA particles in the composite ranged from 0.48 +/- 0.06 to 0.55 +/- 0.02 and 5.090 +/- 0.545 to 6.366 +/- 0.289 microm(-1), respectively. Due to the relatively small changes in Vv and Sv, a macroporosity could be characterized for the biocomposite. X-ray diffraction and infrared spectroscopy showed that the sintered bone was composed essentially of HA with minimum additional groups such as surface calcium hydroxide, surface and crystal water, free carbon dioxide and possibly brushite. Mass spectrometry detected carbonates at A and B sites of HA, and weakly bound to the structure. Human osteoblasts adhered and spread on both the HA particle surface and the collagen fibers, which seemed to guide cells between adjacent particles. The biocomposite studied has several characteristics considered as ideal for its use as a scaffold for osteoconduction and osteoinduction.

Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors

IntroductionPlatelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed.MethodsPRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure.ResultsOur method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only <0.3% from the initial red blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF.ConclusionsOur study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool from different donors with high reproducibility. These findings support the use of PRP in therapies aiming for tissue regeneration, and its content characterization will allow us to understand and improve the clinical outcomes.

Sponges (Porifera) model systems to study the shift from immortal to senescent somatic cells: the telomerase activity in somatic cells

Sponges (Porifera) represent the lowest metazoan phylum, characterized by a pronounced plasticity in the determination of cell lineages. In a first approach to elucidate the molecular mechanisms controlling the switch from the cell lineage with a putative indefinite growth capacity to senescent, somatic cells, the activity of the telomerase as an indicator for immortality has been determined. The studies were performed with the marine demosponges Suberites domuncula and Geodia cydonium. It was found that the activity for the telomerase in the tissue of both sponges is high; a quantitative analysis revealed that the extract from S. domuncula contained 10.3 TPG units per 5000 cell equivalents and the one from G. cydonium 8.3 TPG units; hence the activity reached approximately 30-20% of the activity seen in telomerase-positive reference cells. In contrast, dissociated spherulous cells from G. cydonium, after an incubation period of 24 h, contained no detectable telomerase activity. From earlier studies it is known that isolated sponge cells do not proliferate. Based on these findings it is assumed that the separation of the senescent sponge cell lineage from the immortal germ/somatic cell lineage is triggered by the loss of contact with cell adhesion factors. First evidence is included which suggests that the final progress of the senescent, telomerase-negative cells to cell death is caused by apoptosis.

Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice

We evaluated peripheral nerve regeneration using a tubular nerve guide of resorbable collagen filled with either bone marrow-derived cells (BMDCs) in Dulbeccos cell culture medium (DMEM) or with DMEM alone (control). The control group received just the culture medium (vehicle). The left sciatic nerves of ten isogenic mice were transected and the tubular nerve guides were sutured to the end of the proximal and distal nerve stumps. Motor function was tested at 2, 4 and 6 weeks after surgery using the walking track test. The pawprints were analyzed and the print lengths (PL) were measured to evaluate functional recovery. After 6 weeks, mice were anesthetized, perfused transcardially with fixative containing aldehydes, and the sciatic nerves and tubes were dissected and processed for scanning and transmission electron microscopy. Scanning electron microscopy of the collagen tube revealed that the tube wall became progressively thinner after surgery, proving that the tube can be resorbed in vivo. Quantitative analysis of the regenerating nerves showed that the number of myelinated fibers and the myelin area were significantly increased in the experimental group. Also, motor function recovery was faster in animals that received the cell grafts. These results indicate that the collagen tube filled with BMDCs provided an adequate and favorable environment for the growth and myelination of regenerating axons compared to the collagen tube alone.

Bone marrow subendosteal microenvironment harbours functionally distinct haemosupportive stromal cell populations

In adult animals, bone marrow is the major site of blood cell production, which is controlled by interactions between the local stroma and blood cell progenitors. The endosteal/subendosteal environment comprises bone-lining and adjacent reticular cells and sustains haemopoietic stem cell (HSC) self-renewal, proliferation and differentiation. We have questioned the specific role of each of these stroma cells in controlling HSC fate. We have isolated two distinct stroma-cell populations containing subendosteal reticulocytes (F-RET) and osteoblasts (F-OST) from periosteum-free fragments of murine femurs by a two-step collagenase-digestion procedure. Both populations produce similar extracellular matrix (collagen I, laminin, fibronectin, decorin), except for collagen IV, which is low in F-OST. They also express osteogenic markers: osteopontin, osteonectin, bone sialoprotein and alkaline phosphatase (ALP). The quantity and activity of ALP are however higher in F-OST. When co-cultured with bone marrow mononuclear cells or lineage-negative haemopoietic progenitors, F-OST stroma induces low proliferation and high maintenance of early haemopoietic progenitors, whereas F-RET stroma induces high short-term proliferation and differentiation. Analysis by reverse transcription/polymerase chain reaction has revealed higher levels of Jagged-1 expression by F-OST cells than by the F-RET population. Thus, two adjacent stroma cells (subendosteal and endosteal) play distinct roles in controlling the stem-cell capacity and fate of HSC and probably contribute distinctly to HSC niche formation.

Protein synthesis and secretion in human mesenchymal cells derived from bone marrow, adipose tissue and Wharton’s jelly

IntroductionDifferent mesenchymal stromal cells (MSC) have been successfully isolated and expanded in vitro and nowadays they are tested in clinical trials for a wide variety of diseases. Whether all MSC express the same cell surface markers or have a similar secretion profile is still controversial, making it difficult to decide which stromal cell may be better for a particular application.MethodsWe isolated human mesenchymal stromal cells from bone marrow (BM), adipose tissue (AT) and Wharton’s jelly (WJ) and cultured them in fetal bovine serum supplemented media. We evaluated proliferation, in vitro differentiation (osteogenic, adipogenic and chondrogenic potential), expression of cell surface markers and protein secretion using Luminex and ELISA assays.ResultsCell proliferation was higher for WJ-MSC, followed by AT-MSC. Differences in surface expression markers were observed only for CD54 and CD146. WJ-MSC secreted higher concentrations of chemokines, pro-inflammatory proteins and growth factors. AT-MSC showed a better pro-angiogenic profile and secreted higher amounts of extracellular matrix components and metalloproteinases.ConclusionsMesenchymal stromal cells purified from different tissues have different angiogenic, inflammatory and matrix remodeling potential properties. These abilities should be further characterized in order to choose the best protocols for their therapeutic use.

Transendocardial autologous bone marrow mononuclear cell injection in ischemic heart failure: postmortem anatomicopathologic and immunohistochemical findings.

Background—Cell-based therapies for treatment of ischemic heart disease are currently under investigation. We previously reported the results of a phase I trial of transendocardial injection of autologous bone marrow mononuclear (ABMM) cells in patients with end-stage ischemic heart disease. The current report focuses on postmortem cardiac findings from one of the treated patients, who died 11 months after cell therapy. Methods and Results—Anatomicopathologic, morphometric, and immunocytochemical findings from the anterolateral ventricular wall (with cell therapy) were compared with findings from the interventricular septum (normal perfusion and no cell therapy) and from the inferoposterior ventricular wall (extensive scar tissue and no cell therapy). No signs of adverse events were found in the cell-injected areas. Capillary density was significantly higher (P<0.001) in the anterolateral wall than in the previously infarcted tissue in the posterior wall. The prominent vasculature of the anterolateral wall was associated with hyperplasia of pericytes, mural cells, and adventitia. Some of these cells had acquired cytoskeletal elements and contractile proteins (troponin, sarcomeric &agr;-actinin, actinin), as well as the morphology of cardiomyocytes, and appeared to have migrated toward adjacent bundles of cardiomyocytes. Conclusions—Eleven months after treatment, morphological and immunocytochemical analysis of the sites of ABMM cell injection showed no abnormal cell growth or tissue lesions and suggested that an active process of angiogenesis was present in both the fibrotic cicatricial tissue and the adjacent cardiac muscle. Some of the pericytes had acquired the morphology of cardiomyocytes, suggesting long-term sequential regeneration of the cardiac vascular tree and muscle.

Osteoblasts and Bone Marrow Mesenchymal Stromal Cells Control Hematopoietic Stem Cell Migration and Proliferation in 3D In Vitro Model

Background Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow–derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs. Methodology/Principal Findings A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34+ cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34+ cells was decreased. Conclusions/Significance Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation.