Mesenchymal progenitor cells from canine fetal tissues: yolk sac, liver, and bone marrow

During fetal development, mesenchymal progenitor (MP) cells are co-localized in major hematopoietic territories, such as yolk sac (YS), bone marrow (BM), liver (LV), and others. Studies using mouse and human MP cells isolated from fetus have shown that these cells are very similar but not identical to adult mesenchymal stem cells (MSC). Their differentiation potential is usually restricted to production of highly committed osteogenic and chondrogenic precursors. Such properties of fetal MP cells can be very useful for tissue regeneration, when a great number of committed precursors are required. The objectives of this study were to isolate and characterize MP cells from canine YS, BM, and LV in early and late stages of fetal development. Gestational stage was identified, and cell culture conditions were evaluated for efficient isolation of canine MP cells. All canine fetal MP cells expressed vimentin, nestin, and CD44 proteins. Cytokeratin 18 expression was observed in BM-and LV-MP cells, and vascular endothelial (VE)-cadherin expression was observed only in YS-MP cells. A small number of MP cells (5%) from LV and YS expressed Oct3/4 protein. The differentiation potential of canine fetal MP cells varied significantly: YS- and BM-MP cells differentiated into bone and cartilage, whereas LV-MP cells differentiation was limited to osteogenic fate. None of the canine fetal MP cells were able to differentiate into adipose cells. Our data suggest that canine fetal MP cells are an appropriate in vitro model to study MP biology from hematopoietic territories and they are a source of committed osteogenic and chondrogenic precursors for regenerative medicine.

New Source of Muscle-Derived Stem Cells with Potential for Alveolar Bone Reconstruction in Cleft Lip and/or Palate Patients

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in non-immunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.

The effect of oestrogen and alendronate therapies on postmenopausal bone loss around osseointegrated titanium implants

Objectives This study evaluated the influence of oestrogen deficiency and its therapies on bone tissue around osseointegrated implants. Methods Implants were placed in 66 female rats tibiae. The animals were assigned into five groups: control (CTL), sham, ovariectomy (OVX), oestrogen (EST), and alendronate (ALE). While CTL was sacrificed 60 days after implant placement, other groups were subjected to ovariectomy or sham surgery according to group and euthanized after 90 days. Blood and urine samples were collected at sacrifice day for osteocalcin (OCN) and deoxypyridinoline (DPD) quantification. Densitometry of femur and lumbar vertebrae was performed in order to evaluate rats` skeletal impairment. Non-decalcified sections were referred to fluorescent and light microscopy for analyses of mineral apposition rate (MAR), eroded and osteoclastic surfaces, bone-to-implant contact (BIC), and bone area fraction occupancy (BAFO). Results Results from the OVX group showed significantly lower bone mineral density (BMD), BIC, BAFO, and MAR, while OCN, deoxipiridinoline, eroded surface and ostecoclastic surface were increased compared with the other groups of the study. ALE reduced OCN and DPD concentrations, MAR, osteoclastic and eroded surfaces, and no difference was in BIC and BAFO relative to SHAM. EST and CTL showed similar results to SHAM for measurements. Conclusions Oestrogen deficiency exerted a negative influence on bone tissue around implants, while oestrogen replacement therapy and alendronate were effective against its effects. Although alendronate therapy maintained the quantity of bone around implants, studies evaluating bone turnover kinetics are warranted. To cite this article:Giro G, Coelho PG, Pereira RMR, Jorgetti V, Marcantonio E Jr, Orrico SRP. The effect of oestrogen and alendronate therapies on postmenopausal bone loss around osseointegrated titanium implants.Clin. Oral Impl. Res. 22, 2011; 259-264.doi: 10.1111/j.1600-0501.2010.01989.x.

Evaluation of Maxillary Alveolar Reconstruction Using a Resorbable Collagen Sponge with Recombinant Human Bone Morphogenetic Protein-2 in Cleft Lip and Palate Patients

Introduction: A resorbable collagen matrix with recombinant human bone morphogenetic protein (rhBMP-2) was compared with traditional iliac crest bone graft for the closure of alveolar defects during secondary dental eruption. Methods: Sixteen patients with unilateral cleft lip and palate, aged 8 to 12 years, were selected and randomly assigned to group 1 (rhBMP-2) or group 2 (iliac crest bone graft). Computed tomography was performed to assess both groups preoperatively and at months 6 and 12 postoperatively. Bone height and defect volume were calculated through Osirix Dicom Viewer (Pixmeo, Apple Inc.). Overall morbidity was recorded. Results: Preoperative and follow-up examinations revealed progressive alveolar bone union in all patients. For group 1, final completion of the defect with a 65.0% mean bone height was detected 12 months postoperatively. For group 2, final completion of the defect with an 83.8% mean bone height was detected 6 months postoperatively. Dental eruption routinely occurred in both groups. Clinical complications included significant swelling in three group 1 patients (37.5%) and significant donor-site pain in seven group 2 patients (87.5%). Conclusions: For this select group of patients with immature skeleton, rhBMP-2 therapy resulted in satisfactory bone healing and reduced morbidity compared with traditional iliac crest bone grafting.

Refractory Angina Cell Therapy (ReACT) Involving Autologous Bone Marrow Cells in Patients Without Left Ventricular Dysfunction: A Possible Role for Monocytes

Autologous bone marrow mononuclear cell (BMMC) transplantation has emerged as a potential therapeutic option for refractory angina patients. Previous studies have shown conflicting myocardium reperfusion results. The present study evaluated safety and efficacy of CellPraxis Refractory Angina Cell Therapy Protocol (ReACT). in which a specific BMMC formulation was administered as the sole therapy for these patients. The phase I/IIa noncontrolled, open label. clinical trial, involved eight patients with refractory angina and viable ischemic myocardium, without left ventricular dysfunction and who were not suitable for conventional myocardial revascularization. ReACT is a surgical procedure involving a single series of multiple injections (40-90 injections, 0.2 ml each) into ischemic areas of the left ventricle. Primary endpoints were Canadian Cardiovascular Society Angina Classification (CCSAC) improvement at 18 months follow-up and myocardium ischemic area reduction (assessed by scintigraphic analysis) at 12 months follow-up, in correlation with a specific BMMC formulation. Almost all patients presented progressive improvement in angina classification beginning 3 months (p = 0.008) postprocedure which was sustained at 18 months follow-up (p = 0.004), as well as objective myocardium ischemic area reduction at 12 months (decrease of 84.4%, p < 0.004). A positive correlation was found between monocyte concentration and CCSAC improvement (r = -0.759, p < 0.05). Improvement in CCSAC, followed by correlated reduction in scintigraphic myocardium ischemic area, strongly suggests neoangiogenesis as the main stem cell action mechanism. The significant correlation between number of monocytes and improvement strongly supports a cell-related effect of ReACT. ReACT appeared safe and effective.

Bone Marrow Mononuclear Cell Therapy Led to Alveolar-Capillary Membrane Repair, Improving Lung Mechanics in Endotoxin-Induced Acute Lung Injury

The aim of this study was to test the hypothesis that bone marrow mononuclear cell (BMDMC) therapy led an improvement in lung mechanics and histology in endotoxin-induced lung injury. Twenty-four C57BL/6 mice were randomly divided into four groups (n = 6 each). In the acute lung injur;y (ALI) group, Escherichia coli lipopolysaccharide (LPS) was instilled intratracheally (40 mu g, IT), and control (C) mice received saline (0.05 ml, IT). One hour after the administration of saline or LPS, BMDMC (2 x 10(7) cells) was intravenously injected. At day 28, animals were anesthetized and lung mechanics [static elastance (E(st)), resistive (Delta P(1)), and viscoelastic (Delta P(2)) pressures] and histology (light and electron microscopy) were analyzed. Immunogold electron microscopy was used to evaluate if multinucleate cells were type II epithelial cells. BMDMC therapy prevented endotoxin-induced lung inflammation, alveolar collapse, and interstitial edema. In addition, BMDMC administration led to epithelial and endothelial repair with multinucleated type II pneumocytes. These histological changes yielded a reduction in lung E(st), Delta P(1), and Delta P(2) compared to ALI. In the present experimental ALI model, the administration of BMDMC yielded a reduction in the inflammatory process and a repair of epithelium and endothelium, reducing the amount of alveolar collapse, thus leading to an improvement in lung mechanics.

Understanding the impact of divalent cation substitution on hydroxyapatite: An in vitro multiparametric study on biocompatibility

Hydroxyapatite (HA), a stable and biocompatible material for bone tissue therapy, may present a variable stoichiometry and accept a large number of cationic substitutions. Such substitutions may modify the chemical activity of HA surface, with possible impact on biocompatibility. In this work, we assessed the effects of calcium substitution with diverse divalent cations (Pb(2+), Sr(2+), Co(2+), Zn(2+), Fe(2+), Cu(2+), or Mg(2+)) on the biological behavior of HA. Physicochemical analyses revealed that apatite characteristics related to crystallinity and calcium dissolution/uptake rates are very sensitive to the nature of cationic substitution. Cytocompatibility was evaluated by mitochondrial activity, membrane integrity, cell density, proapoptotic potential, and adhesion tests. With the exception of Zn-HA, all the substituted HAs induced some level of apoptosis. The highest apoptosis levels were observed for Mg-HA and Co-HA. Cu-HA was the only material to impair simultaneously mitochondrial activity, membrane integrity, and cell density. The highest relative cell densities after exposure to the modified HAs were observed for Mg-HA and Zn-HA, while Co-HA significantly improved cell adhesion onto HA surface. These results show that changes on surface dissolution caused by cationic substitution, as well as the increase of metal species released to biological media, were the main responsible factors related to alterations on HA biocompatibility. (C) 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 98A: 351-358, 2011.

Mesenchymal Stem Cells and Bovine Bone Mineral in Sinus Lift Procedures-An Experimental Study in Sheep

Introduction: New reconstructive and less invasive methods have been searched to optimize bone formation and osseointegration of dental implants in maxillary sinus augmentation. Purpose: The aim of the presented ovine split-mouth study was to compare bovine bone mineral (BBM) alone and in combination with mesenchymal stem cells (MSCs) regarding their potential in sinus augmentation. Material and Methods: Bilateral sinus floor augmentations were performed in six adult sheep. BBM and MSCs were placed into the test side and only BBM in the contra-lateral control side of each sheep. Animals were sacrificed after 8 and 16 weeks. Augmentation sites were analyzed by computed tomography, histology, and histomorphometry. Results: The initial volumes of both sides were similar and did not change significantly with time. A tight connection between the particles of BBM and the new bone was observed histologically. Bone formation was significantly (p = 0.027) faster by 49% in the test sides. Conclusion: The combination of BBM and MSCs accelerated new bone formation in this model of maxillary sinus augmentation. This could allow early placement of implants.

In Vivo Comparison of Hard Tissue Regeneration with Human Mesenchymal Stem Cells Processed with Either the FICOLL Method or the BMAC Method

Objective: To compare new bone formation in maxillary sinus augmentation procedures using biomaterial associated with mesenchymal stem cells (MSCs) separated by two different isolation methods. Background: In regenerative medicine open cell concentration systems are only allowed for clinical application under good manufacturing practice conditions. Methods: Mononuclear cells, including MSCs, were concentrated with either the synthetic poylsaccharid (FICOLL) method (classic open system-control group, n = 6 sinus) or the bone marrow aspirate concentrate (BMAC) method (closed system-test group, n = 12 sinus) and transplanted in combination with biomaterial. A sample of the cells was characterized by their ability to differentiate. After 4.1 months (SD +/- 1.0) bone biopsies were obtained and analyzed. Results: The new bone formation in the BMAC group was 19.9% (90% confidence interval [CI], 10.9-29), and in the FICOLL group was 15.5% (90% CI, 8.6-22.4). The 4.4% difference was not significant (90% CI, -4.6-13.5; p = 0.39). MSCs could be differentiated into osteogenic, chondrogenic, and adipogenic lineages. Conclusion: MSCs harvested from bone marrow aspirate in combination with bovine bone matrix particles can form lamellar bone and provide a reliable base for dental implants. The closed BMAC system is suited to substitute the open FICOLL system in bone regeneration procedures.

Calcium Hydroxide Promotes Cementogenesis and Induces Cementoblastic Differentiation of Mesenchymal Periodontal Ligament Cells in a CEMP1-and ERK-Dependent Manner

Periodontal tissue engineering is a complex process requiring the regeneration of bone, cementum, and periodontal ligament (PDL). Since cementum regeneration is poorly understood, we used a dog model of dental pulpal necrosis and in vitro cellular wounding and mineralization assays to determine the mechanism of action of calcium hydroxide, Ca(OH)(2), in cementogenesis. Laser capture microdissection (LCM) followed by qRT-PCR were used to assay responses of periapical tissues to Ca(OH)(2) treatment. Additionally, viability, proliferation, migration, and mineralization responses of human mesenchymal PDL cells to Ca(OH)(2) were assayed. Finally, biochemical inhibitors and siRNA were used to investigate Ca(OH)(2)-mediated signaling in PDL cell differentiation. In vivo, Ca(OH)(2)-treated teeth formed a neocementum in a STRO-1- and cementum protein-1 (CEMP1)-positive cellular environment. LCM-harvested tissues adjacent to the neocementum exhibited higher mRNA levels for CEMP1, integrin-binding sialoprotein, and Runx2 than central PDL cells. In vitro, Ca(OH)(2) and CEMP1 promoted STRO-1-positive cell proliferation, migration, and wound closure. Ca(OH)(2) stimulated expression of the cementum-specific proteins CEMP1 and PTPLA/CAP in an ERK-dependent manner. Lastly, Ca(OH)(2) stimulated mineralization by CEMP1-positive cells. Blocking CEMP1 and ERK function abolished Ca(OH)(2)-induced mineralization, confirming a role for CEMP1 and ERK in the process. Ca(OH)(2) promotes cementogenesis and recruits STRO-1-positive mesenchymal PDL cells to undergo cementoblastic differentiation and mineralization via a CEMP1- and ERK-dependent pathway.

Lineage-Negative Bone Marrow Cells Protect Against Chronic Renal Failure

Progressive renal failure continues to be a challenge. The use of bone marrow cells represents a means of meeting that challenge. We used lineage-negative (Lin(-)) cells to test the hypothesis that Lin(-) cell treatment decreases renal injury. Syngeneic Fischer 344 rats were divided into four groups: sham ( laparotomy only, untreated); Nx (five-sixth nephrectomy and untreated); NxLC1 (five-sixth nephrectomy and receiving 2 x 10(6) Lin(-) cells on postnephrectomy day 15); and NxLC3 (five-sixth nephrectomy and receiving 2 x 10(6) Lin(-) cells on postnephrectomy days 15, 30, and 45). On postoperative day 16, renal mRNA expression of interleukin (IL)-1 beta, tumor necrosis factor-alpha, and IL-6 was lower in NxLC rats than in Nx rats. On postnephrectomy day 60, NxLC rats presented less proteinuria, glomerulosclerosis, anemia, renal infiltration of immune cells, and protein expression of monocyte chemoattractant protein-1, as well as decreased interstitial area. Immunostaining for proliferating cell nuclear antigen showed that, in comparison with sham rats, Nx rats presented greater cell proliferation, whereas NxLC1 rats and NxLC3 rats presented less cell proliferation than did Nx rats. Protein expression of the cyclin-dependent kinase inhibitor p21 and of vascular endothelial growth factor increased after nephrectomy and decreased after Lin(-) cell treatment. On postnephrectomy day 120, renal function (inulin clearance) was significantly better in Lin(-) cell-treated rats than in untreated rats. Lin(-) cell treatment significantly improved survival. These data suggest that Lin(-) cell treatment protects against chronic renal failure. STEM CELLS 2009; 27: 682-692

Comparison of the Effects of Electrical Field Stimulation and Low-Level Laser Therapy on Bone Loss in Spinal Cord-Injured Rats

Objective: This study investigated the effects of low-level laser therapy (LLLT) and electrical stimulation (ES) on bone loss in spinal cord-injured rats. Materials and Methods: Thirty-seven male Wistar rats were divided into four groups: standard control group (CG); spinal cord-injured control (SC); spinal cord-injured treated with laser (SCL; GaAlAs, 830 nm, CW, 30mW/cm, 250 J/cm(2)); and spinal cord-injured treated with electrical field stimulation (SCE; 1.5 MHz, 1: 4 duty cycles, 30 mW, 20 min). Biomechanical, densitometric, and morphometric analyses were performed. Results: SC rats showed a significant decrease in bone mass, biomechanical properties, and morphometric parameters (versus CG). SCE rats showed significantly higher values of inner diameter and internal and external areas of tibia diaphyses; and the SCL group showed a trend toward the same result (versus SC). No increase was found in either mechanical or densitometric parameters. Conclusion: We conclude that the mentioned treatments were able to initiate a positive bone-tissue response, maybe through stimulation of osteoblasts, which was able to determine the observed morphometric modifications. However, the evoked tissue response could not determine either biomechanical or densitometric modifications.

Survival and Neuronal Differentiation of Mesenchymal Stem Cells Transplanted into the Rodent Brain Are Dependent upon Microenvironment

Introduction: The successful integration of stem cells in adult brain has become a central issue in modern neuroscience. In this study we sought to test the hypothesis that survival and neurodifferentiation of mesenchymal stem cells (MSCs) may be dependent upon microenvironmental conditions according to the site of implant in the brain. Methods: MSCs were isolated from adult rats and labeled with enhanced-green fluorescent protein (eGFP) lentivirus. A cell suspension was implanted stereotactically into the brain of 50 young rats, into one neurogenic area (hippocampus), and into another nonneurogenic area (striatum). Animals were sacrificed 6 or 12 weeks after surgery, and brains were stained for mature neuronal markers. Cells coexpressing NeuN (neuronal specific nuclear protein) and GFP (green fluorescent protein) were counted stereologically at both targets. Results: The isolated cell population was able to generate neurons positive for microtubule-associated protein 2 (MAP2), neuronal-specific nuclear protein (NeuN), and neurofilament 200 (NF200) in vitro. Electrophysiology confirmed expression of voltage-gated ionic channels. Once implanted into the hippocampus, cells survived for up to 12 weeks, migrated away from the graft, and gave rise to mature neurons able to synthesize neurotransmitters. By contrast, massive cell degeneration was seen in the striatum, with no significant migration. Induction of neuronal differentiation with increased cyclic adenosine monophosphate in the culture medium before implantation favored differentiation in vivo. Conclusions: Our data demonstrated that survival and differentiation of MSCs is strongly dependent upon a permissive microenvironment. Identification of the pro-neurogenic factors present in the hippocampus could subsequently allow for the integration of stem cells into nonpermissive areas of the central nervous system.

Increased Levels of NOTCH1, NF-kappa B, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

As previously shown, higher levels of NOTCH1 and increased NF-kappa B signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow ( BM). Differences between BM and UCB cell composition also account for this finding. The CD133 marker defines a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purified human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a significantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells ( CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a significantly higher frequency ( than expected by chance) of NF-kappa B-binding sites (BS), including potentially novel NF-kappa B targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all significantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-kappa B, and other important TFs on more primitive HSC sets.

Mesenchymal Stem Cells Derived From Canine Umbilical Cord Vein-A Novel Source for Cell Therapy Studies

The canine model provides a large animal system to evaluate many treatment modalities using stem cells (SCs). However, only bone marrow ( BM) protocols have been widely used in dogs for preclinical approaches. BM donation consists of an invasive procedure and the number and differentiation potential of its mesenchymal stem cells (MSCs) decline with age. More recently, umbilical cord was introduced as an alternative source to BM since it is obtained from a sample that is routinely discarded. Here, we describe the isolation of MSCs from canine umbilical cord vein (cUCV). These cells can be obtained from every cord received and grow successfully in culture. Their multipotent plasticity was demonstrated by their capacity to differentiate in adipocytic, chondrocytic, and osteocytic lineages. Furthermore, our results open possibilities to use cUCV cells in preclinical trials for many well-characterized canine model conditions homologs to human diseases.