Implantation Of Bone Marrow Stromal Cells Research Articles

Co-transplantation of adipose and bone marrow derived stromal cells for treatment of osteonecrosis of femoral head

Osteonecrosis of femoral head (ONFH) is irreversible disease which treatment is almost depending on total joint replacement. The synergistic effect by co-transplantation of adipose-derived stromal cells (ASCs) in enhancing the osteogenic differentiation of bone marrow stromal cells (BMSCs) proved in critical sized calvarial defect model of rat in our previous study. In this study, porcine ASCs and BMSCs were co-transplanted to ONFH model of minipig. In the results of X-rays and CT, defect of cells transplantation group was complete healed up with new bone tissue while control group had unfilled bone defect. Histological findings also corroborated the radiological findings. We observed very rapid and good quality osteogenesis in combined ASCs and BMSCs implantation group compared with control group.

Pre-clinical studies of bone regeneration with human bone marrow stromal cells and biphasic calcium phosphate.

IntroductionRepair of large bone defects remains a significant clinical challenge. Bone marrow stromal cells (BMSCs), a subset of which is known as bone marrow-derived mesenchymal stem cells, show therapeutic potential for bone regeneration. However, their isolation, expansion and implantation will need to be conducted under good manufacturing practices (GMP) at separate locations. An investigation which mimics this clinical scenario where large bone defects shall be regenerated is required before clinical trials can be initiated.MethodsSeven batches of 100 million human ex-vivo expanded BMSCs from five donors were transported fresh in syringes from a GMP facility in Germany to France. BMSCs were mixed with biphasic calcium phosphate (BCP) biomaterial prior to subcutaneous implantation in nude mice. The capacity of BMSCs in unison with BCP to regenerate critical sized cranial bone defects was also evaluated. BMSCs expressing luciferase were used to assess the viability and bio-distribution of implanted cells. In situ hybridization, using the human-specific repetitive Alu sequence, was performed for the identification of human cells in explants.ResultsEight weeks after implantation of BMSCs, mineralized bone containing mature bone marrow territories was formed in ectopic sites and in calvaria defects. Significant loss of cell viability was observed by bioluminescence imaging and only 1.5 percent of the initial number of transplanted cells remained after 37 days. After eight weeks, while explants were comprised primarily of host cells, there were also human cells attached along the periphery of BCP and embedded in osteocyte lacunae dispersed throughout the newly formed bone matrix.ConclusionsThis study demonstrates the safety and efficacy of BMSC/BCP combinations and provides crucial information for the implementation of BMSC therapy for bone regeneration.

Repair of articular cartilage defects with a novel injectablein situforming material in a canine model

We developed an ultra-purified in situ forming gel as an injectable delivery vehicle of bone marrow stromal cells (BMSCs). Our objective was to assess reparative tissues treated with autologous BMSCs implanted using the injectable implantation system into osteochondral defects in a canine model. Forty-eight osteochondral defects in the patella groove of the knee joint were created in 12 adult beagle dogs (two defects in each knee). The defects were divided into a defect group (n = 16), an acellular novel material implantation (material) group (n = 16), and a BMSCs implantation using the current vehicle system (material with BMSCs) group (n = 16). The reparative tissues at 16 weeks postoperatively were assessed through gross, histological, and mechanical analyses. The reparative tissues of the material with BMSCs group were substituted with firm and smooth hyaline-like cartilage tissue that was perfectly integrated into the host tissues. This treatment group obviously enhanced the subchondral bone reconstruction. The compressive modulus of the reparative tissues was significantly higher in the material with BMSCs group than the other groups. This study demonstrated that the implantation of BMSCs using our novel in situ forming material induced a mature hyaline-like cartilage repair of osteochondral defects in a canine model.

Clinical and histopathological observations of autologous bone marrow stromal cells implantation on the regeneration of sciatic nerve neurotmesis in rabbit

The peripheral nerve injuries (PNI) is due to stretching and laceration. Therefore, the epineural repair technique is currently accepted as a standard method for peripheral nerve repair. Thirty-two (32) rabbits were divided equally into perineural nerve sutures as control group (CG) and bone marrow stromal cells (BMSCs) as the treatment group (TG). The aim of this study was to investigate the effect of the autologous BMSCs implantation on the regeneration of sciatic nerve neurotmesis in rabbits. There was significant improvement (p<0.05) of the motor and sensory functions in the TG animals as compared to the CG. Histopathological examinations of the proximal nerve stump in the CG showed mild adherence surrounding an anastomosed area, few vacuolation and plenty of collagen at the peri and epineurium. The anastomosed site showed the degeneration, disorientation and giant cell surrounding the stitches, while intraneurial scar tissues were seen in the distal segment at the 112 th post operation day (POD). The histopathology of TG showed the activated Schwann cells, good myelination, minimum scar tissue, good orientation and remarkable angiogenesis at the 112 th POD. BMSCs are capable of improving the motor and sensory functions and significantly promote the regeneration of sciatic nerve neurotmesis in rabbits.

Fate of Bone Marrow Stromal Cells in a Syngenic Model of Bone Formation

Bone marrow stromal cells (BMSCs) have been demonstrated to induce bone formation when associated to osteoconductive biomaterials and implanted in vivo. Nevertheless, their role in bone reconstruction is not fully understood and rare studies have been conducted to follow their destiny after implantation in syngenic models. The aim of the present work was to use sensitive and quantitative methods to track donor and recipient cells after implantation of BMSCs in a syngenic model of ectopic bone formation. Using polymerase chain reaction (PCR) amplification of the Sex determining Region Y (Sry) gene and in situ hybridization of the Y chromosome in parallel to histological analysis, we have quantified within the implants the survival of the donor cells and the colonization by the recipient cells. The putative migration of the BMSCs in peripheral organs was also analyzed. We show here that grafted cells do not survive more than 3 weeks after implantation and might migrate in peripheral lymphoid organs. These cells are responsible for the attraction of host cells within the implants, leading to the centripetal colonization of the biomaterial by new bone.

Ectopic Bone Induction by BMP-loaded Collagen Scaffold and Bone Marrow Stromal Cell Sheet

19 INTRODUCTION Bone inductive therapies have been developed using tissue engineering with three essential factors, i.e., osteogenic cells, osteoinductive growth factor and a scaffold for bone-forming cells. Bone marrow stromal cells (BMSCs) include multipotent mesenchymal stem cells. BMSCs could differentiate into several cell-types including osteoblasts , and implantation of BMSCs with various biological scaffolds enables to induce a new bone in vivo. Grafting with calcium alginate to alveolar bone defects in dogs accelerated bone regeneration. Bruder et al. reported that the combined implantation with hydroxyapatite/β-tricalcium phosphate ceramics and bone marrow-derived mesenchymal stem cells facilitated bone formation: They insisted that a ceramic scaffold carrying cell source could be primarily biocompatible and be able to provide mechanical stiffness compared to the ceramics scaffold per se. A biological scaffold may play an Ectopic Bone Induction by BMP-loaded Collagen Scaffold and Bone Marrow Stromal Cell Sheet

A cellular implantation system using an injectable ultra‐purified alginate gel for repair of osteochondral defects in a rabbit model

We developed a novel cellular implantation system using an in situ forming ultra-purified alginate gel with quite low endotoxity. The aims of this study were to determine the superiority of chondrogenic potential of bone marrow stromal cells (BMSCs) cultured in the purified alginate gel compared with a commercial grade gel, and to assess reparative tissues treated with BMSCs implanted using the developed system into cartilage defects in rabbit knees. The effects of each alginate gel on cellular proliferation and chondrogenesis of rabbit BMSCs were determined by in vitro assessments. Using our purified alginate gel, a novel vehicle system for injecting BMSCs into osteochondral defects without periosteal patch was successfully established in this animal models. The in vitro analyses demonstrated that the purification of alginate significantly enhanced the cellular proliferation and chondrogenic differentiation of BMSCs. The in vivo assessments suggested that the implantation of BMSCs with the developed system using the purified alginate gel histologically and mechanically improved the reparative tissue of osteochondral defects. This system using the purified alginate gel shows the clinical potential for arthroscopically injectable implantation of BMSCs for the treatment of cartilaginous lesions.

Functional and Morphological Maturation of Implanted Neonatal Cardiomyocytes as a Comparator for Cell Therapy

Knowledge of the rate of development of immature cardiomyocytes after implantation into a host heart is important for studies using cell therapy. To assess this functionally, we have implanted rat neonatal cardiomyocytes (NCMs) in normal and infarcted rat heart and re-isolated them for functional assessment. Maturation of implanted bone marrow stromal cells (BMSCs) was compared under similar conditions. NCMs from green fluorescent protein (GFP) transgenic rats were implanted into adult normal or infarcted rat hearts and re-isolated after 1, 2, or 4 weeks by standard enzymatic digestion. BMSCs labeled with DiI and iron oxide were implanted into rats with myocardial infarction and cells re-isolated 1, 2, 5, 6, and 16 weeks later. GFP-labeled myocytes approaching the adult morphology were detected 2 weeks after implantation of NCMs, but were significantly shorter than adult host myocytes and had reduced contractility. By 4 weeks after implantation, re-isolated GFP-labeled myocytes were close to the adult phenotype in contractile characteristics, although still significantly shorter. Infarction of the host did not alter the rate of maturation of implanted cells. After implantation of BMSCs, small numbers of functional DiI-labeled myocytes were re-isolated from 4/11 animals but were more mature than expected from the NCM studies. This adds evidence that BMSC-derived cardiomyocytes were not a result of transdifferentiation. The maturation rate of implanted NCMs represents a benchmark against which to evaluate the likely rate of formation of fully functional cardiomyocytes from implanted cells.

Parathyroid hormone mediates bone growth through the regulation of osteoblast proliferation and differentiation

PTH (1–34) is the only FDA-approved anabolic agent for osteoporosis treatment in the U.S., but its mechanisms are not completely understood. This study investigated PTH effects on osteogenic cells at various stages of differentiation and proliferation using an engineered bone growth model in vivo. Ossicles were generated from bone marrow stromal cells (BMSCs) implanted in immunocompromised mice. Three weeks of PTH (40 µg/kg/day) or vehicle treatment initiated 1 day, 1, 2, or 3 weeks after BMSC implantation resulted in an anabolic response in PTH-treated implants (via histomorphometry and microCT) in all treatment groups. A novel in vivo tracking strategy with luciferase tagged BMSCs and weekly bioluminescent imaging of ossicles revealed increased donor cell proliferation in PTH-treated ossicles. The greatest increase occurred during the first week, and the activity remained elevated in PTH-treated implants over time. Zoledronic acid (ZA) was combined with PTH to delineate interactive mechanisms of these bone active agents. Combining ZA with PTH treatment reduced the PTH-mediated increase in luciferase BMSC activity, serum osteocalcin, and serum tartrate resistant acid phosphotase-5b (TRAP-5b) but ZA did not reduce the PTH-induced increase in total bone. Since zoledronic acid reduced PTH-induced proliferation without reducing bone volume, these data suggest that combining PTH and bisphosphonate therapy warrants further investigation in the treatment of bone disorders.

Abstract 1112: Re-isolation and Functional Characterization of Cardiomyocytes after Implantation of Bone Marrow Stromal Cells or Neonatal Cardiomyocytes in Rat Heart

Background: Replacement of cardiac muscle is a key goal of cell therapy, but uncertainty exists about whether cell implantation strategies actually generate true functioning cardiomyocytes. We therefore aimed to re-isolate implanted bone marrow stromal cells (BMSCs) to determine their functional characteristics ex vivo . Neonatal cardiomyocytes (NCMs), in which the myocyte phenotype was established before implantation, were used as a gold standard. Methods: NCMs from GFP-transgenic rats were injected into the hearts of nude rats (n=4), and re-isolated after 1, 2 or 4 weeks. These were compared with adult myocytes, and with myocytes from 2 and 4 week-old (2W-O and 4W-O) rats. labelled myocytes detected 2 weeks after implantation of DiI and iron-labelled BMSCs (2W-BI) into infarcted rat hearts (n=4) were studied. Results: GFP-labelled myocytes approaching the adult morphology (rod-shaped, with defined sarcomeres) were not detected until 2 weeks after implantation of NCMs (2W-I), and were significantly shorter than adult myocytes in cell length (52 ± 5μm, n=10 vs. 99 ± 9μm, n=9, p&lt;0.001), but the similar length to those from 2W-O (56 ± 3μm, n=15). Contraction amplitude (% shortening) was significantly lower and time to 50 % relaxation (R50) longer for the re-isolated cells compared with adult. By 4 weeks after implantation (4W-I), re-isolated myocytes were close to the adult phenotype, although cell length (64 ± 5μm, n=20, p&lt;0.01) showed immature characteristics. After implantation of BMSCs, DiI-labelled myocytes were detected in only 4/10 rats and then at low levels (0.4–21 per 10,000). Labelled myocytes, isolated 2 weeks after implantation of BMSCs, had similar length (101 ± 20, n=21), contraction amplitude and beat duration to adult myocytes. Conclusions: Implanted NCMs mature within the heart over a period of at least 4 weeks. The adult phenotype of labelled myocytes after BMSC implantation is inconsistent with a process of transdifferentiation and maturation.

Transplantation of bone marrow stromal cells for peripheral nerve repair

Cell transplantation using bone marrow stromal cells (BMSCs) to alleviate neurological deficits has recently become the focus of research in regenerative medicine. Evidence suggests that secretion of various growth-promoting substances likely plays an important role in functional recovery against neurological diseases. In an attempt to identify a possible mechanism underlying the regenerative potential of BMSCs, this study investigated the production and possible contribution of neurotrophic factors by transected sciatic nerve defect in a rat model with a 15 mm gap. Cultured BMSCs became morphologically homogeneous with fibroblast-like shape after ex vivo expansion. We provided several pieces of evidence for the beneficial effects of implanted fibroblast-like BMSCs on sciatic nerve regeneration. When compared to silicone tube control animals, this treatment led to (i) improved walking behavior as measured by footprint analysis, (ii) reduced loss of gastrocnemius muscle weight and EMG magnitude, and (iii) greater number of regenerating axons within the tube. Cultured fibroblast-like BMSCs constitutively expressed trophic factors and supporting substances, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), collagen, fibronectin, and laminin. The progression of the regenerative process after BMSC implantation was accompanied by elevated expression of neurotrophic factors at both early and later phases. These results taken together, in addition to documented Schwann cell-like differentiation, provide evidence indicating the strong association of neurotrophic factor production and the regenerative potential of implanted BMSCs.

Angiogenic potency of bone marrow stromal cells improved by ex vivo hypoxia prestimulation.

To study the angiogenic potency of hypoxia-prestimulated bone marrow stromal cells (BMSCs) when transplanted into acute myocardial infarction models of rats. BMSCs were cultured under hypoxia condition for 24 h. Their expression of VEGF was investigated. The rat acute myocardial infarction models were made by coronary artery ligation and divided into 3 groups at random. In normoxia group, twice-passaged BMSCs were labeled with Bromodeoxyuridine (BrdU) and then implanted into the infarction regions and ischemic border of the recipients in 4 weeks. The rats in hypoxia group were implanted with hypoxia-prestimulated BMSCs. In control group, the model rats received only DMEM medium injection. Six-weeks after AMI, the infarction regions were examined to identify the angiogenesis and the expression of the VEGF. Our results showed that viable cells labeled with BrdU could be identified in the host hearts. The infarction regions in normoxia and hypoxia groups had a greater capillary density and increased VEGF expression than the regions in control group. The capillary density and VEGF expression in hypoxia group were higher than in normoxia group. It is concluded that the enhanced expression of VEGF in BMSCs could be induced by ex vivo hypoxia stimulation. BMSCs implantation promoted the angiogenesis in myocardial infarction tissue via supplying exogenic VEGF. Angiogenic potency of bone marrow stromal cells was improved by ex vivo hypoxia prestimulation though the enhanced VEGF expression.

Bone marrow stromal cells improve cardiac performance in healed infarcted rat hearts.

Postinfarct congestive heart failure is one of the leading causes of morbidity and mortality in developed and developing countries. The main purpose of this study was to investigate whether transplantation of bone marrow stromal cells (BMSC) directly into the myocardium could improve the performance of healed infarcted rat hearts. Cell culture medium with or without BMSC was injected into borders of cardiac scar tissue 4 wk after experimental infarction. Cardiac performance was evaluated 2 wk after cellular (n = 10) or medium (n = 10) injection by electro- and echocardiography. Histological study was performed 3 wk after treatment. Electrocardiography of BMSC-treated infarcted rats showed electrical and mechanical parameters more similar to those in control than in medium-treated animals: a normal frontal QRS axis in 6 of 10 BMSC-treated and all control rats and a rightward deviation of the QRS axis in all 10 medium-treated animals. BMSC treatment, assessed by echocardiography, improved fractional shortening (39.00 +/- 4.03%) compared with medium-treated hearts (18.20 +/- 0.74%) and prevented additional changes in cardiac geometry. Immunofluorescence microscopy revealed colocalization of 4',6-diamidino-2-phenylindole-labeled nuclei of transplanted cells with cytoskeletal markers for cardiomyocytes and smooth muscle cells, indicating regeneration of damaged myocardium and angiogenesis. These data provide strong evidence that BMSC implantation can improve cardiac performance in healed infarctions and open new promising therapeutic opportunities for patients with postinfarction heart failure.

Bone marrow stromal cell implantation for peripheral nerve repair

Cell therapy using bone marrow stromal cells is a new promising therapy for regenerative medicine. Previous studies demonstrated that local bone marrow stromal cells implantation in the distal stump of transected sciatic nerve of rats promotes early functional recovery. The purpose of this study was to expand on the preliminary research by investigating the long-term efficacy of bone marrow stromal cells using the same experimental setting. Functional test and histological studies demonstrate that bone marrow stromal cell-treated rats exhibit significant improvement on a walking tract test at day 180 after surgery compared with control rats. Taken together, these data suggest that bone marrow stromal cell therapy is a safe and effective strategy for peripheral nerve injuries.