Autologous Bone Marrow Stromal Cells Research Articles

In Vitro Osteogenic Induction of Bone Marrow Stromal Cells with Encapsulated Gene-Modified Bone Marrow Stromal Cells and In Vivo Implantation for Orbital Bone Repair

Osteogenic induction with either growth factors or genetic modification has limitations due to the short half-life and cost of the former, or safety concerns regarding the latter. The objective of this study was to employ a microcapsulation technique to separate genetically modified and nonmodified bone marrow stromal cells (BMSCs) to establish a cost-effective and biosafe osteogenic induction methodology with functional evaluation in vitro and in vivo in a canine model. Autologous BMSCs were isolated and transduced with adenoviral vectors containing either BMP-2 or vascular endothelial growth factor (VEGF) or were dual transduced followed by encapsulation in alginate microcapsules using an electrostatic bead generator. After cocultured with encapsulated cells, normal autologous BMSCs were analyzed for osteogenic differentiation and seeded onto tricalcium phosphate (TCP) scaffolds for in vivo implantation to repair orbital wall bone defects (12 mm in diameter) in a canine model. In vitro assays showed that the expression of the transduced genes was significantly upregulated, with significantly more transduced proteins released from the transduced cells compared with control cells. Importantly, examination of the BMSCs induced by soluble factors released from the encapsulated cells revealed a significant upregulation of expression of osteogenic markers Runx2, BSP, OPN, and OCN in dual-transduction or induction groups. In addition, dual transduction and induction resulted in the highest increase of alkaline phosphatase activity and mineralization compared with other experimental groups. In vivo assays using CT, micro-CT, and histology further supported the qPCR and western blot findings. In conclusion, encapsulation of genetically modified BMSCs was able to release a sufficient amount of BMP-2 and VEGF, which effectively induced osteogenic differentiation of normal-cultured BMSCs and demonstrated bone repair of the orbital wall defect after implantation with β-TCP in vivo.

Regeneration of human-ear-shaped cartilage by co-culturing human microtia chondrocytes with BMSCs

Previously, we had addressed the issues of shape control/maintenance of in vitro engineered human-ear-shaped cartilage. Thus, lack of applicable cell source had become a major concern that blocks clinical translation of this technology. Autologous microtia chondrocytes (MCs) and bone marrow stromal cells (BMSCs) were both promising chondrogenic cells that did not involve obvious donor site morbidity. However, limited cell availability of MCs and ectopic ossification of chondrogenically induced BMSCs in subcutaneous environment greatly restricted their applications in external ear reconstruction. The current study demonstrated that MCs possessed strong proliferation ability but accompanied with rapid loss of chondrogenic ability during passage, indicating a poor feasibility to engineer the entire ear using expanded MCs. Fortunately, the co-transplantation results of MCs and BMSCs (25% MCs and 75% BMSCs) demonstrated a strong chondroinductive ability of MCs to promote stable ectopic chondrogenesis of BMSCs in subcutaneous environment. Moreover, cell labeling demonstrated that BMSCs could transform into chondrocyte-like cells under the chondrogenic niche provided by co-cultured MCs. Most importantly, a human-ear-shaped cartilaginous tissue with delicate structure and proper elasticity was successfully constructed by seeding the mixed cells (MCs and BMSCs) into the pre-shaped biodegradable ear-scaffold followed by 12 weeks of subcutaneous implantation in nude mouse. These results may provide a promising strategy to construct stable ectopic cartilage with MCs and stem cells (BMSCs) for autologous external ear reconstruction.

The effect of bone allografts combined with bone marrow stromal cells on the healing of segmental bone defects in a sheep model

BackgroundThe repair of large bone defects is a major orthopedic challenge because autologous bone grafts are not available in large amounts and because harvesting is often associated with donor-site morbidity. Considering that bone marrow stromal cells (BMSC) are responsible for the maintenance of bone turnover throughout life, we investigated bone repair at a site of a critically sized segmental defect in sheep tibia treated with BMSCs loaded onto allografts. The defect was created in the mid-portion of the tibial diaphysis of eight adult sheep, and the sheep were treated with ex-vivo expanded autologous BMSCs isolated from marrow aspirates and loaded onto cortical allografts (n = 4). The treated sheep were compared with control sheep that had been treated with cell-free allografts (n = 4) obtained from donors of the same breed as the receptor sheep.ResultsThe healing response was monitored by radiographs monthly and by computed tomography and histology at six, ten, fourteen, and eighteen weeks after surgery. For the cell-loaded allografts, union was established more rapidly at the interface between the host bone and the allograft, and the healing process was more conspicuous. Remodeling of the allograft was complete at 18 weeks in the cell-treated animals. Histologically, the marrow cavity was reestablished, with intertrabecular spaces being filled with adipose marrow and with evidence of focal hematopoiesis.ConclusionsAllografts cellularized with AOCs (allografts of osteoprogenitor cells) can generate great clinical outcomes to noncellularized allografts to consolidate, reshape, structurally and morphologically reconstruct bone and bone marrow in a relatively short period of time. These features make this strategy very attractive for clinical use in orthopedic bioengineering.

Dual Delivery of rhPDGF-BB and Bone Marrow Mesenchymal Stromal Cells Expressing the BMP2 Gene Enhance Bone Formation in a Critical-Sized Defect Model

Bone tissue healing is a dynamic, orchestrated process that relies on multiple growth factors and cell types. Platelet-derived growth factor-BB (PDGF-BB) is released from platelets at wound sites and induces cellular migration and proliferation necessary for bone regeneration in the early healing process. Bone morphogenetic protein-2 (BMP-2), the most potent osteogenic differentiation inducer, directs new bone formation at the sites of bone defects. This study evaluated a combinatorial treatment protocol of PDGF-BB and BMP-2 on bone healing in a critical-sized defect model. To mimic the bone tissue healing process, a dual delivery approach was designed to deliver the rhPDGF-BB protein transiently during the early healing phase, whereas BMP-2 was supplied by rat bone marrow stromal cells (BMSCs) transfected with an adenoviral vector containing the BMP2 gene (AdBMP2) for prolonged release throughout the healing process. In in vitro experiments, the dual delivery of rhPDGF-BB and BMP2 significantly enhanced cell proliferation. However, the osteogenic differentiation of BMSCs was significantly suppressed even though the amount of BMP-2 secreted by the AdBMP2-transfected BMSCs was not significantly affected by the rhPDGF-BB treatment. In addition, dual delivery inhibited the mRNA expression of BMP receptor type II and Noggin in BMSCs. In in vivo experiments, critical-sized calvarial defects in rats showed enhanced bone regeneration by dual delivery of autologous AdBMP2-transfected BMSCs and rhPDGF-BB in both the amount of new bone formed and the bone mineral density. These enhancements in bone regeneration were greater than those observed in the group treated with AdBMP2-transfected BMSCs alone. In conclusion, the dual delivery of rhPDGF-BB and AdBMP2-transfected BMSCs improved the quality of the regenerated bone, possibly due to the modulation of PDGF-BB on BMP-2-induced osteogenesis.

Perilesional intrathecal administration of autologous bone marrow stromal cells achieves functional improvement in pigs with chronic paraplegia

Background aimsAt present, on the basis of the great number of preclinical studies and preliminary clinical trials in humans, bone marrow stromal cell (BMSC) transplantation offers promise in the treatment of paraplegia. Nevertheless, there is not enough experience in humans about the best candidates for this type of cell therapy or details about the best parameters or best route of administration. MethodsTwo adult paraplegic pigs with chronic paraplegia were treated only with perilesional intrathecal administration of 40 × 106 autologous BMSC suspended in autologous plasma and followed for 1 year after cell transplantation. ResultsOur study showed clinical improvement, starting at 2 mo after BMSC administration and reaching stabilization at 10 mo. This was associated with recovery of previously abolished somatosensory-evoked potentials. At the end of the study, histological images suggested spinal cord regeneration. ConclusionsOur present findings suggest the possible utility of perilesional intrathecal administration of autologous BMSC in patients with chronic paraplegia.

Comparative study of the osteogenic ability of four different ceramic constructs in an ectopic large animal model

Tissue-engineered constructs combining bone marrow mesenchymal stem cells with biodegradable osteoconductive scaffolds are very promising for repairing large segmental bone defects. Synchronizing and controlling the balance between scaffold-material resorption and new bone tissue formation are crucial aspects for the success of bone tissue engineering. The purpose of the present study was to determine, and compare, the osteogenic potential of ceramic scaffolds with different resorbability. Four clinically relevant granular biomaterial scaffolds (specifically, Porites coral, Acropora coral, beta-tricalcium phosphate and banked bone) with or without autologous bone marrow stromal cells were implanted in the ectopic, subcutaneous-pouch sheep model. Scaffold material resorption and new bone formation were assessed eight weeks after implantation. New bone formation was only detected when the biomaterial constructs tested contained MSCs. New bone formation was higher in the Porites coral and Acropora coral than in either the beta-tricalcium phosphate or the banked bone constructs; furthermore, there was a direct correlation between scaffold resorption and bone formation. The results of the present study provide evidence that, among the biomaterials tested, coral scaffolds containing MSCs promoted the best new bone formation in the present study.

Orbital wall repair in canines with beta‐tricalcium phosphate and induced bone marrow stromal cells

Bone tissue engineering is a new approach for the repair of orbital defects. The aim of the present study was to evaluate prefabricated beta-tricalcium phosphate (β-TCP) combined with autologous bone marrow stromal cells (BMSCs) to repair orbital wall defect in canine models. Defects measuring 10 mm in diameter were created in the orbital medial walls of 12 dogs. The orbits were randomly divided into five groups: group 1, repaired with osteogenesis-induced BMSCs/TCP constructs; group 2, repaired with noninduced BMSCs/TCP constructs; group 3, repaired with β-TCP scaffolds only; group 4, normal group; group 5, negative control (bone defect without treatment). Computed tomography (CT) scanning, gross observation, bone density measurements, micro-CT, and histological observations were performed. In group 1, new bone was observed with only a small amount of residual material, and bony union was achieved 3 months after surgery. In contrast, the constructs showed slow degradation with minimal bone formation in groups 2 and 3. Furthermore, the appearance and bone density of the constructs in group 1 were similar to that of normal bone: the constructs were covered with complete mucosa, and new alveolate plate grew into the ethmoidal sinuses. A large bone defect remained in group 5. This study demonstrated that biologic scaffolds composed of β-TCP and osteogenesis-induced BMSCs have been successfully used to restore bone functionality in animal models, which may provide a potential clinical approach for orbital wall repair and bone regeneration in humans.

Abstract 3305: Sustained production of a soluble IGF-I receptor by gutless adenovirus - transduced host cells protects from tumor growth in the liver .

Abstract The IGF-I receptor (IGF-IR) plays an important role in malignant disease by increasing cancer cell survival and promoting metastasis. It is presently the target of several drugs in clinical trials. Gene therapy is currently being developed for cancer treatment using different nanoparticle vectors designed to deliver therapeutic genes that could induce cell death, increase sensitivity to chemotherapy, block angiogenesis or enhance the anti-cancer immunological response. Previously, we have shown that the implantation of autologous bone-marrow stromal cells producing a soluble form of IGF-IR (sIGFIR) inhibited experimental liver metastasis of several tumor types in mice. Here we evaluated the utility of adenovirus-based gene delivery for generating therapeutically effective plasma levels of this decoy. A third generation gutless adenovirus was selected to deliver the sIGFIR gene because of its reduced immunogenicity and preferential tropism for the liver, the main source of endocrine IGF-1. We found that HEK-293 cells transduced by this, but not by control adenoviruses, secreted soluble receptor protein that blocked IGF-I-induced tumor cell migration, proliferation and survival in vitro. Following virus injection in vivo, viral DNA was detectable by PCR in several host organs, particularly the liver, and this resulted in the production of measurable sIGFIR plasma levels for up to 21 days post injection. In mice producing virus-encoded sIGFIR, experimental liver metastasis was inhibited, indicating that sIGFIR levels were therapeutically effective. The results show that adenovirus-based delivery of inhibitory soluble proteins can provide an effective strategy for blockade of cancer cell growth. Citation Format: Ni Wang, Yifan Lu, Amelie Pilotte, Maxime Pinard, Rénald Gilbert, Bernard Massie, Pnina Brodt. Sustained production of a soluble IGF-I receptor by gutless adenovirus - transduced host cells protects from tumor growth in the liver . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3305. doi:10.1158/1538-7445.AM2013-3305

Sustained production of a soluble IGF-I receptor by gutless adenovirus-transduced host cells protects from tumor growth in the liver

The IGF-I receptor (IGF-IR) has an important role in malignant disease and is the target of several drugs presently in clinical trials. Gene therapy has been explored as cancer treatment, mainly for delivery of genes that induce cell death or enhance the immunological response to cancer. Previously, we have shown that the implantation of autologous bone-marrow stromal cells producing a soluble form of IGF-IR (sIGFIR) inhibited experimental liver metastasis of several tumor types in mice. Here, we evaluated the utility of adenovirus-based gene delivery for generating therapeutically effective plasma levels of this decoy. We constructed a third generation gutless adenovirus expressing sIGFIR and found that HEK-293 cells transduced by this, but not control adenoviruses, secreted soluble receptor protein that blocked IGF-I-induced tumor cell migration, proliferation and survival in vitro. Following virus injection in vivo, viral DNA was detectable by PCR in several host organs, particularly the liver, and this resulted in the production of measurable sIGFIR plasma levels for up to 21 days post injection. In mice producing virus-encoded sIGFIR, experimental liver metastasis was inhibited, indicating that sIGFIR levels were therapeutically effective. The results show that adenovirus-based delivery of inhibitory soluble proteins can provide an effective anticancer strategy.

Retracted] Restoration of Critical‐Sized Defects in the Rabbit Mandible Using Autologous Bone Marrow Stromal Cells Hybridized with Nano‐β‐tricalcium Phosphate/Collagen Scaffolds

Nano‐β‐tricalcium phosphate/collagen (n‐β‐TCP/Col) is considered with good osteoconductivity. However, the therapeutic effectiveness of n‐β‐TCP/Col scaffolds in combination with autologous bone marrow stromal cells (BMSCs) remains to be elucidated for the repair of critical‐sized bone defects. In this study, we found that n‐β‐TCP/Col scaffolds exhibited high biocompatibility in vitro. The introduction of BMSCs expanded in vitro to the scaffolds dramatically enhanced their efficiency to restore critical‐sized bone defects, especially during the initial stage after implantation. Collectively, these results suggest that autologous BMSCs in n‐β‐TCP/Col scaffolds have the potential to be applied in bone tissue engineering.

Safety of Autologous Bone Marrow Stromal Cell Transplantation in Dogs with Acute Spinal Cord Injury

To assess the feasibility and safety of transplantation of autologous bone marrow stromal cell (BMSC) in dogs with acute spinal cord injury (SCI). An open-label single-arm trial. Dogs (n = 7) with severe SCI from T6 to L5, caused by vertebral fracture and luxation. Decompressive and stabilization surgery was performed on dogs with severe SCI caused by vertebral fracture and luxation. Autologous BMSCs were obtained from each dog's femur, cultured, and then injected into the lesion in the acute stage. Adverse events and motor and sensory function were observed for >1 year after SCI. Follow-up was 29-62 months after SCI. No complications (eg, infection, neuropathic pain, worsening of neurologic function) were observed. Two dogs walked without support, but none of the 7 dogs had any change in sensory function. Autologous BMSC transplantation is feasible and safe in dogs with acute SCI. Further studies are needed to determine the efficacy of this therapy.

Bone Substitute Effect on Vascularization and Bone Remodeling after Application of phVEGF165 Transfected BMSC.

VEGF (vascular endothelial growth factor) promotes vascularization and remodeling of bone substitutes. The aim of this study was to examine the effect of distinct resorbable ceramic carriers on bone forming capacities of VEGF transfected bone marrow stromal cells (BMSC). A critical size defect of the radius in rabbits was filled either by a low surface scaffold called beta-TCP (tricalciumphsphate) or the high surface scaffold CDHA (calcium deficient hydroxy-apatite) loaded with autologous BMSC, which were either transfected with a control plasmid or a plasmid coding for phVEGF165. They were compared to unloaded scaffolds. Thus, six treatment groups (n = 6 in each group) were followed by X-ray over 16 weeks. After probe retrieval, the volume of new bone was measured by micro-CT scans and vascularization was assessed in histology. While only minor bone formation was found in both carriers when implanted alone, BMSC led to increased osteogenesis in both carriers. VEGF promoted vascularization of the scaffolds significantly in contrast to BMSC alone. Bone formation was increased in the beta-TCP group, whereas it was inhibited in the CDHA group that showed faster scaffold degradation. The results indicate that the interaction of VEGF transfected BMSC with resorbable ceramic carrier influences the ability to promote bone healing.

Autotransplantation of immobilized stromal bone marrow cells during lumbar spondylosyndesis in an experiment

Aim. To assess the ability of immobilized autologous bone marrow stromal cells to provide an effective repair with the formation of a robust bone block in the area of the interbody defect of the spinal motor segment after total removal of the intervertebral disc. Methods. Studied were the effects of autotransplantation of different cell fractions isolated from the bone marrow (mononuclear, stromal cells and stromal stem cells) on the formation of the anterior bone block during interbody spondylosyndesis in an experiment on 24 mongrel dogs of different sexes (mean weight 18.3 kg, the mean diameter of the lumbar vertebral bodies according to radiographic study 18.6 mm). All animals underwent the crest puncture the right wing of the ilium, with aspiration of 10 ml of bone marrow, conducted was a total diskectomy at the level of the bodies of the VII and VIII lumbar vertebras with replacement of the interbody defect with a porous metal cylinder of the Nickel titanium of an appropriate shape and size. All animals were randomly divided into four groups, 6 in each. In the first group the interbody defect was filled with a porous metal cylinder only, in the second group - with the fraction of bone marrow mononuclear cells, in the third - with the fraction of stromal cells, in the fourth - with the fraction of culture-grown stromal cells. Results. In the first (control) group established were the signs of delayed formation of the anterior bone block, formation of the hypertrophic interbody pseudarthrosis. Noted was the formation of coarse fibrous capsule in the zone of «implant-bone» contact, an excess of cartilage tissue, the small size and low proliferative activity of osteoblasts, hypoplasia of the blood vessels. In the second group determined was the forming artificial bone block with paravertebral hyperostosis, radiologically - microdislocation of the implant with protrusion into the body of the adjacent vertebra and partial osteointegration with the porous structure of the fixer in the contact «implant-bone» zone. Bone formation occurred by the type of secondary osteogenesis with an excess of cartilage tissue. In the third and fourth groups formed was a robust artificial bone block, no signs of implant dislocation were observed. Determined was the full osteointegration of the porous structure of the fixer in the contact «implant-bone» zone. The newly formed bone tissue surrounded the implant in a muft-like fashion and grew into its porous structure, grew into the periosteal zone of adjacent vertebral bodies, well adhering with their surface. Conclusion. The proposed method of correction of osteoreparation disorders can be considered as an alternative to osteoplastic operations with the transplantation of massive bone grafts, as well as in conditions of systemic disorders of the ostepoetic function with a critical shortage of endogenous progenitors of bone cells.

Administration of cultured autologous bone marrow stromal cells into cerebrospinal fluid in spinal injury patients: A pilot study

To determine whether intrathecal administration of cultured autologous bone marrow stromal cells (BMSCs) is safe and feasible for treatment of subacute spinal injury. Five patients with complete tetraplegia due to cervical spinal injury on admission were included. A small amount of bone marrow was obtained during surgery for spinal fusion. BMSCs were cultured, reaching 107-108 cells. The properties and functional efficacy of the BMSCs were verified with surface marker analysis and a neurite extension test. BMSCs were administered by lumbar puncture. The patients were closely observed for 6 months, and the Committee on Effectiveness and Safety of Clinical Treatment (CESCT) evaluated safety. No adverse responses were observed in biochemical and radiographic examinations. The CESCT did not recognize any harmful effects of the transplantation, and concluded it was safe for treatment. The patients were further followed up for 1 to 4 years with no adverse responses. The recovery of American Spinal Injury Association Impairment Scale (AIS) B and C patients at transplantation was rapid and remarkable, but gradual or limited in AIS A patients. This study demonstrated that intrathecal administration of cultured autologous BMSCs is safe and feasible for treatment of spinal cord injury.

骨髄間質細胞を用いた犬の脊髄損傷治療への臨床応用

骨髄間質細胞を用いた犬の脊髄損傷治療への臨床応用

Stem Cell and Tissue Engineering Applications in Orthopaedics and Musculoskeletal Medicine

Stem Cell and Tissue Engineering Applications in Orthopaedics and Musculoskeletal Medicine

Matrix Metalloproteinases (MMPs) Activity in Cultured Canine Bone Marrow Stromal Cells (BMSCs)

Autologous bone marrow stromal cells (BMSCs) infusion therapy improves the hepatic fibrosis. To investigate the mechanism of remission, we evaluated the matrix metalloproteinase (MMP)-2 and -9 activity in canine BMSCs and the effect of pro-inflammatory cytokines on their expression. The activity and the gene expression of MMPs were analyzed by gelatin zymography and quantitative RT-PCR, respectively. The specific gelatinase bands were indicative effect of MMP-2 and -9 in canine BMSCs. MMP-2 expression seemed to be increased by TNF-α and IL-1β while MMP-9 was enhanced by TNF-α and IL-6. These results suggested that remissive effect on liver fibrosis might be partly attributable to the MMP-2 and -9 activity in BMSCs under the inflammatory condition.

Relevance of deep decortication and vascularization in a case of post-traumatic femoral non-union treated with grafts, platelet gel and bone marrow stromal cells

A male patient suffering from non-union of the femoral diaphysis after a traumatic fracture was treated with deep decortication and grafted with lyophilized bone, platelet gel (PG) and autologous bone marrow stromal cells (BMSCs). After 40 days from surgery, he was re-operated, due to fracture secondary displacement, caused by inappropriate load during sports activity. In addition to radiographs, two bone biopsies were retrieved: this allowed for a histological evaluation of the early response of host bone to the graft. To our knowledge, there is no report describing such early tissue response. A clinical-radiographic evaluation of the patient and a histomorphometric analysis of the bone biopsies were performed. An early reparative bone formation was observed adjacent to the osteointegrated graft. Non-resorbed bone chips and large islands of non-vital bone particles, surrounded by fibrous tissue, were observed in a zone of sclerotic diaphyseal bone, that is the process was delayed despite decortication. These findings support the concept, until now evidenced only by imaging, that bone chips added with PG and BMSCs are effective in shortening the healing time in fracture non-union. The clinical relevance of deep decortication and vascularization is emphasized. Therapeutic studies-investigating the results of treatment, Level V.

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.

The study of bone marrow stromal stem cells transplantation via coronary artery after acute myocardial infarction in the dogs

ObjectiveTo investigate the capability of autologous bone marrow stromal stem cells for repair of damaged myocardium through the establishment of acute myocardial infarction model and the inducement of bone marrow...